==== Front ACS Omega ACS Omega ao acsodf ACS Omega 2470-1343 American Chemical Society 10.1021/acsomega.3c01715 Article Seed Priming Modulates Physiological and Agronomic Attributes of Maize (Zea mays L.) under Induced Polyethylene Glycol Osmotic Stress Kakar Hussain Ahmad † Ullah Sami *† Shah Wadood *‡ https://orcid.org/0000-0003-1553-2248 Ali Baber § Satti Sanam Zarif ‡ Ullah Rehman † Muhammad Zahir † Eldin Sayed M. ∥ https://orcid.org/0000-0003-2319-2175 Ali Iftikhar ⊥# Alwahibi Mona S. ∇ Elshikh Mohamed S. ∇ Ercisli Sezai *○◆ † Department of Botany, University of Peshawar, Peshawar 25120, Pakistan ‡ Biological Sciences Research Division, Pakistan Forest Institute, Peshawar 25120, Pakistan § Department of Plant Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan ∥ Future University in Egypt, Center of Research, Faculty of Engineering, New Cairo 11835, Egypt ⊥ University of Swat, Centre for Plant Science and Biodiversity, Charbagh 19120, Pakistan # Department of Genetics and Development, Columbia University Irving Medical Center, New York, New York 10032, United States ∇ Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia ○ Department of Horticulture, Agricultural Faculty, Ataturk Universitesi, Erzurum 25240, Türkiye ◆ HGF Agro, Ata Teknokent, TR-25240 Erzurum, Türkiye * Email: sami_jan69@yahoo.com. * Email: wadood0301@gmail.com. * Email: sercisli@gmail.com. 15 06 2023 27 06 2023 8 25 2278822808 14 03 2023 17 05 2023 © 2023 The Authors. Published by American Chemical Society 2023 The Authors https://creativecommons.org/licenses/by/4.0/ Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/). Drought and osmotic stresses are major threats to agricultural crops as they affect plants during their life cycle. The seeds are more susceptible to these stresses during germination and establishment of seedlings. To cope with these abiotic stresses, various seed priming techniques have broadly been used. The present study aimed to assess seed priming techniques under osmotic stress. Osmo-priming with chitosan (1 and 2%), hydro-priming with distilled water, and thermo-priming at 4 °C were used on the physiology and agronomy of Zea mays L. under polyethylene glycol (PEG-4000)-induced osmotic stress (−0.2 and −0.4 MPa). The vegetative response, osmolyte content, and antioxidant enzymes of two varieties (Pearl and Sargodha 2002 White) were studied under induced osmotic stress. The results showed that seed germination and seedling growth were inhibited under osmotic stress and germination percentage, and the seed vigor index was enhanced in both varieties of Z. mays L. with chitosan osmo-priming. Osmo-priming with chitosan and hydro-priming with distilled water modulated the level of photosynthetic pigments and proline, which were reduced under induced osmotic stress; moreover, the activities of antioxidant enzymes were improved significantly. In conclusion, osmotic stress adversely affects the growth and physiological attributes; on the contrary, seed priming ameliorated the stress tolerance resistance of Z. mays L. cultivars to PEG-induced osmotic stress by activating the natural antioxidation enzymatic system and accumulating osmolytes. King Saud University 10.13039/501100002383 RSP2023R173 document-id-old-9ao3c01715 document-id-new-14ao3c01715 ccc-price ==== Body pmc1 Introduction Drastic shifts in environmental regimes are becoming a hindrance in achieving the growing demand of food and acquiring sustainable agriculture.1,2 Climatic changes lead to fluctuations in temperature,3 droughts, floods, and other environmental calamities, eventually leading to decrease crop productivity.4 The abiotic stresses such as drought,5 extreme temperature, frost, heavy metals,6−15 and salinity16,17 severely impair plant growth and productivity worldwide.18,19 Drought, being the most important environmental stress, severely damages plant growth and development.20 Salt stress leads to an imbalance between antioxidant concentrations and reactive oxygen species (ROS) levels, thus resulting in oxidative stress.21 Salinity-induced production of reactive oxygen species (ROS) causes damage to mitochondria and chloroplasts.22,23 Salt stress adversely affects almost every aspect of the physiology and biochemistry of plants and significantly reduces yield, the most serious threat to agriculture and major environmental factor that limits crop growth and productivity.24,25 Drought and salinity stresses lead to another abiotic stress, the “osmotic stress”. Osmotic stress severely affects plants during their life cycle; it results in leaf chlorosis and antioxidant’s imbalance.24 However, reduction in growth depends upon the duration and the severity of stress.26−29 In this connection, many scientific studies have concluded that osmotic stress hampers the growth of leaves, stems, roots, and total plant dry mass.25,30 Drought stress is a major threat for agricultural crops as the population of the world is increasing at an alarming rate, thus fulfilling their water demand that leads to worsening of the water deficit condition.31 Drought stress is the major cause of crop loss as it reduces yield components, such as reduction in the leaf size and number of grains.32 A major proportion of agriculture land is affected with varying degrees of drought and low atmospheric humidity leading to drought, which is the limiting factor for better plant performance and higher crop yield.33,34 A proper amount of soil moisture is compulsory for plant growth, transpiration, and also for transportation of food prepared in the process of photosynthesis in leaves.35 A number of strategies have been devised to overcome the adverse effects of abiotic stresses, such as the selection in vitro propagation of drought resistant cultivars,36 germplasm,37 and plant breeding methods,38 but all these strategies are expensive. However, an alternative strategy for the possibilities to overcome salt and drought stresses is seed priming. Nowadays, seed priming techniques such as hydro-priming, osmo-priming, thermo-priming, and hormonal priming have been used to enhance emergence of roots and shoots, attaining vigorous plants, and better drought tolerance in many field crops,39 such as wheat, maize, chickpea,40 sunflower,41 and cotton.42 Salinity is the buildup of soluble salts by which saline soils are formed, and the concentration of salt in soil is above the normal levels.43 Salinity is one of the most serious factors, which limits the growth and development of plants.44,45 It adversely affects seed germination, plant vigor, and crop yield.25 Salinity may be due to many factors, but some of the adverse effects of salinity have been attributed to increase in sodium and chloride ions in different plant organs; hence, these ions create the critical conditions for plant survival by interpreting different vital plant mechanisms.46,47 Sodium and chloride are the major ions, which cause many physiological disorders in citrus and limit plant growth and productivity.48 Excess of these salts also enhances the osmotic potential of the soil matrix as a result of which water intake by plants is restricted.49 Salinity stress reduces the chlorophyll content of sensitive species more in comparison to tolerant species.50,51 The most negative effect on seed germination is the presence of salts in the soil.52 It has been reported that not only do the differences in plant response to the amount of salt available in soil and irrigation water depend on plant species, but crop development stages and seedling growth stages are also the most vulnerable stages in the life cycle of plants.53 Therefore, these stages are focused and taken into consideration when the salt tolerance potential of a plant is determined.54 Over 6% of the world’s total land area and 20% of the irrigated land area are affected by salinity stress. Salinity has reached a level of 19.5% in all the irrigated lands (out of 230 million hectares of irrigated land, 45 million hectares are salt-affected soils) and 2.1% in dry lands worldwide. Almost 20% of the cultivated area of the world and half of the world’s irrigated lands are stressed with salinity.55 The condition in which water is deficit due to high levels of salinity or drought is known as osmotic stress, and it creates ion toxicity and disturbs ionic balance.56 Osmotic stresses affect plants during their life cycle as seeds are mostly susceptible to these stresses between sowing and seedling establishment.57 Germination and seedling growth of the plants decrease due to nutritional imbalance, similarly in saline conditions due to an external osmotic potential that prevents water uptake or due to the toxic effects of Na+ and Cl– ions or both on the germination of seeds.58 Salinity and drought stress reduce the plant growth and development through specific ion effects, nutritional imbalance, low osmotic potential of soil solutions, and combination of all these factors.59 Osmotic stress can affect various major plant processes like photosynthesis, protein synthesis, and lipid metabolisms. Generally, salt stress causes both osmotic stress and ionic stress.60 The osmotic effect initially reduces the ability of the plant to absorb water.61 Several minutes after the initial decrease in leaf growth, a gradual growth recovery takes place until a new steady state is reached, depending on the salt concentration outside the root.25 Osmotic stress disturbs plants’ physiological and biochemical processes due to water stress conditions, which is related to a decrease in rate of photosynthesis, closing of stomata, ultimately interrupting photosynthetic pigments and protein formation.62 A reduction in net photosynthetic rate under drought stress conditions is also related to disturbances in biochemical processes of a non-stomatal nature, caused by oxidation of chloroplast, lipids, and changes in the formation of pigments and proteins.63,64 In seed priming, seeds are pre-soaked in distilled water or osmotic solutions. Seed priming is a simple, cost-effective, and compelling approach employed for the enhancement of swift seed germination, early seedling growth, and improved yield under normal and stressed conditions.65 Priming is a form of seed preparation in which seeds are pre-soaked before planting.66 To enhance the resistance of plants to abiotic stresses, the seed priming technique is being used. In this technique, the seeds are soaked in various solutions or exposed to varying degrees of temperatures prior to sowing. Seed priming with organic and inorganic compounds, antioxidants, and hormones have insured an extensive survivability in crop plants under osmotic stress.65 Seed priming is an affordable, economical, and effective scientific procedure for the improvement of seed germination, early seedling, and yield under osmotic stress conditions.67 The chitosan α,β-(1,4)-glucosamine polymer is a safe, natural, and cheap polysaccharide and is produced from chitin, which is the major structural component of the fungus cell wall and the exoskeleton of arthropods.68,69 Chitosan can be used as plant fertilizer as it promotes seed germination, enhances germination percentage, and can modulate the responses of plants to abiotic stresses.70,71 Chitosan priming improves maize germination and seedling growth in relation to physiological changes under low temperature stress; it is used as osmo-priming to decrease the adverse effect of abiotic stress. Chitosan is obtained by deacetylation of chitin.72 The present research work was aimed to study the effect of various priming techniques on Zea mays L. under induced PEG osmotic stress. The effects of chitosan as osmo-priming, distilled water as hydro-priming, and thermo-priming on the physiological and agronomic performance of two cultivars of Z. mays L. were assessed, and a comparison was done between these priming techniques under polyethylene glycol (PEG-4000)-induced osmotic stress. The efficacy of priming techniques in regulating the key metabolic activities improves the osmotic stress tolerance capacity of maize cultivars subjected to varying levels of induced (PEG) osmotic stress conditions. 2 Materials and Methods 2.1 Soil Elemental Analysis The soil texture class was calculated as silt loam. Soil pH was 6.0, electrical conductivity was 2.41 ds/m,73 soil nitrogen (N) content was 2.05 g/kg, organic carbon (C) content was 20.5 g/kg, and potassium (K) available was 90.5 mg/kg.74 2.2 Area of Study and Experimental Design The pot experiment was conducted at the green house of the Department of Botany, University of Peshawar, Pakistan during the month of February 2018. Peshawar is located in the Iranian plateau having tropical climatic conditions. It is the largest and capital city of the Khyber Pakhtunkhwa province of Pakistan (Figure 1). The temperature of Peshawar ranges from 5 °C (in January) to 39 °C (in June). The total area of Peshawar is 1257 km2 with an elevation of 340 m/L, 115.49 feet. The images in Figure 2 depict four land use classes including vegetation, water bodies, urban area, and barren land during the years 1996, 2003, and 2016. Figure 1 Spatial map of the study area.75 Figure 2 Land cover of Peshawar since 1996, 2003, and 2016.75 Seeds of two cultivars (Pearl and Sargodha) of maize (Z. mays L.) were collected from the National Agriculture Research Centre, Islamabad (NARC). The temperatures of Peshawar in the month of February were 20 and 7 °C (high/low), humidity was 61%, and light duration was 10.9 h/day. Before sowing, the seeds were surface-sterilized with 70% ethanol and 0.1% mercuric chloride solution. After surface sterilization, the seeds were rinsed with distilled water. In addition, identical sized and smooth surfaces seeds were selected for the proposed trial. Ten pre germinated seeds of each cultivar (Pearl and Sargodha) both primed and non-primed were sown in pots. Watering of pots was done during the whole growing season regularly. For better growth of seedlings, the pots were exposed to sunlight and kept free from weeds by uprooting weeds periodically. Out of the total 90 pots, 30 pots were taken as the control group and the remaining 60 pots were applied with different levels of osmotic stresses (−0.2 and −0.4 MPa) using polyethylene glycol (PEG) 4000. Three replicates of each treatment were exposed to osmotic stress. During the entire experiment, all the standard practices were done from time to time. The plants were then harvested and frozen for the evaluation of physiological and agronomic studies in the laboratory. 2.3 Osmo-Priming For osmo-priming, the seeds were primed with chitosan (1 and 2% solution) for 3 h followed by washing with distilled water thrice and kept for a period of 2 days in the oven for the purpose of drying at 26 ± 2 °C.76 2.4 Thermo-Priming Thermo-priming of seeds was done by keeping the seeds at 4 °C for 1 h in the freezer. The seeds were then washed with distilled water and kept in the oven for a period of 2 days for the purpose of drying at 26 ± 2 °C.77 2.5 Hydro-Priming For hydro-priming, the seeds were put in distilled water and soaked for 24 h. The seeds were then filtered and kept for 2 days in the microwave oven at a temperature of 26 °C for drying.77 2.6 Induction of Osmotic Stress Osmotic stress was induced using polyethylene glycol (PEG) 4000. PEG solution (20 mL) was directly given to pots after sowing of seeds. PEG 4000 solution was prepared using the standard procedure in ref (78). PEG (14 g) was dissolved in 100 mL of distilled water to induce −0.2 MPa pressure. PEG 4000 (28 g) was dissolved in 100 mL of distilled water to induce −0.4 MPa pressures.79 2.7 Agronomic Characteristics 2.7.1 Absolute Growth Rate (AGR) Absolute growth rate is the total growth rate per unit of time. After 10 days of induction of osmotic stress, three replicates of each treatment were taken to find the mean of each measurement with the help of the following formulas. Absolute growth rate (AGR) was calculated with the help of the formula recommended in ref (80). where H1 and H2 are the plant heights (cm). W1 and W2 are the plant dry weights at time t1 and t2, respectively. 2.7.2 Relative Growth Rate (RGR) Relative growth rate is the rate of growth with respect to its initial size. Relative growth rate (RGR) of plant growth was calculated with the help of the formula as described in ref (80). where W1 and W2 are the plant dry weights (g) during time t1 and t2, respectively. Loge is the natural logarithm (logarithm to the base of 2.3026). Plant relative growth rate is shown in g/plant/day. 2.7.3 Net Assimilation Rate (NAR) NAR is the rate of an increase in dry weight per unit of leaf area. NAR was calculated using the equation arranged in ref (80). It refers to any increase in the dry matter of plant per unit of its assimilatory surface area of per unit of time. where A1 and A2 are the surface areas of leaves and W1 and W2 are the total plant dry matters at t1 and t2 time, respectively. 2.7.4 Crop Growth Rate (CGR) Crop growth rate is the gain of dry matter production of the crop in a given land per unit of time. The crop growth rate was determined using the formula described in ref (81). The samples were kept in the oven up to 3 days at 30 °C, and the dry weight was calculated. where W1 and W2 are the plant dry weights during time T1and T2, respectively. 2.7.5 Leaf Area Ratio (LAR) The leaf area ratio is the ratio between the areas of leaf lamina to the total biomass of plants. The leaf area ratio (LAR) was determined by the method established in ref (81). Length and width of leaves were measured with the help of a measuring scale. The sample in the oven at 30 °C for 48 h and plant dry mass (g) were determined. 2.7.6 Root Shoot Ratio (RSR) The root shoot ratio is the ratio of root weight to the plant top weight. The root shoot ratio (RSR) was reported as per the formula suggested in ref (81). The samples were kept in the oven for 48 h, and dry masses (g) of both root and shoot were determined. 2.7.7 Percent Moisture Content (PMC) The percent moisture content is the percentage of the amount of moisture present in plant parts like shoot, leaf, root, and soil. The moisture content percentage of shoot, leaf, root, and soil was calculated as the formula suggested in ref (81). 2.7.8 Final Emergence Percentage (FEP) Final emergence percentage is the percentage of seeds germinated of the total number of seeds initially sowed. FEP was calculated using the formula as given in ref (81). It is the percentage of total seeds emerged. 2.7.9 Seed Vigor Index (SVI) The seed vigor index is the total activities of a seed during germination and seedlings. The seed vigor index was determined using the method in ref (82). 2.7.10 Coefficient of Velocity of Germination (CVG) The coefficient of velocity of germination indicates the rapidity of germination of seeds. CVG represent germination velocity. If the time that is required for germination is low, then the value of the CVG will be greater. When all the seeds germinate, we will have highest value of CVG.82 where N is the number of seeds that is germinated per day and T represents the time period, which is considered in days from seed sowing. 2.7.11 Leaf Area Index (LAI) The leaf area index is the quantification of the leaf area under canopy. The leaf area was determined using the formula proposed in ref (83). 2.7.12 Mean Emergence Time (MET) Mean emergence time is the total time taken by a seed to germinate. Mean emergence time shows how many inhabitants have emerged faster. The greater the germinated inhabitants, the lower the mean germination time.83 where f shows the numbers of seeds germinated on x day. 2.7.13 Timsen Germination Index (TGI) The Timsen germination index is the germination of seeds per day or in a given time. The TGI indicates the number of seeds germinated on each day. It was determined using the equation used in ref (83). where G is the principal germination percentage per day whereas T is the entire germination period. 2.8 Physiological and Biochemical Attributes 2.8.1 Total Chlorophyll Content Determination (TCC) The photosynthetic pigments were quantified by following refs (84) and (85). These pigments were extracted by homogenizing 0.1 g of fresh leaves with 6 mL of 80% ethanol. The extract was centrifuged, and the supernatant was taken in test tubes. A spectrophotometer (752 N UV–vis, Beijing, China) was used to evaluate the optical density of chlorophyll a and b and carotenoids at 663, 645, 510, and 480 nm. 2.8.2 Estimation of the Soluble Protein Content (SPC) Bovine serum albumin (BSA), as described by Mendez and Kwon,86 was used as a reference to assess the protein content. The fresh leaves (0.1 g) were crushed in a mortar and pestle with 1 mL of phosphate buffer (pH 7.5) and centrifuged for 10 min at 3000 rpm. The total volume of the supernatant (0.1 mL) in test tubes was increased to 1 mL by adding distilled water. Reagent C (solution a and b in a 50:1 ratio) (solution a: 2% Na2CO3, 1% Na-K, 0.4% 0.1 N NaOH; solution b: 0.5% CuSO4·5H2O in dH2O) (1 mL) was added and mixed for 10 min, and then, 0.1 mL of reagent D (Folin phenol: distilled water in a 1:1 ratio) was added. The different concentrations (20, 40, 60, 80, 320, and 640 mg) of the BSA solution were prepared, and then, the absorbance of all samples was measured at 650 nm after 30 min of incubation. 2.8.3 Quantification of the Total Proline Content (TPC) The proline content in shoots was measured using the technique proposed by Parveen and Siddiqui.87 Fresh shoot material (0.2 g) was crushed in 3 mL of 3% sulfosalicylic acid and stored at 5 °C overnight. The obtained suspension was centrifuged for 5 min at 3000 rpm. The supernatant (2 mL) was blended with an acidic ninhydrin reagent after centrifugation. This reagent was prepared by dissolving 1.25 g of ninhydrin in 20 mL of phosphoric acid (6 M) and 30 mL of glacial acetic acid (1 M H3PO4 = 3 N H3PO4) with constant stirring. The reagent was kept stable for 24 h. The tubes carrying the contents were heated for 1 h in a water bath at 100 °C. After cooling, the mixture was extracted with 4 mL of toluene in a separate funnel. At 520 nm, optical density was determined using toluene as blank. where K = 17.52, dilution factor = 2, and fresh weight = 0.5 g. 2.8.4 Quantification of the Total Soluble Sugar Content (TSC) Total soluble sugars (TSS) were calculated using the Grad’s method.88 The fresh leaves (0.1 g) were homogenized with 3–5 mL of 80% ethanol to eliminate all traces of soluble sugars and centrifuged for 10 min at 10,000 rpm. The supernatant was collected and processed to calculate TSS. A freshly prepared anthrone solution (3 mL) and 0.1 mL of alcoholic extract were mixed in test tubes. All test tubes were heated for 12 min in boiling water and then iced for 10 min before being incubated for 20 min at 25 °C. The optical density of the solution was measured at 625 nm using a spectrophotometer (752 N UV–vis, Beijing, China). The total soluble sugars were estimated in μg/mL of fresh weight using the glucose standard curve. To generate a glucose standard curve, a stock solution of glucose was prepared in various concentrations (0, 20, 40, 60, and 100 mg), and optical density was measured at 625 nm. After absorbance, the regression model was used to generate a glucose standard curve. 2.8.5 Quantification of Antioxidants The antioxidants (POD, SOD, and APX) were evaluated in accordance with ref (89). The fresh leaf samples (0.2 g) were crushed in a 2 mL extraction buffer (potassium phosphate, pH 7.5) and ascorbic acid (1 mM) to determine the APX level. The crushed materials were centrifuged for 20 min at 4 °C and 13,000 rpm. The OD was obtained at 290 nm to evaluate APX. A standard curve was used to measure the activity in units/mg of proteins by estimating the decrement of ascorbate. To estimate SOD, the plant leaves were crushed in 4 mL of solution (1 g of PVP, 0.0278 g of Na2EDTA) and centrifuged at 10,000 rpm. A reaction mixture (400 μL of H2O + 350 μL of phosphate buffer + 100 μL of methionine + 50 μL of NBT + 50 μL of enzyme extract + 50 μL of riboflavin) was prepared to measure the activity of the SOD enzyme. The mixture was then exposed to light for 15 min, with the decrease in absorbance measured at 560 nm. A blank was made by omitting the enzyme extract. The activities of SOD were then calculated and expressed in milligrams per milligram of the total soluble protein. Using a precooled mortar and pestle, freshly procured plant leaves (0.20 g) were crushed in 3 mL of 100 mM phosphate buffer (PB) for the POD assay. To separate the homogenate, the sample extract was centrifuged at 4 °C and 10,000 rpm for 15 min. To determine peroxidase, an OD at 470 nm was obtained. One unit of POD is defined as the amount of enzyme that increases by 0.100 absorbance at 436 nm/min. The methodology of ref (90) was followed for the estimation of CAT. Leaves (0.5 g) were homogenized in 1.0 mL of phosphate buffer, which is followed by addition of H2O2 (1 mL) and phosphate buffer (3.0 mL). The absorbency value was then recorded at 240 nm. 2.8.6 Statistical Analysis Statistical analysis was done by using IBM SPSSS Statistics 26, Excel, and ORIGIN 2021 PC Corporation. ANOVA with least significant difference (LSD) and principle component analysis (PCA) was applied to analyze the data. The post hoc test was used for significant difference and expressed in the form alphabetical letters on bars of figures. 3 Results 3.1 Agronomic Characters The results of agronomic characteristics presented in Tables 1–4 included mean germination time (MGT), germination index (GI), absolute growth rate (AGR), relative growth rate (RGR), relative water content (RWC), leaf area ratio (LAR), root shoot ratio (RSR), moisture content percentage (MCP), and seed vigor index (SVI), and all these attributes showed high values with chitosan priming (1 and 2%) under induced polyethylene glycol (PEG) osmotic stress in both of the studied cultivars of Z. mays L. In pearl variety, osmo-priming proved to be effective, while in Sargodha 2002 White, variety treatment with hydro-priming showed significant (p < 0.05) results for the above-mentioned agronomic attributes. Table 1 Effect of Osmo-Priming, Thermo-Priming, and Hydro-Priming on Absolute Growth Rate, Relative Growth Rate, Net Assimilation Rate, and Leaf Area Index under Polyethylene Glycol-Induced Osmotic Stressa variety treatment treatment AGR1 AGR2 RGR NAR Pearl osmotic stress control 0.13 ± 0.01 0.31 ± 0.06 0.98 ± 0.04 1.85 ± 0.04 0.2 PEG 0.17 ± 0.01 0.43 ± 0.11 1.68 ± 0.10 4.90 ± 0.01 0.4 PEG 0.18 ± 0.02 0.34 ± 0.07 2.32 ± 0.85 6.04 ± 2.55 osmo-priming 1% chitosan 0.23 ± 0.01 0.67 ± 0.10 5.02 ± 0.93 15.2 ± 3.94 1% chitosan + 0.2 PEG 0.19 ± 0.02 0.54 ± 0.09 5.42 ± 0.15 16.9 ± 1.26 1% chitosan + 0.4 PEG 0.14 ± 0.02 0.31 ± 0.03 1.69 ± 0.48 2.92 ± 0.96 osmo-priming 2% chitosan 0.20 ± 0.01 0.21 ± 0.04 1.15 ± 0.27 1.97 ± 0.68 2% chitosan + 0.2 PEG 0.12 ± 0.03 0.25 ± 0.03 2.02 ± 0.64 4.39 ± 1.64 2% chitosan + 0.4 PEG 0.18 ± 0.02 0.36 ± 0.14 3.18 ± 1.21 8.87 ± 4.29 thermo-priming 4 °C 0.14 ± 0.01 0.24 ± 0.03 1.55 ± 0.30 3.13 ± 0.99 4 °C + 0.2 PEG 0.14 ± 0.01 0.25 ± 0.03 1.46 ± 0.15 2.59 ± 0.24 4 °C + 0.4 PEG 0.11 ± 0.01 0.27 ± 0.01 2.57 ± 0.44 5.61 ± 1.29 hydro-priming hydro 0.13 ± 0.01 0.22 ± 0.02 1.56 ± 0.77 3.00 ± 1.58 hydro + 0.2 PEG 0.13 ± 0.00 0.21 ± 0.03 0.83 ± 0.14 1.23 ± 0.29 hydro + 0.4 PEG 0.10 ± 0.01 0.09 ± 0.02 0.36 ± 0.05 0.46 ± 0.11 Sargodha 2002 White osmotic stress control 0.13 ± 0.02 0.22 ± 0.05 4.74 ± 1.40 13.4 ± 6.66 0.2 PEG 0.17 ± 0.01 0.32 ± 0.02 5.47 ± 0.31 9.08 ± 3.79 0.4 PEG 0.17 ± 0.02 0.22 ± 0.02 6.29 ± 0.30 15.7 ± 1.78 osmo-priming 1% chitosan 0.22 ± 0.01 0.25 ± 0.02 4.75 ± 0.44 16.1 ± 2.99 1% chitosan + 0.2 PEG 0.23 ± 0.02 0.22 ± 0.05 6.78 ± 1.60 21.6 ± 7.31 1% chitosan + 0.4 PEG 0.26 ± 0.02 0.26 ± 0.02 6.41 ± 1.50 26.6 ± 9.63 osmo-priming 2% chitosan 0.26 ± 0.01 0.25 ± 0.01 5.18 ± 0.51 20.5 ± 3.31 2% chitosan + 0.2 PEG 0.26 ± 0.01 0.19 ± 0.01 6.00 ± 0.83 18.0 ± 4.42 2% chitosan + 0.4 PEG 0.27 ± 0.03 0.22 ± 0.02 5.87 ± 0.49 19.1 ± 3.20 thermo-priming 4 °C 0.17 ± 0.01 0.22 ± 0.03 4.92 ± 0.58 13.5 ± 1.95 4 °C + 0.2 PEG 0.19 ± 0.02 0.18 ± 0.02 5.87 ± 0.54 18.4 ± 4.64 4 °C + 0.4 PEG 0.22 ± 0.02 0.19 ± 0.02 7.27 ± 0.86 24.0 ± 8.72 hydro-priming hydro 0.18 ± 0.04 0.18 ± 0.03 5.14 ± 0.44 16.2 ± 1.07 hydro + 0.2 PEG 0.20 ± 0.02 0.24 ± 0.01 7.35 ± 2.02 26.8 ± 8.32 hydro + 0.4 PEG 0.16 ± 0.01 0.18 ± 0.01 5.67 ± 0.67 15.8 ± 2.78 a AGR = absolute growth rate, RGR = relative growth rate, NAR = net assimilation rate. Table 2 Effect of Osmo-Priming, Thermo-Priming, and Hydro-Priming on Crop Growth Rate, Leaf Area Ratio, Root Shoot Ratio, and Percent Moisture Contents under Polyethylene Glycol (PEG)-Induced Osmotic Stressa variety treatment   CGR LAR RSR PMC Pearl osmotic stress control 0.02 ± 0.00 288.23 ± 18.86 9.02 ± 44.04 7.37 ± 1.29 0.2 PEG 0.04 ± 0.00 243.0 ± 19.1 2.68 ± 0.68 8.88 ± 1.13 0.4 PEG 0.06 ± 0.02 236.7 ± 97.2 3.14 ± 2.25 8.92 ± 2.53 osmo-priming 1% chitosan 0.12 ± 0.02 101.4 ± 11.4 0.46 ± 0.09 4.71 ± 0.36 1% chitosan + 0.2 PEG 0.13 ± 0.00 102.7 ± 1.92 0.34 ± 0.13 5.09 ± 0.34 1% chitosan + 0.4 PEG 0.04 ± 0.01 227.4 ± 54.4 2.02 ± 1.62 9.97 ± 1.54 osmo-priming 2% chitosan 0.03 ± 0.01 264.7 ± 46.6 0.82 ± 0.14 14.23 ± 2.2 2% chitosan + 0.2 PEG 0.05 ± 0.02 234.7 ± 70.5 0.78 ± 0.48 8.54 ± 1.90 2% chitosan + 0.4 PEG 0.08 ± 0.03 188.9 ± 69.7 0.55 ± 0.26 9.74 ± 3.76 thermo-priming 4 °C 0.04 ± 0.01 225.6 ± 36.9 0.56 ± 0.18 8.75 ± 0.50 4 °C + 0.2 PEG 0.03 ± 0.00 243.4 ± 38.0 0.55 ± 0.14 10.23 ± 1.2 4 °C + 0.4 PEG 0.06 ± 0.01 169.0 ± 20.1 0.25 ± 0.03 7.78 ± 0.51 hydro-priming hydro 0.04 ± 0.02 340.30 ± 102 1.16 ± 0.45 12.13 ± 2.8 hydro + 0.2 PEG 0.02 ± 0.00 317.2 ± 39.5 0.73 ± 0.10 14.11 ± 2.2 hydro + 0.4 PEG 0.01 ± 0.00 610.3 ± 47.8 1.95 ± 0.23 23.48 ± 3.2 Sargodha 2002 White osmotic stress control 0.11 ± 0.03 78.73 ± 12.8 0.15 ± 0.01 2.75 ± 0.07 0.2 PEG 0.13 ± 0.01 63.09 ± 7.07 0.12 ± 0.01 2.70 ± 0.25 0.4 PEG 0.15 ± 0.01 63.03 ± 2.68 0.06 ± 0.00 2.64 ± 0.17 osmo-priming 1% chitosan 0.11 ± 0.01 84.68 ± 6.70 0.12 ± 0.02 3.21 ± 0.07 1% chitosan + 0.2 PEG 0.16 ± 0.04 61.44 ± 7.92 0.14 ± 0.02 2.80 ± 0.36 1% chitosan + 0.4 PEG 0.15 ± 0.04 73.07 ± 13.4 0.23 ± 0.04 3.15 ± 0.04 osmo-priming 2% chitosan 0.12 ± 0.01 83.80 ± 4.24 0.14 ± 0.01 3.08 ± 0.14 2% chitosan + 0.2 PEG 0.14 ± 0.02 63.76 ± 4.78 0.20 ± 0.02 2.86 ± 0.06 2% chitosan + 0.4 PEG 0.14 ± 0.01 68.11 ± 2.66 0.17 ± 0.02 2.50 ± 0.06 thermo-priming 4 °C 0.12 ± 0.01 75.98 ± 7.33 0.17 ± 0.01 2.98 ± 0.11 4 °C + 0.2 PEG 0.14 ± 0.01 74.22 ± 3.00 0.17 ± 0.01 3.23 ± 0.19 4 °C + 0.4 PEG 0.17 ± 0.02 52.90 ± 2.32 0.15 ± 0.01 2.76 ± 0.15 hydro-priming hydro 0.12 ± 0.01 81.57 ± 7.50 0.17 ± 0.03 2.98 ± 0.24 hydro + 0.2 PEG 0.18 ± 0.05 69.76 ± 13.3 0.15 ± 0.04 2.99 ± 0.12 hydro + 0.4 PEG 0.14 ± 0.02 70.53 ± 5.21 0.16 ± 0.03 3.07 ± 0.07 a CGR = crop growth rate, LAR = leaf area ratio, RSR = root shoot ratio, PMC = percent moisture content. Table 3 Effect of Osmo-Priming, Thermo-Priming, and Hydro-Priming on Final Germination Percentage, Seed Vigor Index, Coefficient of Velocity of Germination, and Timson Germination Index under Polyethylene Glycol (PEG)-Induced Osmotic Stressa variety treatment   FEP SVI-1 SVI-2 CVG Pearl osmotic stress control 73.33 ± 2.98 835.94 ± 38.09 9.080 ± 5.79 1.50 ± 0.13 0.2 PEG 46.67 ± 2.98 633.78 ± 77.58 13.92 ± 3.16 0.70 ± 0.08 0.4 PEG 53.33 ± 2.98 902.61 ± 102.6 37.2 ± 18.01 0.62 ± 0.11 osmo-priming 1% chitosan 43.33 ± 2.98 776.67 ± 49.12 63.0 ± 10.32 0.58 ± 0.10 1% chitosan + 0.2 PEG 53.33 ± 2.98 1016.39 ± 85.6 97.00 ± 6.39 0.95 ± 0.04 1% chitosan + 0.4 PEG 56.67 ± 5.96 931.78 ± 158.1 25.5 ± 11.64 1.19 ± 0.10 osmo-priming 2% chitosan 66.67 ± 2.98 1096.11 ± 92.3 19.45 ± 5.73 1.06 ± 0.16 2% chitosan + 0.2 PEG 70.00 ± 5.16 970.94 ± 53.07 42.8 ± 16.84 1.18 ± 0.13 2% chitosan + 0.4 PEG 76.67 ± 2.98 1501.3 ± 132.7 77.1 ± 30.12 1.42 ± 0.06 thermo-priming 4 °C 63.33 ± 2.98 855.67 ± 150.0 27.530 ± .96 0.97 ± 0.07 4 °C + 0.2 PEG 66.67 ± 2.98 1029.28 ± 19.0 25.6 ± ±4.89 1.01 ± 0.10 4 °C + 0.4 PEG 63.33 ± 2.98 996.94 ± 54.98 53.1 ± 10.05 1.15 ± 0.03 hydro-priming hydro 70.00 ± 5.16 836.72 ± 128.4 35.5 ± 27.20 0.90 ± 0.06 hydro + 0.2 PEG 66.67 ± 2.98 868.11 ± 127.4 9.480 ± 1.50 1.25 ± 0.14 hydro + 0.4 PEG 66.67 ± 2.98 845.94 ± 103.7 4.430 ± 0.10 1.35 ± 0.03 Sargoda 2002 White osmotic stress control 63.33 ± 2.98 908.06 ± 104.1 116.7 ± 34.0 1.18 ± 0.05 0.2 PEG 60.00 ± 0.00 953.33 ± 12.92 123.0 ± 8.63 1.23 ± 0.10 0.4 PEG 60.00 ± 0.00 1036.33 ± 105. 150.7 ± 6.63 1.11 ± 0.05 osmo-priming 1% chitosan 63.33 ± 2.98 1084.33 ± 89.1 115.3 ± 12.8 1.33 ± 0.01 1% chitosan + 0.2 PEG 63.33 ± 2.98 1150.33 ± 95.9 175.6 ± 46.8 1.20 ± 0.12 1% chitosan + 0.4 PEG 63.33 ± 2.98 1215.06 ± 41.3 155.7 ± 35.9 1.08 ± 0.05 osmo-priming 2% chitosan 60.00 ± 0.00 1030.00 ± 84.6 120.1 ± 13.7 1.18 ± 0.05 2% chitosan + 0.2 PEG 63.33 ± 2.98 1033.61 ± 71.3 150.5 ± 17.4 1.16 ± 0.05 2% chitosan + 0.4 PEG 66.67 ± 2.98 1123.39 ± 37.9 157.1 ± 19.3 1.12 ± 0.07 thermo-priming 4 °C 56.67 ± 2.98 862.33 ± 53.09 109.6 ± 17.6 1.19 ± 0.04 4 °C + 0.2 PEG 70.00 ± 0.00 1237.06 ± 98.2 165.6 ± 15.1 1.50 ± 0.06 4 °C + 0.4 PEG 66.67 ± 2.98 1228.33 ± 26.9 195.1 ± 14.7 1.51 ± 0.04 hydro-priming hydro 56.67 ± 2.98 854.44 ± 60.74 115.0 ± 4.47 1.34 ± 0.12 hydro + 0.2 PEG 63.33 ± 2.98 1031.06 ± 16.6 183.5 ± 49.6 1.35 ± 0.07 hydro + 0.4 PEG 63.33 ± 2.98 1071.89 ± 49.7 146.6 ± 25.7 1.44 ± 0.12 a FEP = final emergence percentage, SVI = seed vigor index, CVG = coefficient of velocity of germination. Table 4 Effect of Osmo-Priming, Thermo-Priming, and Hydro-Priming on Leaf Area Index, Mean Emergence Time, and Timson Germination Index under Polyethylene Glycol (PEG)-Induced Osmotic Stressa variety treatment   LAI MET TGI Pearl osmotic stress control 6.97 ± 0.15 0.39 ± 0.02 2.55 ± 0.12 0.2 PEG 9.98 ± 0.15 0.58 ± 0.04 1.74 ± 0.11 0.4 PEG 8.69 ± 1.21 0.65 ± 0.06 1.57 ± 0.16 osmo-priming 1% chitosan 11.97 ± 1.20 0.66 ± 0.06 1.55 ± 0.15 1% chitosan + 0.2 PEG 13.82 ± 0.52 0.50 ± 0.01 2.02 ± 0.06 1% chitosan + 0.4 PEG 7.93 ± 1.19 0.43 ± 0.01 2.31 ± 0.08 osmo-priming 2% chitosan 6.87 ± 1.22 0.48 ± 0.04 2.12 ± 0.17 2% chitosan + 0.2 PEG 8.98 ± 1.34 0.45 ± 0.03 2.24 ± 0.15 2% chitosan + 0.4 PEG 9.84 ± 2.39 0.40 ± 0.01 2.48 ± 0.04 thermo-priming 4 °C 7.97 ± 0.59 0.50 ± 0.01 2.02 ± 0.06 4 °C + 0.2 PEG 8.45 ± 0.35 0.49 ± 0.03 2.07 ± 0.11 4 °C + 0.4 PEG 10.27 ± 0.53 0.45 ± 0.01 2.24 ± 0.04 hydro-priming hydro 8.48 ± 0.71 0.51 ± 0.02 1.95 ± 0.08 hydro + 0.2 PEG 6.18 ± 0.53 0.43 ± 0.03 2.33 ± 0.15 hydro + 0.4 PEG 5.34 ± 0.39 0.41 ± 0.01 2.43 ± 0.04 Sargodha 2002 White osmotic stress control 8.39 ± 2.04 0.44 ± 0.01 2.29 ± 0.07 0.2 PEG 8.65 ± 1.28 0.43 ± 0.02 2.33 ± 0.09 0.4 PEG 9.85 ± 0.76 0.46 ± 0.02 2.19 ± 0.06 osmo-priming 1% chitosan 9.75 ± 0.30 0.42 ± 0.00 2.40 ± 0.02 1% chitosan + 0.2 PEG 9.54 ± 1.52 0.44 ± 0.02 2.29 ± 0.13 1% chitosan + 0.4 PEG 10.36 ± 1.14 0.47 ± 0.01 2.14 ± 0.06 osmo-priming 2% chitosan 10.60 ± 0.88 0.44 ± 0.01 2.26 ± 0.08 2% chitosan + 0.2 PEG 9.35 ± 1.31 0.45 ± 0.01 2.24 ± 0.06 2% chitosan + 0.4 PEG 9.91 ± 1.01 0.45 ± 0.01 2.21 ± 0.06 thermo-priming 4 °C 9.11 ± 0.89 0.44 ± 0.01 2.29 ± 0.07 4 °C + 0.2 PEG 10.78 ± 1.10 0.39 ± 0.01 2.57 ± 0.06 4 °C + 0.4 PEG 9.51 ± 1.28 0.39 ± 0.01 2.57 ± 0.04 hydro-priming hydro 10.16 ± 0.12 0.41 ± 0.02 2.45 ± 0.14 hydro + 0.2 PEG 11.09 ± 0.75 0.41 ± 0.01 2.43 ± 0.07 hydro + 0.4 PEG 9.93 ± 1.39 0.40 ± 0.02 2.52 ± 0.11 a LAI = leaf area index, MET = mean emergence time, TGI = Timson germination index. According to the results, significant effects were found in variety × treatment (Table 5). AGR (absolute growth rate) values were recorded as 0.093 and 0.06 with a significant level (p < 0.001), AGR in osmotic stress treatment was 0.566 (p < 0.001), and treatment × variety was at 0.36 (p < 0.0036). RGRs (relative growth rates) were reported to be 64.126 and 53.683 (p < 0.0426). LAI (leaf area index) values over treatment were not significant but were having a significant difference in variety × treatment (p < 0.0204). Osmotic stressed and seed priming with chitosan were found to be significant in terms of CGR (crop growth rate) (p < 0.0461); likewise, the LAR (leaf area ratio) was highly significant (p < 0.0001), PMC (percent moisture content) shows a high level of significance (p < 0.001), MET (mean emergence time) showed significant values (p < 0.0001), SVI (seed vigor index) showed non-significant values (p < 0.0551), and the values of CVG (coefficient of velocity of germination) and TGI (Timson germination index) showed a significant difference (p < 0.001). Table 5 Analysis of Variance of Measured Traits under Polyethylene Glycol-Induced Osmotic Stress in Z. mays L.a trait source SS Df MS F P AGR-1 treatment 0.093 14 0.007 5.9709 0.0000*** variety 0.06 1 0.06 54.234 0.0000*** treatment × variety 0.049 14 0.003 3.1252 0.0011*** error 0.067 60 0.001     AGR-2 treatment 0.566 14 0.04 4.2837 0.0000*** variety 0.191 1 0.191 20.292 0.0000*** treatment × variety 0.36 14 0.026 2.7255 0.0036** error 0.566 60 0.009     RGR treatment 64.126 14 4.58 1.9142 0.0426** variety 312.966 1 312.966 130.78 0.0000*** treatment × variety 53.683 14 3.834 1.6024 0.1048 error 143.574 60 2.393     LAI treatment 93.911 14 6.708 1.5339 0.1268 variety 23.226 1 23.226 5.3110 0.0247** treatment × variety 132.308 14 9.45 2.1609 0.0204** error 262.389 60 4.373     CGR treatment 0.036 14 0.003 1.8836 0.0467** variety 0.18 1 0.18 130.86 0.0000*** treatment × variety 0.03 14 0.002 1.5797 0.1117 error 0.083 60 0.001     LAR treatment 30,8190.9 14 22013.63 4.0364 0.0001*** variety 744,962.4 1 744962.4 136.59 0.0000*** treatment × variety 294,442.7 14 21031.62 3.8563 0.0001*** error 327,227.1 60 5453.785     RSR treatment 102.923 14 7.352 2.3353 0.012** variety 51.68 1 51.68 16.416 0.0001*** treatment × variety 104.061 14 7.433 2.3611 0.0111** error 188.885 60 3.148     PMC treatment 447.23 14 31.945 4.1842 0.0000*** variety 1215.286 1 1215.286 159.17 0.0000*** treatment × variety 425.166 14 30.369 3.9778 0.0001*** error 458.082 60 7.635     MET treatment 0.161 14 0.012 5.1354 0.0000*** variety 0.084 1 0.084 37.475 0.0000*** treatment × variety 0.158 14 0.011 5.0372 0.0000*** error 0.135 60 0.002     FEP treatment 2528.889 14 180.635 0.9264 0.0000*** variety 1.111 1 1.111 0.0303 0.0000*** treatment × variety 1982.222 14 141.5873 3.8615 0.0001*** error 2200 60 36.667     SVI-1 treatment 1,541,522 14 110108.7 3.9055 0.0001*** variety 296,296.5 1 296296.5 50210.1 0.0019*** treatment × variety 669,494.8 14 47821.06 1.6962 0.0804 error 1,691,575 60 28192.92     SVI-2 treatment 40,003.51 14 2857.394 1.8274 0.0551* variety 268,768.7 1 268768.7 171.88 0.0000*** treatment × variety 21,746.3 14 1553.307 0.9934 0.0000*** error 93,818.6 60 1563.643     CVG treatment 2.033 14 0.145 5.136 0.0000*** variety 0.961 1 0.961 33.981 0.0000*** treatment × variety 2.04 14 0.146 5.1527 0.0000*** error 1.697 60 0.028     TGI treatment 2.443 14 0.175 4.8614 0.0000*** variety 1.265 1 1.265 35.236 0.0000*** treatment × variety 2.344 14 0.167 4.6639 0.0000*** error 2.154 60 0.036     a AGR = absolute growth rate, RGR = relative growth rate, LAI = leaf area index, CGR = crop growth rate, LAR = leaf area ratio, RSR = root shoot ratio, PMC = percent moisture content, MET = mean emergence time, FEP = final emergence percentage, SVI = seed vigor index, CVG = coefficient of velocity of germination, TGI = Timson germination index. * is significant up to p = 0.05, ** is significant at p = 0.01, and *** is significant at p = 0.001. 3.2 Physiological and Biochemical Attributes 3.2.1 Photosynthetic Pigments (Chlorophyll a and b and Carotenoids) To monitor the plant stress, we need an accurate estimation of the chlorophyll content, which is different in different plant species. The results indicated that the concentrations of the chlorophyll and carotenoid content of Z. mays L. were affected under induced polyethylene glycol (PEG) stress (Figures 3–7). Osmo-priming with chitosan and hydro-priming with water increased the chlorophyll and carotenoid content. The chlorophyll content was decreased under stress conditions. On the contrary, it was reported that hydro-priming treatment under 0.4 MPa induced osmotic stress of polyethylene glycol and the chlorophyll and carotenoid content were increased. After hydro-priming, osmo-priming with chitosan (2%) showed the maximum value of the chlorophyll content. Results suggested that osmotic stress provided in the form of polyethylene glycol (PEG) reduced the growth responses by decreasing the chlorophyll content; osmo-priming with chitosan can adjust ion homeostasis caused by PEG. Figure 3 Effect of osmo-priming, thermo-priming, and hydro-priming on the chlorophyll “a” content of Z. mays L. under polyethylene glycol-induced osmotic stress (mean ± standard error). Letters indicating least significant difference among the mean values at p ≤ 0.05. Figure 4 Effect of osmo-priming, thermo-priming, and hydro-priming on the chlorophyll “b” content of Z. mays L. under polyethylene glycol-induced osmotic stress (mean ± standard error). Letters indicating least significant difference among the mean values at p ≤ 0.05. Figure 5 Effect of osmo-priming, thermo-priming, and hydro-priming on the total chlorophyll content of Z. mays L. under polyethylene glycol-induced osmotic stress (mean ± standard error). Letters indicating least significant difference among the mean values at p ≤ 0.05. Figure 6 Effect of osmo-priming, thermo-priming, and hydro-priming on the chlorophyll a/b ratio content of Z. mays L. under polyethylene glycol-induced osmotic stress (mean ± standard error). Letters indicating least significant difference among the mean values at p ≤ 0.05. Figure 7 Effect of osmo-priming, thermo-priming, and hydro-priming on the total carotenoid content of Z. mays L. under polyethylene glycol-induced osmotic stress (mean ± standard error). Letters indicating least significant difference among the mean values at p ≤ 0.05. 3.2.2 Total Sugar Content The total sugar content of Z. mays L. subjected to osmo-priming with chitosan (1 and 2%), thermo-priming at 4 °C, and hydro-priming with water under polyethylene glycol (PEG)-induced osmotic stress was found to be non-significant (p < 0.05) in both the varieties. Osmo-priming with chitosan 2% was effective in terms of the total sugar content (Figure 8). Figure 8 Effect of osmo-priming, thermo-priming, and hydro-priming on the total sugar content of Z. mays L. under polyethylene glycol-induced osmotic stress (mean ± standard error). Letters indicating least significant difference among the mean values at p ≤ 0.05. 3.2.3 Total Protein Content The total protein content of Z. mays L. subjected to varying levels of osmotic stress with applied treatments, concentration, and their interactive effect showed significant differences (p < 0.05). The maximum value of protein was reported in control of osmo-priming with chitosan 2% in pearl variety followed by thermo-priming (Figure 9). Figure 9 Effect of osmo-priming, thermo-priming, and hydro-priming on the total protein content of Z. mays L. under polyethylene glycol-induced osmotic stress (mean ± standard error). Letters indicating least significant difference among the mean values at p ≤ 0.05. 3.2.4 Total Proline Content The total proline content of Z. mays L. subjected to osmotic stress with applied treatment and concentrations showed significant differences (p < 0.05). The maximum value was reported in thermo-priming of pearl variety. The interactive effect of treatment into the concentration also showed significant differences (p < 0.05). Thermo-priming was effective under 0.2 MPa of induced polyethylene glycol osmotic stress (Figure 10). Figure 10 Effect of osmo-priming, thermo-priming, and hydro-priming on the total proline content of Z. mays L. under polyethylene glycol-induced osmotic stress (mean ± standard error). Letters indicating least significant difference among the mean values at p ≤ 0.05. 3.2.5 Antioxidant Enzymes (SOD, POD, APX, and CAT) The superoxide dismutase (SOD) enzyme content of Z. mays L. subjected to osmo-priming with chitosan (1 and 2%), thermo-priming at 4 °C, and hydro-priming under polyethylene glycol (PEG)-induced osmotic stress showed non-significant differences (p < 0.05) (Figure 11). Peroxidase (POD) enzyme activity of Z. mays L. subjected with chitosan osmo-priming (1 and 2%), thermo-priming at 4 °C, and hydro-priming under polyethylene glycol (PEG)-induced osmotic stress was significant (p < 0.05). The maximum value of POD was found with hydro-priming in both the varieties followed by osmo-priming with chitosan 1% (Figure 12). Figure 11 Effect of osmo-priming, thermo-priming, and hydro-priming on the superoxide dismutase enzyme (SOD) content of Z. mays L. under polyethylene glycol-induced osmotic stress (mean ± standard error). Letters indicating least significant difference among the mean values at p ≤ 0.05. Figure 12 Effect of osmo-priming, thermo-priming, and hydro-priming on the peroxidase enzyme (POD) content of Z. mays L. under polyethylene glycol-induced osmotic stress (mean ± standard error). Letters (a–h) indicating least significant difference among the mean values at p ≤ 0.05. The ascorbate peroxidase (APX) enzyme content under osmo-priming with chitosan (1 and 2%), thermo-priming at 4 °C, and hydro-priming with water under polyethylene glycol (PEG)-induced osmotic stress showed a significant increase (p < 0.05). The maximum level of APX was found in osmo-priming with chitosan 2% in the controlled group as well as in stressed conditions in both the varieties followed by osmo-priming with chitosan 1% (Figure 13). Activity of the catalase (CAT) enzyme content indicated a significant increase (p < 0.05) as the maximum concentration was reported in osmo-priming with chitosan 1% followed by osmo-priming with chitosan 2%. The interactive effects of treatment, concentration, and variety were found to be non-significant at p < 0.05 (Figure 14). Figure 13 Effect of osmo-priming, thermo-priming, and hydro-priming on the ascorbate peroxidase enzyme (APX) content of Z. mays L. under polyethylene glycol-induced osmotic stress (mean ± standard error). Letters indicating least significant difference among the mean values at p ≤ 0.05. Figure 14 Effect of osmo-priming, thermo-priming, and hydro-priming on the catalase enzyme (CAT) content of Z. mays L. under polyethylene glycol-induced osmotic stress (mean ± standard error). Letters indicating least significant difference among the mean values at p ≤ 0.05. 3.3 Analysis of Variance of Measured Trait under Polyethylene-Induced Osmotic Stress in Z. mays L. Analysis of variance (ANOVA) for chlorophyll a, chlorophyll b, and total chlorophyll content revealed significant differences (p < 0.05) in treatment (A), concentration (B), and variety (C). Moreover, the interactive effect of treatment and concentration (AxB) and treatment and variety (AxC) was significant (p < 0.05) in chlorophyll b and the concentration and variety were found to be significant (p < 0.05) in chlorophyll b and total chlorophyll content, under polyethylene glycol-induced osmotic stress (Table 6). Table 6 Mean Square of the ANOVA for Chlorophyll a, Chlorophyll b, and Total Chlorophyll Content of Z. mays L. under Polyethylene Glycol-Induced Osmotic Stressa sources degree of freedom chlorophyll a chlorophyll b total chlorophyll content treatment (A) 4 0.999* 0.724* 3.347* concentration (B) 2 0.854* 0.508NS 2.586* AxB 8 0.226NS 0.527* 1.393NS variety (C) 1 0.928* 2.196NS 5.975* AxC 4 0.295NS 0.248* 1.024NS BxC 2 0.246NS 1.14* 2.444* AxBxC 8 0.132NS 0.263NS 0.736NS error 60 0.195 0.207 0.711 coefficient of variance (%)   33.79% 29.94% 29.83% a * = significant (p < 0.05), NS = non-significant (p > 0.05). Chlorophyll ratio a/b, total carotenoid contents, and total sugar contents revealed significant differences (p < 0.05) in treatment (A), concentration (B), and variety (C). Moreover, the interactive effect of treatment and variety was also found to be significant (p < 0.05) in terms of the total carotenoid content and total sugar contents. Total sugar contents show high significant values, i.e., (p < 0.001) in terms of variety (C) (Table 7). Table 7 Mean Square of the ANOVA for Chlorophyll Ratio “a/b”, Total Carotenoid Content, and Total Sugar Content of Z. mays L. under Polyethylene-Induced Osmotic Stressa sources degree of freedom chlorophyll ratio “a/b” total carotenoid content total sugar content treatment (A) 4 0.043NS 1.666* 0.146* concentration (B) 2 0.07NS 2.375* 0.054NS AxB 8 0.033NS 0.452NS 0.022NS variety (C) 1 0.103NS 0.124NS 0.59*** AxC 4 0.038NS 1.454* 0.116* BxC 2 0.066NS 0.033NS 0.005NS AxBxC 8 0.011NS 0.626NS 0.038NS error 60 0.047 0.371 0.027 coefficient of variance (%)   33.79% 24.58% 36.00% a * = significant (p < 0.05), NS = non-significant (p > 0.05). The total proline content revealed significant differences (p < 0.05) in treatment (A) and concentration (B). Moreover, the interactive effect of treatment and concentration (AxB) in terms of the total proline content showed significant differences (p < 0.0001). The total protein content and antioxidant enzyme superoxide dismutase were non-significant (Table 8). Table 8 Mean Square of the ANOVA of the Total Protein Content, Total Proline Content, and Superoxide Dismutase Enzyme of Z. mays L. under Polyethylene-Induced Osmotic Stressa sources degree of freedom total protein content total proline content superoxide dismutase treatment (A) 4 0.036NS 0.339* 0.016NS concentration (B) 2 0.143NS 0.476* 0.009NS AxB 8 0.202NS 0.659*** 0.012NS variety (C) 1 0.526* 0.005NS 0.005NS AxC 4 0.155NS 0.035NS 0.017NS BxC 2 0.119NS 0.159NS 0.017NS AxBxC 8 0.129NS 0.161NS 0.01NS error 60 0.116 0.1 0.012NS coefficient of variance (%)   33.79% 38.08% 12.07% a * = significant (p < 0.05), NS = non-significant (p > 0.05). Activity of ascorbate peroxidase (APX) and catalase (CAT) revealed significant differences (p < 0.05) in treatment (A), concentration (B), and variety (C). Moreover, the interactive effect was also significant (p < 0.001) in terms of treatment and concentration (AxB) in these two antioxidant enzymes. The peroxidase enzyme showed significant differences (p < 0.05) only in the interactive effect of treatment and concentration (AxB), treatment and variety (AxC), and treatment, concentration, and variety (AxBxC) (Table 9). Table 9 Means Square of the ANOVA of Peroxidase, Ascorbate Peroxidase, and Catalase Enzyme of Z. mays L. under Polyethylene Glycol-Induced Osmotic Stressa sources degree of freedom peroxidase ascorbate peroxidase catalase treatment (A) 4 0.374NS 0.922*** 0.813*** concentration (B) 2 0.256NS 1.088*** 0.475** AxB 8 0.459* 0.744*** 0.896*** variety (C) 1 0.228NS 0.078NS 0.002NS AxC 4 0.519* 0.039NS 0.017NS BxC 2 0.098NS 0.078NS 0.006NS AxBxC 8 0.381* 0.123* 0.072NS error 60 0.175 0.063 0.042 coefficient of variance (%)   33.79% 28.27% 16.42% a * = significant (p < 0.05), NS = non-significant (p > 0.05). 3.4 Principle Component Analysis Based on the Correlation Matrix of Biological Components The results of principle component analysis are based on 12 characters and represented that the first three components enclosed overall 59.753% of the total variation. The PC1 explained 29.910% of complete variance, which were significantly correlated with chlorophyll b, chlorophyll ratio, sugar, protein, and proline. The PC1 was particularly related to growth responses. The PC2 result explained 19.230% of the total variance, which was particularly correlated with chlorophyll a, total chlorophyll, chlorophyll a/b ratio, POD, SOD, and APX. The PC2 was related to the plant chlorophyll content and antioxidant enzymes. The PC3 was accounted with 10.613% of the whole variation, and the important variations under PC3 were carotenoids, soluble proteins, SOD, and APX enzymes. This indicated that PC3 was correlated to antioxidant enzymes and osmolytes. There were variations that were related with parameters among each other and were independent with variation of other components; consequently, we plotted three components in rotated space component design (Figure 15). There was a positive correlation found in PC1 and PC2, which included growth response and osmolytes. There was also a positive correlation found in PC1 and PC3, including growth response and antioxidant enzymes, respectively. There was no correlation found in PC2 and PC3 as we know that PC2 was related to growth response and PC3 was correlated with antioxidant enzymes (Table 10). Figure 15 Component plot in the rotated space: principle component analysis (PCA) based on the correlation matrix of biological components. Table 10 Eigenvalues, Variation Explained (%), Cumulative Values (%), and Coefficient of Determination of the First Three Principle Components Based on the Correlation Matrix of Biological Components in Z. mays L.a     variance % components   component eigenvalues individual cumulative PC1 PC2 PC3 PC4 PC5 chlorophyll a 3.589 29.910 29.910 0.959 0.050 0.089 0.072 0.172 chlorophyll b 2.308 19.230 49.139 0.910 0.106 0.255 0.040 0.239 total chlorophyll 1.274 10.613 59.753 0.975 0.083 0.096 0.057 0.046 chlorophyll ratio 1.177 9.810 69.563 0.103 0.118 0.613 0.036 0.751 carotenoids 1.134 9.447 79.010 0.816 0.135 0.222 0.128 0.001 sugar 0.766 6.386 85.396 0.158 0.396 0.640 0.021 0.367 protein 0.620 5.163 90.559 0.264 0.090 0.506 0.367 0.534 proline 0.542 4.514 95.073 0.026 0.753 0.063 0.300 0.037 SOD 0.323 2.693 97.766 0.109 0.190 0.124 0.814 0.162 POD 0.253 2.109 99.875 0.141 0.428 0.270 0.502 0.089 APX 0.015 0.125 100.000 0.208 0.745 0.070 0.019 0.160 CAT 2.876 2.396 100.000 0.211 0.865 0.049 0.104 0.013 a SOD = superoxide dismutase, POD = peroxidase, APX = ascorbate peroxidase, CAT = catalase. 4 Discussion Osmotic stress affects plants during their life cycle, and seeds are mostly susceptible to these stresses between sowing and seedling establishment.91 The germination rate and seedling growth are influenced by nutritional imbalance and osmotic stress.58 The chitosan α,β-(1,4)-glucosamine polymer is a safe, natural, and cheap polysaccharide and is produced from chitin, which is the major structural component of the fungus cell wall and the exoskeleton of arthropods.68,69 Chitosan is used as plant fertilizer as it promotes seed germination and enhances germination percentage.70 Maize is the most important cereal crop and is grown all over the world.92 It is estimated that the demand of the maize crop will double in the developing countries.93 The present study was aimed at determining the effect of osmo-priming with chitosan (1 and 2%), thermo-priming at 4 °C, and hydro-priming on two varieties (Pearl and Sargodha 2002 White) of Z. mays L. under polyethylene glycol (PEG)-induced osmotic stress. In the present study, the agronomic parameters were adversely affected by polyethylene glycol (PEG)-induced osmotic stress. The same results were reported in ref (94) in maize cultivar under (PEG)-induced osmotic stress. The seed priming technique proved to be useful (Table 1) that showed a significant difference in terms of absolute growth rate (AGR) under osmo-priming with chitosan 2% followed by hydro-priming. The relative growth rate (RGR) was also having a significant impact under osmo-priming with 1% chitosan with the highest value in pearl variety. Crop growth rate (CGR) was high under osmo-priming with 1% chitosan followed by hydro-priming in both the varieties. Similar results were reported in ref (95) in different cultivars of wheat under water deficit conditions. Osmo-priming, hydro-priming, and thermo-priming improved (AGR) and (RGR); hence, our findings were in agreement with the results of ref (96). The percent moisture content (PMC) was influenced by the seed priming technique under induced osmotic stress. In hydro-priming, the PMC values were high in comparison with the controlled group. Osmo-priming with chitosan also shows PMC values greater than control conditions in the pearl variety of Z. mays L. under induced osmotic stress conditions. Osmotic stress reduced the PMC, thus confirming the results reported in ref (97) in Z. mays L. and Gossypium hirsutum L. under osmotic stress conditions. Chitosan promotes growth of cabbage (Brassica oleracea L. var. Capitata L.) callus in vitro,98 and chitin oligosaccharide increased the chitinase activity of rice.99 Chitosan priming on maize seed germination attributes and early seedling has a pronounced effect on growth under osmotic stress induced by PEG.67 Final germination percentage (FGP) was increased in osmo-priming with chitosan (2%) followed by controlled group, and parallel results were reported in ref (77) in wheat. They reported that chitosan priming has the potential to increase the FGP in wheat plant. Under PEG stress, chitosan as osmo-priming proved to be effective in terms of final germination percentage (FGP) and mean emergence time (MET) in wheat plant.79 This increase in seed germination rate with seed priming is due to certain biochemical changes like enzyme activation and metabolism or imbibition.100 Chitosan improves the germination of seeds under water deficit conditions in maize76 and in rice.101 Seed priming with chitosan showed a pronounced increase in seed germination percentage, activity of lipase enzyme, gibberellic acid, and indole acetic acid (IAA) levels in peanut.70 Additionally, seed priming probably permitted some repairs of membrane degradation damage and produced better germination patterns and higher vigor levels in comparison with unprimed seeds.102 In the present study, seeds primed with chitosan had better germination characteristics and seedlings grew more quickly when exposed to osmotic stress. It is clear that chitosan may be useful in high-stress situations. Application of chitosan enhances plant germination in drought-stressed regimes.101 The findings in Table 3 indicated that osmo-priming with chitosan (2%) increased the seed vigor index (SVI) in both the studied varieties of Z. mays L., and similar results were reported in ref (100) in Carum copticum plants, suggesting that with increasing chitosan concentration, the seed vigor index also improved. Chitosan is used as osmo-priming to decrease the adverse effects of oxidative stress. Chitosan is obtained by deacetylation of chitin; it promotes root and shoot growth in Raphanus sativus L. and accelerates flower timing and number in Passiflora edulis.(103) The vigor enhancement of maize seedlings with chitosan priming is reported in ref (104). Under drought stress regimes, cytokinesis and cell elongation are closely associated with the reduction in growth characteristics such as AGR, RGR, and MET. This decrease is thought to be caused by reduced photosynthesis and stomatal closure that ultimately causes the leaves to shrink.104 The low water content, reduced turgidity, wilting, closing of stomata, and eventually a decrease in cell expansion and growth are signs of a water deficit state. Plant growth is affected in different ways by both internal and external causes.105 It has been noted that in Populus species, root length decreased under osmotic stress.106 The primary cause of poor photosynthesis and low crop yield in water-scarce environments is reduced leaf area.107 Plants contain substantial amounts of carotenoids that serve as non-enzymatic scavengers of active oxygen species.108 Drought causes the chlorophyll breakdown and accelerates the leaf senescence.109 The concentrations of the chlorophyll and carotenoid content of Z. mays L. were adversely affected under induced polyethylene glycol PEG osmotic stress. Osmo-priming with chitosan and hydro-priming with water increased the chlorophyll and carotenoid content. Normally, under stress conditions, plants decrease the chlorophyll content.110,111 In the present study, chlorophyll a, chlorophyll b, total chlorophyll content, and chlorophyll ratio a/b were studied in both the varieties (Pearl and Sargodha 2002 White) of Z. mays L. under polyethylene glycol-induced osmotic stress. The chlorophyll content showed a marked decrease under water deficit conditions in maize. The chlorophyll content also decreased under osmotic stress conditions (0.2 and 0.4 MPa) as previously reported in ref (111). It was reported to be high in hydro-priming under 0.4 MPa of induced PEG osmotic stress. After hydro-priming, osmo-priming with chitosan (2%) showed maximum levels of the chlorophyll content. Similar results were reported in ref (112). The photosynthetic attributes of Z. mays L. were decreased under stress conditions.113 The seed priming technique with chitosan (1 and 2%) improved the chlorophyll content of Z. maysL. under stress conditions, and similar results were reported in ref (114) by studying the effects of foliar application of chitosan in maize and soybean (Figures 3–7). Soluble solutes (sugar and protein content) mitigate the lethal effects of drought stress and maintain ionic balance in cells. The total soluble sugar content (Figure 8) significantly increased under osmo-priming with chitosan (2%), and chitosan improved the sugar content of both the varieties of maize, thus confirming the same findings made in ref (115). According to Wang et al.,116 it appears that the lesser amount of soluble sugars produced in response to melatonin-seed priming, particularly under drought stress, may be related to the ameliorative impact of melatonin on drought stress, which creates a favorable environment and prevents the plant from receiving any stress signals. In our study, there was no significant effect on the sugar content, but for osmo-priming with chitosan, the production of sugar increases non-significantly. Proline production in the leaves is thought to play a significant role in the plant’s ability to respond to abiotic stress situations.117,118 The osmo-protective ability of proline under stress regimes is well recognized.119 Proline plays a critical part in the mechanism of osmotic adjustment in many crops under severely stressed conditions, and a rise in proline levels in plants during drought stress is thought to be a sign of drought stress resistance. Proline oxidase (PROX) and −glutamyl kinase (−GK), two significant enzymes, control the amount of proline in plants.120 The total proline content of Z. mays L. under induced PEG stress was studied, and the maximum value was reported in the controlled group of osmo-priming with chitosan (2%) followed by thermo-priming at 4 °C (Figure 10) in both the varieties. Chitosan improved the levels of the proline content, and parallel results were reported in ref (121) by studying the effect of chitosan under osmotic stress conditions in Carthamus tinctorius L. A marked spike was noted in the proline content when water deficit conditions dominated the plants.112 This increase can be correlated with the tolerance of plants under stress conditions.122 To combat ROS-induced cellular damage, plant cells have a sophisticated enzymatic antioxidant system.123−125 Non-enzymatic components like carotenoids, glutathione, and tocopherols work in conjunction with antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) in the elimination of ROS and other free radicals produced under osmotic stress regimes.126 Proteins, lipids, and nucleic acids are all attacked by ROS, and the extent of the damage relies on how well the antioxidative scavenging systems balance ROS generation.127−130 The maximum concentration of POD was found with hydro-priming in both the varieties followed by osmo-priming with chitosan 1%. Ascorbate peroxidase (APX) activity under osmo-priming with chitosan (1 and 2%), thermo-priming at 4 °C, and hydro-priming with water under polyethylene glycol (PEG)-induced osmotic stress showed a significant increase (p < 0.05). The maximum level of APX was found in osmo-priming with chitosan 2% in control as well as in stress conditions in both the varieties followed by osmo-priming with chitosan 1%. The catalase (CAT) content of Z. mays L. indicated a significant increase (p < 0.05) as the maximum concentration was reported in osmo-priming with chitosan 1% followed by osmo-priming with chitosan 2% (Figures 11–14). The plant antioxidant defense mechanism precisely regulates the balance between ROS generation and consumption when growth conditions are ideal.131 Chitosan contains certain anti-oxidative qualities and can serve as the ROS scavenger by boosting the antioxidant capacity of plant cells.76 Chitosan has been shown to boost the peroxidase activity in the roots of date palm, indicating that it is an exogenous inducer of defense responses.132 CAT, SOD, and POD activities increased significantly in the primed seeds, demonstrating a similar defense response caused by chitosan in this series of tests. In a prior study, it was found that chitosan treatments, either as a priming agent or growing media, resulted in enhanced CAT, POD, and SOD enzyme activity in low-temperature challenged maize.76,133 Upon fast desiccation, the embryonic axes of pedunculate oak (Quercus robour) seeds showed a considerable rise in POD activity and a concurrent decrease in total phenolic compounds. Based on these findings, they hypothesized that POD uses phenolic compounds as electron donors in the embryonic axis cells to scavenge H2O2. Catanea sativa embryonic axes under injury and desiccation have demonstrated a similar mechanism.134 5 Conclusions In conclusion, it was evident that physiological and agronomic traits of both the varieties of Z. mays L. were affected adversely under polyethylene glycol-induced stress conditions. Osmo-priming with chitosan (1 and 2%), thermo-priming at 4 °C, and hydro-priming with distilled water proved to be effective in modulating agronomic and physiological attributes of maize cultivars under induced PEG osmotic stress. Moreover, activities of antioxidant enzymes including peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) were markedly improved under stress regimes with seed priming. In addition, osmo-priming with chitosan could be utilized to adjust the ionic differences of seeds in the soil under osmotic stress conditions. Importantly, in the present era of inevitable changing climatic shifts, fulfilling the increasing food demand of the rapidly expanding population is a daunting challenge for the scientific community as well as for the agriculturists; keeping in mind the findings of the present research study and previous related literature, the seed priming technique could open a new avenue to a sustainable agricultural system by increasing the production and improving the abiotic stress tolerance potential of economically important crops. In addition, the seed priming technique could further be extended to replace the conventional agricultural practices; however, investigating and ensuring its biosafety in living systems should be taken into consideration; therefore, there is a dire need for further studies to ensure its safe applicability for sustainable production of crops. Data Availability Statement All data generated or analyzed during this study are included in this published article. Author Contributions Conceptualization, investigation, and methodology: H.A.K. and W.S.; data curation: R.U., Z.M., S.U., and B.A.; formal analysis: S.U. and B.A.; funding acquisition: S.E., M.S.E., M.S.A., S.M.E., and I.A.; project administration and validation: R.U. and Z.M.; resources and supervision: S.U.; software: S.Z.S., H.A.K., and W.S.; visualization: S.Z.S., B.A., R.U., and Z.M.; writing–original draft: H.A.K., W.S., and B.A.; writing–review and editing: H.A.K., W.S., S.M.E., I.A., S.U., B.A., M.S.E., S.E., M.S.A., R.U., and Z.M. The authors declare no competing financial interest. Notes The seedlings of the maize were taken from the University of Peshawar, Pakistan, where the experiment was performed. All the experiments were performed in accordance with relevant guidelines and regulations. Acknowledgments The authors extend their appreciation to the researchers’ supporting project number (RSP2023R173), King Saud University, Riyadh, Saudi Arabia. ==== Refs References Amna ; Ali B. ; Azeem M. A. ; Qayyum A. ; Mustafa G. ; Ahmad M. A. ; Javed M. T. ; Chaudhary H. J. Bio-Fabricated Silver Nanoparticles: A Sustainable Approach for Augmentation of Plant Growth and Pathogen Control. In Sustainable Agriculture Reviews 53; Springer, 2021; pp. 345–371. Javed M.A. ; Khan M.N. ; Ali B. ; Wahab S. ; Din I.U. ; Razak S.A. Positive and Negative Impacts of Biochar on Microbial Diversity. In: Fahad S. , Danish S. , Datta R. , Saud S. , Lichtfouse E. , Eds.; Sustainable Agriculture Reviews 61; Springer, 2023; pp. 310–330, 10.1007/978-3-031-26983-7_14 Saeed S. ; Ullah A. ; Ullah S. ; Noor J. ; Ali B. ; Khan M. N. ; Hashem M. ; Mostafa Y. S. ; Alamri S. Validating the Impact of Water Potential and Temperature on Seed Germination of Wheat (Triticum Aestivum L.) via Hydrothermal Time Model. Life 2022, 12 , 983 10.3390/life12070983.35888073 Shah W. ; Ullah S. ; Ali S. ; Idrees M. ; Khan M. N. ; Ali K. ; Khan A. ; Ali M. ; Younas F. Effect of Exogenous Alpha-Tocopherol on Physio-Biochemical Attributes and Agronomic Performance of Lentil (Lens Culinaris Medik.) under Drought Stress. PLoS One 2021, 16 , e0248200 10.1371/journal.pone.0248200.34358230 Dola D. B. ; Mannan M. A. ; Sarker U. ; Mamun M. A. A. ; Islam T. ; Ercisli S. ; Saleem M. H. ; Ali B. ; Pop O. L. ; Marc R. A. Nano-Iron Oxide Accelerates Growth, Yield, and Quality of Glycine Max Seed in Water Deficits. Front. Plant Sci. 2022, 13 , 992535 10.3389/fpls.2022.992535.36160973 Hassan S. ; Khurshid Z. ; Bhat S. A. ; Kumar V. ; Ameen F. ; Ganai B. A. Marine Bacteria and Omic Approaches: A Novel and Potential Repository for Bioremediation Assessment. J. Appl. Microbiol. 2022, 133 , 2299–2313. 10.1111/jam.15711.35818751 Rizvi A. ; Zaidi A. ; Ameen F. ; Ahmed B. ; AlKahtani M. D. F. ; Khan M. S. Heavy Metal Induced Stress on Wheat: Phytotoxicity and Microbiological Management. RSC Adv. 2020, 10 , 38379–38403. 10.1039/D0RA05610C.35693041 Meena R. A. A. ; Sathishkumar P. ; Ameen F. ; Yusoff A. R. M. ; Gu F. L. Heavy Metal Pollution in Immobile and Mobile Components of Lentic Ecosystems—a Review. Environ. Sci. Pollut. Res. 2018, 25 , 4134–4148. 10.1007/s11356-017-0966-2. Al-Homaidan A. A. ; Al-Otaibi T. G. ; El-Sheikh M. A. ; Al-Ghanayem A. A. ; Ameen F. Accumulation of Heavy Metals in a Macrophyte Phragmites Australis: Implications to Phytoremediation in the Arabian Peninsula Wadis. Environ. Monit. Assess. 2020, 192 , 1–10. 10.1007/s10661-020-8177-6. Bhat S. A. ; Cui G. ; Li W. ; Ameen F. ; Yaseera N. ; Wei Y. ; Li F. Fate of Bio-Contaminants in Soil Systems and Available Remediation Methods. In Fate of Biological Contaminants During Recycling of Organic Wastes; Elsevier, 2023; pp. 213–227, 10.1016/B978-0-323-95998-8.00014-5. Govarthanan M. ; Selvankumar T. ; Mythili R. ; Srinivasan P. ; Ameen F. ; AlYahya S. A. ; Kamala-Kannan S. Biogreen Remediation of Chromium-Contaminated Soil Using Pseudomonas Sp.(RPT) and Neem (Azadirachta Indica) Oil Cake. Int. J. Environ. Sci. Technol. 2019, 16 , 4595–4600. 10.1007/s13762-018-2136-6. Sabreena ; Hassan S. ; Bhat S. A. ; Kumar V. ; Ganai B. A. ; Ameen F. Phytoremediation of Heavy Metals: An Indispensable Contrivance in Green Remediation Technology. Plants 2022, 11 , 1255 10.3390/plants11091255.35567256 Ma J. ; Ali S. ; Saleem M. H. ; Mumtaz S. ; Yasin G. ; Ali B. ; Al-Ghamdi A. A. ; Elshikh M. S. ; Vodnar D. C. ; Marc R. A. ; Rehman A. ; Khan M. N. ; Chen F. ; Ali S. Short-Term Responses of Spinach (Spinacia Oleracea L.) to the Individual and Combinatorial Effects of Nitrogen, Phosphorus and Potassium and Silicon in the Soil Contaminated by Boron. Front. Plant Sci. 2022, 13 , 983156 10.3389/fpls.2022.983156.36212291 Ma J. ; Saleem M. H. ; Ali B. ; Rasheed R. ; Ashraf M. A. ; Aziz H. ; Ercisli S. ; Riaz S. ; Elsharkawy M. M. ; Hussain I. ; Alhag S. K. ; Ahmed A. E. ; Vodnar D. C. ; Mumtaz S. ; Marc R. A. Impact of Foliar Application of Syringic Acid on Tomato (Solanum Lycopersicum L.) under Heavy Metal Stress-Insights into Nutrient Uptake, Redox Homeostasis, Oxidative Stress, and Antioxidant Defense. Front. Plant Sci. 2022, 13 , 950120 10.3389/fpls.2022.950120.36092395 Ma J. ; Saleem M. H. ; Yasin G. ; Mumtaz S. ; Qureshi F. F. ; Ali B. ; Ercisli S. ; Alhag S. K. ; Ahmed A. E. ; Vodnar D. C. ; Hussain I. ; Marc R. A. ; Chen F. Individual and Combinatorial Effects of SNP and NaHS on Morpho-Physio-Biochemical Attributes and Phytoextraction of Chromium through Cr-Stressed Spinach (Spinacia Oleracea L.). Front. Plant Sci. 2022, 13 , 973740 10.3389/fpls.2022.973740.36061765 Shahid M. ; Ameen F. ; Maheshwari H. S. ; Ahmed B. ; AlNadhari S. ; Khan M. S. Colonization of Vigna Radiata by a Halotolerant Bacterium Kosakonia Sacchari Improves the Ionic Balance, Stressor Metabolites, Antioxidant Status and Yield under NaCl Stress. Appl. Soil Ecol. 2021, 158 , 103809 10.1016/j.apsoil.2020.103809. Jan M. ; Anwar-Ul-Haq M. ; Javed T. ; Hussain S. ; Ahmad I. ; Ashraf Sumrah M. ; Iqbal J. ; Hussain Babar B. ; Hafeez A. ; Aslam M. ; Tahir Akbar M. ; Aziz M. ; Alharbi K. ; Ullah I. Response of Contrasting Rice Genotypes to Zinc Sources under Saline Conditions. Phyton (B. Aires) 2023, 92 , 1361–1375. 10.32604/phyton.2023.026620. Sharma S. ; Rana V. S. ; Rana N. ; Sharma U. ; Gudeta K. ; Alharbi K. ; Ameen F. ; Bhat S. A. Effect of Organic Manures on Growth, Yield, Leaf Nutrient Uptake and Soil Properties of Kiwifruit (Actinidia Deliciosa Chev.) Cv. Allison. Plants 2022, 11 , 3354 10.3390/plants11233354.36501392 Shahzadi E. ; Nawaz M. ; Iqbal N. ; Ali B. ; Adnan M. ; Saleem M. H. ; Okla M. K. ; Abbas Z. K. ; Al-Qahtani S. M. ; Al-Harbi N. A. ; Marc R. A. Silicic and Ascorbic Acid Induced Modulations in Photosynthetic, Mineral Uptake, and Yield Attributes of Mung Bean (Vigna Radiata L. Wilczek) under Ozone Stress. ACS Omega 2023, 13971 10.1021/acsomega.3c00376.37091383 Shao H.-B. ; Chu L.-Y. ; Jaleel C. A. ; Manivannan P. ; Panneerselvam R. ; Shao M.-A. Understanding Water Deficit Stress-Induced Changes in the Basic Metabolism of Higher Plants–Biotechnologically and Sustainably Improving Agriculture and the Ecoenvironment in Arid Regions of the Globe. Crit. Rev. Biotechnol. 2009, 29 , 131–151. 10.1080/07388550902869792.19412828 Foyer C. H. ; Noctor G. Oxidant and Antioxidant Signalling in Plants: A Re-evaluation of the Concept of Oxidative Stress in a Physiological Context. Plant, Cell Environ. 2005, 28 , 1056–1071. 10.1111/j.1365-3040.2005.01327.x. Apel K. ; Hirt H. Reactive Oxygen Species: Metabolism, Oxidative Stress, and Signal Transduction. Annu. Rev. Plant Biol. 2004, 55 , 373–399. 10.1146/annurev.arplant.55.031903.141701.15377225 Smirnoff N. Ascorbate, Tocopherol and Carotenoids: Metabolism, Pathway Engineering and Functions. Antioxid. React. Oxygen Species Plants 2007, 2005 , 53–86. 10.1002/9780470988565.ch3. Ashraf M. ; Akram N. A. ; Arteca R. N. ; Foolad M. R. The Physiological, Biochemical and Molecular Roles of Brassinosteroids and Salicylic Acid in Plant Processes and Salt Tolerance. Crit. Rev. Plant Sci. 2010, 29 , 162–190. 10.1080/07352689.2010.483580. Munns R. ; Tester M. Mechanisms of Salinity Tolerance. Annu. Rev. Plant Biol. 2008, 59 , 651–681. 10.1146/annurev.arplant.59.032607.092911.18444910 Akram N. A. ; Saleem M. H. ; Shafiq S. ; Naz H. ; Farid-ul-Haq M. ; Ali B. ; Shafiq F. ; Iqbal M. ; Jaremko M. ; Qureshi K. A. Phytoextracts as Crop Biostimulants and Natural Protective Agents—A Critical Review. Sustainability 2022, 14 , 14498 10.3390/su142114498. Hussain S. S. ; Rasheed M. ; Hamzah Saleem M. ; Ahmed Z. I. ; Hafeez A. ; Jilani G. ; Alamri S. ; Hashem M. ; Ali S. Salt Tolerance in Maize with Melatonin Priming to Achieve Sustainability in Yield on Salt Affected Soils. Pak. J. Bot. 2023, 55 , 19 10.30848/pjb2023-1(27). Khan M. A. ; Adnan M. ; Basir A. ; Fahad S. ; Hafeez A. ; Saleem M. H. ; Ahmad M. ; Gul F. ; Durrishahwar F. ; Subhan F. Impact of Tillage and Potassium Levels and Sources on Growth, Yield and Yield Attributes of Wheat. Pak. J. Bot. 2023, 55 , 321 10.30848/PJB2023-1(30). Zhang X. ; Goldberg M. ; Tarpley D. ; Friedl M. A. ; Morisette J. ; Kogan F. ; Yu Y. Drought-Induced Vegetation Stress in Southwestern North America. Environ. Res. Lett. 2010, 5 , 24008 10.1088/1748-9326/5/2/024008. Taiz L. ; Zeiger E. ; Møller I. M. ; Murphy A. Plant Physiology and Development; Sinauer Associates Incorporated, 2015. Yandigeri M. S. ; Meena K. K. ; Singh D. ; Malviya N. ; Singh D. P. ; Solanki M. K. ; Yadav A. K. ; Arora D. K. Drought-Tolerant Endophytic Actinobacteria Promote Growth of Wheat (Triticum Aestivum) under Water Stress Conditions. Plant Growth Regul. 2012, 68 , 411–420. 10.1007/s10725-012-9730-2. Talebi R. ; Fayaz F. ; Naji A. M. Effective Selection Criteria for Assessing Drought Stress Tolerance in Durum Wheat (Triticum Durum Desf.). Gen. Appl. Plant Physiol. 2009, 35 , 64–74. Szilagyi L. Influence of Drought on Seed Yield Components in Common Bean. Bulg. J. Plant Physiol. 2003, 2003 , 320–330. Bajehbaj A. A. The Effects of NaCl Priming on Salt Tolerance in Sunflower Germination and Seedling Grown under Salinity Conditions. Afr. J. Biotechnol. 2010, 9 , 1764 10.5897/AJB10.1019. Kaydan D. ; Yagmur M. ; Okut N. Effects of Salicylic Acid on the Growth and Some Physiological Characters in Salt Stressed Wheat (Triticum Aestivum L.). Tarim Bilimleri Derg. 2007, 13 , 114–119. Ashraf M. ; Bokhari M. H. ; Chishti S. N. Variation in Osmotic Adjustment of Accessions of Lentil (Lens Culinaris Medic.) in Response to Drought Stress. Acta Bot. Neerl. 1992, 41 , 51–62. 10.1111/j.1438-8677.1992.tb01310.x. Singh D. ; Dhillon T. S. ; Javed T. ; Singh R. ; Dobaria J. ; Dhankhar S. K. ; Kianersi F. ; Ali B. ; Poczai P. ; Kumar U. Exploring the Genetic Diversity of Carrot Genotypes through Phenotypically and Genetically Detailed Germplasm Collection. Agronomy 2022, 12 , 1921 10.3390/agronomy12081921. Tahir O. ; Ali S. ; Bangash K. ; Ibrahim M. ; Shahab S. ; Khattak S. H. ; Din I. U. ; Khan M. N. ; Hafeez A. ; Wahab S. ; Ali B. ; Makki R. M. ; Harakeh S. Evaluation of Agronomic Performance and Genetic Diversity Analysis Using Simple Sequence Repeats Markers in Selected Wheat Lines. Sustainability 2023, 15 , 293 10.3390/su15010293. Iqbal M. ; Ashraf M. Seed Treatment with Auxins Modulates Growth and Ion Partitioning in Salt-stressed Wheat Plants. J. Integr. Plant Biol. 2007, 49 , 1003–1015. 10.1111/j.1672-9072.2007.00488.x. Kaur S. ; Gupta A. K. ; Kaur N. Effect of Osmo-and Hydropriming of Chickpea Seeds on Seedling Growth and Carbohydrate Metabolism under Water Deficit Stress. Plant Growth Regul. 2002, 37 , 17–22. 10.1023/A:1020310008830. Bailly C. ; Benamar A. ; Corbineau F. ; Côme D. Antioxidant Systems in Sunflower (Helianthus Annuus L.) Seeds as Affected by Priming. Seed Sci. Res. 2000, 10 , 35–42. 10.1017/S0960258500000040. Casenave E. C. ; Toselli M. E. Hydropriming as a Pre-Treatment for Cotton Germination under Thermal and Water Stress Conditions. Seed Sci. Technol. 2007, 35 , 88–98. 10.15258/sst.2007.35.1.08. Levy Y. ; Syvertsen J. Irrigation Water Quality and Salinity. Effects in Citrus Trees. Hortic. Rev. (Am. Soc. Hortic. Sci.) 2010, 30 , 37–82. Ali B. ; Hafeez A. ; Javed M. A. ; Afridi M. S. ; Abbasi H. A. ; Qayyum A. ; Batool T. ; Ullah A. ; Marc R. A. ; Al Jaouni S. K. ; Alkhalifah D. H. M. ; Selim S. Role of Endophytic Bacteria in Salinity Stress Amelioration by Physiological and Molecular Mechanisms of Defense: A Comprehensive Review. S. Afr. J. Bot. 2022, 151 , 33–46. 10.1016/j.sajb.2022.09.036. Ali B. ; Wang X. ; Saleem M. H. ; Sumaira ; Hafeez A. ; Afridi M. S. ; Khan S. ; Zaib-Un-Nisa ; Ullah I. ; Amaral Júnior A. T. d. ; Alatawi A. ; Ali S. PGPR-Mediated Salt Tolerance in Maize by Modulating Plant Physiology, Antioxidant Defense, Compatible Solutes Accumulation and Bio-Surfactant Producing Genes. Plants 2022, 11 , 345 10.3390/plants11030345.35161325 Zekri M. Strategies to Manage Salinity Problem in Citrus. In Proceedings of International Society of Citriculture; 2004; pp. 639–643. Raveh E. ; Levy Y. Analysis of Xylem Water as an Indicator of Current Chloride Uptake Status in Citrus Trees. Sci. Hortic. (Amsterdam) 2005, 103 , 317–327. 10.1016/j.scienta.2004.06.007. Kamal A. ; Qureshi M. S. ; Ashraf M. Y. ; Hussain M. Salinity Induced Changes in Some Growth and Physio-Chemical Aspects of Two Soybean [Glycine Max (l.) Merr.] Genotypes. Pak. J. Bot. 2003, 35 , 93–97. García-Sánchez F. ; Jifon J. L. ; Carvajal M. ; Syvertsen J. P. Gas Exchange, Chlorophyll and Nutrient Contents in Relation to Na+ and Cl– Accumulation in ‘Sunburst’Mandarin Grafted on Different Rootstocks. Plant Sci. 2002, 162 , 705–712. 10.1016/S0168-9452(02)00010-9. Saleem A. ; Zulfiqar A. ; Ali B. ; Naseeb M. A. ; Almasaudi A. S. ; Harakeh S. Iron Sulfate (FeSO4) Improved Physiological Attributes and Antioxidant Capacity by Reducing Oxidative Stress of Oryza Sativa L. Cultivars in Alkaline Soil. Sustainability 2022, 14 , 16845 10.3390/su142416845. Khan M. A. ; Shirazi M. U. ; Khan M. A. ; Mujtaba S. M. ; Islam E. ; Mumtaz S. ; Shereen A. ; Ansari R. U. ; Ashraf M. Y. Role of Proline, K/Na Ratio and Chlorophyll Content in Salt Tolerance of Wheat (Triticum Aestivum L.). Pak. J. Bot. 2009, 41 , 633–638. Yagmur M. ; Kaydan D. ; Okut N. Alleviation of Salinity Stress during Seed Germination in Wheat (Triticum Aestivum) by Potassium Applications;8 2011. Azeem M. ; Pirjan K. ; Qasim M. ; Mahmood A. ; Javed T. ; Muhammad H. ; Yang S. ; Dong R. ; Ali B. ; Rahimi M. Salinity Stress Improves Antioxidant Potential by Modulating Physio-Biochemical Responses in Moringa Oleifera Lam. Sci. Rep. 2023, 13 , 2895 10.1038/s41598-023-29954-6.36807545 Ghoulam C. ; Foursy A. ; Fares K. Effects of Salt Stress on Growth, Inorganic Ions and Proline Accumulation in Relation to Osmotic Adjustment in Five Sugar Beet Cultivars. Environ. Exp. Bot. 2002, 47 , 39–50. 10.1016/S0098-8472(01)00109-5. Chinnusamy V. ; Jagendorf A. ; Zhu J.-K. Understanding and Improving Salt Tolerance in Plants. Crop Sci. 2005, 45 , 437–448. 10.2135/cropsci2005.0437. Khan A. A. Preplant Physiological Seed Conditioning. Hortic. Rev. (Am. Soc. Hortic. Sci.) 2010, 13 , 131–181. Flowers T. J. Improving Crop Salt Tolerance. J. Exp. Bot. 2004, 55 , 307–319. 10.1093/jxb/erh003.14718494 Murillo-Amador B. ; López-Aguilar R. ; Kaya C. ; Larrinaga-Mayoral J. ; Flores-Hernández A. Comparative Effects of NaCl and Polyethylene Glycol on Germination, Emergence and Seedling Growth of Cowpea. J. Agron. Crop Sci. 2002, 188 , 235–247. 10.1046/j.1439-037X.2002.00563.x. Ashraf M. Some Important Physiological Selection Criteria for Salt Tolerance in Plants. Flora-Morphol., Distrib. Funct. Ecol. Plants 2004, 199 , 361–376. 10.1078/0367-2530-00165. Ueda A. ; Kanechi M. ; Uno Y. ; Inagaki N. Photosynthetic Limitations of a Halophyte Sea Aster (Aster Tripolium L) under Water Stress and NaCl Stress. J. Plant Res. 2003, 116 , 63–68. 10.1007/s10265-002-0070-6. Yasmeen S. ; Wahab A. ; Saleem M. H. ; Ali B. ; Qureshi K. A. ; Jaremko M. Melatonin as a Foliar Application and Adaptation in Lentil (Lens Culinaris Medik.) Crops under Drought Stress. Sustainability 2022, 14 , 16345 10.3390/su142416345. Asma ; Hussain I. ; Ashraf m. y. ; Saleem M. H. ; Ashraf M. A. ; Ali B. ; Shereen A. ; Farid G. ; Ali M. ; Shirazi M. U. ; Saleem A. Alleviating Effects of Salicylic Acid Spray on Stage-Based Growth and Antioxidative Defense System in Two Drought-Stressed Rice (Oryza Sativa L.) Cultivars. Turk. J. Agric. For. 2023, 47 , 79–99. 10.55730/1300-011X.3066. Al-Huqail A. A. ; Saleem M. H. ; Ali B. ; Azeem M. ; Mumtaz S. ; Yasin G. ; Marc R. A. ; Ali S. Efficacy of Priming Wheat (Triticum Aestivum) Seeds with a Benzothiazine Derivative to Improve Drought Stress Tolerance. Funct. Plant Biol. 2023, 10.1071/FP22140. Miller G. ; Suzuki N. ; Ciftci-Yilmaz S. ; Mittler R. Reactive Oxygen Species Homeostasis and Signalling during Drought and Salinity Stresses. Plant, Cell Environ. 2010, 33 , 453–467. 10.1111/j.1365-3040.2009.02041.x.19712065 Salam A. ; Afridi M. S. ; Javed M. A. ; Saleem A. ; Hafeez A. ; Khan A. R. ; Zeeshan M. ; Ali B. ; Azhar W. ; Sumaira ; Ulhassan Z. ; Gan Y. Nano-Priming against Abiotic Stress: A Way Forward towards Sustainable Agriculture. Sustainability 2022, 14 , 14880 10.3390/su142214880. Ahmad I. ; Khaliq T. ; Ahmad A. ; Basra S. M. A. ; Hasnain Z. ; Ali A. Effect of Seed Priming with Ascorbic Acid, Salicylic Acid and Hydrogen Peroxide on Emergence, Vigor and Antioxidant Activities of Maize. Afr. J. Biotechnol. 2012, 11 , 1127–1132. 10.5897/AJB11.2266. Hameed A. ; Afzal I. ; Iqbal N. Seed Priming and Salinity Induced Variations in Wheat (Triticum Aestivum L.) Leaf Protein Profile. Seed Sci. Technol. 2010, 38 , 236–241. 10.15258/sst.2010.38.1.25. Radman R. ; Saez T. ; Bucke C. ; Keshavarz T. Elicitation of Plants and Microbial Cell Systems. Biotechnol. Appl. Biochem. 2003, 37 , 91–102. 10.1042/BA20020118.12578556 Kurita K. Chitin and Chitosan: Functional Biopolymers from Marine Crustaceans. Mar. Biotechnol. 2006, 8 , 203–226. 10.1007/s10126-005-0097-5. Zhou Y. G. ; Yang Y. D. ; Qi Y. G. ; Zhang Z. M. ; Wang X. J. ; Hu X. J. Effects of Chitosan on Some Physiological Activity in Germinating Seed of Peanut. J. Peanut Sci. 2002, 31 , 22–25. Dzung N. A. ; Thang N. T. ; Suchiva V. K. ; Chandrkrachang S. ; Methacanon P. ; Peter M. G. Effect of Oligoglucosamine Prepared by Enzyme Degradation on the Growth of Soybean. Adv. Chitin Sci. 2002, 5 , 463–467. Alves N. M. ; Mano J. F. Chitosan Derivatives Obtained by Chemical Modifications for Biomedical and Environmental Applications. Int. J. Biol. Macromol. 2008, 43 , 401–414. 10.1016/j.ijbiomac.2008.09.007.18838086 Jackson M. L. Soil Chemical Analysis; Pentice Hall of India Pvt. Ltd.: New Delhi, India 1973, 498 , 151–154. Nelson L. B. ; Heidel H. Soil Analysis Methods as Used in the Iowa State College Soil Testing Laboratory; 1952. Khan I. ; Javed T. ; Khan A. ; Lei H. ; Muhammad I. ; Ali I. ; Huo X. Impact Assessment of Land Use Change on Surface Temperature and Agricultural Productivity in Peshawar-Pakistan. Environ. Sci. Pollut. Res. 2019, 26 , 33076–33085. 10.1007/s11356-019-06448-5. Guan Y. ; Hu J. ; Wang X. ; Shao C. Seed Priming with Chitosan Improves Maize Germination and Seedling Growth in Relation to Physiological Changes under Low Temperature Stress. J. Zhejiang Univ., Sci., B 2009, 10 , 427–433. 10.1631/jzus.B0820373.19489108 Hameed A. ; Sheikh M. A. ; Hameed A. ; Farooq T. ; Basra S. M. A. ; Jamil A. Chitosan Priming Enhances the Seed Germination, Antioxidants, Hydrolytic Enzymes, Soluble Proteins and Sugars in Wheat Seeds. Agrochimica 2013, 57 , 97–110. Harris D. ; Raghuwanshi B. S. ; Gangwar J. S. ; Singh S. C. ; Joshi K. D. ; Rashid A. ; Hollington P. A. Participatory Evaluation by Farmers of On-Farm Seed Priming in Wheat in India, Nepal and Pakistan. Exp. Agric. 2001, 37 , 403–415. 10.1017/S0014479701003106. Hameed A. ; Sheikh M. A. ; Hameed A. ; Farooq T. ; Basra S. M. A. ; Jamil A. Chitosan Seed Priming Improves Seed Germination and Seedling Growth in Wheat (Triticum Aestivum L.) under Osmotic Stress Induced by Polyethylene Glycol. Philipp. Agric. Sci. 2014, 97 , 294–299. Ghule P. L. ; Dahiphale V. V. ; Jadhav J. D. ; Palve D. K. Absolute Growth Rate, Relative Growth Rate, Net Assimilation Rate as Influenced on Dry Matter Weight of Bt Cotton. Int. Res. J. Agric. Econ. Stat. 2013, 4 , 42–46. Shah A. N. ; Yang G. ; Tanveer M. ; Iqbal J. Leaf Gas Exchange, Source–Sink Relationship, and Growth Response of Cotton to the Interactive Effects of Nitrogen Rate and Planting Density. Acta Physiol. Plant. 2017, 39 , 1–10. 10.1007/s11738-017-2402-0. Bina F. ; Bostani A. Effect of Salinity (NaCl) Stress on Germination and Early Seedling Growth of Three Medicinal Plant Species. Adv. Life Sci. 2017, 4 , 77–83. Chuyong G. B. ; Acidri T. Light and Moisture Levels Affect Growth and Physiological Parameters Differently in Faidherbia Albida (Delile) A. Chev. Seedlings. Acta Physiol. Plant. 2017, 39 , 1–6. 10.1007/s11738-017-2410-0. Gu J. ; Zhou Z. ; Li Z. ; Chen Y. ; Wang Z. ; Zhang H. Rice (Oryza Sativa L.) with Reduced Chlorophyll Content Exhibit Higher Photosynthetic Rate and Efficiency, Improved Canopy Light Distribution, and Greater Yields than Normally Pigmented Plants. Field Crops Res. 2017, 200 , 58–70. 10.1016/j.fcr.2016.10.008. El-Esawi M. A. ; Alaraidh I. A. ; Alsahli A. A. ; Alzahrani S. M. ; Ali H. M. ; Alayafi A. A. ; Ahmad M. Serratia Liquefaciens KM4 Improves Salt Stress Tolerance in Maize by Regulating Redox Potential, Ion Homeostasis, Leaf Gas Exchange and Stress-Related Gene Expression. Int. J. Mol. Sci. 2018, 19 , 3310 10.3390/ijms19113310.30355997 Mendez R. L. ; Kwon J. Y. Effect of Extraction Condition on Protein Recovery and Phenolic Interference in Pacific Dulse (Devaleraea Mollis). J. Appl. Phycol. 2021, 33 , 2497–2509. 10.1007/s10811-021-02467-3. Parveen A. ; Siddiqui Z. A. Zinc Oxide Nanoparticles Affect Growth, Photosynthetic Pigments, Proline Content and Bacterial and Fungal Diseases of Tomato. Arch. Phytopathol. Plant Prot. 2021, 54 , 1519–1538. 10.1080/03235408.2021.1917952. Grad W. E. ; Kandil S. H. ; Kenawy E. ; Massoud M. I. The Potential of Sugarcane Bagasse Polymer Composite for Sustainable of Stevia Rebaudiana Productivity under Deficit Irrigation. SVU-Int. J. Agric. Sci. 2021, 3 , 22–36. 10.21608/svuijas.2021.62483.1081. Ali B. ; Hafeez A. ; Ahmad S. ; Javed M. A. ; Sumaira ; Afridi M. S. ; Dawoud T. M. ; Almaary K. S. ; Muresan C. C. ; Marc R. A. ; Alkhalifah D. H. M. ; Selim S. Bacillus Thuringiensis PM25 Ameliorates Oxidative Damage of Salinity Stress in Maize via Regulating Growth, Leaf Pigments, Antioxidant Defense System, and Stress Responsive Gene Expression. Front. Plant Sci. 2022, 13 , 921668 10.3389/fpls.2022.921668.35968151 Velikova V. ; Yordanov I. ; Edreva A. Oxidative Stress and Some Antioxidant Systems in Acid Rain-Treated Bean Plants: Protective Role of Exogenous Polyamines. Plant Sci. 2000, 151 , 59–66. 10.1016/S0168-9452(99)00197-1. Faryal S. ; Ullah R. ; Khan M. N. ; Ali B. ; Hafeez A. ; Jaremko M. ; Qureshi K. A. Thiourea-Capped Nanoapatites Amplify Osmotic Stress Tolerance in Zea Mays L. by Conserving Photosynthetic Pigments, Osmolytes Biosynthesis and Antioxidant Biosystems. Molecules 2022, 27 , 5744 10.3390/molecules27185744.36144480 Farhad W. ; Saleem M. F. ; Cheema M. A. ; Hammad H. M. Effect of Poultry Manure Levels on the Productivity of Spring Maize (Zea Mays L.). J. Anim. Plant Sci. 2009, 19 , 122–125. Rosegrant M. W. ; Msangi S. ; Ringler C. ; Sulser T. B. ; Zhu T. ; Cline S. A. International Model for Policy Analysis of Agricultural Commodities and Trade (IMPACT): Model Description; 2008. Khayatnezhad M. ; Gholamin R. ; Jamaatie-Somarin S. H. ; Zabihi-Mahmoodabad R. Effects of Peg Stress on Corn Cultivars (Zea Mays L.) at Germination Stage. World Appl. Sci. J. 2010, 11 , 504–506. Almaghrabi O. A. Impact of Drought Stress on Germination and Seedling Growth Parameters of Some Wheat Cultivars. Life Sci. J. 2012, 9 , 590–598. Mouradi M. ; Bouizgaren A. ; Farissi M. ; Makoudi B. ; Kabbadj A. ; Very A.-A. ; Sentenac H. ; Qaddoury A. ; Ghoulam C. Osmopriming Improves Seeds Germination, Growth, Antioxidant Responses and Membrane Stability during Early Stage of Moroccan Alfalfa Populations under Water Deficit. Chil. J. Agric. Res. 2016, 76 , 265–272. 10.4067/S0718-58392016000300002. Murungu F. S. ; Nyamugafata P. ; Chiduza C. ; Clark L. J. ; Whalley W. R. Effects of Seed Priming and Water Potential on Germination of Cotton (Gossypium Hirsutum L.) and Maize (Zea Mays L.) in Laboratory Assays. S. Afr. J. Plant Soil 2005, 22 , 64–70. 10.1080/02571862.2005.10634683. Hirano S. The Activation of Plant Cells and Their Self-Defense Function against Pathogens in Connection with Chitosan. J. Agric. Chem. Soc. Jpn. 1988, 1238 10.1271/nogeikagaku1924.62.1238. Shibuya N. ; Ito Y. ; Kaku H. Receptor for Chitin Oligosaccharide Elicitor in Rice. Chem. Regul. Plants 1996, 31 , 125–133. Ajouri A. ; Asgedom H. ; Becker M. Seed Priming Enhances Germination and Seedling Growth of Barley under Conditions of P and Zn Deficiency. J. Plant Nutr. Soil Sci. 2004, 167 , 630–636. 10.1002/jpln.200420425. Boonlertnirun S. ; Sarobol E. D. ; Meechoui S. ; Sooksathan I. Drought Recovery and Grain Yield Potential of Rice after Chitosan Application. Agric. Nat. Resour. 2007, 41 , 1–6. Songlin R. ; Qingzhong X. Effects of Chitosan Coating on Seed Germination and Salt-Tolerance of Seedling in Hybrid Rice (Oryza Sativa L.). Zuowu Xuebao 2002, 28 , 803–808. Utsunomiya N. ; Kinai H. Effect of Chitosan-Oligosaccharides Soil Conditioner on the Growth of Passionfruit. J. Jpn. Soc. Hortic. Sci. 1994, 64 , 176–177. Shao C. X. ; Hu J. ; Song W. J. ; Hu W. M. Effects of Seed Priming with Chitosan Solutions of Different Acidity on Seed Germination and Physiological Characteristics of Maize Seedling. J. Zhejiang Univ. (Agric. Life Sci.) 2005, 31 , 705–708. Zaheer M. S. ; Raza M. A. S. ; Saleem M. F. ; Khan I. H. ; Ahmad S. ; Iqbal R. ; Manevski K. Investigating the Effect of Azospirillum Brasilense and Rhizobium Pisi on Agronomic Traits of Wheat (Triticum Aestivum L.). Arch. Agron. Soil Sci. 2019, 1554 10.1080/03650340.2019.1566954. Yin C. ; Wang X. ; Duan B. ; Luo J. ; Li C. Early Growth, Dry Matter Allocation and Water Use Efficiency of Two Sympatric Populus Species as Affected by Water Stress. Environ. Exp. Bot. 2005, 53 , 315–322. 10.1016/j.envexpbot.2004.04.007. Kramer P. J. ; Boyer J. S. Water Relations of Plants and Soils; Academic press, 1995. Ali B. ; Wang X. ; Saleem M. H. ; Azeem M. A. ; Afridi M. S. ; Nadeem M. ; Ghazal M. ; Batool T. ; Qayyum A. ; Alatawi A. ; Ali S. Bacillus Mycoides PM35 Reinforces Photosynthetic Efficiency, Antioxidant Defense, Expression of Stress-Responsive Genes, and Ameliorates the Effects of Salinity Stress in Maize. Life 2022, 12 , 219 10.3390/life12020219.35207506 Bibi S. ; Ullah S. ; Hafeez A. ; Khan M. N. ; Javed M. A. ; Ali B. ; Din I. U. ; Bangash S. A. K. ; Wahab S. ; Wahid N. ; Zaman F. ; Alhag S. K. ; el-Rahim I. H. A. A. ; Ahmed A. E. ; Selim S. Exogenous Ca/Mg Quotient Reduces the Inhibitory Effects of PEG Induced Osmotic Stress on Avena Sativa L. Braz. J. Biol. 2024, 84 , e264642 10.1590/1519-6984.264642. Ali J. ; Jan I. ; Ullah H. ; Fahad S. ; Saud S. ; Adnan M. ; Ali B. ; Liu K. ; Harrison M. T. ; Hassan S. ; Kumar S. ; Khan M. A. ; Kamran M. ; Alwahibi M. S. ; Elshikh S. M. Biochemical Response of Okra (Abelmoschus Esculentus L.) to Selenium (Se) under Drought Stress. Sustainability 2023, 15 , 5694 10.3390/su15075694. Manivannan P. ; Jaleel C. A. ; Kishorekumar A. ; Sankar B. ; Somasundaram R. ; Sridharan R. ; Panneerselvam R. Changes in Antioxidant Metabolism of Vigna Unguiculata (L.) Walp. by Propiconazole under Water Deficit Stress. Colloids Surf., B 2007, 57 , 69–74. 10.1016/j.colsurfb.2007.01.004. Anjum S. A. ; Wang L. ; Farooq M. ; Khan I. ; Xue L. Methyl Jasmonate-induced Alteration in Lipid Peroxidation, Antioxidative Defence System and Yield in Soybean under Drought. J. Agron. Crop Sci. 2011, 197 , 296–301. 10.1111/j.1439-037X.2011.00468.x. Ali Q. ; Ashraf M. Induction of Drought Tolerance in Maize (Zea Mays L.) Due to Exogenous Application of Trehalose: Growth, Photosynthesis, Water Relations and Oxidative Defence Mechanism. J. Agron. Crop Sci. 2011, 197 , 258–271. 10.1111/j.1439-037X.2010.00463.x. Khan W. M. ; Prithiviraj B. ; Smith D. L. Effect of Foliar Application of Chitin and Chitosan Oligosaccharides on Photosynthesis of Maize and Soybean. Photosynthetica 2002, 40 , 621–624. 10.1023/A:1024320606812. Sara K. ; Hossein A. ; Masoud S. J. ; Hassan M. Effects of Water Deficit and Chitosan Spraying on Osmotic Adjustment and Soluble Protein of Cultivars Castor Bean (Ricinus Communis L.). J. Stress Physiol. Biochem. 2012, 8 , 160–169. Wang X. ; Xiang Y. ; Zhou B. ; Zhang Y. ; Wu J. ; Hu R. ; Liu L. ; Song J. ; Qu J. Enhanced Photocatalytic Performance of Ag/TiO2 Nanohybrid Sensitized by Black Phosphorus Nanosheets in Visible and near-Infrared Light. J. Colloid Interface Sci. 2019, 534 , 1–11. 10.1016/j.jcis.2018.09.013.30196196 Mehmood S. ; Khatoon Z. ; Amna ; Ahmad I. ; Muneer M. A. ; Kamran M. A. ; Ali J. ; Ali B. ; Chaudhary H. J. ; Munis M. F. H. Bacillus Sp. PM31 Harboring Various Plant Growth-Promoting Activities Regulates Fusarium Dry Rot and Wilt Tolerance in Potato. Arch. Agron. Soil Sci. 2021, 1–15. 10.1080/03650340.2021.1971654. Dobrá J. ; Vanková R. ; Havlová M. ; Burman A. J. ; Libus J. ; Štorchová H. Tobacco Leaves and Roots Differ in the Expression of Proline Metabolism-Related Genes in the Course of Drought Stress and Subsequent Recovery. J. Plant Physiol. 2011, 168 , 1588–1597. 10.1016/j.jplph.2011.02.009.21481968 Zainab N. ; Amna ; Khan A. A. ; Azeem M. A. ; Ali B. ; Wang T. ; Shi F. ; Alghanem S. M. ; Munis M. F. H. ; Hashem M. ; Alamri S. ; Latef A. A. H. A. ; Ali O. M. ; Soliman M. H. ; Chaudhary H. J. PGPR-Mediated Plant Growth Attributes and Metal Extraction Ability of Sesbania Sesban l. In Industrially Contaminated Soils. Agronomy 2021, 11 , 1820 10.3390/agronomy11091820. Ahmad P. ; Jaleel C. A. ; Sharma S. Antioxidant Defense System, Lipid Peroxidation, Proline-Metabolizing Enzymes, and Biochemical Activities in Two Morus Alba Genotypes Subjected to NaCl Stress. Russ. J. Plant Physiol. 2010, 57 , 509–517. 10.1134/S1021443710040084. Mahdavi B. ; Modarres Sanavy S. A. M. ; Aghaalikhani M. ; Sharifi M. ; Dolatabadian A. Chitosan Improves Osmotic Potential Tolerance in Safflower (Carthamus Tinctorius L.) Seedlings. J. Crop Improv. 2011, 25 , 728–741. 10.1080/15427528.2011.606354. Demiral T. ; Türkan I. Does Exogenous Glycinebetaine Affect Antioxidative System of Rice Seedlings under NaCl Treatment?. J. Plant Physiol. 2004, 161 , 1089–1100. 10.1016/j.jplph.2004.03.009.15535118 Ali S. ; Ullah S. ; Khan M. N. ; Khan W. M. ; Razak S. A. ; Wahab S. ; Hafeez A. ; Khan Bangash S. A. ; Poczai P. The Effects of Osmosis and Thermo-Priming on Salinity Stress Tolerance in Vigna Radiata L. Sustainability 2022, 14 , 12924 10.3390/su141912924. Hasanuzzaman M. ; Hossain M. A. ; Fujita M. Nitric Oxide Modulates Antioxidant Defense and the Methylglyoxal Detoxification System and Reduces Salinity-Induced Damage of Wheat Seedlings. Plant Biotechnol. Rep. 2011, 5 , 353–365. 10.1007/s11816-011-0189-9. Shah W. ; Zaman N. ; Ullah S. ; Nafees M. Calcium Chloride Enhances Growth and Physio-Biochemical Performance of Barley (Hordeum Vulgare L.) under Drought-Induced Stress Regimes: A Future Perspective of Climate Change in the Region. J. Water Clim. Change 2022, 13 , 3357–3378. 10.2166/wcc.2022.134. Ahmad P. ; Sarwat M. ; Bhat N. A. ; Wani M. R. ; Kazi A. G. ; Tran L.-S. P. Alleviation of Cadmium Toxicity in Brassica Juncea L.(Czern. & Coss.) by Calcium Application Involves Various Physiological and Biochemical Strategies. PLoS One 2015, 10 , e0114571 10.1371/journal.pone.0114571.25629695 Afridi M. S. ; Ali S. ; Salam A. ; César Terra W. ; Hafeez A. ; Sumaira ; Ali B. ; AlTami S. M. ; Ameen F. ; Ercisli S. ; Marc R. A. ; Medeiros F. H. V. ; Karunakaran R. Plant Microbiome Engineering: Hopes or Hypes. Biology (Basel) 2022, 11 , 1782 10.3390/biology11121782.36552290 Afridi M. S. ; Javed M. A. ; Ali S. ; De Medeiros F. H. V. ; Ali B. ; Salam A. ; Sumaira ; Marc R. A. ; Alkhalifah D. H. M. ; Selim S. ; Santoyo G. New Opportunities in Plant Microbiome Engineering for Increasing Agricultural Sustainability under Stressful Conditions. Front. Plant Sci. 2022, 13 , 899464 10.3389/fpls.2022.899464.36186071 Menezes-Benavente L. ; Kernodle S. P. ; Margis-Pinheiro M. ; Scandalios J. G. Salt-Induced Antioxidant Metabolism Defenses in Maize (Zea Mays L.) Seedlings. Redox Rep. 2004, 9 , 29–36. 10.1179/135100004225003888.15035825 Wahab A. ; Abdi G. ; Saleem M. H. ; Ali B. ; Ullah S. ; Shah W. ; Mumtaz S. ; Yasin G. ; Muresan C. C. ; Marc R. A. Plants’ Physio-Biochemical and Phyto-Hormonal Responses to Alleviate the Adverse Effects of Drought Stress: A Comprehensive Review. Plants 2022, 11 , 1620 10.3390/plants11131620.35807572 Hameed A. ; Bibi N. ; Akhter J. ; Iqbal N. Differential Changes in Antioxidants, Proteases, and Lipid Peroxidation in Flag Leaves of Wheat Genotypes under Different Levels of Water Deficit Conditions. Plant Physiol. Biochem. 2011, 49 , 178–185. 10.1016/j.plaphy.2010.11.009.21159517 Hassni M. ; El Hadrami A. ; El Hadrami I. ; Barka E. A. ; Daayf F. Chitosan, Antifungal Product against″ Fusarium Oxysporum″ f. Sp.″ Albedinis″ and Elicitor of Defence Reactions in Date Palm Roots; Firenze University Press, 2004, 1000–1010. Pukacka S. ; Malec M. ; Ratajczak E. ROS Production and Antioxidative System Activity in Embryonic Axes of Quercus Robur Seeds under Different Desiccation Rate Conditions. Acta Physiol. Plant. 2011, 33 , 2219–2227. 10.1007/s11738-011-0761-5. Roach T. ; Beckett R. P. ; Minibayeva F. V. ; Colville L. ; Whitaker C. ; Chen H. ; Bailly C. ; Kranner I. Extracellular Superoxide Production, Viability and Redox Poise in Response to Desiccation in Recalcitrant Castanea Sativa Seeds. Plant. Cell Environ. 2010, 33 , 59–75. 10.1111/j.1365-3040.2009.02053.x.19843255