Introduction

Salinity is generally defined as the presence of excessive amount of soluble salts in the soil solution (Sotiroupolous, 2007). It has become a very serious problem for crop production particularly in arid and semi-arid regions (Archibold, 1995). There are two main types of salinity based on their sources: Primary salinity which results from weathering of minerals and soil derived from saline parent rock (Ashraf, 2004), while secondary salinity is caused by human interference such as irrigation, deforestation, fertilizer application, overgrazing or intensive cropping (Sposito, 1989; Ashraf, 2004). Soil salinization is a serious problem in the entire world and it has grown substantially causing loss in crop productivity (Fao, 2000). It is a major constraint limiting agricultural productivity on nearly 20% of the cultivated and irrigated area worldwide (Zheng et al., 2001). Salinity affects almost every aspect of the physiology and biochemistry of plants and significantly reduces yield. High exogenous salt concentrations affect seed germination, water deficit, ion imbalance of the cellular ions resulting in ion toxicity and osmotic stress (Khan and Panda, 2008). Salt stress has been reported to cause an inhibition of growth and development, reduction in photosynthesis, respiration and protein synthesis in sensitive species (Meloni et al., 2003; Pal et al., 2004).

The most important process that is affected in plants growing under saline conditions is photosynthesis. Reduced photosynthesis under salinity is not only attributed to stomata closure leading to a reduction in intercellular CO₂ concentration, but also to non-stomata factors. There is strong evidence that salt affects photosynthetic enzymes, chlorophylls and carotenoids (Stepien and Klobus, 2006). Salt also affects photosynthetic parameters, including osmotic and leaf water potential, transpiration rate, leaf temperature, and relative leaf water content (Ashraf and Foolad, 2007). Salt tolerance is generally a sustained growth of the plant in the soil environment impregnated with NaCl or other salt combinations (Mori et al., 2006).

Early studies conducted under controlled conditions reported that salt injury in rice plant is caused by both osmotic imbalance and accumulation of chloride ions (Clarkson and Hanson, 1980). Recent studies however, indicated that injury is due to the excessive sodium (Na) uptake, and chloride, being essentially a neutral anion, is tolerated over a wide range of concentrations (Clarkson and Hanson, 1980). Na-K imbalance adversely affects grain yield (Devitt and Grieve, 1981). The typical mechanism of salinity tolerance in rice is the exclusion or reduction of Na uptake and increased absorption of K to maintain a good Na-K balance in the shoot.

There are extensive studies of salinity effects on rice based on NaCl as a predominant salt. The understanding of the quantitative effects of salinity on rice and critical thresholds of responses, especially with respect to the different salt and growth stages is still limited. Hence, this study investigated the effect of salinity stress on some physiological traits with the aim of developing information templates for other cultivars in future breeding programmes.

1 Materials and Methods

1.1 Experimental varieties

Seeds of four different rice varieties; IR20, IR29, NERICA1 and POKKALLI (a salt tolerant check) were utilized in the experiment. The cultivars were collected from the African Rice Centre, International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria.

1.2 Source of soil

The soil was collected behind the Department of Botany, Obafemi Awolowo University, Ile-Ife.

2 Pre planting Analysis

The soil was analyzed at the Department of Soil Science, Faculty of Agriculture, Obafemi Awolowo University, Ile-Ife. The following physical and chemical properties of the soil were determined: particle size distribution (sand, silt and clay) and textural class, pH, organic carbon, organic matter, total nitrogen, available phosphorus, calcium, magnesium, potassium, sodium, exchangeable acidity and effective cation exchange capacity. The soil was then air dried and then transferred into 72 plastic pots (about 21 cm in diameter and 17 cm in depth) each with three holes of approximately 3 mm in diameter bored at the bottom to enhance drainage during the course of the experiment.

3 Screenhouse Experiment

This experiment was conducted in the screenhouse. This is to protect the plants from direct rainfall contaminations, trampling and to avoid being destroyed by rodents as the plants develop. The average daily temperature, relative humidity and light intensity were also taken and recorded.

3.1 Preparation of salt solution

Salinity levels using pure salts was prepared with the following equation: EC = TDS/640 (Meot-Duros and Magne, 2008)

EC = Electrical conductivity

TDS = Concentration of soluble salts in mg/L

3.2 Nutrient solution preparation and salinization

Nutrient solution was prepared following the method described by Yoshida et al. (1976) and was salinized by adding NaCl and Na₂SO₄ at various concentrations.

3.3 Experimental design

A factorial experiment (4 x 4 x 2 x 3) laid out in a randomized complete block design (RCBD) was used. Factors of the experiment include Salt type (NaCl and Na₂SO₄), Salinity concentration (0, 5, 10 and 15 ds/m) while distilled water (0 ds/m) served as the control, Rice cultivars (IR20, IR29, POKKALI and NERICA 1) and growth stages (vegetative, flowering and grain-filling).

3.4 Application of nutrient solution

Each pot was supplied with 200 ml of appropriate nutrient in the morning and 200 ml of distilled water in the evening until the period of salinization. Plants were grown up to 14 d in normal nutrient solution to allow for sufficient growth before salinization. Sampling was done two weeks after salinity and at one week interval.

4 Determination of Germination Parameter

Speed of Germination (SG) and Final Germination Percent (FG%) was calculated following the procedure of Ellis and Robert (1981) and Ruan et al. (2002).

SG = N1/D1+N2/D2+…..+Ni/Di (Ellis and Robert, 1981).

FG% = S/T X 100 (Ruan et al., 2002).

Where, S is the number of germinated seeds, T is the total number of seeds and Ni is the number of germinated seeds per day (Di).

5 Measurement of Morphological Parameters

The following qualitative and quantitative morphological characters will be determined from the study.

5.1 Plant height

The seedlings were tagged and the main culms were measured at interval of 14 days using the metre rule. Plant height was measured from the base of the plant (top of the soil) to the tip of the tallest leaf.

5.2 Determination of root and shoot dry weight

Seedling shoot of ten randomly selected seedlings from each replication will be measured at the time of harvest (14 days after treatment application) with a metre rule. Shoot dry weight and root dry weight (10 seedlings) was recorded after oven drying at 90^°C for 3 days

6 Relative Growth Rate (RGR)

RGR= 1/Ws x dWs/dt, where Ws is plant dry weight, dWs is change in dry weight and dt is time interval

This measured the dry matter accumulated at a particular time regardless of the assimilatory surface present; measured in g/d (Evans, 1972).

7 Leaf Weight Ratio (LWR)

LWR = W₁/W_S

LWR defines assimilation in terms of leaf density: the unit is g^-1 . W₁ is the leaf dry weight and Ws is plant dry weight at harvest.

8 Results

8.1 Threshold of salinity tolerance

Threshold of salinity tolerance of four rice (Oryza sativa L.) cultivars across different salts is shown in Figure 1. Threshold (critical limit of salinity tolerance) for NaCl is at salinity concentration of 10 ds/m while that of Na₂SO₄ is at 5 ds/m. Beyond these limits, there is a reduction in the tolerance level of the cultivars across the different salts.

8.2 Final germination percentage (FGP)

In the salt free regime, the germination percentages of all varieties were greater than 90% under both salinity treatments (Table 1). At the salinity level of 5 ds m^-1, under Na₂SO₄ treatment, a higher germination percentage of more than 90% was observed from the varieties IR 20 and POKALLI, which were statistically similar with two other varieties, NERICA1 and POKKALI under NaCl treatment.      

8.3 Growth traits

Means of some growth traits under different concentrations of the two salts across four rice varieties for salinity tolerance is shown in Table 2. Percentage difference among the growth traits ranges from 28-78% respectively. The greatest reduction was observed in root shoot ratio (RSR42) at the 42^nd day (vegetative stage). Means of some growth traits under different concentrations of NaCl and Na₂SO₄ salts across four rice varieties for salinity tolerance is shown in Table 3. The greatest reduction of 67% was observed in relative growth rate at the grain filling stages under Na₂SO₄ treatment. The means of the growth traits reduce with increase in salinity levels. Shoot dry weight (SDW) was greatly reduced at the vegetative stage (42^nd day) while shoot height reduction occurs at the reproductive stage (56^th day).

8.4 Shoot height

There was a gradual increase in the shoot height of the four rice cultivars under salt free condition from the 28^th day till the end of the experiment (Figure 2). In addition, the shoot heights of IR20, POKKALI and IR29 followed similar trend with a steady increase between the 28^th and 42^nd day (vegetative stage) and with heights ranging between 32 cm and 52 cm respectively. Figure 3 shows the shoot heights of rice cultivars under stress (5 ds/m). the shoot heights of IR29 (70 cm) and NERICA 1 (55 cm) were significantly higher under NaCl treatment as compared with Na₂SO₄ treatment where no significant effects were observed. POKKALI recorded the highest shoot height of approximately 92 cm under NaCl treatment. All the rice cultivars reached their peak on the 70^th day. Similar trends were observed in the shoot heights of the cultivars under both treatments. However, unlike at 5 ds/m, the shoot heights of IR29 (70 cm) and NERICA 1 (62 cm) were significantly higher under Na₂SO₄ stress at 10 ds/m as compared with NaCl stress (Figure 4). The shoot heights of NERICA 1 (30 cm) were significantly lower under NaCl and Na₂SO₄ treatment (Figure 5). However, IR20, POKKALI and IR29 followed similar trend in their growth pattern.

8.5 Fresh weight

There was a rapid increase in the fresh weight of POKKALI under salt free condition from the 28^th day until maximum fresh weight of approximately 22 g was attained on the 63^rd day during the grain filling stage (Figure 6). Rice cultivars growing in 5 ds/m Na₂SO₄ promoted better fresh weight than NaCl stress and there were continuous increase in the fresh weight of rice cultivars under NaCl and Na₂SO₄ stresses (5 ds/m) until maximum fresh weights were attained (Figure 7). IR29 attained a maximum fresh weight of about 6 g on the 56^th day (reproductive stage) while IR20 and NERICA 1 recorded fresh weights of approximately 6.5 g on the 49^th day (vegetative stage). This was followed by sharp decreases till the end of the experiment. However, under Na₂SO₄ treatment, significant difference in the fresh weight of IR29 was observed between the 49^th and 63^rd day. This was followed by a sharp decrease till the end of the experimental period. Similar patterns of fresh weight increases were found in 10 and 15 ds/m of the Na salts throughout the experiment (Figures 8; Figure 9). IR20 recorded a fresh weight of approximately 7.2 g on the 42^nd day (vegetative stage). However, a fresh weight of approximately 8 g was recorded in IR29 and NERICA 1 on the 63^rd day (grain filling stage). IR20 (NaCl) and IR29 (Na₂SO₄) at 15 ds/m stresses showed exceptional fresh weight increases at 56^th day than other treatments.

9 Discussions

Threshold is intuitively a critical parameter for assessing plant salt tolerance differs across salt. Cultivars that are subjected to NaCl stress had their critical limit at 10 ds/m while the threshold of Na₂SO₄ is 5 ds/m. Hence, tolerance level of rice cultivars differs across salt and plant developmental stages. Beyond these limits, there is a considerable change in the response level from tolerance to sensitivity.

The most critical stage in seedling establishment is usually considered as seed germination which consequently determines the successful crop production (Khan et al., 2005). Understanding the responses of plants at these critical stages is particularly important for elucidating the mechanisms of salt resistance or sensitivity in plants and their survival. In this study, germination was completely inhibited for all varieties (POKKALI, IR 20, IR 29 and NERICA 1) at higher salinity concentrations of 15 ds/m in both treatments (NaCl and Na₂SO₄). Inhibition of germination due to salinity as suggested in previous reports (Kumer et al., 2009) was attributed to a decrease in water content that affected the synthesis of hydrolytic enzymes limiting the hydrolysis of food reserves from storage tissues to the developing embryo. The result of these findings is also in conformity with the report of Ashraf et al. (2000), that salinity stress reduced the rate of germination due to reduced water potential and slower rate of imbibition. Salinity alters the imbibition of water by seeds due to lower osmotic potential of germination media (Khan et al., 2005), causes toxicity which changes the activity of enzymes of nucleic acid metabolism (Bor et al., 2003), disturbs hormonal balance and reduces the utilization of seed reserves (Carden et al., 2010). Seed germination is one of the most fundamental and vital phases in the growth cycle of plants that determine plant establishment and the yield of crops. High level of salt stress inhibits the germination of seeds while lower level of salinity induces a state of dormancy (Joseph et al., 2010). The decreasing tendency of speed of germination (SG) due to increasing salinity was in conformity with the reports of others (Khan et al., 2005; James et al., 2000). Parida and Das (2005), reported that the reduction of SG at high salt levels might be mainly due to osmotic stress. Also, Parida and Das (2005) opined that salinity reduces plant growth due to osmotic stress and ionic effects on soil solution. However Mistra et al. (2005) was of the opinion that high accumulation of sodium ions (Na⁺) in saline soils causes lowering of water potential and thus makes the plant unable to extract water from soil due to osmotic stress. Meanwhile, this study did not conform to the result of Joseph et al. (2010) which reported that seed germination did not occur at 15 ds/m. The variability in salinity tolerance among rice varieties at germination have been reported (Hodges, 2003; Parida and Das, 2005). IR 20 had high germination percent (GP) but low germination energy percent (GE%) which possibly suggest that the seeds might had germinated earlier. However, the varieties with high GE% value in both treatments at specific salinity level obviously would be of higher competitive advantage than other varieties having lower GE% value for capturing environmental and edaphic resources. Even though salinity delays germination, higher salt concentrations will eventually reduce the percentage of germinated seeds (Haq et al, 2002).

Reduction in seedling height is a common phenomenon of many crop plants grown under saline conditions (Hasegewa, 2000). Also plant height under Na₂SO₄ was more suppressed than plant height under NaCl at each specific salt concentration level in both treatments. This however does not conform to the report of Ramohya et al. (2004). Islam et al. (2003) also observed the differences in plant height of rice varieties with different salinity levels. The higher plant height observed in IR20 under both treatments as compared to other varieties might be due to more inhibitory effect of NaCl salinity to root growth compared to that of shoot (Gong et al., 2006). Grant et al. (2000) earlier reported that plant height progressively decreased with increase in salinity levels because high concentration of soluble salt in soil and osmotic pressure creates disturbance in uptake of water and other nutrients. Ghoilam et al. (2002) reported that high salinity may inhibit the root and shoot elongation due to slowing down the water uptake by the plant. Also Gama et al., 2007 indicated that salinity can rapidly inhibit the root growth and its capacity to water uptake and essential mineral nutrition from soil. However the inhibition of root and shoot elongation as reported by Khan et al. (2005) were based on NaCl salinity. Meanwhile, in this study, shoot height elongations were more pronounced under NaCl than Na₂SO₄ treatment. This could be due to the more inhibitory effect of NaCl than Na₂SO₄ to water uptake and distribution of essential mineral nutrients. Flowers (2004) reported that the reduction in root and shoot development may be due to toxic effects of NaCl as well as unbalanced nutrient uptake by the seedlings. Saline conditions inhibit plant growth to variable extent either by reducing plant osmotic potential or because of specific ion toxicity (Flowers et al., 1991). The observed reduction in plant height of NERICA1 in both treatments might be might be due to decrease in water potential, high ion concentrations of Na⁺, Cl^- and SO₄^2- accumulation in leaves interfering metabolic processes; photosynthesis and protein synthesis. Higher concentrations of these ions in the root zone suppressed the uptake of other essential ions like K⁺ and Ca²⁺ thus resulting in reduced plant growth (Fedoroff, 2006). Significant reduction in plant height for various cultivars exposed to salinity levels was also reported by Khan et al. (2005). Thus, salt stress application had overall substantial negative effect on all visual growth parameters.

Root and shoot growth of all rice cultivars measured in terms of fresh weight and dry weight were considerably reduced at the grain filling stages. The findings in this study revealed that rice roots and shoot exhibited significant reductions under stress.

10 Conclusions

In this study, germination was completely inhibited for all varieties at the highest salinity concentrations in both treatments. This was attributed to a decrease in osmotic potential that affected the synthesis of hydrolytic enzymes limiting the hydrolysis of food reserves from storage tissues to the developing embryo. Specifically, IR20 had a higher germination percent than other cultivars. Growth analysis revealed that the cultivars tolerated Na₂SO₄ more than NaCl.

This study concludes that different traits are responsible for salinity tolerance in rice cultivars subjected to different salinity stress.

Acknowledgement

I wish to acknowledge the painstaking efforts of my supervisors; Prof. A. A. Adelusi and Dr. R. O. Akinwale, both of Obafemi Awolowo University, Ile-Ife, Nigeria, for guiding me through the course of this study.

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