Evaluation of Physiological and Biochemical Parameters of Some Wheat (Triticum aestivum) Genotypes under Salinity Stress

Seeds of wheat genotypes KH-65, KRL-210, KRL-99, PBW-343 and PBW-373 were kindly provided by the Indian Institute of Wheat and Barley Research (IIWBR) Karnal and Central Soil Salinity Research Institute (CSSRI), Karnal, Haryana, India. 
 
The seeds of wheat genotypes were sterilized by 0.1% HgCl₂ aqueous medium for a time period of 5 minutes and then rinsed, thrice. The sterilized seeds were imbibed in distilled water for two hours and allowed to germinate in autoclaved sand. These were then transferred to hydroponic culture media for 48 hours prior to their saline treatment in a growth chamber. The seedlings at three-leaf-stage were treated with 50, 100, 150, 200, 250 and 300mM NaCl prepared in half strength of modified Hoagland solution (Jones, 1982). The plants were allowed to grow for 48 hours at 20°C under 16 hours light and 8 hours dark photoperiod per day at 2000 lux (Wang et al., 2008) and the plantlets were then harvested.
 
The roots and shoots of seedlings were weighed accurately, within ± 0.01mg, using an analytical balance (METTLER TOLEDO, Model: ML204 /A01) and the average of triplicate measurements was taken. The lengths of shoots or roots of seedlings were measured in cm within ±0.1mm using a standard meter scale. Total chlorophylls and carotenoids concentrations and their percent loss in studied wheat genotypes were determined, spectrophotometrically as described by Costache et al., 2012 and Hussain et al., 2006. Proline levels were measured according to Bates et al., 1973. Modified method of Heath and Packer (1968) was used for the evaluation of lipid peroxidation levels by measuring MDA (malonyldialdehyde) content. Hydrogen Peroxide (H₂O₂) concentrations were obtained by using a method of Velikova et al., (2000).
 
The observed physiological and biochemical data were subjected to statistical analysis by using ANOVA. All the variables in the recorded data are presented as mean ± standard deviation. The data when subjected to one-way ANNOVA was found statistically significant. Principal component analysis (PCA) was done to find the independent predictor of analysis with the help of SPSS-16.

Effect of salinity stress on plant phenotypic parameters
 
Physiological parameters i.e. shoot heights, root lengths, shoot weights and root weights of plantlets in studied wheat genotypes treated with varying salinity levels at 48 hours of growth are compared in Table 1 to 4. Significantly differential effects in their values were observed on varying salinity stress level (p<0.0001) and these parameters were negatively affected by increasing salinity stress. These parameters were considered at 5% level of percentage decrease. In shoot height (Table 1), KH-65 shows the least variation with increasing stress levels. At the same time two cultivars KRL-210 and KRL-99 show the significant reduction at 150mM and 100mM respectively. However, the sensitive cultivars i.e. PBW-343 and PBW-373 show the critical levels at 50mM only. Similarly, critical decrease for root length (Table 2) was shown at 100mM in KRL-210 and KRL-99 whereas PBW-343 and PBW-373 shows same decrease at 50mM. Plant weight is most sensitive character in relation to salt stress in both shoot (Table 3) and root (Table 4). The cultivars showed almost the same trend. KRL-99, PBW-343 and PBW-373 achieved the 5% decrease at initial level of stress application i.e. at 50mM in both cases. Shoot weight shows same level in KH-65 and KRL-210 at 150mM and 100mM treatment respectively whereas in root same level was found at 100mM for both KH-65 and KRL-210. The observed decrease in these physiological parameters upon enhancing salinity level may be due to the toxic effects caused by osmotic stress in the plants, poor nutrient uptake and hampered water uptake process (Datta et al., 2009).  Bilkis et al., (2016) reported similar salinity effects on some physiological and agronomic traits of wheat and as per the findings of Byrt et al., (2018) root growth of most of the crop plants is inhibited by soil salinity. The results indicate that salt sensitivity of the studied wheat genotypes are in the order: PBW-373 > PBW-343 > KRL-99 > KRL-210 > KH-65. Whereas, PBW-343 and PBW-373 showed high susceptibility to salinity stress even at very low salt concentrations. On the contrary, the genotype KH-65 exhibits highest tolerance even upto 300 mM salinity level.
 

 

 

 

 
Effect of salinity stress on plant physiological and biochemical parameters
 
Estimates of chlorophyll, carotenoids, lipid peroxidation, proline and hydrogen peroxide with varying salt concentration at 48 hours in seedlings of studied wheat genotypes are presented in Fig 2(a) to 2(e), respectively. Chlorophyll as well as carotenoid degradations in the plantlet seedlings at salt levels upto 300 mM were found in the order: PBW-373 > PBW-343 > KRL-99 > KRL-210 > KH-65. As suggested by Ibrahim et al., 2017 and Zhao et al., 2007, higher activity of enzyme chlorophyllase and the decreased rate of chlorophyll synthesis might be responsible for decrease in levels of two pigments at higher salinity concentrations. Similar results on chlorophyll degradation under salinity stress were also reported by other researchers (Sairam et al., 2005; Yildiz and Terzi, 2013). Except chlorophyll and carotenoid content all other biochemical test (lipid peroxidation, proline and hydrogen peroxide) were performed at 0, 100, 200 and 300mM salinity levels to obtain significant difference.   
 

 
Lipid peroxidation level in plants is estimated through MDA measurement is an indicator of oxidative stress under salinity conditions.  Higher the MDA concentration in a plant more is the oxidative stress as well as its susceptibility towards salinity (Ashraf et al., 2010; Khaliq et al., 2015). Lipid peroxidation levels in KH-65, KRL-210 and KRL-99 are of same level but are lower by 15% and 50% compared to PBW-343 and PBW-373, respectively. Lipid peroxidation has also been used as an essential parameter in grading salinity tolerant genotypes in other crops such as sorghum and barley and concluded similar relationship of lipid peroxidation level with plant tolerance towards salinity (Brankova et al., 2005). Based upon above observation, salinity sensitivity of different wheat lines have been graded as PBW-373 > PBW-343 > KRL-99 > KRL-210 > KH-65.
 
Different metabolic products accumulated inside plants upon exposure to stress conditions play role plant metabolism e.g., amino acids, precursors of proteins. Proline an important plant osmolyte produced more in stressed conditions provide tolerance to the plants by decreasing oxidative stress (Hayat et al., 2012). Under salinity, proline level increases in cytosol to adjust osmotic imbalance by scavenging ROS produced inside durum wheat (Annunziata et al., 2017). It is also believed that proline being a chaperone molecule, improves enzymatic activities by protecting protein structures (Ashraf and Foolad, 2007; Szabados and Savour’e, 2009). Proline level in plants is significantly affected by salinity stress (p <.00001). Higher accumulation of proline in seedlings of salinity-tolerant varieties than salinity-susceptible ones of rice have been reported by Abdelaziz et al., (2018). Our observation on proline accumulation in seedlings of studied wheat genotypes and their salinity tolerance levels may be put in the order as: KH-65 > KRL-210 > KRL-99 > PBW-343 > PBW-373.
 
Hydrogen peroxide (H₂O₂) a signaling molecule, generated from plant metabolism is involved in plant development and abiotic responses. Being as ROS, its production increases during stress conditions. Due to its oxidative nature, it imparts toxic effects in plants (Petrov and Breusegem, 2012). Niu and Liao (2016) studied that under stressed conditions H₂O₂ affects plant processes therefore have impact on plant health. For each studied genotype, the observed H₂O₂ level increases with the increasing salt concentration and a lower level of H₂O₂ in plants may be correlated to their higher tolerance to the salinity stress (Caverzan et al., 2016). Ali et al., (2017) have also reported higher production of H₂O₂ levels upon salinity stress in some wheat plantlets. Hydrogen peroxide levels with increasing salinity concentrations at 48 hours inside seedlings of studied wheat genotypes were found in order: PBW-373 > PBW-343 > KRL-99 > KRL-210 > KH-65.
 
The principal component analysis (PCA) was used as a statistical tool to predict the most contributing factor (Jolliffe, 2002) for comparing levels of salinity tolerance or sensitivity of the studied wheat genotypes. Results of PCA loading plots obtained from biochemical data of five wheat cultivars subjected to salinity stress are presented in Fig 3.  It was found that total chlorophyll and carotenoid content are the most susceptible factor for stress in this investigation.
 

The physiological and biochemical parameters under salinity stress in seedlings of wheat genotypes: KH-65, KRL-210, KRL-99, PBW-343 and PBW-373 have been evaluated to assess their sensitivity upto 300mM. A significant correlation was seen among the studied parameters with increasing soil salinity levels. Based upon the observed physiological and biochemical data for the wheat lines, salinity tolerance was found in the order: KH-65 > KRL-210 > KRL-99 > PBW-343 > PBW-373. Therefore, it can be hypothesized that observed varying responses of the wheat genotypes to the salinity stress may be due to their genetic variations and the tolerant lines can be used or farming under salt stress conditions for higher yields.