Shoot regeneration and cryopreservation of shoot tips of apple (Malus) by encapsulation–dehydration

Here, we report an efficient and widely applicable method for cryopreservation of Malus shoot tips by encapsulation–dehydration using adventitious shoots. Shoots were induced from leaf segments cultured on a shoot induction medium containing 2–3 mg L^?1 thidiazuron, depending on genotype, and 0.5 mg L^?1 indole-3-butyric acid. Shoot tips (3 mm in length) containing six leaf primordia excised from 11-wk-old adventitious shoots were encapsulated and precultured with 0.5 M sucrose for 5 d, followed by air-drying for 6 h prior to direct immersion in liquid nitrogen. With our protocol, we obtained a mean organogenesis rate of 100%, a mean of 4.5 adventitious shoots per explant (leaf segment), and a mean shoot recovery of 57.0% from cryopreserved shoot tips in four Malus species. Inter-simple sequence repeat (ISSR) analysis did not reveal any polymorphic bands in regenerants recovered from either leaf segments or cryopreserved shoot tips of ‘Gala’. To the best of our knowledge, this is the first report on cryopreservation of Malus shoot tips using adventitious shoots derived from leaf segments and is the most widely applicable protocol so far reported for cryopreservation of Malus. Establishment of this protocol provides an alternative means for cryopreservation of Malus.     

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.     

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.     

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.     

Shoot tip culture and cryopreservation for eradication of Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from apple rootstocks ‘M9’ and ‘M26’

This study attempted to eradicate Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from virus‐infected in vitro shoots of apple rootstocks ‘M9’ and ‘M26’ using shoot tip culture and cryopreservation. In shoot tip culture, shoot tips (0.2 mm in length) containing two leaf primordia failed to show shoot regrowth. Although shoot regrowth rate was the highest in the largest shoot tips (1.0 mm in length) containing four leaf primordia, none of the regenerated shoots was virus‐free. Shoot tips (0.5 mm in length) containing two and three leaf primordia produced 100% and 10% of ASPV‐free shoots, respectively, while those (1.0 mm) containing four leaf primordia were not able to eradicate ASPV. ASGV could not be eradicated by shoot tip culture, regardless of the size of the shoot tips tested. In cryopreservation, shoot tips (0.5 mm in length) containing two leaf primordia did not resume shoot growth. Although 1.0‐mm and 1.5‐mm shoot tips gave similarly high ASPV‐free frequencies, the latter had much higher shoot regrowth rate than the former. Very similar results of shoot regrowth and virus eradication by shoot tip culture and cryopreservation were observed in both ‘M9’ and ‘M26’. Histological observations showed that only cells in upper part of apical dome and in leaf primordia 1–3 survived, while other cells were damaged or killed, in shoot tips following cryopreservation. Virus immunolocalization found ASPV was not detected in upper part of apical dome and leaf primordia 1 and 2, but was present in lower part of apical dome, and in leaf primordium 4 and more developed tissues in all samples tested. ASPV was also detected in leaf primordium 3 in about 16.7% and 13.3% samples tested in ‘M9’ and ‘M26’. ASGV was observed in apical dome and leaf primordia 1–6, leaving only a few top layers of cells in apical dome free of the virus. Different abilities of ASPV and ASGV to invade leaf petioles and shoot tips were also noted.     

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 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 of small leaf squares-bearing adventitious buds of <Emphasis Type="Italic">Lilium</Emphasis> Oriental hybrid ‘Siberia’ by vitrification

We report a new cryopreservation method for Lilium Oriental hybrid ‘Siberia’. Adventitious buds were induced from leaf segments cultured for 12 days on adventitious bud induction medium composed of half-strength Murashige and Skoog medium (MS) supplemented with 1 mg L^?1 α-naphthalene acetic acid and 0.5 mg L^?1 thidiazuron. Small leaf squares (SLSs, 3?×?4 mm), each bearing at least one adventitious bud, were cut from leaf segments, precultured on medium with 0.5 M sucrose for 1 day, and then treated for 20 min with a loading solution containing 0.4 M sucrose and 2 M glycerol, followed by exposure to plant vitrification solution 2 for 7 h at 0 °C. Dehydrated SLSs were directly immersed in liquid nitrogen for 1 h. Cryopreserved SLSs were re-warmed in MS medium containing 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for recovery. With this procedure, 85% survival and 72% shoot regrowth were achieved following cryopreservation. The use of SLSs bearing adventitious buds for cryopreservation reported in the present study eliminates the time-consuming and labour-intensive step of shoot tip excision, and has great potential to facilitate cryopreservation in other plant species.     

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 grapevine (Vitis vinifera L.) in vitro shoot tips

In this work, we compared the efficiency of encapsulation-dehydration and droplet-vitrification techniques for cryopreserving grapevine (Vitis vinifera L.) cv. Portan shoot tips. Recovery of cryopreserved samples was achieved with both techniques; however, droplet-vitrification, which was used for the first time with grapevine shoot tips, produced higher regrowth. With encapsulationdehydration, encapsulated shoot tips were precultured in liquid medium with progressively increasing sucrose concentrations over a 2-day period (12 h in medium with 0.25, 0.5, 0.75 and 1.0 M sucrose), then dehydrated to 22.28% moisture content (fresh weight). After liquid nitrogen exposure 37.1% regrowth was achieved using 1 mm-long shoot tips and only 16.0% with 2 mm-long shoot tips. With droplet-vitrification, 50% regrowth was obtained following treatment of shoot tips with a loading solution containing 2 M glycerol + 0.4 M sucrose for 20 min, dehydration with half-strength PVS2 vitrification solution (30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% dimethylsulfoxide and 0.4 M sucrose in basal medium) at room temperature, then with full strength PVS2 solution at 0°C for 50 min before direct immersion in liquid nitrogen. No regrowth was achieved after cryopreservation when shoot tips were dehydrated with PVS3 vitrification solution (50% (w/v) glycerol and 50% (w/v) sucrose in basal medium).     

Cryopreservation of encapsulated shoot primordia induced in horseradish (Armoracia rusticana) hairy root cultures

Shoot primordia induced inArmoracia rusticana Gaertn. Mey. et Scherb. (horseradish) hairy root cultures were successfully cryopreserved by two cryogenic procedures. Encapsulated shoot primordia were precultured on solidified Murashige-Skoog medium supplemented with 0.5M sucrose for 1 day and then dehydrated with a highly concentrated vitrification solution (PVS2) for 4 h at 0°C prior to a plunge into liquid nitrogen. The survival rate of encapsulated vitrified primordia amounted to 69%. In a revised encapsulation-dehydration technique, the encapsulated shoot primordia were precultured with a mixture of 0.5M sucrose and 1M or 1.5M glycerol for 1 day to induce dehydration tolerance and then subjected to air-drying prior to a plunge into liquid nitrogen. The survival rate of encapsulated dried primordia was more than 90%, and the revived primordia produced shoots within 2 weeks after plating. A long-term preservation of shoot primordia was also achieved by the technique. Thus, this revised encapsulation-dehydration technique appears promising as a routine method for the cryopreservation of shoot primordia of hairy roots.Abbreviations PVS2 Vitrification solution - LN liquid nitrogen - BA 6-benzyladenine - NAA -naphthalene-acetic acid - MS Murashige and Skoog (1962) medium     

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.     

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.     

Antioxidant and cytotoxic activity of bioactive phenolic metabolites isolated from the yeast-extract treated cell culture of apple

Buds of in vitro-grown shoots of two purple-fleshed potato genotypes were successfully cryopreserved by encapsulation-vitrification (Encap-vitri) and droplet-vitrification (Drop-vitri). Optimal time durations of exposure to PVS2 for shoot regrowth of cryopreserved buds were 5–7 h and 6 h for ‘E03-2677’ and for ‘Blue Congo’, respectively, in Encap-vitri, and 30–50 min and 40 min for the former and the latter, respectively, in Drop-vitri. Higher rates of shoot regrowth were obtained in 1.5–2.0 mm-buds than in 1.0–1.4 mm-ones in Encap-vitri for ‘E03-2677’ and ‘Blue Congo’, while opposite results were found in Drop-vitri. In ‘Blue Congo’, only apical shoot tips survived and developed into shoots, with one shoot produced in one cryopreserved bud in Encap-vitri and Drop-vitri. In ‘E03-2677’, survival and shoot regrowth patterns were similar to those of ‘Blue Congo’ in Encap-vitri. However, both apical and axillary shoot tips survived and developed into two shoots in one bud in Drop-vitri. In ‘E03-2677’, histological observations revealed only apical shoot tips survived following Encap-vitri, while both apical and axillary shoot tips survived following Drop-vitri. Vegetative growth in shoots regenerated from Encap-vitri and Drop-vitri after 3 weeks of post-thaw culture was significantly lower than that from control, but markedly increased after 6 months of post-thaw culture. In both ‘E03-2677’ and ‘Blue Congo’, number of microtubers per shoot, per vessel and ≥3 mm in diameter were significantly greater in shoots regenerated from Encap-vitri than in those from the control. Assessments by ISSR and RAPD of genetic stability did not find any polymorphic bands in regenerants recovered from Encap-vitri and Drop-vitri. To the best of our knowledge, this is the first report on cryopreservation of purple-fleshed potato by vitrification-based procedures. Results reported here would provide valuable basic and technical information on cryopreservation of purple-fleshed potato.     

Low-temperature preincubation enhances survival and regeneration of cryopreserved Saussurea involucrata callus

Saussurea involucrata Kar. et Kir. is one of the most well-known Chinese medicinal plants, and it is utilized for a variety of medical conditions. Due to the overexploitation of this endangered species, it is crucial to develop methods for both conservation and propagation. To address this issue, we have developed and optimized a simple and effective vitrification process for the cryopreservation of S. involucrata callus tissue. The optimized method consisted of a 3-d incubation period on medium containing 0.3 M sucrose, transfer to a plant vitrification solution (PVS2) containing 30% (v/v) glycerol, 15% (v/v) ethylene glycol, 15% (v/v) dimethylsulfoxide, and 0.4 M sucrose first at 60% PVS2 for 40 min, then at 100% PVS2 for 60 min, followed by immediate immersion and storage in liquid nitrogen. To thaw the tissue, tissues were rewarmed at 40°C for 2 min. This method resulted in a survival rate of approximately 56% and a regrowth rate of approximately 40%. Survival and regrowth were significantly improved by the addition of a low-temperature preincubation step. Incubating the calli at 4°C for 12 d prior to initiating the optimized cryopreservation protocol increased the survival rate of the tissue to 75%, increased the regrowth rate to 60%, and more than doubled the number of regenerated shoots per explant. Following cryopreservation, greater than 90% of the regenerated shoots formed complete plantlets, and 81% of the regenerated plantlets survived and grew vigorously under greenhouse conditions.     

In vitro conservation and cryopreservation of mature pistachio (Pistacia vera L.) germplasm

As genetic erosion of pistachio (Pistacia vera L.) has been occurring in the Mediterranean, Central and West Asia and North Africa, experiments were conducted to conserve two cultivars (‘Atl?’ and ‘Siirt’) of mature pistachio germplasm by assessing both medium- and long-term conservation techniques. In medium-term conservation, our results showed that it was feasible to conserve both cultivars in the form of either microshoots or encapsulated shoot apices up to 12 months at 4°C in the dark. As regards long-term conservation, encapsulation-dehydration and droplet-vitrification techniques were assessed for cryopreservation of cold-hardened and osmoprotected shoot apices of mature ‘Atl?’ cultivar. Among the methods tested, 13.6% of regrowth was achieved with incubation of explants in the droplets of vitrification solution for 150 min at 0°C followed by direct immersion in liquid nitrogen (LN), rapidly thawed and then cultured on Murashige and Skoog’s (MS) medium containing 1 mg L^?1 BA and 0.5 mg L^?1 GA_3. The developed droplet-vitrification technique appeared as a promising procedure for long-term preservation of shoot apices of mature pistachio germplasm. Moreover, assesment of genetic fidelity by Random Amplified Polymorphic DNA analysis (RAPD) revealed out high levels of genetic stability between donor plant and cryopreserved plants (similarity indexes between 0.959 and 0.973) after they were subcultured for at least 3 months. The detected low level of genetic instability could be due to the toxic effect of PVS2 and regeneration phase. The optimized conservation techniques, especially slow growth storage, could be applied to preserve other Pistacia species.     

Cryopreservation of Teucrium polium L. shoot-tips by vitrification and encapsulation-dehydration

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.     

Cryopreservation of wild Shih (<Emphasis Type="Italic">Artemisia herba</Emphasis>-<Emphasis Type="Italic">alba</Emphasis> Asso.) shoot-tips by encapsulation-dehydration and encapsulation-vitrification

Artemisia herba-alba, called Shih is a medicinal herbal plant found in the wilds. The biodiversity of this plant is heavily subjected to loss because of heavy grazing, land cultivation and collection by people to be used in folk medicine. In the current study, two cryopreservation dependent techniques to conserve the shoot-tips of in vitro grown Shih were evaluated: encapsulation- dehydration and encapsulation- vitrification. Shoot-tips of Shih were encapsulated into sodium-alginate beads. In encapsulation- dehydration, the effect of sucrose concentration (0.5, 0.75 or 1.0 M) and dehydration period (0, 2, 4 or 6 h) under sterile air-flow on survival and regrowth of encapsulated shoot tips were studied. Maximum survival (100%) and regrowth (27%) rates were obtained when encapsulated unfrozen Artemisia herba-alba shoot tips were pretreated with 0.5 M sucrose for 3 days without further air dehydration. After cryopreservation the highest survival (40%) and regrowth (6%) rates were achieved when Artemisia herba-alba shoot tips were pretreated with 1.0 M sucrose for 3 days without further air dehydration. Viability of Artemisia herba-alba shoot tips decreased with increased dehydration period. In encapsulation-vitrification, the effect of dehydration of encapsulated Artemisia herba-alba shoot tips with 100% PVS2 for various dehydration durations (10, 20, 30, 60 or 90 min) prior to freezing was studied. After cryopreservation the dehydration of encapsulated and vitrified shoot tips with 100% PVS2 for 30 min resulted in 68% survival and 12% regrowth rates. Further conservation techniques must be evaluated to increase both survival and regrowth percentages.     

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 somatic embryos from <Emphasis Type="Italic">Petiveria alliacea</Emphasis> L. by different techniques based on vitrification

Petiveria alliacea L. is a medicinal plant originating from the Amazon region. This study describes an efficient cryopreservation protocol for somatic embryos (SEs) produced from roots of P. alliacea based on the comparison of vitrification, encapsulation-dehydration, and D cryo-plate techniques. With the vitrification technique, SEs treated with PVS2 solution (0.4 M sucrose, 3.3 M glycerol, 2.4 M ethylene glycol, and 1.9 M DMSO) for 30 min displayed high viability (85%) and intermediate proliferation recovery (about 12 adventitious SEs produced from original SEs [SEs/SE] after 90 d of culture). With the encapsulation-dehydration technique, lower viability (70%) and very low proliferation recovery (about two SEs/SE) were achieved with cryopreserved SEs dehydrated for 10 min in a laminar air flow cabinet. The D cryo-plate technique led to high viability (85%) and proliferation recovery (19 SEs/SE) of cryopreserved SEs after 90 min dehydration. In the experimental conditions tested, the D cryo-plate method was the most efficient technique for cryopreservation of P. alliacea SEs.