Journal of Experimental Botany, Vol. 53, No. 368, pp. 551-558,
March 1, 2002
© 2002 Oxford University Press
Original Papers |
Drying rate and dehydrin synthesis associated with abscisic acid-induced dehydration tolerance in Spathoglottis plicata orchidaceae protocorms
Department of Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260
Received 19 July 2001; Accepted 5 November 2001
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Abstract |
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Dehydration tolerance of in vitro orchid protocorms was investigated under controlled drying conditions and after abscisic acid (ABA) pretreatment. Protocorms were obtained by germinating seeds on Murashige and Skoog (MS) medium containing 10% (v/v) coconut water, 2% (w/v) sucrose and 0.8% (w/v) agar, and were dehydrated in relative humidities (RH) ranging from 7% to 93% at 25 °C. The critical water content of dehydration tolerance was determined, using the electrolyte leakage method. Drying rate affected the critical water content. Slow drying under high RH conditions achieved the greatest tolerance to dehydration. ABA pretreatment decreased the drying rate of protocorms, and increased dehydration tolerance. Improved tolerance to dehydration after ABA treatment was correlated with the effect of ABA on drying rate of protocorms. When critical water content of protocorms dried under different RH was plotted as a function of actual drying rate, no significant difference in tolerance to dehydration was observed between ABA-treated and control protocorms. ABA pretreatment and dehydration of orchid protocorms induced the synthesis of dehydrin, especially under the slow drying conditions. ABA pretreatment also promoted dry matter accumulation such as carbohydrates and soluble proteins and increased the concentration of K+ and Na+ ions in protocorms. The ABA-induced decrease in drying rate was correlated with lower osmotic potential, the enhanced maturity of protocorms and the accumulation of dehydrin in protocorms during pretreatment.
Key words: Abscisic acid, critical water content, dehydrin, desiccation tolerance, drying rate, soluble sugars, Spathoglottis plicata, water stress.
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Introduction |
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Abscisic acid (ABA) plays a crucial role in higher plants in their response to various environmental stresses and serves as a regulatory link between stress factors and plant responses (Seemann and Sharkey, 1987
; Chandler and Robertson, 1994). Physiological studies and molecular analysis have demonstrated that ABA may regulate the adaptation of plants to environmental stresses (Stewart and Voetberg, 1985; Skriver and Mundy, 1990). The increase in endogenous ABA concentration under water stress conditions, or the application of exogenous ABA is often accompanied by the expression of a number of specific genes in plant tissues (Gomez et al., 1988; Mundy and Chua, 1988; Parcy et al., 1994), and with enhanced synthesis of dehydration-related proteins (Gomez et al., 1988; Blackman et al., 1991, 1992; Bray, 1988; Beardmore and Charest, 1995) and other compatible solutes such as proline, glycine-betaine, cyclitols, and soluble carbohydrates (Hanson et al., 1985; Stewart and Voetberg, 1985; Blackman et al., 1992; Sun et al., 1999). In seed development and maturation, ABA also plays an important role in the acquisition of desiccation tolerance (Quatrano, 1987; Blackman et al., 1991, 1992; Meurs et al., 1992). Exogenously applied ABA was able to induce desiccation tolerance in somatic embryos (Park et al., 1988; Bochicchio et al., 1991; Etienne et al., 1993) and immature zygotic embryos (Wakui et al., 1994; Blackman et al., 1992). It is thought that the synthesis of dehydration-related proteins and small molecular weight compatible solutes would help plant tissues to tolerate severe desiccation, possibly by stabilizing cellular structures and protecting metabolic functions from stress disruption.
However, in these earlier studies, actual drying rates of control tissues and ABA-treated or pre-conditioned tissues were not quantified. It is known that ABA could reduce plant water loss under water stress conditions. For example, ABA could regulate the behaviour of leaf stomata (Munns and King, 1988; MacRobbie, 1990). The effect of drying rate on plant desiccation tolerance has also been well documented. Slow drying significantly increases desiccation tolerance of somatic embryos and immature zygotic embryos or seeds (Blackman et al., 1992; Etienne et al., 1993; Li et al., 1999). In these studies, slow drying was believed to allow plant tissues to acquire desiccation tolerance during the period of prolonged slow dehydration. By contrast, slow drying was found to be detrimental to desiccation tolerance of many recalcitrant (desiccation-sensitive) seeds and embryos (Farrant et al., 1985; Pammenter et al., 1998). More recently, it was reported that there is actually an optimal drying rate for a plant tissue to achieve its maximum desiccation tolerance (Liang and Sun, 2000). Therefore, drying rate appears to modify significantly the ability of plant tissues to withstand dehydration stress. These earlier studies indicate that ABA-induced dehydration tolerance may be associated with changes in drying rate in plant tissues. As far as is currently known, there have been no reports about the effect of ABA treatment on tissue drying rate under controlled conditions.
The objective of the present study is to test the hypothesis that ABA-induced dehydration tolerance is related to the change of drying rate in plant tissues. The relationship between dehydration tolerance and drying rate, and the effect of ABA pretreatment on drying rate of orchid protocorms have been investigated. These data suggest that the improvement of dehydration tolerance in orchid protocorms by ABA pretreatment is largely associated with a decrease in drying rate of the tissue.
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Materials and methods |
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Plant materials
Spathoglottis plicata plants were grown in containers with garden soil. Orchid flowers were hand-pollinated. Healthy capsules were harvested 30 d after pollination. The capsules were surface-sterilized in 20% (v/v) commercial bleach (5.75% NaOCl) for 10 min and rinsed with autoclaved distilled water 3–5 times. The capsules were aseptically cut open and seeds were germinated in Murashige and Skoog (MS) medium with 2% sucrose, 10% coconut water and 0.8% agar (pH 5.2). Seeds were cultured at 25 °C with 16 h photoperiod. Protocorms reached an average diameter of 2 mm before ABA pretreatment and drying studies.
ABA pretreatment
A stock solution of ABA was made in 95% alcohol, filter-sterilized and added to autoclaved MS medium. The final concentration of ABA in the medium was 5 or 10 mg l-1. ABA pretreatment was carried out at 25 °C with 16 h photoperiod. Changes in protocorm weight, chlorophyll content, carotenoid content, osmotic potential, and concentrations of K+, Na+ and Ca2+ ions were determined during ABA pretreatment.
Determination of tissue water content and osmotic potential
Cultured protocorms were collected and washed clean with distilled water. Protocorms were then blot-dried with tissue paper. Water content of samples were determined gravimetrically after drying for 24 h at 85 °C, and expressed on a dry weight basis (g water g-1 dry mass). Osmotic potential in the protocorm was determined using a dew point microvoltmeter (Wescor, Logan, UT, USA). Protocorms were frozen in Eppendorf tubes at -80 °C for 1 d. Samples were thawed at room temperature for 1 h before measurement. The microvoltmeter was calibrated with standard NaCl solutions, and the osmotic potential of protocorms calculated according to the standard curve from a series of NaCl solution. To investigate the possible osmotic adjustment during drying, the amount of water lost during drying were recorded, and dried protocorms were rehydrated to the original water content by adding back the same amount of lost water. After equilibration, osmotic potential of rehydrated protocorms was measured.
Determination of chlorophyll, carotenoid, K+, Na+ and Ca2+ content
Contents of chlorophyll and carotenoid in protocorms were extracted with 80% aqueous acetone solution and determined spectrophotometrically. The concentrations of K+, Na+ and Ca2+ ions in protocorms were determined using a flame photometer. Weighed samples were first boiled in deionized water for 30 min to extract the soluble K+, Na+ and Ca2+ from the tissues. Extraction solutions were then diluted to the optimal range for the ion concentration measurement.
Determination of dehydration tolerance
The dehydration tolerance of cultured protocorms was examined at 25 °C in a wide range of drying rates. To maintain a constant drying rate, protocorms (
180 mg) were dried under specific relative humidity (RH) in sealed containers (Sun and Gouk, 1999). Saturated solutions of the following salts were used to control various RH conditions (in brackets): KNO3 (93%), MgSO4, (89%), K2CrO4 (87%), KCl (85%), NaCl (75%), (NH4)NO3 (63%), MnCl2 (56%), Ca(NO3)2 (52%), K2CO3 (43%), MgCl2 (32%), K acetate (23%), and NaOH (7%). Glycerol solutions at concentrations of 32% (w/w), 36%, 52%, and 64% were also used to generate relative humidities of 91%, 90%, 80%, and 70%. For each drying experiment, 15–20 independent samples were prepared for repeated sampling to eliminate RH fluctuation that was caused by opening containers. Dehydration tolerance was expressed by the critical water content that was determined using the electrolyte leakage method. After dehydration to different water contents, the samples were incubated and gently shaken in 20 ml deionized water for 1 h. The amount of electrolytes leaked from protocorms was then measured with a conductivity meter. The total amount of electrolytes in the tissues was determined after the sample was boiled for 20 min in caped vials and cooled to room temperature. The percentage electrolyte leakage was used as an indicator for dehydration damage. The water content below which the percentage electrolyte leakage increased sharply was calculated (according to Sun and Leopold, 1993), and taken as the critical water content. The critical water content determined by the electrolyte leakage method was further confirmed, using the protocorm survival test after dehydration. To test the survival of protocorms that were dried to different water contents, protocorms were rehydrated and cultured on 0.8% agar/MS medium containing 2% sucrose and 10% coconut water. The percentage of protocorm survival was recorded after 14 d of culture.
Determination of carbohydrate content
Soluble carbohydrates were extracted with 50% ethanol containing 100 µg PGS (phenyl-
-D-glucoside) as an internal standard at 70 °C in a water bath for 60 min. Extraction was then repeated three times with 50% ethanol (without PGS). Extracts were pooled and dried under vacuum, redissolved in 1 ml distilled water, and passed through a column containing Dowex 50W, IRA-94 and PVPP (polyvinylpolypyrrolidone). Filtrate was collected and freeze-dried. Carbohydrates were derivatized with 1 ml of trimethylsilylimidazole (TMSI) dissolved in pyridine (1:4 v/v) at 90 °C for 1 h. Soluble carbohydrates were separated with a 15 m fused capillary HP-1 column (0.32 mm ID, 0.25 µm film thickness) (Hewlett-Packard, USA).
Western blot of dehydrin-related proteins
One gram of fresh protocorms was homogenized with 5 ml of extraction buffer containing 50 mM TRIS-HCl, pH 7.5, 50 mM KCl and 1.0 mM phenylmethylsulphonyl fluoride (PMSF) at 4 °C. Homogenate was centrifuged at 15000 g for 20 min. The supernatant was desalted using a 10-DG column (Bio-Rad Laboratories, Hercules, CA, USA). Protein concentration was determined by the Bio-Rad protein assay using bovine serum albumin as a standard. Proteins (30 µg) were separated by 15% SDS gel, and then transferred to a nitrocellulose membrane using the mini trans-blot electrophoretic transfer cell. Non-specific binding sites were blocked with 3% gelatin in PBS (phosphate buffer with NaCl and KCl, pH 7.4) buffer for 3 h at room temperature. The nitrocellulose membrane was incubated for 2 h at room temperature with primary antibody (rabbit anti-dehydrin), which was raised from a peptide with the sequence CTGEKKGIMDKIKEKLPGQH (Close et al., 1993). The membrane was washed with PBS before it was incubated with goat anti-rabbit IgG alkaline phosphatase conjugate (5000 g dilution with 2% gelatin in PBS) for 2 h. The second antibody was detected using nitroblue-tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP). The relative quantity of dehydrin in the images of Western blots was determined, using the Bio-Rad image analysis software ‘Quantity One’. The images were digitized, and the density of dehydrin bands was integrated. The relative changes in dehydrin content after dehydration and ABA treatment were expressed over dehydrin content in fresh control protocorms (i.e. using the dehydrin content of fresh control protocorms as 100%).
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Results |
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Effects of drying rate on dehydration tolerance of protocorms
Figure 1
shows representative drying curves of orchid protocorms at a constant RH of 7%, 80% and 93%. When the water content of protocorms was expressed on dry weight basis (g water g-1 dry matter), the loss of water from the tissue followed the first-order-kinetics at all RH levels ranging from 7% to 93%, until the protocorms almost achieved equilibrium with the salt solutions. Therefore, these drying curves could be described by a simple exponential function, WC= exp(–ßt), where was the initial water content, ß was the rate constant of water loss, and t was time of drying. In the present study, the rate constant of water loss was used to express the drying rate quantitatively.
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Figure 2A
shows the change of electrolyte leakage in protocorms after drying to different water contents at 7%, 70% and 89% RH. The electrolyte leakage of protocorms did not increase much during the early stages. In each case, dehydration damage, as measured by electrolyte leakage, increased significantly after the protocorms were dried to below a threshold water content. This critical water content was used to express the degree of dehydration tolerance in protocorms. The survival test was also used to confirm the dehydration tolerance of protocorms. Figure 2B showed the good correlation between electrolyte leakage and survival of protocorms dried at 43% RH. Because of its simplicity, the electrolyte leakage method was used as a routine method for dehydration tolerance measurements in the present study. Figure 3 shows the effects of RH on drying rate and dehydration tolerance of orchid protocorms. Drying rate changed greatly under different RH conditions, with the ß-value ranging from 0.20 to 0.01 h-1. The critical water content of protocorms was also affected by RH conditions. RH between 7% and 60% did not significantly affect dehydration tolerance. However, slow drying at higher RH enhanced tolerance to water loss of protocorms. Critical water content of protocorms decreased from 3.0 g g-1 DW to 1.5 g g-1 DW as RH increased from 60% to 93%.
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Effects of ABA pretreatment on growth and development of protocorms
Figure 4 shows the relative increases in fresh weight and dry weight of protocorms during ABA pretreatment. During the pretreatment of 2 weeks, fresh weight and dry weight of control protocorms increased by 200% and 280%, respectively. The addition of ABA at concentrations of 5 mg l-1 and 10 mg l-1 inhibited protocorm growth. The fresh weight of ABA-treated protocorms increased only by 20–30%; however, dry weight increased by 80–100%. ABA treatment appeared to enhance the maturation of protocorms, as indicated by the change of colour. The content of chlorophyll a and carotenoid decreased by more than 40%, relative to control protocorms, after 7 d pretreatment with 10 mg l-1 ABA (Table 1). As a result of the change in the growth and development pattern, control and ABA-treated protocorms were in a different physiological status. ABA treatment for 7 d at 10 mg l-1 resulted in (a) a decrease in osmotic potential from -0.81±0.03 MPa to -1.01±0.04 MPa, (b) an increase in K+ and Na+ concentrations in the tissue by 12–18%, (c) an increase of soluble carbohydrate concentration by more than 80%, of which 90% was sucrose, and (d) an increase in soluble protein content by 39% (Table 1). All of these physiological parameters showed significant differences statistically.
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Effects of ABA pretreatment on drying rate and dehydration tolerance of protocorms
ABA pretreatment altered the response of protocorms to drying. Figure 5 shows the effects of ABA pretreatment on drying rate and critical water content of protocorms. After ABA pretreatment, the drying rate of protocorms was reduced to approximately 50% as compared to control protocorms. The dehydration tolerance of protocorms, as indicated by the critical water content, increased after ABA pretreatment. Interestingly, when critical water content of protocorms dried at different RH was plotted as a function of actual drying rate, there was no significant difference between control and ABA-treated protocorms in their responses to drying (Fig. 6). This result suggests that ABA pretreatment might enhance the tolerance of protocorms to water stress through the reduction in the rate of water loss.
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Effects of drying rate and ABA pretreatment on the synthesis of dehydrin
ABA pretreatment induced the synthesis of several new dehydrins. Fresh protocorms that were cultured in control MS medium showed only one visible protein band that was immunologically related to dehydrin (30 kDa) in the Western blot, whereas ABA-pretreated protocorms contained six additional bands at molecular weights around 27, 22, 19, 16, 13.5, and 12 kDa (Fig. 7A). To investigate dehydrin synthesis of protocorms in response to the rate of drying, control and ABA-pretreated protocorms were dried under both fast and slow drying conditions. Since ABA pretreatment affected the drying rate of protocorms (Fig. 5
), control and ABA-pretreated protocorms were dried at different RH conditions that achieved the same drying rate. Under the fast drying conditions, control and ABA-pretreated protocorms were dried at RH of 67% and 7.2%, respectively, that achieved the same drying rate of 0.09 h-1; whereas under slow drying conditions protocorms were dried at RH of 87% and 75% which achieved the same drying rate of 0.028 h-1. Drying resulted in the synthesis of new dehydrins in control protocorms, and increased the content of existing dehydrins (Fig. 7A). Drying rate affected dehydrin synthesis greatly during dehydration, and slow drying promoted dehydrin synthesis, particularly in control protocorms (Fig. 7A). Figure 7B shows the relative increase of total dehydrin content in protocorms after ABA pretreatment and drying under fast and slow drying conditions. Fast and slowly-dried protocorms led to 300% and 700% increase in dehydrin content relative to fresh control tissues. ABA-pretreated protocorms were less responsive to dehydration. Fast and slow drying resulted in, respectively, only 100% and 180% increase in dehydrin content over ABA-pretreated fresh protocorms.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The response of in vitro orchid protocorms to dehydration was investigated under a wide range of drying conditions (drying rates) in the present study. The critical water content of dehydration tolerance was a function of drying rates for both control and ABA-pretreated protocorms (Figs 3
, 5, 6). One hypothesis that proposes the beneficial effect of slow drying for embryonic tissues is that slow drying could permit the tissue to synthesize certain protective substances in response to dehydration. The data of the present study supports this hypothesis. Protocorms were damaged at high critical water content during rapid drying at low RH between 7% and 60% (Fig. 3). In these conditions, protocorms would lose 50% of their tissue water in less than 5 h and, therefore, there is probably insufficient time for the tissues to adapt to the severe dehydration stress. In order to examine the relationship between the acquisition of dehydration tolerance and slow drying rate further, the time needed for protocorms to lose 50% of their tissue water under different drying conditions was calculated (Fig. 8). The plot of critical water content against the time of 50% water loss indicates clearly the presence of a period for the adaptation or acquisition of dehydration tolerance in protocorms. ABA pretreatment has been known to enhance dehydration tolerance of embryonic tissues (Park et al., 1988; Etienne et al., 1993; Wakui et al., 1994; Li et al., 1999). Therefore, it was not surprising that ABA pretreatment decreased the critical water content of protocorms (Fig. 5). What was surprising was the observation that there was no significant difference between the control and ABA pretreatment, when dehydration tolerance of protocorms was compared at the same drying rate (Figs 6, 8). The increase in dehydration tolerance after ABA pretreatment appeared to be due mainly to the reduction in water loss from protocorms, allowing the tissues more time for the acquisition of dehydration tolerance.
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The reduction in water loss from protocorms was one of the most notable changes after ABA pretreatment (Fig. 5A
). A number of developmental, biochemical and physiological changes might be associated with the decrease in drying rate. Developmental changes included the maturity, size and shape of protocorms. The maturity of protocorms was accelerated during ABA pretreatment (Table 1), and the increased thickness of the cuticle might well contribute to the reduction of drying rate. The shape of protocorms did not change much during ABA pretreatment. The change in protocorm size was significant (Fig. 4), but would probably not decrease the drying rate. ABA-pretreated protocorms were smaller than control protocorms (Fig. 4), and therefore actually had a higher surface-to-volume ratio, which should facilitate the drying of protocorms. Biochemical and physiological factors included the increase in ion concentration, solute synthesis and osmotic regulation during ABA pretreatment. Significant osmotic adjustment occurred in protocorms during ABA pretreatment. The osmotic potential of ABA-treated protocorms decreased from -0.81 MPa to -1.01 MPa in 7 d (Table 1). A major part of this adjustment was attributed to the accumulation of soluble sugars, especially sucrose, and the increased concentration of K+ and Na+ ions in the tissue. The increases in K+, Na+ and sucrose concentrations could contribute to this osmotic adjustment as much as 12%, 28% and 31%, respectively. Altogether they contributed 71% of osmotic adjustment. Additional contributions might come from the ABA-induced synthesis of other solutes and certain proteins. The content of soluble proteins in ABA-treated protocorms increased by 39% (Table 1). The relative contribution of soluble proteins to the osmotic adjustment could not be estimated, because soluble proteins do not behave like simple solutes osmotically. However, the extent of osmotic adjustment (-0.20 MPa) was unlikely to be able to influence the drying rate of protocorms significantly and so any possible additional osmotic adjustment during drying was also investigated. Dried protocorms were rehydrated to the original water content for osmotic potential measurement. There was insignificant osmotic adjustment during drying in either control or ABA-pretreated protocorms (Table 1). Besides osmotic adjustment, compatible solutes may increase dehydration tolerance through other mechanisms. ABA pretreatment significantly increased the content of soluble sugars in the protocorms, especially sucrose. These carbohydrates were found to be a good protectant against dehydration stress in desiccation-tolerant plant tissues (Blackman et al., 1992; Sun et al., 1994, 1999; Pelah et al., 1997; Sun and Leopold, 1997). In an ongoing study, the preculture of protocorms in medium with a high sucrose content (10%, w/v) greatly increased the dehydration tolerance of protocorms. However, sucrose treatment did not affect the drying rate of protocorms (data not shown).
The effect of ABA pretreatment on protein synthesis in protocorms was investigated. The increase of soluble protein content in ABA-treated protocorms by 39% (Table 1) led to the hypothesis that certain proteins may modulate water loss in tissues during dehydration. Recently, Walters et al. reported that desiccation-related LEA proteins had strong water-binding capacity (Walters et al., 1997). ABA-pretreatment has been known to induce the synthesis of a variety of similar proteins (Ried and Walker-Simmons, 1990; Skriver and Mundy, 1990; Blackman et al., 1991, 1992; Chandler and Robertson, 1994). In the present study, it was observed that ABA pretreatment induced the synthesis of six new dehydrins, and resulted in a 130% increase in total dehydrin content over control protocorms (Fig. 7). It remains unclear how ABA-induced dehydrin reduces water loss in protocorms. Drying rate affected both dehydration tolerance and dehydrin synthesis (Fig. 7). The drying rate experiment on dehydrin synthesis in control and ABA-pretreated protocorms showed a significant correlation (r=-0.89, n=6) between critical water content and dehydrin content in dried protocorms. The good correlation, however, did not necessarily suggest a cause–effect relationship between the accumulation of dehydrin and dehydration tolerance.
In conclusion, the present study showed that dehydration tolerance of orchid protocorms was a function of drying rate. Slow drying and ABA pretreatment increased the dehydration tolerance of protocorms and the synthesis of dehydrin. The improvement of dehydration tolerance after ABA pretreatment was associated with the decrease in drying rate of protocorms.
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Acknowledgments |
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The research was supported by a grant from the National University of Singapore to WQS (RP-960366). We thank Dr Andreas Richter (University of Vienna) for his assistance in GC/MS identification of two carbohydrates, and Professor Timothy J Close (University of California at Riverside) for his generous gift of dehydrin antibody.
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Notes |
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1 Present address and to whom correspondence should be sent: LifeCell Corporation, One Millennium Way, Branchburg, NJ 08876, USA. Fax: +19089471085. E-mail: wsun{at}lifecell.com
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