Winrhizo pro

After 15 days of treatment, five seedlings were randomly picked from each treatment, and the selected plants were cut from the same part, divided into above-ground and below-ground parts, any water on the plant surface was dried with absorbent paper and the fresh weight was measured. The samples were then placed in an electric thermostatic drying oven (Heratherm™ General Protocol Ovens, 51028148, Thermo Scientific™, Waltham, MA, USA) set to 105 °C for 30 min. After adjusting the temperature to 80 °C, the material was dried to a constant weight before measuring the dry weight. For root morphology measurements, the whole root system of a plant was scanned with a root system scanner (Epson Perfection V800 Photo, B11B223201, Epson America, Inc., Los Alamitos, CA, USA). The analysis was done using a root scanner (WinRhizo PRO, version 2017, Regent Instruments Inc., Quebec City, QC, Canada), and parameters such as the total root length, total surface area, total volume and average diameter were read [25 (link)]. Root vigor was determined by the triphenyl tetrazolium chloride (TTC) method [26 ]. Fe³⁺ reductase activity was determined according to the method of Ekmekcioglu C [27 (link)]. Three biological replicates for each treatment were set in treatments.

After treatment, the plant roots were immediately scanned in an aqueous solution or were preserved in a 50% ethyl alcohol solution in 50 ml Falcon flasks. The root system scanning was performed using a specialized root scanner (STD4800 Scanner) and WinRHIZO Pro software (Regent Instruments). The roots were cut using sharp scissors in order to separate them and then placed on a waterproof tray in water (Regent Instruments). The roots were spread out on the trays in order to avoid any overlapping lateral roots and to ensure a random distribution. The parameters that were generated using the WinRHIZO system included the total length of the root system (cm), the root system surface (cm²), the root system volume (cm³) and the average root diameter (mm). Statistical analyses of the parameters that characterize the root systems were performed using an ANOVA analysis (P < 0.05) followed by a Tukey’s honest significant difference test (Tukey HSD test).

We removed the entire soil from the PVC pipes and separated the root samples. Then, the roots were gently washed under tap water and were kept in plastic bags containing 10—15 ml of water to preserve moisture in the root samples. We captured root images using a scanner (Expression 12000XL, Epson, Japan). The collected root samples were placed in a transparent tray (30 cm long × 20 cm wide, it was made with the acryl) containing clean water for scanning (Supplementary Figure 1). Then, we saved the scanned root images as 2D and analyzed them using WinRHIZO Pro software (Regent Instruments Inc., Canada).

The seedlings of wheat plants after seed germination were transferred to the Hoagland solution containing 0, 25, 50, 100, 200, 300 mM NaCl for 10 d until sampling. The samples were dried in an oven at 65℃ for 72 h until constant weight to measure the dry weight of roots and shoots. The roots of fresh wheat seedlings were scanned using an EPSON scanner (EPSON perfect V700), and the roots images were analyzed with WinRHIZO Pro™ (Version 2019a, Regent Instruments, Canada) to characterize total root length, root tip number, root surface area, root volume, and average root diameter. Each sample contains three independent biological replicates.

Plants were assessed at 59 DAS. At which time, the shoots were cut at ground level and roots were gently washed free of soil (five replicate plants per treatment, two plants of the same pot were averaged as one replicate). A small fraction of roots from each pot were kept in 70% ethanol for mycorrhizal evaluation (see below for details). Shoots were oven-dried at 75 °C to constant weight to determine shoot dry weight (mg plant⁻¹). Root samples were scanned with a flatbed scanner (Epson Perfection V800, USA) in grayscale at 300 dpi; root images were analyzed using WinRHIZO Pro (2009b, Regent Instruments, Montreal, QC, Canada) to generate morphology values (i.e., root length, average root diameter, root surface area and root volume). After scanning, root dry weight (mg plant⁻¹) was measured after oven-drying as above. Specific root length was calculated as total root length/root dry weight (root length per unit mass). The root dry weight and root volume were used to calculate root tissue density (root mass per volume). Root to shoot ratio was calculated from root dry weight and shoot dry weight.

Plant roots were imaged using a scanner after exposure to low-B for 20 days. Total root length, root volume and root surface area were measured using the root image analysis software Win-RHIZO Pro (Regent Instruments, QC, Canada).