Osmotic and ionic effects of NaCl on germination, early seedling ...

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Osmotic and ionic effects of NaCl on germination, early seedling growth, and ion content of Atriplex halimus (Chenopodiaceae) Mohammed Bajji, Jean-Marie Kinet, and Stanley Lutts

Abstract: The effects of salt and osmotic stresses on the germination processes in seeds of the perennial halophyte species Atriplex halimus L. were compared using iso-osmotic concentrations of NaCl and mannitol. The lowest stress intensity delayed germination, while higher doses of NaCl and mannitol reduced final germination percentages. No significant difference occurred between the effects of these solutes on germination percentages or seedling dry weights. At an external osmotic potential of –0.7 MPa, however, the water content of mannitol-treated seedlings was reduced compared to that of seedlings that developed from NaCl-exposed seeds. The K, Mg, and Pi content decreased in seedlings that developed from mannitol-treated seeds while calcium concentration was strongly reduced in those arising from NaCl-treated seeds. Inhibited seeds were able to germinate at levels similar to those of the control after rinsing in deionized water and imbibition in control conditions. Seedlings produced from NaCl pre-treated seeds had a lower Ca and a higher Na content than control seedlings. The effect of salinity on the germination phase of development is mainly due to its osmotic component, and inhibition of germination is reversible. Both salt and osmotic stresses may have an impact on the mobilization of minerals from the seeds to the young seedling, but this effect does not have any consequence on growth processes analysed on a short-term basis. Key words: Atriplex halimus, halophyte, osmotic stress, recovery of seed germination, salinity. Résumé : Les effets du sel et d’un stress osmotique sur la germination de graines de l’espèce halophyte vivace Atriplex halimus L. ont été comparés en utilisant des concentrations iso-osmotiques de NaCl et de mannitol. La plus faible intensité de stress induit un retard de germination, alors que de plus fortes intensités réduisent le pourcentage final de germination. Aucune différence n’est observée entre les effets des deux solutés sur le pourcentage de germination ou le poids sec des jeunes plantules. À un potentiel osmotique externe de –0,7 MPa, cependant, les teneurs en eau des plantules issues de graines traitées au mannitol sont plus faibles que celle des plantules issues de graines exposées au NaCl. Le contenu en K, Mg et Pi diminue au niveau de plantules issues de graines exposées au mannitol, alors que les teneurs en Ca sont réduites chez les plantules issues de graines exposées au NaCl. Les graines inhibées sont capables de germer après rinçage à l’eau déminéralisée et imbibition en conditions « témoin ». Les plantules issues de graines pré-traitées au NaCl ont une teneur plus faible en Ca et plus élevée en Na. Les effets de la salinité sur la germination sont principalement dus à la composante osmotique de ce stress et l’inhibition de la germination est réversible. Les stress salin et osmotique peuvent avoir un impact sur la mobilisation des éléments minéraux de la graine vers les jeunes plantules mais ces effets n’ont pas de conséquence à court terme sur les processus de croissance. Mots clés : Atriplex halimus, halophyte, stress osmotique, recouvrement de la germinabilité des graines, salinité. [Traduit par la Rédaction]

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Introduction Atriplex halimus L. is a perennial shrub that grows throughout the Mediterranean region, in the Middle East, northern Africa, and southern Europe. Some saltbushes of the genus Atriplex produce good-quality forage on degraded salt lands. Atriplex halimus has a low leaf oxalate content (Ellern et al. 1974) associated with a high protein concentraReceived 13 July 2001. Published on the NRC Research Press Web site at http://canjbot.nrc.ca on 15 March 2002. M. Bajji, J.-M. Kinet, and S. Lutts.1 Université Catholique de Louvain, Unité de Botanique générale, Laboratoire de Cytogénétique, Place Croix-du-Sud 5 (bte 13), B-1348 Louvain-la-Neuve, Belgium. 1

Corresponding author (e-mail: [email protected]).

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tion (El-Shatnawi and Mohawesh 2000). As a consequence, A. halimus could be considered as a promising species for the reclamation of degraded lands where excessive salinity and low moisture level are the main factors limiting plant growth but where there is also a need to provide animals with forage, especially in critical drought periods. In field conditions, plantations of A. halimus are usually established from plants produced from cuttings and multiplied in nurseries (Bouzid and Papanastasis 1994). This method is expensive and time consuming compared with direct sowing. However, despite the fact that some agricultural practices have been suggested to improve its efficiency (Bouzid and Papanastasis 1996), direct sowing cannot be recommended, because several uncontrolled factors may drastically reduce germination percentages in natural environments. This has been mainly investigated in other species of Atriplex, such as Atriplex patula (Ungar 1996), Atriplex

DOI: 10.1139/B02-008

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prostrata (Keiffer and Ungar 1997; Katembe et al. 1998; Egan and Ungar 1999), Atriplex triangularis (Khan and Ungar 1984), Atriplex nummularia (Campbell and Matthewson 1992; Peluc and Parera 2000), Atriplex griffithii (Khan and Rizvi 1994; Khan and Ungar 1997; Khan et al. 2000; Ungar and Khan 2001), and Atriplex cordobensis (Giusti and Grau 1983), but data concerning A. halimus are surprisingly scarce (Osman and Ghassali 1997). Germination is a crucial stage in the life history of plants, and salt tolerance during germination is critical for the establishment of plants that grow in saline soils. Even if growth appears slightly stimulated by moderate NaCl doses in some halophyte species, especially in the genus Atriplex (Bajji et al. 1998; Koheil et al. 1992; Matoh et al. 1986; Khan et al. 2000), seeds of most halophytes attain their maximum germination in the absence of NaCl and are very sensitive to elevated salinity at the germination and early establishment phases (Ungar 1978; Khan and Ungar 1984; Foderaro and Ungar 1997; Khan and Ungar 1996). Salinity may affect germination through an osmotic component that compromises water uptake and through an ionic component linked to the accumulation of Na and Cl. Thus, discrimination between the relative importance of both components is an absolute prerequisite for a better understanding of salt stress effects on germination processes. Another weakness of most studies dealing with the impact of salinity upon the initial stages of plant’s life is that they mainly consider germination percentages and rarely pay attention to the nutritional status of the young seedlings during the early phases of growth. Saline habitats experience moisture and soil salinity fluctuations throughout the year. In these environments, seed germination occurs during a season of high precipitation, when soil salinity is reduced (Khan and Rizvi 1994; Pujol et al. 2000). It may thus be hypothesized that the ability of the seed bank to remain quiescent during exposure to high salt level and to germinate just immediately after a reduction of salinity may be an important aspect for the ability of halophyte species to efficiently colonize their environment. Evaluating the effect of a pre-incubation in stress conditions on the subsequent behavior of young seedlings growing in control conditions thus appears to be crucial but, once again, has not been frequently considered. The objectives of the present study are (ii) to compare the effect of salt (NaCl) and iso-osmotic concentrations of mannitol on germination processes in A. halimus, (ii) to quantify ion content in relation to growth responses of young seedlings exposed to stress conditions, and (iii) to analyze these parameters on seeds allowed to germinate in control conditions after a pre-incubation in a stressful media.

and 5% (w/v) calcium hypochlorite for 20 min. They were then rinsed three times with sterile deionized water. All germination tests were carried out in 9-cm Petri dishes lined with two sheets of filter paper (Whatman No. 41) imbibed with 4 mL of sterile test solution and sealed with parafilm to avoid loss of water. NaCl and mannitol were used at four iso-osmotic concentrations corresponding to osmotic potentials (Ψs) of 0 (control), –0.7 (150 mM NaCl and 268 mM mannitol), –1.4 (300 mM NaCl and 505 mM mannitol), –2.1 (450 mM NaCl and 745 mM mannitol), and –2.9 MPa (600 mM NaCl and 950 mM mannitol), estimated by a vapor pressure osmometer (Wescor 5500). For the stress period, six replicates of 40 seeds per treatment were incubated at 25°C in the dark (Martínez 2001), and the number of germinated seeds was determined at 12-h intervals. Germination was scored when a 2-mm radicle had emerged from the seed coat. After 120 h, final germination percentages were recorded and seedling fresh weights (FW) immediately determined. To determine the impact of the stress treatment on mineral mobilization from the endosperm, all seedlings were separated from the remaining seeds (seed coat and remaining endosperm). Seedling dry weights (DW) were evaluated after 48 h in an oven at 80°C. The FW and DW thereafter served to estimate seedling water content (WC) on a FW basis according to the formula (FW – DW)/FW. Cations (Na, K, Mg, Fe, and Ca) and Pi seedling contents were estimated by an inductively coupled argon plasma emission spectrophotometer (Jobin-Yvon JY 48) after digestion of 15 mg dry matter with 35% nitric acid. For the recovery period, ungerminated seeds were removed from the Petri dishes, rinsed three times (10 min each) with sterile deionized water, and put into new 9-cm Petri dishes lined with two sheets of filter paper (Whatman No 41) imbibed with 4 mL of sterile deionized water. The final germination percentage of these seeds and their seedling growth (FW, DW, and WC) and ion content were determined, as for the stress period, after 120 h as mentioned above. The recovery percentages were determined by the following formula: (a – b)/(c – b) × 100, where a is the total number of seeds germinated after transfer to distilled water, b is the total number of seeds germinated in stress conditions, and c is the total number of sown seeds. Germination, seedling DW, WC, and ion content data were analyzed using analysis of variance (ANOVA II). Percent germination data and water content were arcsine transformed before statistical analysis to ensure homogeneity of variance. The experiment was repeated three times with similar trends. Results presented hereafter are from one single experiment.

Materials and methods

Results

Atriplex halimus fruits (seeds with enclosing bracts) were harvested in 1995 from wild plants in the Kairouan region, Tunisia. They were collected randomly from the whole population to get an adequate representation of genetic diversity. Fruits were stored in plastic jars at room temperature and used for germination studies. After removal of the bracts, the seeds were surface sterilized for 30 s in 97% ethanol, followed by treatments in 0.8% (v/v) formaldehyde for 40 min

Germination was rapid with almost all seeds germinating in control conditions (Fig. 1). It was similarly affected by NaCl and mannitol. At the lowest stress intensity (–0.7 MPa), germination was delayed but statistically reached similar final percentages to those recorded in controls. At higher stress intensities, final germination percentages were drastically reduced. In both NaCl and mannitol treatments, they were lower than 10% in response to an external Ψ s of –2.1 and –2.9 MPa. © 2002 NRC Canada



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40

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0 0

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120 0

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72

96

120

Time (h)

Fig. 2. Final germination percentages (mean ± SE) of seeds of Atriplex halimus exposed to iso-osmotic concentrations of NaCl and mannitol (Ψs = 0 (control), –0.7, –1.4, –2.1, and –2.9 MPa). Germination was recorded after 120 h of treatment (n (number of batches per treatment) = 4, 40 seeds per batch). Ungerminated seeds in the most stressful conditions were then rinsed in distilled water and allowed to germinate in control conditions during a recovery period of 120 h. During this recovery period, the number of seeds in each batch varied depending on the germination percentages during the previous stress treatment.

Recovery

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Osmotic potential (MPa) In both cases, however, this inhibition was reversible, since ungerminated seeds rinsed in deionized water and then incubated in control conditions were able to germinate at a level similar to that of control seeds (Fig. 2). Moreover, germina-

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Fig. 3. Fresh weight of young seedlings (mean ± SE) of Atriplex halimus exposed to iso-osmotic concentrations of NaCl and mannitol (Ψs = 0 (control), –0.7, –1.4, –2.1, and –2.9 MPa). For the stress period, values were recorded 120 h after the beginning of seeds exposure to test solutions. For the recovery period, ungerminated seeds during the previous period were then rinsed in distilled water and allowed to germinate in control conditions for 120 h. The number of seedlings in each treatment varied depending on the germination percentages.

0 -0.7 -1.4

Fig. 1. Percent germination (mean ± SE), as a function of time, of Atriplex halimus seeds exposed to iso-osmotic solutions of NaCl and mannitol. Osmotic potential of solutions were 0 (control), –0.7, –1.4, –2.1, and –2.9 MPa.

Osmotic potential (MPa) tion kinetics of these seeds during the recovery period was similar to that of control seeds (results not shown). At the end of the stress period, seedling fresh weight could not be determined in response to an external Ψs of –2.1 and –2.9 MPa, because of the dramatic reduction in germination percentages. In response to an external Ψ s of –0.7 MPa, seedling fresh weight was not reduced in the NaCl treatment but decreased by more than 55% in response to an iso-osmotic dose of mannitol (Fig. 3). When Ψ s was decreased to –1.4 MPa, seedling fresh weight was reduced in both treatments but still remained significantly higher in the presence of NaCl than in the presence of mannitol. Such differences in fresh weights were due mainly to strong variations in the water content which, for example, was not affected at –0.7 MPa in the NaCl treatment compared with controls, while in mannitol-treated seedlings, it was drastically reduced (Table 1). No significant difference was recorded in the dry weight of seedlings exposed to either NaCl or mannitol; mean values in response to –0.7 MPa were 0.315 ± 0.024 mg and 0.303 ± 0.017 mg for NaCl and mannitol treatment, respectively, and in response to –1.4 MPa, values of 0.293 ± 0.014 mg and 0.297 ± 0.027 mg, respectively, were recorded. Seedlings that developed from stressinhibited seeds, which germinated after rinsing in unstressed conditions, exhibited growth responses similar to those of untreated controls, indicating that the stressinduced inhibition of germination was fully reversible. © 2002 NRC Canada



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Table 1. Water content (in %) of young seedlings (mean ± SE) of Atriplex halimus exposed to iso-osmotic concentrations of NaCl and mannitol (Ψs = 0 (control), –0.7, –1.4, –2.1, and –2.9 MPa). Ψs (MPa) Stress period 0 –0.7 –1.4 –2.1 –2.9 Recovery period –1.4 to >0 –2.1 to >0 –2.9 to >0

NaCl

Mannitol

93.9a 93.8a 85.8b nd nd

93.8a 83.1b 75.5c nd nd

92.9a 93.6a 93.6a

93.1a 93.4a 93.3a

Note: For the stress period, values were recorded 120 h after beginning the exposure of seeds to test solutions. For the recovery period, ungerminated seeds during the previous period were then rinsed in distilled water and allowed to germinate in control conditions for 120 h. The number of seedlings in each treatment varied depending on the germination percentages. Means followed by the same letter are not significantly different at P = 0.05. nd, not determined because of drastic inhibition of germination.

Mineral concentrations of seedlings analysed at the end of the stress and recovery periods are shown in Fig. 4. The sodium content of young seedlings expressed on a dry weight basis increased in response to NaCl treatments but was unexpectedly higher in the presence of 150 mM NaCl (–0.7 MPa) compared with 300 mM NaCl (–1.4 MPa). It is noteworthy that seedlings produced from inhibited seeds, which germinate after the stress relief, contained a higher amount of sodium than the controls. Potassium content appeared slightly decreased only in the response to the highest dose of mannitol. Pi and Mg concentrations decreased in stressed seedlings, the deleterious effects of mannitol being slightly more important than those of iso-osmotic concentrations of NaCl. Seedlings analysed at the end of the recovery period in control conditions and, thus, produced from seeds previously exposed to both stressing agents had a lower Pi content than those obtained from non-treated seeds. This is not the case for the Mg level, which was similar to that of controls. An important difference between the two stressing agents was observed for seedling Ca content: this element strongly decreased in seedlings obtained directly from NaCl-treated seeds (especially at the lowest NaCl dose). The Ca content decreased to a lesser extent in mannitoltreated seedlings. The Ca concentration of seedlings analyzed at the end of the recovery period was significantly influenced by the nature of the stressing agent previously used during stress exposure: seedlings obtained from NaCltreated seeds exhibited a lower concentration of Ca than controls, while those produced from mannitol-treated seeds presented a significantly higher content than nontreated material, except when the highest stress intensity (–2.9 MPa) was used for pre-treatment. Similarly, while mannitol treatment reduced iron content of young seedlings, a higher concentration of this element was found in seedlings that developed from mannitol pretreated seeds than in controls.

Discussion Halophyte species may have very different demands in terms of environmental conditions for optimal germination processes. Alternating temperatures clearly stimulate germination in Atriplex griffithii, Atriplex glabriuscula, and Atriplex lancinata (Ignaciuk and Lee 1980; Khan and Ungar 1984; Khan and Rizvi 1994). Light is another important regulatory environmental signal in the germination of many halophytes. Khan and Weber (1986) found that germination responses of Salicornia pacifica were adversely affected in the dark. Light also stimulates germination of Haloxylon recuvrum and Triglochin maritima (Khan and Ungar 1997). In the present work, we observed that A. halimus germinated to almost 100% in the dark and in constant temperature, thus showing that seeds from this species have no particular requirement in terms of thermoperiod or light intensity. Inhibition of seed germination in the genus Atriplex is a well-known phenomenon often attributed to the presence of saponin or other inhibitory compounds such as abscisic acid (Giusti and Grau 1983). According to several authors, the presence of high amounts of soluble salts in the bracts may be another cause of inhibition of germination in natural conditions (Silcock et al. 1990; Campbell and Mathewson 1992; Ungar and Khan 2001). In A. nummularia, bracteoles may accumulate up to 11.2% of NaCl (Uchiyama 1987), and this concentration was reported to be as high as 31% in Atriplex inflata Muell (Beadle 1952). According to Ungar and Khan (2001), soluble substances present in bracts may lead to an osmotic potential as low as –4.8 MPa. In the present study, we show that seeds with bracts removed germinated to almost 100%, thus demonstrating that there is no mechanical inhibition of germination linked to seed coat impermeability in A. halimus, in contrast to what has been previously reported in Atriplex repanda (Lailhacar and Laude 1975), Atriplex cordobenis (Aiazzi and Arguëllo 1992) or A. nummularia (Peluc and Parera 2000). Salinity stress had a direct negative impact on the germination of seeds from which the bracts were removed. Although a moderate NaCl dose (150 mM) has a positive effect on seedling growth (Bajji et al. 1998), this was clearly not the case for germination, which was delayed in the presence of this salt concentration, although not affecting the final germination percentage (Fig. 1). Higher doses of NaCl clearly reduced the final germination percentage. Several authors estimate that, in species belonging to the genus Atriplex (Ignaciuk and Lee 1980) as well as in other halophyte species (see Ungar 1978 and references therein), the osmotic component of salt stress is the main factor responsible for the inhibition of germination. During seed imbibition, a first phase is associated with water movement into the apoplasm: this physical process is a function of a matrix potential and is not a direct function of external osmotic potential value. During a second phase, water moves through plasma membranes, and this flux depends on the difference in Ψ s between the seed cells and the external medium. Both Na and Cl may cross membranes and then contribute to water uptake by decreasing the internal osmotic potential (Dodd and Donovan 1999; Almansouri et al. 2001) in contrast to polyethylene glycol, which has been used in © 2002 NRC Canada



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Fig. 4. Mineral (Na, K, Pi, Mg, Ca, Fe) concentration of young seedlings (mean ± SE) of Atriplex halimus exposed to iso-osmotic concentrations of NaCl and mannitol (Ψs = 0 (control), –0.7, –1.4, –2.1, and –2.9 MPa). For the stress period, values were recorded 120 h after the beginning of seeds exposure to test solutions. For the recovery period, ungerminated seeds during the previous period were then rinsed in distilled water and allowed to germinate in control conditions during 120 h. The number of seedlings in each treatment varied depending on the germination percentages. DW, dry weight. 3500

NaCl

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Fe (µmol/g DW)

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-1.4 -> 0 -2.1 -> 0 -2.9 -> 0

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numerous studies devoted to water stress effects (PérezMolphe-Balch et al. 1996; Tobe et al. 2000; Almansouri et al. 2001) and is a nonpenetrating solute that drastically inhibits water uptake. To determine the specific effect of ion toxicity of NaCl, the comparative use of mannitol may be of special interest; since it slowly penetrates the tissues, it can mimic the salt contribution to internal osmotic potential decrease, but it has no toxic effect and is not metabolized by tissues, at least in chenopods. The fact that, in terms of germination percentages, mannitol and NaCl had the same effect supports the hypothesis that ion toxicity was not the main cause of germination inhibition in our work. However, a contrasting effect of the two compounds was recorded during the subsequent stage of early seedling growth at an external Ψs of –0.7 MPa. Indeed, at this stress intensity, mannitol was more detrimental than NaCl, but it is interesting to note that deleterious mannitol effect influenced only the water status and not the dry weight of young seedlings. For higher doses, we hypothesize that the proportion of osmoticum penetrating in germinating structures is not sufficient to ensure seed imbibition in a condition of a very low external Ψs. This would explain why the water content of seedlings was strongly reduced at –1.4 MPa and why germination was almost completely inhibited at higher doses. During germination, reserves within the storage tissues of the seeds are mobilized by hydrolytic enzymes, such as amylases, lipases, and proteases, to support seedling growth (Lai et al. 1995). It is clear, however, that minerals present in the seeds may also be mobilized to cover metabolic requirements during the early stages of seedling growth, when root hairs of expanding radicles are not yet functional and when there is not yet a transpiration stream to help in ion translocation to aerial parts. In our work, the use of deionized water as control may appear as an oversimplification of what really occurs in normal soil conditions, but it allows us to determine the impact of the stress treatment on mineral mobilization from the endosperm to the developing seedling. Indeed, except for Na (and Cl, which was not quantified in the present study), all elements present in seedlings arise from seed mobilization. It is interesting to note that Na concentration of young seedlings produced from seeds exposed to 300 mM NaCl (–1.4 MPa) is lower than that of seedlings exposed to 150 mM (–0.7 MPa). The underlying cause for such a difference is not clear. Considering the difference in the final germination percentages (Fig. 2) as well as the very high intraspecific variability occurring in A. halimus, we may not exclude that a selection process occurred at –1.4 MPa and that only seeds that were able to limit sodium uptake during imbibition were able to germinate. The dry weight of the seedlings, however, was the same in the two cases (–0.7 and –1.4 MPa) suggesting once again that, although it penetrates into the seeds and subsequently in seedlings, Na by itself had no direct injury effects and that its influence on germination should be regarded in terms of its osmotic effect. Thus, the situation in this halophyte species may be quite different from what occurs in a glycophyte species such as Triticum durum Desf. where NaCl is more detrimental to early seedling growth than iso-osmotic concentrations of mannitol and where salt stress affects germination through its ionic component (Almansouri et al. 2001).

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However, NaCl drastically reduced the Ca content of young seedlings, and this effect was statistically higher than the effect observed in mannitol-treated material (Fig. 4). Calcium is a very important element for plant metabolism, and it is specifically involved in adaptative responses to salt stress (Bressan et al. 1998). Keiser et al. (1995) demonstrated that inadequate seed Ca concentrations are associated with reduced seed quality in soybean (Glycine max) in relation to an increased membrane permeability leading to an important sugar leakage and compromising early seedling growth. In our experiment, the salt-induced decrease in Ca had no impact on growing processes of young seedlings; however, we used only a very short-term exposure, and we may not exclude that the physiological and biochemical consequences of a Ca deficiency would have appeared in further stages of growth. The composition of soil solution in natural field conditions may undoubtedly somewhat mitigate the effect of a poor Ca mobilization to elongating tissues as shown by Keiser et al. (1995). An opposite trend was observed for K, which decreased in response to mannitol but remained unaffected in the presence of NaCl. It is interesting, however, to show that the mineral status of the young seedlings produced from pre-treated seeds and analysed at the end of the recovery period may be very different from control seedlings. This is true for Na, suggesting that the rinsing procedure did not fully eliminate Na that might have penetrated into the seeds during the incubation period. It may also be hypothesized (and still need to be demonstrated in the future) that Na had crossed the membranes and had a symplastic localization compromising its complete removal during rinsing. In contrast, K, Ca, Mg and Fe were higher in seedlings developing from mannitol pre-treated seeds than in controls, while Pi was lower. The underlying reasons for an increase in mineral content of seedlings after pre-exposure are unknown. A lower Ca concentration was recorded in seedlings after a NaCl pre-treatment. Such an effect may result from an increase in membrane permeability during pretreatment leading to electrolyte leakage, which may differ from an element to another (Keiser et al. 1995; Bajji et al. 2001). In our short-term experiment, however, no detrimental effects on early seedling growth seems to result from the modification of mineral status. Inhibition of germination at high NaCl dose may have an important ecological significance; germination is not possible in the summer time, when evaporation at the substrate surface leads to very high Na concentrations, which would hamper seedling growth. In contrast, germination would be possible in spring or autumn after rainfall. Several Atriplex species, such as A. patula and A. prostrata, exhibit an obvious seed dimorphism, since they produce either heavy brown seeds or small dark seeds (Khan and Ungar 1984; Katembe et al. 1998). The former seeds are able to take up water rapidly, have a limited dormancy, and are less sensitive to NaCl, while the small dark seeds may remain dormant for a longer period and are more sensitive to NaCl. This is not the case in A. halimus, and although variability may occur in seed size, we never detected such a clear seed dimorphism. Therefore, the ability of the seeds to remain quiescent when conditions are not optimal for germination is of paramount importance. If favourable conditions only occur transiently, such as after rainfall in spring, germination and early seedling growth © 2002 NRC Canada



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during this recovery period should be as rapid as possible. Several authors reported that seeds of halophyte species are indeed able to germinate after a long period of incubation in salt stress conditions (Ungar 1978; Keiffer and Ungar 1997; Katembe et al. 1998; Weber and D’Antonio 1999; Gul and Weber 1999; Pujol et al. 2000; Tobe et al. 2000). In some instances, seeds pre-incubated in salt conditions and then transferred to control medium germinated at a higher percentage (Keiffer and Ungar 1997; Weber and D’Antonio 1999) or more quickly (Pujol et al. 2000) than untreated control seeds. This, however, is not a general rule in halophyte species and a negative effect of a pre-incubation on the kinetics of germination has also been reported (Tobe et al. 2000). In A. halimus, a pre-incubation treatment in the presence of a high salt concentration (>300 mM NaCl) had no impact on the germination percentages during subsequent exposure to control conditions. The kinetics of germination was not affected during the recovery period, and there was no effect on seedling growth in contrast to the negative impact of a pre-exposure to NaCl on the radicle elongation of Kalidium caspicum (Tobe et al. 2000). We also showed that a similar absence of effect in A. halimus was valid in response to a pre-exposure to mannitol.

Acknowledgements This study was supported by European Union (INCO program, Convention No. ERB IC18-CT98-0390). The authors wish to thank Professor S. Bouzid (University of Tunis) for kindly providing the seeds.

References Aiazzi, M.T., and Arguëllo, J.A. 1992. Dormancy and germination studies on dispersal units of Atriplex cordobensis (Gondoger and Stucker) (Chenopodiaceae). Seed Sci. Technol. 20: 401–407. Almansouri, M., Kinet, J.-M., and Lutts, S. 2001. Effect of salt and osmotic stresses on germination in durum wheat (Triticum durum Desf.). Plant Soil, 231: 243–254. Bajji, M., Kinet, J.-M., and Lutts, S. 1998. Salt stress effects on roots and leaves of Atriplex halimus L., and their corresponding callus culture. Plant Sci. 137: 131–142. Bajji, M., Kinet, J.-M., and Lutts, S. 2001. The use of electrolyte leakage method for assessing cell membrane stability as a water stress tolerance test in durum wheat. Plant Growth Regul. In press. Beadle, N.C.W. 1952. Studies in halophytes. I. The germination of the seed and establishment of the seedling of five species of Atriplex in Australia. Ecology, 33: 49–62. Bouzid, S.M., and Papanastasis, V.P. 1994. Intra and interspecific competition of hay crops Medicago arborea and Atriplex halimus as a function of seedling density. Agr. Med. 124: 62–69. Bouzid, S.M., and Papanastasis, V.P. 1996. Effects of seeding rate and fertilizer on establishment and growth of Atriplex halimus and Medicago arborea. J. Arid Environ. 33: 109–115. Bressan, R.A., Hasegawa, P.M., and Pardo, J.M. 1998. Plants use calcium to resolve salt stress. Trends Plant Sci. 3: 411–412. Campbell, E.E., and Matthewson, W.J. 1992. Optimizing germination in Atriplex nummularia (Lind.) for commercial cultivation. S. Afr. J. Bot. 58: 478–481.

303 Dodd, G.L., and Donovan, L.A. 1999. Water potential and ionic effects on germination and seedling growth of two cold desert shrubs. Am. J. Bot. 86: 1146–1153. Egan, T.P., and Ungar, I.A. 1999. The effects of temperature and seasonal changes on the germination of two salt marsh species, Atriplex prostrata and Salicornia europaea, along a salinity gradient. Int. J. Plant Sci. 160: 861–867. Ellern, S.J., Samish, Y.B., and Lachover, D. 1974. Salt and oxalic acid content of leaves of the saltbush Atriplex halimus in the northern Negev. J. Range Manag. 27: 267–271. El-Shatnawi, M.K., and Mohawesh, Y.M. 2000. Seasonal chemical composition of saltbush in semiarid grasslands of Jordan. J. Range Manag. 53: 211–214. Foderaro, M.A., and Ungar, I.A. 1997. Growth and survival of Polygonum aviculare L. at a brine-contaminated site in southeastern Ohio. Am. Midl. Nat. 138: 140–152. Giusti, L., and Grau, A. 1983. Inhibidores de la germinacion en Atriplex cordobensis Gand et Stucker (Chenopodiaceae). Lilloa, 36: 143–149. Gul, B., and Weber, D.J. 1999. Effect of salinity, light, and temperature on germination of Allenrolfea occidentalis. Can. J. Bot. 77: 240–246. Ignaciuk, R., and Lee, J.A. 1980. The germination of four annual strand-line species. New Phytol. 84: 581–591. Katembe, W.J., Ungar, I.A., and Mitchell, J.P. 1998. Effect of salinity on germination and seedling growth of two Atriplex species (Chenopodiaceae). Ann. Bot. (London), 82: 167–175. Keiffer, C.H., and Ungar, I.A. 1997. The effect of extended exposure to hypersaline conditions on the germination of five inland halophyte species. Am. J. Bot. 84: 104–111. Keiser, J.R., Mullen, R.E., and Hinz, P.N. 1995. Effects of Ca2+and Mg2+-enriched germination media on germination and sugar leakage of Ca-deficient soybean seed. Can. J. Plant Sci. 75: 343–346. Khan, A.M., and Rizvi, Y. 1994. Effect of salinity, temperature, and growth regulators on the germination and early seedling growth of Atriplex griffithii var. stocksii. Can. J. Bot. 72: 475–479. Khan, M.A., and Ungar, I.A. 1984. Seed polymorphism and germination responses to salinity stress in Atriplex triangularis Willd. Bot. Gaz. 145: 487–494. Khan, M.A., and Ungar, I.A. 1996. Influence of salinity and temperature on the germination of Haloxylon recurvum. Ann. Bot. (London), 78: 547–551. Khan, M.A., and Ungar, I.A. 1997. Effects of light, salinity and thermoperiod on the seed germination of halophytes. Can. J. Bot. 75: 835–841. Khan, M.A., and Weber, D.J. 1986. factors affecting seed germination in Salicornia pacifica var. utahensis. Am. J. Bot. 73: 1163–1167. Khan, M.A., Ungar, I.A., and Showalters, A.M. 2000. Effects of salinity on growth, water relations and ion accumulation of the subtropical perennial halophyte, Atriplex griffithii var. stocksii. Ann. Bot. (London), 85: 225–232. Koheil, M.A.H., Hilal, S.H., El-Alfy, T.S., and Leistner, E. 1992. Quaternary ammonium compounds in intact plants and cell suspension cultures of Atriplex semibaccata and A. halimus during osmotic stress. Phytochemistry, 31: 2003–2008. Lai, FM., Lecouteux, CG., and McKersie, B.D. 1995. Germination of alfalfa (Medicago sativa L.) seeds and desiccated somatic embryos. I. Mobilization of storage reserves. J. Plant Physiol. 145: 507–513. Lailhacar, DS., and Laude, H.M. 1975. Improvement of seed germination in Atriplex repanda Phil. J. Range Manag. 26: 491–494. © 2002 NRC Canada



304 Martínez, JP. 2001. Mechanisms of resistance to water stress at plant and cellular levels in Atriplex halimus. Ph.D. thesis, Université catholique de Louvain, Louvain-la-Neuve, Belgium. Matoh, T., Watanabe, J., and Takahashi, E. 1986. Effects of sodium and potassium salts on the growth of a halophyte Atriplex gmelini. Soil Sci. Plant Nutr. 32: 451–459. Osman, A.E., and Ghassali, F. 1997. Effects of storage conditions and presence of fruiting bracts on the germination of Atriplex halimus and Salsola vermiculata. Exp. Agric. 33: 149–155. Peluc, S.I., and Parera, C.A. 2000. Germination improvement of Atriplex nummularia (Chenopodiaceae) by pericarp elimination. Seed Sci. Technol. 28: 559–566. Pérez-Molphe-Balch, E., Gidekel, M., Segura-Nieto, M., HerreraEstrella, L., and Ochoa-Alejo, N. 1996. Effects of water stress on plant growth and root proteins in three cultivars of rice (Oryza sativa) with different levels of drought tolerance. Physiol. Plant. 96: 284–290. Pujol, J.A., Calvo, J.F., and Ramirez-Díaz, L. 2000. Recovery of germination from different osmotic conditions by four halophytes from southern Spain. Ann. Bot. (London), 85: 279–286.

Can. J. Bot. Vol. 80, 2002 Silcock, R.G., Williams, L.M., and Smith, F.Y. 1990. Quality and storage characteristics of the seed of important native pasture species in south-west Queensland. Aust. Rangeland J. 12: 14–20. Tobe, K., Li, X., and Omasa, K. 2000. Seed germination and radicle growth of a halophyte, Kalidium caspicum (Chenopodiaceae). Ann. Bot. (London), 85: 391–396. Uchiyama, Y. 1987. Salt tolerance of Atriplex nummularia. Tech. Bull. Trop. Agric. Res. Cent. No. 22. pp. 1–69. Ungar, I.A. 1978. Halophyte seed germination. Bot. Rev. 44: 233–264. Ungar, I.A. 1996. Effect of salinity on seed germination, growth, and ion accumulation of Atriplex patula (Chenopodiaceae). Am. J. Bot. 83: 604–607. Ungar, I.A., and Khan, A.M. 2001. Effect of bracteoles on seed germination and dispersal of two species of Atriplex. Ann. Bot. (London), 87: 233–239. Weber, E., and D’Antonio, C.M. 1999. Germination and growth responses of hybridizing Carpobrotus species (Aizoaceae) from costal California to soil salinity. Am. J. Bot. 86: 1257–1263.

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