AOBPreview originally published online on January 3, 2006
Annals of Botany 2006 97(3):453-459; doi:10.1093/aob/mcj049
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Effect of Growth Phase on Survival of Bromegrass Suspension Cells Following Cryopreservation and Abiotic Stresses
1 Genetic Diversity Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, Japan 305-8602 and 2 Department of Plant Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5A8
* For correspondence. E-mail isikawam{at}affrc.go.jp
Received: 14 September 2005 Returned for revision: 11 November 2005 Accepted: 22 November 2005 Published electronically: 3 January 2006
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ABSTRACT |
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 TOPFOOTNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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• Background and Aims Cryopreservation is a practical method of preserving plant cell cultures and their genetic integrity. It has long been believed that cryopreservation of plant cell cultures is best performed with cells at the late lag or early exponential growth phase. At these stages the cells are small and non-vacuolated. This belief was based on studies using conventional slow prefreezing protocols and survival determined with fluorescein diacetate staining or 2,3,5-triphenyltetrazolium chloride assays. This classical issue was revisited here to determine the optimum growth phase for cryopreserving a bromegrass (Bromus inermis) suspension culture using more recently developed protocols and regrowth assays for determination of survival.
• Methods Cells at different growth phases were cryopreserved using three protocols: slow prefreezing, rapid prefreezing and vitrification. Stage-dependent trends in cell osmolarity, water content and tolerance to freezing, heat and salt stresses were also determined. In all cases survival was assayed by regrowth of cells following the treatments.
• Key Results Slow prefreezing and rapid prefreezing protocols resulted in higher cell survival compared with the vitrification method. For all the protocols used, the best regrowth was obtained using cells in the late exponential or early stationary phase, whereas lowest survival was obtained for cells in the late lag or early exponential phase. Cells at the late exponential phase were characterized by high water content and high osmolarity and were most tolerant to freezing, heat and salt stresses, whereas cells at the early exponential phase, characterized by low water content and low osmolarity, were least tolerant.
• Conclusions The results are contrary to the classical concept which utilizes cells in the late lag or early exponential growth phase for cryopreservation. The optimal growth phase for cryopreservation may depend upon the species or cell culture being cryopreserved and requires re-investigation for each cell culture. Stage-dependent survival following cryopreservation was proportionally correlated with the levels of abiotic stress tolerance in bromegrass cells.
Key words: Bromegrass (Bromus inermis), cryopreservation, freezing, heat, salt, vitrification, prefreezing, culture growth phase, suspension cultures, regrowth, viability assay, osmolarity
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INTRODUCTION |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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In the last two decades, plant cell cultures have been utilized for genetic transformation, in vitro multiplication and as experimental systems for basic research. Problems associated with cell cultures are that the cultures or their specific characters can be lost due to prolonged subculturing, contamination, somaclonal variation, intensive and high labour requirement, and potential loss due to incubation failure. Cryopreservation has attracted attention as a method to preserve these cultures and maintain their genetic integrity safely (Grout, 1995
).
Currently, four main methods are available for cryopreservation of plant cell cultures: conventional slow prefreezing, vitrification, rapid prefreezing and desiccation (with or without encapsulation in alginate beads) (Ishikawa, 1994). In these methods, the specimen is dehydrated either by extracellular freezing, using high-concentration cryoprotective solutions or desiccation prior to exposure to ultra low temperatures. The ability to tolerate extreme dehydration aids in improving survival following cryopreservation. In the pioneer era of cryopreservation studies, Sugawara and Sakai (1974) and Withers and Street (1977) described the importance of culture growth phases using sycamore cells. Their results demonstrated that the late lag or early log (exponential) phase, during which cells are small in size, rich in cytoplasm and less vacuolated, results in optimum survival using traditional slow prefreezing protocols. These results are reported in standard textbooks (Kartha, 1985; Grout, 1995). Since then, only a few studies have focused on this issue. It is of interest to determine whether this hypothesis on growth phase dependency of cell survival is applicable to more recently developed methods of cryopreservation, such as vitrification and rapid prefreezing.
Many studies, including the classical papers on cryopreservation (Sugawara and Sakai, 1974; Withers and Street, 1977), utilized fluorescein diacetate (FDA) staining or 2,3,5-triphenyltetrazolium chloride (TTC) reduction assays to determine survival of cells. For as yet unknown reasons, FDA and TTC assays conducted immediately after thawing often give erroneously high survival values compared with regrowth assays (Ishikawa et al., 1996). It is generally agreed that cell regrowth is a more reliable measure of cell survival.
In cryopreservation protocols, cells are exposed to combinations of cryoprotective chemicals and dehydration treatments prior to being submersed in liquid nitrogen (LN2). The amounts of water and osmolytes in the cells are crucial for cryopreservation. If cells at a particular growth phase are tolerant to abiotic stress, it is highly likely that the cells may have a high survival rate following a freeze–thaw protocol in cryopreservation. There have been few studies conducted on this line of reasoning.
The issue of culture growth phases for successful cryopreservation was revisited here using a bromegrass suspension culture with the following objectives: (1) to determine using regrowth assays which culture growth phase is optimum for cryopreserving a cell suspension culture using three protocols: slow prefreezing, rapid prefreezing and vitrification; (2) to characterize the cells at various growth phases showing high or low survival following cryopreservation in terms of water content and osmolarity; and (3) to determine the relationship between cryopreservation survival and tolerance to other abiotic stresses.
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MATERIALS AND METHODS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Cultured cells
A non-embryogenic suspension culture of smooth bromegrass (Bromus inermis Leyss ‘Manchar’) was maintained as previously described (Ishikawa et al., 1990
). Briefly, cultures were initiated by transferring 1·3 g of cells to 50 mL of fresh Erickson's (ER) medium (pH 5·8, 0·5 ppm 2,4-D) and incubated on a rotary shaker (80 rpm) at 25 °C unless otherwise noted. Cells of various incubation periods (0–14 d from subculture) were harvested by filtering through a nylon mesh of 80 µm. Cells were blotted on sterilized filter paper and processed for cryopreservation. All experiments were replicated three times or more.
Cryopreservation using a slow prefreezing (two-step) method
The protocol was basically as previously described (Ishikawa et al., 1996). Briefly, bromegrass cells (usually 0·2–0·3 g) were dispensed into conical centrifuge tubes (10 mL, graduated glass). One-millilitre aliquots of a cryoprotectant cocktail, CSP1, comprising sucrose (10 %, w/v), dimethyl sulfoxide (DMSO; 10 %, w/v), and glycerol (5 %, w/v), were directly added to the cells and incubated at 25 °C for 30 min. Thereafter, the final volume was reduced to 0·5 mL and the cells in the tubes were equilibrated at –8 °C for 20 min, aseptically ice-inoculated and held for a further 15 min to ensure freezing was completed. The cells were cooled linearly at 0·3 °C min–1 to –30 °C in a programmable freezer and after 5 min the cells were immersed directly into LN2. Following storage in LN2 for 1 h, the tubes were rapidly rewarmed in water maintained at 40 °C. The cryoprotectant solution was diluted five-fold by dropwise addition of 3 % (w/v) sucrose over 15 min at 25 °C and processed for viability assays. Non-frozen control cells treated with the cryoprotectant only were diluted and processed in the same manner.
Rapid prefreezing method
The rapid prefreezing protocol was performed as described previously (Ishikawa et al., 1991; Ishikawa, 1992). Briefly, bromegrass cells (approx. 0·2 g) were dispensed into conical centrifuge tubes (10 mL, graduated glass). Aliquots (0·3 mL) of cryoprotectant cocktail, RPF2, comprising sucrose (5 %, w/v), glycerol (30 %, w/v) and CaCl2 (10 mM), were directly added to the cells and incubated at 25 °C for 3 or 5 min. The cells in the tubes were directly placed at –30 °C (without ice-inoculation) for 30 min (the RPF2 solution was completely frozen in about 20 min at –30 °C) prior to immersion into LN2. Following storage in LN2 for 1 h, the tubes were rapidly rewarmed by immersion in water at 37 °C for 2–3 min. Immediately thereafter, the cells in RPF2 were rapidly diluted by addition of 2 mL of 1·2 M sucrose containing 10 mM CaCl2 and then incubated for 10 min before being blotted on filter paper for regrowth assay. Non-frozen control cells treated with the cryoprotectant only were diluted and processed in the same manner.
Vitrification method
Vitrification procedures were performed as previously reported (Ishikawa et al., 1996). Briefly, cells (approx. 0·15–0·2 g) were pre-incubated in 1 mL CSP1 for 30 min at 25 °C (followed by complete removal of CSP1) prior to treatment with 0·3 mL of a vitrification solution [PVS2: 15 % (w/v) ethylene glycol, 30 % (w/v) glycerol, 15 % (w/v) DMSO, 0·4 M sucrose] (Sakai et al., 1990) for 1–5 min at 25 °C. The cells in the tubes were immediately submerged in LN2 for 1 h. Following rapid thawing in water at 40 °C, the cells were diluted by direct addition of 2 mL of 1·2 M sucrose. Then, the cells were further diluted with 2 mL of liquid ER medium over 10 min, centrifuged at 1000 rpm for 3 min and processed for regrowth assays. Non-frozen control cells treated with CSP1 and PVS2 were diluted in the same manner.
Regrowth assays
Viability of the cryopreserved and treated control cells was determined by regrowth as described previously (Ishikawa et al., 1995, 1996). Briefly, the cryopreserved cells were blotted on filter paper and incubated on fresh semi-solid ER medium (two pieces of filter paper with cells on 25 mL medium) at 25 °C. Untreated control cells and cells killed at 100 °C were cultured in the same manner. The cells were harvested after 9 d of culture and washed with distilled water. Dry weight (d. wt) of the cells was determined by oven-drying at 70 °C for 2 d. Survival rates were calculated from the d. wt of the untreated control, killed control and freeze–thawed cells as described previously (Ishikawa et al., 1995).
Determination of cell culture growth
The cells from a flask were harvested and washed with 250 mL of distilled water and blotted on a paper towel for 90 s, and the fresh weight (f. wt) was determined.
Determination of water content and osmolarity
Approximately 2–3 g of blotted cells were transferred to a disposable syringe fitted with glass wool at the tip and centrifuged at 3000 rpm for 5 min to remove intercellular water. Some of the centrifuged cells were weighed for f. wt determination and oven-dried at 70 °C for 2 d to determine d. wt. Water content was expressed as (g H2O/g d. wt) unless otherwise noted. The remaining cells (about 1 g) in the syringe were used to determine osmolarity.
The syringe containing the cells was directly submerged in LN2 and thawed at 25 °C. The cell sap was squeezed from the freeze-thawed cells in the syringe and used to determine osmolarity with a Wescor vapour pressure osmometer.
Determination of freezing, heat and salt tolerance
Freezing, heat and salt tolerance of bromegrass cells grown at 25 °C for various time periods from an initial 1 g of cell inoculum was determined as previously described (Ishikawa et al., 1995). Briefly, harvested cells were washed with 250 mL of distilled water, and approx. 0·3 g f. wt of cells were placed in test tubes with 0·5 mL of presterilzed water (triplicates for each test temperature or treatment). In freeze tests, the cells were ice-nucleated at –2 °C by touching the test tubes with dry ice and held at –3 °C overnight before being cooled at 2 °C h–1 to –12 °C, then at 5 °C h–1 to –40 °C. The cells were thawed at 4 °C overnight. Salt stress was imposed by incubating the cells in 0·2 M NaCl solution for 16 h at 25 °C. To determine heat tolerance, the cells were held at 46 °C for 2 h. Following stress treatments, survival was estimated by regrowth assays as described previously (Ishikawa et al., 1995). Freezing tolerance was represented as LT50, the lethal temperature at which there was a 50 % decrease in the survival as compared with the non-stressed and 100 % killed control. Growth, water content and osmolarity of the cells in this set of experiments were determined as described above.
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RESULTS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Effect of growth phase on survival following cryopreservation by slow prefreezing
Highest regrowth after 30 min incubation with cryoprotectant CSP1 was obtained with bromegrass cells subcultured for 8–10 d (Fig. 1). Highest regrowth of CSP1-treated cells after exposure to LN2 following prefreezing to –30 °C was attained with 10-d-old cells, whereas 4- and 14-d-old cells showed least regrowth.
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Effect of growth phase on survival following cryopreservation by rapid prefreezing
In this cryopreservation protocol, bromegrass cells were incubated in RPF2 solution for either 3 or 5 min at 25 °C, placed directly at –30 °C and then submerged in LN2. The best regrowth of cells (incubation time in RPF2 of 3 min) following cryopreservation was obtained with 12-d-old subcultured cells followed by 10-d-old cells (Fig. 2A). When the period of incubation in RPF2 was increased to 5 min, the best regrowth was attained with cells subcultured for 10 d followed by cells subcultured for 12 d (Fig. 2B). Four-day-old subcultured cells had the least regrowth irrespective of the pre-incubation time (3 or 5 min) at 25 °C.
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Effect of growth phase on survival following cryopreservation by vitrification
Regrowth of bromegrass cells freeze-thawed after incubation for 1–5 min in vitrification solution PVS2 following pretreatment with CSP1 for 30 min is shown in Fig. 3A. Generally, regrowth was lower compared with for cells cryopreserved using the two methods described above. The optimal incubation time in PVS2 for cryopreservation was 1 min in most cases; longer incubation periods resulted in reduced survival. This is due to the toxicity of PVS2; similar decreases in survival were observed with non-cryopreserved cells incubated in PVS2 for increasing time periods (data not shown). Using this cryopreservation protocol, the best regrowth (40–45 %) was obtained with 8- to 10-d-old subcultured cells followed by 12-d-old cells (Fig. 3B). Cells subcultured for 2–6 and 14 d had the least regrowth following the freezing treatment.
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Changes in the water content and osmolarity during cell culture
The effect of subculture time on cell growth, water content and cell osmolarity when 50 mL of ER medium was inoculated with 1·3 g cells is shown in Fig. 4. Growth of bromegrass cells during the 14-d subculture period was divided into six growth phases: late lag (2–4 d), early exponential (4–6 d), middle exponential (6–8 d), late exponential (8–10 d), early stationary (10–12 d) and stationary phase (12–14 d) (Fig. 4A). Cell growth, as determined by the f. wt of the cells, increased almost linearly from day 4 to day 10 and thereafter remained constant. Cell osmolarity on day 2 was 347 mOsm, decreased to 209 mOsm on day 4, steadily increased to 396 on day 10 and slowly decreased to 326 mOsm on day 16 of subculturing (Fig. 4B). Cell water content on day 2 was 4·3 g H2O/g d. wt, increased steadily to 7·5 on day 10 and thereafter decreased to 5·4 g H2O/g d. wt on day 16 of subculture (Fig. 4C).
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Effect of growth phase on tolerance to abiotic stresses
The effect of culture growth phase on freezing, heat and salt tolerance is shown in Fig. 5. The cell inoculum mass was smaller (1 g) than that used in the cryopreservation studies (1·3 g) and therefore cell growth was slightly delayed (Fig. 5D compared with Fig. 4A). Although the interval used in determining these traits was not as frequent as used in Fig. 4, an obvious tendency was that cells at the late exponential and early stationary phase (10·5–14 d old) were the most tolerant to freezing, heat and salt stresses compared with cells at the late lag or early exponential phase (3·5–6·5 d old). Cells at the late exponential or early stationary phase were characterized by a high water content and high osmolarity compared with those at the early exponential phase (Fig. 5).
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DISCUSSION |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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In this study, the effect of culture growth phases on cryopreservation survival was examined using cells cultured from non-embryogenic bromegrass suspensions. Three cryopreservation protocols were employed: slow prefreezing (conventional two-step) using CSP1 as a cryoprotectant, rapid prefreezing using RPF2 and vitrification using PVS2. No preculture with high osmoticum, which usually requires an additional 2–4 d, was used as this complicates interpretation of the experimental results. Preliminary experiments showed that such precultures did not improve cryopreservation survival of bromegrass cells (data not shown).
The optimal growth phase for cryopreservation of the bromegrass culture was the late exponential (8–10 d old) or early stationary phase (10–12 d old). The late lag (2–4 d old) or early exponential phase (4–6 d old) resulted in lowest survival (Figs 1–4). Therefore, the traditional hypothesis that the late lag or early exponential growth phase is optimal for cryopreservation does not appear to be valid for bromegrass cell cultures. The optimal growth phase may vary depending upon cell cultures, culture conditions or species from which the cells are derived. The traditional generalization regarding which growth phase should be used for cryopreservation of cell suspension cultures should be reconsidered for each cell culture.
The classical studies of Sugawara and Sakai (1974) and Withers and Street (1977) demonstrated that sycamore suspension cultured cells in the late lag or early log (exponential) phases resulted in the highest survival using slow prefreezing cryopreservation protocols. At these stages, cells are small, rich in cytoplasm and less vacuolated, characteristics presumably similar to those of cold-hardy plant cells (Levitt, 1980; Sakai and Larcher, 1987), and are considered to be optimal for cryopreservation. Their results have since been repeatedly cited in textbooks (Kartha, 1985; Grout, 1995). In the majority of studies published subsequently, cell cultures at the late lag or early exponential phase were used (Reuff et al., 1988; Heszky et al., 1990; Panis et al., 1990; Aguilar et al., 1993; Engelmann et al., 1994; Kuriyama et al., 1996; Luo and Widholm, 1997; Moran et al., 1999), whereas in a few studies, cells at the time of subculture were used (Goldner et al., 1991; Laine et al., 1992). Unfortunately in many of these studies, no data were presented on the effect of growth phases, and thus what growth phase was optimal for cryopreservation remained unknown. Only a few studies on cryopreservation have determined the effect of growth phase on survival: the late lag or early exponential phase proved best in cultures of millet (Lu and Sun, 1992) and Taxus (Kim et al., 2001), whereas the mid exponential phase was optimal for cultures of rice (Sala et al., 1979) and Papaver (Friesen et al., 1991). In most of these reports, including the classical studies of Sugawara and Sakai (1974) and Withers and Street (1977), FDA or conventional TTC assays were used for comparing cell survival at different growth phases. In some cases (e.g. Sugawara and Sakai, 1974), cryopreservation survival was calculated as the percentage of a control treated with cryoprotectants (not that of an untreated control).
In the present study, regrowth was used to determine growth phase dependency of survival. Regrowth capability is an essential trait in determining cell preservation and more reliably represents cell survival than do FDA (intactness of plasma membranes) and TTC (intactness of mitochondria) assays (Ishikawa et al., 1995). For as yet unknown reasons, FDA and conventional TTC assays conducted immediately after thawing often give erroneously high survival scores compared with regrowth assays performed later (Ishikawa et al., 1996). In some studies, FDA staining was not correlated with regrowth or there was a long lag phase before growth resumed (e.g. Kim et al., 2001). It is possible that the growth phase dependency of cryopreservation survival observed with FDA or conventional TTC assays may not necessarily represent regrowth capability.
Bromegrass cells at the late exponential phase and early stationary phases are characterized by high cell water content and high cell osmolarity (Fig. 4). Because the water content was determined with centrifuged cells, contamination of intercellular water is negligible. This implies that cells at these stages were larger in size or more vacuolated (verified from microscopic observations: data not shown) and contained high osmoticum. These cells were most tolerant to freezing (without cryoprotectants), heat and salt stresses (Fig. 5). Cells at the early exponential phase were characterized by low water content and low osmolarity (Fig. 4) and were very sensitive to freezing, heat and salt stresses (Fig. 5). These cells were presumably smaller in size or less vacuolated and contained low osmoticum. Theoretically, cells with high osmolarity are less plasmolysed as a result of the salt or cryoprotectant treatments (e.g. PVS2, 8 M or 7994 mOsm) and less dehydrated owing to extracellular freezing (Chen et al., 1984). A small reduction in cell volume results in less rehydration upon thawing or recovery. High levels of osmolytes protect macromolecules and membranes from injuries during abiotic stress treatments (Levitt, 1980; Sakai and Larcher, 1987). These conditions would allow a reduction in strains resulting from exposure to salt and heat stresses and freezing–thaw cycles with or without cryoprotectants, which may contribute to the high survival of cells during the late exponential or early stationary phase.
Withers (1978), in comparing different cell cycle stages for cryopreservation, found that sycamore cells entering the G1 phase had the highest survival rate as determined by TTC and FDA assays. Bromegrass cells in the late exponential or early stationary phase are presumably no longer dividing and may possibly be arrested in the G1 or G0 phases of the cell cycle. This possibility has yet to be elucidated.
A study of chilling tolerance in mung bean cell cultures revealed that cells at the early exponential phase were most sensitive, whereas cells at the late exponential phase were most tolerant to chilling stress (Yoshida et al., 1993). There were concomitant changes in the chilling sensitivity of tonoplast proton pumps, which are considered to be the primary site of chilling injury in mung bean cells. The detailed mechanisms governing the tolerance of bromegrass cells to various stresses and cryopreservation during the growth cycle have yet to be elucidated.
Bromegrass cells at the late exponential and early stationary phases were recently reported to give the highest frequency of genetic transformation using Agrobacterium (Nakamura and Ishikawa, 2006). Cells at these phases may be more tolerant to biotic stress as well.
Among the three cryopreservation protocols applied here to bromegrass cultures, the slow prefreezing method using CSP1 and the rapid prefreezing method using RPF2 resulted in better survival following a freeze–thaw cycle than the vitrification protocol using CSP1/PVS2 at all growth phases. Slow prefreezing (with CSP1) and rapid prefreezing (with RPF2) protocols were tested for the cryopreservation of non-embryogenic plant cell suspension cultures, such as of tobacco BY-2 (Nagata et al., 1992), rice OC (Baba et al., 1986), Arabidopsis T87 (Axelos et al., 1992), soybean and carrot. In these studies, cell cultures at the late exponential or early stationary phase were used and no sophisticated preculture treatments were employed. High regrowth following a freeze–thaw cycle was obtained in tobacco BY-2 with the slow prefreezing method, whereas in soybean cell cultures good survival was achieved only with the rapid prefreezing method (Ishikawa et al., 2000). Rice, Arabidopsis and carrot cell cultures were successfully cryopreserved using either of the protocols. Recovered cryopreserved cells, transferred to routine liquid cultures, regained their original cell characteristics after two cycles of subculture, comparable with non-cryopreserved cells. These cell cultures (tobacco, rice, soybean, Arabidopsis) have been cryopreserved in the vapour phase (–170 °C) of LN2 in the Genebank of the National Institute of Agrobiological Sciences, Ibaraki, since 1998 to check the effect of long-term storage (Ishikawa et al., 2000).
In this study, from practical and physiological viewpoints, the issue of culture growth phases on cryopreservation of bromegrass cells was re-examined using regrowth as an estimation of survival. The late exponential phase or early stationary phase proved best for survival, whereas the early exponential phase was the least optimal using either of the slow freezing, rapid freezing or vitrification protocols. This is contrary to the classical concept. The culture growth phase issue may be species dependent. Cells at the optimal growth phase for cryopreservation were characterized by a high water content and high osmolarity. At this growth phase, cells were also tolerant to freezing, heat and salt stresses.
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ACKNOWLEDGEMENTS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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The authors would like to thank Ms T. Kitashima, H. Nakatani and A. Oda, NIAS, Ibaraki, for their technical assistance. This research was partly supported by an OECD fellowship fund to M.I.
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FOOTNOTES |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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These authors contributed equally to the work. 
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LITERATURE CITED |
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