CryoLetters 30 (3), 291-299 (2009) © CryoLetters,
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DEVELOPMENT OF ALTERNATIVE LOADING SOLUTIONS IN DROPLET-VITRIFICATION PROCEDURES Haeng-Hoon Kim1*, Yoon-Geol Lee1, Sang-Un Park2, Sheong-Chun Lee3, Hyung-Jin Baek1, Eun-Gi Cho1 and Florent Engelmann4,5 1
National Academy of Agricultural Science, RDA, Suwon 441-707, Korea. Chungnam National University, Daejon, 305-764, Korea. 3 Sunchon National University, Suncheon, 540-742, Korea. 4 Institut de recherche pour le développement (IRD), UMR DIAPC, BP 64501, 34394 Montpellier cedex 5, France. 5 Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy. *Corresponding author email:
[email protected] 2
Abstract In plant vitrification protocols, the loading treatment, which involves treating the explants with a moderately concentrated cryoprotectant solution, precedes dehydration of explants with highly concentrated vitrification solutions in order to reduce the toxicity which can be induced by their direct exposure to such highly concentrated solutions. This study aimed at developing alternative loading solutions composed of mixtures of glycerol and sucrose at various concentrations. Differential scanning calorimetry runs of loading solutions and of loaded and dehydrated explants were performed to assay thermal events occurring during cooling and warming. These loading solutions were applied to two model species, viz. garlic and chrysanthemum which were cryopreserved using a droplet-vitrification procedure. The loading treatment proved to be beneficial to both garlic and chrysanthemum and increased recovery of cryopreserved explants. However, response to the loading solutions tested varied between the two model species employed: with garlic, all the loading solutions had a similar effect, whereas survival of chrysanthemum shoot tips was significantly influenced by the composition of the loading solution employed. A loading solution comprising 1.9 M glycerol and 0.5 M sucrose was the most effective. The loading treatment may thus act as an osmotic stress neutralizer and/or induce the physiological adaptation of tissues and cells, including membranes, to both dehydration and freezing. Keywords: chrysanthemum, garlic, droplet-vitrification, loading solution. INTRODUCTION Vitrification-based cryopreservation procedures involve dehydration with highly concentrated cryoprotectant mixtures (vitrification solutions, VSs) prior to immersion in liquid nitrogen (LN) in order to eliminate most or all freezable water from explants. A loading treatment prior to dehydration with highly concentrated VSs generally increases recovery of 291
cryopreserved explants, and is thus applied in many of plant vitrification procedures, even though the protection mechanism involved is not clearly understood (1, 6, 9, 15). In plant vitrification protocols, the loading treatment involves incubating explants in a cryoprotectant solution (loading solution, LS) with a lower concentration than that of VSs in order to reduce the toxicity which can be induced by direct exposure of explants to highly concentrated VSs. Even though the loading treatment established by Nishizawa et al. (9) and Matsumoto et al. (6), which consists of exposing the samples for a short time period (20-60 min) to a LS including 2 M glycerol + 0.4 M sucrose in liquid culture medium has been applied to many plant species, a number of diverse loading treatments have been experimented for freezing plant samples. These treatments include notably exposing samples to LSs consisting of 1 M glycerol + 0.8 M sucrose for sweet potato, 2 M glycerol + 0.5 M sucrose for garlic (4), 2 M glycerol + 0.6 M sucrose for sweet potato (1) and garlic (3). Following the original procedure of Rall and Fahy (10), diluted VSs have also been used as LSs, e.g. 20-60% PVS2 (12, 13, 16). Loading treatments of explants for longer periods (overnight) with LSs at lower concentrations (0.5 M glycerol + 0.3 M sucrose) proved to be beneficial in the case of wasabi (7). Loading samples with cryoprotectants primarily serves to minimize injury during the dehydration step, more specifically to prevent dehydration-induced destabilization of cellular membranes and possibly of proteins, as in non-acclimated protoplasts (14). Another purpose of the loading step is to increase the solute concentration of the cytosol, so that it will vitrify during quenching in the cryogenic fluid (14). Nishizawa et al. (9) also noted that loading appears to be effective in inducing freeze-dehydration tolerance or dehydration tolerance. During the short loading treatment, cells are plasmolysed due to intense dehydration, but little permeation of glycerol takes place in the cytosol (7). Matsumoto et al. (7) noted that the protective effect of LSs in the periprotoplasmic space may be due to mitigation of osmotic stress induced by the severe dehydration with PVS2. Sakai indicated that the protective effect of a brief incubation of explants with LS might be a result of the concentration of cytosolic cryoprotectants accumulated during preculture with sucrose, and of the protective effect of plasmolysis (11). Takagi also pointed out that loading treatment is useful to minimize the adverse effects of VSs, especially when it is applied to shoot apices of species which are sensitive to PVS2 (15). The present study aimed at developing alternative plant LSs composed of glycerol and sucrose, which were employed in a droplet-vitrification procedure. Differential scanning calorimetry (DSC) runs of LSs and of loaded and dehydrated samples were performed to study the thermal events occurring during the freeze-thaw cycle. The LSs selected were applied to samples of two model plant species, i.e. large bulbous garlic shoot apices (3 mm in diameter), which are tolerant to VSs and smaller chrysanthemum shoot tips (1.2-1.5 mm in length), which are sensitive to VSs. MATERIALS AND METHODS Plant materials The Korean garlic cv. Danyang was used in this study. Bulb scales were planted in the field in Suwon, Korea in September 2006 with mulching provided by black plastic film. Bulbs were harvested in June 2007, left to dry in the plastic greenhouse for 4 weeks and then stored in a cold room at 4°C with 65-7 % relative humidity (RH) for 3 to 6 months until sampling. In vitro grown shoot tips of chrysanthemum (Dendranthema grandiflora T. cv. peak) were the starting material of the experiment. They were sampled from nodal segments, which had been maintained in vitro for 4 years, with subcultures every 6-7 weeks on MS medium (8) with 0.15 mg l-1 indole acetic acid (IAA), 0.05 mg l-1 zeatin, 0.1 M sucrose and 2.2 g l-1 292
Phytagel at 24 1°C, with a light intensity of 50 µE m-2 s-1 and under a 16 h light/8 h dark photoperiod. Loading solutions Several alternative plant LSs, including various concentrations of glycerol and sucrose in culture medium were designed. To prepare these LSs, 13.7-24.0 % sucrose was dissolved in 5-strength MS medium (8) stock. After adding 9.2-23.0% glycerol, the final volume was adjusted with deionized water, pH was adjusted to 5.8 and the solution was filter-sterilized. The composition of the LSs studied is listed in Table 1. Table 1. Composition and total concentration of the loading solutions used in this study. Loading solutions
Composition (M, glycerol + sucrose)
Composition (% w/v, glycerol + sucrose)
Total concentration (% w/v)
C1 C2 C3 C4 C6 C7 C8 C9
1.0 + 0.7 2.0 + 0.7 1.6 + 0.4 1.9 + 0.5 2.2 + 0.6 2.0 + 0.4 2.5 + 0.5 2.4 + 0.7
9.2 + 24.0 18.4 + 22.3 15.0 + 15.0 17.5 + 17.5 20.0 + 20.0 18.4 + 13.7 23.0 + 17.1 22.5 + 22.5
33.2 40.7 30.0 35.0 40.0 32.1 40.1 45.0
Remarks
30 % PVS3 35 % PVS3 40 % PVS3
45% PVS3
Thermal analysis The thermal analysis system used in this investigation was a DSC822 (TA8000 MettlerToledo, GMbh, Switzerland) incorporating a heat flux module, LN cooling system and an Epson TAS811 workstation employing STARe Software. For DSC analysis, the formulated LSs were weighed on an analytical balance (12-13 mg, precision ± 1µg) and placed individually in 40 µl aluminum pans for scanning. Garlic shoot apices incubated in LSs for 40 min, then dehydrated with PVS3 for 150 min, were sampled and placed individually in 100 µl aluminum pans for scanning. The average individual fresh weight of shoot apices was 21-30 mg. Chrysanthemum shoot tips incubated in LSs for 40 min, then dehydrated with PVS3 for 60 min, were sampled and placed individually in 40 µl aluminum pans for scanning. The average fresh weight of two shoot tips was 12-13 mg. Scans involved cooling with a scanning rate of 10°C min-1 from 25 to -85°C, followed by isothermal hold at -85°C for 2 min to allow sample equilibration. Samples were then heated to 25°C using a linear scanning rate of 10°C min-1. Analyses were performed using an average of 4-6 samples for each experimental condition. The thermograms obtained from each run were analyzed using the DSC evaluation software, to obtain onset temperatures of crystallization and of ice melting, and enthalpies. Enthalpies were determined from the area of the peaks above or below the interpolated baseline. Cryopreservation of garlic shoot apices Garlic clove shoot apices were cryopreserved using the method of Kim et al. (2, 4). Shoot apices were extracted from garlic cloves using a metallic cork borer with a 3 mm diameter. Basal plates were cut until they were 0.8 to 1.1 mm thick; the upper parts of the apices were then trimmed to a size of 3 x 4 mm. The explants employed for freezing consisted of the meristematic dome, the surrounding leaf primordia and a basal part. Explants were inoculated on MS medium with 0.15 mg l-1 indole acetic acid (IAA), 0.2 mg l-1 zeatin, 0.3 M sucrose and 2.2 g l-1 phytagel and precultured at 10°C for 3-4 days, under a 16 h light/8 h dark 293
photoperiod, with a light intensity of 50 µE m-2 s-1. Explants were incubated in diverse LSs containing glycerol + sucrose in MS medium for 40 min at 24 1°C. They were dehydrated in 30 ml PVS3 solution (9) for 150 min with continuous shaking (90 rpm) at 24 1°C. Just before plunging in LN, five drops (5 µl each) of VS were placed on aluminum foil strips (7 x 20 mm). One explant was put in each of the five VS drops and then the foil strips were directly plunged in LN. After a few min, foil strips were transferred to 2 ml cryovials previously filled with LN (two foil strips per vial) and stored in a LN tank for no less than a day. To determine recovery, explants from 4-5 cryovials (i.e. 40-50 explants) were treated as follows: for warming, foil strips were retrieved from the cryovials and plunged in pre-heated (40°C) unloading solution (0.8 M sucrose) for 30 s, after which an equal volume of unloading solution was added. Explants were incubated in the unloading solution at 24 1°C for 50 min to facilitate unloading. Explants were retrieved from the unloading solution and incubated on MS medium with 0.15 mg l-1 IAA, 0.2 mg l-1 zeatin, 0.3 M sucrose and 2.0 g l-1 phytagel with 0.1 M sucrose under standard conditions. Survival was evaluated after 10 days by counting the number of shoots which were green and swollen (≥ 3 mm in length). Regeneration was evaluated 30 days after cryopreservation by counting the number of shoots which had developed leaves (≥ 13 mm in length). Cryopreservation of chrysanthemum shoot tips Axillary shoot tips (1.2-1.5 mm in length) isolated from 3-4 day cultured nodal segments of in vitro chrysanthemum plantlets, which had been subcultured every 6-7 weeks, were used in this study. Isolated shoot tips were precultured in liquid MS medium at 24±1°C with progressively increasing sucrose concentration, with the following sequence: 0.3 M sucrose for 27 h, 0.5 M sucrose for 18 h and 0.7 M sucrose for 8 h. After loading at room temperature for 40 min with the LSs tested, shoot tips were dehydrated with 10 ml PVS3 for 60 min at room temperature with continuous shaking (90 rpm). The cooling and warming procedures were the same as those described above for garlic apices. For growth recovery, shoot tips were post-cultured on semi-solid MS medium supplemented with 0.15 mg l-1 IAA + 0.2 mg l-1 zeatin + 0.05 mg l-1 gibberellic acid (GA3) + 0.1 M sucrose + 2.0 g l-1 phytagel at 24±1°C under low light intensity for 7 days and then transferred to standard culture conditions. Survival was evaluated 14 days after cryopreservation by counting the number of shoot tips which were green and swollen (≥ 3 mm). Regeneration was counted after 48 days when shoots had developed leaves (≥ 10 mm). In all experiments, 12-14 shoot tips were used per experimental condition and experiments were replicated 4-5 times. Statistical analysis Results are presented in percentages with their standard deviation. Data were analyzed by analysis of variance (ANOVA), following arcsin transformation, with Duncan’s multiple range tests (DMRT), using the SAS 8.1 software. RESULTS Thermal properties of loading solutions The osmolarity (Osm), endothermic enthalpy (Ensol) and onset temperature (Onsol) of the LSs tested and Ensol of garlic shoot apices loaded with LSs (Ensam) were measured in terms of total concentration (TC) and glycerol to sucrose ratio (GSR) of the LSs. The parameters of the LSs, including TC and GSR, significantly (P < 0.0001) affected Osm, Ensol, Onsol and Ensam of the LSs tested (Table 2), indicating that their design was appropriate. 294
Thermal properties of the LSs were mostly affected by both TC and Osm rather than GSR. Total concentration (TC) of the LSs studied significantly (P < 0.0001) affected Osm, Ensol, Onsol and Ensam, with correlation coefficients of 0.86, 0.95, -0.94 and 0.86, respectively. Ensol was clearly grouped by TC, i.e. 45 % < 40-40.7% < 32-35 % < 30%. While GSR did not affect Ensol, Onsol nor Ensam, it significantly (P < 0.0001) affected Osm with a correlation coefficient of 0.51. Ensam had high correlation with Osm, ensol and onsol (r = 0.85, 0.79 and -0.87, respectively), indicating thermal properties of garlic shoot apices loaded with LSs were similar to those of the LSs tested. Endothermic enthalpies of garlic shoot apices loaded with LSs studied (Ensam) were distributed between -129.7 Jg-1 FW (C9) and 194.5 Jg-1 FW (C3). Table 2. Osmolarity, endothermic enthalpy of LSs tested and of garlic shoot apices loaded with LSs for 40 min related to some parameters of the LSs. TC (% w/v) 33.2 40.7 30.0 35.0 40.0 32.1 40.1 45.0
Osm Ensol Onsol Ensam (Osmol) (Jg-1 FW) (Jg-1 FW) (Jg-1 FW) C1 0.38 0.92±0.06f -137.6±3.4c -12.8±0.1a -193.0±3.7e c b d C2 0.83 1.44±0.01 -117.9±6.0 -16.8±0.2 -148.2±7.8b e d a C3 1.00 1.16±0.02 -149.4±7.3 -12.9±0.4 -194.5±4.1e c c c -165.2±6.7c C4 1.00 1.39±0.01 -134.2±5.9 -15.2±0.4 b b de C6 1.00 1.58±0.01 -122.7±4.4 -17.5±0.4 -149.7±2.4b d c b C7 1.34 1.25±0.00 -139.9±7.0 -14.2±0.5 -174.2±7.6d C8 1.35 1.60±0.00b -116.1±3.1b -17.9±0.5e -170.9±7.0d a a f C9 1.00 1.83±0.04 -100.2±4.4 -20.1±0.5 -129.7±5.2a P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 LS, loading solution; TC, total concentration; GSR, glycerol to sucrose ratio; Osm, osmolarity (osmol) of 50 %-diluted LSs; Ensol, endothermic enthalpies of LS tested; Onsol, onset temperature of recrystallization of the LS tested; Ensam, endothermic enthalpies of garlic shoots apices loaded with LS for 40 min; MC, moisture content (fresh weight basis) of garlic shoot apices loaded with LS for 40 min. LS
GSR
Table 3. Endothermic enthalpies (Jg-1 FW) of LSs, of garlic shoot apices and chrysanthemum shoot tips after preculture (PC), loading (LD) and dehydration (DH). Enthalpy (Jg-1 FW) Treatment LS
Loading solution
Garlic
Chrysanthemum b
C3 C4 C6
-149.4±7.3 -134.2±5.9a -122.7±4.4a P < 0.01 PC-LD Non-LD -245.5±4.7d -181.9±23.7b c C3 -194.5±4.1 -117.8±3.9a b C4 -165.2±6.7 -118.1±18.1a a C6 -149.7±2.4 -101.5±12.9a P < 0.0001 P < 0.05 PC-LD-DH Non-LD -0.2±0.2 -0.2±0.2 C3 -0.2±0.3 -0.0±0.0 C4 -0.4±0.3 -1.2±2.4 C6 -0.2±0.3 -0.6±1.2 ns ns LS, loading solution; PC, preculture with 0.3 M sucrose solid medium for 3 days at 10°C for garlic or with 0.3 M – 0.5 M – 0.7 M sucrose solution for 27 h, 18 h and 8 h, respectively, for
295
chrysanthemum; LD, incubation with LSs tested for 40 min; DH, dehydration with PVS3 for 150 min for garlic or 60 min for chrysanthemum. Non-LD, loading control.
Thermal properties of three LSs (C3, C4, C6, i.e. 30-40 % PVS3) were compared during the course of the droplet-vitrification procedure (Table 3). Even though significant differences were observed in LS (P < 0.01) and PC-LD (P < 0.0001 for garlic, P < 0.05 for chrysanthemum), no difference was observed in PC-LD-DH. Furthermore, the endothermic enthalpy of non-loaded and dehydrated explants (PC-DH) was not significantly different from that of loaded and dehydrated explants (PC-LD-DH), which implies that the lower regeneration percentage of cryopreserved garlic shoot apices (Control in Table 4) may not be due to crystallization injury but more likely to the osmotic stress incurred by exposure to PVS3. Even though the damage possibly caused by osmotic stress in non-loaded explants did not significantly decrease recovery of dehydrated controls (-LN), it significantly (P < 0.0001) decreased recovery of cryopreserved (+LN) explants. Overall, the same pattern was observed in chrysanthemum shoot tips. After loading with C4 for 40 min (LD), moisture content (MC) of precultured (PC) garlic shoot apices decreased from 82.7% to 72.1%; it further decreased to 37.6% after dehydration with PVS3 for 150 min (DH). Endothermic enthalpy decreased from -245.5 Jg-1 FW (PC) to -165.2 Jg-1 FW (LD), then to -0.4 Jg-1 FW after dehydration (DH). Application to garlic shoot apices Loading did not affect survival and regeneration of dehydrated control (-LN) garlic shoot apices (Table 4). After cryopreservation, non-loaded explants showed 84.5% survival and 82.7% regeneration, which was significantly (P < 0.0001) lower than that of loaded samples. However, no significant difference was observed in survival and regeneration of cryopreserved (+LN) explants incubated with the LSs tested, indicating that loading treatment did not affect recovery of cryopreserved garlic shoot apices. This result highlights the importance of incubating garlic shoot apices with one of the LSs tested, whatever its composition, before exposure to PVS3. Table 4. Survival (Surv, %) and regeneration (Rege, %) of control (-LN) and cryopreserved (+LN) garlic shoot apices incubated for 40 min with the LSs tested, followed by dehydration with PVS3 for 150 min. -LN
+LN
Loading solution Control C1 C2 C3 C4 C6 C7 C8 A9
Surv (%)
Rege (%)
Surv (%)
Rege (%)
100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 ns
96.9±4.4 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 100.0±0.0 ns
84.5±1.4b 100.0±0.0a 100.0±0.0a 98.9±2.3a 100.0±0.0a 98.9±1.9a 100.0±0.0a 100.0±0.0a 100.0±0.0a P < 0.0001
82.7±3.0b 100.0±0.0a 100.0±0.0a 98.9±2.3a 100.0±0.0a 100.0±0.0a 98.5±2.6a 100.0±0.0a 100.0±0.0a P < 0.0001
Application to chrysanthemum shoot tips The composition of the LS employed significantly (P < 0.001) affected regeneration of dehydrated control (-LN) chrysanthemum shoot tips, while it did not affect survival (Table 5). 296
Regeneration of dehydrated controls (-LN) significantly decreased when the concentration of LS was below 30% (C3) or above 45% (C9), and GSR was below 1.0 (C1 0.38, C2 0.83). Significantly (P < 0.001) lower regeneration of cryopreserved (+LN) explants incubated with these loading solutions was observed, implying damage in cryopreserved explants may be due to osmotic shock rather than to crystallization injury. Significant differences were observed in survival (P < 0.05) and regeneration (P < 0.0001) of cryopreserved (+LN) explants incubated in the various LSs tested. The highest regeneration was observed with C4 (35% PVS3) followed by C6 (40% PVS3) in both dehydrated control (LN) and cryopreserved (+LN) explants, indicating that equilibrium in LS with the appropriate glycerol and sucrose percentage was beneficial. These results imply that selecting the appropriate LS is a prerequisite for successful cryopreservation of chrysanthemum shoot tips. Table 5. Survival (Surv, %) and regeneration (Rege, %) of dehydrated control (-LN) and cryopreserved (+LN) chrysanthemum shoot tips incubated for 40 min in LSs followed by dehydration with PVS3 for 60 min. Vitrification solution C1 C2 C3 C4 C6 C7 C8 A9 F > Pr
Surv (%) 94.9±5.9 95.1±5.7 94.0±4.1 98.3±3.3 96.7±6.7 98.3±3.3 93.9±7.1 90.1±8.2 ns
-LN Rege (%) 74.2±6.2c 74.7±2.3c 72.6±4.6c 90.0±7.8a 88.6±8.9ab 85.8±5.0ab 79.1±4.5bc 69.2±9.1c P < 0.001
+LN Surv (%) 81.1±2.7b 81.3±3.4b 82.2±4.2b 89.2±4.6a 86.2±6.9ab 84.1±4.0ab 79.7±2.6b 80.1±2.4b P < 0.05
Rege (%) 39.0±4.0e 55.4±4.5b 40.4±5.5de 65.3±5.3a 56.5±3.2b 53.9±4.8b 52.4±2.9bc 46.5±4.2cd P < 0.0001
DISCUSSION Some alternative plant LSs composed of glycerol and sucrose were designed in this work. The osmolarity and thermal properties of the LSs were predominantly affected by the total concentration (30-45% w/v) of the LSs tested rather than by the glycerol to sucrose ratio (0.38-1.35). The thermal properties of three LSs were compared during the course of the dropletvitrification procedure of garlic shoot apices. The loading treatment significantly (P < 0.0001) affected recovery of cryopreserved explants compared to non-loaded samples, whatever the LS tested. In non-loaded explants, even though damage, possibly induced by osmotic stress, did not significantly decrease recovery of control samples, it significantly (P < 0.0001) decreased recovery of cryopreserved samples. Even though significant differences were observed in endothermic peaks between explants loaded with the different LSs tested, no significant difference was detected in endothermic peaks between dehydrated samples. Furthermore, the endothermic enthalpy of non-loaded and dehydrated explants was not significantly different from that of loaded and dehydrated explants, indicating that loading treatment did not affect ice blocking properties of the dehydrated samples. This is supported by the previous observation that a loading treatment with 2 M glycerol + 0.4 M sucrose (C7) did not significantly affect the concentration of glycerol and sucrose influxed into the apices nor the moisture content of garlic shoot apices dehydrated with PVS3 (5). Therefore, the damage observed in non-loaded cryopreserved garlic shoot apices may not be due to crystallization, because of the insufficient influx of 297
cryoprotectants and efflux of water, but rather due to osmotic stress caused by direct exposure of shoot apices to PVS3. Therefore, the loading treatment may thus act as an osmotic stress neutralizer and/or induce a physiological adaptation of tissues and cells, including membranes, before both dehydration and freezing. Unlike with garlic, the composition of LSs significantly affected regeneration of both dehydrated and cryopreserved chrysanthemum shoot tips. A lower recovery was observed when explants were incubated with LSs with both lower (30%) and higher (45%) total concentration of cryoprotectants and lower GSR (0.7 M sucrose). The highest regeneration was observed with C4 (35%-diluted PVS3 solution) in both dehydrated control and cryopreserved explants. Even though a loading treatment was beneficial for both garlic and chrysanthemum to increase recovery of cryopreserved explants, the response of these two materials to the LSs tested was different: the composition of the LS had no effect on recovery of garlic apices, whereas recovery of chrysanthemum shoot tips was significantly influenced by the composition of the LSs tested. The lower recovery of cryopreserved chrysanthemum seems due to sensitivity to cytotoxicity of the VS rather than to ice crystallization damage, since dehydrated chrysanthemum shoot tips displayed lower endothermic peaks compared to dehydrated garlic apices. Therefore, an appropriate LS should be selected for plant species which are highly sensitive to cytotoxicity of the VSs. The beneficial effect of the loading treatment may be explained in several ways. Firstly, the loading treatment may (biophysically) neutralize osmotic stress of plant samples by exposure to a moderately concentrated osmoticum, before severe dehydration with highly concentrated VSs. As pointed out by Steponkus et al. (14), the addition of cryoprotectants (LS) also serves to mitigate the dehydration-induced destabilization of cellular membranes and possibly proteins during dehydration. Secondly, the hypothesis of an increase in the solute concentration of cytosolic cryoprotectants needs to be clarified, whether it is the concentration of cytosolic cryoprotectants accumulated during preculture (11) or the increase of the total amount of cryoprotectants before freezing (14). The concentration of cytosolic cryoprotectants should not be the effect of the loading treatment only, but predominantly that of dehydration, since it would be better facilitated during dehydration with highly concentrated VSs than with LSs. For the same reason, cell plasmolysis should also be an effect of dehydration, rather than that of loading treatment. Even though the amount of glycerol and sucrose in garlic shoot apices dehydrated with PVS3 for 150 min following loading treatment was not significantly increased compared to non-loaded explants (5), in certain cases, a loading treatment preceding a short dehydration period with a toxic VS (e.g. PVS2) may increase the total amount of cryoprotectants before freezing. Thirdly, loading treatment may allow cryoprotectants to enter within the inner parts of tissues/organs and/or to stabilize samples biochemically, especially when dehydration duration is short due to the toxicity of VSs. In conclusion, both the protective mechanism of LSs and the appropriate composition of LSs may depend on the nature of VS toxicity, i.e. osmotic and/or biochemical. Selecting the appropriate LS is crucial for plant species which are sensitive to VSs, especially when samples are large and/or very sensitive to biochemical toxicity of the VSs, which limits the duration of the dehydration period. Acknowledgements: This work was supported by NAAS and a grant (2008040103406001) from BioGreen 21 Program, Rural Development Administration, Korea.
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