Chickpea (Cicer arietinum L.) is among the earliest human grown grain crops.Today chickpea ranks third in world production after beans (Phaseolus  spp.) and field pea (Pisum sativum L.) among food legumes [1]. Besides being an important food source for humans and animals, the crop also  plays an important role in the maintenance of soil fertility, particularly in arid regions [2]. Nowadays, the crop is grown on an area of 14.56 million ha in the world with a total yield of 11.5 million ton , so we see a strong trend to increase interest in chickpea cultivation worldwide, especially in the countries of North America, Australia, Europe [3]. Soil salinity is one of the main constraints in the growth of chickpea. The soil salinity is assumed to be one of the key factors contributing to acritical decrease in crop productivity [4] . Chickpea is often grown not only in the arid climatic conditions of the Asian and African areas, but also on salt soils or on salt water irrigated soils in India, Pakistan and Australia [5,6]

Chickpea is especially susceptible to salt stress at the reproductive stage of development,  [7] with crop roots suffering (Tejera et al., 2006) leading to lower productivity  [8,9]. Salinity stress cause a decrease in plant height, crop growth rate and total plant biomass respectively by 20, 15 and 28 % [10]. Saline water irrigation significantly compromises almost all of the crop's physiological mechanisms, resulting in worse production [11].

In Chickpea germination of seeds is delayed and reduced, and vegetative plant growth is suppressed under saline conditions [12,13], which varies from germination to vegetation and from fruiting to podding; at the same time, chickpea tolerance for salinity differs from one genotype to another [14]. Many have very high germination potential and seedling growth in salt soil, while others germinate well in salt soil, but with very low seedling growth [15,16] demonstrated that there is no difference in seed germination in the conditions of salt stress relating to the form of chickpea (kabuli or desi). However, [17] demonstrated that the form of chickpea kabuli was more prone to increased soil salinity (0.9–4.9 ds/m) than the form desi. Salinity had a significant impact on seed  germination and dry weight seedling [18] found that, the salt concentration had a major impact on seed germination and seedling growth parameters, these characteristics decreased dramatically as the salt concentration increased from 2 to 20 dsm^-[19] reported that, no effect of NaCl treatment on germination frequency was observed; however, a dramatic decline in early seedling growth was observed as NaCl concentration increases.  [20]  reported that, salinity stress decreased all of the parameters examined with increased concentration of NaCl in common bean, except for mean germination time.  [21] recorded that, salinity significantly delayed germination at 300-400 NaCl for germination, emergence of seedlings, fresh and dry weight of both shoots and roots, and seed yield decreased within growing salinity. Germination percentage and velocity, plumulus length, radical length and dry seedling weight were higher in control [22]. 

A lot of researchers reported similar results with different crops [23-25].This study aimed to investigate the effects of different concentrations of NaCl on Chickpea seed germination.

Germination conditions.

This experiment aimed to characterize the range of salinity tolerated by Chickpea (Cicer arietinum L.) at the germination stage. Seeds, selected for homogeneity, were germinated at 25 ^ᵒC in the dark in 12-cm Petri dishes lined with filter paper moistened with five levels of NaCl: 0.0, 50, 100, 200 and 300 mM; 50 seeds per dish. At intervals seeds and emerging seedlings were transferred under dim light to new dishes lined with filter papers, saturated with the experimental solutions to prevent buildup of salt. Germination was monitored daily for 12 days from sowing and three replicates seeds were considered germinating when the radical was emerged to a length of 2 mm. After approaching steady germination percentage in the test solutions, the non-germinated seeds were transferred from the high salt solutions to distilled water to monitor recovery of germination from salt stress, and the number of seeds germinating after transfer was counted daily for 11 days. 

Definitions, calculations and statistical analysis.

Final cumulative germination percentage and final percentage recovery of germination from salinity stress were arcsine transformed before performing statistical analysis to ensure homogeneity of variance. Data were analyzed using SPSS version 22. The effect of temperature on seed germination in the  in the  experiment and of  salinity stress and their interaction on seed germination in the third experiment were assessed using  two-way ANOVA respectively. Mean separation was performed using the Duncan's multiple range test at p < 0.05.

According to [26] the germination parameters estimated in this work were grouped into five categories - taking into account the different notations and expressions in the literature. These are the germinability or final germination percentage, rate or speed, times, uniformity and synchrony of germination. The following parameters were estimated.

Germinability or germination capacity. It is the final cumulative germination percentage and was calculated as the total number of germinants at the end of germination period as a percentage of the total number of seeds.

Rate or speed of germination. Estimated by using several calculations as follows:

Mean daily germination (MDG) or Daily germination speed (DGS). Calculated as:

MDG = _.                (% d^-1)

The cumulative germination % is the number of germinants as a percentage of the total number of seeds.

Peak value (PV) is the maximum MDG, or the maximum quotient derived by dividing daily the accumulated number of germinants by the corresponding number of days; i.e., the mean daily germination of the most vigorous component of a seed lot.

Germination value (GV). Called the Czabator index of germination velocity, was calculated as:

       GV = PV ×  final MDG                                       (% d^-1)

Timson index of germination velocity. Calculated as:

Timson index =  =     (% d^-1)

Where G₁, G₂, G₃, G_i and G_n are the cumulative number of germinants at the first, second, third, i^th and final time respectively and T is the total germination period; that is  to sum the cumulative germination % for certain intervals and divide by the final germination period.

Speed of accumulated germination (SAG). Calculated as:

SAG =Σ = + + + ………..+               (% d^-1)

Germination rate index (GRI), also called speed of germination was calculated as:

GRI = Σ  =  + + + ………..+                   (% d^-1)

=  +   + + ………..+

The germination  index (GI), also called the "germination rate" is  a measure of both  percentage and speed of  germination and assigns maximum arithmetic weight to embryos or  seeds  that germinate  first and  less weight  to those that germinate  later.

GI = Σg_i × (T-j)

Where g_i is the daily germination percentage or number of newly germinated seeds at time t_i,T is the total period of germination and j = i-1; that is

GI = Σg_i × (T-i+1)= g₁×T + g₂ × (T-1) + G₃ × (T-2) + …... + g_n×1          (%.d)

The coefficient of germination (CG), also called the coefficient of velocity of germination(CVG), or Kotowski coefficient of germination was calculated using the following formula:

CVG =                                     (d ^-1)

Where g_i is the  number of newly germinants at times t_i.

It is the reciprocal of MGT  multiplied by 100 = (1/MGT) ×100

This coefficient, which is the reciprocal of the mean germination time, was used to calculate the mean germination rate or , which in turn was used to calculate the weighted mean germination rate () 

=  =                 (d^-1)

Weighted mean germination rate (WMGR or). Calculated using the mean germination rate ( of each replicate and its variance (S²_V) as follows:

=(d^-1)

where Wj =              S²_j =()⁴ × S²_t         and              n_j =

Mean germination time (MGT or  ), also called mean emergence time (MET) or mean length of incubation time (MLIT) or mean days for germination (Mdays), is a measure of the average length of time required for maximum germination of a seed lot. It is one of the measures of time of germination and can be employed also as an inverse measure of speed of germination, and was calculated according to the following equation:

MGT = =                                                  (d)

Where g_i is the number of seeds newly germinated, or the daily germination percentage at time t_i from sowing, not the cumulative germination %, and N is the total number of germinants or the final cumulative germination percentage.

The variance of germination time (S²_t) was calculated according to the following formula:

  S²_t =                                                             (d²)

S²_t was used in the calculation of the coefficient of variation of germination time (CV_t); one of the measures of germination uniformity.

Germination times:

The first day of germination (FDG) is the time of first germination or the time of germination of the faster or most vigorous seeds.

The last day of germination (LDG) is the time of last germination or the time of germination of the slower or the least vigorous seeds.

Time spread of germination (TSG) is the time elapsing between FDG and LDG and was calculated as:    TSG = (LDG - FDG) + 1

Mean germination time (MGT or), was mentioned above among the indices of rate of germination. 

T₁₀ or time to 10% germination is a measure of the lag period between imbibition and onset of germination.

Uniformity of germination:

The coefficient of uniformity of germination (CUG) measures the variability among seeds in relation to the mean germination time of the sample and was calculated as:

CUG =                                                          (d^-2)

Where g_i is the number of newly germinated seeds on time t_ifrom sowing and is the mean germination time. High values would be associated with concentrated germination in time. 

The coefficient of variation of the germination time (CV_t) is another measure of the germination uniformity or variability in relation to the mean germination time and was calculated as:

CV_t =                                                                 %

Where S_t is the standard deviation of the germination time and the mean germination time.

Synchrony of germination was estimated using the synchronization index (), calculated as:

= - Σf_i × log₂f_i                   (bit)                  and  f_i =

where f_i is the  relative frequency of germination and g_i the number of seeds germinated on day i. Low values of  indicate more synchronized germination.

The recovery from salinity stress was calculated using the following formula of Khan and Ungar (1984):

Percent recovery =

where a is the total number of seeds germinated after being transferred to distilled water, b is the total number of seeds germinated in saline solution and c is the total number of seeds. In other words, the recovery percentage is the number of newly germinated seeds after transfer to water (a-b) as a percentage of the number of seeds transferred (those non-germinated in the saline solution (c-b).

The threshold and critical salinity levels for a specific process (germination index or embryonic growth) are defined as those levels leading to 5 and 50 % reductions respectively

Salt response of Chickpea (Cicer arietinum L.) during germination. 
This experiment aimed to specify the range of NaCl salinity tolerated by Chickpea (Cicer arietinum L.)  during germination and to characterize the effect of salinity stress on germination parameters of this species. A wide range of salinity has been examined in this experiment (0.0-300 mM NaCl). Time course of germination revealed progressive reduction in magnitude and speed, with prolonged lag of germination with the increase in salinity level from 0.0 to 300 mM NaCl (Figure 1). The FGP was reduced from 91% in non-salinized seeds to 15% in seeds treated with 300 mM NaCl. Similarly, speed of germination was sharply reduced under the impact of salinity. The speed of germination, in terms of GV and Timson index was sharply reduced from 84.2 and 60 % d^-1, respectively in non-salinized seeds to 3.7 and 11 % d^-1, respectively in seeds treated with 300 mM NaCl as shown in Table (1). 

Uniformity and synchrony of germination exhibited a threshold of 50 mM NaCl, beyond which germination uniformity was sharply increased (CVt lowered by from 34.6% at 0.0 mM NaCl to 12.7% at 300 mM NaCl) and germination synchrony was substantially increased ( lowered from 2.79 bit at 50 mM NaCl to 1.09 bit at 300 mM NaCl) as shown in Table (1). 

The first day of germination was non-significantly affected by increasing salinity up to 50 mM NaCl, but it was doubled as salinity level further increased to 300 mM NaCl. LDG was maintained  (by 10%) with the increase in salinity from 0.0 to 50 mM NaCl, followed by 25% reduction upon Increasing salinity pretreatment from 100 to 300 mM . TSG was significantly higheraffected by increasing salinity from 0.0 to 100 mM NaCl but was sharply shortened (by 87%) with further increase in salinity up to 300 mM NaCl. T₁₀ was almost doubled with the increase in salinity from 150 to 300 mM NaCl as shown in Table (1). 

Figure 1. Time course of germination of Cicer arietinum L. seeds under the impact of increasing levels of NaCl salinity.  Each value is the mean of 3 replicates.

Table 1. Germination parameters of Cicer arietinum L seeds under the impact of increasing levels of NaCl salinity. Each value is the mean of 3 replicates ± SE

 Means followed by the same letter are non-significantly different at P ≤ 0.05. 

*T₁₀ was calculated using the mean germination percentages of the time course of germination curves; therefore, they are not followed by SE.

Uniformity of recovery increased (CVt decreased by 41%) as salinity pretreatment exceeded a threshold of 150 mM NaCl up to 300 mM NaCl. On the other hand, synchrony of recovery was reduced ( increased by 26%) upon increasing salinity pretreatment from 100 to 150 mM NaCl, with no further change at higher salinity pretreatment. Increasing salinity pretreatment from 150 to 300 mM doubled FDG without effect on LDG; consequently, TSG was maintained at 9 days across the range of salinity pretreatment from 100 to 200 mM NaCl but was shortened to 8 days (11% decrease) upon further increase in salinity pretreatment up to 300 mM NaCl as shown in Table (2). 

Figure 2. Time course of germination recovery from salt stress of Cicer arietinum L. seeds. Seeds were incubated with 100-300 mM NaCl for 12 days and non-germinated seeds were transferred to distilled water to monitor recovery from salt stress for a period of 11 days.

Table 2. Parameters of germination recovery from salt stress of Cicer arietinum L. seeds. Seeds were incubated with 100-300 mM NaCl for 12 days and non-germinated seeds were transferred to distilled water to assess recovery from salt stress.  Each value is the mean of 3 replicates ± SE.

Means followed by the same letter are non-significantly different at P ≤ 0.05. 

*T10 was calculated using the mean germination percentages of the time course of germination curves; therefore, they are not followed by SE.

The widespread occurrence of Cicer arietinum L. in diverse habitats of varying levels of soil salinity gives the impression that the plant is salt-resistant.  Nevertheless, the severe reduction in germination percentage and germination speed with prolonged lag period by moderate salinity level of 100 mM NaCl, with almost cessation of germination at 300 mM NaCl suggests that Cicer arietinum L is a salt-sensitive species during germination according to the classification adopted. Seed germination is delayed and reduced, and vegetative plant growth is suppressed under saline conditions in Chickpea [12,13]. The reduction in germination efficiency under salt stress was more severe in terms of germination speed than in germination magnitude (the final germination percentage). This is in agreement with the behavior of Vicia faba [27] , and supports the conclusion that germination speed represents a more reliable measure of salt injury during germination than does the magnitude of germination. 

The reduced germination efficiency, expressed as reduced percentage and speed of germination, under the impact of salinity stress was associated with substantial lag of germination (increased T₁₀) and reduced germination uniformity (increased CV_t) and germination synchrony (increased ). However, whereas germination percentage increased in proportion with the increase in germination speed in almost a linear pattern, the increases in CV_t and (i.e. the reductions in germination uniformity and germination synchrony, respectively) approached a limit beyond germination speed of 23% day^-1   [5,6]. The effect of salinity on uniformity and synchrony of germination seems to be species specific and dose-dependent; leading either to non-significant changes in soybean [28] and Physalis peruviana [29] or to reduction in Moringa oleifera [30]  and Senna spectabilis [31]. In Elymus farctus, moderate salinity led to limited lowering in germination uniformity, which was followed by sharp increase at high salinity, and this was accompanied with increased germination synchrony at high salinity [32] . Whereas plant fresh weight was reduced under the impact of water stress [33].

The changes in times of germination of Cicer arietinum L. under salt stress suggest that salinity might delay start of germination but accelerates its termination with a consequent shortening of the time spread of germination. Similar response has been reported in Elymus farctus, particularly in absence of nutrients [32]. However, increasing salinity might delay both the onset and termination of germination, in wheat [32,34] 

The lower recovery percentage, which was associated with higher speed of recovery compared with the corresponding parameters of non-treated seeds, along with the limited improvement in recovery percentage with the increase in the level of salinity pretreatment might refer to a toxic ion effect of the low salinity levels on seed viability but a priming effect of high salinity, probably via an osmotic effect. In addition, these results suggest that salinity might exert a toxic effect on a portion of the seed population but the surviving portion will be kept ready to recover at higher speed and lower uniformity relative to their non-treated counterparts. The toxic ion effect on seed germination is expected at low salt levels, where the salt ions can diffuse into the seed and damage the embryo but the priming osmotic effect is expected at high salt levels [30]

Generally, restoration of germination efficiency after release of salt stress points to an osmotic effect, but low recovery means specific ion toxicity. [35] reported that the reduced germination recovery upon prolonged duration of seed soaking in saline medium which was related to uptake of Na⁺ and release of K⁺ from seeds under salt stress. The specific toxic effect of salt ions is likely to emerge in glycophytes whereas the osmotic effect is more likely in halophytes and salt-resistant species. Seeds of halophytes, due to their unique metabolic machinery, usually recover completely when salt stress is released indicating an osmotic effect [36,37]. On the other hand, the high recovery of seeds from abiotic stress compared with the original germinability means a priming effect of the stress pretreatment, which can be manipulated to enhance seed germination and plant growth, especially in poor quality seeds or under stressful environments [38] In addition, the present finding suggest, that salinity pretreatment seems to enhance start of recovery upon release of stress, with marginal effect on its termination and consequent prolongation in the TSG during recovery.

Chickpea germination and initial growth are strongly affected by salinity stress. The most susceptible stage is germination. Chickpea can be cultivated without any considerable decrease of its growth and development in slightly saline conditions . while the crop is exposed to strong suppression by the higher degrees of salinity. Therefore, we conclude that the crop has to be cultivated at the lands with no or slight salinity level.

Acknowledgments

The author is grateful to Botany Department, Faculty of Science, Zintan University, Zintan City, Libya, and Prof. Dr. Helal Ragab Moussa for help their during this  study.

The authors declare that they have no conflict of interest

No funding sources

The study was approved by the Zintan University, Zintan City, Libya.

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