DEVELOPMENT OF A CRYOPRESERVATION PROCEDURE USING ALUMINIUM CRYO-PLATES

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1 CryoLetters 32 (3), (2011) CryoLetters, DEVELOPMENT OF A CRYOPRESERVATION PROCEDURE USING ALUMINIUM CRYO-PLATES Shin-ichi Yamamoto 1, Tariq Rafique 2, Wickramage Saman Priyantha 3, Kuniaki Fukui 1, Toshikazu Matsumoto 4 and Takao Niino 1* 1 Genebank, National Institute of Agrobiological Sciences (NIAS), Kannondai 2-1-2, Tsukuba , Japan. 2 Plant Genetic Resource Programme (IABGR), National Agricultural Research Center, Islamabad, Pakistan. 3 Regional Agricultural Research and Development Center, Makandura, Gonawila, Sri Lanka. 4 Faculty of Life and Environmental Science, Shimane Univ., Matsue, Shimane , Japan. *Corresponding author niinot@affrc.go.jp Abstract A cryopreservation procedure using an aluminium cryo-plate was successfully developed using in vitro-grown Dalmatian chrysanthemum (Tanacetum cinerariifolium) shoot tips. Shoot cultures were cold-hardened at 5 C on MS medium containing 0.5 M sucrose over a period of 20 to 40 days. Shoot tips with basal plate ( x 1.0 mm) were dissected from shoot cultures and precultured at 5 C for 2 days on MS medium containing 0.5 M sucrose. Precultured shoot tips were placed on aluminium cryo-plates (7 mm x 37 mm x 0.5 mm) with 10 wells (diameter 1.5 mm, depth 0.75 mm) and embedded in alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30 or 60 min in 25 ml pipetting reservoirs filled with loading solution (2 M glycerol M sucrose). For dehydration, the loading solution was replaced with PVS 7M vitrification solution (30% (w/v) glycerol, 19.5% (w/v) ethylene glycol and 0.6 M sucrose in liquid MS basal medium), which was applied for 40 min. After rapid immersion in liquid nitrogen, shoot tips attached to the cryo-plates were rewarmed by immersion in cryotubes containing 2 ml 1 M sucrose solution. Using this procedure, regrowth of cryopreserved shoot tips of line 28v-75 reached 77%. This protocol was successfully applied to six additional lines, with high regrowth percentages ranging from 65% to 90%. By contrast, the modified vitrification protocol tested as a reference produced only moderate regrowth percentages. This new method displays many advantages and will facilitate large scale cryostorage in genebank. Abbreviations: BA, benzyladenine; LN, liquid nitrogen; MS, Murashige and Skoog; PVP, polyvinyl pyrolidone; PVS, plant vitrification solution; LS, loading solution. Key words: aluminium plate, cryo-plate, Dalmatian chrysanthemum (Tanacetum cinerariifolium), vitrification, vitrification solution. 256

2 INTRODUCTION Cryopreservation is an ideal method for long-term conservation of plant germplasm because this method requires a minimum of storage space, labour and maintenance. Cryopreservation techniques are now used in an increasing number of institutes around the world (9). However, implementation of cryopreservation techniques in genebanks still takes place in a limited number of situations. The droplet-vitrification (DV) technique, a combination of droplet-freezing and solutionbased vitrification, has been notably used for routine cryopreservation of Musa (13), Korean garlic and chrysanthemum (5, 6) germplasm collections. DV produces high recovery, because it allows direct contact of explants with liquid nitrogen (LN), thus facilitating their rapid cooling and warming (7). However, this method needs skilful manipulation and includes cumbersome steps such as osmoprotective and dehydration treatments, transfer of samples on aluminium foil strips and transfer of foil strips in cryovials. Thus, simple and user-friendly cryopreservation procedures are required to facilitate large scale cryobanking. Dalmatian chrysanthemum ( Jochugiku in Japanese) is one of the many valuable medicinal species cultivated in Japan. It is an herbaceous perennial plant, which produces the natural insecticide pyrethrin. In the NIAS Genebank Project, 40 Dalmatian chrysanthemum lines are maintained in the field. Cryostorage is now needed to reduce their maintenance cost. Cryopreservation of Dalmatian chrysanthemum using an air dehydration method following preculture with 0.55 M sucrose and DMSO incubation has been reported by Hitmi et al. (5). In this paper, we developed an aluminium cryo-plate device for vitrification of Dalmatian chrysanthemum in vitro shoot tips, with the objective of establishing an efficient and simple cryopreservation procedure, thereby facilitating genebank cryostorage of this important germplasm. MATERIALS AND METHODS Plant material and tissue culture protocol The Dalmatian chrysanthemum (Tanacetum cinerariifolium) plants used in this study were obtained from the National Agricultural Research Center for Western Region, sub-bank of NIAS Genebank Project, in Japan. The line 28v-75 (registered name) was used for most experiments. Six other lines of Dalmatian chrysanthemum were also tested. The apical shoots excised from in vivo explants were surface-sterilized in 70% ethanol for 1 min followed by 0.5% sodium hypochlorite for 10 min, then rinsed twice in sterile distilled water. Shoot tips with basal plates (1 1 mm) were excised from the shoots and cultured on 60 ml of solid 1/2 strength Murashige and Skoog medium (1/2 MS; 8) in culture bottles (80 mm 100 mm). This medium was supplemented with 0.9 µm benzyladenine (BA), 3.0% (w/v) sucrose, 0.2 % (w/v) polyvinyl pyrolidone (PVP) and 0.9% (w/v) agar (Wako Pure Chemical Industries, Japan) (9). The first subculture was performed after 50 days, then the stock in vitro plantlets obtained were subcultured every 2 months on full strength MS medium with 0.9 μm BA, 3.0% (w/v) sucrose, 0.2% (w/v) PVP and 0.9% (w/v) agar (MS medium). Cultures were incubated at 25 1 C with a 16 h light/8 h dark photoperiod under white fluorescent light (52 μmol m 2 s 1 ) (standard conditions). Preconditioning and preculture of plant materials to be cryopreserved Apical shoot sections of 5 mm in length were cut and plated on 20 ml solid MS medium in Petri dishes (90 mm 20 mm), and cultured for 3 weeks under standard conditions. Shoots were then cold-hardened at 5 C on MS medium containing 0.5 M sucrose, 0.9 μm BA, 0.2% (w/v) PVP and 0.9% (w/v) agar with a 8 h light/16 h dark photoperiod under white 257

3 fluorescent light (26 µmol m 2 s 1 ) over a period of 20 to 40 days. Shoot tips ( mm) were then dissected from the shoots and precultured for 2 days on the same medium as above under the same cold-hardening conditions. Modified vitrification procedure Precultured shoot tips were placed in 2 ml plastic cryotubes (Wheaton Science Products, NJ, USA) and osmoprotected with 1 ml loading solution (LS; 12) containing 2 M glycerol to 1.6 M sucrose in liquid MS basal medium for 30 min at 25 C. After LS had been removed, shoot tips were dehydrated in 1 ml PVS2 (14) or plant vitrification solution 7M (PVS 7M) at 25 C for various periods. The PVSs were replaced once during treatment with 1 ml fresh PVS. After dehydration, the PVS was drained from the cryotubes and uncapped cryotubes containing shoot tips were directly plunged in LN using a cryo-cane and kept for at least 30 min at -196 C. PVS2 (14) contains 0.4 M sucrose, 30% (w/v) glycerol, 15% (w/v) ethylene glycol and 15% (w/v) dimethylsulfoxide in liquid MS basal medium (ph 5.8). Its total molarity is 8.0 M and its glass transition temperature is about -115 C. PVS 7M contains 30% (w/v) glycerol, 19.5% (w/v) ethylene glycol and 0.6 M sucrose in liquid MS basal medium (ph 5.8). Its total molarity is 7.0 M and its glass transition temperature is about C. The glass transition temperatures of these PVSs were measured using a differential scanning calorimeter (Q200, TA Instruments, Japan). Both PVSs were autoclaved for 20 min at 121 C before use. For regeneration, uncapped cryotubes were retrieved from LN and LN was poured out of the cryotubes. After 15 s, 2 ml 1 M sucrose solution in MS basal medium were poured in the cryotubes for rapid rewarming at room temperature. After 15 min, shoot tips were transferred on semi-solid standard MS medium. Post-rewarming regrowth (regrowth level) was evaluated after 4 weeks of culture at 25 C under standard conditions by counting the number of shoot tips that developed normal shoots. Three replicates of 10 shoot apices were tested in each experimental treatment. Results of the replicates are shown as averages ±SEM. Statistical analysis was performed using the Tukey test and significant differences (P<0.05) are indicated by different letters. Vitrification using aluminium cryo-plate (V-Cryo-plate) procedure Aluminium cryo-plates were developed in order to establish a simple, reproducible and reliable vitrification protocol. The two types of cryo-plates used (7 mm 37 mm 0.5 mm, Fig. 1A) were made of aluminium with 10 wells, and they were designed to fit in 2 ml cryotubes. On cryo-plate N 1, the wells had a diameter of 1 mm and a depth of 0.5 mm (Fig. 1A left). On cryo-plate N 2, the wells had a diameter of 1.50 mm and a depth of 0.75 mm (Fig. 1A right). The successive steps of the V-Cryo-plate procedure were the following: 1. Place an aluminum cryo-plate under the microscope in a Petri dish (9 cm ) and pour sodium alginate solution (N 1: 1 μl; N 2: 2 μl) in the wells of the aluminium plate using a micropipette. The alginate solution contains 2% (w/v) sodium alginate in calcium-free MS basal medium with 0.4 M sucrose. 2. Place the precultured shoot tips in the wells one by one with the tip of a scalpel blade and slightly press the shoot tips to make them fit in the plate s wells. 3. Pour the alginate solution (1 μl) over the shoot tips in order to cover them completely. In the case of Cryo-plate N 2, this step may skipped. 4. Pour the calcium chloride solution drop wise on the section of the aluminium plate where the shoot tips are located until they are covered and wait for 15 min to achieve complete 258

4 polymerization (Fig. 1B). The calcium solution contains 0.1 M calcium chloride in MS basal medium with 0.4 M sucrose. 5. Remove the calcium solution from the cryo-plate by sucking it gently with a micropipette. Shoot tips adhere to the cryo-plate through the alginate gel distributed in the wells (Fig. 1J, K). 6. Place the cryo-plate with shoot tips in a 25 ml pipetting reservoir (Matrix Tech. Co. NH, USA) filled with about 20 ml loading solution (LS). Shoot tips are osmoprotected at 25 C for 30 min. The LS solution contains 2 M glycerol to 1.6 M sucrose in liquid MS basal medium. 7. Remove the cryo-plate from LS and place it in a 25 ml pipetting reservoir filled with PVS2 or PVS 7M (about 20 ml). Shoot tips are dehydrated at 25 C for various durations (Fig. 1C). 8. After dehydration, transfer the cryo-plate in a 2 ml cryotube, which is held on a cryo-cane, and directly plunge it in LN where it is kept for at least 30 min (Fig. 1D-F). 9. Retrieve the cryotube from LN, take the cryo-plate with shoot tips out of the cryotube and immerse it in 2 ml 1 M sucrose solution contained in a 2 ml cryotube (Fig.1G-I). Shoot tips are incubated in this solution for 15 min at room temperature, and then transferred on solid MS medium. Post-rewarming regrowth (level) is evaluated after 4 weeks of culture at 25 C under standard conditions. Figure 1. Cryopreservation procedure using the aluminum cryo-plate. A: Aluminium cryo-plate N 1 (left) and N 2 (right). B: Calcium solution is poured on the cryo-plate. C: Dehydration with PVS 7M. D: The cryo-plate is inserted in a 2 ml cryotube. E-F: Immersion and storage in LN. G-I: Retrieval from LN, rewarming and dilution of VS. J-K: Cryo-plate with shoot tips (scale in mm). J K Measurement of cooling and warming rates Cooling and warming rates were measured with a platinum thermoelement sensor and data acquisition instrumentation. The temperature signals were recorded on a memory card by a wave thermo NR-1000 instrument (Keyence Co., Osaka, Japan). 259

5 RESULTS Optimization of the modified vitrification protocol Preliminary experiments (data not shown) had indicated that a day cold hardening treatment of in vitro plantlets ensured high regrowth after cryopreservation. Cold-hardened shoot tips were dissected from the shoots and precultured for 2 days on MS medium containing 0.5 M sucrose, 0.9 μm BA, 0.2% (w/v) PVP. To determine the optimal duration of exposure to PVS2 and PVS 7M, shoot tips were treated with both PVSs for different periods before immersion in LN. Exposure to PVSs produced time-dependent post-rewarming regrowth (Table 1). The highest regrowth was obtained for cryopreserved shoot tips treated with PVS 7M for min at 25 C. Regrowth after PVS2 treatment was low for all durations tested. In an attempt to increase osmoprotection and dehydration tolerance, cold-hardened and precultured shoot tips were osmoprotected in LS containing 2 M glycerol and 0.6 to 1.6 M sucrose in liquid MS basal medium for 30 min at 25 C. The highest regrowth of cryopreserved shoot tips was obtained after treatment with LS containing 2 M glycerol and 1.4 M sucrose (Table 2). Table 1. Effect of duration of exposure to PVS2 and PVS 7M on regrowth (%) of cryopreserved shoot tips. Duration of exposure Regrowth (% ± SEM) to PVSs PVS2 PVS 7M 40 min 16.7 ± 5.8 a 50.0 ± 10.0 b 50 min 26.7 ± 5.8 a 53.3 ± 5.8 b 60 min 13.3 ± 5.8 a 20.0 ± 10.0 a Note: Shoots were cold-hardened at 5 C for 37 days. Shoot tips were excised, precultured for 2 days at 5 C on MS with 0.5 M sucrose, loaded in 2 M glycerol +1.2 M sucrose for 30 min at 25 C and exposed to PVSs for min at 25 C. Ten shoot tips were tested for each of the three replicates per experimental condition. In columns, data followed by different letters are significantly different at the 95 % probability level. Table 2. Effect of sucrose concentration in LS solution on regrowth (%) of cryopreserved shoot tips. Sucrose concentration in LS Regrowth (% ± SEM) 0.6 M 25.0 ± 5.8 b 0.8 M 30.0 ± 8.2 b 1.0 M 37.5 ± 12.6 ab 1.2 M 40.0 ± 8.2 ab 1.4 M 50.0 ± 10.2 a 1.6 M 30.0 ± 8.2 b Note: Shoots were cold-hardened at 5 C for 30 days. Shoot tips were excised, precultured for 2 days at 5 C on MS with 0.5 M sucrose, loaded in each LS solutions for 30 min at 25 C and exposed to PVS 7M for 50 min at 25 C. Ten shoot tips were tested for each of the three replicates. Data followed by different letters are significantly different at the 95 % probability level. 260

6 Based on these results, the optimal modified vitrification procedure for cryopreservation of Dalmatian chrysanthemum shoot tips was as follows: 1. Apical shoots were cut and plated on standard solid MS medium and cultured for 3 weeks at 25 C under standard conditions. 2. Shoots were cold-hardened at 5 C on MS medium containing 0.5 M sucrose, 0.9 μm BA and 0.2% (w/v) PVP 0.9% (w/v) for 20 to 40 days. 3. Shoot tips were dissected and precultured at 5 C for 2 days on solid MS medium containing 0.5 M sucrose. 4. Precultured shoots were placed in 2 ml plastic cryotubes and osmoprotected with 1 ml LS containing 2 M glycerol and 1.4 M sucrose for 30 min at 25 C. 5. Shoot tips were dehydrated in 1 ml PVS 7M at 25 C for 30 min. The PVS 7M was replaced with 1 ml fresh PVS 7M once during treatment. 6. After dehydration, the PVS 7M was drained from cryotubes, and uncapped cryotubes containing the shoot tips treated were directly plunged in LN using a cryocane. However, this procedure was not user-friendly, mainly because small shoot tips suspended in large quantities of culture medium were difficult to manipulate. It was thus judged necessary to develop a new and simpler procedure, which could be used for large scale cryobanking. Vitrification procedure using aluminum cryo-plates with wells (V-Cryo-plate procedure) Cryopreservation procedures should be standardized so that they can be implemented by all genebank staff. We thus developed two aluminum cryo-plates (No. 1 and No. 2), which were employed for performing all steps of the vitrification procedure. Cryo-plate No. 1 had 10 wells of 1.0 mm diameter and cryo-plate No. 2 had 10 wells of 1.5 mm diameter. Shoot tips to be cryopreserved were trapped in the wells with the Na alginate solution. The cryo-plates with shoot tips were immersed in LS and PVS for the required durations, then transferred to cryotubes, which were plunged in LN. This procedure allowed retention of shoot tips in LS and PVS and damage with forceps and cumbersome pipetting was avoided. For rewarming, the cryo-plates were retrieved from LN and immersed in cryotubes filled with 2 ml of 1 M sucrose solution. The cooling and warming rates of shoot tips on the plates were 79.5 C s -1 and 75.4 C s -1, respectively. In this V-Cryo-plate procedure, shoot tips were attached by alginate gel to the cryo-plates. It is important that the shoot tips adhere steadily to the cryo-plate during the whole procedure, including rewarming. Following tests performed by seven persons, cryo-plate N 2 was selected for further experiments. Indeed, most shoot tips came away from cryo-plate N 1 during the cryopreservation procedure, whereas most of them remained attached to cryo-plate N 2. An experiment was performed to determine the optimal duration of treatment of shoot tips with PVS 7M using cryo-plate N 2. The highest regrowth percentage of cryopreserved shoot tips was achieved after 40 min dehydration with PVS 7M (Table 3). 261

7 Table 3. Effect of duration of exposure to PVS 7M on regrowth (%) of control (-LN) and cryopreserved (+LN) shoot tips using aluminium cryo-plate N 2. Duration of Regrowth (% ± SEM) exposure to PVS -LN +LN 30 min 80.0 ± 10.0 a 40.0 ± 10.0 b 40 min 76.7 ± 5.8 a 76.7 ± 11.5 a 50 min 60.0 ± 10.0 ab 50.0 ± 10.0 b 60 min 60.0 ± 0.0 ab 40.0 ± 10.0 b 70 min 40.0 ± 10.0 bc 20.0 ± 0.0 bc Note: Shoots were cold-hardened at 5 C for 30 days on MS with 0.5 M sucrose. Shoot tips were excised, precultured for 2 days at 5 C on MS with 0.5 M sucrose, loaded in 2.0 M glycerol and 1.4 M sucrose solution for 30 min at 25 C and exposed to PVS 7M for min at 25 C. Ten shoot tips were tested for each of the three replicates. In columns, data followed by different letters are significantly different at the 95 % probability level. Among the three durations of exposure to LS containing 2.0 M glycerol and 1.4 M sucrose tested, regrowth of cryopreserved shoot tips was higher after 30 and 60 min treatment durations (Table 4). Table 4. Effect of duration of exposure to LS solution containing 2.0 M glycerol and 1.4 M sucrose on regrowth (%) of cryopreserved Dalmatian chrysanthemum shoot tips using aluminium cryo-plate N 2. Duration of exposure Regrowth (% ± SEM) to LS 30 min 73.3 ± 5.8 a 60 min 80.0 ± 10.0 a 90 min 36.7 ± 5.8 b Note: Shoots were cold-hardened at 5 C for 30 days on MS with 0.5 M sucrose. Shoot tips were excised, precultured for 2 days at 5 C on MS with 0.5 M sucrose, loaded in 2.0 M glycerol and 1.4 M sucrose solution for 30 min at 25 C and exposed to PVS 7M for min at 25 C. Ten shoot tips were tested for each of the three replicates. Data followed by different letters are significantly different at the 95 % probability level. Based on these results, the optimal V-cryo-plate procedure for cryopreservation of Dalmatian chrysanthemum shoot tips was as follows. 1. Five mm shoots were sampled from stock cultures, plated on solid MS medium and cultured for 3 weeks at 25 C. 2. Shoots were cold-hardened at 5 C with an 8 h light/16 h dark photoperiod for days on MS with 0.5 M sucrose. 3. Shoot tips ( mm) were dissected from the shoots and precultured at 5 C for 2 days on MS with 0.5 M sucrose. 4. Precultured shoot tips were placed on N 2 aluminium cryo-plates (7 mm x 37 mm x 0.5 mm) with 10 wells (diameter 1.5 mm, depth 0.75 mm) and fixed on the plates using calcium alginate gel. 5. Cryo-plates with shoot tips were put in a 25 ml pipetting reservoir filled with 20 ml LS solution containing 2 M glycerol and 1.4 M sucrose for min at 25 C. 6. Cryo-plates were taken out of LS and transferred to a 25 ml pipetting reservoir filled with 262

8 20 ml PVS 7M for 40 min at 25 C. 7. After dehydration, the cryo-plates were transferred to 2 ml cryotubes, which were directly plunged in LN. 8. For rewarming the cryotubes were retrieved from LN, the cryo-plates were taken out of cryotube and immersed for 15 min at room temperature in 2 ml 1 M sucrose solution contained in 2 ml cryotubes. The shoot tips were then transferred to solid standard MS medium. Using the optimal procedure established in this study, we tested the regrowth rates of shoots from 6 lines of Dalmatian chrysanthemum. Regrowth was relatively high in all lines, ranging from 65% to 90%, with an average of 76.7% for the 6 lines (Table 5). Surviving shoot tips resumed growth within three days of plating and developed a normal shoot without intermediary callus formation (Fig. 2). Table 5. Regrowth (%) of shoot tips from six Dalmatian chrysanthemum lines cryopreserved using aluminium cryo-plate No.2 Line Regrowth (% ± SEM) Line Regrowth (% ± SEM) 19v-KO ± v ± v ± v ± v ± v ± 7.1 Note: Shoots were cold-hardened at 5 C for 35 days on MS with 0.5 M sucrose. Shoot tips were excised, precultured for 2 days at 5 C on MS with 0.5 M sucrose, loaded in 2.0 M glycerol and 1.4 M sucrose solution for min at 25 C and exposed to PVS 7M for 40 min at 25 C. Ten shoot tips were tested for each of the three replicates. Figure 2. Regrowth of cryopreserved Dalmatian chrysanthemum shoot tips 5 days (left) and 30 days (right) after rewarming. Scale bars in mm. DISCUSSION In conventional vitrification procedures including DV, small size shoot tips are suspended in the various solutions employed, which have to be removed and added by repeated pipetting. This often results in damage and loss of shoot tips during the course of the cryopreservation protocol. Moreover, vitrification procedures require a precise control of duration of treatment with vitrification solutions, due to the narrow range of optimal treatment durations. In a DV procedure, dehydrated shoot tips need also to be transferred on aluminium 263

9 strips just before immersion in LN. All these disadvantages may impede the implementation of cryostorage in genebanks using DV procedures. By contrast, the vitrification protocol developed in this work, the so-called V-Cryo-plate procedure has many advantages as follows: 1. Handling of shoot tips throughout the procedure is very easy and quick because only the cryo-plate is manipulated. 2. The possibility of injuring and losing shoot tips is considerably reduced compared with other methods. 3. Shoot tips can be easily and efficiently treated with LS and PVS as they do not float in the solutions or adhere to the wall of the cryotubes. 4. Cooling and warming are also performed very easily by immersing the cryo-plate in LN (cooling) and the 1.0 M sucrose solution (warming). 5. Very high regrowth percentages can be obtained using this V-Cryo-plate procedure. 6. This new method is much less laborious than other methods. 7. All staff can implement this procedure after a little practice with mounting of shoot tips on the cryo-plates. In the vitrification method, steps such as preconditioning, preculture, osmoprotection (loading treatment), exposure to PVSs, and post-rewarming manipulations are vital for successful cryopreservation (10, 11). In any cryogenic protocol, cells and tissues to be cryopreserved must be in a physiologically optimal status in order to acquire dehydration tolerance and to produce vigorous growth recovery (1, 16). Hirai and Sakai (2) indicated that the success of cryopreservation by vitrification depends on preconditioning meristems to induce osmotolerance to PVS2. For cryopreservation of potato shoot tips by encapsulationvitrification, LS solution (a mixture of 2 M glycerol plus 0.6 M sucrose) was effective in increasing osmotolerance to PVS2 (2). These authors also mentioned that for cryopreservation of sweet potato shoot tips, a higher sucrose concentration (1.6 M) in LS and a longer period of osmoprotection (3 h at 25 C) were necessary to increase osmotolerance (4). However, direct exposure of shoot tips to PVS2 caused harmful effects due to osmotic stress (3). In our V-Cryo-plate procedure, shoot tips are encapsulated in calcium alginate gel, which might mitigate the chemical toxicity of cryoprotectant solutions. When developing alternative LSs to be employed in a droplet-vitrification procedure, Kim et al. (7) indicated that the loading treatment might act as an osmotic stress neutralizer and/or induce a physiological adaptation of tissues and cells before both dehydration and vitrification. They also pointed out that an appropriate LS should be selected for plant species, which are highly sensitive to the cytotoxicity of PVSs, in order to reduce their detrimental effect. In the case of Dalmatian chrysanthemum, a LS solution with a high sucrose concentration was effective in increasing osmotolerance to PVS 7M. Meristematic cells, which have been treated with LS are osmotically dehydrated and might be plasmolyzed to a considerable extent (15). These cells have then to be dehydrated with PVSs until they reach the optimal cytosolic concentration required for vitrification (2). The key to successful cryopreservation by vitrification lays with the establishment of precisely controlled dehydration procedures, while preventing injury by chemical toxicity or excessive osmotic stress during treatment with PVS. The V-Cryo-plate procedure established in this work renders the application of a vitrification protocol to fragile shoot tips possible. We are currently employing this technique for cryostoring 40 Dalmatian chrysanthemum lines and we are also applying it to other plant species. In conclusion, this is the first report of the utilization of the V-Cryo-plate method, which is a combination of DV and encapsulation-vitrification protocols. This method appears to be promising for facilitating large scale cryobanking in genebanks. 264

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