Biochar Application for Alleviating Nickel Stress and Enhancing Growth, Photosynthetic Pigments, and Antioxidant Defense Mechanisms in Sorghum

Biochar Application for Alleviating Nickel Stress and Enhancing Growth, Photosynthetic Pigments, and Antioxidant Defense Mechanisms in Sorghum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Biochar Application for Alleviating Nickel Stress and Enhancing Growth, Photosynthetic Pigments, and Antioxidant Defense Mechanisms in Sorghum Ayesha Arshad, Athar Mahmood, Safura Bibi, Muhammad Mansoor Javaid, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6536755/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Increased nickel (Ni) concentrations in plant cells induce physiological, metabolic, and cellular changes, resulting in serious damage to the plants. Among the various strategies to mitigate Ni toxicity in plants, the use of biochar (BC) is highly effective. Biochar enhances soil remediation by immobilizing Ni, reducing its bioavailability, and improving overall soil health. This research aimed to evaluate the effectiveness of BC in alleviating Ni stress in sorghum. The BC was applied at 0, 2.5 and 5 g per kg of soil and Ni concentration was kept at 0, 50 and 100 ppm. The results indicated that the treatment of BC at 5 g enhanced the root and shoot length, fresh weight, and dry weight of both shoot and root, but the application of Ni reduced all assessed growth parameters. Under Ni stress conditions, a concentration of 5 g of biochar enhanced the photosynthetic pigments chlorophyll a , chlorophyll b , total chlorophyll, chlorophyll ratio, and carotenoids. At 5 g, biochar lowered the amounts of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) by increasing the activity of the antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). However, Ni stress increased the amounts of MDA and H 2 O 2 . The application of 5 g of BC under Ni stress enhanced the uptake of Ca²⁺ and K⁺ while reducing Na⁺ accumulation. In conclusion, the application of BC at 5g per kg of soil enhanced plant growth, photosynthetic pigments, Ni absorption, antioxidants, ionic contents, and reduced oxidative stress indications, thereby mitigating Ni stress conditions. Biological sciences/Biochemistry Biological sciences/Biological techniques Biological sciences/Physiology Biological sciences/Psychology Sorghum Antioxidant Biochar Nickel Ionic content Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Introduction Sorghum ( Sorghum bicolor L.), a member of the Poaceae family, is a perennial, summer-growing plant cultivated for multiple purposes, including grain production, animal feed, sugar extraction, and bioenergy. Almost 50 percent of the sorghum crop is used for human diet all over the world. Out of this about 90% of sorghum crop is used for animal nourishment in USA 1 . Sorghum is the fifth most important cereal crop globally, cultivated across 44 million acres in 99 countries. Sorghum grows best in well-drained soils with moderate organic matter and a pH of 6–7.5. However, heavy metal contamination harms its growth. Sorghum mainly accumulates metals in its roots, but higher concentrations increase their movement to the shoots, further reducing plant health 2 . Human activities, such as mining and industrialization, are responsible for the release of heavy metals that have caused changes in the natural ecosystem. Metals that pose a threat to human health include lead, arsenic, cadmium, Ni, and mercury. Ni ranks as the 24th most common element in the earth crust 3 . Industrial locations that have high levels of sewage sludge release substantial quantities of Ni into nearby soils. The concentration of Ni in soil may vary from 3 to 100 ppm. Ni exists in several forms in soil, including inorganic crystalline minerals, on inorganic cation exchange surfaces, on organic cation surfaces, as a free ion, water-soluble, and as a chelated complex. Ni is a threat to human well-being and has the potential to cause cancer and other disorders if ingested via contaminated food 4 . Ni induces deleterious morphological, physiological, or biochemical impacts on living organisms. In plants, they inhibit seedling development, limit root elongation, reduce transpiration, suppress chlorophyll synthesis, inhibit cell division, and ultimately hinder plant growth 5 . Ni is beneficial to plants in small amounts, supporting growth and metabolism, but its excessive accumulation can be toxic. High Ni concentrations disrupt key physiological processes, impairing nutrient uptake, enzyme activity, and photosynthesis. Extreme Ni exposure eventually reduces crop yield and productivity, making it difficult to grow sorghum sustainably in contaminated soils. Ni toxicity in cereals, especially sorghum, negatively impacts shoot development, resulting in stunted growth, chlorosis, and decreased biomass 5 .Therefore, it is important to remove Ni through the soil to lessen the negative effects on plants. Researchers have recommended organic modification to stabilize heavy metal in the soil during the restoration process. Biochar is a carbon-rich solid material produced by the thermal decomposition of organic biomass at relatively low temperatures under limited oxygen conditions. 6 . Recent studies show that adding biochar to soil enhances its agricultural qualities and efficiently eliminates both organic and inorganic pollutants because of its high absorption capacity. BC functional groups help make it an effective modification for overcoming metals in soil because they give the metals active sites to attach, which decreases their mobility in the soil 7 . Sorghum shows better root shoot function and development when treated with biochar among metal-contaminated soils because BC efficiently reduces heavy metals and lowers their bioavailability 8 . Sorghum, unlike many other crops, takes advantage of BC protective properties, particularly when used in high concentrations. As a consequence, it may be used as a useful tactic to increase agricultural resilience in soils that are polluted with heavy metals such as Ni. Within the framework of organic nature alterations, BC has shown impressive effects in boosting the adsorption of metal in soils, thereby lowering their bioavailability 9 . Studies on the impact of BC on crop production are still ongoing. Through assessing its effects on plant growth and development, this study explores its potential to reduce Ni-induced stress in sorghum. When subjected to Ni toxicity, it evaluates the morphological, biochemical, and physiological responses of sorghum to BC amendment. The goal of this research is to add the increasing field of knowledge and set the framework for further studies with the objective of improving biochar application techniques for heavy metal-stressed sustainable crop production. Materials and Methods Experimental material and site The experiment was carried out in the summer seasons of 2023–2024 at the Botanical Garden of community college PARS, University of Agriculture Faisalabad, Pakistan (31.3992° N, 73.0313° E, at a height of approximately 184 meters above mean ground level. These sites were used to assess the effectiveness of biochar in mitigating Ni-induced stress. The Sorghum-2011 variety was obtained from the Fodder Research Institute of the Ayub Agricultural Research Institute (AARI) in Faisalabad, Pakistan Experimental design and treatment application Table 1 provides the physicochemical parameters of the soil at the experiment location. The soil has been collected, dried in the air, and sieved to remove any debris. The initial water content of the soil was determined by weighing 100 g of new soil, which was subsequently oven-dried at 105°C for 24 hours, and the soil dry mass was measured. Soil moisture contents were measured as: Table 1 Soil physicochemical traits and soil irrigation treatments of experimental site Parameters Value References pH 7.5–8.0 48 Electrical Conductivity (EC) 1.2–1.5 dS/m 49 Organic Matter 0.8–1.0% 50 Nitrogen (N) 0.05–0.07% 51 Available Phosphorus (P) 10–15 mg/kg 52 Available Potassium (K) 160–180 mg/kg 53 Bulk Density 1.1–1.3 g/cm³ 54 Soil Texture Loamy to clay loam 55 Cation Exchange Capacity (CEC) 18–22 cmol/kg 56 Nickel (Ni) Concentration 10–50 mg/kg 25 . Soil fresh weight - soil dried weight / soil fresh weight) x 100 The experiment used standard plastic pots measuring 9.5 cm in diameter and 25.5 cm in height, filled with dry soil for planting. To lessen the effect of environmental variability, a total of 27 pots were dispersed using a completely randomized block design, with three replicates of each treatment (Fig. 1 ). These pots were rotated on a regular basis. After a week of seedling and chosen pots were treated with nickel stress consisting of three levels: 0 ppm, 50 ppm, and 100 ppm ( Fig. 2 ) . To address the nutritional needs of the plants, Hoagland nutrient solution was administered at half strength. Furthermore, BC1 (0 g), BC2 (2.5 g), and BC3 (5 g) were the three levels of biochar application that were utilized. Prior to seeding, biochar was added to the top layer of soil. After three to four weeks of seedling growth, data from a number of morpho-physiological, biochemical, ionic, and enzymatic parameters were examined. Morphological characteristics After the experiment, the plants were carefully collected, and the roots were properly rinsed with distilled water in order to eliminate any dirt particles that had adhered to them. Growth characteristics, such as leaf area, length of shoot, root length, and accumulation of biomass, were measured to evaluate the impact of interventions on plant development 10 . Leaf area per plant (mm²) was measured using leaf area meter (MENTION MODEL HERE). A digital weighing scale was used to record the fresh weights of roots and shoots immediately after harvest. Plant samples were dried in an oven at 62°C for two weeks until a consistent weight was reached to measure the dry weight of the plant 11 . Physiological parameters Chlorophyll contents The chlorophyll contents were determined using the method provided by Davis 12 and Arnon 13 . Each replication yielded two plants, each with one fully developed leaf. A 0.5 g leaf sample was mashed with a pestle and mortar before applying 5 mL of acetone with a concentration of 80% to extract the pigments. The extract was kept at 10°C overnight before centrifugation at 1400 revolutions per minute for 5 minutes. The absorbance of the supernatant was measured with the help of a spectrophotometer at 480 nm, 645 nm, and 663 nm to measure carotenoids, chlorophyll b, and chlorophyll a, accordingly 14 by using formula given below. Chl. a (mg/g) = [12.7 (OD663) – 2.69 (OD645)] * V/1000* W Chl. b ( mg/g) =[22.9 (OD645- 4.68(OD663O)] * V/1000*W Chl a/b (mg/g) = Divided chlorophyll a value with chlorophyll b Carotenoid (mg/g) = OD480 + 0.114*OD663-0.638*OD645/2500 Total Chl. = [20.2(OD645)-8.02(OD663)] *V/1000 * W Biochemical parameters Assessment of enzymatic antioxidants Fresh leaf samples were used to measure the enzymatic antioxidant activity. A leaf sample weighing 0.25 g has been homogenized in a pestle and mortar with 5mL of potassium buffer with phosphate. The homogenate was then transferred to a 2 mL eppendorf tube and subsequently centrifuged at 12,000 rpm for thirty minutes at 4°C 15 . The supernatant containing the enzyme extract was removed from contaminants and kept at 20°C for subsequent analysis. Digestion method (ion test and Ni uptake) The concentration of mineral ions in the roots was determined using the digestion method described by Wolf 16 . Dried root samples from each replication were digested in 2.5 mL of concentrated H₂SO₄ at room temperature in digestion flasks. Subsequently, 4 mL of 35% H₂O₂ was added, and the mixture was heated at 350°C until a clear colorless solution was obtained 17 . The digested solution was then filtered and diluted with distilled water to a final volume of 50 mL. Ion concentrations were analyzed using a flame photometer. Super oxide dismutase Activity (SOD) According to Giannopolitis and Ries 18 method Superoxide dismutase (SOD) activity was assessed using fresh leaf samples. A 0.25 g leaf sample was homogenized in 5 mL of 50 mM potassium phosphate buffer using a mortar and pestle. The homogenate was then processed for enzymatic activity analysis. Centrifuge the sample at a speed of 14000 rpm for a duration of 15 minutes. Create a solution of potassium phosphate buffer at a concentration of 20 mM. Dissolve 8.7 g of K 2 HPO 4 and 6.8 g of KH 2 PO 4 in 250 mL of distilled water, each in separate containers. Combine the ingredients separately, and then combine both mixtures together to get a total volume of 500 mL with a pH of 7.4. Prepare a blank solution by adding 50 µl of buffer instead of the sample and 50 µl of distilled water. After adding riboflavin to the blank cuvette, add it to the others. Expose to light for 15 minutes, then analyze using a spectrophotometer 19 . Mix 400 µl of distilled water, 250 µl of 200 mM KP buffer, 100 µl of L. methionine, 100 µl of Triton, 50 µl of NBT, 50 µl of enzyme extract, and 50 µl of riboflavin to make the reaction mixture. Then, measure the absorbance at 560 nm. Record the blank reading last, ensuring it is higher than the samples. Peroxidase (POD) Activity According to Chance and Maehly 20 fresh leaf sample weighing 0.25 g was homogenized in 5 mL of 50 mM potassium phosphate buffer. The supernatant was then separated for enzymatic analysis by centrifuging the mixture for 15 minutes at 4°C and 14,000 rpm. In 250 mL of water that had been distilled, 8.7 g of K₂HPO₄ and 6.8 g of KH₂PO₄ were individually dissolved to create a 20 mM potassium phosphate buffer. In order to get the required buffer concentrations and pH balance for enzymatic activity tests, the solutions were subsequently combined. Mix the components individually, then combine them altogether to obtain a 500 mL volume having a pH of 7.4. Make a solution of a potassium phosphate buffer with a concentration of 50 mM 21 . A 750 µL of Guaiacol was diluted with distilled water to a total volume of 50 mL. Prepare a solution by mixing 100 µL of hydrogen peroxide (22.8 µL in 5 mL water) with 50 µL of enzyme extract, and measure the absorbance at 450 nm. Catalase activity (CAT) Catalase (CAT) activity was determined using the method of Chance and Maehly 20 . Fresh leaf samples (0.25 g) were homogenized in 5 mL of 50 mM potassium phosphate buffer using a mortar and pestle. The homogenate was then centrifuged at 14,000 rpm for 15 minutes at 4°C to obtain the supernatant for enzymatic analysis. A 20 mM potassium phosphate buffer solution was prepared by dissolving the appropriate amounts of K₂HPO₄ and KH₂PO₄ in distilled water, ensuring proper pH adjustment for enzymatic analysis. Dissolve 8.7 g of K 2 HPO 4 and 6.8 g of KH 2 PO 4 in 250 mL of distilled water individually. Combine the two mixtures separately, then combine them together to get a total volume of 500 mL with a pH of 7.4. To produce a solution of H 2 O 2 with a concentration of 0.059 M, measure out 29 µl of H 2 O 2 and transfer it into falcon tubes 22 . Dilute the solution with pure water until the volume reaches 5 mL. Combine 1.9 mL of KP buffer (50mM), 100 µl of H 2 O 2 (0.059 M) and100 µl of enzyme extract, and measure the absorbance at 240 nm. Malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2) content Malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) contents were measured by using the method of Alexieva, et al. 23 to assess oxidative stress in plant tissues. Fresh leaf samples (0.25 g) were homogenized in 5 mL of 5% trichloroacetic acid (TCA). The TCA solution was prepared by dissolving 5 g of TCA in 100 mL of distilled water. For the TCA-TBA reagent, 0.5 g of thiobarbituric acid (TBA, 0.5%) was mixed with 20 g of TCA (20%) and diluted to a final volume of 100 mL with distilled water. Combine 0.5 µl of extract with 0.5 µl of TBA-TCA solution. Then, place it in a water bath for 15 minutes. Cool and measure the absorbance at 532 nm and 600 nm 24 . Homogenize 0.25 g of leaf sample in 1% TCA and centrifuge at 12,000 rpm for 15 minutes. To prepare a 1 M solution of KI, dissolve 165.9 g in enough water to make a final volume of 1 L. Prepare TCA 0.1% by dissolving 0.1 g of TCA in distilled water, resulting in a volume of 100 mL. Measure 5 mL of extract and combine it with 0.5 mL of KP buffer (50 mM, pH 7), followed by the addition of 1 mL of potassium iodide (1 M). Use distilled water as the blank and turn the mixture to measure the absorbance at 390 nm. Statistical analysis A statistical analysis of variance (ANOVA) was performed on all experimental parameters using a completely randomized design (CRD) with three replicates. The combined effect of Ni and BC was assessed through two-way ANOVA, followed by an LSD test at a 5% significance level. Morpho-physiological parameters were analyzed using COSTAT software to determine significant differences among mean values and interactions. Additionally, relationships between morphological, biochemical, and physiological traits were examined using correlation analysis, principal component analysis (PCA), and clustered heatmaps generated in R (version 4.1). These analyses provided insights into the associations among plant traits in response to Ni stress and biochar application. Results Growth parameters The result in Figs. 3 and 4 illustrated the effects of Ni stress on various plant growth parameters, such as shoot, root length, leaf number, leaf area, plant height and leaf area index, across different biochar application levels (0, 2.5, and 5g per kg of soil). All measured parameters show a significant decrease in plant growth as the Ni concentration rises from 0 ppm to 100 ppm. Biochar application significantly reduced this adverse effect. Plants without biochar (0g BC) exhibited a significant decrease in growth under Ni stress, especially at a concentration of 100 ppm. Plants treated with 5 g of biochar demonstrate the longest shoots and roots, even in the presence of high Ni concentrations, indicating that biochar promotes shoot and root growth under metal toxicity as well. The quantity of leaves per plant and the overall leaf area and leaf area index exhibits a similar trend. When exposed to Ni stress, plants treated with 5g BC exhibit a higher leaf number and increased leaf area, and leaf area index in contrast to those having no biochar or lower biochar concentrations. Ni stress significantly affects plant height, with the 5g BC treatment showed the largest plant growth at all Ni concentrations. Conversely, plants without biochar exhibited a significant decrease in height under higher Ni stress. Ni stress resulted in a reduction in shoot and root fresh and dry weights, total dry mater and as well as plant biomass. The BC treatment mitigated the detrimental impacts of Ni stress by enhancing fresh and dry masses of the shoot and root, total dry matter and plant biomass as well. The study demonstrated the beneficial effects of BC at both 2.5 g and 5 g concentrations in reducing Ni toxicity, with the 5 g concentration demonstrating the highest efficiency. ANOVA analysis showed that there was no significant interaction between the Ni and BC treatments. The statistical effects of Ni stress, the application of BC, and their interaction (Ni × BC) on the morphological characteristics of Sorghum bicolor are summarized in Table 3 . The majority of parameters, such as plant height (PH), root length (RL), shoot length (SL), leaf area (LA), and biomass (PB), showed highly significant differences (p < 0.001) under both Ni and BC treatments. The interaction effect (Ni × BC) was also significant for various traits, confirming the role of biochar in mitigating Ni-induced stress. Table 2 Biochemical attributes of sorghum ( sorghum bicolor L. ) with application of biochar grown under Ni stress conditions. *Significant at p < 0.05%, **Significant at p < 0.01%; ***Significant at p 0.05%; RNa = Root Sodium = RK, Root Potassium, RCa = Root Calcium, SNa + = Shoot Sodium, SK + = Shoot potassium, SCa2 + = Shoot calcium, NiR = Nickel root, NiS = Nickel shoot, SOD = superoxide, POD = peroxidase, CAT = Catalase, CHLR = Chlorophyll ratio TCH = Total chlorophyll, df = degrees of freedom, H₂O 2 = hydrogen peroxide, MDA = Malondialdehyde Ni = Nickel stress, BC = Biochar, LSD = least significant difference. Source RNa + RK + RCa 2+ SNa + SK + SCa 2+ NiR NiS SOD POD CAT CHLR TCHL H 2 O 2 MDA Ni 84.2*** 514.9*** 448*** 63.5*** 475.6*** 475.2*** 91.9*** 46.9*** 79.5*** 684.2*** 26.7*** 8.9* 0.001*** 0.072*** 0.0028*** BC 168.2*** 564.1*** 584.4*** 253.8*** 644.8*** 615.3*** 8.6*** 4.6*** 11.8*** 82.97*** 3.5*** 7.7* 9.99*** 0.04*** 4.32*** Ni *BC 3.9ns 86.7*** 84.2** 0.23ns 71.9** 77.03*** 0.09*** 0.23* 1.3** 5.3*** 0.23* 1.4ns 3.99*** 0.005*** 3.28* Error 2.4 10.4 14.7 4.6 11.3 7.8 0.006 0.06 0.2 0.7 0.08 1.8 3.4 0.001 9.03 LSD Ni 1.52 3.19 3.78 2.105 3.32 2.74 0.075 0.236 0.421 0.7695 0.268 0.004 5.74 0.034 0.0029 LSD BC 1.52 3.19 3.78 2.105 3.32 2.74 0.075 0.236 0.421 0.7695 0.268 0.004 5.74 0.034 0.0029 LSD Ni*BC 1.02 7.39 8.06 5.55 8.01 7.37 0.08 0.28 0.81 0.68 0.44 0.012 1.725 0.0256 4.091 Table 3 Morpho-physiological attributes of sorghum ( sorghum bicolor L. ) with application of biochar grown under Ni stress conditions. Source PH RL SL NOL LA SFW SDW RFW RDW CHLa CHLb CAR PB TDW LAI Ni 536.8*** 168.6*** 125.2*** 13.4*** 5936.8*** 6.2*** 4.7*** 124.9*** 0.3*** 9.98*** 0.001*** 9.8*** 47.9 *** 7.16*** 256.33*** BC 602.5*** 101.7*** 182.8*** 44.5*** 1086.3*** 14.2*** 15.2*** 159.9*** 0.9*** 8.7*** 0.0014*** 7.2*** 34.11*** 23.04*** 196.33*** Ni *BC 61.4** 2.1*** 1.4*** 0.3ns 3.2*** 0.51* 0.2*** 20.5*** 0.03** 5.7* 7.3** 7.4*** 1.21*** 0.08*** 0.33*** Error 12.3 0.2 0.1 0.5 0.4 0.2 0.01 1.9 0.006 1.7 1.3 9.6 0.02 0.01 0.06 LSD Ni 3.47 0.38 0.28 0.68 0.561 0.61 0.093 1.342 0.078 1.28 0.005 9.66 0.157 0.10 0.24 LSD BC 3.47 0.38 0.28 0.68 0.56 0.61 0.093 1.342 0.078 1.28 0.005 9.66 0.157 0.10 0.24 LSD Ni*BC 9.06 0.24 0.17 1.95 1.08 1.80 0.087 3.47 0.096 3.63 7.50 2.76 0.18 0.0764 0.123 *Significant at p ≤ 0.05%, **Significant at p ≤ 0.01%, ***Significant at p 0.05%; PH = plant height, RL = Root length, SL = Shoot length, NOL = No of leaves, LA = Leaf area, SFW = Shoot fresh weight, SDW = Shoot dry weight, RFW = Root fresh weight, RDW = Root dry weight, Chla = chlorophyll a, Chlb = Chlorophyll b, Car = Carotenoids, PB = Plant biomass, TDW = Total dry weight, LAI = Leaf area index, df = degrees of freedom, Ni = Nickel stress, BC = Biochar, LSD = least significant difference. Photosynthetic pigments The result in Fig. 5 showed how different amounts of Ni and BC affect several photosynthesis-related pigments, such as chlorophyll a , chlorophyll b , total chlorophyll, the chlorophyll a/b ratio, and carotenoids. As the Ni concentration increased, the chlorophyll a content drops. The use of BC minimized the adverse effects of Ni on chlorophyll concentration, with 5g BC showing height effects at every Ni concentration. At 0 ppm Ni, 2.5g BC has the greatest chlorophyll a concentration. However, when the Ni concentration rises, the 5g BC treatment showed higher chlorophyll a content. Similar to chlorophyll a , the concentration of chlorophyll b falls as Ni concentrations increase. The 5 g BC sample has the highest concentration of chlorophyll b for all the Ni concentrations applied. The total chlorophyll content exhibited a similar pattern to chlorophyll a and b , wherein the concentration of pigment decreases as the levels of Ni rise. Across all Ni concentrations, the 5 g BC formulation consistently displayed the highest total chlorophyll content. The ratio is higher in treatments with 5 g BC when they are exposed to Ni stress. The carotenoid content declines as the Ni concentration increases. Furthermore, 0 ppm Ni combined with 2.5 g BC yields the maximum carotenoid concentration, while 100 ppm Ni combined with 0 g BC leads to the lowest carotenoid. At 5 g, BC contributes to the maintenance of elevated carotenoid levels in the presence of Ni stress. Analysis of variance table (ANOVA) showed that the interaction between all treatments of biochar under Nickel stress was almost highly significant (Table 2 , 3 ). Biochemical parameters Enzymatic antioxidant SOD, POD and CAT The activity of superoxide dismutase (SOD) increases as the concentration of Ni increased for all BC treatments. After 5 g BC treatment, the greatest superoxide dismutase (SOD) activity observed under 100 ppm Ni. The SOD, POD activity raised with increased concentration of Ni. The POD activity reaches its peak at 100 ppm Ni and 5g BC conditions. Greater doses of BC and Ni consistently show an upward trend in POD activity. The CAT activity also exhibits a positive correlation with elevated Ni concentrations and BC treatments. Overall, the results in Fig. 6 showed that adding both biochar and Ni to the samples increased the activity of antioxidant enzymes (SOD, POD, and CAT). Maximum enzyme activity at the highest concentrations of both biochar (5g) and Ni (100 ppm) was observed. H 2 O 2 and MDA The results in Fig. 7 showed the impact of Ni stress and BC application on plant oxidative stress, measured through malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) levels. Under moderate Ni stress (50 ppm Ni), MDA levels increase; however, plants treated with 2.5g and 5g BC, exhibit a lesser increase compared to those without BC, suggesting that biochar reduces oxidative damage. Exposure to 50 ppm Ni resulted in increased H 2 O levels, with the highest concentrations noted in plants without BC. Plants treated with BC at 2.5g and 5g per kg of soil exhibited lower H₂O₂ levels relative to the untreated group. At 100 ppm Ni, H₂O₂ levels rise significantly, especially in the untreated group. However, BC effectively lowers these levels. The results indicated that biochar application mitigates oxidative stress in plants by decreasing MDA and H₂O₂ levels, particularly at elevated Ni concentrations. Table 2 shows how oxidative stress markers, hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), are affected in Sorghum bicolor by Ni stress and the use of biochar (BC). The maximum accumulation was seen at 100 ppm Ni without BC, and Ni stress markedly (p < 0.001) raised H₂O₂ and MDA levels. However, the application of BC (2.5 g or 5 g) dramatically decreased MDA and H₂O₂ levels, suggesting that it has a protective function in reducing oxidative stress. BC effectiveness in reducing Ni-induced oxidative damage was confirmed by the Ni × BC interaction, which likewise revealed a substantial (p < 0.05) decrease in these stress indicators. Ions Na + , Ca 2+ , and K + content The results in Fig. 8 indicated a significant interaction between BC application and Ni stress regarding the accumulation of essential ions (K + , Ca 2+, Na + ) in the roots and shoots of the plant. BC application significantly increases potassium (K + ) accumulation in both roots and shoots. The effect is particularly evident at the highest biochar dose (5g BC), where potassium concentrations reach their maximum, even in the presence of increasing Ni stress. In the absence of biochar (, K + levels are significantly reduced, and Ni stress seems to reduce K + accumulation. BC application significantly increases calcium (Ca 2+ ) concentrations in both roots and shoots. Despite the level of Ni stress, the maximum amount of accumulation of calcium occurs at the highest level of the BC (5 g). This suggested that biochar plays a key role in improving calcium absorption, allowing plants to maintain high Ca²⁺ levels under stressful circumstances. Sodium (Na⁺) buildup is greatly increased by Ni stress, especially in roots. This impact is lessened by the addition of charcoal; greater dosages of BC (5 g) resulted in reduced concentrations of sodium across both root and shoot. This suggests that when plants are under Ni stress, biochar reduces sodium toxicity. The statistical evaluation These results are further demonstrated in Table 2 , which shows that biochar significantly affects all ion concentrations (p < < 0.001). K⁺, Ca²⁺, and Na⁺ levels are strongly impacted by Ni stress, and this stress response is mostly controlled by the relationship between biochar and nickel (Ni x BC). By facilitating the accumulation of essential ions (K⁺ and Ca²⁺) and mitigating the adverse effects of excessive sodium, biochar helps plants cope with Ni-induced stress. Ni uptake in root and shoot The results in Fig. 9 illustrate how Ni stress and biochar application affect Ni (Ni) uptake in plant roots and shoots. Both root and shoot tissues demonstrated a distinct trend of increased Ni uptake corresponding to elevated Ni stress levels. Biochar incorporation significantly improved Ni absorption in both plant tissues. Plants treated with 5 g of BC (5 g) demonstrate the highest Ni uptake, followed by those treated with 2.5 g of BC (2.5 g). Results indicated that Ni uptake by the roots consistently exceeds that of the shoots across all stress and treatment levels. The pattern of increased uptake with biochar addition is the same for the roots. The NiR attained a value of 91.9g, whereas NiS reaches 46.9g, indicated root's enhanced ability to accumulate Ni in comparison to the shoot. The results showed that biochar, especially at higher concentrations, increased the uptake of Ni in both shoot and root tissues under Ni stress. Table 2 showed how much nickel (Ni) is absorbed by sorghum roots (NiR) and shoots (NiS) when exposed to Ni stress and biochar. Both roots and shoots accumulated more Ni under Ni stress (p < 0.001), with roots regularly absorbing more (91.9 g) than shoots (46.9 g). Applying biochar improved Ni absorption even more; plants treated with 5 g BC showed the greatest uptake. The considerable (p < 0.05) impact of the Ni × BC interaction further supported the idea that biochar encourages Ni absorption, especially in root tissues. Correlation The correlation maps in Fig. 10 show the connections between different aspects of sorghum plants and soil that are stressed by Ni and how biochar helps them. The first map shows strong positive connections between many stress-related variables, such as the amount of Ni taken up by shoots (NUS), superoxide dismutase (SOD), and peroxidase (POD). This suggests that plants' oxidative stress responses are heightened when Ni stress is high. Root dry weight (RDW), chlorophyll content (Chl), and root sodium (RNa), on the other hand, strongly negatively correlate with stress markers such as malondialdehyde (MDA). This means that increased oxidative stress from Ni toxicity significantly decreases sorghum plant growth and health. This pattern demonstrated that Ni stress negatively impacts sorghum plant, as seen by the significant negative associations detected. Principal component analysis Principal component analysis was used to assess the impact of biochar on sorghum plants under Ni stress conditions. Biplot Fig. 11 arranges the treatments based on their performance along two main components, which account for the majority of the dataset's variation. The first two plots show that Dim1 is responsible for 85.5% of the variation. This makes it the main axis for looking at how Ni stress and biochar affect the sorghum plants. The samples designated "Ni0BC0" (high Ni stress without biochar) are located far from the center, suggesting that Ni stress significantly influences characteristics such as root dry weight (RDW), chlorophyll content (Chl), and root sodium (RNa). Samples "Ni2BC1" and "Ni1BC2," demonstrating the incorporation of biochar, move the data points closer to the plot's center, signifying a reduction in the negative effects of Ni toxicity. This indicates that biochar successfully alleviates Ni stress and enhances the assessed parameters. Biplot Fig. 12 indicates that Dim1 accounts for 73.3% of the variation, while Dim2 contributes 23.1%, emphasizing both primary and secondary effects. Ni-stressed samples without biochar, like "Ni0BC0", are located far from the center, indicating significant stress. Nevertheless, biochar-treated samples, such as "Ni2BC2," concentrate toward the center, indicating enhanced plant conditions. Biochar has a positive effect on growth, as seen by its favorable correlation with characteristics like length of root (RL) and dry weight of the shoot (SDW), which are more closely correlated with samples treated with biochar. Applying biochar also lowers stress markers like superoxide dismutase (SOD) and malondialdehyde (MDA), which may indicate a reduction in Ni-induced stress caused by oxidative stress. As a mitigating agent for Ni-induced toxicity, biochar enhances critical growth factors and reduces stress indicators. Heatmaps The heatmaps illustrate the relationship between Ni stress and the quantity of biochar applied as a mitigating measure. Each heatmap is paired using hierarchical clustering, which arranges comparable answers along the X-axis (which is thought to represent sample points or attributes) and the Y-axis (which is thought to represent various methods or environmental circumstances). The color gradient, transitioning from blue to red, signifies the level of stress or mitigation, with blue indicating reduced stress (more effective mitigation) and red indicating increased stress (less effective mitigation). The first heatmap Fig. 13 displays a pronounced red color in the second row, signifying elevated Ni-induced stress without a lot of biochar reduction. Bipilot through the rows, the color gradually transitions from red to orange and blue, demonstrating the effective reduction of Ni-induced toxicity by biochar, particularly in the upper regions of the heatmap, which could indicate higher concentrations of biochar. The clustering shows big differences between Ni treatments with and without biochar, which means that different amounts of biochar have different effects on reducing damage. The second heatmap Fig. 14 displays concentrated red areas in the third row, indicating continuous Ni toxicity under certain circumstances, despite the presence of biochar. Nonetheless, the central and lower regions exhibit a transition from red to lighter colors, indicating some mitigation by biochar under these circumstances. From the blue color in the bottom regions, the clustering shows that some treatments, probably those with higher biochar concentrations, consistently show lower stress responses. The third heatmap (bottom) shows significant red coloring in the top rows, likely indicating untreated samples or those with low biochar concentrations, where Ni-induced stress is most prevalent. As the levels go down, the red transitions to yellows and blues, signifying enhanced mitigation with rising biochar concentrations. The clear difference in the clustering shows that biochar significantly lowers stress levels, separating treatments with higher biochar concentrations into separate groups with lower overall stress. These heatmaps show that Ni is a significant stressor, with red regions representing elevated toxicity in treatments lacking or containing little biochar. Biochar efficiently mitigates this toxicity, seen by changing to lighter colors (yellow and blue) in regions with elevated biochar concentrations. The structure of clustering supports the finding that different biochar concentrations lead to different stress responses, with higher concentrations lowering stress over time during treatments. Discussion Nickel contamination has become common in soil and water, reducing sustainable plant development and production on a global scale. BC is an innovative strategy for treating Ni-contaminated soil 25 . Biochar has the capability to immobilize Ni in soil and water via ion exchange, physical adsorption, and surface area attraction. Biochar showed notable mitigation ability for nickel in the agricultural sector 26 . The present research found that when sorghum plants were under Ni stress, their shoot and the length of the root significantly decreased. When wheat's Ni concentration increased to 25 to 50 µg, there was a noticeable reduction in the length of the roots and shoots 27 . Similar results have been shown in pea, rice, and Indian mustard plants, suggesting that elevated Ni stress causes a reduction in plant height. A nutritional deficit under Ni stress circumstances may have contributed to the length loss by decreasing nutrient uptake and overall plant length. The fresh and dry weights of the shoots and roots, including the leaf area, decreased in previous studies on the sorghum under Ni stress 28 . Studies have shown that as heavy metal absorption or tissue buildup increases, the division of mitotic cells in root meristematic cells usually decreases. In particular, Ni can get through the endodermal barrier into root cells and then build up in the pericycle, which lowers the plant's dry and fresh biomass 28 . Moreover, Ni stress suppresses the proton pump, which lowers the rate of cell elongation and division and, eventually, causes a reduction in leaf area. However, by increasing the total length, dry and fresh weights of roots and shoots, and by increasing leaf area during Ni stress conditions, BC administration lessened the negative impacts of Ni stress. Plant length and productivity are improved by the application of BC, which also raises the useful potassium (K) content and the amount of phosphorus and nitrogen in soils. Additionally, an increase in the number of cells increases the leaf area per plant 29 . According to the current study, Ni dramatically lowered the amounts of photosynthetic pigments. Nickel stress may prevent essential minerals like iron and magnesium from being absorbed, which might impact several phases of chlorophyll synthesis and lower the amount of chlorophyll in the impacted plants 5 . In addition, Ni stress damages the chain of electron transport, decreases membrane permeability, and hinders CO₂ fixation, all of which lower the rate of photosynthesis 30 . Using BC raised the amounts of carotenoid, total chlorophyll, chlorophyll a, and chlorophyll b in this experiment. The application of BC significantly increased the rate of photosynthesis and the amount of chlorophyll by 27.1 percent and 16.1 percent, respectively, according to documented results. The increased level of chlorophyll content in leaves is attributed to biochar's capacity to improve nitrogen availability in the soil, hence increasing nitrogen concentration in plant leaves 31 . In this study, enzymatic antioxidant activity increased under Ni stress conditions. Prior research has shown an elevation in CAT, SOD and POD activity in response to Ni stress in many crops, including maize, rice, and wheat 32 . Heavy metal stress induces the production of reactive oxygen species (ROS) in plant tissues, prompting an increase in the activity of antioxidant enzymes inside cytosolic cells to mitigate the production of ROS and enhance the plant's response mechanisms. The application of BC promotes the production of antioxidants in sorghum leaves. It was elucidated that BC enhances antioxidant synthesis, efficiently scavenging reactive oxygen species (ROS) and increasing oxidative stress tolerance in plants 33 . Nickel stress elevated H 2 O 2 and MDA levels in sorghum plants. Vicia sativa, tomato, and rice plants have shown an increase in MDA and H 2 O 2 content during Ni stress 34 . However, biochar absorbed reactive oxygen species and improved Ni tolerance in sorghum plants. The enhanced activity of antioxidant enzymes may effectively detoxify reactive oxygen species (ROS) and increase plant stress tolerance 35 . Current discoveries on sorghum indicate that same results have been seen in other crops, whereby biochar has shown the capacity to mitigate heavy metal stress and improve ion absorption 36 . For example, in maize ( Zea mays ), the application of biochar enhanced Na +, K⁺ and Ca²⁺ absorption under cadmium (Cd) and lead (Pb) stress, hence promoting growth and resistance to stress, similar to the effects shown in sorghum under nickel (Ni) stress 37 . Furthermore, studies on rice ( Oryza sativa ) indicated that biochar promotes the formation of Na + , K⁺ and Ca²⁺, mitigating the adverse effects of arsenic (As) stress, similar to biochar's function in preserving ion homeostasis in sorghum exposed to Ni stress 38 . When zinc (Zn) stress occurs in sunflowers (Helianthus annuus), biochar has been demonstrated to improve the availability of vital nutrients including Na⁺, K⁺, and Ca²⁺. This is in contrast to the defensive strategies used in sorghum. These illustrations show that biochar consistently increases the concentration of essential ions in a variety of crop species, hence boosting their resistance to various heavy metal stressors, such as the Ni stress 38 . Higher concentrations of biochar, particularly at 5 g BC, were observed to be associated with increased absorption of nickel (Ni) in both roots and shoots. According to earlier studies, biochar can alter the pH level and cation exchange ability of soil, which facilitates plants' uptake of heavy metals such as mercury like Ni 39 . Research using sorghum has demonstrated that the addition of biochar significantly boosts the absorption of heavy metals such as cadmium and lead. This is because biochar helps the soil retain nutrients and improves its structure, which makes it simpler for plants to consume the metals 40 . Researchers have shown comparable results in other crops, such as wheat and rice, where biochar increases the accumulation of heavy metals, highlighting its function in improving the ability of different crops to absorb pollutants 41 . Current study data indicate that roots are the main source of heavy metals, suggesting a defense strategy to reduce toxicity in vital tissues. This is consistent with the observed pattern of greater Ni absorption in roots as opposed to shoots. Studies on sorghum show that, like other agricultural crops, the plant exhibits increased heavy metal buildup in its root system, particularly when biochar is applied 8 . The present finding emphasizes how important roots are for storing heavy metals during stressful situations. According to the results, biochar functions as a buffer to increase plant tolerance by promoting Ni buildup and perhaps reducing the harmful effects of heavy metals. Research indicates that sorghum productivity and resiliency are enhanced by the addition of biochar to polluted soils, even in the presence of heavy metal stress. Together, the findings highlight the potential use of biochar in remediation techniques for a range of crops, including sorghum. The findings demonstrated its ability to improve heavy metal absorption in the area surrounding the roots, enabling efficient contamination control and extraction 42 . Higher concentrations of biochar, particularly at 5 g BC, are associated with increased absorption of Ni in both the roots and shoots. Additional data supports previous studies showing that biochar may alter the soil's pH level and capacity for cation exchange, facilitating plants' uptake of heavy metals like Ni 43 . Based on sorghum study findings, using biochar significantly improves the absorption of heavy metals like lead and cadmium by enhancing the soil's composition and nutrient retention, which in turn increases the availability of metals to plants. Researchers have shown comparable results in other crops, such as wheat and rice, where biochar increases the accumulation of heavy metals, highlighting its function in improving the ability of different crops to absorb pollutants 41 .. Like other cereal crops, sorghum is extremely sensitive to heavy metal stress, especially Ni, which can have a negative impact on nutrient transport, water absorption, and root growth. Similar trends have been observed in other crops under Ni stress, including maize, wheat, and rice, where high Ni concentrations affect phloem function, reduce xylem growth, and damage root structure 30 . Like sorghum, biochar helps these crops absorb more water and nutrients and thickens their roots, which lessens the negative impacts of heavy metals. Similarly to the pattern that was seen in the vascular tissues and roots of sorghum in this study, applying biochar to maize enhances the morphology of root structures and increases the plant's resistance to metal toxicity 44 . Wheat and rice show better root function and development when treated with biochar among metal-contaminated soils because biochar efficiently reduces heavy metals and lowers their bioavailability. According to the study findings, sorghum, unlike many other crops, takes advantage of biochar's protective properties, particularly when used in high concentrations. As a consequence, it may be used as a useful tactic to increase agricultural resilience in soils that are polluted with heavy metals of various kinds 45 . The results demonstrate that the presence of Ni stress, particularly at increased levels (Ni 100 ppm), had detrimental impacts on sorghum structure by decreasing epidermal thickness and preventing vascular bundle formation, which in turn reduced the xylem and phloem tissue efficiency 46 . Because nickel plays a crucial role in plant growth, decreased nickel levels (Ni 50 ppm) showed positive benefits. By strengthening vascular integrity, especially in the phloem and xylem, and boosting water retention, biochar, when used at 2.5 g or 5 g per kg of soil, reduced nickel-induced stress. The interaction between nickel (NixBC) and biochar demonstrates that biochar can lessen nickel stress 46 . The sorghum shoots' form improved most noticeably with the BC at 5g along with 50 ppm Ni treatment. This is comparable to what researchers have discovered in other crops, such as rice and maize, wherein biochar has been demonstrated to enhance water retention and lessen the negative impacts of heavy metals, both of which aid in plant growth under stress. When treated with biochar, maize under nickel stress exhibits better root and shoot growth as well as less oxidative damage. Biochar is a crucial component of sustainable agriculture in heavy metal-contaminated areas because of its potential to enhance nutrient absorption and water control in the face of Ni stress, which may be linked to a variety of crops 47 . Conclusion Nickel (Ni) toxicity in sorghum plants causes ionic imbalance, slowed development, reduced photosynthetic pigments, decreased activity of antioxidants, and altered osmolyte accumulation. Yet BC treatment dramatically reduced these adverse effects by increasing metabolite levels, boosting nutrient absorption, and lowering oxidative stress. Among the treatments studied, 5 g BC per kg of soil was particularly beneficial, since it not only effectively immobilized Ni but also increased the generation of free antioxidants, boosting the plant's resistance to Ni stress. BC reduced sodium ion (Na⁺) buildup and increased food absorption, promoting ionic equilibrium. Furthermore, biochar lowered oxidative stress markers such as malondialdehyde (MDA) and hydrogen peroxide, or H₂O₂, by increasing the activity of key antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). These biochemical enhancements improved photosynthetic efficiency, chlorophyll concentration, and total plant development. Furthermore, BC increased the soil's structure and availability of nutrients, allowing for sustainable sorghum growth in Ni-contaminated soils. The observed effects of biochar application demonstrate its promise as an ecologically friendly technique for reducing metal stress in plants. These findings indicate that biochar can play an important role in increasing plant tolerance to metal toxicity while also supporting sustainable agriculture methods. Future research needs to concentrate on long-term uses of biochar, how it interacts with other soil modifications, and its influence on soil microbes. At the end, using 5 g of biochar per kg of soil is a potential way to eliminate Ni-induced stress in sorghum, leading to improved plant health, increased yield, and long-term crop production in metal-polluted conditions. Declarations Author’s Contribution AA, S.B; Conducted investigations and drafted paper, AM, S.B; supervised research, Conception and design, analyses and interpretation of the data, AM, MMJ, MAN, SHQ, MH and LA; analyses and interpretation of the data Drafting of paper; Application of Statistics Analyses and Software, FAA; Provide Resources and Revising it critically for intellectual content, AM, MH, MAN, SHQ, MMJ, FAA; revised intellectual content; and the final approval to be published. All authors agree to be accountable for all aspects of the work. All authors reviewed the manuscript. Ethics approval and consent to participate N/A Consent for publication All Authors give consent to publish data. Availability of data and materials All data generated or analysed during the current study are included in this article. Competing interests The authors declare no conflict of interests. Funding The authors extend their appreciation to the Deanship of Research and Graduate Studies at King Khalid University for funding this work through large group research under grant number RGP 2/216/46. Acknowledgments The authors extend their appreciation to the Deanship of Research and Graduate Studies at King Khalid University for funding this work through large group research under grant number RGP 2/216/46. References Ameen, M., Mahmood, A., Shahzad, A. N., Zia, M. A. & Javaid, M. M. Sorghum's potential unleashed: A comprehensive exploration of bio-energy production strategies and innovations. Bioresour Technol. Rep. 27 , 101906. https://doi.org/10.1016/j.biteb.2024.101906 (2024). Shahid, M. et al. Chromium speciation, bioavailability, uptake, toxicity and detoxification in soil-plant system: A review. Chemosphere 178 , 513–533. https://doi.org/10.1016/j.chemosphere.2017.03.074 (2017). Mishra, S. et al. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6536755","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":452826362,"identity":"096a9d15-94ed-400e-8d96-3022cdcd128f","order_by":0,"name":"Ayesha Arshad","email":"","orcid":"","institution":"University of Agriculture Faisalabad","correspondingAuthor":false,"prefix":"","firstName":"Ayesha","middleName":"","lastName":"Arshad","suffix":""},{"id":452826363,"identity":"a5674cac-99cb-4c7b-a1fa-18eb7107ccf2","order_by":1,"name":"Athar Mahmood","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIiWNgGAWjYFACHghlAGZXMDCwNzAwSMAEsAI2FC1ngPgASVp424jQwj+/9+DnCoZt8uZihw8+eDvvsDwPA/PB2zwMd4xxaZE4xpcseYbhtuHO2WnJhnO3HTbsYWBLtuZheGaG02HHeAwkGxhuM264nWMmzbvtMON+Bh4zaR6Gwza4dMgf4zH+CdRiD9Ey57B9DwP/N7xaDI7xmIFsSYRoaTic2MPAwwbSgtNhhsdyzCwbDG4nb7gN9MucY+nJPcxsxpZzDJ7h9L7c4TPGNxsqbttuuJ188MGbGmvbHvbmhzfeVNwxbMDpf7DzkDnMYJEDeDVgBWRoGQWjYBSMguEKAHKDVLi78mhlAAAAAElFTkSuQmCC","orcid":"","institution":"University of Agriculture Faisalabad","correspondingAuthor":true,"prefix":"","firstName":"Athar","middleName":"","lastName":"Mahmood","suffix":""},{"id":452826364,"identity":"be36d0b0-e33b-4f72-b2a9-751adf22237e","order_by":2,"name":"Safura Bibi","email":"","orcid":"","institution":"University of Agriculture Faisalabad","correspondingAuthor":false,"prefix":"","firstName":"Safura","middleName":"","lastName":"Bibi","suffix":""},{"id":452826365,"identity":"c43691dd-82d2-4ee8-93ae-c560f01db160","order_by":3,"name":"Muhammad Mansoor Javaid","email":"","orcid":"","institution":"University of Sargodha","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Mansoor","lastName":"Javaid","suffix":""},{"id":452826366,"identity":"b40e3231-3802-430e-906e-c4c5f27be6fe","order_by":4,"name":"Liaqat Ali","email":"","orcid":"","institution":"The Islamia University of Bahawalpur","correspondingAuthor":false,"prefix":"","firstName":"Liaqat","middleName":"","lastName":"Ali","suffix":""},{"id":452826367,"identity":"39b394db-8a12-4041-84c9-21db0aaeee5f","order_by":5,"name":"Muhammad Ather Nadeem","email":"","orcid":"","institution":"University of Sargodha","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Ather","lastName":"Nadeem","suffix":""},{"id":452826368,"identity":"d8beeffc-0fce-44c5-befa-36b1c4ec9b9a","order_by":6,"name":"Sameer H. Qari","email":"","orcid":"","institution":"Al-Jumum University College, Umm Al-Qura University","correspondingAuthor":false,"prefix":"","firstName":"Sameer","middleName":"H.","lastName":"Qari","suffix":""},{"id":452826369,"identity":"1a6e4141-ed0e-4471-8c80-8bfc0f2b41fc","order_by":7,"name":"Faizah Amer Altihani","email":"","orcid":"","institution":"King Khalid University","correspondingAuthor":false,"prefix":"","firstName":"Faizah","middleName":"Amer","lastName":"Altihani","suffix":""},{"id":452826370,"identity":"9656a78a-9933-4523-9ae2-ce691a7cd1b7","order_by":8,"name":"Mohamed Hashem","email":"","orcid":"","institution":"Assiut University","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"","lastName":"Hashem","suffix":""}],"badges":[],"createdAt":"2025-04-26 19:53:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6536755/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6536755/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82331207,"identity":"a2bfcbee-a91e-4a59-999d-a241615b0a07","added_by":"auto","created_at":"2025-05-09 07:20:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1049318,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A.B): \u003c/strong\u003eGraphical Illustration of experimental Layout\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6536755/v1/5158d7607fbdd70f2851fce9.png"},{"id":82332720,"identity":"e74b9ec0-53ea-4e1a-84de-05f1c016dff5","added_by":"auto","created_at":"2025-05-09 07:36:38","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2313494,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental material site and treatment application\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6536755/v1/24109cfe5640cf8e453b2144.jpeg"},{"id":82332203,"identity":"bb95d3ee-e510-41ea-8bdb-4b3ee629301c","added_by":"auto","created_at":"2025-05-09 07:28:38","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":739154,"visible":true,"origin":"","legend":"\u003cp\u003eThe graphs showing the shoot fresh weight\u003cem\u003e \u003c/em\u003e(A), shoot dry weight (B), root fresh weight\u003cem\u003e \u003c/em\u003e(C), root dry weight (D), total dry matter (E), and plant biomass (F) of sorghum seedlings, and various level of biochar (0g, 2.5g and 5g) and nickel stress at various level of (0ppm, 50ppm, and 100ppm).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6536755/v1/8b0235a05cb4efaa0a20bc0f.jpeg"},{"id":82332721,"identity":"b640f729-4953-466c-a06b-f8c82ddbce03","added_by":"auto","created_at":"2025-05-09 07:36:38","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":357435,"visible":true,"origin":"","legend":"\u003cp\u003eThe graphs showing the shoot length\u003cem\u003e \u003c/em\u003e(A), root length\u003cem\u003e \u003c/em\u003e(B), number of leaves plant\u003csup\u003e-1\u003c/sup\u003e (C), leaf area (D), plant height (E), and leaf area index (F) of sorghum seedlings, and various level of biochar (0g, 2.5g and 5g) and nickel stress at various level of (0ppm, 50ppm, and 100ppm).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6536755/v1/5bf908a9f39e77cbc23cc384.jpeg"},{"id":82331211,"identity":"8ef6e74f-b9f7-4da7-a2f0-91974dbe0b2a","added_by":"auto","created_at":"2025-05-09 07:20:38","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":354287,"visible":true,"origin":"","legend":"\u003cp\u003eThe graphs showing the photosynthetic pigments contents; chlorophyll \u003cem\u003ea \u003c/em\u003e(A), chlorophyll \u003cem\u003eb\u003c/em\u003e (B), total chlorophyll (C), total chlorophyll ratio (D) and carotenoids (E) of sorghum seedlings, and various level of biochar (0g, 2.5g and 5g) and nickel stress at various level of (0ppm, 50ppm, and 100ppm).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6536755/v1/f193d89542e6f530819baa58.jpeg"},{"id":82332204,"identity":"aa8d0063-0fbf-469e-9b6a-8fe5ba80feb1","added_by":"auto","created_at":"2025-05-09 07:28:38","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":178377,"visible":true,"origin":"","legend":"\u003cp\u003eThe graphs showing the enzymatic antioxidants; SOD\u003cem\u003e \u003c/em\u003e(A), POD (B), and CAT (C), of sorghum seedlings, and various level of biochar (0g, 2.5g and 5g) and nickel stress at various level of (0ppm, 50ppm, and 100ppm).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6536755/v1/d6877c355b1bcb43967ec837.jpeg"},{"id":82331208,"identity":"67eeb115-625f-4ad6-ad93-3dc6c0d1689b","added_by":"auto","created_at":"2025-05-09 07:20:38","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":129869,"visible":true,"origin":"","legend":"\u003cp\u003eThe graphs showing the hydrogen peroxide\u003cem\u003e \u003c/em\u003e(A), shoot MDA\u003cem\u003e \u003c/em\u003e(B), of sorghum seedlings, and various level of biochar (0g, 2.5g and 5g) and nickel stress at various level of (0ppm, 50ppm, and 100ppm).\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6536755/v1/3a60fcddd486304529945a3f.jpeg"},{"id":82331218,"identity":"78cff1fe-69eb-4977-afd9-8fdc57f7d04e","added_by":"auto","created_at":"2025-05-09 07:20:38","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":350657,"visible":true,"origin":"","legend":"\u003cp\u003eThe graphs showing the root potassium\u003cem\u003e \u003c/em\u003e(A), root calcium\u003cem\u003e \u003c/em\u003e(B), root sodium\u003cem\u003e \u003c/em\u003e(C), shoot potassium (D), shoot calcium (E) and shoot sodium (F) content of sorghum seedlings, at various level of biochar (0g, 2.5g and 5g) and Nickel stress at various level (0ppm, 50ppm and 100ppm)\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6536755/v1/423bd25e727f3c16f4ef0dd0.jpeg"},{"id":82331213,"identity":"dd46b8b9-2d34-402f-af82-a1b874a7bb12","added_by":"auto","created_at":"2025-05-09 07:20:38","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":145304,"visible":true,"origin":"","legend":"\u003cp\u003eThe graphs showing the uptake of Ni by shoot\u003cem\u003e \u003c/em\u003e(A), and uptake of Ni by root\u003cem\u003e \u003c/em\u003e(B), of sorghum seedlings, and various level of biochar (0g, 2.5g and 5g) and nickel stress at various level of (0ppm, 50ppm, and 100ppm).\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6536755/v1/1cb63553725885c3046025b5.jpeg"},{"id":82331237,"identity":"e2cefbb2-c915-499e-a275-28989d6345d4","added_by":"auto","created_at":"2025-05-09 07:20:38","extension":"jpeg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":674589,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation plot of \u0026nbsp;Morphological attributes: \u0026nbsp;NOL (Number of leaves), PBM (Plant biomass), PH (Plant height),\u0026nbsp;SDW (Shoot dry weight), SFW (Shoot fresh weight), RDW (Root dry weight), RFW (Root fresh weight), SL (Shoot length), RL (Root length), LA (Leaf area), LAI (Leaf area index), Physiological attributes: Chlorophyll \u003cem\u003ea\u003c/em\u003e (Chla), Chlorophyll \u003cem\u003eb\u003c/em\u003e (Chlb), Carotenoids (CAR), Total chlorophyll (TChl), Ratio of Chlorophyll (ChlR), Biochemical attributes: Shoot Sodium(SNa\u003csup\u003e+\u003c/sup\u003e), Shoot Potassium(SK\u003csup\u003e+\u003c/sup\u003e), Shoot Calcium(SCa\u003csup\u003e+2\u003c/sup\u003e), Root Sodium(RNa\u003csup\u003e+\u003c/sup\u003e), Root Potassium(RK\u003csup\u003e+\u003c/sup\u003e), Root Calcium(RCa\u003csup\u003e+2\u003c/sup\u003e), Nickel Root (NiR), Nickel Shoot (NiS),\u0026nbsp; Malondialdehyde (MDA), Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), Superoxide dismutase (SOD), Catalase (CAT), Peroxidase (POD) \u0026nbsp;attributes of sorghum under nickel stress condition.\u003c/p\u003e","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6536755/v1/789ca60df8c435ce832484f7.jpeg"},{"id":82332207,"identity":"a4a435b7-ae54-4082-97a9-102fefe10b84","added_by":"auto","created_at":"2025-05-09 07:28:38","extension":"jpeg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":335266,"visible":true,"origin":"","legend":"\u003cp\u003ePCA-Biplot of Morphological attributes: NOL (Number of leaves), RDW (Root dry weight), RFW (Root fresh weight), SL (Shoot length), RL (Root length), Physiological attributes: Chlorophyll \u003cem\u003ea\u003c/em\u003e (Chla), Chlorophyll \u003cem\u003eb\u003c/em\u003e (Chlb), Carotenoids (CAR), Total chlorophyll (TChl), Ratio of Chlorophyll (ChlR), Biochemical attributes: Shoot Sodium(SNa\u003csup\u003e+\u003c/sup\u003e), Shoot Potassium(SK\u003csup\u003e+\u003c/sup\u003e), Shoot Calcium(SCa\u003csup\u003e+2\u003c/sup\u003e), Root Sodium(RNa\u003csup\u003e+\u003c/sup\u003e), Root Potassium(RK\u003csup\u003e+\u003c/sup\u003e), Root Calcium(RCa\u003csup\u003e+2\u003c/sup\u003e) \u0026nbsp;attributes of sorghum under nickel stress condition.\u003c/p\u003e","description":"","filename":"floatimage11.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6536755/v1/abc125521e469b2369bc766e.jpeg"},{"id":82331242,"identity":"fca670a5-0e4c-4e13-9d71-aa062f2cff81","added_by":"auto","created_at":"2025-05-09 07:20:39","extension":"jpeg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":437517,"visible":true,"origin":"","legend":"\u003cp\u003ePCA-Biplot of Morphological attributes: \u0026nbsp;PB (Plant biomass), PH (Plant height),\u0026nbsp;SDW (Shoot dry weight), TDW (Total dry weight), SL (Shoot length), RL (Root length), LA (Leaf area), LAI (Leaf area index), Biochemical attributes: Nickel Root (NiR), Nickel Shoot (NiS), Malondialdehyde (MDA), Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), Superoxide dismutase (SOD), Catalase (CAT), Peroxidase (POD) attributes of sorghum under nickel stress condition.\u003c/p\u003e","description":"","filename":"floatimage12.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6536755/v1/28016ced5e7107cafa71edc2.jpeg"},{"id":82332215,"identity":"5d9f49b9-7ed3-4651-a436-c83bdb7eb7f1","added_by":"auto","created_at":"2025-05-09 07:28:39","extension":"jpeg","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":173311,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap showing interaction of \u0026nbsp;Morphological attributes: NOL (Number of leaves), RDW (Root dry weight), RFW (Root fresh weight), SL (Shoot length), RL (Root length), Physiological attributes: Chlorophyll \u003cem\u003ea\u003c/em\u003e (Chla), Chlorophyll \u003cem\u003eb\u003c/em\u003e (Chlb), Carotenoids (CAR), Total chlorophyll (TChl), Ratio of Chlorophyll (ChlR), Biochemical attributes: Shoot Sodium(SNa\u003csup\u003e+\u003c/sup\u003e), Shoot Potassium(SK\u003csup\u003e+\u003c/sup\u003e), Shoot Calcium(SCa\u003csup\u003e+2\u003c/sup\u003e), Root Sodium(RNa\u003csup\u003e+\u003c/sup\u003e), Root Potassium(RK\u003csup\u003e+\u003c/sup\u003e), Root Calcium(RCa\u003csup\u003e+2\u003c/sup\u003e) \u0026nbsp;attributes of sorghum under nickel stress condition.\u003c/p\u003e","description":"","filename":"floatimage13.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6536755/v1/53e0234a01fb62c1f012b69b.jpeg"},{"id":82331231,"identity":"3fe1040f-4a0b-41c3-b398-20b1bd396335","added_by":"auto","created_at":"2025-05-09 07:20:38","extension":"jpeg","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":167885,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap showing interaction of Morphological attributes:\u0026nbsp; PB (Plant biomass), PH (Plant height),\u0026nbsp;SDW (Shoot dry weight), TDW (Total dry weight), SL (Shoot length), RL (Root length), LA (Leaf area), LAI (Leaf area index), Biochemical attributes: Nickel Root (NiR), Nickel Shoot (NiS), Malondialdehyde (MDA), Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), Superoxide dismutase (SOD), Catalase (CAT), Peroxidase (POD) attributes of sorghum under nickel stress condition.\u003c/p\u003e","description":"","filename":"floatimage14.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6536755/v1/7031fc34d56dab2b5d5ac801.jpeg"},{"id":86211973,"identity":"638b2f52-1e46-4f00-a0c1-699180b81fc9","added_by":"auto","created_at":"2025-07-08 04:47:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8582022,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6536755/v1/c595db51-f072-4454-98ba-cf4f4dfcc657.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biochar Application for Alleviating Nickel Stress and Enhancing Growth, Photosynthetic Pigments, and Antioxidant Defense Mechanisms in Sorghum","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSorghum (\u003cem\u003eSorghum bicolor\u003c/em\u003e L.), a member of the Poaceae family, is a perennial, summer-growing plant cultivated for multiple purposes, including grain production, animal feed, sugar extraction, and bioenergy. Almost 50 percent of the sorghum crop is used for human diet all over the world. Out of this about 90% of sorghum crop is used for animal nourishment in USA \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Sorghum is the fifth most important cereal crop globally, cultivated across 44\u0026nbsp;million acres in 99 countries. Sorghum grows best in well-drained soils with moderate organic matter and a pH of 6\u0026ndash;7.5. However, heavy metal contamination harms its growth. Sorghum mainly accumulates metals in its roots, but higher concentrations increase their movement to the shoots, further reducing plant health \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHuman activities, such as mining and industrialization, are responsible for the release of heavy metals that have caused changes in the natural ecosystem. Metals that pose a threat to human health include lead, arsenic, cadmium, Ni, and mercury. Ni ranks as the 24th most common element in the earth crust \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Industrial locations that have high levels of sewage sludge release substantial quantities of Ni into nearby soils. The concentration of Ni in soil may vary from 3 to 100 ppm. Ni exists in several forms in soil, including inorganic crystalline minerals, on inorganic cation exchange surfaces, on organic cation surfaces, as a free ion, water-soluble, and as a chelated complex. Ni is a threat to human well-being and has the potential to cause cancer and other disorders if ingested via contaminated food \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNi induces deleterious morphological, physiological, or biochemical impacts on living organisms. In plants, they inhibit seedling development, limit root elongation, reduce transpiration, suppress chlorophyll synthesis, inhibit cell division, and ultimately hinder plant growth \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Ni is beneficial to plants in small amounts, supporting growth and metabolism, but its excessive accumulation can be toxic. High Ni concentrations disrupt key physiological processes, impairing nutrient uptake, enzyme activity, and photosynthesis. Extreme Ni exposure eventually reduces crop yield and productivity, making it difficult to grow sorghum sustainably in contaminated soils. Ni toxicity in cereals, especially sorghum, negatively impacts shoot development, resulting in stunted growth, chlorosis, and decreased biomass \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.Therefore, it is important to remove Ni through the soil to lessen the negative effects on plants.\u003c/p\u003e \u003cp\u003eResearchers have recommended organic modification to stabilize heavy metal in the soil during the restoration process. Biochar is a carbon-rich solid material produced by the thermal decomposition of organic biomass at relatively low temperatures under limited oxygen conditions. \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Recent studies show that adding biochar to soil enhances its agricultural qualities and efficiently eliminates both organic and inorganic pollutants because of its high absorption capacity. BC functional groups help make it an effective modification for overcoming metals in soil because they give the metals active sites to attach, which decreases their mobility in the soil\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSorghum shows better root shoot function and development when treated with biochar among metal-contaminated soils because BC efficiently reduces heavy metals and lowers their bioavailability\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Sorghum, unlike many other crops, takes advantage of BC protective properties, particularly when used in high concentrations. As a consequence, it may be used as a useful tactic to increase agricultural resilience in soils that are polluted with heavy metals such as Ni. Within the framework of organic nature alterations, BC has shown impressive effects in boosting the adsorption of metal in soils, thereby lowering their bioavailability\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eStudies on the impact of BC on crop production are still ongoing. Through assessing its effects on plant growth and development, this study explores its potential to reduce Ni-induced stress in sorghum. When subjected to Ni toxicity, it evaluates the morphological, biochemical, and physiological responses of sorghum to BC amendment. The goal of this research is to add the increasing field of knowledge and set the framework for further studies with the objective of improving biochar application techniques for heavy metal-stressed sustainable crop production.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental material and site\u003c/h2\u003e \u003cp\u003eThe experiment was carried out in the summer seasons of 2023\u0026ndash;2024 at the Botanical Garden of community college PARS, University of Agriculture Faisalabad, Pakistan (31.3992\u0026deg; N, 73.0313\u0026deg; E, at a height of approximately 184 meters above mean ground level. These sites were used to assess the effectiveness of biochar in mitigating Ni-induced stress. The Sorghum-2011 variety was obtained from the Fodder Research Institute of the Ayub Agricultural Research Institute (AARI) in Faisalabad, Pakistan\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental design and treatment application\u003c/h3\u003e\n\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e provides the physicochemical parameters of the soil at the experiment location. The soil has been collected, dried in the air, and sieved to remove any debris. The initial water content of the soil was determined by weighing 100 g of new soil, which was subsequently oven-dried at 105\u0026deg;C for 24 hours, and the soil dry mass was measured. Soil moisture contents were measured as:\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSoil physicochemical traits and soil irrigation treatments of experimental site\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eValue\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReferences\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.5\u0026ndash;8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElectrical Conductivity (EC)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.2\u0026ndash;1.5 dS/m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrganic Matter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8\u0026ndash;1.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitrogen (N)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.05\u0026ndash;0.07%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAvailable Phosphorus (P)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u0026ndash;15 mg/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAvailable Potassium (K)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e160\u0026ndash;180 mg/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBulk Density\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.1\u0026ndash;1.3 g/cm\u0026sup3;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoil Texture\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLoamy to clay loam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCation Exchange Capacity (CEC)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18\u0026ndash;22 cmol/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNickel (Ni)\u003c/p\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u0026ndash;50 mg/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSoil fresh weight - soil dried weight / soil fresh weight) x 100\u003c/p\u003e \u003cp\u003eThe experiment used standard plastic pots measuring 9.5 cm in diameter and 25.5 cm in height, filled with dry soil for planting. To lessen the effect of environmental variability, a total of 27 pots were dispersed using a completely randomized block design, with three replicates of each treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e These pots were rotated on a regular basis. After a week of seedling and chosen pots were treated with nickel stress consisting of three levels: 0 ppm, 50 ppm, and 100 ppm \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. To address the nutritional needs of the plants, Hoagland nutrient solution was administered at half strength. Furthermore, BC1 (0 g), BC2 (2.5 g), and BC3 (5 g) were the three levels of biochar application that were utilized. Prior to seeding, biochar was added to the top layer of soil. After three to four weeks of seedling growth, data from a number of morpho-physiological, biochemical, ionic, and enzymatic parameters were examined.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eMorphological characteristics\u003c/h3\u003e\n\u003cp\u003eAfter the experiment, the plants were carefully collected, and the roots were properly rinsed with distilled water in order to eliminate any dirt particles that had adhered to them. Growth characteristics, such as leaf area, length of shoot, root length, and accumulation of biomass, were measured to evaluate the impact of interventions on plant development \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLeaf area per plant (mm\u0026sup2;) was measured using leaf area meter (MENTION MODEL HERE). A digital weighing scale was used to record the fresh weights of roots and shoots immediately after harvest. Plant samples were dried in an oven at 62\u0026deg;C for two weeks until a consistent weight was reached to measure the dry weight of the plant \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003ePhysiological parameters\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eChlorophyll contents\u003c/h2\u003e \u003cp\u003eThe chlorophyll contents were determined using the method provided by Davis \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and Arnon \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Each replication yielded two plants, each with one fully developed leaf. A 0.5 g leaf sample was mashed with a pestle and mortar before applying 5 mL of acetone with a concentration of 80% to extract the pigments. The extract was kept at 10\u0026deg;C overnight before centrifugation at 1400 revolutions per minute for 5 minutes. The absorbance of the supernatant was measured with the help of a spectrophotometer at 480 nm, 645 nm, and 663 nm to measure carotenoids, chlorophyll b, and chlorophyll a, accordingly \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e by using formula given below.\u003c/p\u003e \u003cp\u003eChl. a (mg/g) = [12.7 (OD663) \u0026ndash; 2.69 (OD645)] * V/1000* W\u003c/p\u003e \u003cp\u003eChl. b ( mg/g) =[22.9 (OD645- 4.68(OD663O)] * V/1000*W\u003c/p\u003e \u003cp\u003eChl a/b (mg/g)\u0026thinsp;=\u0026thinsp;Divided chlorophyll a value with chlorophyll b\u003c/p\u003e \u003cp\u003eCarotenoid (mg/g)\u0026thinsp;=\u0026thinsp;OD480\u0026thinsp;+\u0026thinsp;0.114*OD663-0.638*OD645/2500\u003c/p\u003e \u003cp\u003eTotal Chl. = [20.2(OD645)-8.02(OD663)] *V/1000 * W\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical parameters\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eAssessment of enzymatic antioxidants\u003c/h2\u003e \u003cp\u003eFresh leaf samples were used to measure the enzymatic antioxidant activity. A leaf sample weighing 0.25 g has been homogenized in a pestle and mortar with 5mL of potassium buffer with phosphate. The homogenate was then transferred to a 2 mL eppendorf tube and subsequently centrifuged at 12,000 rpm for thirty minutes at 4\u0026deg;C \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The supernatant containing the enzyme extract was removed from contaminants and kept at 20\u0026deg;C for subsequent analysis.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eDigestion method (ion test and Ni uptake)\u003c/h3\u003e\n\u003cp\u003eThe concentration of mineral ions in the roots was determined using the digestion method described by Wolf \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Dried root samples from each replication were digested in 2.5 mL of concentrated H₂SO₄ at room temperature in digestion flasks. Subsequently, 4 mL of 35% H₂O₂ was added, and the mixture was heated at 350\u0026deg;C until a clear colorless solution was obtained \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The digested solution was then filtered and diluted with distilled water to a final volume of 50 mL. Ion concentrations were analyzed using a flame photometer.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSuper oxide dismutase Activity (SOD)\u003c/h2\u003e \u003cp\u003eAccording to Giannopolitis and Ries \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e method Superoxide dismutase (SOD) activity was assessed using fresh leaf samples. A 0.25 g leaf sample was homogenized in 5 mL of 50 mM potassium phosphate buffer using a mortar and pestle. The homogenate was then processed for enzymatic activity analysis. Centrifuge the sample at a speed of 14000 rpm for a duration of 15 minutes. Create a solution of potassium phosphate buffer at a concentration of 20 mM. Dissolve 8.7 g of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e and 6.8 g of KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e in 250 mL of distilled water, each in separate containers. Combine the ingredients separately, and then combine both mixtures together to get a total volume of 500 mL with a pH of 7.4. Prepare a blank solution by adding 50 \u0026micro;l of buffer instead of the sample and 50 \u0026micro;l of distilled water. After adding riboflavin to the blank cuvette, add it to the others. Expose to light for 15 minutes, then analyze using a spectrophotometer \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMix 400 \u0026micro;l of distilled water, 250 \u0026micro;l of 200 mM KP buffer, 100 \u0026micro;l of L. methionine, 100 \u0026micro;l of Triton, 50 \u0026micro;l of NBT, 50 \u0026micro;l of enzyme extract, and 50 \u0026micro;l of riboflavin to make the reaction mixture. Then, measure the absorbance at 560 nm. Record the blank reading last, ensuring it is higher than the samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePeroxidase (POD) Activity\u003c/h2\u003e \u003cp\u003eAccording to Chance and Maehly \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e fresh leaf sample weighing 0.25 g was homogenized in 5 mL of 50 mM potassium phosphate buffer. The supernatant was then separated for enzymatic analysis by centrifuging the mixture for 15 minutes at 4\u0026deg;C and 14,000 rpm. In 250 mL of water that had been distilled, 8.7 g of K₂HPO₄ and 6.8 g of KH₂PO₄ were individually dissolved to create a 20 mM potassium phosphate buffer. In order to get the required buffer concentrations and pH balance for enzymatic activity tests, the solutions were subsequently combined. Mix the components individually, then combine them altogether to obtain a 500 mL volume having a pH of 7.4. Make a solution of a potassium phosphate buffer with a concentration of 50 mM \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. A 750 \u0026micro;L of Guaiacol was diluted with distilled water to a total volume of 50 mL. Prepare a solution by mixing 100 \u0026micro;L of hydrogen peroxide (22.8 \u0026micro;L in 5 mL water) with 50 \u0026micro;L of enzyme extract, and measure the absorbance at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCatalase activity (CAT)\u003c/h2\u003e \u003cp\u003eCatalase (CAT) activity was determined using the method of Chance and Maehly \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Fresh leaf samples (0.25 g) were homogenized in 5 mL of 50 mM potassium phosphate buffer using a mortar and pestle. The homogenate was then centrifuged at 14,000 rpm for 15 minutes at 4\u0026deg;C to obtain the supernatant for enzymatic analysis. A 20 mM potassium phosphate buffer solution was prepared by dissolving the appropriate amounts of K₂HPO₄ and KH₂PO₄ in distilled water, ensuring proper pH adjustment for enzymatic analysis. Dissolve 8.7 g of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e and 6.8 g of KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e in 250 mL of distilled water individually. Combine the two mixtures separately, then combine them together to get a total volume of 500 mL with a pH of 7.4. To produce a solution of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e with a concentration of 0.059 M, measure out 29 \u0026micro;l of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and transfer it into falcon tubes \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDilute the solution with pure water until the volume reaches 5 mL. Combine 1.9 mL of KP buffer (50mM), 100 \u0026micro;l of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (0.059 M) and100 \u0026micro;l of enzyme extract, and measure the absorbance at 240 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMalondialdehyde (MDA) and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2)\u003c/sub\u003e content\u003c/h2\u003e \u003cp\u003eMalondialdehyde (MDA) and hydrogen peroxide (H₂O₂) contents were measured by using the method of Alexieva, et al. \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e to assess oxidative stress in plant tissues. Fresh leaf samples (0.25 g) were homogenized in 5 mL of 5% trichloroacetic acid (TCA). The TCA solution was prepared by dissolving 5 g of TCA in 100 mL of distilled water. For the TCA-TBA reagent, 0.5 g of thiobarbituric acid (TBA, 0.5%) was mixed with 20 g of TCA (20%) and diluted to a final volume of 100 mL with distilled water. Combine 0.5 \u0026micro;l of extract with 0.5 \u0026micro;l of TBA-TCA solution. Then, place it in a water bath for 15 minutes. Cool and measure the absorbance at 532 nm and 600 nm \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHomogenize 0.25 g of leaf sample in 1% TCA and centrifuge at 12,000 rpm for 15 minutes. To prepare a 1 M solution of KI, dissolve 165.9 g in enough water to make a final volume of 1 L. Prepare TCA 0.1% by dissolving 0.1 g of TCA in distilled water, resulting in a volume of 100 mL. Measure 5 mL of extract and combine it with 0.5 mL of KP buffer (50 mM, pH 7), followed by the addition of 1 mL of potassium iodide (1 M). Use distilled water as the blank and turn the mixture to measure the absorbance at 390 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eA statistical analysis of variance (ANOVA) was performed on all experimental parameters using a completely randomized design (CRD) with three replicates. The combined effect of Ni and BC was assessed through two-way ANOVA, followed by an LSD test at a 5% significance level. Morpho-physiological parameters were analyzed using COSTAT software to determine significant differences among mean values and interactions. Additionally, relationships between morphological, biochemical, and physiological traits were examined using correlation analysis, principal component analysis (PCA), and clustered heatmaps generated in R (version 4.1). These analyses provided insights into the associations among plant traits in response to Ni stress and biochar application.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eGrowth parameters\u003c/h2\u003e\n \u003cp\u003eThe result in Figs. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e illustrated the effects of Ni stress on various plant growth parameters, such as shoot, root length, leaf number, leaf area, plant height and leaf area index, across different biochar application levels (0, 2.5, and 5g per kg of soil). All measured parameters show a significant decrease in plant growth as the Ni concentration rises from 0 ppm to 100 ppm. Biochar application significantly reduced this adverse effect. Plants without biochar (0g BC) exhibited a significant decrease in growth under Ni stress, especially at a concentration of 100 ppm. Plants treated with 5 g of biochar demonstrate the longest shoots and roots, even in the presence of high Ni concentrations, indicating that biochar promotes shoot and root growth under metal toxicity as well. The quantity of leaves per plant and the overall leaf area and leaf area index exhibits a similar trend. When exposed to Ni stress, plants treated with 5g BC exhibit a higher leaf number and increased leaf area, and leaf area index in contrast to those having no biochar or lower biochar concentrations. Ni stress significantly affects plant height, with the 5g BC treatment showed the largest plant growth at all Ni concentrations. Conversely, plants without biochar exhibited a significant decrease in height under higher Ni stress.\u003c/p\u003e\n \u003cp\u003eNi stress resulted in a reduction in shoot and root fresh and dry weights, total dry mater and as well as plant biomass. The BC treatment mitigated the detrimental impacts of Ni stress by enhancing fresh and dry masses of the shoot and root, total dry matter and plant biomass as well. The study demonstrated the beneficial effects of BC at both 2.5 g and 5 g concentrations in reducing Ni toxicity, with the 5 g concentration demonstrating the highest efficiency. ANOVA analysis showed that there was no significant interaction between the Ni and BC treatments. The statistical effects of Ni stress, the application of BC, and their interaction (Ni \u0026times; BC) on the morphological characteristics of \u003cem\u003eSorghum bicolor\u003c/em\u003e are summarized in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. The majority of parameters, such as plant height (PH), root length (RL), shoot length (SL), leaf area (LA), and biomass (PB), showed highly significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) under both Ni and BC treatments. The interaction effect (Ni \u0026times; BC) was also significant for various traits, confirming the role of biochar in mitigating Ni-induced stress.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\n \u003cdiv class=\"colspec\" align=\"char\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eBiochemical attributes of sorghum (\u003cem\u003esorghum bicolor\u003c/em\u003e L.\u003cem\u003e)\u003c/em\u003e with application of biochar grown under Ni stress conditions. *Significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05%, **Significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.01%; ***Significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.001%; ns at p\u0026thinsp;\u0026gt;\u0026thinsp;0.05%; RNa\u0026thinsp;=\u0026thinsp;Root Sodium\u0026thinsp;=\u0026thinsp;RK, Root Potassium, RCa\u0026thinsp;=\u0026thinsp;Root Calcium, SNa\u0026thinsp;+\u0026thinsp;=\u0026thinsp;Shoot Sodium, SK\u0026thinsp;+\u0026thinsp;=\u0026thinsp;Shoot potassium, SCa2\u0026thinsp;+\u0026thinsp;=\u0026thinsp;Shoot calcium, NiR\u0026thinsp;=\u0026thinsp;Nickel root, NiS\u0026thinsp;=\u0026thinsp;Nickel shoot, SOD\u0026thinsp;=\u0026thinsp;superoxide, POD\u0026thinsp;=\u0026thinsp;peroxidase, CAT\u0026thinsp;=\u0026thinsp;Catalase, CHLR\u0026thinsp;=\u0026thinsp;Chlorophyll ratio TCH\u0026thinsp;=\u0026thinsp;Total chlorophyll, df\u0026thinsp;=\u0026thinsp;degrees of freedom, H₂O\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;hydrogen peroxide, MDA\u0026thinsp;=\u0026thinsp;Malondialdehyde Ni\u0026thinsp;=\u0026thinsp;Nickel stress, BC\u0026thinsp;=\u0026thinsp;Biochar, LSD\u0026thinsp;=\u0026thinsp;least significant difference.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRNa\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRK\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRCa\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSNa\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSK\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSCa\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNiR\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNiS\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSOD\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePOD\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCAT\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCHLR\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTCHL\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMDA\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNi\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e84.2***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e514.9***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e448***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e63.5***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e475.6***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e475.2***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e91.9***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e46.9***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e79.5***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e684.2***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26.7***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.9*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.001***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.072***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0028***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eBC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e168.2***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e564.1***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e584.4***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e253.8***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e644.8***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e615.3***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.6***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.6***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.8***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e82.97***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.5***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.7*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.99***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.04***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.32***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNi *BC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.9ns\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e86.7***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e84.2**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.23ns\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e71.9**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e77.03***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.09***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.23*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.3**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.3***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.23*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.4ns\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.99***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.005***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.28*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eError\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLSD Ni\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.075\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.236\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.421\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.7695\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.268\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.034\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0029\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLSD BC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.075\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.236\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.421\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.7695\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.268\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.034\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0029\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLSD Ni*BC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.725\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.091\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\u0026nbsp;\n \u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMorpho-physiological attributes of sorghum (\u003cem\u003esorghum bicolor\u003c/em\u003e L.\u003cem\u003e)\u003c/em\u003e with application of biochar grown under Ni stress conditions.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePH\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRL\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSL\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNOL\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eLA\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSFW\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSDW\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRFW\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRDW\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCHLa\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCHLb\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCAR\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePB\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTDW\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLAI\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNi\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e536.8***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e168.6***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e125.2***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.4***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e5936.8***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.2***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.7***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e124.9***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.3***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.98***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.8***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47.9\u003c/p\u003e\n \u003cp\u003e***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.16***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e256.33***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e602.5***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e101.7***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e182.8***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e44.5***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1086.3***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.2***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.2***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e159.9***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.7***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0014***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.2***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.11***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.04***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e196.33***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNi *BC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e61.4**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.1***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.4***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.3ns\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e3.2***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.51*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.5***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.03**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.7*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.3**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.4***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.21***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.08***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.33***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eError\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLSD Ni\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e0.561\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.093\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.342\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.078\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.157\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLSD BC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e0.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.093\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.342\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.078\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.157\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLSD Ni*BC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e1.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.087\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.096\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0764\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.123\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"17\"\u003e*Significant at p\u0026thinsp;\u0026le;\u0026thinsp;0.05%, **Significant at p\u0026thinsp;\u0026le;\u0026thinsp;0.01%, ***Significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.001%, ns at p\u0026thinsp;\u0026gt;\u0026thinsp;0.05%; PH\u0026thinsp;=\u0026thinsp;plant height, RL\u0026thinsp;=\u0026thinsp;Root length, SL\u0026thinsp;=\u0026thinsp;Shoot length, NOL\u0026thinsp;=\u0026thinsp;No of leaves, LA\u0026thinsp;=\u0026thinsp;Leaf area, SFW\u0026thinsp;=\u0026thinsp;Shoot fresh weight, SDW\u0026thinsp;=\u0026thinsp;Shoot dry weight, RFW\u0026thinsp;=\u0026thinsp;Root fresh weight, RDW\u0026thinsp;=\u0026thinsp;Root dry weight, Chla\u0026thinsp;=\u0026thinsp;chlorophyll a, Chlb\u0026thinsp;=\u0026thinsp;Chlorophyll b, Car\u0026thinsp;=\u0026thinsp;Carotenoids, PB\u0026thinsp;=\u0026thinsp;Plant biomass, TDW\u0026thinsp;=\u0026thinsp;Total dry weight, LAI\u0026thinsp;=\u0026thinsp;Leaf area index, df\u0026thinsp;=\u0026thinsp;degrees of freedom, Ni\u0026thinsp;=\u0026thinsp;Nickel stress, BC\u0026thinsp;=\u0026thinsp;Biochar, LSD\u0026thinsp;=\u0026thinsp;least significant difference.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003ePhotosynthetic pigments\u003c/h2\u003e\n \u003cp\u003eThe result in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e showed how different amounts of Ni and BC affect several photosynthesis-related pigments, such as chlorophyll \u003cem\u003ea\u003c/em\u003e, chlorophyll \u003cem\u003eb\u003c/em\u003e, total chlorophyll, the chlorophyll \u003cem\u003ea/b\u003c/em\u003e ratio, and carotenoids. As the Ni concentration increased, the chlorophyll a content drops. The use of BC minimized the adverse effects of Ni on chlorophyll concentration, with 5g BC showing height effects at every Ni concentration. At 0 ppm Ni, 2.5g BC has the greatest chlorophyll a concentration. However, when the Ni concentration rises, the 5g BC treatment showed higher chlorophyll \u003cem\u003ea\u003c/em\u003e content.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eSimilar to chlorophyll \u003cem\u003ea\u003c/em\u003e, the concentration of chlorophyll \u003cem\u003eb\u003c/em\u003e falls as Ni concentrations increase.\u003c/p\u003e\n \u003cp\u003eThe 5 g BC sample has the highest concentration of chlorophyll \u003cem\u003eb\u003c/em\u003e for all the Ni concentrations applied. The total chlorophyll content exhibited a similar pattern to chlorophyll \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e, wherein the concentration of pigment decreases as the levels of Ni rise. Across all Ni concentrations, the 5 g BC formulation consistently displayed the highest total chlorophyll content. The ratio is higher in treatments with 5 g BC when they are exposed to Ni stress. The carotenoid content declines as the Ni concentration increases. Furthermore, 0 ppm Ni combined with 2.5 g BC yields the maximum carotenoid concentration, while 100 ppm Ni combined with 0 g BC leads to the lowest carotenoid. At 5 g, BC contributes to the maintenance of elevated carotenoid levels in the presence of Ni stress. Analysis of variance table (ANOVA) showed that the interaction between all treatments of biochar under Nickel stress was almost highly significant (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e,\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eBiochemical parameters\u003c/h2\u003e\n \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\n \u003ch2\u003eEnzymatic antioxidant\u003c/h2\u003e\n \u003cdiv id=\"Sec21\" class=\"Section4\"\u003e\n \u003ch2\u003eSOD, POD and CAT\u003c/h2\u003e\n \u003cp\u003eThe activity of superoxide dismutase (SOD) increases as the concentration of Ni increased for all BC treatments. After 5 g BC treatment, the greatest superoxide dismutase (SOD) activity observed under 100 ppm Ni. The SOD, POD activity raised with increased concentration of Ni. The POD activity reaches its peak at 100 ppm Ni and 5g BC conditions. Greater doses of BC and Ni consistently show an upward trend in POD activity. The CAT activity also exhibits a positive correlation with elevated Ni concentrations and BC treatments. Overall, the results \u003cstrong\u003ein\u003c/strong\u003e Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e showed that adding both biochar and Ni to the samples increased the activity of antioxidant enzymes (SOD, POD, and CAT). Maximum enzyme activity at the highest concentrations of both biochar (5g) and Ni (100 ppm) was observed.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and MDA\u003c/h2\u003e\n \u003cp\u003eThe results \u003cstrong\u003ein\u003c/strong\u003e Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e showed the impact of Ni stress and BC application on plant oxidative stress, measured through malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) levels. Under moderate Ni stress (50 ppm Ni), MDA levels increase; however, plants treated with 2.5g and 5g BC, exhibit a lesser increase compared to those without BC, suggesting that biochar reduces oxidative damage. Exposure to 50 ppm Ni resulted in increased H\u003csub\u003e2\u003c/sub\u003eO levels, with the highest concentrations noted in plants without BC. Plants treated with BC at 2.5g and 5g per kg of soil exhibited lower H₂O₂ levels relative to the untreated group. At 100 ppm Ni, H₂O₂ levels rise significantly, especially in the untreated group. However, BC effectively lowers these levels. The results indicated that biochar application mitigates oxidative stress in plants by decreasing MDA and H₂O₂ levels, particularly at elevated Ni concentrations.\u003c/p\u003e\n \u003cp\u003eTable\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e shows how oxidative stress markers, hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), are affected in \u003cem\u003eSorghum bicolor\u003c/em\u003e by Ni stress and the use of biochar (BC). The maximum accumulation was seen at 100 ppm Ni without BC, and Ni stress markedly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) raised H₂O₂ and MDA levels. However, the application of BC (2.5 g or 5 g) dramatically decreased MDA and H₂O₂ levels, suggesting that it has a protective function in reducing oxidative stress. BC effectiveness in reducing Ni-induced oxidative damage was confirmed by the Ni \u0026times; BC interaction, which likewise revealed a substantial (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decrease in these stress indicators.\u003c/p\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003eIons Na\u003csup\u003e+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, and K\u003csup\u003e+\u003c/sup\u003e content\u003c/h2\u003e\n \u003cp\u003eThe results in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e indicated a significant interaction between BC application and Ni stress regarding the accumulation of essential ions (K\u003csup\u003e+\u003c/sup\u003e, Ca\u003csup\u003e2+,\u003c/sup\u003e Na\u003csup\u003e+\u003c/sup\u003e) in the roots and shoots of the plant. BC application significantly increases potassium (K\u003csup\u003e+\u003c/sup\u003e) accumulation in both roots and shoots. The effect is particularly evident at the highest biochar dose (5g BC), where potassium concentrations reach their maximum, even in the presence of increasing Ni stress. In the absence of biochar (, K\u003csup\u003e+\u003c/sup\u003e levels are significantly reduced, and Ni stress seems to reduce K\u003csup\u003e+\u003c/sup\u003e accumulation. BC application significantly increases calcium (Ca\u003csup\u003e2+\u003c/sup\u003e) concentrations in both roots and shoots. Despite the level of Ni stress, the maximum amount of accumulation of calcium occurs at the highest level of the BC (5 g). This suggested that biochar plays a key role in improving calcium absorption, allowing plants to maintain high Ca\u0026sup2;⁺ levels under stressful circumstances. Sodium (Na⁺) buildup is greatly increased by Ni stress, especially in roots. This impact is lessened by the addition of charcoal; greater dosages of BC (5 g) resulted in reduced concentrations of sodium across both root and shoot. This suggests that when plants are under Ni stress, biochar reduces sodium toxicity.\u003c/p\u003e\n \u003cp\u003eThe statistical evaluation These results are further demonstrated in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, which shows that biochar significantly affects all ion concentrations (p \u0026lt; \u0026amp;lt; 0.001). K⁺, Ca\u0026sup2;⁺, and Na⁺ levels are strongly impacted by Ni stress, and this stress response is mostly controlled by the relationship between biochar and nickel (Ni x BC). By facilitating the accumulation of essential ions (K⁺ and Ca\u0026sup2;⁺) and mitigating the adverse effects of excessive sodium, biochar helps plants cope with Ni-induced stress.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003eNi uptake in root and shoot\u003c/h2\u003e\n \u003cp\u003eThe results in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e illustrate how Ni stress and biochar application affect Ni (Ni) uptake in plant roots and shoots. Both root and shoot tissues demonstrated a distinct trend of increased Ni uptake corresponding to elevated Ni stress levels. Biochar incorporation significantly improved Ni absorption in both plant tissues. Plants treated with 5 g of BC (5 g) demonstrate the highest Ni uptake, followed by those treated with 2.5 g of BC (2.5 g). Results indicated that Ni uptake by the roots consistently exceeds that of the shoots across all stress and treatment levels. The pattern of increased uptake with biochar addition is the same for the roots. The NiR attained a value of 91.9g, whereas NiS reaches 46.9g, indicated root\u0026apos;s enhanced ability to accumulate Ni in comparison to the shoot. The results showed that biochar, especially at higher concentrations, increased the uptake of Ni in both shoot and root tissues under Ni stress.\u003c/p\u003e\n \u003cp\u003eTable\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e showed how much nickel (Ni) is absorbed by sorghum roots (NiR) and shoots (NiS) when exposed to Ni stress and biochar. Both roots and shoots accumulated more Ni under Ni stress (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with roots regularly absorbing more (91.9 g) than shoots (46.9 g). Applying biochar improved Ni absorption even more; plants treated with 5 g BC showed the greatest uptake. The considerable (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) impact of the Ni \u0026times; BC interaction further supported the idea that biochar encourages Ni absorption, especially in root tissues.\u003c/p\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003eCorrelation\u003c/h2\u003e\n \u003cp\u003eThe correlation maps in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e show the connections between different aspects of sorghum plants and soil that are stressed by Ni and how biochar helps them. The first map shows strong positive connections between many stress-related variables, such as the amount of Ni taken up by shoots (NUS), superoxide dismutase (SOD), and peroxidase (POD). This suggests that plants\u0026apos; oxidative stress responses are heightened when Ni stress is high. Root dry weight (RDW), chlorophyll content (Chl), and root sodium (RNa), on the other hand, strongly negatively correlate with stress markers such as malondialdehyde (MDA). This means that increased oxidative stress from Ni toxicity significantly decreases sorghum plant growth and health. This pattern demonstrated that Ni stress negatively impacts sorghum plant, as seen by the significant negative associations detected.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003ePrincipal component analysis\u003c/h2\u003e\n \u003cp\u003ePrincipal component analysis was used to assess the impact of biochar on sorghum plants under Ni stress conditions. Biplot Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e arranges the treatments based on their performance along two main components, which account for the majority of the dataset\u0026apos;s variation. The first two plots show that Dim1 is responsible for 85.5% of the variation. This makes it the main axis for looking at how Ni stress and biochar affect the sorghum plants. The samples designated \u0026quot;Ni0BC0\u0026quot; (high Ni stress without biochar) are located far from the center, suggesting that Ni stress significantly influences characteristics such as root dry weight (RDW), chlorophyll content (Chl), and root sodium (RNa). Samples \u0026quot;Ni2BC1\u0026quot; and \u0026quot;Ni1BC2,\u0026quot; demonstrating the incorporation of biochar, move the data points closer to the plot\u0026apos;s center, signifying a reduction in the negative effects of Ni toxicity. This indicates that biochar successfully alleviates Ni stress and enhances the assessed parameters. Biplot Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e indicates that Dim1 accounts for 73.3% of the variation, while Dim2 contributes 23.1%, emphasizing both primary and secondary effects. Ni-stressed samples without biochar, like \u0026quot;Ni0BC0\u0026quot;, are located far from the center, indicating significant stress. Nevertheless, biochar-treated samples, such as \u0026quot;Ni2BC2,\u0026quot; concentrate toward the center, indicating enhanced plant conditions. Biochar has a positive effect on growth, as seen by its favorable correlation with characteristics like length of root (RL) and dry weight of the shoot (SDW), which are more closely correlated with samples treated with biochar. Applying biochar also lowers stress markers like superoxide dismutase (SOD) and malondialdehyde (MDA), which may indicate a reduction in Ni-induced stress caused by oxidative stress. As a mitigating agent for Ni-induced toxicity, biochar enhances critical growth factors and reduces stress indicators.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n \u003ch2\u003eHeatmaps\u003c/h2\u003e\n \u003cp\u003eThe heatmaps illustrate the relationship between Ni stress and the quantity of biochar applied as a mitigating measure. Each heatmap is paired using hierarchical clustering, which arranges comparable answers along the X-axis (which is thought to represent sample points or attributes) and the Y-axis (which is thought to represent various methods or environmental circumstances). The color gradient, transitioning from blue to red, signifies the level of stress or mitigation, with blue indicating reduced stress (more effective mitigation) and red indicating increased stress (less effective mitigation).\u003c/p\u003e\n \u003cp\u003eThe first heatmap Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e displays a pronounced red color in the second row, signifying elevated Ni-induced stress without a lot of biochar reduction. Bipilot through the rows, the color gradually transitions from red to orange and blue, demonstrating the effective reduction of Ni-induced toxicity by biochar, particularly in the upper regions of the heatmap, which could indicate higher concentrations of biochar. The clustering shows big differences between Ni treatments with and without biochar, which means that different amounts of biochar have different effects on reducing damage.\u003c/p\u003e\n \u003cp\u003eThe second heatmap Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003e displays concentrated red areas in the third row, indicating continuous Ni toxicity under certain circumstances, despite the presence of biochar. Nonetheless, the central and lower regions exhibit a transition from red to lighter colors, indicating some mitigation by biochar under these circumstances. From the blue color in the bottom regions, the clustering shows that some treatments, probably those with higher biochar concentrations, consistently show lower stress responses. The third heatmap (bottom) shows significant red coloring in the top rows, likely indicating untreated samples or those with low biochar concentrations, where Ni-induced stress is most prevalent. As the levels go down, the red transitions to yellows and blues, signifying enhanced mitigation with rising biochar concentrations. The clear difference in the clustering shows that biochar significantly lowers stress levels, separating treatments with higher biochar concentrations into separate groups with lower overall stress.\u003c/p\u003e\n \u003cp\u003eThese heatmaps show that Ni is a significant stressor, with red regions representing elevated toxicity in treatments lacking or containing little biochar. Biochar efficiently mitigates this toxicity, seen by changing to lighter colors (yellow and blue) in regions with elevated biochar concentrations. The structure of clustering supports the finding that different biochar concentrations lead to different stress responses, with higher concentrations lowering stress over time during treatments.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eNickel contamination has become common in soil and water, reducing sustainable plant development and production on a global scale. BC is an innovative strategy for treating Ni-contaminated soil \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Biochar has the capability to immobilize Ni in soil and water via ion exchange, physical adsorption, and surface area attraction. Biochar showed notable mitigation ability for nickel in the agricultural sector \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe present research found that when sorghum plants were under Ni stress, their shoot and the length of the root significantly decreased. When wheat's Ni concentration increased to 25 to 50 \u0026micro;g, there was a noticeable reduction in the length of the roots and shoots\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Similar results have been shown in pea, rice, and Indian mustard plants, suggesting that elevated Ni stress causes a reduction in plant height. A nutritional deficit under Ni stress circumstances may have contributed to the length loss by decreasing nutrient uptake and overall plant length. The fresh and dry weights of the shoots and roots, including the leaf area, decreased in previous studies on the sorghum under Ni stress\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eStudies have shown that as heavy metal absorption or tissue buildup increases, the division of mitotic cells in root meristematic cells usually decreases. In particular, Ni can get through the endodermal barrier into root cells and then build up in the pericycle, which lowers the plant's dry and fresh biomass\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Moreover, Ni stress suppresses the proton pump, which lowers the rate of cell elongation and division and, eventually, causes a reduction in leaf area. However, by increasing the total length, dry and fresh weights of roots and shoots, and by increasing leaf area during Ni stress conditions, BC administration lessened the negative impacts of Ni stress. Plant length and productivity are improved by the application of BC, which also raises the useful potassium (K) content and the amount of phosphorus and nitrogen in soils. Additionally, an increase in the number of cells increases the leaf area per plant\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAccording to the current study, Ni dramatically lowered the amounts of photosynthetic pigments. Nickel stress may prevent essential minerals like iron and magnesium from being absorbed, which might impact several phases of chlorophyll synthesis and lower the amount of chlorophyll in the impacted plants\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In addition, Ni stress damages the chain of electron transport, decreases membrane permeability, and hinders CO₂ fixation, all of which lower the rate of photosynthesis\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Using BC raised the amounts of carotenoid, total chlorophyll, chlorophyll a, and chlorophyll b in this experiment. The application of BC significantly increased the rate of photosynthesis and the amount of chlorophyll by 27.1 percent and 16.1 percent, respectively, according to documented results. The increased level of chlorophyll content in leaves is attributed to biochar's capacity to improve nitrogen availability in the soil, hence increasing nitrogen concentration in plant leaves \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, enzymatic antioxidant activity increased under Ni stress conditions. Prior research has shown an elevation in CAT, SOD and POD activity in response to Ni stress in many crops, including maize, rice, and wheat \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Heavy metal stress induces the production of reactive oxygen species (ROS) in plant tissues, prompting an increase in the activity of antioxidant enzymes inside cytosolic cells to mitigate the production of ROS and enhance the plant's response mechanisms. The application of BC promotes the production of antioxidants in sorghum leaves. It was elucidated that BC enhances antioxidant synthesis, efficiently scavenging reactive oxygen species (ROS) and increasing oxidative stress tolerance in plants \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNickel stress elevated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and MDA levels in sorghum plants. Vicia sativa, tomato, and rice plants have shown an increase in MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content during Ni stress \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, biochar absorbed reactive oxygen species and improved Ni tolerance in sorghum plants. The enhanced activity of antioxidant enzymes may effectively detoxify reactive oxygen species (ROS) and increase plant stress tolerance \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCurrent discoveries on sorghum indicate that same results have been seen in other crops, whereby biochar has shown the capacity to mitigate heavy metal stress and improve ion absorption \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. For example, in maize (\u003cem\u003eZea mays\u003c/em\u003e), the application of biochar enhanced Na\u003csup\u003e+,\u003c/sup\u003e K⁺ and Ca\u0026sup2;⁺ absorption under cadmium (Cd) and lead (Pb) stress, hence promoting growth and resistance to stress, similar to the effects shown in sorghum under nickel (Ni) stress \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Furthermore, studies on rice (\u003cem\u003eOryza sativa\u003c/em\u003e) indicated that biochar promotes the formation of Na\u003csup\u003e+\u003c/sup\u003e, K⁺ and Ca\u0026sup2;⁺, mitigating the adverse effects of arsenic (As) stress, similar to biochar's function in preserving ion homeostasis in sorghum exposed to Ni stress \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. When zinc (Zn) stress occurs in sunflowers (Helianthus annuus), biochar has been demonstrated to improve the availability of vital nutrients including Na⁺, K⁺, and Ca\u0026sup2;⁺. This is in contrast to the defensive strategies used in sorghum. These illustrations show that biochar consistently increases the concentration of essential ions in a variety of crop species, hence boosting their resistance to various heavy metal stressors, such as the Ni stress\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHigher concentrations of biochar, particularly at 5 g BC, were observed to be associated with increased absorption of nickel (Ni) in both roots and shoots. According to earlier studies, biochar can alter the pH level and cation exchange ability of soil, which facilitates plants' uptake of heavy metals such as mercury like Ni\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Research using sorghum has demonstrated that the addition of biochar significantly boosts the absorption of heavy metals such as cadmium and lead. This is because biochar helps the soil retain nutrients and improves its structure, which makes it simpler for plants to consume the metals\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Researchers have shown comparable results in other crops, such as wheat and rice, where biochar increases the accumulation of heavy metals, highlighting its function in improving the ability of different crops to absorb pollutants\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Current study data indicate that roots are the main source of heavy metals, suggesting a defense strategy to reduce toxicity in vital tissues. This is consistent with the observed pattern of greater Ni absorption in roots as opposed to shoots. Studies on sorghum show that, like other agricultural crops, the plant exhibits increased heavy metal buildup in its root system, particularly when biochar is applied\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe present finding emphasizes how important roots are for storing heavy metals during stressful situations. According to the results, biochar functions as a buffer to increase plant tolerance by promoting Ni buildup and perhaps reducing the harmful effects of heavy metals. Research indicates that sorghum productivity and resiliency are enhanced by the addition of biochar to polluted soils, even in the presence of heavy metal stress. Together, the findings highlight the potential use of biochar in remediation techniques for a range of crops, including sorghum. The findings demonstrated its ability to improve heavy metal absorption in the area surrounding the roots, enabling efficient contamination control and extraction\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHigher concentrations of biochar, particularly at 5 g BC, are associated with increased absorption of Ni in both the roots and shoots. Additional data supports previous studies showing that biochar may alter the soil's pH level and capacity for cation exchange, facilitating plants' uptake of heavy metals like Ni\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Based on sorghum study findings, using biochar significantly improves the absorption of heavy metals like lead and cadmium by enhancing the soil's composition and nutrient retention, which in turn increases the availability of metals to plants. Researchers have shown comparable results in other crops, such as wheat and rice, where biochar increases the accumulation of heavy metals, highlighting its function in improving the ability of different crops to absorb pollutants\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e..\u003c/p\u003e \u003cp\u003eLike other cereal crops, sorghum is extremely sensitive to heavy metal stress, especially Ni, which can have a negative impact on nutrient transport, water absorption, and root growth. Similar trends have been observed in other crops under Ni stress, including maize, wheat, and rice, where high Ni concentrations affect phloem function, reduce xylem growth, and damage root structure\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Like sorghum, biochar helps these crops absorb more water and nutrients and thickens their roots, which lessens the negative impacts of heavy metals. Similarly to the pattern that was seen in the vascular tissues and roots of sorghum in this study, applying biochar to maize enhances the morphology of root structures and increases the plant's resistance to metal toxicity\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWheat and rice show better root function and development when treated with biochar among metal-contaminated soils because biochar efficiently reduces heavy metals and lowers their bioavailability. According to the study findings, sorghum, unlike many other crops, takes advantage of biochar's protective properties, particularly when used in high concentrations. As a consequence, it may be used as a useful tactic to increase agricultural resilience in soils that are polluted with heavy metals of various kinds\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe results demonstrate that the presence of Ni stress, particularly at increased levels (Ni 100 ppm), had detrimental impacts on sorghum structure by decreasing epidermal thickness and preventing vascular bundle formation, which in turn reduced the xylem and phloem tissue efficiency\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Because nickel plays a crucial role in plant growth, decreased nickel levels (Ni 50 ppm) showed positive benefits. By strengthening vascular integrity, especially in the phloem and xylem, and boosting water retention, biochar, when used at 2.5 g or 5 g per kg of soil, reduced nickel-induced stress. The interaction between nickel (NixBC) and biochar demonstrates that biochar can lessen nickel stress\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe sorghum shoots' form improved most noticeably with the BC at 5g along with 50 ppm Ni treatment. This is comparable to what researchers have discovered in other crops, such as rice and maize, wherein biochar has been demonstrated to enhance water retention and lessen the negative impacts of heavy metals, both of which aid in plant growth under stress. When treated with biochar, maize under nickel stress exhibits better root and shoot growth as well as less oxidative damage. Biochar is a crucial component of sustainable agriculture in heavy metal-contaminated areas because of its potential to enhance nutrient absorption and water control in the face of Ni stress, which may be linked to a variety of crops\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eNickel (Ni) toxicity in sorghum plants causes ionic imbalance, slowed development, reduced photosynthetic pigments, decreased activity of antioxidants, and altered osmolyte accumulation. Yet BC treatment dramatically reduced these adverse effects by increasing metabolite levels, boosting nutrient absorption, and lowering oxidative stress. Among the treatments studied, 5 g BC per kg of soil was particularly beneficial, since it not only effectively immobilized Ni but also increased the generation of free antioxidants, boosting the plant's resistance to Ni stress. BC reduced sodium ion (Na⁺) buildup and increased food absorption, promoting ionic equilibrium. Furthermore, biochar lowered oxidative stress markers such as malondialdehyde (MDA) and hydrogen peroxide, or H₂O₂, by increasing the activity of key antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). These biochemical enhancements improved photosynthetic efficiency, chlorophyll concentration, and total plant development. Furthermore, BC increased the soil's structure and availability of nutrients, allowing for sustainable sorghum growth in Ni-contaminated soils. The observed effects of biochar application demonstrate its promise as an ecologically friendly technique for reducing metal stress in plants. These findings indicate that biochar can play an important role in increasing plant tolerance to metal toxicity while also supporting sustainable agriculture methods. Future research needs to concentrate on long-term uses of biochar, how it interacts with other soil modifications, and its influence on soil microbes. At the end, using 5 g of biochar per kg of soil is a potential way to eliminate Ni-induced stress in sorghum, leading to improved plant health, increased yield, and long-term crop production in metal-polluted conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor’s Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAA, S.B; Conducted investigations and drafted paper, AM, S.B; supervised research, Conception and design, analyses and interpretation of the data, AM, MMJ, MAN, SHQ, MH and LA; analyses and interpretation of the data Drafting of paper; Application of Statistics Analyses and Software, FAA; Provide Resources and Revising it critically for intellectual content, AM, MH, MAN, SHQ, MMJ, FAA; revised intellectual content; and the final approval to be published. All authors agree to be accountable for all aspects of the work. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll Authors give consent to publish data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during the current study are included in this article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors extend their appreciation to the Deanship of Research and Graduate Studies at King Khalid University for funding this work through large group research under grant number RGP 2/216/46.\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eThe authors extend their appreciation to the Deanship of Research and Graduate Studies at King Khalid University for funding this work through large group research under grant number RGP 2/216/46.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmeen, M., Mahmood, A., Shahzad, A. 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Evaluating models to estimate cation exchange capacity of calcareous soils. \u003cem\u003eGeoderma\u003c/em\u003e \u003cb\u003e400\u003c/b\u003e, 115221. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.geoderma.2021.115221\u003c/span\u003e\u003cspan address=\"10.1016/j.geoderma.2021.115221\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Sorghum, Antioxidant, Biochar, Nickel, Ionic content","lastPublishedDoi":"10.21203/rs.3.rs-6536755/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6536755/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIncreased nickel (Ni) concentrations in plant cells induce physiological, metabolic, and cellular changes, resulting in serious damage to the plants. Among the various strategies to mitigate Ni toxicity in plants, the use of biochar (BC) is highly effective. Biochar enhances soil remediation by immobilizing Ni, reducing its bioavailability, and improving overall soil health. This research aimed to evaluate the effectiveness of BC in alleviating Ni stress in sorghum. The BC was applied at 0, 2.5 and 5 g per kg of soil and Ni concentration was kept at 0, 50 and 100 ppm. The results indicated that the treatment of BC at 5 g enhanced the root and shoot length, fresh weight, and dry weight of both shoot and root, but the application of Ni reduced all assessed growth parameters. Under Ni stress conditions, a concentration of 5 g of biochar enhanced the photosynthetic pigments chlorophyll \u003cem\u003ea\u003c/em\u003e, chlorophyll \u003cem\u003eb\u003c/em\u003e, total chlorophyll, chlorophyll ratio, and carotenoids. At 5 g, biochar lowered the amounts of malondialdehyde (MDA) and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) by increasing the activity of the antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). However, Ni stress increased the amounts of MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The application of 5 g of BC under Ni stress enhanced the uptake of Ca\u0026sup2;⁺ and K⁺ while reducing Na⁺ accumulation. In conclusion, the application of BC at 5g per kg of soil enhanced plant growth, photosynthetic pigments, Ni absorption, antioxidants, ionic contents, and reduced oxidative stress indications, thereby mitigating Ni stress conditions.\u003c/p\u003e","manuscriptTitle":"Biochar Application for Alleviating Nickel Stress and Enhancing Growth, Photosynthetic Pigments, and Antioxidant Defense Mechanisms in Sorghum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-09 07:20:33","doi":"10.21203/rs.3.rs-6536755/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"401f71a5-6a6b-47e9-b62c-45bba7c2c4af","owner":[],"postedDate":"May 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48158139,"name":"Biological sciences/Biochemistry"},{"id":48158140,"name":"Biological sciences/Biological techniques"},{"id":48158141,"name":"Biological sciences/Physiology"},{"id":48158142,"name":"Biological sciences/Psychology"}],"tags":[],"updatedAt":"2025-07-08T04:38:47+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-09 07:20:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6536755","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6536755","identity":"rs-6536755","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}