Journal of Experimental Botany, Vol. 51, No. 344, pp. 635-643,
March 2000
© 2000 Oxford University Press
A study of water relations in neem (Azadirachta indica) seed that is characterized by complex storage behaviour
1 Centre National de Semences Forestières (CNSF), BP 2682, Ouagadougou, Burkina Faso
2 Department of Plant Sciences, Laboratory of Plant Physiology, Wageningen University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands
Received 13 May 1999; Accepted 28 October 1999
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Abstract |
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Neem (Azadirachta indica) seed is reputed to have limited tolerance to desiccation, to be sensitive to chilling and imbibitional stress, and to display intermediate storage behaviour. To understand this behaviour the properties of water in seed tissues were studied. Water sorption isotherms showed that at similar relative humidity (RH), the water content was consistently higher in axes than in cotyledons, mainly due to the elevated lipid content (51%) in the cotyledons. Using differential scanning calorimetry, melting transitions of water were observed at water contents higher than 0.14 g H2O g-1 DW in the cotyledons and 0.23 g H2O g-1 DW in the axes. Beside melting transitions of lipid, as verified by infrared spectroscopy, changes in heat capacity were observed which shifted with water content, indicative of glass-to-liquid transitions. State diagrams are given on the basis of the water content of seed tissues, and also on the basis of the RH at 20 °C. Longevity was considerably improved, and the sensitivity to chilling/subzero temperatures was reduced when axis and cotyledons were dehydrated to moisture contents
of approximately 0.05 g H2O g-1 DW. However, longevity during storage at very low water contents was limited. A possible mechanism for the loss of sensitivity to chilling/subzero temperatures at low water contents is discussed. The results suggest that dry neem seeds in the glassy state have great potential for extended storability, also at subzero temperatures. Key words: Azadirachta indica, chilling tolerance, desiccation tolerance, glassy state, seed storage.
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Introduction |
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Seed storage longevity depends on intrinsic properties of the species and on external factors during storage such as temperature, relative humidity (RH) and, to a lesser extent, composition of the gaseous atmosphere (Priestley, 1986
). To survive long-term storage in the dehydrated state, seeds have to be able to withstand desiccation to low water contents. A large group of so-called orthodox seeds have this ability, whereas another group of mainly tropical seeds, designated recalcitrant, are damaged during drying, often in the range of 0.4–1.0 g H2O g-1 DW (Hong et al., 1996). Empirical models on the basis of moisture content and temperature have been constructed for the storage behaviour of orthodox seeds, which can predict the viability of a seed lot over time at a broad range of different water contents and storage temperatures (Roberts, 1972; Justice and Bass, 1978; Ellis and Roberts, 1980).
Deterioration of orthodox seeds during storage is largely determined by the physical state and properties of water (see Walters, 1998, for a review). The physical state of water in seeds has been characterized by the motional and thermal properties of intracellular water on the basis of thermodynamic considerations. Degradative reactions may be controlled by the chemical potential of water and the availability of water for chemical reactions, which can be described by equilibrium thermodynamics (Vertucci and Roos, 1990, 1993). Also, water can be regarded in terms of viscous glasses. A glass is defined as a meta-stable state that resembles a solid, brittle material, but retains the disorder and physical properties of the liquid phase (Franks et al., 1991). The slow molecular diffusion in such glasses renders chemical reactions improbable, which is considered to enhance long-term stability substantially (Slade and Levine, 1991). There is considerable support for the existence of glasses in dry orthodox seeds (Williams and Leopold, 1989; Bruni and Leopold, 1991; Williams et al., 1993; Leopold et al., 1994; Sun et al., 1994). Seed metabolism is minimized in such a glass and storage longevity is maximized, particularly at low temperatures. By contrast, the non-glassy, liquid phase allows an accelerated physical and chemical deterioration of seeds (Sun and Leopold, 1993; Sun et al., 1994), probably as a result of an increased molecular mobility (Buitink et al., 1998).
For the recalcitrant seeds in which water properties appear not to be linked with desiccation tolerance (Pammenter et al., 1991, 1993) no predictive models have been proposed. The relatively high water content at which recalcitrant seeds start to lose viability is far above that at which a glassy state can exist at room temperature and non-freezable water is lost. Many recalcitrant tropical seeds are chilling-sensitive and rapidly lose viability even if stored at relatively high moisture contents. Due to the active metabolism in the hydrated state, viable recalcitrant seeds cannot be stored for long-term periods. The exact causes of recalcitrant seed storage behaviour remain to be ascertained (Vertucci and Farrant, 1995), although orthodox seeds feature typical changes in key compounds during the acquisition of desiccation tolerance that do not occur to the same extent in recalcitrant seeds, e.g. the increase in oligosaccharide content (Horbowicz and Obendorf, 1994; Steadman et al., 1996).
A third, intermediate class of seed behaviour has been recognized, which is characterized by an intermediate moisture limit below which the seeds cannot survive dehydration (Ellis et al., 1990, 1991). Seeds of neem, an economically important multipurpose tropical tree species, have limited tolerance to desiccation and do not store well for extended periods of time, e.g. years. Consequently, these seeds were considered as displaying intermediate behaviour (Gamené et al., 1996; Hong et al., 1996; Poulsen, 1996; Sacandé et al., 1996, 1997; Hong and Ellis, 1998), although their behaviour also has been categorized as orthodox (Tompsett and Kemp, 1996). In addition, the seeds are chilling-sensitive above 0.09 g H2O g-1 seed DW (Sacandé et al., 1998). At and below 0.09 g H2O g-1 DW, the seeds lose this chilling sensitivity, but become sensitive to imbibitional stress, which may partly explain their reduced tolerance to dehydration. The water contents at which neem seeds experience the above-mentioned problems is well in the range of water contents in which equilibrium thermodynamics of water and glasses can play a role. A study of seed-water relations in terms of cytoplasmic glassy state and water properties is therefore expected to give more insight in the unusual behaviour of intermediate neem seeds during dehydration and storage.
The present paper explores the properties of water in dehydrating neem seeds. Water sorption isotherms for excised embryonic axes and cotyledons were constructed, and glass transitions were studied from thermal events recorded by differential scanning calorimetry (DSC). The results are discussed in relation to desiccation tolerance and storage longevity.
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Materials and methods |
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Plant material
Fully mature neem (Azadirachta indica A. Juss.) seeds were harvested from yellow fruits in July 1997 (see Sacandé et al., 1997
, for the staging of seed development). The fruits were picked by hand from more than 25 trees in the Ouagadougou region (Burkina Faso). The preparation of the seeds was carried out locally on the day of harvesting. The fruits were soaked in water and rubbed with sand to depulp. The seeds were then cleaned with water and dried in the shade on a grid for 2 d. Seeds surrounded by an intact endocarp were selected and sent by air cargo to Wageningen, the Netherlands, in cotton bags, arriving a week after harvest.
Determination of moisture content and viability
Excised embryonic axes (5) and cubes (3 of approximately 3 mm3) of cotyledons were exposed to the atmospheres above different saturated salt solutions or P2O5 (<1% RH) in closed containers at 20 °C for at least 1 week (Vertucci and Roos, 1993) (ZnCl2 [5.5% RH], KOH [8% RH], LiCl [13% RH], K-acetate [25% RH], MgCl2 [32% RH], Ca(NO3)2 [53% RH], NaNO2 [64% RH], NaCl [75% RH], KCl [85% RH], and KNO3 [91% RH]). Samples taken from the containers were rapidly sealed in Perkin Elmer aluminium DSC pans that were weighed, then punctured and oven-dried at 96 °C for 48 h, and weighed again, allowing determination of the water content. Water contents were expressed on a dry weight basis.
Seeds were stored in open storage conditions at four different RHs, -10% (established in a commercial cabinet), and 32%, 53% and 75% RH (established above saturated salt solutions)—for 1 month or 1 year at 20 °C. Also, seeds were dehydrated above the saturated salt solutions for approximately 3 weeks to obtain a range of water contents. They were then packed into laminated aluminium foil packets that were hermetically sealed, and stored for an additional 3 months at a range of temperatures. Seeds were allowed to germinate at 30/20 °C (day/night) (according to Sacandé et al., 1998). Final germination was scored on the basis of radicle protrusion (2 cm; ISTA, 1993).
Total lipid analysis
Total lipids were extracted from the seed tissues with chloroform-methanol using the modified Folch's method (described in Hamilton et al., 1992), and the amounts were determined gravimetrically.
Fourier transform infrared spectroscopy (FTIR)
FTIR spectra were recorded on a Perkin-Elmer 1725 IR-spectrometer (Perkin-Elmer, Beaconsfield, Buckinghamshire, UK), equipped with a liquid nitrogen-cooled mercury/cadmium/telluride detector and a Perkin-Elmer microscope as described previously (Wolkers and Hoekstra, 1995). Oil pressed out of seed axes or cotyledons was placed between two diamond windows that were tightly mounted into a temperature-controlled brass cell. Temperature control of the sample in the instrument was with a computer-controlled device that activated a liquid nitrogen pump, in conjunction with a power supply for heating of the cell. The temperature of the sample was recorded using two PT-100 elements that were located close to the sample windows. The temperature dependence of the FTIR spectra was studied in the range between -60 °C and 60 °C, starting with the lowest temperature. Spectra were recorded every minute at temperature increments of 1.5 °C min-1. The instrument was purged of water vapour with a Balston dry air generator (Balston, Maidstone, Kent, England).
The spectral region between 3000 and 2800 cm-1 was selected and second derivative spectra were calculated [19 points smoothing factor; calculated using the Infrared Data Manager Analytical Software, version 3.5 (Perkin Elmer)]. The position of the symmetric CH2 stretching vibration band of the oil around 2852 cm-1 was determined from these second derivative spectra.
Differential scanning calorimetry
Melting transitions and changes in heat capacity of the samples were determined using a DSC (Pyris 1 DSC, Perkin-Elmer, Norwalk, CT, USA), calibrated for temperature with Indium (156.6 °C) and methylene chloride (-95 °C) standards and for energy with Indium (28.54 J g-1). Axes and cotyledons (5–10 mg) with different water contents were hermetically sealed in Perkin Elmer aluminium pans. After cooling the sample at 10 °C min-1 to -100 °C and keeping this temperature for 10 min, scans were recorded from -100 °C to 120 °C at a rate of 10 °C min-1. The enthalpy (
H) of the melting transition of water was determined from the area under the peak. Second order (glass) transitions were assessed as a sudden, stepwise increase in specific heat. The mid-point of the temperature range over which the change in specific heat occurred was used to determine the glass-to-liquid transition temperature (Tg). All thermograms were analysed using Pyris Software for Windows (Perkin-Elmer Thermal Analysis). Baselines were determined using an empty pan, and all thermograms were baseline-corrected. After recording the thermograms, water contents of the samples were determined as described above.
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Results |
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Water sorption isotherms
Water sorption isotherms of excised axes and cotyledon cubes at 20 °C are shown in Fig. 1
. Water contents were consistently higher in the axes than in the cotyledons for the same RH. Because lipids are inaccessible to water but, nevertheless, contribute to the dry weight, the difference in water absorption between axes and cotyledonary tissue might be explained by a possible difference in lipid content. Therefore, the amounts of lipids in both tissues were analysed. They were 51% and 14% for the cotyledons and the axes, respectively, indicating that the cotyledons contained 3.7 times more lipid than the axes. Recalculating the water content for zero amount of lipid showed that the cotyledons absorbed more water from the vapour phase under similar RH conditions than did the axes (dotted curves in Fig. 1).
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Desiccation tolerance and storage longevity
Fresh seeds (0.37 g H2O g-1 DW; 98% germination) were exposed to a range of RH conditions at 20 °C. After 1 month of storage the seeds were allowed to germinate (Fig. 2). Germination decreased with decreasing RH, to reach a value of 40% at 10% RH (0.038 g H2O g-1 DW seed). After 1 year of storage, only the seeds exposed to 75% RH completely lost their germination capacity, and the germination percentages of the seeds exposed to 53%, 32% and 10% RH decreased further, the 53% RH conditions giving the best survival.
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The effects of a range of temperatures during 3 months of hermetic storage on the germination of seeds having different water contents is shown in Fig. 3
. It is clear that the seeds are chilling sensitive at water contents above 0.048 g H2O g-1 embryo DW. In addition, the seeds of 0.048 H2O g-1 embryo DW resisted temperatures as low as -20 °C to a certain extent. Fresh seeds (0.493 g H2O g-1 embryo DW) had lost viability at all the temperatures tested. After 1 month under these conditions the fresh seeds had higher viability at 15 °C and 10 °C than at 5 °C (data not shown). After 1 year of storage, the seeds having embryo moisture contents of 0.074 or 0.099 g H2O g-1 DW completely lost viability at 5 °C and 20 °C, but with some survival at 10 °C and 15 °C (data not shown). Note that the moisture content of the seed is greatly influenced by the pericarp (45% of the seed dry weight), that has a high sorption of water. Thus, under conditions of equilibrium RH, the water content of the seed is considerably higher than that of the embryo.
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2-test).
Thermal transitions
Thermal transitions in neem axes and cotyledons of different water contents were determined by DSC from heating thermograms. Figure 4 shows a selection of endotherms of axes and cotyledons in which several transitions are noticeable. At high water contents, a broad endothermic peak was observed. The area and the temperature of this peak decreased with decreasing water contents, indicating that these peaks were water melting transitions. At high water contents a sharp peak was observed superimposed on the broad peak. This pattern indicates the melting transition of pure water probably arising from water vapour condensing inside the DSC pans. The onset temperatures of the broad melting transitions were considerably lower in the seed tissues than in pure water or dilute solutions, as previously found for other biological systems (Vertucci, 1990).
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The amount of unfrozen water can be calculated from the
H of the melting endotherms versus the water content relationships for the dehydrating axes and cotyledons (Fig. 5A, B). The areas under the peaks were determined from thermograms similar to those in Fig. 4. Two linear parts in the plots were apparent. The intersection with the horizontal line indicates the water content below which there is only unfrozen water left. These water contents were 0.23 and 0.14 g H2O g-1 DW for axes and cotyledons, respectively. Correction for zero oil revealed that the critical water content was almost similar in both tissues (0.27–0.29 g H2O g-1 DW). The H g-1 H2O of the melting transitions, calculated from the slopes, was approximately 300 J g-1 H2O for both axes and cotyledons. The lines drawn represent the least-squares best fit (r2=0.998 for all regressions). The transition enthalpies of the axes and cotyledons below these critical water contents remained approximately 15 J g-1 H2O (Fig. 5A) and 30 J g-1 H2O (Fig. 5B), respectively. These enthalpies are due to the melting of lipids as outlined next.
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Below the water content at which freezable water could not be observed, some first order thermal events were still visible (Fig. 4). These events occurred at the same temperature irrespective of how far the tissues were dehydrated. A detailed thermogram is shown in Fig. 6A
for axes and in Fig. 7A for cotyledons. The nature of the compounds causing these thermal events was verified by FTIR analysis of the symmetric CH2 stretching band of the oil that was pressed out of the specimens. Wave number versus temperature plots are presented in Figs 6B and 7B for axes and cotyledons, respectively. The peaks in the thermograms corresponded well with the discrete shifts in these plots, indicating that the thermal events seen in the thermograms of the dry specimens are indeed due to lipid melting.
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Beside first order lipid transitions, other features are visible in thermograms of cotyledons having different water contents (Fig. 8
). At low water contents, a shift in the baseline was observed above 40 °C, indicating a second order transition. The temperature at which the mid-point of this transition occurred shifted to lower values with increasing water contents. Such a shift with water content is indicative that the transition observed is caused by melting of a glass (Leprince and Walters-Vertucci, 1995). In samples with elevated water contents the glass-to-liquid transition (Tg) was detected below -50 °C. Similar shifts were observed in scans of the axes (data not shown). In the thermograms of cotyledons with water contents below 0.04 g H2O g-1 DW, a small first order transition of unknown origin was present around 30 °C, possibly due to the melting of a lipid (marked with an asterisk in Fig. 8). The heating thermogram of cotyledons containing 0.146 g H2O g-1 DW shows some additional features, that succeed the melting of the glassy state. The exothermic event, marked d, is indicative of a devitrification event (Leprince and Walters-Vertucci, 1995). Just after this devitrification event, a first order transition due to the melting of water is apparent (w).
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The mid-points of the temperature range over which the change in specific heat occurred for a range of water contents in both axes and cotyledons are presented in Fig. 9
. Tg increased with decreasing water content. The values of the state diagram of the axes were higher than those of the cotyledons. Between approximately -20 °C and +30 °C, Tg was undetectable because of interference with the lipid melting. With drying, Tg reached values as high as 70 °C at a moisture content of approximately 0.025 g H2O g-1 DW. In the case of samples having water contents of >0.3 g H2O g-1 DW, a second glass transition was observed at a constant temperature of -45 °C (thermograms not shown), following a devitrification event, representing Tg' (melting of the maximally freeze-concentrated cytoplasmic solute; Leprince and Walters-Vertucci, 1995). The phase diagram of water in the cotyledons also is presented in Fig. 9. The relationship of Tg versus RH at 20 °C is shown in the inset to Fig. 9. To this end, the fitted state diagrams of Fig. 9 were combined, with the fitted sorption curves of Fig. 1, with the curves shown as the result of the calculations.
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Discussion |
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Neem seeds are reputed to have limited desiccation tolerance and a relatively short storage longevity (Gamené et al., 1996
; Hong and Ellis, 1998; Sacandé et al., 1996; 1998). Because they can tolerate an equilibrium RH of 10% (Fig. 2) and storage at -20 °C (Fig. 3), albeit with a considerable decrease in germination percentage, their behaviour has orthodox features rather than typically recalcitrant or intermediate characteristics. Although the general opinion is that neem seed behaviour is intermediate, it also has been categorized as orthodox (Tompsett and Kemp, 1996). Nevertheless, stored seeds are difficult to handle, because they are sensitive to chilling/subzero temperatures above 0.048 g H2O g-1 embryo DW (Fig. 3) and extremely sensitive to imbibitional damage when dry (Sacandé et al., 1998). These characteristics may have contributed to the complex storage behaviour.
The water sorption isotherms of cotyledons and embryonic axes of neem (Fig. 1) had the characteristic sigmoidal shape as found previously for other seeds (Vertucci and Leopold, 1984). The shoulder of the isotherms has been associated with the presence of structural water (Chung and Pfost, 1967; Mahama and Silvy, 1982; Vertucci and Leopold, 1987). The lower equilibrium moisture contents of the cotyledons compared with those of the axes cannot be solely explained on account of differences in lipid content. After correction to zero oil content, the cotyledons had a higher equilibrium moisture content than the axes (Fig. 1, dashed curves).
The thermograms (made by DSC) obtained with neem axes and cotyledons were comparable to those found for other seeds or seed tissues (Vertucci, 1990; Pammenter et al., 1991; Leprince and Walters-Vertucci, 1995). The non-frozen water limit in axes and cotyledons was 0.23 g H2O g-1 DW and 0.14 g H2O g-1 DW, respectively (Fig. 4). Correction to zero lipid contents elevated these values to 0.27 and 0.29 g H2O g-1 DW, respectively, which are in the range reported for other seed tissues, both desiccation tolerant or intolerant (Vertucci, 1990; Pammenter et al., 1993).
Neem seeds have been successfully cryopreserved at liquid nitrogen temperature when they were first dried to 0.09 g H2O g-1 DW for the entire seed and 0.23 g H2O g-1 DW for isolated axis (Berjak and Dumet, 1996). It was shown that isolated embryos with a water content 
0.37 g H2O g-1 DW died when exposed to liquid nitrogen, but that they survived to a high degree when desiccated to 0.13 or 0.04 g H2O g-1 DW (Chaudhury and Chandel, 1991). These water contents are in the range at which no freezable water is expected (Fig. 5), given the fact that the intact embryo mainly consists of cotyledon tissue (99.5%). Even with some freezable water present in the tissue, liquid nitrogen storage may be successful if freezing rates are fast enough to avoid intercellular ice formation (Vertucci, 1989; Wesley-Smith et al., 1992).
Beside melting transitions of water, the thermograms showed first order transitions that could be attributed to different classes of lipids, as evidenced by temperature-dependent FTIR data of the symmetric CH2 stretch of the neutral lipids pressed out of the specimens (Figs 6, 7). Inspection of the thermograms (Fig. 8) further revealed second order transitions that shifted with water content, suggestive of the presence of cytoplasmic glasses (Williams and Leopold, 1989; Leopold et al., 1994; Leprince and Walters-Vertucci, 1995). The presence of glasses has been suggested to reduce ageing rates of stored seeds (Leopold et al., 1994; Sun, 1997), and this suggestion has been verified for pollen (Buitink et al., 1998).
From the mid-Tg values obtained at different water contents, state diagrams were constructed (Fig. 9). The curve separating the glassy state from the liquid phase for the axes was higher than that for the cotyledons. With such curves at hand, one could elaborate on the possible significance of water relations with respect to long-term storage and chilling/subzero sensitivity of neem or other intermediate seeds. An interesting finding was the loss of the sensitivity to chilling and subzero temperatures of seeds that were stored at embryo water contents of 0.048 g H2O g-1 DW (Fig. 3). An indication that dehydration can alleviate chilling stress could be derived from the storage data on neem seeds in Hong and Ellis (Hong and Ellis, 1998). Inspection of the state/phase diagrams in Fig. 9 revealed that both the cotyledons and the axes were in the glassy state under these conditions. The question is whether the glass might be involved in the loss of this chilling sensitivity. To answer such a question, the possible cause of chilling sensitivity must be considered first. In tropical plant species the phase transition temperature of membranes has been shown to occur around 10–15 °C, which is considerably above that of chilling-tolerant plant species (Crowe et al., 1989). Cooling below the phase transition temperature leads to gel phase, often followed by phase separations (Platt-Aloia and Thomson, 1987). The last phenomenon, in particular, is not easily restored upon return to temperatures above the phase transition and may lead to extensive leakage. Also in neem seedlings, the membranes have an elevated transition temperature (10 °C; Sacandé, unpublished results). An explanation for the loss of sensitivity to chilling/subzero temperatures 0.048 g H2O g-1 embryo DW could be that the transition temperature was depressed to below that of the hydrated control, or that phase separations did not occur. The first possibility is not very likely, since this has never been observed in vivo in other dry systems (Hoekstra and Golovina, 1999). However, when, at low water contents, molecules other than water (e.g. sugars) are hydrogen bonded to the membrane components and at the same time to the glass, lateral diffusion of membrane components might be restricted. Thus, phase separations may be prevented, and chilling/subzero sensitivity may be abolished.
To enable predictions on favourable storage conditions when seeds are equilibrated at certain RH, curves were constructed of Tg versus RH at 20 °C (Fig. 9, inset). At 20 °C and 75% RH, for example, both the cotyledons and the axes are expected to be out of the glassy state. Under these conditions, seed longevity is limited to a maximum of 6 months (Sacandé et al., 1998; Fig. 2). A considerable extension of the storage longevity (at least 2 years) is reached when both tissues are in the glassy state, e.g. around 50% RH at 20 °C. This also applies to seeds stored at an even lower RH (Fig. 2). The amount of water in these samples keeps the cytoplasm below Tg, but above the level at which the structural stability of the cells would be compromised. The critical water content below which the seeds will not germinate is difficult to determine, because such dry seeds are extremely sensitive to imbibitional stress. This stress can at least partly be alleviated by imbibition at elevated temperatures (25–35 °C) (Sacandé et al., 1998).
In conclusion, the significant loss of viability upon drying and cold storage (this paper; Sacandé et al., 1998) indicates that neem seed still poses more problems than an orthodox seed type would do. With the use of state/phase diagrams as shown in Fig. 9, a better insight was obtained in the peculiar behaviour of neem seeds. Correlating the germination results in Figs 2 and 3 with the state diagrams in Fig. 9 suggests that cautious manipulation may enable seed lots of neem to survive reasonably for a number of years when the seeds are sufficiently dehydrated and in the glassy state. Thus, characterization of water properties in neem seeds illustrates that there is a potential for development of efficient methods for storage of tropical seeds displaying intermediate storage behaviour.
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Acknowledgments |
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The financial support by the Dutch Organization for the Advancement of Tropical Research (WOTRO) is gratefully acknowledged. We wish to thank Dr Olivier Leprince and two anonymous reviewers for their critical comments on the manuscript.
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Notes |
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3 To whom correspondence should be addressed in The Netherlands. Fax: +31 317 484740. E-mail:moctar.sacande{at}algem.pf.wau.nl
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