Screening of Rice Accessions for Tolerance to Drought and Salt Stress Using Morphological and Physiological Parameters

Drought and salinity are the most widespread soil problems, posing a big threat to food security in rice growing regions. The present study evaluated the performance of eleven rice genotypes using morphological and physiological parameters, under induced drought and salinity conditions. The seedlings were raised in 5 kg of homogenous soil in plastic bags in the greenhouse. For the drought experiment, each bag was watered with 200 ml of water twice daily until plants reached the five-leaf stage when watering was suspended for 2 weeks for the drought stressed plants but not suspended for the control plants. The experiment was a 2 × 11 factorial and the set up was ar-ranged using the completely randomized design with three replications. Data were taken on Plant height, Number of tillers, leaf length, Number of green leaves, Number of dead leaves, Leaf rolling score (LRS) and Rate of water loss. The salinity experiment was set up in a similar manner except that the plants were irrigated twice a day for 2 weeks with 200 ml of treatment solution containing either 0 mM NaCl or 75 mM and data were collected on plant height, number of tillers, shoot fresh weight, shoot dry weight, Na + and K + concentrations, relative water content and chlorophyll content. Data from both ex-periments were subjected to Analysis of variance test using the GenStat soft-ware 10 th edition and the means separated using least significant difference test. Individual 55) were clearly susceptible. FARO 44 is the only genotype that showed tolerance to both drought and salinity. The identified drought and salinity tolerant rice genotypes from this study can be recommended as genetic sources for future breeding programs for drought and salinity tolerance in rice.


Introduction
Rice (Oryza sativa L.) is presently one of the prominent cereal crops, accounting for more than half of human caloric intake globally and valued for its nutritional benefits. It is high in fibre, contains vitamin E and some minerals such as potassium, calcium, magnesium, selenium, zinc and iron and has low cholesterol and sodium contents, hence its choice as a healthy and affordable source of energy [1]. Modelling simulations estimate that agricultural production will need to double by 2050, especially with regards to high-demand staple foods such as rice, in order to sustain the growing population [2]. In Nigeria, rice has a critical role to play because it is a staple, and the country consumes about 7 million tons of rice a year. Although Nigeria is one of the major producers of rice in SSA and has the potential to combat food insecurity presently facing the country, the country has exhibited less expansion in rice production. Over the years, the Nigerian government has introduced various policies and made concerted efforts towards increasing rice production in the country, but not much success has been recorded because of low yields. The low productivity in agricultural crops including rice is mostly attributed to various abiotic stresses. The major abiotic stresses which affect rice production in Nigeria include salt toxicity, drought and Nutrient deficiency [3]. They negatively influence the survival, biomass production and yield of rice, which is a major threat to food security worldwide [4].
Drought stress affects approximately 23 million ha of rainfed rice worldwide.
The situation is expected to worsen under the prevailing climate change [5] [6].
Drought stress results when water loss from the plant exceeds the ability of roots to absorb water and when the plant's water content is reduced enough to interfere with normal plant processes. Without adequate water, biological processes, such as photosynthesis, are greatly reduced. Reduced photosynthesis means reduced plant growth, including root growth, which leads to a reduction in yield.
The extent to which drought affects yield depends on its intensity and the time of occurrence within the crop growth cycle [7]. Its major impact is reported to occur during the flowering and grain filling phases, resulting in significant yield losses [8]. under low rainfall conditions and in areas without an appropriate irrigation system such as many parts of Nigeria. Furthermore, given the increasing scarcity of water resources, and competition for them, irrigation is not a practical option for alleviating drought in most of the rainfed areas. Drought management strategies therefore need to focus on maximizing extraction of available soil moisture and the efficiency of its use in crop establishment, growth, biomass and grain yield [9].
Features associated with drought-stressed plants include changes in root morphology, root penetrability and distribution; leaf rolling, reduced leaf area, stomatal closure, early flowering, early seed maturity, osmotic adjustment and increased production of Abscisic acid (ABA) [10] [11]. These can guide in the screening for drought-tolerant rice cultivars.
Soil salinity is a complex phenotypic and physiological phenomenon in plants, imposing ion imbalance or disequilibrium, ionic and osmotic stress, inducing oxidative stress and negatively influencing metabolic activities in crop plants, hence minimizing the productivity of crop plants [12]. Worldwide, more than 80 million hectares of irrigated land (representing 40% of total irrigated land) have already been rendered toxic to plants by salt [13]. Area under salt stress is rapidly increasing due to a combination of factors which include climate change, rising sea levels, excessive irrigation without proper drainage in inlands and underlying rocks rich in harmful salts amongst others. Salt stress leads to severe inhibition of plant growth and development, membrane damages, ion imbalances due to Na + and Cl − accumulation, enhanced lipid peroxidation and increased production of reactive oxygen species like superoxide radicals, hydrogen peroxide and hydroxyl radicals [14]. It has been estimated that if the current situation of increasing salinity stress would persist, it could result in the loss of 50% of present agricultural lands by 2050 [15].

Screening for Drought Tolerance
Seeds of eleven (11) rice varieties were pre-germinated at 28˚C by soaking in water for 2 days in labeled 50 ml falcon tubes wrapped in aluminum foil. Thereafter, five rice seedlings per variety were transferred to plastic bags containing 5 kg of homogenous soil and grown in the greenhouse. Each bag was watered with 200 ml of water twice daily until plants reached the five-leaf stage when watering was suspended for 2 weeks for the drought stressed plants, watering was not discontinued for the control plants. Data for growth parameters were taken at the end of the 2 weeks watering suspension (Zhang et al. [28] with slight modifications). The experiment was a 2 × 11 factorial and the set up was arranged using the completely randomized design with three replications. Tolerance to drought was evaluated based on the following parameters: Plant height, Number of tillers, leaf length, Number of green leaves, Number of dead leaves, Leaf rolling score (LRS) and Rate of water loss. Leaf rolling score was recorded at mid-day, 15 days after stress inducement using the scale described by [29], from 1 (fresh flat leaves) to 5 (tightly rolled leaves). To detect rate of water loss under dehydration conditions, flag leaves were detached from plants and exposed to air at room temperature (approximately 24˚C) and weighed at 0, 0.5, 1, 2, 3, 4, 5, and 6 hours after their detachment from the plant. This was done in 3 replications and the means used to calculate water loss rates as the percentage of initial fresh weight. Bar charts were used to highlight the effects in all cases. Data were subjected to analysis of variance test using the GenStat soft-ware 10 th edition and the means separated using least significant difference test. Individual stress response index (ISRI) for each parameter was calculated by dividing the trait value for a parameter under stress for a given variety by the trait value for that parameter under control. The ISRIs for all the parameters studied were added together to give the Cumulative stress response index (CSRI) for each variety [16]. The CSRI was divided by the number of parameters to obtain the mean which was used along with leaf rolling score and number of dead leaves in grouping the varieties into Highly tolerant, Tolerant, Moderately tolerant and Susceptible. The ISRI could not be meaningfully calculated for number of dead leaves because some varieties recorded zero dead leaves so the means recorded were used directly. Similarly, the leaf rolling scores were used directly because the value for control was 1.00 in all the varieties.

Screening for Salt Tolerance
Seeds were germinated as described in Section 2.2; thereafter five (5) rice seedlings per genotype were transplanted to labeled plastic bags containing approximately 5 kg of homogeneous soil obtained from the botanical garden of the University of Calabar. Three weeks after germination, the plants were irrigated for 2 weeks with 200 ml of treatment solution A (0 mM NaCl) or B (75 mM NaCl). The experiment was set up in a completely randomized design with three replications.

Evaluation of Morphological Parameters
After the two-week salinity treatment, data were taken on the following morphological parameters: Plant height, Number of tillers, Number of leaves, Number of dead leaves, shoot fresh weight and shoot dry weight. Shoot fresh weights of the plants were taken immediately after harvesting them from the greenhouse; thereafter they were oven-dried for 48 hrs at 80˚C and the shoot dry weights recorded.

Estimation of Physiological Parameters
The relative water content was estimated from shoot fresh and dry weights using the equation: where SFW = shoot fresh weight, SDW = shoot dry weight.
Chlorophyll was extracted from leaf tissues of control and salt treated plants, and quantified as follows: the third leaf from the base were systematically har- (Labtech, India). Chlorophyll content was then calculated as described by [30].
Chlorophyll was extracted from leaf tissues of control and salt treated plants where A663 = Absorbance value at 663 nm, A645 = Absorbance value at 645 nm, V = Volume of solvent used in ml, W = Weight of sample in mg.
Na + and K + concentrations were determined according to the method of Roy et al. [31], with slight modifications. Briefly, shoot and root tissues from the plants were harvested and dried at 80˚C for 48 h using UN75 plus oven (Memmert, Germany). Dry tissues were digested in 1% nitric acid overnight at 85˚C.
Na + and K + concentrations were determined using an Atomic absorption spectrophotometer (Cecil, England).

Data Analysis and Stress Response Characterization
Morphological and Physiological data were subjected to analysis of variance tests with the means separated using Least Significance Difference tests. Mean stress response indices were calculated as described in Experiment 1.

Screening for Drought Tolerance
The mean effects of drought stress (2 weeks

1) Number of dead leaves
When subjected to drought conditions, a significant (p < 0.001) reduction in number of dead leaves was observed in the rice genotypes compared with the control plants (Supplementary Table S1 & Table S2). FARO Figure 1).

2) Number of green leaves
Under drought conditions, significant (p < 0.001) differences in number of green leaves produced were observed in the rice genotypes compared with the control plants (Supplementary Table S1 & Table S2). NERICA 2 and NERICA 8 produced more green leaves than control and were thus the least affected by  Table S1).

3) Leaf length
Significant (p < 0.001) differences in leaf length were observed in the rice genotypes subjected to drought stress (Supplementary  Figure 3).  Figure 4). Leaf rolling score was significantly higher (p < 0.001) in stressed plants compared to control (Supplementary Table S1 & Table S2).

5) Number of tillers
There was a general reduction in the number of tillers produced under drought stress for all the genotypes except for NERICA 8 and NERICA 2 which produced more tillers than the corresponding control plants ( Figure 5) and had  Figure 6). Under stress conditions, NERICA 8, was the tallest with height of 51.33cm and this did not deviate much from control at ISRI value of 0.95 ( Figure 6).

7) Rate of water loss
Faro 44 had the highest water retention capacity by retaining 80% -100% of its moisture in the first four hours and after 6 hours it was able to hold over 50% of its water. On the other hand, Faro 55 immediately lost 45% of its moisture in 30 min and only 26.9% moisture content was observed after just 6 hours ( Figure   7).

Screening for Salt Tolerance
The mean effect of salt toxicity on growth parameters of eleven rice varieties is presented in Figures 8-15.

1) Plant height (PH)
A significant reduction (p < 0.001) in plant height was observed in some of the salt treated genotypes compared with their controls (Supplementary Table S3 &           Figure 10).

4) Number of tillers
Salt treatment caused a highly significant reduction (p < 0.001) in number of tillers in the treated genotypes compared with their controls (Supplementary   Figure 11).

5) Shoot Fresh Weight to Shoot Dry Weight ratio (SFW/SDW)
Data on SFW/SDW ratio showed no significant difference (p > 0.05) among the genotypes and also between the control and treated plants (Supplementary  Figure 12).

6) Shoot Sodium (Na + ) content
The concentration of sodium in the shoot of the rice genotypes were significantly (p < 0.001) increased by the salinity stress conditions (Supplementary Table S3 & Table S4). Under salinity conditions the maximum Na + concentration was observed in FARO 63 (3.36 µmol Na + /g DW) while the minimum was in NERICA 4 (1.99 µmol Na + /g DW) ( Figure 13).

7) Shoot Potassium (K + ) concentration
There was a general increase in the concentration of shoot K + under saline conditions except for NERICA 8, NERICA 2 and FARO 52 ( Figure 11, Supplementary Plant height and leaf length in the drought stressed plants were reduced compared to their control except for NERICA 5, NERICA 2 and NERICA 4 ( Figure   6, Table 2). Significant reduction in both parameters for drought stressed susceptible rice plants were also reported by Farooq et al. [36] and Kumar et al. [19]. Several researchers in an attempt to explain the reduction in plant height under drought stress have attributed it to the limited cell length and reduced green leaves which act as source for carbon assimilation [17]. In the present study, all the accessions with reduced plant height also had reduced number of green leaves to further corroborate the assertion. Decreased leaf elongation under water stress is similarly caused by reduced cell expansion arising from reduction in photosynthesis. Pandey and Shukla [37] had attributed reduction in leaf area in rice under water stress to reduced photosystem 11 (PS11) activity which results in rapid declines in cell division or cell size, sometimes leading to death of the cells. Loretto et al. [38] also added that the activity of the photosynthetic electron transport chain is affected by changes in PS11 under drought conditions.
Nooden [39] reported that drought causes early senescence of the leaves Tillering determines grain yield in rice plants. Reduced tiller number was reported as the major cause of yield reduction under drought stress during vegetative phase of rice growth [40]. In the present study, there was no reduction in the number of tillers for NERICA 8, NERICA 2 and FARO 44 under drought stress which further confirms their tolerance to drought. On the other hand, there was a 33% reduction in tillering for FARO 64 pointing to its susceptibility to drought stress. Similar record has been observed by Pantuwan, et al. [41] who noticed 52% to 81% decrease in number of tillers in rice plants under drought stress. Tiller abortion has been noticed in previous reports [17].
Significant variation was observed in Leaf rolling score (LRS) among the genotypes under drought stress ( Table 2). LRS has been identified as a standard parameter for estimation of drought stress [20] [42] [43] [44]. FARO 44 and NERICA 8 had the smallest LRS of 1.33 each (Figure 4), which indicates their resistance to drought stress conditions. On the other hand, FARO 64 had the highest LRS score of 4.33 which indicates its susceptibility to drought conditions. Leaf rolling is caused by loss of turgor and poor osmotic adjustment in rice plants [43]. It is a defensive mechanism for reducing net radiation load on the leaf [21].
For water retention, FARO 44 had the best performance which could be seen as a confirmation of its drought tolerance quality (Figure 7). Water retention ability indicates the degree of hydration in cells and tissues which is crucial for optimization of growth processes in plants. The ability to retain higher relative water content under drought condition is a tolerance mechanism for water scarcity in rice [45]. When the means of the Individual Stress Response Indices for all the parameters were taken into consideration, NERICA 8, NERICA 5, NERICA 2 and FARO 44 were clearly tolerant to drought stress which further confirms the tolerance report on FARO 44 by Afiukwa et al. [1].

Salinity Tolerance
In this study, tolerance to salinity stress was estimated using morpho-physiologic traits in the eleven rice accessions. Morphological parameters such as shoot dry weight (SDW), shoot fresh weight (SFW), and tillering are reported to be significantly correlated with salt tolerance at different stages of growth and can be used as indicators for estimation of salt tolerance [22]. In this study, salinity caused reduction in plant height in many of the rice accessions (to different levels) compared with their controls which is consistent with reports by Chinnusamy et al. [23]. There was, however, no reduction in the plant height of RAM  [24] of the rice plants which eventually reflects on the total yield of the plant. Salinity caused reduction in the number of tillers per plant with maximum reduction (70%) observed in FARO 55 while RAM 137 had the minimum reduction (4%), ( Figure  15). This result also indicates the tolerance of RAM 137 to salinity conditions and the sensitivity of FARO 55. The finding is in agreement with Zeng and Shannon [25] and Tanveer-Ul-Haq et al. [24] who also observed a significant reduction in tillering in rice plants exposed to salt. In the present study, it was observed that salinity caused a significant reduction in SFW and SDW. According to Munns et al. [46], this reduction in biomass could be attributed to decreased water potential of the rooting medium and growth inhibition caused by the salinity stress. Ashraf and Sawar [47] further attributed the reduction in plant biomass under salt stress to imbalances in the uptake of mineral nutrients due to competition with the excess influx of Na + . In this study, the lowest reduction in SFW was noticed in FARO 44 (6.5%) while the maximum reduction was observed in FARO 55 (67%). Na + increased significantly under salinity conditions in all the accessions. FARO 44, RAM 137 and NERICA 4 had the least accumulation of Na + under salinity conditions which indicates their good tolerance level to salt stress, while FARO 55, FARO 64 and FARO 63 had the highest accumulation of Na + signifying their sensitivity to salinity stress. Salt tolerant varieties generally maintain lower concentrations of Na + in their shoot than those of salt sensitive varieties under excess salt conditions [48]. Indeed, Munns and Tester [12] noted that the ability of crops to maintain low cytosolic levels of Na + in leaves is taken to be one of the major determining factors for salt tolerance. Higher concentrations of Na + are harmful to plant growth and could be responsible for reduction in plant biomass [24]. The relationship between Na + and K + concentration in plants follows that a good supply of K + to plants could reduce the rate of injury as a result of high Na + concentration under salinity conditions [24]. A positive relationship between high K + /Na + and salinity tolerance has been established by some workers [26].
Total leaf chlorophyll content was significantly reduced under salinity condi-  (Table 3). This result is in agreement with reports by Uyoh et al. [29] and Kargbo et al. [49] on the status of FARO 61. Most of the varieties used in these studies were different. Also, the parameters analyzed were different except for shoot length and biomass which may also account for the differences observed.

Conclusion
The present study evaluated the performance of eleven rice genotypes using morphological and physiological growth parameters, under induced drought and salinity conditions. Generally, significant differences were obtained (p <  Means denoted with the same letter and within each column are not significantly different (p ≥ 0.05). LSD = Least Significant Difference. NS = Not significantly different. PH = plant height, SDW = shoot dry weight, SDS = salinity damage score, SFW = shoot fresh weight, K + = potassium content, Na + = sodium content. *, = significant at ≤0.05 ** = significant at ≤0.01. N.S = not significant. SDW = shoot dry weight, SDS = salinity damage score, SFW = shoot fresh weight, K + = potassium content, Na + = sodium content.