Materials of Study
Surface soil samples (0-30 cm) were collected from the experimental farm of Government College Women University Faisalabad, then air dried, crashed and sieved to pass through a 2-mm sieve. Soil characters were investigated according to Sparks et al. 40 and the results are presented in Table 1.
Wheat seeds (Triticum aestivum L., cv. Lasani-2008) were obtained from certified seed dealer of the Government of Punjab, Pakistan. All the seeds were disinfected with 95% ethanol followed by 70% sodium hypochlorite solution washing. Finally, the seeds were rinsed three times with distilled water. The use of plants in the present study complies with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. For the production of timber waste biochar, timber waste was collected from regional timber market. The timber waste was first sun-dried for a week and then pyrolyzed at 390°C for 80 min in a pyrolizer. Then, the timber waste biochar (BC) was crushed in a grinder and sieved through a 2 mm sieve and the fine powder was stored. Some major timber waste biochar properties were (Table 2).
Plant material, experimental design and growth conditions
A pot experiment was conducted at the Botanical Garden, Government College Women University Faisalabad, Pakistan to test the hypothesis of the study. Soil portions equivalent to 8 kg soil were mixed thoroughly with one of the following treatments: 0% (control, no timber waste biochar) and 2% biochar (equivalent to 160 g biochar per 8 kg of soil) were packed uniformly in plastic pots (28 cm diameter × 20 cm height). These pots were arranged in complete randomized block design, and each treatment was replicated trice. Seven seeds were sown in each pot, and five healthy seedlings were left by thinning at 15 days after planting till the end of the incubation period (35 days after planting). All plants were watered optimally at 75 WHC (75% of the water holding capacity) until three weeks of sowing; thereafter one group of pots (drought-stressed) were watered at only 35 WHC (35% of the water holding capacity), while the other group was watered at 75 of WHC. On the other hand, a foliar application of 24-epibrassinolide was sprayed on stressed plants at either 0 (distilled water), 1 (BR1) or 3 (BR2) μmol concentrations per pot three times with one day interval after the three weeks period of sowing.
Plant harvesting and growth attributes
Plants were harvested after 35 days of sowing and their root and shoot fresh weights were determined immediately using a digital weighing balance. Root and shoot lengths were recorded using the measuring tape. Three plant samples were selected randomly from each treatment then oven-dried at 65°C for 72 h to determine their dry weights. Other fresh materials were stored at -30°C for further fresh analysis.
The chlorophyll a, b and total contents as well as carotenoids pigments were estimated in fresh leaf samples following the Arnon protocol 41. To determine photosynthetic pigments, a 0.1 g sample was placed in 8 mL of 95% acetone then incubated overnight at 4°C. Color intensity was recorded at 646, 663 and 450 nm using spectrophotometer (UV-2550; Shimadzu, Kyoto, Japan).
Estimation of H2O2 contents was done following Mukherjee and Choudhari technique 42. In this method, 0.1 g leaf sample was extracted in 10 mL cold acetone, centrifuged at 10,000 rpm and then 4 mL titanium reagent and 5 mL of concentrated ammonium solution was added to the reaction mixture. The mixture was then centrifuged at 10,000 rpm for 5 min and the precipitate was dissolved in 10 mL of 2 N H2SO4. The residue was again centrifuged to remove suspended particles. Optical density was recorded at 415 nm against blank by spectrophotometer (UV-2550; Shimadzu, Kyoto, Japan).
Measurement of malondialdehyde and electrolyte leakage
Chloroplast’s lipid peroxidation was determined by estimating malondialdehyde (MDA) contents following thiobarbituric acid (TBA) reaction by Heath and Packer method 43. The electrolyte leakage (EL) was determined following Anjum et al. 44 protocol.
Estimation of proline and non-enzymatic antioxidants
To estimate osmolytes, i.e., proline and other non-enzymatic antioxidants, 50 mg dried plant samples were extracted in 10 mL ethanol (80%), then filtered followed by re-extraction in ethanol (10 mL). A final volume of 20 mL was maintained by mixing the two samples. The obtained extracted solution was used to estimate proline 45, flavonoids and anthocyanin 46, phenolics 47, ascorbic acid 48, proteins 49 and glycine betaine 50 contents.
Ca, Na, P and K ion concentrations
Molybdate/ascorbic acid blue method was used for P determination 51 then measured by spectrophotometer (UV-2550; Shimadzu, Kyoto, Japan). K ion concentrations in plant extracts were measured by flame photometer while Ca and Na concentrations in these extracts were estimated using Atomic Absorption Spectrum (AAS; Shimadzu instruments, Inc., Spectra AA-220, Kyoto, Japan).
Statistical analysis was conducted by using the two-way analysis of variance to find significance of applied treatments in drought stress. All the treatment means were compared by LSD test at 5% level of significance (P < 0.05). Logarithmic transformations for data normalization were carried out before analysis, where necessary. Pearson’s correlation analysis was performed to compute associations among various analyzed variables. The heat-map conceits between measured variables were also calculated by using the Origin software.