Search Results (24)

Ginger (Zingiber officinale), a globally grown and economically valuable plant, has inadequate research on germplasm cryopreservation, and droplet-vitrification is yet to be applied. The present study established an efficient droplet-vitrification protocol for Z. officinale ‘Yunnan Xiaohuangjiang’. The droplet-vitrification procedure was as follows: excise 1.5–2.0 mm shoot tips with 3–4 leaf primordia from five-week-old cultures, preculture on MS medium with 0.25 M sucrose for 1 d, treat with MS liquid medium with 2 M glycerol and 0.4 M sucrose for 20 min, dehydrate with PVS2 plus 0.1 M ascorbic acid at 0 °C for 20 min, plunge into LN for 1 h, thaw in MS liquid medium with 1.2 M sucrose for 20 min, post-culture on shoot recovery medium (MS with 0.1 g/L GA₃) in the dark for 3 d. Histological and ultrastructural analyses revealed that PVS + ascorbic acid-treated shoot tips exhibited numerous living cells with small vacuoles in the apical dome, leaf primordia, and basal parts. Genetic stability results showed that the plantlets regenerated from cryopreserved shoot tips had no genetic variation. This is the first report on ginger cryopreservation via droplet-vitrification, providing technical support for ginger germplasm cryopreservation and virus elimination cryotherapy in ginger. Full article

Rhodomyrtus psidioides (G.Don) Benth. (Myrtaceae) is a critically endangered rainforest species from the east coast of Australia, where populations have severely and rapidly declined due to the effects of repeated myrtle rust infection. With very limited material available in the wild and freezing-sensitive seeds that have prevented storage in a seed bank, ex situ conservation of this exceptional species has proven difficult. Material from a seed orchard grown at the Australian Botanic Garden Mount Annan was successfully used to initiate three new accessions into tissue culture from cuttings, and to undertake cryopreservation experiments using a droplet-vitrification (DV) protocol for both seeds and cultured shoot tips. Use of seedling material for tissue culture initiation was very effective, with a 94–100% success rate for semi-hardwood explants and a 50–62% success rate for softwood explants. Although no survival of seeds after cryopreservation was observed, seeds of R. psidioides showed some tolerance of desiccation and exposure to cryoprotective agents. Regeneration after cryopreservation using a DV protocol was demonstrated in only one shoot tip precultured on basal medium containing 0.4 M sucrose and incubated in PVS2 for 20 min prior to immersion in liquid nitrogen. These results demonstrate the value of living collections in botanic gardens for conservation research, highlight the importance of germplasm choice for tissue culture initiation, and demonstrate the potential of cryobiotechnologies for the ex situ conservation of exceptional plant species. Full article

Low-temperature (LT) stress seriously affects the distribution, seedling survival, and grain yield of maize. At the seedling emergence stage, maize’s coleoptile is one of the most sensitive organs in sensing LT signaling and, in general, it can envelop young leaves to protect them from LT damage. In addition, brassinolides (BRs) have been shown to enhance LT tolerance from various species, but the effects of BRs on coleoptiles in maize seedlings under LT stress are unclear. Therefore, in this study, the pre-cultured coleoptiles of Zheng58 seedlings were treated with or without 2.0 μM 24-epibrassinolide (EBR) at 25 °C and 10 °C environments for five days to analyze their physiological and transcriptomic changes. Physiological analysis showed that a 10°C LT stress increased the content of glucose (0.43 mg g⁻¹ FW), sucrose (0.45 mg g⁻¹ FW), and starch (0.76 mg g⁻¹ FW) of Zheng58 coleoptiles compared to a 25°C environment. After the coleoptiles were exposed to a 2.0 μM EBR application under 10°C temperature for five days, the contents of these three sugars continued to increase, and reached 2.68 mg g⁻¹ FW, 4.64 mg g⁻¹ FW, and 9.27 mg g⁻¹ FW, respectively, indicating that sugar signaling and metabolism played key roles in regulating LT tolerance in the coleoptiles of maize seedlings. Meanwhile, a transcriptome analysis showed that 84 and 15 differentially expressed genes (DEGs) were enriched in the sucrose and starch metabolism and photosynthesis pathways, respectively, and multiple DEGs involved in these pathways were significantly up-regulated under LT stress and EBR stimulation. Further analysis speculated that the four DEGs responsible for sucrose-phosphate synthetase (SPS, i.e., Zm00001d048979, probable sucrose-phosphate synthase 5 and Zm00001d012036, sucrose-phosphate synthase 1), sucrose synthase (SUS, Zm00001d029091, sucrose synthase 2 and Zm00001d029087, sucrose synthase 4) were crucial nodes that could potentially link photosynthesis and other unknown pathways to form the complex interaction networks of maize LT tolerance. In conclusion, our findings provide new insights into the molecular mechanisms of exogenous EBR in enhancing LT tolerance of maize seedlings and identified potential candidate genes to be used for LT tolerance breeding in maize. Full article

The objective of this work was to assess the suitability of the Droplet-vitrification protocol previously developed with Agave peacockii shoot tips for the cryopreservation of six Agave species. Shoot tips were precultured for 1 day on a medium with 0.3 M sucrose in the dark, loaded in a solution with 1.6 M glycerol and 0.4 M sucrose for 20 min, and dehydrated by exposure to Plant Vitrification Solution 2 (PVS2) at 0 °C for 20 min. Complementary studies using histological analysis, Differential scanning calorimetry (DSC), and evaluation of morphological characteristics in cryo-derived plants were performed. Survival rates ranged from 84% to 100% and from 76% to 97% before and after cryopreservation regardless of the Agave species belonging to two taxonomic subgenera. Thermal analysis of shoot tips subjected to the successive steps of the Droplet-vitrification protocol identified ice crystal formation after loading treatment and glass transition after osmotic dehydration with PVS2. The average glass transition temperature (Tg) was −55.44 °C based on the results of four Agave species. The histological studies showed the anatomical differences that could be found in the meristematic structures depending on the loss of apical dominance. This is the most advanced research on cryopreservation of Agave shoot tips. Full article

Cryopreservation, storing biological material in liquid nitrogen (LN, −196 °C), offers a valuable option for the long-term conservation of non-orthodox seeds and vegetatively propagated species in the sector of agrobiodiversity and wild flora. Although the large-scale cryobanking of germplasm collections has been increasing worldwide, the wide application of cryopreservation protocols in wild flora is hampered by difficulties in vitro propagation and a lack of universal cryopreservation protocols, among others. This study established a systematic approach to developing an in vitro culture and droplet-vitrification cryopreservation procedure for shoot tips of Scrophularia kakudensis. The standard procedure includes a two-step preculture with 10% sucrose for 31 h and with 17.5% sucrose for 16 h, osmoprotection with loading solution C4-35% (17.5% glycerol + 17.5% sucrose, w/v) for 30 min, cryoprotection with A3-80% (33.3% glycerol + 13.3% dimethyl sulfoxide + 13.3% ethylene glycol + 20.1% sucrose, w/v) at 0 °C for 60 min, and cooling and rewarming using aluminum foil strips. After unloading, a three-step regrowth procedure starting with an ammonium-free medium with growth regulators was essential for developing normal plantlets from cryopreserved shoot tips. Liquid overlay on the gelled medium two weeks after inoculation resulted in vigorous growth during subcultures. Moreover, liquid overlay increased LN regeneration by up to 80%, i.e., 23% higher than no liquid overlay. Full article

Over 30 years of plant vitrification, droplet vitrification (DV) of in vitro propagules and slow freezing of dormant buds are typical methods of large-scale cryobanking worldwide. One-step sucrose preculture and Plant Vitrification Solution 2 (PVS2) cryoprotection in solution-based vitrification often face unacceptably low regeneration, and the results are on a case-by-case basis depending on the plant species, like a blind test. The absence of a universal protocol applicable across all plant diversity is considered one of the limiting factors. For wild flora, limits of source material available and difficulties in in vitro propagation make it worse to re-optimize the protocol steps for new species. Since cryoprotectant toxicity is the most crucial barrier to the vitrification of organized explants, selecting alternative plant vitrification solutions (PVS) based on the cytotoxicity of cryoprotectants is vital. This review proposes the concept of donor plant vigor (DPV), which refers to the donor plant properties that determine the potential to regenerate normal plantlets under various cryopreservation procedures. DV is a multi-stage procedure with many factors from stage (1) material preparation to (2) pre-liquid nitrogen (pre-LN) (preculture, osmoprotection, cryoprotection), (3) LN (cooling), (4) warming conditions (rewarming, unloading), and (5) regrowth. Since the cytotoxicity of PVS is a primary limiting factor in DV approaches, DPV is crucial for coping with the toxicity of PVS. The DPV is innate and can be maximized with appropriate material preparations, i.e., vigorously growing in subcultures aided by a liquid overlay on top of the gelled medium, selecting proper explants, optimizing the two-step preculture conditions, and media supplements. Developing the DV protocol starts with testing the material with a tentative standard protocol, which includes a two-step preculture (10% sucrose for 31 h and 17.5% sucrose for 16 h), osmoprotection with C4-35%, cryoprotection with A3-80% (60 min at 0 °C), cooling, and rewarming using aluminum foil strips. Using a three-step regrowth initially with ammonium-free regrowth medium, regrowth of shoot tips in one plate following the successive stages of the tentative standard protocol for shoot tips, i.e., fresh, PC, OP, CP (LNC), and LN, is a valuable tool to characterize the sensitivity of the material and to standardize the procedure by tuning the cryoprotection and cytotoxicity of cryoprotectants. A-series PVS (A3-90%, A3-80%, A3-70%) and B-series PVS (PVS3, B5-85%) can be tested based on the DPV. These alternative PVSs have been applied in over 30 pieces of literature with an 8.5~67.3% increase in LN regeneration compared to PVS2 and Plant Vitrification Solution 3 (PVS3) treatments. Using this approach as an alternative to blind condition screening would be influential in broadening the cryopreservation of diverse wild species and problem materials. Full article

Cryopreservation in liquid nitrogen (LN, −196 °C) is a unique option for the long-term conservation of threatened plant species with non-orthodox or limitedly available seeds. In previous studies, a systematic approach was used to develop a droplet-vitrification (DV) cryopreservation protocol for Postemon yatabeanus shoot tips that includes preculture with 10% sucrose, osmoprotection with C4-35%, cryoprotection with A3-80% vitrification solution, and a three-step regrowth starting with the ammonium-free medium. The tricarboxylic acid (TCA) cycle is a crucial component of plant cell metabolism as it is involved in redox state regulation and energy provision. We hypothesized that organic acids (OAs) associated with the TCA and its side reactions indirectly indicate metabolism intensity and oxidative stress development in shoot tips under the cryopreservation procedure. In this study, the contents of 14 OAs were analyzed using gas chromatography–tandem mass spectrometry (GC-MS/MS) in P. yatabeanus shoot tips in a series of treatments including individual steps of the DV procedure, additional stress imposed by non-optimum protocol conditions (no preculture, no osmoprotection, various vitrification solution composition, using vials instead of aluminum foils, etc.) and regrowth on different media with or without ammonium or growth regulators. The possible relation of OA content with the total cryoprotectant (CPA) concentration and shoot tips regeneration percentage was also explored. Regeneration of cryopreserved shoot tips reduced in descending order as follows: standard protocol condition (91%) > non-optimum vitrification solution (ca. 68%) > non-optimum preculture (60–62%) > regrowth medium (40–64%) > no osmoprotection, cryopreservation in vials (28–30%). Five OAs (glycolic, malic, citric, malonic, and lactic) were the most abundant in the fresh (control) shoot tips. The dynamic pattern of OAs during the DV procedure highly correlated (r = 0.951) with the total CPA concentration employed: it gradually increased through the preculture, osmoprotection, and cryoprotection, peaked at cooling/rewarming (6.38-fold above control level), and returned to the fresh control level after 5 days of regrowth (0.89-fold). The contents of four OAs (2-hydroxybutyric, 3-hydroxypropionic, lactic, and glycolic) showed the most significant (10-209-fold) increase at the cooling/rewarming step. Lactic and glycolic acids were the major OAs at cooling/rewarming, accounting for 81% of the total OAs content. The OAs were categorized into three groups based on their dynamics during the cryopreservation protocol, and these groups were differently affected by protocol step modifications. However, there was no straightforward relationship between the dynamics of OAs and shoot tip regeneration. The results suggest that active modulation of OAs metabolism may help shoot tips to cope with osmotic stress and the chemical cytotoxicity\ of CPAs. Further intensive studies are needed to investigate the effect of cryopreservation on cell primarily metabolism and identify oxidative stress-related biomarkers in plant materials. Full article

The objective of this study is to assess the suitability of vitrification cryo-plate (V cryo-plate) and dehydration cryo-plate (D cryo-plate) methods for the long-term conservation of eight autochthonous Prunus domestica L. genotypes originating from the Balkan Peninsula region. In vitro shoot tips were briefly pre-cultured for 1 day at 23 °C in the dark on a medium containing 0.3 M sucrose and then embedded in calcium alginate gel within the wells of the aluminum cryo-plates. In the V cryo-plate protocol, dehydration was carried out at room temperature using the following vitrification solutions: original plant vitrification solution 2 (PVS2) and 90% PVS2 solution (for 20 and 40 min) and plant vitrification solution 3 (PVS3) (for 60 and 80 min). In the D cryo-plate protocol, desiccation was performed for 2, 2.5, or 3 h over silica gel at 23 °C. The effect of different treatments was evaluated by monitoring the regrowth of both non-frozen and cryo-preserved explants. After cryo-preservation, five genotypes achieved regrowth rates over 40% in at least one of the applied protocols, while two genotypes showed regrowth rates of around 10%. A significant improvement in regrowth success for all genotypes using both cryo-plate methods was achieved by pre-culturing shoot tips for 7 days on a medium containing 0.5 M sucrose in complete darkness at 4 °C. Shoots regenerated from cryo-preserved explants were further monitored in vitro. By the third subculture, they had not only regained but had even exceeded the multiplication capacity (index of multiplication, length of axial, and lateral shoots) of shoots regenerated from dissection controls. Following multiplication, the cryo-preserved shoots were successfully rooted and rooting ability was assessed by monitoring the percentage of rooting, number and length of roots, and height of rooted plantlets. Full article

Cryopreservation is considered the safe and efficient strategy for the long-term conservation of embryogenic cultures. The objective of this study was to cryopreserve the embryogenic tissues of hybrid larch to overcome the result raised by rapid growth rates of conifer embryogenic cultures necessitating frequent sub-culturing. We systematically evaluated several parameters, including the pre-culture method (liquid or solid), osmoprotectant type (DMSO, sucrose, or PEG6000), duration of cryoprotection (1–3 h), and thawing temperature (4 °C, 25 °C, or 40 °C). After one month of cryopreservation, we assessed the regeneration efficiency and maturation ability of both cryo-preserved and non-cryopreserved tissues. Our optimized protocol involves pre-culturing embryonic tissue on the solid medium with 0.4 M sorbitol for 48 h, followed by treatment with 10% DMSO, 0.4 M sucrose, and 15% PEG6000 for 1 h on ice, and immersion in liquid nitrogen with rapid thawing at 40 °C. Notably, the use of solid media during pre-culturing was crucial to enhancing the success rate of cryopreservation. Using protocol optimization, we achieved high embryogenic tissue survival rates of over 80% without affecting the ability of somatic embryogenesis. This work provides a comprehensive set of steps for routine cryopreservation of embryogenic tissues for long-term conservation in hybrid larch, along with sample protocols for cryopreservation of larch. The results demonstrate that vitrification is a reliable method for preserving embryogenic tissues of hybrid larch with broader implications for the cryopreservation of other plant species. Further optimization and standardization of protocols across different species would ensure the preservation of genetic diversity and facilitate future research in plant biotechnology that benefits human health, food security, and environmental sustainability. Full article

Oil palm (Elaeis guineensis) is the highest oil-yielding commercially grown perennial tree. Oil palm germplasm conservation and in vitro clonal propagation strengthened the world’s efforts to ensure future food security. Cryopreservation provides long-term storage for germplasm. The storage of plant material at cryogenic temperatures (−196 °C) following dehydration causes cryoinjury. The cryotolerance mechanism has rarely been studied in oil palm zygotic embryos (ZE) and embryogenic calli (EC). A simple and effective cryopreservation method was established for ZE. ZE surrounded by endosperm was air-dried for 3 days without any complicated chemical pre-treatments before cryopreservation, while the viability rate and following germination rate could reach up to 96.67% and 90.88%, respectively. As for EC, the preferred method could be pre-culture in liquid MS medium with 0.3 M sucrose for 12 h and PVS2 treatment for 5 min prior to cryopreservation, and the viability rate reached 68.33%. SSR markers were used to verify the genetic stability after cryopreservation. In addition, changes in enzyme activities (CAT, POD, and SOD) showed a consistent trend with H₂O₂ production among ZE samples, indicating that these antioxidants were involved in ROS scavenging. Furthermore, differently expressed genes (DEGs) related to ROS, osmotic, and cold stress responses were selected for correlation network analysis. Most genes involved in ROS production (RBOH, PAO, and PRX) and ROS scavenging (APX, PER, SOD, CAT, GPX, and AOX) showed higher expression levels in EC, suggesting that EC was more sensitive to oxidative stress than ZE. The cryotolerance mechanism was further summarized accordingly. These results contributed to cryopreservation methods and provided a better understanding of cryotolerance in oil palm. Full article

Cryopreservation approaches have been implemented in gene banks as a strategy to back up plant genetic resource collections that are vegetatively propagated. Different strategies have been employed to effectively cryopreserve plant tissue. There is little information on the cellular processes and molecular adjustments that confer resilience to the multiple stresses imposed during a cryoprotocol. In the present work, the cryobionomics of banana (Musa sp.), a non-model species, was investigated through the transcriptomic approach using RNA-Seq. Proliferating meristems of in vitro explants (Musa AAA cv ‘Borjahaji’) were cryopreserved using the droplet-vitrification technique. Transcriptome profiling analysis of eight cDNA libraries including the bio-replicates for T0 (stock cultures (control tissue), T1 (high sucrose pre-cultured), T2 (vitrification solution-treated) and T3 (liquid nitrogen-treated) meristem tissues was carried out. The raw reads obtained were mapped with a Musa acuminata reference genome sequence. A total of 70 differentially expressed genes (DEGs) comprising 34 upregulated and 36 downregulated were identified in all three phases as compared to control (T0). Among the significant DEGs (>log FC 2.0), during sequential steps, 79 in T1, 3 in T2 and the 4 in T3 were upregulated and 122 in T1, 5 in T2 and 9 in T3 were downregulated. Gene ontology (GO) enrichment analysis showed that these significant DEGs were involved in the upregulation of biological process (BP-170), cellular component (CC-10) and molecular function (MF-94) and downregulation of biological process (BP-61), cellular component (CC-3) and molecular function (MF-56). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that DEGs were involved in the biosynthesis of secondary metabolites, glycolysis/gluconeogenesis, MAPK signaling, EIN 3-lke 1 protein, 3-ketoacy-CoA synthase 6-like, and fatty acid elongation during cryopreservation. For the first time, a comprehensive transcript profiling during four stages of cryopreservation in banana were carried out, which will pave the way for devising an effective cryopreservation protocol. Full article

Cryopreservation, storing biological material in liquid nitrogen (LN, −196 °C), offers a valuable option for the long-term conservation of non-orthodox seeds and vegetatively propagated species in the sector of agrobiodiversity and wild flora. Although large-scale cryobanking of germplasm collections has been increasing worldwide, the wide application of cryopreservation protocol is hampered by a lack of universal cryopreservation protocols, among others. This study established a systematic approach to developing a droplet-vitrification cryopreservation procedure for chrysanthemum shoot tips. The standard procedure includes two-step preculture with 10% sucrose for 31 h and with 17.5% sucrose for 16 h, osmoprotection with loading solution C4-35% (17.5% glycerol + 17.5% sucrose, w/v) for 40 min, cryoprotection with alternative plant vitrification solution A3-80% (33.3% glycerol + 13.3% dimethyl sulfoxide + 13.3% ethylene glycol + 20.1% sucrose, w/v) at 0 °C for 60 min, and cooling and rewarming using aluminum foil strips. After unloading, a three-step regrowth procedure starting with an ammonium-free medium with 1 mg L⁻¹ gibberellic acid (GA₃) and 1 mg L⁻¹ benzyl adenine (BA) followed by an ammonium-containing medium with and without growth regulators was essential for the development of normal plantlets from cryopreserved shoot tips. A pilot cryobanking of 154 accessions of chrysanthemum germplasm initiated with post-cryopreservation regeneration of 74.8%. This approach will facilitate the cryobanking of the largest Asteraceae family germplasm as a complementary long-term conservation method. Full article

In order to simplify the experimental procedure and treatment procedure, we preserved the embryonic callus (EC) of Fraxinus mandshurica more efficiently. In this paper, we established a method for cryopreservation of EC of F. mandshurica by vitrification. EC was subcultured for 7–10 days (d). Vigorous EC with good growth conditions were selected, and cryopreservation was performed by vitrification. The best pre-culture method was to pre-culture EC on 0.5 mol·L⁻¹ sucrose medium for 3 d, load and culture in the liquid woody plant medium (WPM) supplemented with 2 mol·L⁻¹ glycerol and 0.4 mol·L⁻¹ sucrose for 60 min, then dehydrate in 2 mL of plant vitrification solution 2 (PVS2) (30% glycerol + 15% dimethyl sulfoxide (DMSO) + 15% ethylene glycol + 0.4 mol·L⁻¹ sucrose + liquid WPM). EC was rewarmed in a 40 °C water bath for 2 min after cooling in liquid nitrogen. The procedure for cryopreservation of F. mandshurica EC by the vitrification method established in this experiment is relatively reliable. The results from the present study provide a technical reference for improving the cryopreservation of F. mandshurica EC. Full article

Picea pungens (Engelm.), known for its blue-green needles, has become a likable ornamental species in northeast China since 2000. Nonetheless, a lack of propagation methods that can maintain genetic fidelity and develop seedlings at a large scale prevents the further expansion of the species. Somatic embryogenesis (SE), paired with cryopreservation technologies, may provide a valid alternative. Picea pungens SE is not new, but its practical application has been limited due to low efficiencies in SE initiation and maturation as well as a lack of effective cryopreservation technology. In this study, experiments were carried out to overcome the limitations by modifying culture media. For initiation, the efficiency was enhanced by adjusting concentrations of 2.4-dichlorophenoxy acetic acid (2,4-D), 6-benzyl amino–purine (6-BA) or sucrose supplemented to the induction medium. The concentrations of 4.0 mg/L 2,4-D, 2 mg/L 6-BA, and 5 to 10 g/L sucrose were found optimal in maximizing initiation efficiency. For maturation, the efficiency, expressed as the number of mature somatic embryos per gram of fresh mass cultured (E/gFM), varied greatly with the choices of the basal medium and concentration of abscisic acid (ABA) of the maturation medium. Based on our results, the judicial choices were using the DCR medium as the basal medium and 10 mg/L ABA. The maturation efficiency could also be improved by adjusting the maturation medium’s osmotic pressure by manipulating the concentrations of carbohydrate and Gelrite and culture density. While the maturation medium, using sucrose as carbohydrate source or supplemented with a low (<8 g/L) Gelrite concentration, facilitated maturation, optimal selections were truly genotype-dependent. Our results also suggest that, while the optimal culture density varied with genotype, in general it is needless to culture more than 100 mg embryogenesis tissues per dish (size: 10 × 1.5 cm). Based on this study, the optimum pretreatment for embryogenesis tissue cryopreservation was culturing the tissues on the proliferation medium with 0.4 mol/L sorbitol for 24 h, followed by treatment with 5% Dimethyl sulfoxide. This study significantly improved the initiation (achieved a frequency of 0.56) and embryo maturation efficiencies (achieved 1030 E/gFM) and established an effective preculturing protocol for cryopreservation (recovered 1354 E/gFM) for the species. The protocols developed here, paired with the available ones for other SE steps in the literature, form a well-refined SE technology intended for commercial application to Picea pungens. Full article

This study provides alternative approaches toward ex situ conservation by means of in vitro seed germination and the multiplication of Penthorum chinense Pursh using nodal explants. An overlay of a liquid medium on top of a gelled medium significantly increased the growth of shoots and roots, while the presence of activated charcoal or growth regulators (benzyl adenine and α-naphthaleneacetic acid) decreased the growth. Shoot tips of in vitro plantlets were cryopreserved using a droplet-vitrification method. The standard procedure included preculture with 10% sucrose for 31 h and with 17.5% sucrose for 17 h, osmoprotection with loading solution C4-35% (17.5% glycerol + 17.5% sucrose, w/v) for 20 min, cryoprotection with alternative plant vitrification solution (PVS) A3-70% (29.2% glycerol + 11.7% DMSO + 11.7% EG + 17.4% sucrose, w/v) at 0 °C for 30 min, cooling the samples in liquid nitrogen using aluminum foil strips and rewarming by plunging into pre-heated (40 °C) unloading solution (35% sucrose) for 40 min. A three-step regrowth procedure starting with ammonium-free medium followed by ammonium-containing medium with and without growth regulators was essential for the regeneration of cryopreserved shoot tips. The species was found to be very sensitive to the chemical cytotoxicity of permeating cryoprotectants during cryoprotection and to ammonium-induced oxidant stress during initial regrowth steps. Improvement of donor plant vigor by using apical sections and liquid overlay on top of the solid medium for propagation, improved shoot tip tolerance to osmotic stress and increased post-cryopreservation regeneration up to 64% were observed following PVS B5-85% (42.5% glycerol + 42.5% sucrose) treatment for 60 min. The systematic approach used in this study enables fast optimization of the in vitro growth and cryopreservation procedure for a new stress-sensitive wild plant species. Full article