Drought stress affected different morphological attributes of plants, and 19 percent reduction was recorded in shoot lengths of both canola cultivars as compared to control (Fig. 1-a). The application of MET and SA caused a significant increase in plant height in both normal and stressed conditions. MET priming appeared to be most effective treatment of all under both normal and drought-stressed conditions, as an increase of 22% in plant height was recorded in MET primed (T1), super canola plants compared with non-treated drought-stressed plants (Fig. 1-a). Similarly, plant height was improved by 17% in plants treated with MET priming combined with foliar spray of SA (T7). Shoot fresh and dry biomass also show a declining trend when plants were exposed to drought and a reduction of about 14% in fresh biomass and 17% in dry weight was recorded (Fig. 1-c, d). Application of plant growth regulators proved beneficial in reducing the negative effects of drought shoot fresh and dry biomass. ME priming showed better results among all treatments where about 46% better fresh biomass and 40% better dry biomass was recorded in both canola cultivars under drought stress (Fig. 1-c, d). These results were statistically non-significant from the plants treated with combined application of melatonin priming and salicylic acid foliar spray. Overall, V1 showed better growth under drought than V2.
Photosynthetic rate showed a significant reduction (43%) after canola cultivars were exposed to drought. Seed priming treatments with plant growth regulators improved the photosynthetic rate of both cultivars under normal as well as stressed conditions (Fig. 2). Seed priming treatment with MET (T1) caused a significant increase of 43% in photosynthetic rate, followed by those plants which were treated with combined ME and SA (T5) priming (Fig. 2-a). A similar sort of drought stress caused reduction in transpiration rate (73%) and stomatal conductance (55%) was recorded in both canola cultivars (Fig. 2-b, c). Seed priming treatment with MET (T1) and SA (T3) showed better results and improved transpiration rates and stomatal conductance under drought in both canola cultivars (Fig. 2-b, c). Foliar spray of SA (T4) and combined foliar treatment of SA and MET (T6) slightly improved all photosynthetic attributes in stressed environment, but their impact was not as significant as of all other treatments involved. Among the cultivars, V1 showed better results than V2.
Declining trend was recorded in water and osmotic potential values of canola plants after exposure to drought stress (Fig. 3-a, b). Seed priming and foliar spray application of both plant growth regulators improved water potential and osmotic potential under both controlled and stressed conditions. Maximum water and osmotic potential values under normal and drought-stressed environments were recorded in MET primed plants (T1), followed by combined treatment of MET priming and SA foliar spray (T7). Combined foliar spray treatment (T6) of both growth regulators didn’t show as significant effect on these parameters as shown by other treatments involved (Fig. 3-a, b).
An increase in compatible solutes concentration i.e., soluble proteins, free amino acids, and total soluble sugars were recorded after drought stress treatment in canola plants (Fig. 4). An increase of 15% was recorded in protein contents under drought stress, compared to water-treated plants. These Proteins contents were further increased by MET and SA application, and the maximum increase was recorded in MET primed (T1) plants (Fig. 4-a), where an increase of 25% in protein contents was recorded under drought, compared to non-treated drought-stressed plants (T0). Similarly, about 20% more free amino-acid contents were recorded in drought stress, as plants accumulate more amino acids to cope with drought stress. Amino-acid contents were further enhanced by seed priming treatment of MET combined with foliar spray of SA (T7), where about 34% more amino-acid activity was found compared with non-treated (T0) water-stressed plants (Fig. 4-b). A slight increase (5%) was recorded in total soluble sugar contents of drought-stressed cultivars, compared to well-watered plants. Hormonal priming treatment further enhanced these contents, and MET priming treatment combined with SA foliar spray (T7) proved to be most effective under drought stress. Foliar spray of SA (T4) and combined foliar treatment of SA and MET (T6) slightly improved all biochemical attributes under water deficit conditions, but their impact was not as significant as of all other treatments involved (Fig. 4-c). Among the cultivars, V1 showed better biochemical activity than V2.
Antioxidant levels i.e., Peroxidase (POD), Superoxide Dismutase (SOD), and catalase (CAT) activity increased in drought exposed canola plants, compared to well-watered plants (Fig. 5). An increase of 23% in POD levels was observed in drought-stressed canola cultivars. Application of plant growth regulators further enhanced POD contents under both normal and stressed conditions. Maximum results were shown by combined treatment of MET as seed priming and SA as foliar spray (T7) under both normal and drought stress conditions (Fig. 5-a). These results were statistically non-significant from MET priming treated plants (T1). Similarly, an increase of 20% in SOD contents was recorded in drought-stressed canola plants. MET priming combined with SA foliar spray (T7) further enhanced these contents and about 17% increase under normal and 28% increase under stressed conditions was recorded (Fig. 5-b). CAT contents also showed a significant increase (27%) after drought exposure (Fig. 5-c). MET priming (T1) further increased catalase contents by 35% under stressed environment and by 14% under normal conditions. Foliar spray of SA (T4) and combined foliar treatment of SA and MET (T6) slightly improved SOD, POD and CAT levels in stressed environment, but their impact was not as significant as of all other treatments involved (Fig. 5-a, b, c). Among the cultivars, V1 showed better antioxidant activity than V2.
Yield attributes of canola showed significant reduction after exposure to drought stress, where about 30% less siliqua per plant and 18% and 30% lower number of grains per siliqua were recorded in Super and Faisal canola respectively, as compared to control (Fig. 6-a, b). However, a considerable increase (49 and 47%) in siliqua per plant and an increase of 40 and 56% in the number of grains per siliqua was recorded in Super (V1) and Faisal canola (V2) respectively, after treating plants with MET and SA under drought. Maximum no. of siliqua per plant under drought conditions were shown by MET priming treated plants (T1), followed by plants treated with SA (T3) priming (Fig. 6-a). Similarly, the maximum number of grains per siliqua was recorded in those plants which were treated with MET priming combined with foliar spray of SA (T7) in a stressed environment (Fig. 6-b). Foliar spray of SA (T4) and combined foliar treatment of SA and MET (T6) slightly improved plant yield attributes in both controlled and stressed environment, but their impact was not as significant as of all other treatments involved. Among the cultivars, V1 showed a slightly better yield than V2.
Relationship and multivariate analysis
In order to verify the relationship between different studied attributes, we have plotted Pearson correlation and principal component analysis (PCA-biplot) (Fig. 7-8). This analysis disclosed correlation (positive and negative) among various parameters of canola cultivars under drought and normal conditions. It has been noticed that drought stress imposed significant impact on all the parameters. Fig. 7(a-b) showed significantly positive correlations among biochemical attributes, enzymatic antioxidant, and plant water relations both under drought and control conditions. On the other side, biochemical parameters showed a negative correlation with root length and stomatal conductance. Plant height and fresh weights were positively impacted by water relation attributes, but transpiration rate showed negative relationship with them. Overall transpiration rate (E) had negatively correlation with all other attributes under control conditions but showed almost zero relationship with number of grain per silique and number of seed per plant, while photosynthetic rate had shown positive correlation both under drought as well as controlled conditions due to application of melatonin and salicylic acid (Fig. 7a, b).
Our Pearson’s correlation among various attributes of both canola varieties have been further validated by PCA-Biplot. Fig. 8 represents PCA-Biplot of different attributes in canola plants subjected to drought stress with foliar and primed seed treatments of growth regulators. Principal component analysis executed the degree of association between variables under drought and controlled environment. Both dimensions of PCA i.e., Dim-1 and Dim-2 jointly elaborated 82.6% variability in the dataset. They showed obvious and marked separation of attributes under stressed and non-stressed conditions. Dim-1 showed 44.9%, while Dim-2 observed 37.7% variation to the total variance. There was a clear separation in studied parameters under normal and drought stress. Biochemical and antioxidant traits showed more relationship and impact under drought while growth attributes showed better relation in controlled conditions (Fig. 8).