CRYOPRESERVATION OF THE CONCHOCELIS OF PORPHYRA (RHODOPHYTA) BY APPLYING A SIMPLE PREFREEZING SYSTEM1

The conchocelis cells of four strains of Porphyra yezoensis Udea and four other Porphyra species were cryopreserved in liquid nitrogen (LN) using a programmable freezer or a simple prefreezing system, which consisted of a styrofoam box and a deep-freezer at ?40° C. The cells differed in their freezing tolerance but survived maximally when prefrozen to ?40° C in a cryoprotective solution composed of 10% dimethylsulfoxide and 0.5 M sorbitol in 50% seawater. The cryopreservation was successfully performed by applying the simple prefreezing system as well as by a programmable freezer. Conchocelis cells thawed from the LN temperature formed colonies and retained the ability to form conchospores that grew into gametophytic thalli. This technique using a simple prefreezing system will accelerate the spread of Porphyra 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     

Cryopreservation of apple (Malus×domestica Borkh.) shoot tips following encapsulation-dehydration or encapsulation-vitrification

Axillary shoot tips of apple cv. Golden Delicious isolated from shoot cultures were successfully cryopreserved using the encapsulation-dehydration technique. After encapsulation in alginate gel, embedded shoot tips were dehydrated by exposure to a sterile air flow before being frozen in liquid nitrogen and subsequent slow thawing. A preculture on modified MS medium containing 0.75 M sucrose followed by 6 h of dehydration (21% residual water) led to the highest shoot regrowth of frozen, coated shoot tips (83.7%). Among the sugars tested, sucrose and sorbitol presented the best cryoprotective effect. Four other scion apple varieties and rootstocks were also successfully cryopreserved. Axillary shoot tips of five apple (Malus×domestica Borkh.) scion and rootstock cultivars were cryopreserved using the encapsulation-vitrification technique. Using a one-step freezing method, we successfully cryopreserved axillary shoot tips without the requirement of a cold hardening pretreatment of the shoot cultures. Cryopreserved shoot tips treated with aqueous cryoprotective mixture IV containing 180% (w/v) sucrose and 120% (v/v) ethylene glycol showed the highest shoot regrowth rates, which varied from 64% to 77%, depending on the cultivar. Received: 29 July 1999 / Revision received: 24 September 1999 / Accepted: 26 November 1999     

Effect of loading and vitrification solutions on survival of cryopreserved oil palm polyembryoids

The present study evaluates the effect of six loading solutions and five vitrification solutions (VS) and their time of exposure on the survival of oil palm (Elaeis guineensis) polyembryoids in liquid nitrogen (LN). In vitro grown polyembryoids of oil palm were successfully cryopreserved by vitrification with 45% survival. Individual polyembryoids, isolated from 2-month old culture, were precultured in liquid Murashige and Skoog medium supplemented with 0.5 M sucrose for 12 h and treated with a mixture of 10% (w/v) dimethyl sulphoxide (DMSO) plus 0.7 M sucrose for 30 min. Polyembryoids were then subjected to plant vitrification solution-2 (PVS2) (30% (w/v) glycerol plus 15% (w/v) EG plus 15% (w/v) DMSO plus 0.4 M sucrose) exposure for 5 min at 26 ± 2°C and subsequently plunged into LN. Thawed polyembryoids resumed growth within 8 days of culture and shoot development was recorded at 25 days of growth. Scanning electron micrograph revealed that successful regeneration of cryopreserved polyembryoids was due to stabilization of cellular integrity through optimum VS exposure.     

Cryopreservation of in vitro-grown shoot tips of grapevine by encapsulation-dehydration

In vitro-grown shoot tips of the LN33 hybrid (Vitis L.) and cv. Superior (Vitis vinifera L.) were successfully cryopreserved by encapsulation-dehydration. Encapsulated shoot tips were precultured stepwise on half-strength MS medium supplemented with increasing sucrose concentrations of 0.25, 0.5, 0.75 and 1.0 M for 4 days, with one day for each step. Following preculture, encapsulated shoot tips were dehydrated prior to direct immersion in liquid nitrogen for 1 h. After thawing, cryopreserved shoot tips were post-cultured on a post-culture medium for survival. An optimal survival of cryopreserved shoot tips was achieved when encapsulated shoot tips were dehydrated to 15.6 and 17.6% water content for the LN33 hybrid and cv. Superior, respectively. Comparison between the effects of dehydration with silica gel and by air drying on cryopreserved shoot tips, showed that survival was dependent on water content, not on dehydration method. The thawing method markedly affected survival of cryopreserved shoot tips, and thawing at 40 °C for 3 min was found best. No callus formation and fastest shoot elongation were obtained when cryopreserved shoot tips were post-cultured on the post-culture medium composed of half-strength MS supplemented with 1 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. With these optimized parameters, 60 and 40% survival of cryopreserved shoot tips were obtained for the LN33 hybrid and cv. Superior, respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Germplasm conservation of Guazuma crinita,a useful tree in the Peru-Amazon,by the cryopreservation of in vitro-cultured multiple bud clusters

In vitro-cultured adventitious bud clusters of Guazuma crinita Mart. were successfully cryopreserved by the one-step vitrification method. Small segments (1.0-1.5 mm³) cut from adventitious bud clusters were exposed to a cryoprotectant mix solution containing (w/v), 25 glycerol, 15 sucrose, 15 ethylene glycol, 13 dimethyl sulfoxide, and 2 polyethylene glycol, at 25 °C for 15-60 min prior to storage in liquid nitrogen. After rapid warming (37 °C), the segments were treated with woody plant medium containing 40 (w/v) sucrose for 20 min at 25 °C, and then transferred to recovery-growth medium. High survival rates (about 80) were achieved without any cold hardening and/or pre-culturing treatments, and about 30 of the surviving cryopreserved explants regenerated plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

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 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     

Cryopreservation of human fetal liver hematopoietic stem/progenitor cells using sucrose as an additive to the cryoprotective medium

IntroductionHuman fetal liver (HFL) is a valuable source of hematopoietic stem/progenitor cells (HSCs) for the treatment of various hematological disorders. This study describes the effect of sucrose addition to a cryoprotective medium in order to reduce the Me₂SO concentration during cryopreservation of HFL hematopoietic cell preparations.MethodsHuman fetal liver (HFL) cells of 8–12 weeks of gestation were cryopreserved with a cooling rate of 1 °C/min down to −80 °C and stored in liquid nitrogen. The cryoprotectant solutions contained 2% or 5% Me₂SO (v/v) with or without sucrose at a final concentration of 0.05, 0.1, 0.2 or 0.3 M. The metabolic activity of HFL cells was determined using the alamar blue assay. For the determination of the number and survival of hematopoietic progenitors present, cells were stained with CD34 (FITC) and 7-AAD, and analyzed by flow cytometry. The colony-forming activity of HFL hematopoietic stem/progenitor cells after cryopreservation was assessed in semisolid methylcellulose.ResultsThe addition of sucrose to the cryoprotective medium produced a significant reduction in HFL cell loss during cryopreservation. The metabolic activity of HFL cells, cryopreserved with 5% Me₂SO/0.3 M sucrose mixture was comparable to cryopreservation in 5% Me₂SO/10% FCS. Although the inclusion of sucrose did not affect the survival of CD34⁺ cells in HFL after cryopreservation it did improve the functional capacity of hematopoietic stem/progenitor cells.ConclusionThe inclusion of sucrose as an additive to cryoprotective media for HFL cells enables a reduction in the concentration of Me₂SO, replacing serum and increasing the efficiency of cryopreservation.     

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.     

Survival and genetic stability of Picea abies embryogenic cultures after cryopreservation using a pregrowth-dehydration method

A vitrification method enabled efficient cryopreservation of embryogenic tissue (ETs) of Norway spruce (Picea abies L.) at ?196 °C in liquid nitrogen (LN). Correctly formed, normal somatic embryos were generated from ETs that had been thawed after removal from LN. The pregrowth-dehydration method involved preculture of ETs with sucrose (0.25–1.00 M) in the presence or absence of 10 μM abscisic acid (ABA), followed by air-drying for 2 h and rapid freezing in LN. Pretreatment of ETs with both sucrose and ABA promoted ET growth after preculture and thawing more effectively than treatment with sucrose alone. Survival of ETs after thawing from LN using both sucrose and ABA was 54.4 % compared to pretreatment with sucrose alone which was 20 %. Addition of ABA in the preculture medium also improved the ability of ETs to form cotyledonary stage somatic embryos. The somatic embryos, which had normal shoot and root apices and the correct number of cotyledons, were indistinguishable from regenerants obtained from control cultures. Genetic analysis of control and cryopreserved ETs, as well as somatic embryos derived from cryopreserved ETs, indicated that the cryopreservation method had no effect on any of the five microsatellite loci (SpAGC1, SpAGC2, SpAGG3, SpAC1H8, and SpAC1F7) tested. The cryopreservation protocol outlined should enable the long-term storage of valuable clones of Norway spruce in LN, potentially for hundreds of years.     

Induction and identification of tetraploid <Emphasis Type="Italic">Hedychium coronarium</Emphasis> through thin cell layer culture

We describe an encapsulation–dehydration procedure with prefreezing steps for the cryopreservation of rhizome bud explants of Asparagus officinalis L. cv. Morado de Huétor. With this procedure, survival of Rhizome buds was at least 84 and 42% developed to complete plantlets at 8 weeks. Flow cytometry and EST-SSR molecular markers were used to assess genetic stability of the regenerated material. Effects of preculture time in a medium rich in sucrose and prefreezing treatments (0 °C or/and ??20 °C) on plant recovery were evaluated. Rhizome Buds of the “Morado de Huétor” landrace were incubated in preculture medium (MS?+?0.3 M sucrose) for 48 h, encapsulated in alginate beads and desiccated until a water content of 35%, prefrozen for one hour at 0 °C plus one hour at ? 20 °C, followed by cryopreservation in liquid nitrogen, and then were rewarmed and recovered in ARBM medium for 6 weeks and finally incubated in ARBM-0 for 4 weeks. Analyses of ploidy and molecular stability of plantlets recovered from cryopreserved rhizome buds of two selected genotypes showed no differences compared with the mother plants. Cryopreservation of RB explants of A. officinalis with this Encapsulation–Dehydration procedure will be useful in long-term preservation programs.     

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.     

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 in vitro-grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration

Summary In vitro-grown apical meristems of wasabi (Wasabia japonica Matsumura) were successfully cryopreserved by vitrification. Excised apical meristems precultured on solidified M S medium containing 0.3M sucrose at 20°C for 1 day were loaded with a mixture of 2M glycerol and 0.4M sucrose for 20 min at 25°C. Cryoprotected meristems were then sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) for 10 min at 25°C prior to a plunge into liquid nitrogen. After rapid warming, the meristems were expelled into 2 ml of 1.2M sucrose for 20 min and then plated on solidified culture medium. Successfully vitrified and warmed meristems remained green after plating, resumed growth within 3 days, and directly developed shoots within two weeks. The average rate of normal shoot formation amounted to about 80 to 90% in the cryopreserved meristems. This method was successfully applied to three other cultivars of wasabi. This vitrification procedure promises to become a routine method for cryopreserving meristems of wasabi.Abbreviations BA 6-benzylaminopurine - DMSO dimethylsulfoxide - EG ethylene glycol - LN liquid nitrogen - MS medium Murashige and Skoog medium (1962) - PVS2 vitrification solution     

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 'Troyer' citrange [Poncirus trifoliata (L.) Raf. × Citrus sinensis (L.) Osbeck.] by encapsulation-dehydration

. In vitro-grown shoot tips excised from preconditioned stock shoots of 'Troyer' citrange were successfully cryopreserved by encapsulation-dehydration. Optimal survival of cryopreserved shoot tips was achieved when encapsulated shoot tips were dehydrated to 17.1% water content. The sucrose concentration in the preconditioning medium significantly influenced the growth and dry matter percentage of the stock shoots as well as subsequent survival of the cryopreserved shoot tips. Maximal growth of stock shoots was obtained in sucrose concentrations in the range of 0.15 M to 0.29 M, while the dry matter percentage increased as sucrose concentration increased up to 0.44 M. The survival of cryopreserved shoot tips increased from 40% to approximately 80% as the sucrose concentration for stock shoots increased from 0.09 M to 0.22 M or 0.29 M. The benzyladenine concentration in the post-culture medium significantly affected the survival and regrowth of the cryopreserved shoot tips. Survival of the shoot tips was lowest when they were post-cultured on benzyladenine-free medium. However, high benzyladenine concentrations (3-4 µM) induced callus formation. Optimal recovery was obtained in post-culture medium containing 2 µM benzyladenine and 0.05 µM !-naphthalene acetic acid. The extraction of shoot tips from alginate beads greatly improved the regrowth of cryopreserved shoot tips.     

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 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 of in vitro-grown apical meristems of lily by vitrification

Apical meristems from adventitious buds induced by culturing of bulb-scale segments of Japanese Pink Lily (Lilium japonicum Thunb.) were successfully cryopreserved by a vitrification. The excised apical meristems were precultured on a solidified Murashige & Skoog medium, containing 0.3 M sucrose, for 1 day at 25°C and then loaded in a mixture of 2 M glycerol plus 0.4 M sucrose for 20 min at 25°C. Cryoprotected meristems were then sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) at 25°C for 20 min or at 0°C for 110 min prior to a plunge into liquid nitrogen. After rapid warming in a water bath at 40°C, the meristems were placed in 1.8 ml of 1.2 M sucrose for 20 min and then, placed on filter papers over gellan gum-solidified MS medium. The revived meristems resumed growth within 5 days and directly produced shoots. The rate of shoot formation was approximately 80% after 4 weeks. When bulb-scale segments with adventitious buds were cold-hardened at 0°C for more than 7 days before the procedure, the rates of shoot formation were significantly increased. This vitrification method was successfully applied to five other lily cultivars. Thus, this vitrification procedure for cryopreservation appears promising as a routine method for cryopreserving meristems of lily.Abbreviations DMSO dimethylsulfoxide - EG ethylene glycol - LN liquid nitrogen - MS medium Murashige & Skoog (1962) medium - PVS2 vitrification solution