MATERIALS AND METHODS

Seeds germination condition and treatments: The experiment was conducted at the University of Tiaret (35° 23' 17" N, 1° 19' 22" E, altitude of 1080 m northwest of Algeria).

Seeds of common bean (Phaseolus vulgaris L.) cultivars Cocorose (salt sensitive) and Djadida (salt tolerant), were surface sterilized by immersion in 5% (v/v) NaClO₄ solution for 4 min, rinsed four times with sterile distilled water. Twenty seeds per cultivar were placed on filter paper (Whatman N°2) in Petri dishes (9 cm) containing 5 mL of distilled water (control) and 50, 100, 150 and 200 mM of NaCl corresponding to 0.592, 0.767, 0.919 and 1.073 MPa, respectively and allowed to germination at 25°C. Each treatment includes six replicates.

Traits measurements
Kinetic of seed imbibition, germination rate and root length: Water uptake was recorded during 96 h every 12 h. It was calculated according to the following equation:

where,
W₁ was the initial seed weight before imbibition and W₂ was the weight of seeds after water absorption. Seed germination was evaluated after 108 h. Seeds were considered as germinated when the radicle reached 2 mm. The germinated seeds were counted and germination percentage was determined. Also, the radicle length (mm) was measured.

α-Amylases extraction and assay: Extraction of α-amylase was carried out on germinating seeds placed after 48-72 h and their activity was measured. Ten grams of cotyledons were ground in 50 mL of acetate buffer solution (pH 4.8) containing 50 mM of sodium acetate and 20 mM of CaCl₂ (Kaur et al., 1998). The extract was centrifuged at 10000 g for 10 min at 4°C. The supernatant was then heated at 75°C for 15 min to inactivate β-amylase according to Guglielminetti et al. (1995). The extract was used for estimating the activity of α-amylases according to the method described by Almansouri et al. (2001). This technique involved using 1 mL of enzyme extract, supplemented with 0.5 mL of starch solution 1%. It was diluted in acetate buffer containing 0.05 mM of sodium acetate and 0.02 mM of CaCl₂ (pH 4.8). The whole was mixed by vortex and incubated for 15 min at 35°C. The reaction was stopped by adding 0.5 mL of 3,5 Dinitrosalicylic Acid Reagent (DNS). The assembly was placed in a boiling water bath for 5 min and cooled. The absorbance of the reaction mixture was determined at 540 nm in spectrophotometer (Jenway 73 series model spectrophotometer) against maltose as standard. The α-amylase activity was determined in μ moles of reducing sugars formed min^-1 g^-1 FW.

Determination of soluble sugars contents: Soluble sugar content was evaluated on 100 mg of cotyledons of germinating seeds (72 h) from all treatments (0, 50, 100, 150 and 200 mM of NaCl). The cotyledons were homogenized on 5 mL of 80% ethanol for 24 h (Sidari et al., 2008). The extract obtained was diluted 10 times with 80% ethanol. From the resulting solution, 2 mL were mixed with 4 mL anthrone reagent (200 mg anthrone, 200 mL of sulfuric acid W/W). After stirring, samples were placed in boiling water bath for 10 min and cooled for 30 min in the dark. The light absorption of the samples was estimated at 585 nm using a Jenway 73 series model spectrophotometer.

Soluble sugars contents were determined using glucose standard and expressed in mg g^-1 FW of cotyledons.

Determination of osmotic potential: Cotyledons are placed in Eppendorf tubes, dipped in liquid nitrogen and ground with Retsch MM 400 mixer mill (USA) and centrifuged. Osmotic potential was determined on 10 mL of supernatant using Wescor Vapro 5520 model osmometer (USA).

Statistical analysis: They was performed using two way ANOVA (for p<0.01) from the Statistica 8.0 package. Mean comparison was performed by Duncan test at 5% level of significance.

RESULTS

Effect of salinity and cultivar on water uptake, seed germination and radicle length: Salt treatments and cultivar affected significantly water uptake, seed germination and radical length (Table 1). Moreover, interaction cultivar xsalt treatment was significant. Seed imbibition was not changed by the salt stress intensity (Fig. 1). However, the water uptake by Cocorose was higher than Djadida during different times. For example, 96 h after sowing, the rate of imbibition of the control reached 111.79 and 94.42% for Cocorose and Djadida, respectively. At higher salt treatment (200 mM NaCl), these rates were reduced by 1.82 and 1.35% for Cocorose and Djadida, respectively.

No significant difference was observed for the rate of seed germination between control and treatment at 50 and 100 mM of salt (Fig. 2). In contrast, higher salt treatment reduced markedly this rate (Fig. 2). The radicle length was reduced significantly by the salt stress intensity (Table 2). However, this reduction depended on the concentration applied and genotypes tested (Table 1). Compared to control, salt treatment at 50 mM decreased the length of radicle by 16.8 and 29.42% for Djadida and Cocorose, respectively. This reduction was more pronounced for the treatments 100, 150 and 200 mM for both genotypes (Table 2).

Table 1:	Mean square values and significance of NaCl treatment, genotype and their interaction effects on germination (%) radicle length, osmotic potential, α-amylase activity and soluble sugars content measured on two cultivars bean
*p≤0.05, **p≤0.01, ***p≤0.001, ns: not significant

Fig. 1(a-b):	Effect of salinity on seed water uptake during germination of two cultivars of Phaseolus vulgaris L., (a) Cocorose and (b) Djadida

Djedida was less affected than Cocorose for 50, 100 and 150 mM, while Cocorose showed higher radical length at 200 mM.

Table 2:	Mean values of radicle length and sugar content seeds under different NaCl treatments
Means indicated by different letters (within each NaCl treatment) are significantly different (at 0.05 probability level) by the Duncan comparison test

Fig. 2:	Effect of salinity (NaCl) concentrations (0, 50, 100, 150 and 200 mM) on seed germination (%) of two cultivars of Phaseolus vulgaris L., Cocorose and Djadida

Effect of salinity and cultivar on biochemical traits: All biochemical traits were significantly affected by salt treatment and cultivar. Interaction cultivar xsalt treatment was also significant for all traits except for (Table 1).

Fig. 3 (a-b):	Effect of salinity (NaCl) conc (0, 50, 100, 150 and 200 mM) on activity of α-Amylase in seeds after 48 and 72 h of germination of two cultivars of Phaseolus vulgaris L., (a) Djadida and (b) Cocorose

The activity of α-amylase was affected by salt stress intensity and period of germination (Fig. 3). The impact of salinity was intensified with time of germination in both genotypes and all salt treatments (Fig. 3). In the control treatment, α-Amylase activity from 48-72 h increased by 87 and 81% for Djadida and Cocorose, respectively. Djadida presented higher α-Amylase activity whatever the intensity of salt treatment. Moreover, the difference in α-Amylase activity between 48 and 72 h was more important for Djedida than for Cocorose at higher salt stress intensity (Fig. 3).

Fig. 4:	Effect of salinity (NaCl) conc. (0, 50, 100, 150 and 200 mM) on osmotic potential of seeds of two cultivars of Phaseolus vulgaris L., Djadida and Cocorose

The results indicated that the concentration 50 mM and intensities linked to the time increased α-Amylase activity compared to that of the control. Thus, α-Amylase activity was greater after 48 h of germination than after 72 h. During the first period, it was 17.40 and 9.18%, respectively for Djadida and Cocorose, against 1.72 and 1.87% for the second. Beyond this level, the activity gradually decreased regarding to the concentration of NaCl and the duration of germination. The inhibition of the activity was more pronounced in the treatment of 200 mM and after 72 h of germination time. It was valued at 45.74 and 52.65%, respectively in Djadida and Cocorose.

The results (Table 2) showed that the sugar content of germinating seeds depended on genotype, salt treatments and their interaction. The increase of NaCl concentration induced a relative rise in the sugar content of seeds of both genotypes. Indeed, by comparison with control sugar content increased from 9.43% (50 mM) to 44.23% (200 mM). The range of sugar content augmentation was quite similar for both genotypes.

Higher NaCl content induced highly significant decrease in osmotic potential of germinating seeds and the sugar accumulation (Fig. 2 and Table 2). The two genotypes responded differently to salt treatment. In Djadida, values of osmotic potential markedly decreased with salt stress intensity compared Cocorose (Fig. 4).

DISCUSSION

Seed germination starts with uptake of water by quiescent seed and terminates with elongation of the embryonic axis. The germination is exposed to different environmental stresses, where drought and salinity are the most severe ones. Our results show that salinity levels of 100 and 200 mM caused a marked decrease in the rate of seed germination (Fig. 2).

This result confirmed previous reports (El-Tayeb, 2005; Othman et al., 2006; Jamil et al., 2006; Al-Saady et al., 2013) which showed a reduction in seed rate germination due to higher levels of NaCl. The effect of salinity on radicle length was more marked. Indeed, the increase in salinity reduced the length of the radicle (Table 2).

The α-Amylase activity estimated after 48 and 72 h at low NaCl concentration 50 mM increased. These results coincided with those of Dodd and Donovan (1999), Sidari et al. (2008) and Mei and Song (2008). Beyond this concentration, salinity presented a reducing effect on the activity. Siddiqui and Khan (2011) showed that from 100 mM of NaCl, α-Amylase activity is greatly reduced. These traits provide important criteria to characterize the response of plants to abiotic stress as salinity (Adda et al., 2005; Othman et al., 2006; Jamil et al., 2006). However, cultivars did not responded similarly to salinity which mirrored the existence of broad genetic variability within this species. Moreover, difference in water uptake and seed germination (Fig. 1) were found between Cocorose and Djadida as observed in several species (Othman et al., 2006; Jamil et al., 2006; Al-Saady et al., 2013; Ogiwara and Terashima, 2010). The large genotypic variability observed for studied traits may offer valuable tools to investigate mechanism of salt tolerance during germination.

Seed germination begins with the water uptake by seed and ends with the radicle protrusion and elongation. Barroco et al. (2005) suggested that cell elongation is necessary and can be generally accepted as a sufficient phenomenon for the completion of this process.

During germination, the osmotic potential of the radicle cells becomes more negative because of the accumulation of solutes mainly sugars (Bewley, 1997; Munns, 2002; Othman et al., 2006; Ogiwara and Terashima, 2010). The decrease in osmotic potential would lead to increased water uptake, where resulting turgor and cell extension (Mei and Song, 2008; Jamil et al., 2006).

Higher concentration of salt reduced osmotic potential (and probably water potential) in the medium which limited water absorption by germinating seeds and thus reduced germination as already reposted by Maas et al. (1983) and Al-Saady et al. (2013). Therefore, the rise of salinity was accompanied by a significant decrease in the osmotic potential of cotyledons (Fig. 4). Which leads to maintain water uptake by seeds and rate germination. We suggest that accumulation of sugars in germinating seeds exposed to high NaCl concentrations may explain allow utilization of these sugars in germination process. In fact, they may be used in osmotic adjustment and synthesis of energy necessary for growth (Bewley, 1997; Gill et al., 2003). This osmotic adjustment maintains cell turgor which results in a reduction in the growth rate. The maintain of cell turgor was probably reached by the rise of sugar content which resulted in lowering of the osmotic potential of seeds subjected to salt stress (Table 2). Nevertheless, increase in sugar content was maintained despite the reduction in the α-Amylase activity mainly beyond 100 mM NaCl, These results are in accordance with those reported in wheat same results were shown in several plant species, (Jamil et al., 2006; Othman et al., 2006; Zheng et al., 2008).

CONCLUSION

Increase in NaCl stress affected negatively seed water uptake and α-Amylase activity depending on the concentration of salt. This fact allowed to a decrease of germination level mostly at higher salt treatments. However, soluble sugars content raised significantly which leads to a significant decrease in the osmotic potential of bean seeds during germination. In contrast, this trait was significantly reduced at 150 and 200 mM NaCl. Radicle length growth was the most affected parameter by salinity treatment. These results suggest that for both 50 and 100 mM NaCl treatments, seed germination was not affected by salinity and the α-Amylase activity was increased. Beside, at 150 and 200 mM NaCl an opposite situation for seed germination and α-Amylase activity was observed.

Finally increasing concentrations of NaCl in the medium was accompanied by an increase of the soluble sugar contents in bean seeds which leads to decrease their osmotic potential and therefore maintained water uptake.