Winrhizo pro software

Roots were washed thoroughly by putting on a wire-mesh sieve and using a slow-speed water stream. The longest root length was measured using a centimeter ruler. Each root system was floated on a waterproof Plexiglas tray and scanned using a specialized dual-scan optical scanner (Regent Instruments Inc., Montreal, QC, Canada). The image acquisition parameters were set to “high” resolution (800 × 800 dpi), and the acquired images were analyzed using WinRHIZO Pro software (Version 2009c; Regent Instruments, Montreal, QC, Canada) to obtain cumulative root length, root surface area, root diameter, root volume, number of tips (root tips), number of forks (root forks), and number of crossings.

The winRHIZO optical scanner (Regent Instruments, Inc., Québec, Canada) was used to scan the roots before drying the roots in the first set of experiments. After separating the stem from individual root systems of each plant, roots were washed by placing the pot on sieves and gently spraying with water. Roots images were acquired and analyzed for different parameters of roots using WinRHIZO Pro software (Regent Instruments, Inc.) as described by [35 ,36 ]. The measurements were taken on five different replicate plants in each treatment.

Ten plants per treatment were selected from the five replicates (two plants per replicate), gently shaken to remove the quartz sand on the root surface, and carefully washed with running water. Root quantitative traits were measured from scanned images from a desktop scanner (EPSON Perfection V800 Photo, CA, USA) using the WinRHIZO Pro software (Regent Instruments Inc., Quebec, Canada). Seedling height was measured with a vernier caliper; the root and shoot fresh weights were recorded separately using an electric balance (Type: ML 204; Mettler Toledo Company, Greifensee, Switzerland; measurement accuracy 0.0001 g). Samples were dried at 80 °C to constant weight and the dry weights were recorded.

Root images were acquired using a mirrorless camera (M100, Canon, Japan) with a mini-rhizobox (Supplementary Figure S1). The camera information is detailed in Supplementary Table S1. All root samples were imaged with the lens focus fixed at 22 mm. Additionally, to measure root morphological traits, the collected root images were analyzed using WinRHIZO Pro software (WinRHIZO, Regent Instruments, Inc., Canada). Each root trait is defined in Supplementary Table S2.

Maize (Zea mays L.) seeds were soaked in tap water overnight and fifteen (15) seeds were deposited for each replicate on round filter paper placed in a Petri dish. At least three replicates were used per each experiment. The filters were moistened with aqueous solution of HLS at various concentrations (ranging from 0 to 100 mg HLS L^–1) and the seeds were germinated in the dark at 298 K for 96 h. Thereafter, the plantlets were scanned with a modified flatbed scanner (Epson Perfection V700, Seiko Epson, Corp., Japan) and the length of primary root, lateral seminal roots, total root apparatus and coleoptile was measured by using the WinRhizo Pro software, version 2016 (Regent Instruments, Inc., Canada).

The accessions from the natural population were completely randomly grown and evaluated with three replications. In each replication, three uniform plants per accession were collected from the germination device at 13 days after sowing (DAS). At 6 DAS, 24 plants of an accession were transplanted to one growth device (Additional file 1: Figure S1h). Then three plants per accession were sampled from the growth device at four timepoints, namely, 10 days after transplanting (10 DAT, equal to 16 DAS), three expanding leaves (3 EL), 5 EL, and 7 EL, respectively. In total, 12,600 plants (280 accessions × 3 replicates × 3 plants × 5 timepoints) were sampled. Once the plants were sampled, shoot fresh weight (SFW), root fresh weight (RFW), and primary root length (PRL) were measured manually. The intact roots in a transparent box full of water were scanned with the root scanner (EPSON, 11000XL). The obtained high-resolution root images were analyzed using WinRHIZO-Pro software (Regent Instruments, QC, Canada) to determine total root length (TRL), total root surface (TSA), total root volume (TRV), and total number of roots (TNR). Subsequently, shoot and root samples were dehydrated at 65 °C for a week to determine shoot dry weight (SDW) and root dry weight (RDW).