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. 2006 Jun;97(6):1073–1081. doi: 10.1093/aob/mcl054

Physiological Changes in Gentian Axillary Buds During Two-step Preculturing with Sucrose that Conferred High Levels of Tolerance to Desiccation and Cryopreservation

MITSUTERU SUZUKI 1,2,§, MASAYA ISHIKAWA 2,*,§, HITOSHI OKUDA 3, KATSUJI NODA 4, TADASHI KISHIMOTO 2, TOSHIHIDE NAKAMURA 2, ISAO OGIWARA 5, ISAO SHIMURA 5, TOMOYA AKIHAMA 1
PMCID: PMC2803387  PMID: 16565150

Abstract

• Background and Aims Induction of dehydration tolerance is a key to achieving high survival rates in cryopreservation of plant specimens. It has been reported previously that two-step preculturing with sucrose effectively increased desiccation tolerance in axillary buds of gentian (Gentiana scabra), which allow the buds to survive cryopreservation. This study is aimed at characterizing each step of this preculturing and to elucidate physiological changes induced during this preculturing.

• Methods In standard two-step preculture, excised gentian axillary buds were incubated for 11 d on MS medium with 0·1 m sucrose at 25 °C (first step: mild osmotic stress was given) and the subsequent incubation on MS medium with 0·4 m and 0·7 m sucrose for 1 d each (second step). The levels of abscisic acid (ABA), proline and soluble sugars in gentian buds during the preculture were determined. Effects of various combinations of two-step preculturing and of exogenous ABA and proline were studied.

• Key Results During the first preculture step, there was a transient increase in ABA content peaking on day 4, which declined to a background level at the end of the first and second step preculturing. Proline level increased steadily during the first preculture step and increased further in the second preculture step. Incubating buds with medium containing proline, instead of the two-step preculturing, did not allow them to survive desiccation. Incubating buds with ABA instead of 0·1 m sucrose-preculturing effectively increased desiccation tolerance only when it was followed by the second preculture step. Fluridone, an ABA synthesis inhibitor included in the two-step preculture medium, reduced desiccation tolerance of the buds. The normal first-step preculture increased the levels of soluble sugars 2·4-fold, especially sucrose and raffinose. Buds treated with the second preculture step had greatly increased sucrose levels.

• Conclusions These observations lead to the hypothesis that the first preculture step involves ABA-mediated cellular changes and the second step induces loading of sucrose in the gentian buds.

Keywords: Gentian (Gentiana scabra var. buergeri), abscisic acid (ABA), axillary buds, cryopreservation, desiccation tolerance, raffinose, sucrose, sugars, proline, preculture, dehydration, drying

INTRODUCTION

Cryopreservation has attracted attention as a method for long-term storage of plant genetic resources and cultured plant materials. In the last decade, new cryopreservation techniques for plant specimens have been developed including vitrification and desiccation methods (Grout, 1995). In any of the methods, induction of high levels of dehydration tolerance in the tissue or organ to preserve is an essential step, as the specimen has to endure extreme dehydration in the vitrification solution or severe air-drying before immersion in liquid nitrogen. Such increased tolerance to desiccation is usually achieved by preculturing either with high concentrations of sucrose (e.g. Fabre and Dereuddre, 1990), abscisic acid (ABA) (e.g. Senaratna et al., 1989) or cold-hardening (e.g. Reed, 1993; also see Suzuki et al., 1998; Ishikawa et al., 2005; Suzuki et al., 2005), but is often difficult to induce, thus restricting the applicability of cryopreservation techniques.

In a previous study, using axillary buds of in vitro-grown gentian plants, a novel two-step preculture method for efficiently increasing dehydration tolerance was developed (Suzuki et al., 1998). The preculture comprised two steps: incubation on Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) with 0·1 m sucrose at 25 °C for 11 d (first step) and the subsequent incubation on MS medium with 0·4 m and 0·7 m sucrose for 1 d each (second step). Following this preculturing, gentian axillary buds, either with or without encapsulation, tolerated desiccation to 10 % water content (at least for the short term) and subsequent liquid nitrogen (LN) exposure with high recovery rates (Suzuki et al., 1998; Suzuki et al., 2005). The method is also applicable to vitrification-based cryopreservation (M. Suzuki, T. Akihama, M. Ishikawa, unpubl. res.).

Whether the nature of desiccation tolerance induced in gentian buds by two-step preculturing is similar to long-term desiccation tolerance in orthodox seeds is unknown (Walters et al., 2001). Understanding induction mechanisms of such dehydration tolerance is necessary for optimizing preculture conditions and improving survival in cryopreservation. However, only a few reports have addressed what physiological changes occur during the preculture treatments and their relevance to survival following cryopreservation (Hitmi et al., 1999; Vandenbussche et al., 1999; Reinhoud et al., 2000). In the present paper, some physiological changes in gentian axillary buds induced by each of the two-step preculture treatments are reported in an attempt to clarify the roles of each step in survival following desiccation and cryopreservation.

MATERIALS AND METHODS

Plant materials

In vitro plants of gentian (Gentiana scabra Bunge var. buergeri Maxim.) were propagated from the stem segments with axillary buds as previously described (Suzuki et al., 1998). Briefly, they were cultured on MS medium (Murashige and Skoog, 1962) containing half-strength inorganic salts and 15 g L−1 sucrose (half strength MS medium was used only for subculturing) solidified with 8 g L−1 agar (pH 5·8). The segments were incubated at 25 °C under a 16-h photoperiod (50 µmol s−1 m−2) and subcultured at an interval of 60 d by transplanting nodal segments with axillary buds. After removal of leaves and apical buds from 60-d-old in vitro-grown plantlets, about 2-mm-long nodal stem segments with axillary buds and traces of petioles were excised from the stem and immediately used for preculture experiments. These excised entire nodal stem segments with axillary buds (which contained lateral meristems not actively growing due to the apical dominance) were hereafter referred to as axillary buds or buds.

Preculture procedures using sucrose

The standard preculture procedure comprised two steps (Suzuki et al., 1998) (Fig. 1). In the first step of normal two-step preculturing, axillary buds were precultured on full-strength MS medium containing 0·1 m sucrose and 8 g L−1 agar for 11 d at 25 °C under a 16-h photoperiod (50 µmol s−1 m−2) unless otherwise specified. This step gave mild osmotic stress as sucrose and mineral concentrations were 2·3- and 2-fold the subculture medium, respectively. In the subsequent step, the buds were consecutively cultured on full-strength MS media (+ 8 g L−1 agar) containing 0·4 m sucrose for 1 d, then 0·7 m sucrose for 1 d at 25 °C under the same light condition unless otherwise mentioned. In some experiments to reveal the effects of each preculture step, these steps were either prolonged, reduced, omitted or additional steps added. Precultured axillary buds were subjected to air-drying and cryopreservation.

Fig. 1.

Fig. 1.

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A schematic representation of desiccation and cryopreservation procedures of gentian axillary buds including the two-step preculture used in this study.

Preculture with ABA or proline

In some experiments, instead of the first preculture step with sucrose, gentian buds were incubated in full-strength MS medium (15 g L−1 sucrose, 8 g L−1 agar, pH 5·8) containing 1–10 mg L−1 of (±)-ABA (Sigma) for 3–20 d at 25 °C under a 16 h-photoperiod (50 µmol s−1 m−2). Then the buds were used directly for desiccation or following the normal second preculture step (MS media with 0·4 m or 0·7 m sucrose). Inclusion of ABA (2–10 mg L−1) in media of both steps of the standard two-step preculturing protocol was also tested. When necessary, the buds were precultured in semi-solid full-strength MS medium (+ 15 g L−1 sucrose and 8 g L−1 agar) containing 0·1–1 m proline for 2–4 d, instead of the two-step preculture, and used for desiccation experiments.

Air-drying of buds and LN immersion procedures

After removal of excess medium, the precultured axillary buds were placed on filter paper in a Petri dish and desiccated in a laminar flow chamber for up to 24 h at 25 °C and about 45 % relative humidity (RH). Approximately ten buds were dried for each designated period, transferred into a 10-mL conical glass centrifuge tube, submerged directly in LN and held there for at least 30 min. The water content of the dried buds was determined gravimetrically after oven drying at 70 °C for 48 h.

Determination of the survival (regrowth capability) of axillary buds

The axillary buds immersed in LN were warmed by shaking the tube in a water bath at 37 °C for about 1 min. The buds subjected to desiccation alone or to desiccation and LN immersion, were placed on full-strength MS medium containing 30 g L−1 sucrose and 8 g L−1 agar (pH 5·8) and cultured under a 16 h-photoperiod at 25 °C. The survival (regrowth capability) of the axillary buds was defined as the percentage of nodal segments which produced normal shoots and roots from the axillary buds after about 20 d in vitro.

Determination of proline content

The proline content of gentian buds during the preculture treatment was determined according to Bates et al. (1973). The buds (0·4 g f. wt) precultured for designated periods were collected and homogenized using liquid nitrogen and a pestle and mortar, followed by extraction in 10 mL of 3 % (w/v) sulfosalicilic acid for 10 min. The extract was filtered. A 2-mL aliquot of the extract in a test tube was mixed thoroughly with 2 mL of acid ninhydrin solution and 2 mL of acetic acid, followed by incubation at 100 °C for 1 h. The reaction was stopped by cooling the tube on ice and the solution was vigorously mixed with 4 mL of toluene for 20 s. A520 of the toluene layer was read against toluene as a blank.

Determination of soluble sugars

Soluble sugars were extracted and analysed according to Ogiwara et al. (1998) and Ohtsuka et al. (2004). Approximately 0·7 g (f. wt) of axillary buds at the end of preculture steps were collected and washed five or six times with distilled water for 20 min to remove possible sugar contamination from the preculture medium. The buds were homogenized using liquid nitrogen and extracted three times with 80 % (v/v) ethanol at 80 °C for 20 min, followed by centrifugation at 1600 g for 10 min. The combined supernatant was filtered and dried under vacuum. The dried sample was dissolved in water and passed through a Sep-Pak Plus C18 column (Waters Co., Milford, MA, USA) to remove pigments. The eluate was then passed through a Sep-Pak Plus CM and a Sep-Pak Plus QMA culumn (Waters Co.). Aliquots (2 mL) of the eluate were dried and dissolved in 0·2 mL of water. Following filtering through a membrane filter unit (0·45 µm), the sample was analysed using an HPLC (Shimazu LC-9A) equipped with a Shimazu SCR-101N column, which was eluted with water (0·5 mL min−1) at 40 °C using a refraction detector (RID-6A). The contents of glucose, sucrose, fructose, raffinose and stachyose were determined.

Determination of ABA content

ABA in the axillary buds was extracted, purified and quantified according to Okuda et al. (1995) and Okuda (2000) with some modifications. Briefly, axillary buds (0·2 g f. wt) collected during preculturing were homogenized and extracted with methanol at 5 °C for 20 min three times using d6-ABA as the inner standard. Combined extracts were filtered and evaporated to obtain the aqueous phase after addition of 10 mL of water. The aqueous phase was passed through a membrane filter unit (0·45 µm) and partitioned three times with water-saturated ethyl acetate (1 : 3, v/v) after pH adjustment to 2·5 with 1 n HCl. The organic solvent phase obtained was evaporated, dissolved in 50 % (v/v) methanol and applied to an ODS column (Pheneomenx, LUNA, 5, 4·6 × 250 mm) using an HPLC system (Shimadzu LC-10A). The fraction corresponding to ABA was collected, evaporated, dissolved in n-hexane:ethyl acetate (1 : 1) after methylation with diazomethane and analysed using a GC-MS (Hewlett Packard 5890A) equipped with a Hewlett Packard HP-1 column (30 m × 0·25 mm). The amount of endogenous ABA was calculated from the peak area of m/z 190 relative to that of m/z 194 (the known amount of d6-ABA).

RESULTS

Effect of preculturing conditions on the survival (regrowth capability) of gentian buds exposed to LN following desiccation

When buds precultured in a two-step manner (MS medium containing 0·1 m sucrose for 11 d then MS medium with 0·4 m and 0·7 m sucrose for 1 d each at 25 °C) were air-dried at 25 °C for 0–24 h, there was only a slight decrease in survival (regrowth capacity) until the water content reached 10 % on a fresh weight basis (16 h of drying) (Table 1). Buds exposed to further desiccation showed reduced survival. Survival of buds exposed to LN was first detected when the buds were desiccated to 24 % water content and attained highest survival at 10 % water content. At 10 % water content, 90 % of buds survived both desiccation and LN exposure. Subsequently for evaluating desiccation tolerance of gentian buds treated with various preculturing protocols, buds were desiccated for 16 h to a water content of 10 % (fresh weight basis).

Table 1.

Survival (regrowth capability) of gentian axillary buds air-dried for designated length of time to various water contents and subsequently exposed to liquid nitrogen (LN)

Survival (regrowth capability) (% ± s.e.)
Desiccation period (h) Water content (% f. wt) Desiccation LN
0 70·5 100 ± 0 0 ± 0
1 44·8 96·8 ± 3·9 0 ± 0
4 24·5 88·9 ± 0 6·5 ± 6·5
8 17.4 84·3 ± 5·6 35·2 ± 2·7
12 15·7 85·7 ± 5·9 41·1 ± 6·1
16 10·0 90·4 ± 5·6 90·4 ± 13·0
20 8·2 70·3 ± 5·4 69·3 ± 5·6
24 2·9 53·6 ± 13·0 46·7 ± 13·2
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Axillary buds were precultured in a two-step manner as shown in Fig. 1 before being subjected to desiccation at 25 °C and approx. 45 % RH and subsequently immersed into LN. Ten or more axillary buds were treated for each of three replicates.

The effect of various preculture combinations and duration on desiccation tolerance of gentian buds is summarized in (Fig. 2). Desiccation tolerance was not induced either by the first preculture step with 0·1 m sucrose alone (11 and 21 d) or by the second preculture step with 0·4 m or 0·7 m sucrose (1 d each) alone, suggesting that both preculture steps are required. When the second preculture step (following 11 d of the first preculture step) was performed with only 0·4 m sucrose treatment for 1 d, the induced level of desiccation tolerance was much less (35 % survival) compared with the complete preculture (0·4 m or 0·7 m sucrose for 1 d each: 90 % survival). A prolonged second preculture step (0·4 m for 3 d and 0·7 m for 2 d) did not increase survival after desiccation (Fig. 2). When the buds treated with the complete two-step preculturing were further cultured with 0·4 m sucrose for an additional day, the desiccation tolerance was reduced (72 % survival). When the buds were further transferred to 0·1 m sucrose in the second preculture step, desiccation tolerance was reduced to a low level (5 % survival). When the buds thus incubated were additionally incubated in 0·4 m and 0·7 m sucrose for 1 d each, the level of survival attained was restored almost to the level of the normal two-step preculture. During the first preculture step with MS medium containing 0·1 m sucrose, 8–11 d of incubation was found to be optimal for inducing high survival following desiccation and LN exposure (Fig. 3A).

Fig. 2.

Fig. 2.

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Effect of preculturing conditions using sucrose on the survival of dried gentian axillary buds. Gentian buds were either two-step precultured at 25 °C (standard), or modified as designated in the ordinate before being air-dried to water content 10 % at 25 °C and RH 45 %. The survival (regrowth capability) was evaluated by the ability to develop normal shoots and roots after reculturing for 20 d (for details see the Materials and methods). The standard two-step preculturing comprised incubation on semi-solid MS medium with 0·1 m sucrose for 11 d at 25 °C (Step 1) and subsequently on semi-sold MS medium with 0.4 m and 0.7 m sucrose for 1 d each at 25 °C (Step 2). These steps were either prolonged, reduced, omitted or followed by one to four additional steps (incubation on semi-solid MS medium with 0·1, 0·4 or 0·7 m sucrose for 1 d at 25 °C) to give different final preculture sucrose concentrations. Approximately ten axillary buds were treated for each of the three replicates.

Fig. 3.

Fig. 3.

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Effect of the length of the first preculture step with MS medium containing 0·1 m sucrose on the survival (regrowth capability) of desiccated and cryopreserved gentian axillary buds (A), and changes in the levels of ABA (B) and proline (C) during the first preculture step (MS + 0·1 m sucrose for 11 d at 25 °C) and after the second preculture step (MS + 0·4 m and 0·7 m sucrose for 1 d each at 25 °C). In (A), following the first preculture step for the designated periods shown, the buds were treated with the second preculture step before being desiccated to 10 % water content on a fresh weight basis (DC), then exposed to liquid nitrogen (LN). The data are the mean ± s.e. of triplicates (approx. ten buds for each replicate) for (A) and the mean ± s.e. (n = 3) for (B) and (C).

Changes in the ABA content of buds during the two-step preculturing with sucrose

Gentian buds before preculturing (control) had about 100 ng of ABA per gram fresh weight (Fig. 3B). During the first preculture step with MS medium containing 0·1 m sucrose, there was a 4-fold increase in the ABA level of buds, which was only transient and peaked at 4 d of incubation. The ABA content returned to the basal level at the end of the first preculture step (usually 11 d) and there was no further increase at the end of the second preculture step with MS medium containing 0·4 m sucrose for 1 d and 0·7 m sucrose for 1 d.

Proline content of axillary buds during the two-step preculturing with sucrose

One gram fresh weight of gentian buds before preculturing (control) contained approx. 0·4 µmol of proline (Fig. 3C). The level of proline increased steadily with increasing periods of the first preculture step and reached about three times the original level between 7 and 11 d. Preculture periods longer than 11 d resulted in a decrease in proline levels. Second step-precultured gentian buds, following 11 d of first step-preculturing, had about a 4·5-fold increase in the level of proline compared with the control buds.

Effect of the two-step preculturing on the levels of soluble sugars in gentian buds

There have been few reports that measured soluble sugar content in precultured materials, as a large amount of external sugars contained in the preculture medium would affect the analysis of tiny specimens. To avoid contamination with sugar from the medium, gentian bud samples were washed extensively before extracting sugars. One gram fresh weight of gentian buds before preculturing contained 43·6 µmol (16·2 mg) of soluble sugars: 1·7 µmol (0·3 mg) each of glucose and fructose, 28·9 µmol (9·9 mg) of sucrose and 11·4 µmol (5·7 mg) of raffinose (Fig. 4). Only trace amounts of stachyose were detected in all the treatments. Following the first preculture step, the level of total soluble sugars in gentian buds increased by 2·4-fold (103 µmol, 39·2 mg) compared with the control buds: 5·9 µmol of glucose (3·5-fold increase), 10·1 µmol of fructose (6-fold increase), 47 µmol of sucrose (1·6-fold increase) and 40·2 µmol of raffinose (3·5-fold increase). The increase in the total sugar content was mainly due to increases in raffinose followed by sucrose. After the second preculture, the total soluble sugar content increased to 148 µmol (50 mg). This was mainly due to the increase in sucrose (117 µmol) whilst raffinose content decreased to the level of control buds and the content of glucose and fructose remained similar to those of buds following the first preculture step.

Fig. 4.

Fig. 4.

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Changes in the soluble sugar (glucose, fructose, sucrose and raffinose) content of gentian axillary buds after the first step (MS + 0·1 m sucrose for 11 d at 25 °C) and second step preculturing (MS medium + 0·4 or 0·7 m sucrose for 1 d each at 25 °C). After preculturing, the buds were washed extensively before extraction of soluble sugars, which were analysed with HPLC. Control refers to the gentian buds before preculturing (excised from in vitro shoots). Only trace amounts of stachyose were detected in all the treatments and not shown in the figure. The data presented are the mean ± s.e. of triplicates.

Effect of exogenous ABA treatment as an alternative to the two-step preculturing with sucrose

Since the first preculture step involves a transient increase in ABA content of gentian buds, it was investigated how preculturing with ABA would affect the survival of gentian buds exposed to desiccation for 16 h. Incubation with MS medium (15 g L−1 sucrose) containing ABA (2–10 mg L−1) for 3–20 d did not confer desiccation tolerance in gentian buds (only 12 d incubation data is shown in Table 2). However, when preculturing with ABA (1–10 mg L−1 for 11 d at 25 °C) was followed by second-step preculturing with ABA-free MS medium containing 0·4 m or 0·7 m sucrose for 1 d each, 81–86 % of gentian buds tolerated desiccation to 10 % water content (Table 2). When ABA was included in both the first and second preculture-step media, there was no further improvement but some decline in the survival of gentian buds exposed to air-drying for 16 h (Table 2). When an ABA synthesis inhibitor, fluridone (Rasmussen et al., 1997) was included in the medium for the first- and second-step preculturing, increasing concentrations (0·3–10 mg L−1) of fluridone resulted in progressively decreased survival (54–27 %) after air-drying to 10 % water content (Fig. 5).

Table 2.

Effect of incubation with ABA on the survival (regrowth capability) of gentian axillary buds subjected to air-drying

ABA concentration (mg L−1)
Expt First step Second step Survival (regrowth capability) (% ± s.e.)
A* 2 Not performed 0 ± 0
5 Not performed 0 ± 0
10 Not performed 0 ± 0
B† 1 0 85·8 ± 8·7
10 0 80·8 ± 8·6
C‡ 0 0 90·8 ± 5·8
2 2 57·2 ± 7·8
5 5 82·2 ± 4·8
10 10 67·8 ± 2·0
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*In experiment A, the buds were precultured on MS medium (15 g L−1 sucrose) containing the designated concentration of ABA for 12 d at 25 °C (without second-step preculture) prior to desiccation treatments.

†In experiment B, the buds were precultured on MS medium (15 g L−1 sucrose) containing the designated concentration of ABA for 11 d at 25 °C and subsequently on ABA-free MS medium containing 0·4 m and 0·7 m sucrose for 1 d each at 25 °C (same as the second preculture step) before desiccation treatment.

‡In experiment C, ABA (0–10 mg L−1) was added to the medium of both preculture steps. The buds were precultured on MS media containing ABA and sucrose (0·1 m) for 11 d in the first step and then on media with ABA and sucrose (0·4 and 0·7 m) for 1 d each in the second step at 25 °C prior to desiccation. In all the experiments, the precultured buds were desiccated for 16 h at 25 °C and RH 45 % to 10 % (f. wt basis) water content. The data are the mean ± standard error of triplicates (ten or more buds for each replicate).

Fig. 5.

Fig. 5.

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Effect of fluridone contained in the first and second preculture medium on the survival (regrowth capability) of desiccated gentian axillary buds. The buds were precultured on MS medium containing the designated concentrations of fluridone and sucrose (0·1 m for 11 d in the first step and 0·4 and 0·7 m for 1 d each in the second step) at 25 °C before being desiccated for 16 h at 25 °C and RH 45 % to 10 % (fresh weight basis) water content. The data are the mean ± s.e. of triplicates (ten or more buds for each replicate).

Effect of preculturing with proline on the induction of desiccation tolerance and survival after exposure to LN

Since the proline content steadily increased during two-step preculturing with sucrose, the effect of preculturing with proline was examined to see if this treatment could replace preculturing with sucrose. Buds incubated with MS medium (+15 g L−1 sucrose) with proline (0·1, 0·5 or 1 m) for 2 or 4 d did not tolerate air-drying for 16 h at 25 °C (0 % survival in all cases, data not shown). Incubation with proline medium for 5 d or more resulted in killing of the buds (without desiccation) and seemed toxic to gentian buds.

DISCUSSION

Significance of two-step preculturing with sucrose

Here the two-step preculturing method that has been developed by Suzuki et al. (1998) for efficiently increasing desiccation tolerance in gentian buds is characterized. The preculture procedure consisted of two steps: the first step with 0·1 m sucrose (+ MS medium) for 8–13 d and the second step with successive exposure to 0·4 m and 0·7 m sucrose (+ MS medium) for 1 d each. With this preculture, about 90 % of the axillary buds withstood desiccation to 10 % water content on a fresh weight basis and subsequent immersion in LN. Since this two-step preculturing induces high dehydration tolerance, the method has recently been found to be effective for encapsulation-desiccation based cryopreservation of gentian buds (M. Suzuki et al., 2005) and for cryopreserving gentian buds using vitrification methods (M. Suzuki, T. Akihama, M. Ishikawa unpubl. res.). This preculturing can circumvent the lengthy cold hardening treatment (about 30 d) which otherwise is required for cryopreservation of gentian buds (Kikuchi and Yashiro 1995; Tanaka et al., 2004).

Detailed analysis of preculturing using various combinations revealed that both the first and second steps are required for increasing desiccation tolerance in the axillary buds (Fig. 2). When either of the preculture steps was omitted, the buds did not tolerate desiccation to 10 % water content. Longer incubation periods of either the first step or second step alone were not effective. In the second step, the final concentration of sucrose (0·7 m) was shown to be crucial: transfer of gentian buds to 0·1–0·4 m sucrose greatly reduced the survival following desiccation but subsequent retransfer to 0·7 m sucrose resulted in recovery of high desiccation tolerance. These experiments allow the interdependence and the significance of the two preculture steps to be recognized: the first step giving the latent cell capacity required, which develops into various levels of desiccation tolerance depending upon the final concentration of sucrose in the second step.

Analysis of ABA, proline and soluble sugar contents helped to explain the features of each step of the two-step preculturing. The first preculture step was characterized by a transient increase in ABA content and increases in the content of proline (3-fold) and soluble sugars (2·4-fold), especially raffinose. The second step was characterized by increases in the sucrose content (2·5-fold increases from the levels of the first step). These results allow the speculation that the latent capacity achieved during the first step involves ABA-mediated cellular changes, and enhancement of desiccation tolerance in the second step involves loading of sucrose into gentian bud cells.

Involvement of ABA

The first preculture step, where mild osmotic stress was imposed, accompanied a transient increase in ABA content while ABA remained at low levels in the second preculture step (Fig. 3B). Preculturing gentian buds with ABA (in MS medium + 15 g L−1 sucrose), instead of the first preculture step, was effective in increasing desiccation tolerance only when it was followed by the second preculture step with 0·4 m or 0·7 m sucrose (Table 2). Addition of ABA to the second preculture step was slightly inhibitory (Table 2). Inclusion of fluridone, an ABA synthesis inhibitor, to the preculture medium reduced survival after desiccation by 40–70 % (Fig. 5). These lines of evidence suggest that the first preculture step accompanies ABA-mediated cellular changes, whose effects are manifested as increases in desiccation tolerance following the second preculture step which accompanies loading of sucrose into the gentian bud cells (this step does not need ABA).

An alternative to the first preculture step is cold-hardening treatment (Kikuchi and Yashiro, 1995) and a transient ABA increase is reported to occur during the early stages of cold hardening in potato plants (Chen et al., 1983) and wheat seedlings in vitro (Holappa and Walker-Simmons, 1995). The first preculture step with sucrose and cold hardening treatment may share similar ABA-mediated cellular changes that would confer desiccation tolerance.

Exogenous ABA treatment has been known to induce freezing tolerance in cell cultures of cold hardy plants and cross-adaptation to various abiotic stresses including osmotic stress (Ishikawa et al., 1990; Chandler and Robertson, 1994; Ishikawa et al., 1995; Swamy and Smith, 1999). Thus, preculturing with ABA has been attempted in many species for cryopreservation either as an alternative to sucrose preculture or as an additive to other conditioning treatments. In some plant species and tissues, preculture with ABA worked well (Senaratna et al., 1989; Shimonishi et al., 1991; Na and Kondo, 1996; Ryynanen, 1998) and, in others cases, ABA was not as efficient (Reed, 1993; Ishikawa et al., 1996; Thierry et al., 1999; Vandenbussche et al., 1999; Bian et al., 2002). The present results suggest that ABA preculturing followed by a sucrose loading process (in the absence of ABA) may improve survival.

Involvement of proline

Proline content during the first preculture step increased steadily and reached maximum between days 7 and 11, which approximately coincided with the period when gentian buds showed highest capacity to tolerate desiccation to 10 % water content following the second preculture step (Fig. 3). The result was consistent with previous cryopreservation studies revealing increases in proline content at the end of preculture with 0·3–0·5 m sucrose or mannitol (Hitmi et al., 1999; Reinhoud et al., 2000). Proline has been known to accumulate in response to desiccation and ABA treatment (Delauney and Verma, 1993; Huber, 1974) and is considered to work as a compatible osmolyte and also as a scavenger against reactive oxygen species (Matysik et al., 2002). Studies of Δ-1-pyrroline-5-carboxylate synthetase (P5CS), a key enzyme in proline biosynthesis, in Arabidopsis have revealed that expression of AtP5CS is increased by dehydration, salt and ABA in the whole plant tissues with a concomitant accumulation of proline (Yoshiba et al., 1995, 1999). The increase in proline content during the two-step preculturing is consistent with these recent results at the molecular level and is most probably a part of ABA-mediated cellular changes contributing to the acquired desiccation tolerance in gentian buds. In contrast, incubation of gentian buds with exogenous proline (0·1–1 m) alone seemed toxic and did not increase desiccation tolerance. Combined treatment (ABA and proline) of taro somatic embryos increased desiccation tolerance (Shimonishi et al., 1993). Preculturing with the combination of sucrose and proline has been reported to increase desiccation tolerance in buds of Prunus and currant (Brison et al., 1995; Luo and Reed, 1997). It is possible that proline may contribute to desiccation tolerance in combination with some other factors such as sucrose.

Involvement of soluble sugars

Measuring soluble sugar levels in small samples incubated in high concentrations of sucrose is difficult. The sugar concentrations obtained in this study were much lower than similar data obtained with other materials precultured in 0·5–1·0 m sugars (Dumet et al., 1994; Suzuki et al., 1997; Hitmi et al., 1999). This is probably due to successful prevention of contamination with extracellular sugars by extensive washing of the materials before the analysis. Total soluble sugar content increased by 2·4-fold at the end of the first preculture step which is attributed to increases in all the soluble sugars but particularly in raffinose and sucrose (Fig. 4). Raffinose has been known to increase in response to cold and dehydration in cereals (Koster and Lynch, 1992) and also in the seed maturation process (Brenac et al., 1997). Overexpression of AtGolS2, a gene responsible for the key enzyme in raffinose synthesis, resulted in increased raffinose content and increased drought tolerance in transgenic Arabidopsis (Taji et al., 2002). The observed raffinose increase in gentian buds treated with the first preculture step is consistent with these recent findings and is also a part of ABA-mediated cellular changes.

Figure 4 suggests that the second preculture step is a process of loading sucrose into the bud cells, as there was a 2·5-fold increase in sucrose content from the level of the first preculture step while other sugars remained at almost the same level (glucose and fructose) or even decreased (raffinose). Biophysical comparison of orthodox (desiccation tolerant) and recalcitrant (desiccation sensitive) seeds suggested that the combination of sucrose and raffinose is important in the formation of stable glasses (vitrification) at ambient temperatures (Koster, 1991). Glasses inhibit fusion of membranes (Crowe et al., 1998) and increase the stability of cellular components in a dry state (Buitink and Leprince, 2004). The molar ratio of sucrose : raffinose (8·6 : 1) at the end of the second preculture step is similar to that observed in desiccation-tolerant orthodox seeds (Koster, 1991; Sun et al., 1994). More recently, late embryogenesis-abundant proteins have been suggested to stabilize the glass in the dry state (Walters et al., 1997; Wolkers et al., 2001; Buitink and Leprince, 2004). Exogenously applied ABA-induced heat stable proteins increased desiccation tolerance of meristems (Luo and Reed, 1997). Similar proteins may have been induced during the first preculture step of gentian buds and contributed to the desiccation tolerance acquired, which is yet to be analysed.

For successful cryopreservation, it is necessary to induce high levels of dehydration tolerance (at least for the short term) in the plant cells, tissues and organs for preservation. It has been shown that in gentian buds, dehydration tolerance is efficiently increased by two-step preculturing with sucrose and each of the two steps was indispensable and interdependent. The first step, where mild osmotic stress was given, involved ABA-mediated cellular changes and the second step incorporation of sucrose into the cells. The present results may help establish the optimal preculture conditions in other plant materials for improving survival after dehydration and subsequent cryopreservation.

Acknowledgments

We thank Dr Duncan A. Vaughan (NIAS) for critically reading the manuscript and Ms A. Oda and H. Nakatani (NIAS) for helping to prepare the figures and maintaining the in vitro gentian cultures. M. Suzuki acknowledges the support of JSPS Fellowships for Young Scientists.

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