Cryopreservation of Limonium Shoot Tips and Shoot

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23 Cryopreservation of Limonium Shoot Tips and Shoot Primordia Toshikazu Matsumoto* Shimane Agricultural Technology Center, Ashiwata 2440, Izumo, Shimane 693-0035, Japan Correspondence: * [email protected]

Keywords: encapsulation/dehydration, encapsulation/vitrification, liquid nitrogen, statice, vitrification

ABSTRACT In vitro-grown apical shoot tips of Limonium (hybrid statice; L. altaica Mill. x L. caspium Mill. cv. ‘Blue Symphonet’) were cryopreserved in liquid nitrogen (LN) by following three cryogenic procedures; vitrification, vitrification with encapsulation (encapsulation/vitrification), and 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 to 75%). Bulbous structures consisting of meristematic clumps (designated “shoot primordia”) were induced from a meristematic culture of statice. Cold-hardened, precultured small segments of shoot primordia were successfully cryopreserved in LN by vitrification. Shoot primordia appear promising for large-scale production and cryopreservation for statice.

1. INTRODUCTION 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, shoot tips and somatic embryos. Recent work has been focused on procedures that would eliminate the need for controlled freezing and enable cells and shoot tips to be cryopreserved by direct transfer into liquid nitrogen (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 (Wang and Perl 2006). In any cryogenic procedure, cells and shoot tips must be sufficiently dehydrated to be capable of vitrifying before immersed into LN (Sakai and Yoshida 1967; Fabre and Dereuddre 1990). Thus, it can be hypothesized that shoot tips with acquired dehydration tolerance by sucrose and/or cryoprotection treatments can provide high rate of survival after cryopreservation at –196°C. We compared the survival of shoot tips cooled to –196°C by three different cryogenic protocols under well-optimized conditions using in vitro-grown shoot tips of hybrid statice (Matsumoto et al. 1997). “Shoot primordia” were first reported by Tanaka and Ikeda (1983) with Haplopappus gracilis (Nutt.) Gray. The mass of shoot primordia propagates vegetatively at a very high rate and readily regenerate plantlets by organogenesis. We succeeded to induce in vitro-cultured masses of shoot primordia using hybrid statice and survive them at –196°C by vitrification (Matsumoto et al. 1998a).

2. MATERIALS AND METHODS 2.1. Plant materials for cryopreservation In vitro-grown plantlets of hybrid statice (Limonium Mill. cv. ‘Blue Symphonet’) were used in this study. The 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, Ltd., Osaka, Japan) at pH 5.8. They were subcultured every 35 to 40 days on 5 ml medium, in test tubes (11 mm in diameter) under white fluorescent light (52 μmol· m-2 ·s-1) using a 16 hr photoperiod at 25°C.

2.2. Vitrification Shoot tips of about 1 mm in length dissected from 30 mm long 30 to 40-day-old plantlets were precultured on solidified 1/2 MS medium containing 0.3 M sucrose for 1 day at 25°C. Ten precultured shoot tips were placed in a 1.8 ml cryotube and treated with a mixture of 2.0 M Abbreviations: LN, liquid nitrogen; MS medium, Murashige and Skoog medium; PVS2, vitrification solution

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glycerol and 0.4 M sucrose for 20 min at 25°C for cryoprotection. After removing the solution, 2.0 ml of PVS2 (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 was removed and placed once, then held at 25°C for various lengths of time. The cryotubes with 10 shoot tips and 1.0 ml of PVS2 were plunged into LN and held for at least 60 min. Cryotubes were warmed in 40°C water bath for about 90 sec. After rapid warming, PVS2 was drained and replaced with 1.2 M sucrose solution for 20 min.

2.3. Encapsulation/vitrification Precultured shoot tips 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 shoot tips were dehydrated with PVS2 in a 100 ml glass beaker at 100 rpm on a rotary shaker at 25°C for various lengths of time. Ten encapsulated dehydrated shoot tips were suspended in 0.7 ml PVS2 in 1.8-ml cryotubes and plunged into LN for at least 1 hr.

2.4. Encapsulation/dehydration Shoot tips 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 from a sterile disposable plastic syringe (1 ml) into 100 ml of 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 shoot tip were treated with 0.8 M sucrose solution, or a mixture of 0.8 M sucrose and 0.5 M glycerol, for 16 hr at 25°C before dehydration. The encapsulated shoot tips were subjected to dehydration in a petri dish (9 cm in diameter) containing 50 g dry silica gel held at 25°C for up to 10 hr. After dehydration, about 10 dried shoot tips were placed in a 1.8-ml cryotube and immersed into LN more than 1 hr. Water content of beads and shoot tips were expressed on a fresh weight basis. Dry weight was determined after drying for 100 hr at 80°C.

2.5. Induction of shoot primordia Shoot tips of approximately 1 mm in diameter were dissected from 30-day-old plantlets. They were cultured in vials (30 mm × 200 mm, 30 ml) containing 1/2 MS medium, supplemented with a various concentrations of BA and NAA to make 25 separate media (Table 4). The shoot tips were incubated at 25°C on rotating stages (1 m in diameter) at 2 cycles/min under continuous fluorescent illumination (80 μmol·m-2·s-1). One month after inoculation of shoot tips, numerous dark-green shoot primordia formed and individual masses were 2-3 mm in diameter. A large globular mass of shoot primordia was divided into small segments (2-3 mm in diameter) and subcultured every 2 weeks. In preparation for histological observation of shoot primordia segments, specimens were dehydrated using a graduated ethyl alcohol series to absolute alcohol, and embedded in methacrylate. 10 μm sections were cut and stained with toluidine blue for observation.

2.6. Vitrification of shoot primordia

Table 1 Effect of preculture and cryoprotectant treatments on the shoot formation of vitrified shoot tips of statice cooled to -196°C by vitrification. Preculture Cryoprotectant Shoot formation (%±S.E.) 18.0 ± 6.5 + 42.0 ± 5.0 + 55.3 ± 4.6 + + 76.0 ± 2.0 Preculture: 0.3 M sucrose for 1 day at 25°C, Cryoprotectant treatment: a mixture of 2.0 M glycerol and 0.4 M sucrose for 20 min at 25°C, Dehydration with PVS2: for 15 min at 25°C, Shoot formation (%): percent of shoot tips producing normal shoots 28 days after plating. Approximately 10 shoot tips were tested for each of four replicates.

Shoot primordia segments (about 2 mm in diameter) were cold-hardened in the same medium at 4°C for 10 d by shaking at 2 rpm on rotating stages and then precultured on solidified 1/2 MS medium supplemented with 0.3 M sucrose at 25°C for 16 hr. For cryoprotection, ten hardened and precultured segments were placed in a 1.8 ml cryotube and then treated with a mixture of 2.0 M glycerol and 0.4 M sucrose and held 20 min at 25°C. Segments were sufficiently dehydrated by exposure to PVS2 at 0°C for various lengths of time. Finally, shoot primordia were resuspended in 1 ml of fresh PVS2, directly plunged into LN, and held for more than 1 hr. After rapid warming in a water bath at 40°C for 1 min, PVS2 was drained, replaced with 1.8 ml of 1.2 M sucrose solution, and held for 20 min. Segments were then transferred onto sterilized paper discs in a Petri dish containing the same medium. Shoot formation from vitrified and warmed segments were recorded as the percent of the total number of segments forming normal shoots 21 days after plating.

2.7. Viability and plant growth Vitrified and encapsulated-vitrified samples 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 one day, the beads were transferred onto the same fresh medium in a Petri dish. The encapsulated dried shoot tips were plated onto solidified 1/2 MS medium containing 3% sucrose. One day after plating, some shoot tips were removed from the beads and cultured on the same medium. Recovering shoot tips were observed at weekly intervals. Shoot formation was recorded as percent of total number of shoot tips forming normal shoots 28 days after plating. Ten shoot tips were tested for each of three to four replicates for each experiment.

2.8. Statistical analyses Approximately 10 shoot tips were tested for each of three to four replicates of each experiments above. For each figure and table, standard error or Duncan’s multiple range test were used for the statistical analyses.

3. RESULTS 3.1. Vitrification To enhance the shoot formation, precultured shoot tips with 0.3 M sucrose for 1 day were then treated with a mixture of 2.0 M glycerol and 0.4 M sucrose for 20 min at 25°C before dehydration with PVS2. The effects of preculturing and cryoprotection treatment on shoot formation of vitrified

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Shoot formation (%)

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Fig. 1 Effect of exposure time to PVS2 at 25°C on the shoot formation of shoot tips cooled to –196°C by (A) vitrification and (B) encapsulation/vitrification. Shoot tips were dehydrated with PVS2 at 25°C for various lengths of time prior to cooling (+LN) or without cooling to –196°C (-LN). Excised shoot tips were precultured with 0.3 M sucrose and then (A) treated with 2.0 M glycerol and 0.4 M sucrose for 20 min at 25°C or (B) encapsulated with alginate beads including 2.0 M glycerol and 0.4 M sucrose for 30 min at 25°C before dehydration with PVS2. Approximately 10 shoot tips were tested for each of three to four replicates. Vertical bars represent the standard error.

shoot tips are summarized in Table 1. The shoot tips treated with 2.0 M glycerol and 0.4 M sucrose (cryoprotection treatment) following preculture with 0.3 M sucrose produced the highest shoot formation (76%) after cooled to –196°C. To determine the optimum time of exposure to PVS2 at 25°C, precultured, cryoprotected shoot tips were dehydrated with PVS2 for various lengths of time prior to a plunge into LN. Exposure to PVS2 for various lengths of time resulted in a variable rate of shoot formation (Fig. 1). The highest rate (about 80%) of shoot formation was obtained with shoot tips treated with PVS2 for 15 min at 25°C.

3.2. Encapsulation/vitrification The shoot tips precultured with 0.3 M sucrose for 1 day were encapsulated in alginate-gel beads containing a mixture of 2.0 M glycerol and 0.4 M sucrose. These encapsulated shoot tips were dehydrated with PVS2 at 25°C for different lengths of time prior to a plunge into LN. As shown in Fig. 1, the highest rate of shoot formation at 25°C was obtained in the shoot tips treated with PVS2 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 shoot tips with 0.8 M sucrose or a mixture of 0.8 M sucrose and 0.5 M glycerol for 16 hr. As shown in Table 2, a mixture of 0.8 M sucrose and 0.5 M glycerol produced significantly higher levels of shoot formation than the shoot tips treated with 0.8 M sucrose alone. The shoot tips which were removed from the beads one day after plating showed much higher levels of shoot formation than the shoot tips encapsulated in beads throughout the culturing. Shoot formation of shoot tips cooled to -196°C 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 25°C was greatly different among them: vitrification (15 min), encapsulation/vitrification (50 min), encapsulation/dehydration (420 min).

3.3.1. Induction of shoot primordia

Table 2 Effect of treatment with 0.8 M sucrose or a mixture of 0.8 M sucrose and 0.5 M glycerol on the shoot formation of encapsulated dried shoot tips cooled to -196°C. Treatment Shoot formation (%±S.E.) Shoot tips in beads Removed beadsa 0.8 M sucrose 6.7 ± 3.3 34.3 ± 4.3 0.8 M sucrose + 0.5 M glycerol 46.7 ± 3.3 75.0 ± 5.0 Encapsulated shoot tips containing 0.4 M sucrose were treated with 0.8 M sucrose or a mixture of 0.8 M sucrose and 0.5 M glycerol for 16 hr before dehydration. aThe encapsulated shoot tips in gel beads were removed one day after plating. Shoot formation was determined 28 days after plating. Table 3 Shoot formation of shoot tips of statice cooled to -196°C by three different cryogenic protocols. Cryogenic protocol Shoot formation Time used for (%±S.E.) dehydration (min) Vitrificationa 76.0 ± 2.4 15 Encapsulation/vitrificationb 70.0 ± 5.0 20 73.3 ± 4.7 420 Encapsulation/dehydrationc aPrecultured and cryoprotected shoot tips were dehydrated with PVS2 for 15 min at 25°C prior to a plunge into LN. bPrecultured shoot tips were encapsulated into alginate beads including 2.0 M glycerol and 0.4 M sucrose were then dehydrated with PVS2 for 50 min at 25°C prior to a plunge into LN. cPrecultured shoot tips were encapsulated into alginate beads and then treated with 0.8 M sucrose and 0.5 M glycerol for 16 hr. These shoot tips were dehydrated with dry silica gel (50 g) for 7 hr prior to a plunge into LN. Encapsulated shoot tips in gel beads were removed one day after plating. Table 4 Structure produced by shoot tip culture of statice in 1/2 MS medium modified with several combinations of BA and NAA. Structure produced BA NAA (mg/l) (mg/l) 0 0.01 0.1 1.0 5.0 0 B(10/10)a BC(10/10) SP(10/10) C(10/10) − 0.01 B(8/10) BC(10/10) SP(10/10) C(10/10) − C(10/10) − 0.1 C(10/10) BC(10/10) SP(8/10) C(1/10) − − 1.0 C(10/10) C(4/10) 5.0 − − − − − aNumber of shoot tips developed/number of shoot tips plated. B: branches, SP: shoot primordia, BC: bud clusters, C: callus, ―: No growth or death.

Ten masses of hybrid statice shoot primordia were induced only in 1/2 MS medium that contained a combination of 0.1 mg/l BA with 0 to 0.1 mg/l NAA (Table 4). All other combinations produced precocious branches, bud clusters or callus. The higher concentrations of BA and NAA produced callus and lower concentrations of BA produced bud clusters or branches. During one month of subculture, the mass of shoot primordia increased in mass as much as about 4 times (data is not shown). Histological observation on segments of shoot primordia revealed many small rises shaped like a dome (Fig. 2A). These differentiated structurally into an outer single meristematic cell layer where the inner cell mass was composed of many large parenchyma cells (Fig. 2B). However, distinct bud formation was not observed on the cross sections, as described by others for multiple bud clusters (Banerjee et al. 1991; Kohmura et al. 1992). A mixture of 1% (w/v) sucrose and 0.1 mg/l BA proFloriculture, Ornamental and Plant Biotechnology Volume V ©2008 Global Science Books, UK

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duced the most number of shoots (Table 5). Plants successfully regenerated from a mass of shoot primordia segments within 21 days after transfer to solidified 1/2 MS medium supplemented with 1% sucrose and 0.1 mg/l BA (Fig. 3A). Most of the plants rooted after 30 days of reculturing on solidified hormone free 1/2 MS medium, acclimated, and successfully grew into normal plants in a greenhouse.

3.3.2. Cryopreservation of shoot primordia by vitrification Exposure to PVS2 to 0°C for various lengths of time resulted in a variable rate of shoot formation following vitrification (Fig. 4). The highest rate of shoot formation was obtained when the segments of shoot primordia treated with PVS2 for 60 min at 0°C. Successfully vitrified and warmed segments of shoot primordia turned brown, but recovered within 7 days and eventually developed shoots 20 to 25 days after plating (Fig. 3C).

Fig. 2 (A) Clump of dark-green bulbous (globular) structures consisting of numerous meristematic clusters designated “shoot primordia”. (B) Thin cross section of shoot primordia segment. Bars: A = 5 mm, B = 50 μm.

4. DISCUSSION

Shoot formation (%)

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 shoot tips 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). Fig. 3 Developmental progress of statice through to shoot primordia. (A) Mass of In the vitrification method with or without encapsulation, shoot primordia. (B) Developmental stage of shoot primordia, (C) Whole plantlet. Bar: A, shoot tips are sufficiently dehydrated (osmotically) by exposure to C = 10 mm, B = 2 mm. a highly concentrated vitrification solution (ex. PVS2) which hardly penetrates into the cytosol during the dehydration process 100 prior to a plunge into LN. During the PVS2 dehydration, there is no appreciable influx of additional cryoprotectants into specimens 80 due to differences in the permeability coefficients for water and 60 solutes and a large difference in the activation energies for water and solute permeation. As a result, the specimens remain osmot40 ically concentrated and the increase in the cytosolic concentration required for vitrification is attained by dehydration (Steponkus et 20 al. 1992). 0 In the vitrification method, the direct exposure of cells and 0 20 40 60 80 100 120 shoot tips to PVS2 causes harmful effects due to osmotic stress and chemical toxicity. Thus, to obtain a successful cryopreservaExposure time to PVS2 (min) tion by vitrification is to carefully control the dehydration proceFig. 4 Effect of exposure to PVS2 at 0°C on shoot formation for shoot primordia dure with PVS2. In addition, it is necessary to increase segments cooled to –196°C by vitrification. Precultured and cryoprotected segments dehydration tolerance of cells and shoot tips to be cryopreserved were treated with PVS2 for various lengths of time before immersed into liquid nitrogen. About 10 segments were tested in triplicate for each value. by preconditioning (preculture and cryoprotection treatment) before dehydration. The injurious effects caused by direct exposure to PVS2 can be eliminated or reduced by optimizing the exposure time, adding a gradual amount of PVS2 or a gradual dehydration process followed by dehydrating specimens at 0°C. In the present study, the above injurious effects were effectively overcome by preculturing excised shoot tips on sucrose enriched medium for one day (a significant increase in cell concentration of up to about 0.6 M; Matsumoto et al. 1998b), followed by cryoprotection treatment with a mixture of 2.0 M glycerol and 0.4 M sucrose for about 25 min at 25°C (Note: the cells of shoot tips plasmolysed considerably, but glycerol and sucrose did not penetrate into the cytosol for 25 min as observed through a cytosolic volume change) before dehydration with PVS2 (the plasmolysis proceeds intensively). During preculture on sucrose enriched medium for approximately one day, sugar and proline was greatly increased in the shoot tips (Matsumoto et al. 1998), which, in turn, may enhance 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 in the study that the development tolerance of tobacco cells pre-cultured with 0.3 M mannitol and exposed to a PVS2 for 1 day, appeared to be the combined results of the cell’s responsiveness to mild osmotic stress caused by preculture. In particular, the production of ABA, the accumulation of mannitol during preculture, proline and certain proteins including late embryogenesis Floriculture, Ornamental and Plant Biotechnology Volume V ©2008 Global Science Books, UK

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abundant (LEA). For the many herbaceous plants tested, overnight preculture of excised shoot tips with 0.3 M sucrose appeared to be inconsequential for producing a high level of recovery growth by vitrification. It was further demonstrated that 2.0 M glycerol and 0.4 M sucrose solution for cryoprotection treatment was very effective in increasing tolerance to freeze-dehydration down to -30°C and to dehydration using PVS2 (Sakai et al. 1991; Nishizawa et al. 1992). The protective effect of 2.0 M glycerol and 0.4 M sucrose solution in the cell’s peri-protoplasmic space may be due to mitigation of a large osmotic stress by severe dehydration with PVS2 in addition to some mechanism of action which minimizes the 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 cryoprotected shoot tips were dehydrated with PVS2 at 25°C for 20 min (Fig. 2A) or 50 min (Fig. 2B), 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 shoot tips acquired dehydration tolerance to the PVS2 which optimized the 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 shoot tips cryopreserved by vitrification (Matsumoto and Sakai 1995). In the former technique, encapsulated shoot tips are treated with 0.8 M sucrose for 16 hr to induce dehydration tolerance before air-drying (Fabre and Dureddre 1990). The overnight treatment with 0.8 M sucrose produced a much lower level of recovery growth (45%) than those of vitrified shoot tips (70 to 80%) 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 shoot tips was significantly improved (from 45 to 75%) provided the following two conditions were met: (1) they were treated with a mixture of 0.8 M sucrose and 0.5 M glycerol, and (2) they were removed from beads one 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. 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 shoot tips at the same time. Thus, encapsulation/vitrification method was developed by Matsumoto et al. (1995) and has been applied to many plants; ex. strawberry (Hirai et al. 1998), potato (Hirai et al. 1999), cassava (Charoensub et al. 2004) and gentiana (Tanaka et al. 2004). With this method is easy to handle and treat a large number of shoot tips at the same time. Furthermore, the recovery growth is much earlier than when using encapsulated dried shoot tips. The vitrification method significantly decreased the time used for dehydration and simplified the cryogenic procedures. And this method was successfully applied to about 20 tropical monocotyledonous plants (Thinh 1997) and woody plants (Niino et al. 1992a, 1992b; Kuranuki and Sakai 1995; Niino et al. 1997; Matsumoto et al. 2001; Matsumoto and Sakai 2002). Thus, the vitrification method seems promising for the cryopreservation of shoot tips and somatic embryos. The ability to regenerate whole plants is a vital objective of in vitro tissue culture techniques and is necessary for the application of molecular and somatic genetics for plant improvement and for efficient germplasm conservation. Shoot primordia can propagate vegetatively at a very high rate and readily regenerates plants by organogenesis. Thus, shoot primordia appear promising for mass propagation and the material of cryopreservation for Limonium. However, further studies are necessary to induce and cryopreserve shoot primordia in other cultivars of statice.

5. CONCLUSION To establish an effective and reliable protocol for cryopreservation of in vitro-grown shoot tips of statice, different cryogenic protocols (vitrification, encapsulation/vitrification and encapsulation/dehydration) were tested. These three procedures produced nearly the same levels of growth recovery (70 to 80%) under well-optimized conditions. However, the time used for dehydration at 25°C in vitrification was shortest among three cryogenic procedures. Shoot primordia of statice were induced and cryopreserved by vitrification. It can thus be concluded that the most promising cryogenic procedure of statice shoot tips appears to be vitrification in terms of both its high recovery growth and the simplicity of the procedure.

ACKNOWLEDGEMENTS I would like to express my deep thanks to Prof. A. Sakai for his enthusiastically untired encouragement, expert recommendations and valuable support. The text, tables and figures of this paper were partly cited from our following published papers: HortScience 32, 309-311, 1997, © The American Society for Horticultural Science; Scientia Horticulturae 76, 105-114, 1998, © Elsevier Science; Cryopreservation of Plant Germplasm II, pp 180-195, 2002, © Springer Science and Business Media.

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the techniques of vitrification. Plant Cell Reports 11, 433-437 Kuranuki Y, Sakai A (1995) Cryopreservation of in vitro-grown shoot tips of tea (Camellia sinensis) by vitrification. CryoLetters 16, 345-352 Matsumoto T, Sakai A, Takahashi C, Yamada K (1995) Cryopreservation of in vitro-grown shoot tips of wasabi (Wasabia japonica) by encapsulation-vitrification method. CryoLetters 16, 189-196 Matsumoto T, Sakai A (1995) An approach to enhance dehydration tolerance of alginate-coated dried shoot tips cooled to -196°C. CryoLetters 16, 299-306 Matsumoto T, Sakai A, Nako Y (1998) A novel preculturing for enhancing the survival of in vitro-grown shoot tips of wasabi (Wasabia japonica) cooled to -196°C by vitrification. CryoLetters 19, 27-36 Matsumoto T, Mochida K, Itamura H, Sakai A (2001) Cryopreservation of persimmon (Diospyros kaki Thunb.) by vitrification of dormant shoot tips. Plant Cell Reports 20, 398-402 Matsumoto T, Sakai A (2002) Cryopreservation of axillary shoot tips of in vitro-grown grape (Vitis) by a two-step vitrification protocol. Euphytica 131, 173-175 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15, 473-497 Niino T, Sakai A, Yakuwa H, Nojiri K (1992a) Cryopreservation of in vitro-grown shoot tips of apple and pear by vitrification. Plant Cell, Tissue and Organ Culture 28, 261-266 Niino T, Sakai A, Enomoto S, Magoshi J, Kato S (1992b) Cryopreservation of in vitro-grown shoot tips of mulberry by vitrification. CryoLetters 13, 303-312 Niino T, Tashiro K, Suzuki M, Ohuch S, Magoshi J, Akiyama T (1997) Cryopreservation of in vitro-grown shoot tips of cherry by one-step vitrification. Scientia Horticulturae 70, 155163 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. CryoLetters 13, 379-388 Reinhoud PJ, Schrijnemakers EWM, Iren F, Kijne JW (1995) Vitrification and a heat-shock treatment improve cryopreservation of tobacco cell suspension compared to two-step freezing. Plant Cell, Tissue and Organ Culture 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 Physiology 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 Reports 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 Science 74, 243-248 Sakai A (1995) Cryopreservation of germplasm collection in woody plants. In: Bajaj YPS (Ed) Biotechnology in Agriculture and Forestry (Vol 32) Cryopreservation of Plant Germplasm, Springer-Verlag, Dordrecht, The Netherlands, pp 55-69 Sakai A (1997) Potentially valuable cryogenic procedures for cryopreservation of cultured plant shoot tips. In: Razdan MK, Cocking EC (Eds) Conservation of Plant Genetic Resources in Vitro, Science Publishers, New Hampshire, pp 53-66 Steponkus PL, Langis R, Fujikawa S (1992) Cryopreservation of plant tissues by vitrification. In: Steponkus PL (Ed) Advances in Low Temperature Biology (Vol 1), JAI Press, London, pp 1-61 Tanaka R, Ikeda H (1983) Perennial maintenance of annual Haplopappus gracilis (2n = 4) by shoot tip cloning. Japanese Journal of Genetics 58, 65-70 Tanaka D, Niino T, Isuzugawa K, Hikage T, Uemura M (2004) Cryopreservation of shoot apices of in vitro-grown gentiana plants: a comparison of vitrification and encapsulationvitrification protocols. CryoLetters 25, 167-176 Thinh NT (1997) Cryopreservation of germplasm of vegetatively propagated tropical monocots by vitrification. PhD Thesis, Dept. of Agronomy, Kobe University, Kobe, 187 pp Wang Q, Perl A (2006) Cryopreservation in floricultural plants. In: Teixeira da Silva JA (Ed) Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues (1st Edn, Vol I), Global Science Books, Isleworth, UK, pp 523-539
