Study on Germination of Seven Barley Cultivars (Hordeum vulgare L.) under Salt Stress

The present work aims to study the germination behavior of seven Tunisian varieties of barley (Kounouz, Faiz, Roho, Manel, Lemsi, Rihane and Tej) during germination without or with NaCl. These varieties have been chosen in order to have the greatest possible diversity on the agro-physiologic level and to select tolerant plants to salinity. Barley grains are germinated (in Petri dishes lined with a double layer of filter paper soaked) in a solution of NaCl 0, 50, 100, 150, 200 mM for 7days. Two germination criteria were studied: the radicle breaching and the coleoptile’s emergence. The measured parameters were the kinetics, the germination rate, the final germination and the GSI (Germination Stress Index). The results showed that, daily germination decreased significantly under NaCl. If we consider the two germination criteria, salt exerts a depressive effect on seed germination capacity of barley and slows germination speed. There is also an important intra-specific variability opposite of salinity. The studied barley varieties were classified in descending order of salt tolerance as following: Roho > Faiz > Kounouz > Manel > Rihane > Lemsi > Tej.


Introduction
The cereals yield, especially barley (Hordeum vulgare L.), remains moderate and heavily influenced by biotic and abiotic stresses (Slama et al., 2005).Salinity is a major abiotic stress, some 800 million hectares of land are affected by high levels of salt in the world (Munns, 2005).In Tunisia, saline soils occupy about 23% of the total area with 8.7 million hectares of arable land (FAO, 2008).However, water resources for irrigation in Tunisia are limited and of poor quality, 30% of these resources have a salt content greater than 3 g/L (Cheikh et al., 2008).For cereals, the depressive effect of salt occurs from a critical threshold concentration characteristic of the species or variety (Bouaouina et al., 2000).The plant species response to salt depends on the species itself, its variety, the culture salt concentration and the stage of plant development (Mallek-Maalej et al., 2004).Seed germination is the most critical phase of the plant life, it is a physiological phase which corresponds to the transition from the dormant seeds phase to a reactivation phase of the metabolism (Gallardo et al., 2003).Germination and plant seedling were considered as salt sensitive stages (Munns & Tester, 2008).Indeed, salinity reduces speed germination and exposes seeds to some growth risk (Slama, 2004).The high salt concentrations in the medium affect seriously the seed germination by reducing the bioavailability of water retained by Na + ions causing water deficit (Parida & Das, 2005), which disrupts the absorption of water by seeds leading to a delay in germination process (Jamil et al., 2006).At this osmotic component is added an ionic component characterized by an excess of Na + , Cl -, K + levels and Ca 2+ deficiency (Karimi et al., 2005).Furthermore, salinity caused a disruption of the enzyme systems involved in various physiological functions of the seed; such as decreased activity of polyphenol-oxidases and amylases (Khemiri et al., 2004).Rajaskaran et al. (2000) suggest that tolerance at germination stage is not expressed in the adult stage in several species such as maize and beans.Indeed, salt tolerance at germination stage may be similar or different from that in the adult plant stage.For some species, the seeds that have an ability to germinate under salinity conditions have susceptibility to continue this stress tolerance, during their subsequent growth (Rozema, 1975).
Thus, the identification of salt tolerant cultivars would certainly improve the production zones exposed to salinity risk or irrigated with brackish water and present a high interest in the varietal improvement (Slama, 1996).
The objective of this work was to use a precocity and reliable test to characterize the effect of salt stress on seven Tunisian barley cultivars at the germination stage and to identify the most resistant one.This study would highlight the salinity influence on different characteristic parameters such as germination rate, kinetics and the germination stress tolerance index to identify the degree of salinity tolerance.

Plant Material
Seven cultivars of barley were used in this study.Origin and varieties characteristics are shown in Table 1.

Germination Experiment
Barley seeds were surface-sterilized by soaking in 10% sodium hypochlorite solution for 15 min followed by three washes with sterile distilled water.Seeds were then germinated in 9 cm Petri dishes on filter paper discs moistened with distilled water (control) or NaCl solutions, with 20 seeds in each dish and were incubated in darkness at 25 °C for 7 days.
The experimental design adopted was a randomized block with 3 replications, 7 varieties in both control (NaCl 0 mM) and stressed conditions (NaCl 50, 100, 150 and 200 mM).
The studied germination criteria were the radicle breaching and the coleoptile's emergence.The number of germinated grains was evaluated every 24 hours and germination rate was determined in the 7 th day.

Measurements and Data Collection
The final germination rate was calculated the 7 th day as corrected germination rate (GC) from the formula of Smith and Dobrenz (1987): Where, Nx is the number of sprouts in a medium containing x mM NaCl, and No the number of sprouts with 0 mM NaCl (control).
GC% is the best way to identify the salt concentration of physiological limit of the seed germination.
The germination speed which express the germination energy responsible for the exhaustion of the grain reserve was estimated by the mean time (T50) which corresponds to the germination of 50% of the total grains (Lang, 1965).
The germination kinetics was followed to evaluate germination behaviour of the studied varieties. jas.ccsenet.

The numb calculated
Where, nd

Statisti
The statist error (SE) to be statis      respectively, while seeds of Rihane, Manel, Lemsi and Tej had the low percentage (67, 60, 60, 50%).Thus, Konouz, Faiz, and Roho cultivars are considered as salt-tolerant cultivars.Instead, Rihane, Manel and Lemsi were moderately salt tolerant.However, Tej cultivar is considered as the most sensitive to salt stress.Indeed, results show that even with a low concentration of 100 mM NaCl, Tej shows a reduction of 50% of the germination rate compared to control.The obtained results confirm those of previous studies revealing the depressive effect of salt on germination capacity of the others seeds species such as tomato (Allagui et al., 1994), wheat (Ben Naceur et al., 2001), castor (Raghavaiah et al., 2006) and watermelon (Askri et al., 2007).

Kinetic
Our results showed that Barley was a salt-resistant species at radicle breaching stage (Figure 2) which is consistent with other results with the tolerance variability among cultivars (Shannon, 1984;Srivastava & Jana, 1984).
The decrease in the final germination rate, correspond to an increase in the external osmotic pressure (more negative osmotic pressure) resulted from the increased environment salinity, it disrupts the seed hydration.On the other hand, the high concentration of cations and anions (especially Na + and Cl -) in the environment impede the seed germination by imposing toxicity in seeds (Rajabi & Postini, 2005;Atak et al., 2006).Germination capacity is nearly 100% in control medium, for the seven cultivars.It was little affected at low doses of NaCl (100 mM and at 150 mM) but it was reduced significantly at high concentration of NaCl up to 200 mM for all studied cultivars especially Rihane, Lemsi and Tej where the coleoptile's emergence is completely inhibited (Figure 4B).Mrani-Alaoui et al. (2013) showed that stressed grain germination rate was located below those of control treatment and come up to zero gradually as the NaCl concentration increases.
In all barley studied cultivars, salinity reduces speed germination without affecting the germination capacity; this depressive effect was already reported in barley by El Mekkaoui (1990).The reduction of germination rate may reflect the embryo sensitivity to osmotic effects of NaCl.The solution of 200 mM NaCl leads difficult hydration for the embryo, which would be unable to absorb the amount of water needed to start the germination process (Abdelly, 1992).The delay of trigger germination is the time required to set up mechanisms for osmotic adjustment.The decrease in the rate of coleoptile's emergence under high NaCl concentration was observed in other plants (Bounaqba, 1993) reflecting the slowdown in reserve mobilization to seedling (Prisco et al., 1981).
Results indicated that salinity induced delay and decrease the germination percentage through lower water uptake and higher accumulation of Na + and Cl - (Begum et al., 2010).As salinity enhances osmotic pressure leading to a reduction in water absorbance, division and cell differentiation, which negatively affects metabolic and physiological processes.The consequences were a delay of germination processes followed by prolonged seed germination duration (Kang & Saltveit, 2002).
The study of the germination rates showed that, NaCl provoked an increase of T50 or a decrease of the germination speed for all varieties.For controls plants and those exposed to low NaCl concentrations, the T50 is little or unaffected for both criteria germination.These results corroborate those of Adjel et al. (2013), who demonstrated for twelve barley genotypes that the germination speed decreased from 16.2 to 8.3% at 150 mM.
These delays of germination by increasing of NaCl concentrations are due to a problem of hydration seeds as a result of high osmotic potential.Results could be explained also by the high time necessary to trigger mechanisms to adjust osmotic pressure in seeds tissues (Jaouadi et al., 2010;Salehifar et al., 2010).
Germination stress index (GSI) of different barley cultivars was affected by salt stress (P < 0.05) (Figure 6).GSI was significantly reduced by 200 mM NaCl compared to the control treatment.Our results are consistent with those of Mallek-Maalej et al. (2015).
According to Figures 6 (A) and (B) which shows the differences between GSI studied varieties; the highest value of GSI was registered with Roho, Faiz and Kounouz cultivars.Reduction of germination under salt stress seems to be related to stress intensity and variety (Ben Naceur et al., 2001;Kadri et al., 2009).

Conclusion
The results reported in this study suggest that barley is supposed as sensitive plant to NaCl concentration at germination stage.NaCl, at doses up to 200 mM, slows the rate of barley germination without affecting their ability to germinate.During the phase of coleoptile's emergence, barley cultivars show greater sensitivity to NaCl than during radicle breaching, Roho cultivar is distinguished by its ability to emerge at higher doses of NaCl (200 mM).In absence as in presence of salt, this cultivar has the best germination behaviour.However, Tej and Lemsi cultivars are the most sensitive.Analysis of variance shows highly significant differences in the cultivar and the treatment effect, when we consider the two criteria of germination.According to the tolerance Figures 1 germinatio percentage emergence affected by 200 mM N radicle bre Figures 3 the germin reflecting (germinati The latenc to two day (Figure 4A (Figure 3B than 7 day In salt med were statis grains decr Figure 4 Figure 6.