CryoLetters 27(4), 223-234 (2006) CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK
CRYOPRESERVATION OF POTATO CULTIVATED VARIETIES AND WILD SPECIES: CRITICAL FACTORS IN DROPLET VITRIFICATION Haeng-Hoon Kim1*, Ju-Won Yoon1, Young-Eun Park2, Eun-Gi Cho1, Jae-Keun Sohn3, Tae-San Kim1, and Florent Engelmann4, 5 1
National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, Korea. 2National Institute of Highland Agriculture, RDA, Pyungchang 232-955, Korea. 3Department of Agronomy, Kyungpook National University, Taegu, Korea. 4Cirad, Station de Roujol, 97170 Petit-Bourg, Guadeloupe, French West Indies (present address). 5International Plant Genetic Resources Institute (IPGRI), Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, * Italy. Correspondence:
[email protected] Abstract The applicability of cryopreservation protocols to a broad range of genotypes is a key issue for genebanks. We tried to identify the critical factors causing differences in survival of cryopreserved shoot tips using potato varieties coming from cultivated and wild species. The droplet-vitrification method, a combination of droplet-freezing and solution-based vitrification, was selected from several protocols. High survival after freezing was observed after dehydration with PVS2 for 20 min, cooling shoot tips placed in a droplet of PVS2 solution on aluminum foil strips by immersing the foil strips in liquid nitrogen, warming them by plunging the foil strips into a 0.8 M sucrose solution (at 40°C) for 30 s and unloading in 0.8 M sucrose for 30 min. This optimized protocol was successfully applied to 12 accessions with survival ranging between 64.0 and 94.4%. Keywords: droplet-vitrification, PVS2/PVS3 loading, shoot tips, Solanum tuberosum L. INTRODUCTION Various cryopreservation studies have been carried out on potato, including (ultra)-rapid freezing (2,3,10) and two-step (slow or controlled) freezing (3,12,37,38,39,40), but these methods have limitations, such as low survival, recovery lag phase and callus formation (3,39,40). Recently, vitrification based protocols, such as vitrification (8,32), encapsulationdehydration (4,7,9) and encapsulation-vitrification (14) have been applied to potato germplasm. Though, among these protocols, vitrification (8,19) and droplet-freezing (33,34) have been implemented on a large scale (CIP, Peru; DSMZ, Germany), recovery is variable, reflecting the genetic diversity and responses of accessions to cryopreservation (7,8,14,33). Wild species such as Solanum goniocalyx, S. chaucha, S. stenotomum, showed lower levels of recovery compared to cultivated varieties, possibly due to low growing vigor under in vitro and in field conditions (8,10,34). Vitrification solutions are complex mixtures of cryoprotectants, which have been formulated for their ability to vitrify during cooling. Most vitrification solutions employed for plant materials have been elaborated by Sakai’s, Steponkus’ and Towill’s groups. PVS2, the 223
vitrification solution most commonly employed for cryopreservation of plant species, has a total molarity of 7.8 M and is highly toxic (31). We need to compare various vitrification solutions to understand biophysical mechanisms occurring in explants as well as to select the most appropriate solution(s). During the cooling and warming procedures, rapid heat transfer is needed to avoid freezing injury. With garlic, the combination of rapid cooling by plunging aluminum foil strips on which bulbil primordia had been placed in droplets of cryoprotectant medium and rapid warming by dipping the foil strips in pre-heated (40°C) 0.8 M sucrose solution for 30 s produced higher regeneration than the other cooling and warming combinations tested (unpublished data). The applicability of a cryopreservation protocol to diverse genotypes is of paramount importance to genebank curators for implementing long-term and large scale germplasm conservation programmes. The different survival levels observed within different potato species could be related to the vigor of mother plants or depend on the genotype (8). Therefore, the optimization of the protocol for wild species is a prerequisite for implementing cryopreservation of potato collections in genebanks. Thus, the analysis of factors causing differences in survival between cultivated and wild species during a cryopreservation procedure should be performed. Preliminary (unpublished) studies have shown that the droplet-vitrification method, a combination of the droplet-freezing and vitrification protocol, was highly appropriate for this study. We applied it to one cultivated and one wild potato species to determine the critical factors affecting the survival of cryopreserved shoot tips. In a previous study, survival of cryopreserved shoot tips of both varieties tested had been significantly affected by subculture conditions and frequency, the size of the shoot tips and their location on the plantlet axis, sucrose concentration of the preculture medium and preculture time (unpublished results). In the present work, we compared one cultivated and one wild potato species during the course of a vitrification process including the dehydration, cooling and warming, and unloading steps. MATERIALS AND METHODS In vitro grown potato shoot tips of Solanum tuberosum (var. Dejima, 4X, cultivated) and S. stenotomum (STN13, 2X, wild), which were introduced in vitro by meristem cultures were used in this study. Tissue culture methods and sample preparation For micropropagation, nodal segments consisting of a piece of stem were transferred to Murashige and Skoog basal medium (MS, 24) containing 30 g/l sucrose, 2.2 g/l phytagel (Sigma Co.) without growth regulators and incubated at 24 ± 1°C, under a photoperiod of 16 h light/8 h dark, with a light intensity of 100-140 µmol m-2 s1 and subculture intervals of 7 weeks for var. Dejima and 5 weeks for STN13. Duct system and Gaooze containers (height 13 cm, diameter 9 cm; KSTI Co, Korea) were used to allow ventilation of culture shelves and culture vessels, respectively. After 7 and 5 weeks for var. Dejima and STN13, respectively, axillary shoot tips (1.5-2.0 mm and 1.0-1.5 mm in size for var. Dejima and STN13, respectively) were isolated by dissection from the upper to middle part of the mother-plants. Cryopreservation Isolated shoot tips were precultured in liquid MS medium with 0.3 M sucrose for 16-24 h under the conditions mentioned above. Shoot tips were further precultured in liquid MS 224
medium with 0.7 M sucrose for 7-8 h under the same conditions. No loading treatment was performed, except where mentioned in the experiments. A combination of droplet-freezing (33,34) and solution-based vitrification was employed in this study. Shoot tips (n = 14) were dehydrated in 10 ml PVS2 solution (31; 30 % (w/v) glycerol + 15 % (w/v) ethylene glycol (EG) + 15 % (w/v) dimethyl sulfoxide (DMSO) in MS basal medium with 0.4 M sucrose) for 20 min with continuous shaking (60 rpm). A few min before plunging in liquid nitrogen (LN), seven drops (2.5 µl each) of PVS2 solution were placed on an aluminum foil strip (7 x 20 mm) using a dispenser. One shoot tip was put in each of the seven PVS2 drops and then the foil strip was immediately plunged in LN. After a few min, two foil strips were transferred in one 2 ml cryovial, which had been previously filled with LN and stored in a LN tank for no less than one day. Dehydration was performed with various vitrification solutions, including PVS2, PVS3, Steponkus, and Towill solutions (Table 1) for 20 and 60 min in both varieties. To select the optimal dehydration time, shoot tips were dehydrated with PVS2 for 0-60 min in both varieties. Table 1. Composition of the various vitrification solutions used in the study Solution PVS2 PVS3
Composition Glycerol 30 % + dimethylsulfoxide (DMSO) 15 % + ethylene glycol (EG) 15 % in MS with 0.4 M sucrose Glycerol 50 % + sucrose 50 % in MS
Steponkus EG 50 % + sorbitol 15 % + bovine serum albumin (BSA) 6 % (in MS with 0.4 M sucrose) Towill EG 35 % + DMSO 1 M + polyethylene glycol (PEG) 8000 10 % (in MS with 0.4 M sucrose)
Reference 31 26 19 41
Four different protocols, which have been applied to potato germplasm were compared to optimize these procedures (Table 2). The protocol employed in this study is dropletvitrification B, if not specified otherwise. Three different cryo-containers for shoot-tips, i.e. aluminum foil strips, polypropylene cryovials, propylene straws, were employed to compare the effect of cooling and warming rates (Table 6). A combination of cooling and warming procedures was experimented with (Table 7). During cooling, foil strips were plunged in LN (LN) or in LN slush (S-LN). For warming, foil strips were taken out of the cryovials and immediately plunged in pre-heated unloading solution (0.8 M sucrose (10 ml) at 40°C) for 30 s and 5 ml of the pre-chilled unloading solution were added (Dip). Alternatively, frozen vials were directly immersed into 40°C water for 5 sec to evaporate LN, uncapped and poured 1.5 ml of 0.8 M sucrose (40°C) into the vial. Warmed shoot tips were further incubated for 20 min (Evaporate & Pour). After warming, shoot tips were further incubated in the unloading solution at room temperature (RT) for 30 min to facilitate unloading. Unloading solutions with different sucrose osmolarities (0.3-1.2 M) and different unloading durations (0-60 min) were compared (Table 8). The osmolarity of unloading solutions was 0.42, 1.12, and 2.29 Osm for 0.3 M, 0.8 M, and 1.2 M sucrose, respectively.
225
Table 2. Comparison of the different cryopreservation protocols employed with the potato varieties. Procedure Shoot tips Preculture
Dehydration Cooling
Warming
Unloading Postculture
Droplet-freezing Droplet-vitrification A Droplet-vitrification Vitrification B Apical Axillary Axillary Axillary MSTo medium + 16-20 h with 0.3 M 8 h with 0.3 M 24 h with 0.1 M 3 % sucrose, sucrose, Loading (0.4 sucrose followed by sucrose followed by overnight M sucrose + 2 M 18 h with 0.7 M 5 h with 0.6 M glycerol) for 60 min sucrose sucrose 10 % DMSO for 2 PVS2 for 30 min PVS2 for 20 min PVS2 for 20 min h Plunge the foil Plunge the foil strips Plunge the foil Plunge the strips in LN in nitrogen slush strips in LN propylene straw (2.5 µl) in LN Dip the foil strip in Evaporate LN and Dip the foil strip in Dip the straw in unloading solution then add preheated pre-heated 0.8M ethanol (RT) 1.2 M sucrose (40°C) sucrose (40°C) for for 30 s 30 s MSTo medium for 20 min in 1.2 M 30 min in 0.8 M 30 min in 1.5 Osm 30 min sucrose sucrose sorbitol
Liquid MSTo medium (vitamin free MS + 2 % sucrose) and then transfer to solid medium Designer Schäfer-Menuhr (Operating (PAL/DSMZ, institute) Germany) 33, 34 Reference
2 d in liquid MSTo medium and then transfer to solid medium
Semisolid MS Semisolid MS medium + 0.3 mg/L medium + 0.04 zeatin + 0.05 mg/L mg/L kinetin + 0.1 IAA +0.05 mg/L mg/L GA3 + 2.5 % GA3+ 2.5% sucrose sucrose
Towill (NCGRP, USA)
Kim (NIAB/RDA, Korea)
Steponkus (CIP, Peru)
36
-
8
MSTo: MS medium + 0.5 mg/l indole acetic acid (IAA) +0.5 mg/l zeatin + 0.2 mg/l gibberellic acid (GA3). Solidified LN: Fill a vessel with LN and place under vacuum for 15-20 min. After vacuum treatment (solidification of LN), LN turns into slush and does not boil at room temperature. Pre-heated 0.8 M sucrose (at 40°C): Preheat 0.8 M sucrose solution to 40 °C in a water bath before warming. Upon warming, take off foil strips from cryovials and immediately plunge in 6-7 ml of pre-heated (40°C) 0.8 M sucrose solution for 30 s, after which the same volume of the same unloading solution is added.
Application of the optimized protocol (Droplet-Vitrification B) to a range of varieties Twelve potato varieties, both cultivated and wild species (Table 3) were cryopreserved using the optimized droplet-vitrification protocol after different subculture periods (3-7 weeks). Postculture and survival assessment For recovery, shoot tips were post-cultured on semi solid MS medium (MS + 0.05 mg/l IAA + 0.3 mg/l zeatin + 0.05 mg/l GA3 + 3 % sucrose + 1.8 mg/l phytagel) at 24 ± 1°C under low light intensity for 7 days and then transferred to standard culture conditions. Different post-freezing procedures were applied to compare the different protocols described in Table 2. 226
Unloaded shoot tips were postcultured in liquid medium and then transferred to solid medium (Droplet-freezing, Droplet-vitrification A), or postcultured directly on semi-solid medium (Droplet-vitrification B, Vitrification). Survival was evaluated 14 days after cryopreservation by counting the number of shoot tips that were green and swollen ( 3 mm). In all experiments, 14 shoot tips were used per experimental condition and experiments were replicated 3-4 times. Controls correspond to shoot tips submitted to all treatments except cryopreservation. Table 3. List of a range of potato varieties used in cryopreservation. Donor No. AG14025 AG24004 AG24006 B3 B5 AG54055 STN13 07S0300241 07S0300007 CIP701830 CHC53 CHC67
Scientific name Solanum tuberosum L. S. tuberosum L. S. tuberosum L. S. tuberosum L. S. tuberosum L. S. tuberosum L. S. stenotomum JUZ. et BUK. S. stenotomum JUZ. Et BUK. S. goniocalyx JUZ. et BUK. S. goniocalyx JUZ. Et BUK S. chacoense L. S. chacoense L.
Variety name Cherokee Dejima Superior Superior (GMO) Superior (GMO) Chuncheonjaerae STN13 K6-2 07S0300007 CIP701830 CHC53 CHC67
Description Cultivar Cultivar Cultivar pBIN2 TP/Pbin35s #37-1-1 Landrace Wild Wild Wild Wild Wild Wild
Statistical analysis Results are presented as mean percentages with their standard deviation. Results were analyzed using the ANOVA table with the least significant difference (LSD) and Duncan’s multiple range tests (DMRT), using the SAS 8.1 software. RESULTS Dehydration With variety Dejima, the vitrification solution significantly affected survival in both dehydrated control (LN-, P < 0.05) and cryopreserved (LN+, P < 0.001) shoot-tips (Table 4). With STN13, no significant difference was observed in survival of dehydrated control (LN-) shoot-tips. Among the vitrification solutions and dehydration periods tested, dehydration with PVS2 for 20 min resulted in highest survival with both varieties. Based on the results of Table 4, the effects of dehydration time with PVS2 were further examined. The highest survival of cryopreserved shoot tips was observed after dehydration for 20 min with PVS2 in both varieties (Fig. 1). Thereafter, survival of both dehydrated control (LN-) and cryopreserved (LN+) shoot tips decreased significantly (P < 0.05, P < 0.001, respectively) as the dehydration time increased, possibly due to the cytotoxicity of PVS2 (17,30).
227
Table 4. Effect of vitrification solution and dehydration time on the survival of dehydrated control (LN-) and cryopreserved (LN+) potato shoot tips. Survival (%)
Vitrification solution and dehydration time (min)
Dejima STN13 LNLN+ LNLN+ a a a PVS2 20 100.0±0.0 91.9±9.9 100.0±0.0 86.4±9.4a 60 74.5±9.4b 60.1±8.1bc 97.5±3.5a 80.3±8.3ab ab e a 35.3±7.9 100.0±0.0 83.3±3.9ab PVS3 20 86.7±11.5 60 93.3±11.5ab 63.8±17.0b 83.3±23.6a 81.4±13.7ab a bcd a Steponkus 20 100.0±0.0 55.4±18.1 100.0±0.0 45.8±10.2d c cde a 41.8±16.1 83.3±23.6 41.6±16.4d 60 57.5±5.6 ab de a Towill 20 96.7±5.8 36.5±5.4 100.0±0.0 62.4±6.1c 60 93.3±11.5ab 66.0±10.2b 97.5±3.5a 69.8±2.2bc Pr > F < 0.0030 < 0.0001 ns < 0.0001 Vitrification solution 0.0283 0.0004 ns < 0.0001 Time 0.0002 ns ns ns Vitrification sol.*Time 0.0005 < 0.0001 ns ns Means ± SD followed by the same letter in columns are not different at the 95 % significance level of the ANOVA table with DMRT. ns: non significant.
C-D EGF,H
>
F
Dejima LN100.0±0.0a 94.8±4.5a 100.0±0.0a 91.1±7.8a ns
STN13 LN+ 76.7±1.8ab 67.7±2.4b 83.3±10.4a 47.9±7.0c 0.0059
LN100.0±0.0a 93.3±4.7a 100.0±0.0a 90.2±5.9a ns
LN+ 29.0±2.8c 54.5±1.3b 70.3±1.6a 14.3±4.2d 0.0018
Cooling and warming protocols The cooling and warming devices significantly (P < 0.001) affected survival of cryopreserved (LN+) shoot tips in both varieties (Table 6). The highest survival was observed with foil strips, followed by straws and cryovials, due to the higher cooling and warming rates. Table 6. Survival of potato shoot tips cryopreserved using different devices for cooling and warming. Survival (%, LN+) Dejima STN13 72.9±5.5a 69.8±4.3a 39.6±14.8b 28.4±4.8c 63.1±3.4a 46.8±6.3b 0.0120 0.0096
Device Aluminum foil strip Polypropylene cryovial Propylene straw Pr > F
The warming protocol significantly (P < 0.05 for var. Dejima and P < 0.001 for STN13) affected survival of cryopreserved shoot tips in both varieties (Table 7). Warming of shoot tips by directly dipping the foil strips in pre-heated (40°C) 0.8 M sucrose medium (Dip) resulted in significantly higher survival, regardless of the cooling regime (LN or S-LN). Table 7. Effect of different cooling and warming protocols on the survival of cryopreserved potato shoot-tips. Cooling
Warming
LN
Dip Evaporate & Pour S-LN Dip Evaporate & Pour Pr > F Cooling Warming Cooling * Warming
229
Survival (%) Dejima 90.4±6.6a 62.5±5.9b 87.5±5.9a 75.0±7.1ab 0.0367 ns 0.0112 ns
STN13 78.5±1.3a 51.9±3.2b 75.2±4.5a 55.2±5.0b < 0.0001 ns < 0.0001 ns
Unloading Both sucrose concentration of unloading solution and unloading time significantly (P < 0.001) influenced survival of cryopreserved shoot tips of both varieties studied (Table 8). Unloading control shoot tips showed survival percentages below 30 % in both varieties, indicating that the unloading treatment is indispensable for obtaining high survival of cryopreserved shoot tips. The highest survival was observed after unloading with a 0.8 M sucrose solution for 30 min in both varieties. Table 8. Survival of cryopreserved potato shoot-tips as a function of the sucrose concentration of the unloading solution and of the unloading time employed. Sucrose concentration of unloading solution (M) 0.3
0.8
1.2
Survival (%)
Unloading time (min) 0 10 30 60 10 30 60 10 30 60
Dejima 22.6±9.3c 65.5±11.7ab 62.0±18.3ab 71.0±5.2ab 62.8±16.9ab 80.4±17.1a 78.5±5.6a 53.3±18.0b 58.8±21.2ab 63.8±16.0ab 0.0010 0.0135 0.0002 ns
Pr > F Concentration Time Concentration * Time
STN13 27.1±4.2e 53.2±4.0cd 57.4±19.5bcd 48.3±9.6cd 78.0±8.6a 81.1±9.2a 73.0±10.1ab 41.9±14.0de 65.0±14.0abc 56.0±4.4cd < 0.0001 < 0.0001 0.0006 ns
Application of the optimized protocol to diverse potato genotypes The optimized protocol (Droplet-Vitrification B) was successfully applied to 12 potato varieties including both cultivated and wild species. Survival of cryopreserved shoot tips was significantly (P < 0.05) different among diverse genotypes (Table 9). The optimal subculture time in terms of recovery and the number of shoot tips available for freezing were different among varieties. In some wild species, the number of shoot tips available per mother-plant was quite low, since shoot tips were at different developmental stages or had already elongated. Both immature and elongated shoot tips showed lower survival after cryopreservation.
230
Table 9. Survival of control (LN-) and cryopreserved (LN+) shoot tips of 12 potato varieties. Species S. tuberosum S. tuberosum S. tuberosum S. tuberosum S. tuberosum S. tuberosum S. stenotomum S. stenotomum S. goniocalyx S. goniocalyx S. chacoense S. chacoense
Subculture Shoot tips (weeks) /plantlet Cherokee 4-5 5-6 Dejima 6-7 5-6 Superior 6-7 5-6 Superior (GMO) 6-7 5-6 Superior (GMO) 6-7 5-6 6-7 5-6 Chuncheon-jaerae STN13 5 4-5 K6-2 3 2-3 07S0300007 4 2-3 CIP701830 4 1-2 CHC53 3-4 1-2 CHC67 3-4 3-5 Pr > F Variety
Survival (%) LNLN+ a 100.0±0.0 80.0±0.0abc a 100.0±0.0 94.4±7.9a a 100.0±0.0 85.0±7.1ab a 100.0±0.0 78.7±8.4abc 97.5±3.5a 89.4±0.8a a 100.0±0.0 90.9±0.0a a 100.0±0.0 87.3±3.8ab 91.7±11.8a 72.9±5.6bc 100.0±0.0a 91.7±11.8a a 93.8±8.8 64.0±10.4c a 100.0±0.0 68.2±6.4c 89.4±0.8a 87.1±5.4ab ns 0.0101
DISCUSSION Among the cryopreservation protocols tested, droplet-vitrification, a combination of droplet-freezing and vitrification, showed higher survival than droplet-freezing or vitrification. Within the droplet-vitrification protocol, survival of cryopreserved shoot tips was significantly different depending on the warming regime. The highest survival of cryopreserved potato shoot tips was observed when shoot tips were placed on aluminum foil strips and directly immersed in LN for cooling and directly dipped in pre-heated (40°C) 0.8 M sucrose solution during warming, since faster heat transfer was achieved. Aluminum foil has an efficient thermal conductivity, resulting in quick and uniform heat distribution among explants (15, 20). The differences in survival of cryopreserved shoot tips among protocols seemed mainly due to high cooling and warming rates, since aluminum foil strips provided higher survival over cryovials or straws under the same conditions. The highest cooling rate is measured in the following order: aluminum foil strip in LN slush (400°C/s; 28) >> aluminum foil strips in LN (130°C/s; 36) > specimen straws into LN (60°C/s; 36) > cryovials in LN (6°C/s; 36). To achieve rapid heat transfer during cooling and warming, straws were used, instead of cryovials for freezing mint (19,29) and potato shoot tips (8) using a vitrification protocol. In this study, cooling of potato shoot tips in sealed propylene straws provided higher survival than polypropylene cryovials. The droplet-freezing method at a slow cooling rate (0.5 °C/min) has been applied to cassava shoot tips (15). The droplet-freezing method combined with solution-based vitrification has been applied successfully to papaya (1), Prunus (5), chrysanthemum (11), yam (20), banana (27) sweet potato (28), and potato (Towill, NCGRP, USA, pers. com.). In addition to rapid cooling, warming the foil strips (explants) by direct dipping in preheated (40°C) unloading solution ensured faster warming than evaporating LN and pouring pre-heated (40°C) 0.8 M sucrose solutions into the cryovials, and resulted in significantly 231
higher survival with both cooling regimes tested. It is likely that more rapid warming achieved by direct immersion of foil strips into pre-heated unloading solution (0.8 M sucrose) following droplet-freezing in LN compensates the effect of higher cooling rate of dropletfreezing in LN slush. It is recognized that a high warming rate should be employed to avoid recrystallization of intracellular ice or additional cell dehydration by extracellular ice, when vitrification of samples during cooling is not stable (22). Dumet et al. (2002) reported that rapid thawing by immersion of explants in a sucrose based pre-heated unloading solution resulted in higher survival than dipping the cryovial in a pre-heated water bath. The vitrification solution employed significantly affected survival of cryopreserved shoot tips in both potato varieties. Cryopreserved shoot tips of var. Dejima dehydrated with PVS3 or Towill solution for 20 min showed significantly (P < 0.001) lower survival than STN13, possibly due to relatively larger size since larger explants need longer dehydration time (17, 18). Survival of dehydrated control (LN-) and cryopreserved (LN+) shoot tips of var. Dejima dehydrated with PVS2 or Steponkus solution for 60 min was lower than for 20 min, possibly due to their cytotoxic effect. PVS2 is a highly concentrated vitrification solution and is thus highly toxic to plant tissues (30). Sensitivity to dehydration time varies widely among different species, e.g. from 20 min for persimmon (21) to 120 min for Prunus shoot tips (25). Variety STN13 was less sensitive to cytotoxicity of the vitrification solutions tested than var. Dejima, since there was no significant difference among vitrification solutions and dehydration periods in control (LN-) shoot tips. A 1.2 M sucrose solution has been used as unloading solution in many vitrification protocols (e.g. 13,14,28). Samples are usually washed in 1.2 M sucrose for several min and then transferred to a new medium after one day of postculture (20). No significant difference was observed in survival of cryopreserved garlic shoot tips unloaded with 0.3-1.2 M sucrose and equivalent sorbitol solutions (16). In this study, 0.8 M sucrose resulted in higher survival in comparison with 0.3 M or 1.2 M sucrose with both varieties tested. The efficiency of the unloading solution is influenced by its osmolarity (osmotic stress and efficiency of unloading) and by the duration of its application (accumulation of osmotic stress and amount of cryoprotectants effluxed). The concentration of remaining cryoprotectants after the unloading treatment decreased as the unloading time increased in garlic shoot tips (17,18). Therefore, transferring samples to a new medium after one day of postculture can be avoided, if the unloading time is increased. This study demonstrated the higher applicability of droplet-vitrification in comparison with conventional vitrification protocols, presumably due to the higher cooling and warming rates achieved. Droplet-freezing facilitates faster heat transfer, since the samples/cryoprotectants solutions are in direct contact with LN. This optimized protocol was successfully applied to 12 potato accessions, including wild species. These wild species previously showed poor response to cryo-exposure, because of the low number of shoot tips at the right developmental stage. Only minor changes in the duration of the last subculture before cryopreservation allowed increasing the number of shoot tips per mother plant adequate for cryopreservation. This simplified protocol eliminates the necessity of performing a loading treatment, of using liquid nitrogen slush, and of transferring the explants to a new medium during postculture. This protocol is currently being applied to the Korean in vitro potato collection, which comprises about one thousand genotypes and which is maintained in Pyungchang, Korea by the National Institute of Highland Agriculture, RDA for the large scale implementation of germplasm cryopreservation. Acknowledgements: We are grateful to Mrs. Dong-Bin Ahn and Mrs. Eun-Ju Han for their technical assistance. 232
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.
Ashmore S, Saunders R & Drew R (2000) in Cryopreservation of Tropical GermplasmCurrent Research Progress and Application, (eds) F Engelmann & H Takagi, JIRCAS, Tsukuba & IPGRI, Rome, pp. 453-456. Bajaj YPS (1977) Crop Improvement 4, 48-53. Benson EE, Harding K & Smith H (1989) CryoLetters 10, 323-344. Bouafia S, Jelti N, Lairy G, Blance A, Bonnel & Dereuddre J (1996) Potato Res 39, 6978. Boucaud MT, Brison M, Helliot B & Herve-Paulus V (2002) in Cryopreservation of Plant Germplasm II vol. 50, (eds) LE Towill & YPS Bajaj, Springer-Verlag, Berlin, pp. 287-311. Dumet D, Grapin A, Bailly C & Dorion N (2002) Plant Science 163, 1121-1127. Fabre J & Dereuddre J (1990) CryoLetters 11, 413-426. Golmirzaie A & Panta A (2000) in Cryopreservation of Tropical Plant Germplasm Current Research Progress and Applications, (eds) F Engelmann & H Takagi, JIRCAS, Tsukuba & IPGRI, Rome, pp. 250-254. Grospietsch M, Stodulkova E & Záme ník J (1999) CryoLetters 20, 339-346. Grout BW & Henshaw GG (1978) Annals of Botany 42, 1227-1229. Halmagyi A, Fischer-Kluver G, Mix-Wangner & Schumacher HM (2004) Plant Cell Reports 22, 371-375. Harding K, Benson EE & Smith H (1991) CryoLetters 12, 17-22. Hirai D & Sakai A (2003) Plant Cell Reports 21, 961-966. Hirai D & Sakai A (2000) in Cryopreservation of Tropical Plant Germplasm - Current Research Progress and Applications, (eds) Engelmann F & Takagi H, JIRCAS, Tsukuba & IPGRI, Rome, pp. 205-211. Kartha KK, Leung NL & Mroginski LA (1982) Z Pflanzenphysiologie 107, 133-140. Kim HH, Cho EG, Baek HJ, Kim CY, Keller ERJ & Engelmann F (2004) CryoLetters 25, 5970. Kim HH, Kim JB, Baek HJ, Cho EG, Chae YA & Engelmann F (2004) CryoLetters 25, 91-100. Kim JB, Kim HH, Baek HJ, Cho EG, Kim YH & Engelmann F (2005) CryoLetters 26, 103112. Langis R & Steponkus PL (1990) Plant Physiology 92, 666-671. Leunufna S & Keller ERJ (2003) Plant Cell Reports 21, 1159-1166. Matsumoto T, Mochida K, Itamura H & Sakai A (2003) Plant Cell Reports 20, 398-402. Meryman HT & Williams RJ (1985) in Cryopreservation of Plant Cells and Organs, (ed) KK Kartha, CRC Press, Boca Raton, Florida, pp. 13-48. Moges AD, Shiblira & Karam (2004) In Vitro Cell Devel Biology-Plant 40, 389-395. Murashige T & Skoog F (1962) Physiologia Plantarum 15, 473-497. Niino T, Tashiro K, Suzuki M, Ohuchi S, Magoshi J & Akihama T (1997) Scientia Horticulturae 70, 155-163. Nishizawa S, Sakai A, Amano A & Matsuzawa T (1993) Plant Science 91, 67-73. Panis B, Piette B & Swennen R (2005) Plant Science 168, 45-55. Pennycooke JC & Towill LE (2000) Plant Cell Reports 19, 733-737. Plessis P & Steponkus P (1996) Cryobiology 33, 655-656. Sakai A (1995) in Biotechnology in Agriculture and Forestry, Vol 52. (eds) YPS Bajaj, Springer-velaf, Berlin, Heidelberg, New York, pp. 53-69. Sakai A, Kobayashi S & Oiyama I (1990) Plant Cell Reports 9, 30-33. Sarkar D & Naik P (1998) Annals of Botany 82, 455-461. Schäfer-Menuhr A, Muller E & Mix-Wagner G (1996) Potato Research 39, 507-513. 233
34. Schäfer-Menuhr A, Schumacher HM & Mix-Wagner G (1997) Plant Genetic Resources Newsletter 111, 19-24. 35. Takagi H (2000) in Cryopreservation of Tropical Plant Germplasm- Current Research Progress and Applications, (eds) F Engelmann & H Takagi, JIRCAS, Tsukuba & IPGRI, Rome, pp. 178-193. 36. Towill LE & Bonnart R (2003) CryoLetters 24, 341-346. 37. Towill LE (1981) CryoLetters 2, 373-382. 38. Towill LE (1981) Plant Science Letters 20, 315-324. 39. Towill LE (1983) Cryobiology 20, 567-573. 40. Towill LE (1984) CryoLetters 5, 319-326. 41. Towill LE (1990) Plant Cell Reports 9, 178-180. Accepted for publication 25/6/06
234
