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Cryopreservation of in vitro-grown apical meristems of hybrid statice by three different procedures
Scientia Horticulturae 76 (1998) 105±114
Cryopreservation of in vitro-grown apical meristems of hybrid statice by three different procedures Toshikazu Matsumotoa,*, Chiaki Takahashib, Akira Sakaic, Yoji Nakoa a
Shimane Agricultural Experiment Station, Ashiwata 2440, Izumo, Shimane 693-0035, Japan b Yokota Town Office, Yokota 1037, Nita, Shimane 699-1832, Japan c Asabucho 1-5-23, Kitaku, Sapporo 001-0045, Japan Accepted 7 April 1998
Abstract In vitro-grown apical meristems of hybrid statice (Limonium cv. Blue Symphonet) were cryopreserved by three cryogenic procedures; (1) vitrification with encapsulation, (2) vitrification without encapsulation, and (3) a revised encapsulation/dehydration technique. When dehydration tolerance was well developed by preconditioning and cryogenic procedures were well optimized, these three procedures produced nearly the same levels of growth recovery (70±75%). These results support our theory that the acquisition of dehydration tolerance is sufficient for specimens to survive to cryopreservation. # 1998 Elsevier Science B.V. All rights reserved Keywords: Cryopreservation; Meristems; Statice; Vitri®cation; Encapsulation/vitri®cation; Encapsulation/dehydration
1. Introduction In recent years, cryopreservation has become a very important tool for long-term storage of germplasm and experimental materials with unique Abbreviations: MS medium, Murashige and Skoog medium; DMSO, dimethyl sulfoxide; EG, ethylene glycol; LN, liquid nitrogen; PVS2, vitrification solution * Corresponding author. Tel.: +81 853 22 6650; fax: +81 853 21 8380; e-mail: [email protected] 0304-4238/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved PII S 0 3 0 4 - 4 2 3 8 ( 9 8 ) 0 0 1 2 7 - 7
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attributes using minimal space and maintenance requirements without causing genetic alterations (Sakai, 1995, 1997). The development of a simple and reliable method for cryopreservation would allow for a much more widespread use of cryopreserved cultured cells, meristems and somatic embryos. Recently, simplified cryogenic procedures such as, vitrification (Langis et al., 1990; Sakai et al., 1990) encapsulation/dehydration (Fabre and Dereuddre, 1990) and encapsulation/vitrification (Matsumoto et al., 1995) have been developed. In addition, the number of species or cultivars to be cryopreserved has increased sharply over the last several years. These new procedures replaced freeze-induced cell dehydration at very low temperatures (ÿ308C or ÿ408C) by; (1) removal of the major part of cellular water by exposure to a highly concentrated vitrification solution (7±8 M) or (2) to air-drying at non-freezing temperatures followed by vitrifying upon rapid cooling into liquid nitrogen (LN). In any cryogenic procedure, cells and meristems must be sufficiently dehydrated in order for vitrification to occur upon rapid cooling into LN without undergoing lethal intracellular freezing. Thus, it is proposed that specimens which acquired full tolerance to dehydration produced a high rate of survival after cooling to ÿ1968C. The aim of this study is to substantiate this proposal and to further characterize the three cryogenic procedures using in vitro-grown meristems of a hybrid statice. In recent years, cryopreservation has become a very important tool for the long-term storage of germplasm and experimental materials with unique attributes using a minimum of space and maintenance without genetic alteration (Sakai, 1995). The development of a simple and reliable method for cryopreservation would allow much more widespread use of cryopreserved cultured cells, meristems and somatic embryos. Recent work has focused on procedures that would eliminate the need for controlled freezing and enable cells and meristems to be cryopreserved by direct transfer into LN (Sakai, 1997). Since three simplified cryogenic procedures, (vitrification, Sakai et al., 1990; encapsulation/dehydration, Fabre and Dereuddre, 1990 and encapsulation/vitrification, Matsumoto et al., 1995) have been developed and the number of species to be cryopreserved has increased sharply over the last few years. In any cryogenic procedure, cells and meristems must be sufficiently dehydrated to be capable of vitrifying before immersion into LN (Sakai and Yoshida, 1967; Fabre and Dereuddre, 1990). Thus, it can be hypothesized that meristems with acquired dehydration tolerance by sucrose and/or cryoprotectant treatments can provide a high rate of survival after cryopreservation at ÿ1968C. The aim of this study was to clarify this hypothesis and the characteristics of these different methods. The survival of meristems cooled to ÿ1968C by three different protocols was compared under well-optimized conditions using in vitro-grown meristems of statice.
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2. Materials and methods 2.1. Plant materials In vitro-grown plantlets of hybrid statice (Limonium Mill. cv. Blue Symphonet) were used in the present study. Stock cultures of statice plants were maintained on Murashige and Skoog (1962) basal medium (half strength of ammonium nitrate and potassium nitrate, termed 1/2 MS medium) containing 3% (w/v) sucrose and 0.2% (w/v) gellan gum (Wako Pure Chemical Industries, Osaka, Japan) at pH 5.8. They were subcultured every 35±40 days on 5 ml medium, in test tubes (11 mm in diameter) under white fluorescent light (52 m mol mÿ2 sÿ1) using a 16 h photoperiod at 258C. 2.2. Vitrification Apical meristems of about 1.0 mm in length dissected from 30 mm long 30±40day-old plantlets were precultured on solidified 1/2 MS medium containing 0.3 M sucrose for 1 day at 258C. Ten precultured meristems were placed in a 1.8 ml cryotube and loaded with a mixture of 2.0 M glycerol and 0.4 M sucrose for 20 min at 258C. After removing the solution, 2.0 ml of PVS2 solution (30% (w/v) glycerol, 15% (w/v) EG and 15% (w/v) DMSO in 0.4 M sucrose at pH 5.8, Sakai et al., 1990) was added and gently mixed. PVS2 solution was removed and placed once, then held at 258C for various lengths of time. The cryotubes with 10 meristems and 1.0 ml of PVS2 solution were plunged into LN and held for at least 60 min. Cryotubes were warmed in a 408C water bath for about 90 s. After rapid warming, PVS2 solution was drained and replaced with 1.2 M sucrose solution for 20 min. 2.3. Encapsulation/vitrification Precultured meristems were encapsulated into alginate beads (about 3 mm in diameter) containing 2.0 M glycerol and 0.4 M sucrose. After the surface solution was wiped off on sterile filter papers, the encapsulated meristems were dehydrated with PVS2 solution in a 100 ml glass beaker at 100 rpm on a rotary shaker at 258C for various lengths of time. Ten encapsulated dehydrated meristems were suspended in 0.7 ml PVS2 solution in 1.8 ml cryotubes and plunged into LN for at least 1 h. 2.4. Encapsulation/dehydration Meristems were suspended in calcium-free 1/2 MS medium supplemented with 3% (w/v) Na-alginate solution and 0.4 M sucrose. The mixture was dispensed
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from a sterile disposable plastic syringe (1 ml) into 100 ml of the culture medium that contained 100 mM calcium chloride plus 0.4 M sucrose and held for 30 min. Beads (5 mm in diameter) containing one meristem were treated with 0.8 M sucrose, or 0.8 M sucrose plus 0.5 M glycerol, for 16 h at 258C before dehydration. The encapsulated meristems was subjected to dehydration in a petri dish (9 cm in diameter) containing 50 g dry silica gel held at 258C for up to 10 h. After dehydration, about 10 dried meristems were placed in a 1.8 ml cryotube and immersed into LN for more than 1 h. The water content of beads and meristems was expressed on a fresh weight basis. Dry weight was determined after drying for 100 h at 808C. 2.5. Viability and plant growth Vitrified encapsulated±vitrified meristems were transferred onto solidified 1/2 MS basal medium containing 3% sucrose and 0.2% gellan gum and cultured under standard conditions described above. After 1 day, the beads were transferred onto the same fresh medium in a petri dish. The encapsulated dried meristems were plated onto solidified 1/2 MS medium containing 3% sucrose. One day after plating, some meristems were removed from the beads and cultured on the same medium. Recovering meristems were observed at weekly intervals. Shoot formation was recorded as a percent of the total number of meristems forming normal shoots 28 days after plating. Ten meristems were tested for each of three to four replicates for each experiment. 3. Results 3.1. Vitrification To enhance the shoot formation, precultured meristems with 0.3 M sucrose for 1 day were then loaded with a mixture of 2.0 M glycerol plus 0.4 M sucrose for 20 min at 258C before dehydration with PVS2 solution. The effects of preculturing and loading treatment on shoot formation of vitrified meristems are summarized in Table 1. The meristems loaded with 2.0 M glycerol plus 0.4 M sucrose following preculture with 0.3 M sucrose produced the highest shoot formation (76%) after cooling to ÿ1968C. To determine the optimum time of exposure to PVS2 at 258C, precultured, loaded meristems were dehydrated with PVS2 solution for various lengths of time prior to a plunge into LN. Exposure to PVS2 solution for various lengths of time resulted in a variable rate of shoot formation (Fig. 1). The highest rate (about 75%) of shoot formation was obtained with meristems treated with PVS2 for 15 min at 258C.
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Table 1 Effect of preculture and loading treatments on the shoot formation of vitrified meristems of statice cooled to ÿ1968C by vitrification Preculture
Loading
Shoot formation (%SE)
ÿ ÿ
ÿ ÿ
18.06.5 42.05.0 55.34.6 76.02.0
Preculture: 0.3 M sucrose for 1 day at 258C; Loading treatment: a mixture of 2.0 M glycerol and 0.4 M sucrose for 20 min at 258C; Dehydration with PVS2 solution: for 15 min at 258C; Shoot formation (%): percent of meristems producing normal shoots 28 days after plating. Approximately 10 meristems were tested for each of four replicates.
Fig. 1. Effect of exposure time to PVS2 at 258C on the shoot formation of meristems cooled to ÿ1968C by (A) vitrification and (B) encapsulation/vitrification. Meristems were dehydrated with PVS2 solution at 258C for various lengths of time prior to cooling (LN) or without cooling to ÿ1968C (ÿLN). Excised meristems were precultured with 0.3 M sucrose and then (A) loaded with 2.0 M glycerol plus 0.4 M sucrose for 20 min at 258C or (B) encapsulated with alginate beads including 2.0 M glycerol plus 0.4 M sucrose for 30 min at 258C before dehydration with PVS2 solution. Approximately 10 meristems were tested for each of three to four replicates. Vertical bars represent standard error.
3.2. Encapsulation/vitrification The meristems precultured with 0.3 M sucrose for 1 day were encapsulated in alginate±gel beads containing a mixture of 2.0 M glycerol plus 0.4 M sucrose. These encapsulated meristems were dehydrated with PVS2 solution at 258C for
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different lengths of time prior to a plunge into LN. As shown in Fig. 1, the highest rate of shoot formation at 258C were obtained in the meristems treated with PVS2 solution for 50 min (about 70%). 3.3. Encapsulation/dehydration In the encapsulation/dehydration technique, resistance to dehydration and deep cooling to LN was induced by preculturing encapsulated meristems with 0.8 M sucrose or a mixture of 0.8 M sucrose plus 0.5 M glycerol for 16 h. As shown in Table 2, a mixture of 0.8 M sucrose plus 0.5 M glycerol produced significantly higher levels of shoot formation than meristems treated with 0.8 M sucrose alone. The meristems which were removed from the beads 1 day after plating showed much higher levels of shoot formation than the meristems Table 2 Effect of treatment with 0.8 M sucrose or a mixture of 0.8 M sucrose plus 0.5 M glycerol on the shoot formation of encapsulated dried meristems cooled to ÿ1968C Treatment 0.8 M sucrose 0.8 M sucrose 0.5 M glycerol
Shoot formation (%SE) Meristems in beads
Removed beadsa
6.73.3 46.73.3
34.34.3 75.05.0
Encapsulated meristems containing 0.4 M sucrose were treated with 0.8 M sucrose or a mixture of 0.8 M sucrose plus 0.5 M glycerol for 16 h before dehydration. a The encapsulated meristems in gel beads were removed 1 day after plating. Shoot formation was determined 28 days after plating. Table 3 Shoot formation of apical meristems of statice cooled to ÿ1968C by three different cryogenic protocols Cryogenic protocol
Shoot formation (%SE)
Time used for dehydration (min)
Vitrificationa Encapsulation/vitrificationb Encapsulation/dehydrationc
76.02.4 70.05.0 73.34.7
15 50 420
a
Precultured and loaded meristems were dehydrated with PVS2 solution for 15 min at 258C prior to a plunge into LN. b Precultured meristems were encapsulated into alginate beads including 2.0 M glycerol plus 0.4 M sucrose were then dehydrated with PVS2 for 50 min at 258C prior to a plunge into LN. c Precultured meristems were encapsulated into alginate beads and then treated with 0.8 M sucrose plus 0.5 M glycerol for 16 h. These meristems were dehydrated with dry silica gel (50 g) for 7 h prior to a plunge into LN. Encapsulated meristems in gel beads were removed 1 day after plating.
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encapsulated in beads throughout the culturing. Shoot formation of meristems cooled to ÿ1968C by three different cryogenic protocols was compared. These optimized protocols produced high levels of shoot formation (Table 3). However, the time used for dehydration at 258C was greatly different among them: vitrification (15 min), encapsulation/vitrification (50 min), encapsulation/dehydration (420 min). 4. Discussion For successful cryopreservation, it is essential to avoid lethal intracellular freezing, which occurs during rapid cooling using LN (Sakai and Yoshida, 1967; Sakai, 1995). Thus, in any cryogenic procedure, cells and meristems have to be sufficiently dehydrated to avoid intracellular freezing and to be vitrified upon rapid cooling into LN. Vitrification refers to the physical process by which a highly concentrated cryoprotective solution supercools to very low temperatures and eventually solidifies into a metastable glass without undergoing crystallization at a glass transition temperature (Fahy et al., 1984). In the vitrification method with or without encapsulation, meristems are sufficiently dehydrated (osmotically) by exposure to a highly concentrated vitrification solution (PVS2) which hardly penetrates into the cytosol during the dehydration process prior to a plunge into LN. During the PVS2 treatment, there is no appreciable influx of additional cryoprotectants into specimens due to differences in the permeability coefficients for water and solutes and a large difference in the activation energies for water and solute permeation. As a result, the specimens remain osmotically concentrated and the increase in the cytosolic concentration required for vitrification is attained by dehydration (Steponkus et al., 1992). In the vitrification method, the direct exposure of cells and meristems to a PVS2 solution causes harmful effects due to osmotic stress and chemical toxicity. Thus, to obtain a successful cryopreservation by vitrification, the dehydration procedure needs to be carefully controlled with a PVS2 solution. In addition, it is necessary to increase dehydration tolerance of cells and meristems to be cryopreserved by preconditioning (preculture and loading treatment) before dehydration. The injurious effects caused by direct exposure to PVS2 solution can be eliminated or reduced by optimizing exposure time, adding a gradual amount of PVS2 solution or a gradual dehydration process followed by dehydrating specimens at 08C. In the present study, the above injurious effects were effectively overcome by preculturing excised shoot tips on a sucrose-enriched medium for 1 day (a significant increase in cell concentration of up to about 0.6 M; Matsumoto et al., 1998), followed by a loading treatment with a mixture of 2 M glycerol plus 0.4 M
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sucrose (LD solution) for about 20 min at 258C (Note: the cells of meristems plasmolysed considerably, but glycerol and sucrose did not penetrate into the cytosol for 20 min as observed through a cytosolic volume change) before dehydration with a PVS2 solution (the plasmolysis proceeds intensively). During preculture on the sucrose-enriched medium for approximately 1 day, sugar and proline was greatly increased in the meristems (Matsumoto et al., 1998), which, in turn, enhanced the stability of membranes under conditions of severe dehydration (Crowe et al., 1984). Additionally, Reinhoud et al. (1995) succeeded in the cryopreservation of cultured tobacco-cultured cells by vitrification. It was clearly demonstrated by Reinhoud et al., that the development tolerance of tobacco cells precultured with 0.3 M mannitol and exposed to a PVS2 solution for 1 day, appeared to be the combined results of the cell's responsiveness to mild osmotic stress caused by preculture: in particular, production of ABA, accumulation of mannitol during preculture, proline and certain proteins including late embryogenesis abundant (LEA). For the many herbaceous plants tested, overnight preculture of excised meristems with 0.3 M sucrose appeared to be inconsequential for producing a high level of recovery growth by vitrification. It was further demonstrated that LD solution was very effective in increasing tolerance to freeze-dehydration down to ÿ308C and to dehydration using a PVS2 solution (Sakai et al., 1991; Nishizawa et al., 1992). The protective effect of 2 M glycerol and 0.4 M sucrose in the cell's peri-protoplasmic space may be due to mitigation of a large osmotic stress by severe dehydration with PVS2 solution in addition to some mechanism of action which minimizes injurious membrane changes from severe dehydration (Crowe et al., 1988; Steponkus et al., 1992). It was also suggested that plasmolysis might reduce the generation of mechanical stress on plasma membranes, which might be produced by deformation of the cell wall during extracellular freezing (Jitsuyama et al., 1997). In our experiments, precultured and loaded meristems were dehydrated with PVS2 solution at 258C for 15 min (Fig. 1(A)) or 50 min (Fig. 1(B)), and they survived subsequent rapid cooling and rewarming in the excursion of vitrification procedure with a slight additional decrease in survival. Thus, it can be postulated that the meristems acquired dehydration tolerance to the PVS2 solution which optimized exposure time to improve tolerance to cryopreservation by vitrification. These results support our theory that under well-optimized conditions of cryogenic procedures, acquisition of PVS2 tolerance is sufficient for specimens to survive cryopreservation by vitrification. The encapsulation/dehydration technique was successfully applied to a wide range of materials. However, there are lower rates of survival and later recovery growth when compared to meristems cryopreserved by vitrification (Matsumoto and Sakai, 1995). In the former technique, encapsulated meristems are treated with 0.8 M sucrose for 16 h to induce dehydration tolerance before air-drying
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(Fabre and Dereuddre, 1990). The overnight treatment with 0.8 M sucrose produced a much lower level of recovery growth (7%) than that of vitrified meristems (70±75%) with or without encapsulation. Thus, the treatment with 0.8 M sucrose alone, appears to be insufficient to produce a higher level of recovery growth. In the present study, the recovery growth of encapsulated dried meristems was significantly improved (from 7% to 75%) provided the following two conditions were met: (1) they were treated with a mixture of 0.8 M sucrose plus 0.5 M glycerol, and (2) they were removed from beads 1 day after plating. Thus, the three cryogenic procedures tested, produced nearly the same recovery growth when dehydration tolerance was fully developed and cryogenic procedures were optimized. These results also support our hypothesis. The encapsulation/dehydration technique is easy to handle and alleviates the dehydration process, but is laborious and time-consuming when compared with the vitrification method. In the vitrification method, it is difficult to treat carefully a large number of meristems at the same time. Thus, we developed an encapsulation/vitrification method (Matsumoto et al., 1995). This method is easy to handle and treat a large number of meristems at the same time. Furthermore, recovery growth is much earlier than when using encapsulated dried meristems. The vitrification method significantly decreased the time used for dehydration and simplified the cryogenic procedures. More recently, the vitrification method was successfully applied to about 20 tropical monocotyledonous plants (Thinh, 1997). Thus, the vitrification method seems promising for the cryopreservation of meristems and somatic embryos. References Crowe, J.H., Crowe, J.F., Carpenter, L.M., Aurell-Wistrom, C., Wistrom, C., 1984. Stabilization of dry phospholipid bilayers and proteins by sugars. Plant Cell Rep. 12, 89±94. Crowe, J.H., Crowe, J.F., Carpenter, L.M., Rudolph, A.S., Wistrom, C.A., Spargo, B.J., Anchordoguy, T.J., 1988. Interaction of sugars with membranes. Biochem. Biophys. Acta 947, 367±384. Fabre, J., Dereuddre, J., 1990. Encapsulation±dehydration: A new approach to cryopreservation of Solanum shoot tips. Cryo-Lett. 11, 413±426. Fahy, G.M., MacFarlene, D.R., Angell, C.A., Meryman, H.T., 1984. Vitrification as an approach to cryopreservation. Cryobiology 21, 407±426. Jitsuyama, Y., Suzuki, T., Harada, T., Fujikawa, S., 1997. Ultrastructural study on mechanism of increased freezing tolerance due to extracellular glucose in cabbage cells. Cryo-Lett. 18, 33±44. Langis, R., Schnabel-Preikstas, B.J., Earle, F.D., Steponkus, P.L., 1990. Cryopreservation of carnation shoot tips by vitrification. Cryobiology 27th annual meeting abstract, pp. 657±658. Matsumoto, T., Sakai, A., Takahashi, C., Yamada, K., 1995. Cryopreservation of in vitro-grown meristems of wasabi (Wasabia japonica) by encapsulation±vitrification method. Cryo-Lett. 16, 189±196. Matsumoto, T., Sakai, A., 1995. An approach to enhance dehydration tolerance of alginate-coated dried meristems cooled to ÿ1968C. Cryo-Lett. 16, 299±306.
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Matsumoto, T., Sakai, A., Nako, Y., 1998. A novel preculturing for enhancing the survival of in vitro-grown meristems of wasabi (Wasabia japonica) cooled to ÿ1968C by vitrification. CryoLett. 19, 27±36. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473±497. Nishizawa, S., Sakai, A., Amano, Y., Matsuzawa, T., 1992. Cryopreservation of asparagus (Asparagus officinalis L. Osb.) embryogenic cells and subsequent plant regeneration by a simple freezing method. Cryo-Lett. 13, 379±388. Reinhoud, P.J., Schrijnemakers, E.W.M., Iren, F., Kijne, J.W., 1995. Vitrification and a heat-shock treatment improve cryopreservation of tobacco cell suspension compared to two-step freezing. Plant Cell Tiss. Org. Cult. 42, 261±267. Sakai, A., Yoshida, S., 1967. Survival of plant tissue at super-low temperature. VI. Effects of cooling and rewarming rates on survival. Plant Physiol. 42, 1695±1701. Sakai, A., Kobayashi, S., Oiyama, I., 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Rep. 9, 30±33. Sakai, A., Kobayashi, S., Oiyama, I., 1991. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb.) by a simple freezing method. Plant Sci. 74, 243±248. Sakai, A., 1995. Cryopreservation of germplasm collection in woody plants. In: Bajaj, Y.P.S. (Ed.), Biotechnology in Agriculture and Forestry, vol. 32, Cryopreservation of Plant Germplasm I. pp. 55±69. Sakai, A., 1997. Potentially valuable cryogenic procedures for cryopreservation of cultured plant meristems. In: Razdan, M.K., Cocking, E.C. (Eds.), Conservation of Plant Genetic Resources In Vitro. Science Publishers, New Hampshire, pp. 53±66. Steponkus, P.L., Langis, R., Fujikawa, S., 1992. Cryopreservation of plant tissues by vitrification. In: Steponkus, P.L. (Ed.), Advances in Low Temperature Biology, vol.1. JAI Press, London, pp. 1±61. Thinh, N.T., 1997. Cryopreservation of germplasm of vegetatively propagated tropical monocots by vitrification. Doctoral Thesis, Dep. of Agronomy, Kobe University, Kobe.
Cryopreservation of in vitro-grown apical meristems of hybrid statice by three different procedures Toshikazu Matsumotoa,*, Chiaki Takahashib, Akira Sakaic, Yoji Nakoa a
Shimane Agricultural Experiment Station, Ashiwata 2440, Izumo, Shimane 693-0035, Japan b Yokota Town Office, Yokota 1037, Nita, Shimane 699-1832, Japan c Asabucho 1-5-23, Kitaku, Sapporo 001-0045, Japan Accepted 7 April 1998
Abstract In vitro-grown apical meristems of hybrid statice (Limonium cv. Blue Symphonet) were cryopreserved by three cryogenic procedures; (1) vitrification with encapsulation, (2) vitrification without encapsulation, and (3) a revised encapsulation/dehydration technique. When dehydration tolerance was well developed by preconditioning and cryogenic procedures were well optimized, these three procedures produced nearly the same levels of growth recovery (70±75%). These results support our theory that the acquisition of dehydration tolerance is sufficient for specimens to survive to cryopreservation. # 1998 Elsevier Science B.V. All rights reserved Keywords: Cryopreservation; Meristems; Statice; Vitri®cation; Encapsulation/vitri®cation; Encapsulation/dehydration
1. Introduction In recent years, cryopreservation has become a very important tool for long-term storage of germplasm and experimental materials with unique Abbreviations: MS medium, Murashige and Skoog medium; DMSO, dimethyl sulfoxide; EG, ethylene glycol; LN, liquid nitrogen; PVS2, vitrification solution * Corresponding author. Tel.: +81 853 22 6650; fax: +81 853 21 8380; e-mail: [email protected] 0304-4238/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved PII S 0 3 0 4 - 4 2 3 8 ( 9 8 ) 0 0 1 2 7 - 7
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attributes using minimal space and maintenance requirements without causing genetic alterations (Sakai, 1995, 1997). The development of a simple and reliable method for cryopreservation would allow for a much more widespread use of cryopreserved cultured cells, meristems and somatic embryos. Recently, simplified cryogenic procedures such as, vitrification (Langis et al., 1990; Sakai et al., 1990) encapsulation/dehydration (Fabre and Dereuddre, 1990) and encapsulation/vitrification (Matsumoto et al., 1995) have been developed. In addition, the number of species or cultivars to be cryopreserved has increased sharply over the last several years. These new procedures replaced freeze-induced cell dehydration at very low temperatures (ÿ308C or ÿ408C) by; (1) removal of the major part of cellular water by exposure to a highly concentrated vitrification solution (7±8 M) or (2) to air-drying at non-freezing temperatures followed by vitrifying upon rapid cooling into liquid nitrogen (LN). In any cryogenic procedure, cells and meristems must be sufficiently dehydrated in order for vitrification to occur upon rapid cooling into LN without undergoing lethal intracellular freezing. Thus, it is proposed that specimens which acquired full tolerance to dehydration produced a high rate of survival after cooling to ÿ1968C. The aim of this study is to substantiate this proposal and to further characterize the three cryogenic procedures using in vitro-grown meristems of a hybrid statice. In recent years, cryopreservation has become a very important tool for the long-term storage of germplasm and experimental materials with unique attributes using a minimum of space and maintenance without genetic alteration (Sakai, 1995). The development of a simple and reliable method for cryopreservation would allow much more widespread use of cryopreserved cultured cells, meristems and somatic embryos. Recent work has focused on procedures that would eliminate the need for controlled freezing and enable cells and meristems to be cryopreserved by direct transfer into LN (Sakai, 1997). Since three simplified cryogenic procedures, (vitrification, Sakai et al., 1990; encapsulation/dehydration, Fabre and Dereuddre, 1990 and encapsulation/vitrification, Matsumoto et al., 1995) have been developed and the number of species to be cryopreserved has increased sharply over the last few years. In any cryogenic procedure, cells and meristems must be sufficiently dehydrated to be capable of vitrifying before immersion into LN (Sakai and Yoshida, 1967; Fabre and Dereuddre, 1990). Thus, it can be hypothesized that meristems with acquired dehydration tolerance by sucrose and/or cryoprotectant treatments can provide a high rate of survival after cryopreservation at ÿ1968C. The aim of this study was to clarify this hypothesis and the characteristics of these different methods. The survival of meristems cooled to ÿ1968C by three different protocols was compared under well-optimized conditions using in vitro-grown meristems of statice.
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2. Materials and methods 2.1. Plant materials In vitro-grown plantlets of hybrid statice (Limonium Mill. cv. Blue Symphonet) were used in the present study. Stock cultures of statice plants were maintained on Murashige and Skoog (1962) basal medium (half strength of ammonium nitrate and potassium nitrate, termed 1/2 MS medium) containing 3% (w/v) sucrose and 0.2% (w/v) gellan gum (Wako Pure Chemical Industries, Osaka, Japan) at pH 5.8. They were subcultured every 35±40 days on 5 ml medium, in test tubes (11 mm in diameter) under white fluorescent light (52 m mol mÿ2 sÿ1) using a 16 h photoperiod at 258C. 2.2. Vitrification Apical meristems of about 1.0 mm in length dissected from 30 mm long 30±40day-old plantlets were precultured on solidified 1/2 MS medium containing 0.3 M sucrose for 1 day at 258C. Ten precultured meristems were placed in a 1.8 ml cryotube and loaded with a mixture of 2.0 M glycerol and 0.4 M sucrose for 20 min at 258C. After removing the solution, 2.0 ml of PVS2 solution (30% (w/v) glycerol, 15% (w/v) EG and 15% (w/v) DMSO in 0.4 M sucrose at pH 5.8, Sakai et al., 1990) was added and gently mixed. PVS2 solution was removed and placed once, then held at 258C for various lengths of time. The cryotubes with 10 meristems and 1.0 ml of PVS2 solution were plunged into LN and held for at least 60 min. Cryotubes were warmed in a 408C water bath for about 90 s. After rapid warming, PVS2 solution was drained and replaced with 1.2 M sucrose solution for 20 min. 2.3. Encapsulation/vitrification Precultured meristems were encapsulated into alginate beads (about 3 mm in diameter) containing 2.0 M glycerol and 0.4 M sucrose. After the surface solution was wiped off on sterile filter papers, the encapsulated meristems were dehydrated with PVS2 solution in a 100 ml glass beaker at 100 rpm on a rotary shaker at 258C for various lengths of time. Ten encapsulated dehydrated meristems were suspended in 0.7 ml PVS2 solution in 1.8 ml cryotubes and plunged into LN for at least 1 h. 2.4. Encapsulation/dehydration Meristems were suspended in calcium-free 1/2 MS medium supplemented with 3% (w/v) Na-alginate solution and 0.4 M sucrose. The mixture was dispensed
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from a sterile disposable plastic syringe (1 ml) into 100 ml of the culture medium that contained 100 mM calcium chloride plus 0.4 M sucrose and held for 30 min. Beads (5 mm in diameter) containing one meristem were treated with 0.8 M sucrose, or 0.8 M sucrose plus 0.5 M glycerol, for 16 h at 258C before dehydration. The encapsulated meristems was subjected to dehydration in a petri dish (9 cm in diameter) containing 50 g dry silica gel held at 258C for up to 10 h. After dehydration, about 10 dried meristems were placed in a 1.8 ml cryotube and immersed into LN for more than 1 h. The water content of beads and meristems was expressed on a fresh weight basis. Dry weight was determined after drying for 100 h at 808C. 2.5. Viability and plant growth Vitrified encapsulated±vitrified meristems were transferred onto solidified 1/2 MS basal medium containing 3% sucrose and 0.2% gellan gum and cultured under standard conditions described above. After 1 day, the beads were transferred onto the same fresh medium in a petri dish. The encapsulated dried meristems were plated onto solidified 1/2 MS medium containing 3% sucrose. One day after plating, some meristems were removed from the beads and cultured on the same medium. Recovering meristems were observed at weekly intervals. Shoot formation was recorded as a percent of the total number of meristems forming normal shoots 28 days after plating. Ten meristems were tested for each of three to four replicates for each experiment. 3. Results 3.1. Vitrification To enhance the shoot formation, precultured meristems with 0.3 M sucrose for 1 day were then loaded with a mixture of 2.0 M glycerol plus 0.4 M sucrose for 20 min at 258C before dehydration with PVS2 solution. The effects of preculturing and loading treatment on shoot formation of vitrified meristems are summarized in Table 1. The meristems loaded with 2.0 M glycerol plus 0.4 M sucrose following preculture with 0.3 M sucrose produced the highest shoot formation (76%) after cooling to ÿ1968C. To determine the optimum time of exposure to PVS2 at 258C, precultured, loaded meristems were dehydrated with PVS2 solution for various lengths of time prior to a plunge into LN. Exposure to PVS2 solution for various lengths of time resulted in a variable rate of shoot formation (Fig. 1). The highest rate (about 75%) of shoot formation was obtained with meristems treated with PVS2 for 15 min at 258C.
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Table 1 Effect of preculture and loading treatments on the shoot formation of vitrified meristems of statice cooled to ÿ1968C by vitrification Preculture
Loading
Shoot formation (%SE)
ÿ ÿ
ÿ ÿ
18.06.5 42.05.0 55.34.6 76.02.0
Preculture: 0.3 M sucrose for 1 day at 258C; Loading treatment: a mixture of 2.0 M glycerol and 0.4 M sucrose for 20 min at 258C; Dehydration with PVS2 solution: for 15 min at 258C; Shoot formation (%): percent of meristems producing normal shoots 28 days after plating. Approximately 10 meristems were tested for each of four replicates.
Fig. 1. Effect of exposure time to PVS2 at 258C on the shoot formation of meristems cooled to ÿ1968C by (A) vitrification and (B) encapsulation/vitrification. Meristems were dehydrated with PVS2 solution at 258C for various lengths of time prior to cooling (LN) or without cooling to ÿ1968C (ÿLN). Excised meristems were precultured with 0.3 M sucrose and then (A) loaded with 2.0 M glycerol plus 0.4 M sucrose for 20 min at 258C or (B) encapsulated with alginate beads including 2.0 M glycerol plus 0.4 M sucrose for 30 min at 258C before dehydration with PVS2 solution. Approximately 10 meristems were tested for each of three to four replicates. Vertical bars represent standard error.
3.2. Encapsulation/vitrification The meristems precultured with 0.3 M sucrose for 1 day were encapsulated in alginate±gel beads containing a mixture of 2.0 M glycerol plus 0.4 M sucrose. These encapsulated meristems were dehydrated with PVS2 solution at 258C for
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different lengths of time prior to a plunge into LN. As shown in Fig. 1, the highest rate of shoot formation at 258C were obtained in the meristems treated with PVS2 solution for 50 min (about 70%). 3.3. Encapsulation/dehydration In the encapsulation/dehydration technique, resistance to dehydration and deep cooling to LN was induced by preculturing encapsulated meristems with 0.8 M sucrose or a mixture of 0.8 M sucrose plus 0.5 M glycerol for 16 h. As shown in Table 2, a mixture of 0.8 M sucrose plus 0.5 M glycerol produced significantly higher levels of shoot formation than meristems treated with 0.8 M sucrose alone. The meristems which were removed from the beads 1 day after plating showed much higher levels of shoot formation than the meristems Table 2 Effect of treatment with 0.8 M sucrose or a mixture of 0.8 M sucrose plus 0.5 M glycerol on the shoot formation of encapsulated dried meristems cooled to ÿ1968C Treatment 0.8 M sucrose 0.8 M sucrose 0.5 M glycerol
Shoot formation (%SE) Meristems in beads
Removed beadsa
6.73.3 46.73.3
34.34.3 75.05.0
Encapsulated meristems containing 0.4 M sucrose were treated with 0.8 M sucrose or a mixture of 0.8 M sucrose plus 0.5 M glycerol for 16 h before dehydration. a The encapsulated meristems in gel beads were removed 1 day after plating. Shoot formation was determined 28 days after plating. Table 3 Shoot formation of apical meristems of statice cooled to ÿ1968C by three different cryogenic protocols Cryogenic protocol
Shoot formation (%SE)
Time used for dehydration (min)
Vitrificationa Encapsulation/vitrificationb Encapsulation/dehydrationc
76.02.4 70.05.0 73.34.7
15 50 420
a
Precultured and loaded meristems were dehydrated with PVS2 solution for 15 min at 258C prior to a plunge into LN. b Precultured meristems were encapsulated into alginate beads including 2.0 M glycerol plus 0.4 M sucrose were then dehydrated with PVS2 for 50 min at 258C prior to a plunge into LN. c Precultured meristems were encapsulated into alginate beads and then treated with 0.8 M sucrose plus 0.5 M glycerol for 16 h. These meristems were dehydrated with dry silica gel (50 g) for 7 h prior to a plunge into LN. Encapsulated meristems in gel beads were removed 1 day after plating.
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encapsulated in beads throughout the culturing. Shoot formation of meristems cooled to ÿ1968C by three different cryogenic protocols was compared. These optimized protocols produced high levels of shoot formation (Table 3). However, the time used for dehydration at 258C was greatly different among them: vitrification (15 min), encapsulation/vitrification (50 min), encapsulation/dehydration (420 min). 4. Discussion For successful cryopreservation, it is essential to avoid lethal intracellular freezing, which occurs during rapid cooling using LN (Sakai and Yoshida, 1967; Sakai, 1995). Thus, in any cryogenic procedure, cells and meristems have to be sufficiently dehydrated to avoid intracellular freezing and to be vitrified upon rapid cooling into LN. Vitrification refers to the physical process by which a highly concentrated cryoprotective solution supercools to very low temperatures and eventually solidifies into a metastable glass without undergoing crystallization at a glass transition temperature (Fahy et al., 1984). In the vitrification method with or without encapsulation, meristems are sufficiently dehydrated (osmotically) by exposure to a highly concentrated vitrification solution (PVS2) which hardly penetrates into the cytosol during the dehydration process prior to a plunge into LN. During the PVS2 treatment, there is no appreciable influx of additional cryoprotectants into specimens due to differences in the permeability coefficients for water and solutes and a large difference in the activation energies for water and solute permeation. As a result, the specimens remain osmotically concentrated and the increase in the cytosolic concentration required for vitrification is attained by dehydration (Steponkus et al., 1992). In the vitrification method, the direct exposure of cells and meristems to a PVS2 solution causes harmful effects due to osmotic stress and chemical toxicity. Thus, to obtain a successful cryopreservation by vitrification, the dehydration procedure needs to be carefully controlled with a PVS2 solution. In addition, it is necessary to increase dehydration tolerance of cells and meristems to be cryopreserved by preconditioning (preculture and loading treatment) before dehydration. The injurious effects caused by direct exposure to PVS2 solution can be eliminated or reduced by optimizing exposure time, adding a gradual amount of PVS2 solution or a gradual dehydration process followed by dehydrating specimens at 08C. In the present study, the above injurious effects were effectively overcome by preculturing excised shoot tips on a sucrose-enriched medium for 1 day (a significant increase in cell concentration of up to about 0.6 M; Matsumoto et al., 1998), followed by a loading treatment with a mixture of 2 M glycerol plus 0.4 M
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sucrose (LD solution) for about 20 min at 258C (Note: the cells of meristems plasmolysed considerably, but glycerol and sucrose did not penetrate into the cytosol for 20 min as observed through a cytosolic volume change) before dehydration with a PVS2 solution (the plasmolysis proceeds intensively). During preculture on the sucrose-enriched medium for approximately 1 day, sugar and proline was greatly increased in the meristems (Matsumoto et al., 1998), which, in turn, enhanced the stability of membranes under conditions of severe dehydration (Crowe et al., 1984). Additionally, Reinhoud et al. (1995) succeeded in the cryopreservation of cultured tobacco-cultured cells by vitrification. It was clearly demonstrated by Reinhoud et al., that the development tolerance of tobacco cells precultured with 0.3 M mannitol and exposed to a PVS2 solution for 1 day, appeared to be the combined results of the cell's responsiveness to mild osmotic stress caused by preculture: in particular, production of ABA, accumulation of mannitol during preculture, proline and certain proteins including late embryogenesis abundant (LEA). For the many herbaceous plants tested, overnight preculture of excised meristems with 0.3 M sucrose appeared to be inconsequential for producing a high level of recovery growth by vitrification. It was further demonstrated that LD solution was very effective in increasing tolerance to freeze-dehydration down to ÿ308C and to dehydration using a PVS2 solution (Sakai et al., 1991; Nishizawa et al., 1992). The protective effect of 2 M glycerol and 0.4 M sucrose in the cell's peri-protoplasmic space may be due to mitigation of a large osmotic stress by severe dehydration with PVS2 solution in addition to some mechanism of action which minimizes injurious membrane changes from severe dehydration (Crowe et al., 1988; Steponkus et al., 1992). It was also suggested that plasmolysis might reduce the generation of mechanical stress on plasma membranes, which might be produced by deformation of the cell wall during extracellular freezing (Jitsuyama et al., 1997). In our experiments, precultured and loaded meristems were dehydrated with PVS2 solution at 258C for 15 min (Fig. 1(A)) or 50 min (Fig. 1(B)), and they survived subsequent rapid cooling and rewarming in the excursion of vitrification procedure with a slight additional decrease in survival. Thus, it can be postulated that the meristems acquired dehydration tolerance to the PVS2 solution which optimized exposure time to improve tolerance to cryopreservation by vitrification. These results support our theory that under well-optimized conditions of cryogenic procedures, acquisition of PVS2 tolerance is sufficient for specimens to survive cryopreservation by vitrification. The encapsulation/dehydration technique was successfully applied to a wide range of materials. However, there are lower rates of survival and later recovery growth when compared to meristems cryopreserved by vitrification (Matsumoto and Sakai, 1995). In the former technique, encapsulated meristems are treated with 0.8 M sucrose for 16 h to induce dehydration tolerance before air-drying
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(Fabre and Dereuddre, 1990). The overnight treatment with 0.8 M sucrose produced a much lower level of recovery growth (7%) than that of vitrified meristems (70±75%) with or without encapsulation. Thus, the treatment with 0.8 M sucrose alone, appears to be insufficient to produce a higher level of recovery growth. In the present study, the recovery growth of encapsulated dried meristems was significantly improved (from 7% to 75%) provided the following two conditions were met: (1) they were treated with a mixture of 0.8 M sucrose plus 0.5 M glycerol, and (2) they were removed from beads 1 day after plating. Thus, the three cryogenic procedures tested, produced nearly the same recovery growth when dehydration tolerance was fully developed and cryogenic procedures were optimized. These results also support our hypothesis. The encapsulation/dehydration technique is easy to handle and alleviates the dehydration process, but is laborious and time-consuming when compared with the vitrification method. In the vitrification method, it is difficult to treat carefully a large number of meristems at the same time. Thus, we developed an encapsulation/vitrification method (Matsumoto et al., 1995). This method is easy to handle and treat a large number of meristems at the same time. Furthermore, recovery growth is much earlier than when using encapsulated dried meristems. The vitrification method significantly decreased the time used for dehydration and simplified the cryogenic procedures. More recently, the vitrification method was successfully applied to about 20 tropical monocotyledonous plants (Thinh, 1997). Thus, the vitrification method seems promising for the cryopreservation of meristems and somatic embryos. References Crowe, J.H., Crowe, J.F., Carpenter, L.M., Aurell-Wistrom, C., Wistrom, C., 1984. Stabilization of dry phospholipid bilayers and proteins by sugars. Plant Cell Rep. 12, 89±94. Crowe, J.H., Crowe, J.F., Carpenter, L.M., Rudolph, A.S., Wistrom, C.A., Spargo, B.J., Anchordoguy, T.J., 1988. Interaction of sugars with membranes. Biochem. Biophys. Acta 947, 367±384. Fabre, J., Dereuddre, J., 1990. Encapsulation±dehydration: A new approach to cryopreservation of Solanum shoot tips. Cryo-Lett. 11, 413±426. Fahy, G.M., MacFarlene, D.R., Angell, C.A., Meryman, H.T., 1984. Vitrification as an approach to cryopreservation. Cryobiology 21, 407±426. Jitsuyama, Y., Suzuki, T., Harada, T., Fujikawa, S., 1997. Ultrastructural study on mechanism of increased freezing tolerance due to extracellular glucose in cabbage cells. Cryo-Lett. 18, 33±44. Langis, R., Schnabel-Preikstas, B.J., Earle, F.D., Steponkus, P.L., 1990. Cryopreservation of carnation shoot tips by vitrification. Cryobiology 27th annual meeting abstract, pp. 657±658. Matsumoto, T., Sakai, A., Takahashi, C., Yamada, K., 1995. Cryopreservation of in vitro-grown meristems of wasabi (Wasabia japonica) by encapsulation±vitrification method. Cryo-Lett. 16, 189±196. Matsumoto, T., Sakai, A., 1995. An approach to enhance dehydration tolerance of alginate-coated dried meristems cooled to ÿ1968C. Cryo-Lett. 16, 299±306.
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Matsumoto, T., Sakai, A., Nako, Y., 1998. A novel preculturing for enhancing the survival of in vitro-grown meristems of wasabi (Wasabia japonica) cooled to ÿ1968C by vitrification. CryoLett. 19, 27±36. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473±497. Nishizawa, S., Sakai, A., Amano, Y., Matsuzawa, T., 1992. Cryopreservation of asparagus (Asparagus officinalis L. Osb.) embryogenic cells and subsequent plant regeneration by a simple freezing method. Cryo-Lett. 13, 379±388. Reinhoud, P.J., Schrijnemakers, E.W.M., Iren, F., Kijne, J.W., 1995. Vitrification and a heat-shock treatment improve cryopreservation of tobacco cell suspension compared to two-step freezing. Plant Cell Tiss. Org. Cult. 42, 261±267. Sakai, A., Yoshida, S., 1967. Survival of plant tissue at super-low temperature. VI. Effects of cooling and rewarming rates on survival. Plant Physiol. 42, 1695±1701. Sakai, A., Kobayashi, S., Oiyama, I., 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Rep. 9, 30±33. Sakai, A., Kobayashi, S., Oiyama, I., 1991. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb.) by a simple freezing method. Plant Sci. 74, 243±248. Sakai, A., 1995. Cryopreservation of germplasm collection in woody plants. In: Bajaj, Y.P.S. (Ed.), Biotechnology in Agriculture and Forestry, vol. 32, Cryopreservation of Plant Germplasm I. pp. 55±69. Sakai, A., 1997. Potentially valuable cryogenic procedures for cryopreservation of cultured plant meristems. In: Razdan, M.K., Cocking, E.C. (Eds.), Conservation of Plant Genetic Resources In Vitro. Science Publishers, New Hampshire, pp. 53±66. Steponkus, P.L., Langis, R., Fujikawa, S., 1992. Cryopreservation of plant tissues by vitrification. In: Steponkus, P.L. (Ed.), Advances in Low Temperature Biology, vol.1. JAI Press, London, pp. 1±61. Thinh, N.T., 1997. Cryopreservation of germplasm of vegetatively propagated tropical monocots by vitrification. Doctoral Thesis, Dep. of Agronomy, Kobe University, Kobe.