Cryopreservation of white poplar (Populus alba L.) by vitrification of in vitro-grown shoot tips

 Shoot tips from in vitro-grown, cold-hardened stock plants of white poplar (Populus alba L.) were successfully cryopreserved at –196  °C by one-step vitrification. After preculturing at 5  °C for 2 days on hormone-free MS medium containing different sucrose concentrations, and loading for 20 min with 2 m glycerol and 0.4 m sucrose, shoot tips were treated with the PVS2 vitrification solution and plunged directly into liquid nitrogen. Best survival rate (90%) was obtained when shoot tips were precultured on 0.09 m sucrose, hormone-free MS medium, vitrified by exposure to PVS2 solution for 60 min at 0  °C and, following cryopreservation, rewarmed at 40  °C and washed in 1.2 m sucrose solution for 20 min. Regrowth was improved by plating shoot tips on a gelled MS medium containing 1.5 μm N⁶-benzyladenine plus 0.5 μm gibberellic acid, while shoot rooting was achieved on MS medium containing 3 μm indole-3-butyric acid. Following this procedure, almost 60% rooted shoots were obtained from cryopreserved shoot tips. Received: 1 February 1999 / Revision received: 3 May 1999 · Accepted: 21 May 1999     

Cryopreservation of shoot tips from in vitro plants of sweet potato [Ipomoea batatas (L.) Lam.] by vitrification

 Routine cryopreservation of shoot tips from sweet potato [Ipomoea batatas (L.) Lam] has been hampered by their survival variability after cryogenic exposure. We examined the effects of light conditions on stock plants, sucrose preculture and cryoprotectant loading on survival after vitrification using PVS2 solution. The survival of vitrified sweet potato shoot tips cooled to approximately –208  °C was increased by preculturing with 0.3 M sucrose for 24 h at 22  °C. Survival was also enhanced by excising shoot tips immediately after the 8-h dark photoperiod. The best survival after cryogenic exposure was obtained using 2 M glycerol +0.4 M sucrose for 1 h at 22  °C followed by dehydration with PVS2 for 16 min at 22  °C. Rapid cooling was used and achieved by the immersion of foil strips into partially solidified nitrogen. Successfully vitrified and warmed shoot tips directly developed shoots on a medium containing 1 μM NAA, 0.5 μM BA and 0.1 μM kinetin with only minimum callus formation. Shoot formation occurred in all surviving shoot tips. This procedure shows promise for cryopreserving sweet potato shoot tips. Received: 2 March 1999 / Revision received: 21 September 1999 / Accepted: 29 September 1999     

High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of transgenic papaya lines

In vitro grown shoot tips of transgenic papaya lines (Carica papaya L.) were successfully cryopreserved by vitrification. Shoot tips were excised from stock shoots that were preconditioned in vitro for 45–50-day-old and placed on hormone-free MS medium with 0.09 M sucrose. After loading for 60 min with a mixture of 2 M glycerol and 0.4 M sucrose at 25°C, shoot tips were dehydrated with a highly concentrated vitrification solution (PVS2) for 80 min at 0°C and plunged directly into liquid nitrogen. The regeneration rate was approximately 90% after 2 months post-thawing. Successfully vitrified and warmed shoot tips of three non-transgenic varieties and 13 transgenic lines resumed growth within 2 months and developed shoots in the absence of intermediate callus formation. Dehydration with PVS2 was important for the cryopreservation of transgenic papaya lines. This vitrification procedure for cryopreservation appears to be promising as a routine method for cryopreserving shoot tips of transgenic papaya line germplasm.     

Cryopreservation of Shoot Tips of Tetraploid Potato (Solanum tuberosumL.) Clones by Vitrification

In vitro-grown shoot tips of five tetraploid potato (SolanumtuberosumL.) clones were cryopreserved by vitrification. Excisedshoot tips (0.5–0.7 mm) were pre-cultured on filter paperdiscs over half strength liquid Murashige and Skoog (MS) mediumsupplemented with 8.7 µMGA₃and different combinationsof sucrose (0.3, 0.5 and 0.7M) plus mannitol (0, 0.2 and 0.4M)for 2 d under a 16 h photoperiod at 24 °C. The pre-culturedshoot tips were either successively loaded with 20 and 60% PVS2 solutions or directly exposed to concentrated vitrificationsolution before physical vitrification during liquid nitrogentreatment. The vitrified shoot tips were warmed rapidly andtreated with dilution mixture (MS+1.2Msucrose) for 30 min beforeplating on regrowth medium. Addition of mannitol to the pre-culturemedium improved survival of vitrified shoot tips. Direct dehydrationof pre-cultured shoot tips with concentrated PVS 2 was detrimentalto survival of vitrified shoot tips. Shoot tips pre-culturedon medium containing 0.3Msucrose plus 0.2Mmannitol, and loadedwith 20% PVS 2 for 30 min followed by 15 min incubation in 60%PVS 2 and 5 min incubation in 100% PVS 2 at 0 °C resultedin up to 54% survival after vitrification. About 50% of vitrifiedand warmed shoot tips formed shoots directly. Post-thaw culturingof vitrified shoot tips on medium containing an elevated levelof sucrose (0.2M) under diffuse light for the first week enhancedthe survival rate. Continuous culturing of vitrified shoot tipson high-sucrose medium induced multiple shoot formation.Copyright1998 Annals of Botany Company Solanum tuberosumL., potato, cryopreservation, germplasm conservation,in vitroconservation, meristems, shoot tips, tissue culture, vitrification.     

Cryopreservation of Doritaenopsis suspension culture by vitrification

 Cells of a suspension culture of Doritaenopsis cv. New Toyohashi were placed in a mixture of 2 M glycerol and 0.4 M sucrose for 15 min at room temperature and then dehydrated with a vitrification solution (PVS2) for 1–3 h on ice and plunged into liquid nitrogen. The highest viability (64% by 2,3,5-triphenyltetrazolium chloride stainability) was obtained when the cells were precultured in liquid New Dogashima medium with 0.1 M sucrose and 1.0 mg/l abscisic acid for 1 week at 25  °C in the light. Dehydration by PVS2 was important for the cryopreservation of Doritaenopsis cells. Protocorm-like bodies were induced from cryopreserved cells without morphological variations. Received: 18 January 2000 / Revision received: 16 June 2000 / Accepted: 22 June 2000     

Cryopreservation of chestnut by vitrification of <Emphasis Type="Italic">in vitro</Emphasis>-grown shoot tips

Summary Plants of European chestnut (Castanea sativa) have been consistently recovered from cryopreserved in vitro-grown shoot apices by using the vitrification procedure. Factors found to influence the success of cryopreservation include the source of the shoot tips (terminal buds or axillary buds), their size, the duration of exposure to the cryoprotectant solution, and the composition of the post-cryostorage recovery medium. The most efficient protocol for shoot regrowth employed 0.5–1.0 mm shoot tips isolated from 1 cm-long terminal buds that had been excised from 3–5-wk shoot cultures and cold hardened at 4°C for 2 wk. The isolated shoot tips were precultured for 2d at 4°C on solidified Gresshoff and Doy medium (GD) supplemented with 0.2M sucrose, and were then treated for 20 min at room temperature with a loading solution (2M glycerol+0.4M sucrose) and for 120 min at 0°C with a modified PVS2 solution before rapid immersion in liquid nitrogen (LN). After 1 d in LN, rapid rewarming and unloading in 1.2M sucrose solution for 20 min, the shoot tips were plated on recovery medium consisting of GD supplemented with 2.2 μM benzyladenine, 2.9 μM 3-indoleacetic acid, and 0.9 μM zeatin. This protocol achieved 38–54% shoot recovery rates among five chestnut clones (three of juvenile origin and two of mature origin), and in all cases plant regeneration was also obtained.     

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 in vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn.

In vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn. were successfully cryopreserved by vitrification. Shoot tips (0.5–1 mm) excised from 6-week-old shoots were precultured in hormone-free Woody Plant Medium (WPM) supplemented with 0.2 M sucrose, for 2 days at 4 °C in the dark, and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose, for 20 min at 25 °C. Osmoprotected shoot tips were first dehydrated with 50 % vitrification solution (PVS2), for 30 min at 0 °C, and then placed in 100 % PVS2, for 30 min at 0 °C. The solution was replaced with fresh 100 % PVS2, and the shoot tips were plunged directly into liquid nitrogen. The shoot tips were rewarmed in a water bath at 40 °C for 2 min, and then washed twice, for 10 min at 25 °C, with 1.2 M sucrose solution, before being transferred onto WPM supplemented with 0.5 mg l^?1 N ⁶-benzyladenine, 0.5 mg l^?1 indole-3-acetic acid, 0.2 mg l^?1 zeatin, 20 g l^?1 glucose and 6 g l^?1 Difco Bacto agar. The shoot tips were kept in darkness for 1 week and under dim lighting for another week, before being exposed to standard culture conditions (16 h photoperiod). This protocol was successfully applied to three alder genotypes, with recovery rates higher than 50 %.     

Cryopreservation of in vitro-grown axillary shoot-tip meristems of mint (Mentha spicata L.) by encapsulation vitrification

 Alginate-coated meristems from in vitro-grown axillary buds of mint (Mentha spicata L.) were successfully cryopreserved by vitrification. Excised meristems from nodal segments cold hardened at 4  °C for 3 weeks were encapsulated and osmoprotected by a mixture of 2 M glycerol plus 0.4 M sucrose. These meristems were dehydrated with a highly concentrated vitrification solution (PVS2 solution) for 3 h at 0  °C prior to a plunge into liquid nitrogen. Successfully encapsulated vitrified meristems developed shoots within a week after plating without intermediary callus formation. The average rate of shoot formation amounted to nearly 90%. This procedure was successfully applied to other Mentha species. It was also confirmed that encapsulated vitrified meristems produced a much higher rate of shoot formation than the encapsulated dried meristems. Thus, this revised encapsulation vitrification method appears promising for the cryopreservation of mint and other germplasm. Received: 24 November 1998 / Revision received: 8 February 1999 / Accepted: 26 February 1999     

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 African violet (Saintpaulia ionantha Wendl.) shoot tips

Summary Cryopreservation of African violet via encapsulation-dehydration, vitrification, and encapsulation-vitrification of shoot tips was evaluated. Encapsulation-dehydration, pretreatment of shoot tips with 0.3 M sucrose for 2 d followed by air dehydration for 2 and 4 h resulted in complete survival and 75% regrowth, respectively. Dehydration of encapsulated shoot tips with silica gel for 1 h resulted in 80% survival but only 30% regrowth. Higher viability of shoot tips was obtained when using a step-wise dehydration of the material rather than direct exposure to 100% plant vitrification solution (PVS2). Complete survival and 90% regrowth were achieved with a four-step dehydration with PVS2 at 25°C for 20 min prior to freezing. The use of 2M glycerol plus 0.4M sucrose or 10% dimethyl sulfoxide (DMSO) plus 0.5M sucrose as a cryoprotectant resulted in 55% survival of shoots. The greatest survival (80–100%) and regrowth (80%) was obtained when shoot tips were cryoprotected with 10% DMSO plus 0.5M sucrose or 5% DMSO plus 0.75M sucrose followed by dehydration with 100% PVS2. Shoot tips cryoprotected with 2M glycerol plus 0.4M sucrose for 20 min exhibited complete survival (100%) and the highest regrowth (55%). In encapsulation-vitrification, dehydration of encapsulated and cryoprotected shoot tips with 100% PVS2 at 25°C for 5 min resulted in 85% survival and 80% regrowth.     

High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of rare and endangered plant <Emphasis Type="Italic">Emmenopterys henryi</Emphasis> Oliv.

In vitro-grown shoot tips of Emmenopterys henryi Oliv. were successfully cryopreserved by vitrification. Shoot tips excised from 3-month old plantlets were precultured in a liquid hormone-free Murashige and Skoog (MS) medium supplemented with 0.5 M sucrose for 3 days at 25°C and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose (LS solution) for 40 min at 25°C. Osmo-protected shoot tips were first dehydrated with 60% vitrification solution (PVS₂) for 30 min at 0°C and followed by 100% PVS₂ for 40 min at 0°C. After changing the solution with fresh 100% PVS₂, the shoot tips were directly plunged into liquid nitrogen. After rapid warming in a water-bath at 40°C for 2 min, the shoot tips were washed for 20 min at 25°C with liquid MS medium containing 1.2 M sucrose and then transferred onto solid MS medium supplemented with kinetin 2 mg l⁻¹, α-naphthaleneacetic acid 0.1 mg l⁻¹, 3% (w/v) sucrose and 0.75% (w/v) agar. The shoot tips were kept in the dark for 7 days prior to exposure to the light (12 h photoperiod cycle). Direct shoot elongation was observed in approximately 12 days. The regeneration rate was approximately 75–85%. This method appears to be a promising technique for cryopreserving shoot tips of Emmenopterys henryi Oliv. germplasm.     

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.     

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.     

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.     

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

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

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

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

Adaptation and acclimation of growth and photosynthesis of five Antarctic red algae to low temperatures

Temperature requirements for growth, photosynthesis and dark respiration were determined for five Antarctic red algal species. After acclimation, the stenothermal species Gigartina skottsbergii and Ballia callitricha grew at 0 or up to 5 °C, respectively; the eurythermal species Kallymenia antarctica, Gymnogongrus antarcticus and Phyllophora ahnfeltioides grew up to 10 °C. The temperature optima of photosynthesis were between 10 and 15 °C in the stenothermal species and between 15 and 25 °C in the eurythermal species, irrespective of the growth temperature. This shows that the temperature optima for photosynthesis are located well below the optima from species of other biogeographical regions, even from the Arctic. Respiratory rates rose with increasing temperatures. In contrast to photosynthesis, no temperature optimum was evident between 0 and 25 °C. Partial acclimation of photosynthetic capacity to growth temperature was found in two species. B. callitricha and Gymnogongrus antarcticus acclimate to 0 °C, and 5 and 0 °C, respectively. But acclimation did in no case lead to an overall shift in the temperature optimum of photosynthesis. B. callitricha and Gymnogongrus antarcticus showed acclimation of respiration to 5 °C, and P. ahnfeltioides to 5 and 10 °C, resulting in a temperature independence of respiration when measured at growth temperature. With respect to the acclimation potential of the species, no distinction can be made between the stenothermal versus the eurythermal group. (Net)photosynthetic capacity:respiration (P:R) ratios showed in all species highest values at 0 °C and decreased continuously to values lower than 1.0 at 25 °C. In turn, the low P:R ratios at higher temperatures are assumed to determine the upper temperature growth limit of the studied species. Estimated daily carbon balance reached values between 4.1 and 30.7 mg C g⁻¹ FW day⁻¹ at 0 °C, 16:8 h light/dark cycle, 12–40 μmol m⁻² s⁻¹. Received: 4 November 1999 / Accepted: 7 March 2000     

Cryopreservation of shoot-tips by droplet vitrification applicable to all taro (Colocasia esculenta var. esculenta) accessions

The application of the droplet vitrification cryopreservation technique to taro accessions from a range of Asia Pacific countries is presented. The optimum protocol involves excision of about 0.8 mm shoot-tips from in vitro plants, 20–40 min PVS2 exposure at 0°C followed by rapid plunge into liquid nitrogen. Thawing was done at room temperature (25°C) and shoot-tips inoculated on MS medium with 0.1 M sucrose regenerated into plantlets 4–6 weeks later. This new droplet vitrification protocol improved the mean post-thaw regeneration rates to 73–100% from 21–30% obtained with the previous cryo-vial vitrification protocol.