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.     

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.     

Global DNA methylation profiles of somatic embryos of peach palm (Bactris gasipaes Kunth) are influenced by cryoprotectants and droplet-vitrification cryopreservation

Regrowth capacity and genetic stability of plants recovered following cryopreservation are associated with changes in DNA epigenetics, particularly in DNA methylation levels. In this study, global DNA methylation profiles associated with frequency of regrowth of peach palm (Bactris gasipaes) somatic embryos following cryopreservation using droplet-vitrification were investigated. Somatic embryo clusters (SEC) subjected to plant vitrification solution 3 (PVS3) for different durations (0, 60, 120, 180, and 240 min) were evaluated for regrowth capacity. The highest frequency of regrowth (52.4 %) was obtained when SEC were incubated in PVS3 for 120 min prior to droplet-vitrification cryopreservation. Global DNA methylation profiles were influenced by both cryoprotectants and droplet-vitrification cryopreservation. Incubation of SEC in PVS3 for limited durations not only reduced frequency of regrowth, but also increased DNA methylations levels when compared with proliferating SEC grown in a temporary immersion system. Although SEC subjected to cryopreservation exhibited the highest DNA methylation variation, 120 min SEC incubation in a PVS3 solution resulted in the recovery of initial global methylation profiles after 24 weeks of regrowth.     

Droplet-vitrification methods for apical bud cryopreservation of yacon [Smallanthus sonchifolius (Poepp. and Endl.) H. Rob.]

This study aimed to develop a cryopreservation protocol for the long-term preservation of yacon [Smallanthus sonchifolius (Poepp. and Endl.)], an Andean crop with high fructooligosaccharide content in its tuberous roots. Initially, the cryopreservation protocol was developed using a yacon clone originated from Ecuador classified as ECU 41. Osmotic dehydration of apical buds (2–3 mm long) was carried out by assessing two plant vitrification solutions, PVS2 (15, 30, and 60 min) at 0 °C and PVS3 (30, 45, 60, and 75 min) at 22 °C. After cryopreservation, the apical buds were thawed and placed on MS medium?±?0.1 mg l^?1 N⁶-benzyladenine (BA). The survival rates ranged from 37 to 90% within all treatments, with those subjected to PVS2 and PVS3 for 60 min showing the highest survival rates on MS medium without BA (87 and 90%, respectively). At 12 weeks post cryopreservation, these treatments also provided the highest regrowth rates, both reaching 73% of normally growing (shooting, rooting) plantlets. Survival rates on MS?+?0.1 mg l^?1 BA regrowth medium reached up to 90%; however, regrowth into normally rooted plantlets did not exceed 67% post cryopreservation. The optimized protocols were then applied to 4 additional yacon clones originated from Bolivia and Peru, classified as BOL 22, BOL 23, PER 12, and PER 14. This resulted in survival and regeneration rates ranging between 79.7–94.1% and 66.3–75.4% respectively. Our study shows that optimal cryopreservation protocols for the long-term conservation of yacon can be based on both PVS2 and PVS3 vitrification solutions.

     

Thidiazuron enhances shoot organogenesis from leaf explants of Saussurea involucrata Kar. et Kir

An efficient protocol for the in vitro micrpropagation of Saussurea involucrata Kar. et Kir, an endangered Chinese medicinal plant, was developed. Shoot organogenesis was obtained following culture of leaf explants on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ). After 28 d of culture, 15.6?±?1.4 shoots were regenerated per leaf explant on MS medium containing 0.5 ??M TDZ. After transfer of shoots to a medium containing 5.0 ??M indole-3-acetic acid, approximately 80% of the regenerated shoots formed roots and whole plantlets. After transfer of rooted shoots to the greenhouse, 83% of the regenerated plantlets survived and grew vigorously. The regeneration protocol developed in this study provides a basis for germplasm conservation and for the production of plant material necessary to study the medicinally active components of S. involucrata.     

Cryopreservation of an endangered <Emphasis Type="Italic">Hladnikia pastinacifolia</Emphasis> Rchb. by shoot tip encapsulation-dehydration and encapsulation-vitrification

The objective of the present study was the cryopreservation of monotypic endemic Hladnikia pastinacifolia Rchb. shoot tips from an in vitro culture, via encapsulation-dehydration (ED) or encapsulation-vitrification (EV). For all tested genotypes, the highest rates of shoot regrowth and multiplication were obtained after overnight preculture in 0.4 M sucrose, encapsulation in Murashige and Skoog (MS) medium with 0.4 M sucrose and 1 M glycerol, followed by polymerization in 3% (w/v) Na-alginate in MS with 0.4 M sucrose. Optimal osmoprotection was achieved for ED with 0.4 M sucrose plus 1 M glycerol and for EV with 0.4 M sucrose plus 2 M glycerol. The best dehydration time for ED was 150 min in a desiccation chamber with silica gel, and the best vitrification time for EV was 85 min in plant vitrification solution 2 (PVS2). For ED, dehydration for 150 min resulted in explant water content of 22%. When the encapsulation method was combined with ED, 53% regrowth was achieved, and when it was combined with EV, 64% regrowth was achieved. Both methods could become applicable for the long-term cryopreservation of H. pastinacifolia germplasm, although EV was faster and resulted in better final regrowth success. Genetic stability analysis of cryopreserved plant samples was carried out for two genotypes, using random amplified polymorphic DNA (RAPD) markers to compare the two different cryopreservation protocols. Significant genetic differences between the genotypes were detected and a low level of genomic variation was observed.     

Cryopreservation,early seedling development,and genetic stability of <Emphasis Type="Italic">Oncidium flexuosum</Emphasis> Sims

An efficient cryopreservation protocol was developed for mature seeds of Oncidium flexuosum Sims. Seed morphology, protocorm formation, and early seedling development were also assessed. The effects of phloroglucinol and Supercool X-1000^® as cryoprotectant additives in the vitrification solution were investigated. Dehydration using the plant vitrification solution 2 (PVS2) for 60 and 120 min prior to immersion in liquid nitrogen promoted the highest frequency of in vitro seed germination 6 weeks following culture on half-strength Murashige and Skoog (½ MS) medium. Mature seeds submitted to vitrification for 120 min in PVS2 and 1 % phloroglucinol at 0 °C enhanced germination by 68 %, whereas in PVS2 and 1 % Supercool X-1000^® germination was just moderately enhanced (26 %). In vitro-germinating seedlings developed healthy shoots and roots without the use of plant growth regulators. After 6 months of growth, there were no differences between in vitro- and ex vitro-grown seedlings for various phenotypic characteristics, including shoot length, number of leaves, number and length of roots, and fresh and dry weight. Seedlings were transferred to greenhouse conditions and successfully acclimatized, further developing into normal plants with over 90 % survival. Comparative analysis of seedlings from control and vitrified seeds using flow cytometry indicated that no change in ploidy levels occurred as a result of cryopreservation, therefore maintaining seedlings genetic stability. In this study, vitrification with PVS2 for 120 min with the addition of 1 % phloroglucinol offers a simple, safe, and feasible protocol for cryopreservation of O. flexuosum mature seeds.     

Cryopreservation of immature Parkia speciosa Hassk. zygotic embryonic axes following desiccation or exposure to vitrification solution

This work reports on the cryopreservation of immature zygotic embryonic axes (EA) of petai (Parkia speciosa Hassk.) for the first time. Two cryopreservation protocols, namely desiccation and vitrification method were tested individually using excised EA. Desiccation of EA to lower moisture content (MC) reduced the survival percentage but a drastic decline in survival percentage (~20 %) was recorded at 16 % MC prior to exposure to LN, rendering the EA to be sensitive to desiccation. Cryopreservation of EA after desiccation, irrespective of the MC, did not result in any survival. On the other hand, post-cryopreservation survival was obtained when the EA were exposed to plant vitrification solution-2 (PVS2) for 75–105 min. The best results were obtained when the EA were exposed to PVS2 for 90 min with an average recovery of 55.5 %. EA recovery into whole plantlets was obtained when the EA were cultured on MS medium supplemented with 2 gl^?1 activated charcoal and 0.1 mgl^?1 of the plant growth regulators α-naphthalene acetic acid, 6-benzylaminopurine and gibberellin A₃, each. EA, exposed for less than 75 min and more than 105 min to PVS2, did not show any survival after cryopreservation. The optimization of exposure time is necessary to increase survival. This study has shown that the employment of suitable method is important for conservation using cryopreservation.     

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.     

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.     

Cryoprotectants and components induce plasmolytic responses in sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.) suspension cells

Plant genebanks often use cryopreservation to securely conserve clonally propagated collections. Shoot tip cryopreservation procedures may employ vitrification techniques whereby highly concentrated solutions remove cellular water and prevent ice crystallization, ensuring survival after liquid nitrogen exposure. Vitrification solutions can be comprised of a combination of components that are either membrane permeable or membrane impermeable within the timeframe and conditions of cryoprotectant exposure. In this study, the osmotic responses of sweet potato [Ipomoea batatas (L.) Lam.] suspension cell cultures were observed after treatment with plant vitrification solution 2 [PVS2; 15% (v/v) dimethyl sulfoxide (DMSO), 15% (v/v) ethylene glycol, 30% (v/v) glycerol, 0.4 M sucrose], plant vitrification solution 3 (PVS3; 50% (v/v) glycerol, 50% (w/v) sucrose), and their components at 25 and 0°C, as well as cryoprotectant solution, PGD (10% (w/v) PEG 8000, 10% (w/v) glucose, 10% (v/v) DMSO) at 25°C. At either 25 or 0°C, sweet potato cells plasmolyzed after exposure to PVS2, PVS3, and PGD solutions as well as the PVS2 and PVS3 solution components. Cells deplasmolyzed when the plasma membrane was permeable to the solutes and when water re-entered to maintain the chemical potential. Sweet potato suspension cells deplasmolyzed in the presence of 15% (v/v) DMSO or 15% (v/v) ethylene glycol. Sweet potato plasma membranes were more permeable to DMSO and ethylene glycol at 25°C than at 0°C. Neither sucrose nor glycerol solutions showed evidence of deplasmolysis after 3 h, suggesting low to no membrane permeability of these components in the timeframes studied. Thus, vitrification solution PVS2 includes components that are more membrane permeable than PVS3, suggesting that the two vitrification solutions may have different cryoprotectant functions. PGD includes DMSO, a permeable component, and likely has a different mode of action due to its use in two-step cooling procedures.     

Duration of sucrose preculture is critical for shoot regrowth of in vitro-grown apple shoot-tips cryopreserved by encapsulation-dehydration

A simple and efficient cryopreservation protocol using encapsulation-dehydration was established for in vitro-grown shoot-tips of apple ‘Gala’ (Malus × domestica Borkh.). Shoot-tips, of 2.0 mm in length and with 5–6 leaf primordia, excised from 4-week-old shoot stock cultures, without cold-hardening, were encapsulated into beads, each being about 5 mm in diameter and containing a single shoot-tip. The beads were precultured on MS medium containing 0.5 M sucrose for 7 days. The precultured beads were dehydrated by air-drying to reduce the water content of the beads to about 22–20 % in 5–7 h, followed by a direct immersion in liquid nitrogen for 1 h. Frozen shoot-tips were re-warmed in a water bath at 38 °C for 2 min and post-cultured on a recovery medium for shoot regrowth. This protocol was successfully applied to four Malus species and one hybrid, among which M. micromalus and M. robusta are wild species native to China. The highest and lowest shoot regeneration rates were found in ‘Gala’ (75 %) and ‘Wangshanhong’ (36 %), with a mean shoot regrowth rate of 61 % attained for the seven Malus genotypes tested. Histological studies revealed that shoots could be regenerated in cryopreserved shoot-tips only when many cells in the leaf primordia and most of the cells in the apical dome survived following cryopreservation. Morphologies of the regenerated plantlets were identical to those from the in vitro stock cultures. Therefore, the encapsulation-dehydration procedure developed in the present study should provide a technical support for setting-up Malus cryo-banking in China.     

Cryopreservation: A tool to conserve date palm in Saudi Arabia

The cryostoring of embryogenic tissue of the date palm (Phoenix dactylifera L. cv. Sagai) was examined through dehydrated-encapsulation, vitrification, and vitrification-encapsulation. The most extreme regeneration rate (53.33%) of epitomized, cryostored liquid nitrogen (+LN) treated embryos was observed when pre-embryonic masses were hatched with 0.5 M sucrose for 48 h pursued by 6 h air drying out. The most noteworthy survival rate (80.0%) of epitomized, cryopreserved embryonic cluster came about when calli were hatched with 0.3 or 0.7 M sucrose for 48 h pursued by four hours of lack of hydration, or with 0.5 M sucrose for 48 h without air drying out or with 2 h of air drying out. Following cryopreservation utilizing the embodiment vitrification convention, the most astounding survival (86.7%) as well as the greatest growth (46.7%) was accomplished when the typified vitrified, cryopreserved calli were treated with Vitrification Solution 2 for plants (PVS2) for 60 min at 25 °C. Cryopreservation utilizing the vitrification convention brought about the most extreme recuperation of 53.3%, when vitrified-cryopreserved calli were subjected to PVS2 solution for 30 min at 25 °C. Most extreme (40%) regeneration of vitrified, cryopreserved embryonic calli was seen when these calli were treated with PVS2 solution for 60 min at 25 °C. The outcome got amid this investigation of regrowth after cryopreservation of the cv. Sagai was over the base suitable for a cryo-germplasm bank. Recovery and regrowth were above 30% for all the techniques developed for the cv. Sagai.     

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 pharmaceutically important orchid Dendrobium chrysanthum Wall. ex Lindl. using vitrification based method

Our present study constitutes the successful and efficient protocol for cryopreservation of Dendrobium chrysanthum. D. chrysanthum Wall. ex Lindl. is a pharmaceutically valuable, ornamental epiphytic orchid of temperate and subtropical regions. On account of excellent herbal medicinal value and horticultural importance, D. chrysanthum is becoming rare due to over exploitation. For long-term conservation of this orchid, protocorm-like bodies of D. chrysanthum were excised and used for cryopreservation by encapsulation–vitrification. In this cryogenic procedure, PLBs were initially osmoprotected with a mixture of 0.4 M sucrose and 2 M glycerol, incorporated in the encapsulation matrix (comprising of 3 % (w/v) sodium alginate and 0.1 M CaCl₂). Encapsulated protocorm-like bodies (PLBs) were then precultured on MS liquid medium supplemented with different concentrations of sucrose (0.06, 0.3, 0.5, 0.7 M), and loaded in a loading solution (comprised of 2 M glycerol and 0.4 M sucrose) for different duration to make the precultured PLBs tolerant to plant vitrification solution 2 (PVS2). Subsequently, the PLBs were subjected to PVS2 (Sakai et al. 1990) treatment at different time of exposure (minutes) and temperatures (0 °C and 25 °C). Encapsulated–vitrified PLBs were plunged directly into liquid nitrogen and stored for 1 h. Optimum result (survival 63.2 % and regrowth 59.9 %) was obtained when the beads treated with loading solution for 80 min followed by PVS2 treatment for 100 min. Regenerated plants showed normal morphology as that of control plants.     

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 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 nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification

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.Abbreviations DMSO dimethyl sulfoxide - PVS2 vitrification solution - LN liquid nitrogen - DSC differential scanning calorimeter - BA 6-benzylaminopurine - MT Murashige-Tucker basal medium - INAA naphthaleneacetic acid     

An efficient,widely applicable cryopreservation of Lilium shoot tips by droplet vitrification

We report a straightforward and widely applicable cryopreservation method for Lilium shoot tips. This method uses adventitious shoots that were induced from leaf segments cultured for 4 weeks on a shoot regeneration medium containing 1 mg/l α-naphthaleneacetic acid and 0.5 mg/l thidiazuron. Shoot tips (1.5–2 mm in length) including 2–3 leaf primordia were precultured on Murashige and Skoog (MS; 1962) medium with 0.5 M sucrose for 1 day and then treated with a loading solution containing 0.4 M sucrose and 2 M glycerol for 20 min, followed by a Plant Vitrification Solution 2 (PVS2) treatment for 4 h at 0 °C. Dehydrated shoot tips were transferred onto 2.5 µl PVS2 droplets on aluminum foil strips, prior to a direct immersion into liquid nitrogen for 1 h. Frozen shoot tips were re-warmed in MS medium containing 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for shoot regrowth. Shoot regrowth levels ranged from 42.5 % for L. longiflorum × Oriental ‘Triumphator’ to 87.5 % for L. Oriental hybrid ‘Siberia’, with a mean shoot regrowth level of 67.1 % across the six diverse Lilium genotypes tested. Histological observations found that the survival patterns were similar in cryopreserved shoot tips of ‘Triumphator’ and ‘Siberia’. Assessments using inter-simple sequence repeat markers found no differences in regenerants recovered from the control stock cultures and from cryopreserved shoot tips in ‘Triumphator’ and ‘Siberia’. This Lilium droplet-vitrification cryopreservation method is efficient, simple and widely applicable for the long-term conservation of lily genetic resources.     

Cryopreservation of sour orange (Citrus aurantium L.) shoot tips

Summary The objective of this study was to establish a cryopreservation protocol for sour orange (Citrus aurantium L.). Cryopreservation was carried out via encapsulation-dehydration, vitrification, and encapsulation-vitrification on shoot tips excised from in vitro cultures. Results indicated that a maximum of 83% survival and 47% regrowth of encapsulated-dehydrated and cryopreserved shoot tips was obtained with 0.5M sucrose in the preculture medium and further dehydration for 6 h to attain 18% moisture content. Dehydration of encapsulated shoot tips with silica gel for 2h resulted in 93% survival but only 37% regrowth of cryopreserved shoot tips. After preculturing with 0.5M sucrose, 80% of the vitrified cryopreserved shoots survived when 2M sucrose plus 10% dimethyl sulfoxide (DMSO) was used as a cryoprotectant for 20 min at 25°C. Survival and regrowth of vitrified cryopreserved shoot tips were 67% and 43%, respectively, when 0.4M sucrose plus 2M glycerol was used as a loading solution followed by application of 100% plant vitrification solution (PVS2) for 20 min. Increased duration of exposure to the loading solution up to 60 min increased survival (83%) and regrowth (47%) of cryopreserved shoot tips. With encapsulation-vitrification, dehydration with 100% PVS2 for 2 or 3 h at 0°C resulted in 50 or 57% survival and 30 or 40% regrowth, respectively, of cryopreserved shoot tips.