CryoLetters 30 (3), 232-243 (2009) © CryoLetters, c/o
[email protected]
CRYOPRESERVATION OF GINKGO BILOBA CELL CULTURE: EFFECT OF PRETREATMENT WITH SUCROSE AND ABA -
Lu, Elena V. Popova, Chun-Hua Wu, Eun-Jung Lee, Eun-Joo Hahn and Kee-Yoeup Paek*
Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju 361-763, Korea. *Corresponding author e-mail:
[email protected] Abstract The report describes the impact of preculture with sucrose and sucrose + ABA on desiccation and cryopreservation tolerance of cell cultures of Ginkgo biloba L., an important landscape and medicinal tree. Callus clumps were incubated on MS medium supplemented with high sucrose concentrations (up to 24%, w/v), employed alone or with ABA (2-10 mg l-1) for various durations followed by desiccation for 0-240 min and cryopreservation. The beneficial effect of preculture on regrowth after desiccation without cryopreservation was only observed for the cells with water content of 20% FW and was not influenced by presence of ABA. However, preculture of calli in presence of ABA resulted in a lower desiccation rate as compared with untreated controls and calli pretreated with sucrose alone. In calli precultured with sucrose alone, post-thaw regrowth was occasional regardless of the sugar concentration in the medium, while pretreatment of calli with ABA and sucrose ensured stable regrowth after cryopreservation. The highest post-thaw regrowth of 54% was achieved for calli precultured on medium supplemented with 10% (w/v) sucrose and 2 mg l-1 ABA for 21 days followed by desiccation for 150 min. The different effects of preculture treatments on postthaw regrowth were associated with significant changes in content and in composition of endogenous soluble sugars in calli. Sucrose and glucose accumulated preferentially in ABAprecultured calli, while the fructose content was higher in calli precultured in absence of ABA. The possible role of preculture on desiccation and cryopreservation tolerance of G. biloba cell cultures is discussed. Keywords: Ginkgo biloba, callus, cryopreservation, abscisic acid, sucrose preculture, soluble sugar content
232
INTRODUCTION Ginkgo biloba L., the so called “living fossil”, belongs to the Ginkgoaceae family and Ginkgoales order. It is popular worldwide as a landscape tree and as an important source of medicinal substances, mostly referred to as ginkgolides and bilobalide (17). Cell cultures of G. biloba have been reported to be an effective system for the production of ginkgolides A and B, bilobalide and flavonol aglycones (5, 13, 14, 18), as well as a potential tool for intensive plant propagation (26). Cryopreservation offers an attractive alternative to labour- and fund-consuming maintenance of in vitro cell cultures since samples in liquid nitrogen do not require periodic subcultures and can be stored for decades using minimum space (21). To date, various cryopreservation techniques including controlled rate (programmed) freezing, vitrification, encapsulation-dehydration and encapsulation-vitrification have been developed and successfully applied to undifferentiated cell cultures of numerous plants (1,11,15,23,24). Previously, we achieved successful cryopreservation of Ginkgo biloba calli by desiccation method but post-freeze regrowth did not exceeded 23% (19). Preculture of plant material in presence of osmotically active compounds such as proline, mannitol, sugars and sugar alcohols contributes to plant cell dehydration and chilling tolerance and is thus considered essential for achieving regrowth after cryopreservation (24). However, the precise impact of such pretreatments on the physiological state of the plant material still remains unclear. Preculture on sucroseenriched medium induced a range of physiological response in plant tissues including increase in soluble sugar content (2, 9, 28) and total fatty acids (20, 31), significant changes in protein composition (28, 31) and cell ultrastructure (2). Supplementing ABA in the preculture medium resulted in changes in gene expression (6) as well as in the accumulation of intracellular sugars (10) and dehydrins (3). However, it has been proposed that sucrose and ABA induced different mechanisms of desiccation tolerance in plant cells (29). Previous study showed that preculture on medium supplemented with 10% (w/v) sucrose and 2 mg l-1 ABA for at least 7 days was essential for successful cryopreservation of Ginkgo biloba calli (19). However, the roles of sucrose and ABA in the preculture process were not revealed. In the present study various preculture conditions were screened to improve regrowth of G. biloba calli after cryopreservation and the effect of preconditioning with sucrose and sucrose+ABA on regrowth after dehydration and cryopreservation, on desiccation rates and soluble sugar composition of G. biloba calli is demonstrated.
233
MATERIALS AND METHODS Plant material G. biloba L. seeds collected in North Chungcheong province, Republic of Korea, were used as initial plant material. Calli were induced from in vitro developed cotyledons and maintained on gelrite-solidified MS (16) medium supplemented with 3% (w/v) sucrose and 5 mg l-1 NAA (BM, basal medium) under dark conditions at 25 ± 1°C for two years prior to cryopreservation experiments. Subcultures during maintenance were performed every 3 weeks by transferring friable, yellowish calli to the fresh medium. Callus clumps 3-4 mm in diameter (30-40 mg FW) separated at the end of routine 3week subcultures were incubated on the same medium for 7 days and harvested for preculture treatments and cryopreservation experiments. Preculture treatments To determine the effect of sucrose pretreatment, sucrose concentration in preculture medium was increased, directly or step-wise, from 3% to 10% and 24% (w/v) (Table 1). Calli were incubated on preculture medium for various durations followed by desiccation in the air current of a laminar flow cabinet for 0-120 min and cryopreservation. Control clumps were precultured on BM. In further experiments, 2, 5 and 10 mg l-1 ABA were added in preculture medium containing 10% (w/v) sucrose, and calli were incubated on these media for 7 days prior to desiccation and freezing in liquid nitrogen (LN). As a result of this experiment, medium containing 10% (w/v) sucrose and 2 mg l -1 ABA has been selected. In the final experiment, calli were precultured on medium supplemented with 10% (w/v) sucrose and 2 mg l -1 ABA for 7, 14 and 21 days followed by desiccation for 60-240 min and cryopreservation. Desiccation and freeze-thaw procedures A simple desiccation method was applied to calli. Precultured calli were blotted dry with sterile filter paper (Whatman no. 4, Whatman Ltd., England) and transferred to 5 × 25 mm stripes of filter paper, 4-5 clumps per a stripe. Calli were desiccated in open 9 cm Petri dishes under the laminar air flow cabinet for 0-240 min depending on the experimental scheme. Temperature and relative air humidity during desiccation were controlled to 28 ± 1°C and 11-12%, respectively as determined with a Thermo recorder (TR-72S, T&D, Nagano, Japan), and remained stable among treatments. After desiccation, paper stripes with calli were transferred to BM for regrowth or put in 1.8 ml cryotubes (Nunc, Denmark) at rate of ten to twelve clumps per tube. Tubes were closed, sealed with sticking plaster and plunged directly in LN. After at least one hour, cryotubes 234
were re-warmed in a water bath at 41 ± 2°C for 90 s followed by surface-sterilization with 70% (v/v) ethyl alcohol. Paper stripes with calli were taken out and placed on the surface of Basal medium to allow calli to rehydrate. After 1-2 min, paper strips were removed and calli were placed in the dark at 25 ± 1°C for regrowth. Regrowth Regrowth was estimated as the number of callus clumps which formed new tissues after 5 weeks of reculture on BM, and expressed in percentage. Three to five independent replications were performed for each treatment except for preliminary screening of preculture conditions (Tables 1 and 2) which were performed in two replications; 10-15 callus clumps were treated in each replication. Determination of water content Water content in callus clumps was monitored during desiccation. Calli were weighted and dried in an oven at 104°C for 3 days. Water content was calculated on a fresh weight basis (FW). Three to six measurements were performed for each point of desiccation curves; about sixteen callus clumps were taken for each measurement. Analysis of soluble sugars The content of endogenous soluble sugars in calli was analyzed during preculture treatments. One gram of fresh calli was ground in to a fine powder with LN and extracted with 3 ml deionized water at 60°C for 30 min followed by centrifugation at 10 000 g for 15 min (12). The supernatant, containing the soluble sugar fraction was filtered through a 0.45 µm pore size filter and analyzed by HPLC system (Waters 2690 separation module; Walters, USA) using a high performance carbohydrate column coupled to a reflex Differential Refractometer (RI 410, Walters, USA). An aqueous solution of 80% acetonitrile flowing at 0.8 ml min-1 was used as mobile phase. Glucose, fructose and sucrose peaks were identified and quantified using comparison with Sigma standards. Statistical analysis Data were analyzed using SAS Version 9.1 (SAS, Raleigh, NC). Significance of differences between treatments was analyzed using Duncan`s multiple range test at P ≤ 0.05. Figures present mean values with SE.
235
RESULTS In the regrowth process, two types of calli were formed on the surface of callus clumps subjected to desiccation and cryopreservation (Figure 1). White watered calli exhibited slow growth during further subcultures while yellowish friable calli grew well after cryopreservation and could be used for the regeneration of culture. Figure 1. Regrowth of new tissues from G. biloba callus subjected to cryopreservation, 1.5 months after thawing. Calli were precultured on MS medium supplemented with 10% (w/v) sucrose and 2 mg l-1 ABA for 14 days followed by 110 min desiccation. Bar = 1 mm.
In the first experiment, seven variants of sucrose pretreatment were screened for their ability to improve callus regrowth after desiccation and cryopreservation. Results are presented in Table 1. Among all treatments tested, calli precultured with 10% (w/v) sucrose for 14 and 25 days showed higher regrowth after desiccation for 90 and 120 min. Higher sucrose contents were always detrimental to regrowth even when sugar concentration in the medium was increased step-wise. After cryopreservation, only occasional regrowth of calli was observed following preculture on medium supplemented with 10% (w/v) sucrose for 14 and 25 days (Table 1). In an attempt to improve regrowth after cryopreservation and reduce preculture duration, 2, 5 or 10 mg l-1ABA were added in the preculture medium containing 10% (w/v) sucrose and preculture was performed for one week. Even after such a short preculture, the presence of 2 mg l-1 ABA provided stable regrowth after desiccation and freezing (Table 2). Increasing ABA concentration to 5 or 10 mg l-1 was not beneficial to post-thaw regrowth. Based on these results, in further experiment calli were precultured on medium supplemented with 2 mg l-1 ABA and 10% (w/v) sucrose for various durations (7, 14 or 21 days) followed by desiccation for 60-240 min and cryopreservation (Fig. 2). The optimum desiccation time varied according to preculture treatment and was found to be 110 min for calli precultured for 7 days and 150 min for 14- and 21-day precultured calli. The highest post-thaw regrowth of 54% was achieved for calli precultured for 21 days followed by 150 min desiccation. This regrowth was significantly (P < 0.05) higher than those after 7- and 14-day preculture. After cryopreservation, calli grew well and exhibited the same profiles of the relative DNA content as untreated calli (not shown). 236
Table 1. Regrowth of G. biloba calli after desiccation (-LN) and cryopreservation (+LN) as affected by sucrose and NAA concentration in the medium and preculture duration. Sucrose concentration in the medium was increased directly or step-wise from 3% to 10 and 24% (w/v). Data are shown as no. of clumps with regrowth / total no. of clumps taken for treatment Preculture
LN +/-
Desiccation time (min) 0
20
40
60
80
90
120
18/18
22/22
17/18
33/37
-
30/45
3/47
0/33
0/21
0/18
0/14
-
0/14
0/15
- LN
46/46
36/40
56/61
15/32
6/23
-
0/12
+LN
0/15
0/21
0/17
0/20
0/20
-
-
10% sucrose 7 days ->
- LN
14/14
14/14
13/15
10/13
4/15
-
-
24% 3 days
+LN
0/13
0/15
0/9
0/8
0/10
-
-
10% sucrose 7 days ->
- LN
23/31
7/16
4/14
2/16
-
-
-
24% 10 days
+LN
0/47
0/20
0/14
0/12
-
-
-
10% sucrose 14 days
- LN
16/16
15/15
16/16
15/15
14/18
-
8/43
+LN
0/21
0/28
0/32
0/22
0/18
0/17
1/34
15/15
17/17
16/16
11/11
5/14
-
0/23
0/13
0/18
0/16
0/16
0/15
13/13
13/14
5/14
4/14
3/13
0/13
- LN
Control -1
(3% sucrose + 5 mg l NAA) +LN 10% sucrose 7 days
-1
10% sucrose + 5 mg l NAA - LN
+LN
14 days
10% sucrose 14 days + 24% - LN
-
12h
+LN
0/14
0/14
0/14
0/14
0/12
0/9
10% sucrose 25 days
- LN
17/17
17/17
13/13
27/30
10/24
15/31
1/33
+LN
0/10
0/10
0/17
0/14
0/28
3/50
1/31
Table 2. Effect of ABA concentration in preculture medium in combination with 10% (w/v) sucrose on regrowth of G. biloba calli after desiccation (-LN) and cryopreservation (+LN). Preculture was performed for 7 days. Data are shown as no. of clumps with regrowth / total no. of clumps taken for treatment (%) Desiccation time (min)
ABA conc., mg l-1 2 5 10
80
110
150
-LN
+LN
-LN
+LN
-LN
+LN
17/47
4/32
25/48
7/46
0/31
2/15
(36.2)
(12.3)
(52.1)
(15.2)
(0)
(13.3)
21/35
0/28
15/34
4/25
2/27
0/10
(60.0)
(0)
(44.1)
16.0)
(7.4)
(0)
16/34
1/28
19/29
5/31
4/27
0/27
(47.1)
(3.4)
(65.5)
(16.1)
(14.8)
(0)
237
70
7 days 14 days 21 days
60
Regrowth, %
50 40 30 20 10 0 50
100
150
200
250
Desiccation, min Figure 2. Regrowth of G. biloba calli after cryopreservation as affected by preculture and
Calli showed different rates of water loss depending on sucrose concentration and presence of ABA in preculture medium (Fig. 3). Addition of ABA during preculture resulted in a significant (P < 0.05) reduction of desiccation rate and, as a result, in lower decrease in regrowth compared with preculture with sucrose alone (Fig. 3). However, calculation on dry weight basis (Fig. 4) clearly demonstrated that preculture improved regrowth only
desiccation durations. Calli were precultured on
at water content of 20% FW (0.25 gH2O/gDW) (means in with 10% (w/v) sucrose and 2 mg l-1 ABA for 7, pares control/preculture were 14 and 21 days. significantly differed at P < 0.05). Moreover, there were no significant differences at P < 0.05 in post-desiccation regrowth BM for 7 days then on medium supplemented
between two preculture treatments. The soluble sugar content in calli during preculture was investigated to explain the differences in regrowth observed after freezing depending on the preculture treatments. In control calli, the content of total endogenous soluble sugars showed a noticeable peak on the 14th day followed by a two fold reduction at the third week of culture (Fig. 5). A stable increase in soluble sugar content was observed in calli precultured with sucrose alone. The content in soluble sugars increased significantly (P < 0.05) in calli precultured on medium containing sucrose +ABA compared with 7-days control (Fig. 5). It reached 777.2 mg (g DW)-1 7 days after transferring to preculture medium and remained high after the following 7 days of preculture. Sugar composition was also affected by preculture treatment. Sucrose content increased after 14 days but remained stable during further culturing in control calli and calli precultured with sucrose alone (Fig. 5). By contrast, a sharp increase in sucrose content was observed after 7 days of preculture on medium supplemented with combination of sucrose and ABA. In all treatments, the glucose content increased gradually during preculture; however, this increase was about 4.2 and 2.6 fold higher in calli precultured with a combination of sucrose and ABA than with sucrose alone. The highest fructose content was observed in control calli at the 14th day of preculture, followed by calli from the sucrose and sucrose-ABA pretreatments (Fig. 5). Equal 238
100
80
Water content, %FW
amounts of fructose were found in all treatments at the 21th day of preculture.
Control Sucrose+ABA Sucrose
60
DISCUSSION
40
As a general rule, plant material should be desiccated to water content of 20-30% FW before
20
0 0
20
40
60
80 100 120 140 160 180 200
Regrowth, %
100
cryopreservation (3). However, in our study no regrowth was observed after desiccation to 20% FW in G. biloba calli precultured on BM containing 3% (w/v) sucrose (control). Meantime, a 7-day preculture on medium supplemented with 10% (w/v) sucrose alone or combined with 2 mg l-1 ABA significantly improved regrowth of
80 60 40 20
7 days on the same medium (control) or on
calli after desiccation to 20% FW. These results coincide with findings made by other authors who reported the efficiency of sucrose preculture for induction of desiccation tolerance in plant cells and organs and for their successful cryopreservation (e.g. 25,27,29). Preculture with high sucrose concentrations was found
medium supplemented with 10% (w/v) sucrose
useful
0 0
20
40
60
80 100 120 140 160 180 200
Desiccation, min Figure 3. Curves of water loss and regrowth of G. biloba calli during desiccation as affected by composition of preculture medium. Calli were precultured on BM for 7 days, then for another
for
cryopreservation
-1
by
vitrification procedure applied to various herbaceous and woody plants like gentiana, wasabi and apples (24). For plant materials which are sensitive to desiccation and high sugar content, a step-wise increase in sucrose concentration in the preculture medium was found to be more effective (28, 30). In our study, however, sucrose concentrations higher than 10% (w/v) showed a detrimental effect on callus regrowth even when sugar content was increased gradually.
alone or in combination with 2 mg l ABA.
Preculture with high concentrations of sugars caused a variety of stress-induced 239
Control
10% s uc ros e
10% s uc ros e + ABA
Regrowth, %
100 80 60 40 20 0 1
0.5
0.25
Water content, g H2O/ gDW
Figure 4. Regrowth of G. biloba calli after desiccation to different water contents as
responses in plant cells. Alongside with frequently observed accumulation of intracellular sugars (2, 9, 28), accumulation of specific proteins (4, 28), changes in fatty acid content and double bond index (20, 31) has been recorded in several species during sucrose preculture. The ratio of stigmasterol to sitosterol increased significantly in sucrose-pretreated banana meristems (31). Carpentier et al. (4) used banana cauliflower-like meristems as a model
for proteome analysis and discovered that pretreatment with 0.4 M sucrose Calculated from Fig. 3. caused up- and down-regulation of particular enzymes of the glycolytic pathwas including significant up-regulation of enolase and down-regulation of sucrose synthase. They also observed induction of affected by composition of preculture medium.
specific genes of energy-conserving glycolysis in response to sucrose pretreatment. Abscisic acid has been reported to induce desiccation tolerance in plant tissues (10) and proved to be essential for successful cryopreservation of Arachis, blackberry and raspberry shoot tips (7, 22), and Dendrobium candidum orchid protocorms-like bodies (3). In our research, addition of ABA to sucrose-enriched preculture medium improved post-thaw regrowth of Ginkgo cells compared with sucrose alone. The analysis of soluble sugar content and rates of water loss were performed in an attempt to explain the different post-thaw recovery of calli after preculture with sucrose and sucrose+ABA. Wang et al. (29) demonstrated that different physiological mechanisms were involved in the induction of desiccation tolerance by ABA and sucrose pretreatments in Spathoglottis plicata protocorms. In their study, ABA-induced desiccation tolerance was highly correlated with the reduction of the drying rate. In a similar way, in this study it was shown that supplementing ABA in the preculture medium resulted in slower water loss by G. biloba cells. Preculture on sucrose and sucrose+ABA containing medium led to significant increase in content of total soluble sugars in G. biloba cells. Sugars are known to possess protective properties in plant cells during desiccation (8). It is proposed that at water contents below 20% FW sugars can replace hydration shell and form hydrogen bonds with macromolecules thereby preventing protein denaturation and phase transition in 240
1000
Control Total soluble sugars Sucrose Sucrose+ABA
Glucose 400
800 300
Sugar content, mg/(gDW)
600 200
400
100
200 0
0
7
600
14
21
7
600
Fructose
500
500
400
400
300
300
200
200
100
100
0
14
21
Sucrose
0
7
14
7
21
Preculture duration, days
14
21
Preculture duration, days
Figure 5. Changes in soluble sugar content and composition in G. biloba callus during preculture treatment. Callus clumps were precultured on BM for 7 days then transferred to the same medium (control) and medium supplemented with 10% (w/v) sucrose alone or in combination with 2 mg l-1 ABA.
membranes (cited by 10). This hypothesis could explain the significant increase in regrowth observed with sucrose and sucrose+ABA precultured G. biloba calli compared with control when desiccated to 20% FW. Adding ABA in the preculture medium resulted in significant increase in soluble sugar content in G. biloba cells compared with calli precultured with sucrose alone. The increase in sugar concentration in ABA-precultured calli occurred mostly due to sucrose and glucose, while fructose was preferably accumulated in control calli. In a coincidence with our results, in Spathoglottis plicata protocorms a combination of ABA and sucrose in preculture medium resulted in a higher accumulation of total soluble sugars (mostly due to sucrose) than preculture with sucrose alone (29). In addition to its effect on sugar content, ABA preculture led to increased chlorophyll and caroteniod contents and induced dehydrin synthesis in protocorms but these parameters did not differ between ABA+sucrose and sucrose treatments (29). 241
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