To test for metabolite differences between treatments, metabolite abundances were Hellinger‐transformed (Ramette, 2007 ) and principal component analysis (PCA) was performed with PC‐ORD v6.08 (McCune & Mefford, 1999 ). Data on plant biomass, root system architecture (primary seminal root length, total root length, root surface area), ion content, lipid and metabolite abundance were subjected to statistical analysis by one‐way ANOVA, using JMP software (SAS institute Inc., Cary, NC). Significance was defined as a probability level of the student's t test at p ≤ .05. Total root length and root surface area were performed using WinRHIZO software (Arsenault, Poulcur, Messier, & Guay, 1995 ) based on scanned root images using standard parameters (Regent Instruments Inc., Ontario, CA).
Winrhizo software
Manufactured by Regent Instruments
Sourced in Canada, United States, Japan, United Kingdom
WinRHIZO is a software solution designed for the analysis of root systems. It provides a comprehensive set of tools to capture, measure, and analyze digital images of plant roots.
Automatically generated - may contain errors
Lab products found in correlation
Std 1600, by Regent Instruments (5 mentions)
Ds u3 camera, by Nikon (4 mentions)
Perfection v700, by Epson (3 mentions)
Expression 11000xl, by Epson (3 mentions)
Rhizoscanner, by Regent Instruments (2 mentions)
Expression 1680, by Epson (2 mentions)
Expression 10000xl, by Epson (2 mentions)
Epson1680, by Regent Instruments (2 mentions)
Spad 502, by Konica Minolta (2 mentions)
Expression 10000xl 1, by Epson (2 mentions)
Perfection v700 photo, by Epson (2 mentions)
Jmp software, by SAS Institute (1 mentions)
Perfection v800 photo, by Epson (1 mentions)
Af6000, by Leica (1 mentions)
V750 pro, by Epson (1 mentions)
10000xl scanner, by Epson (1 mentions)
Axio imager z2 microscope, by Zeiss (1 mentions)
Perfection v850, by Epson (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Expression 1600 scanner, by Epson (1 mentions)
105 protocols using winrhizo software
1
Metabolic Profiling and Root Traits Analysis
2
Maize Root Morphology and Architecture
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Morphology and architecture of roots were captured by scanning at 300 dpi using a Hewlett Packard Scan Jet 3c/T optical scanner (Epson Perfection V800 photo). The image was then analyzed with winrhizo software (Regent Instruments, Inc., Quebec, Canada) for the average diameter, total length, and total surface area. Before scanning, roots were physically separated into three different groups (i.e., lateral root, crown root, and primary root) based on the structure of the maize root system. The corresponding root samples were oven‐dried at 65°C for 48 h and weighed. SRL and SRSA were calculated as the root length per unit dry weight and root surface area per unit dry weight, respectively (Comas & Eissenstat, 2009 (link)).
3
Root Morphological Analysis by Scanning
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Root morphological parameters, such as total root length, surface area, total number of tips, forks, crossings, and fine and medium root length, were determined 24, 48, and 72 h from the treatment by computerized scanning (STD 1600, Regent Instruments, Canada) and analyzed using WinRHIZO software (Regent Instruments).
4
Root Morphology and Viability Analysis
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After collection of the exudates, the roots were imaged using a scanner (V750 Pro, Epson) set to high accuracy (800 dpi). The scanned images were analyzed using WinRHIZO software (version 2008a Pro, Regent Instruments Inc., Canada) to obtain root morphology parameters, including root tips (RT), total root length (RL), crossings (RC), average diameter (RAD), forks (RF), surface area (RSA), and volume (RV). Next, the root tips/surface area ratio (NRT/SA) was calculated based on the measured root parameters. Lastly, the dry biomass of the plant was weighed after oven-drying at 70°C for 72 h.
The viability of the RT was determined by staining the roots for 10 min with a fluorescent dye (Pan et al., 2001) comprising fluorescein diacetate (12.5 g mL -1 ) and propidium iodide (5 g mL -1 ). Fluorescence images of the RT were obtained using a fluorescence microscope (AF6000, Leica, Germany).
The viability of the RT was determined by staining the roots for 10 min with a fluorescent dye (Pan et al., 2001) comprising fluorescein diacetate (12.5 g mL -1 ) and propidium iodide (5 g mL -1 ). Fluorescence images of the RT were obtained using a fluorescence microscope (AF6000, Leica, Germany).
5
Root Morphology of PGPR-Inoculated Wheat
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The root morphological study is mostly carried out in PGPRs-inoculated plants under stress conditions to find their role in stress alleviation. To study the effects of PGPRs (MNCN1, LLCG23 and FZB42) on wheat root morphology in the presence of salt stress an automated Rhizoscanner, equipped with WinRHIZO software offered by Regent Instruments Co. was used to calculate different morphological root parameters, i.e., root length, surface area, volume, diameter and number of root tips for each wheat plant. Three wheat plants were randomly selected from each treatment in triplicate after 9 days’ post-inoculation for Rhizoscanning and the average values were calculated for the above parameters to see the effects of each PGPR on wheat root morphology under salt stress.
6
Characterizing Root System Architecture
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The mapping population of 184 RILs along with two parental lines of each replication, one normal seedling at the growth stage V1 was collected to manually measure shoot root traits, such as shoot length (SL), plant height (PH), and tap root length (TRL), with a ruler. For other root traits, including total root length (RL), root surface area (RSA), root volume (RV), and distribution of root length (RD_L), root surface area (RD_S), and thickness (RD_T) in diameter class of 1.0 to 1.5 mm, were evaluated using an Epson Scanner 10000XL (Epson America Inc., CA, USA) and analyzed using WinRhizo software (Regent Instruments Inc., Canada). The detailed information of root diameter distribution is critical to understand the root system architecture and its implication in soil function (Blouin et al., 2007 (link)). The measurements of shoot- and root-related traits were averaged for further analysis in this study as described in Table 1 Supplementary Table S3
7
Wheat Root Morphology under Nitrogen Stress
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Wheat seedlings were grown for 5 d in a nutrient solution that contained 0 mM NH 4 NO 3 , 0.5 mM NH 4 NO 3 , 1.0 mM NH 4 NO 3 , and 1.5 mM NH 4 NO 3 . The primary root tip (1.0 cm) was taken manually from each individual root using a razor blade to investigate the morphology of the elongation zone in primary roots. Root cross sections of 0.5-cm thickness (1.0-1.5 cm distant from the primary root tip) were also taken to observe the diameter of root cross sections. The cross sections were stained for 1 min with Methylene blue solution (0.1% (w/v) in distilled water) before being imaged with Zeiss Axio Imager Z2 microscope.
Roots were collected from taare1 mutant and wild-type wheat seedlings, which were grown for 12 d in a nutrient solution containing 0 mM and 1.5 mM NH 4 NO 3 , individually. The root morphological parameters were analyzed with WinRHIZO software developed by Regent Instruments Canada. In terms of root and shoot ratio, dry weight of roots and shoots were measured under different hydroponic culture solutions for wildtype and taare1 mutant wheat seedlings after 20 d.
Roots were collected from taare1 mutant and wild-type wheat seedlings, which were grown for 12 d in a nutrient solution containing 0 mM and 1.5 mM NH 4 NO 3 , individually. The root morphological parameters were analyzed with WinRHIZO software developed by Regent Instruments Canada. In terms of root and shoot ratio, dry weight of roots and shoots were measured under different hydroponic culture solutions for wildtype and taare1 mutant wheat seedlings after 20 d.
8
Plant Biomass Measurement Protocol
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FW was measured immediately after harvesting. DW was measured after drying for 48h in an oven at 80 °C. Roots were scanned with a digital scanner (Epson, Nagano, Japan) and then analysed with WinRHIZO software (Regent Instruments Inc., Quebec, Canada).
9
Bacterial Inoculation Enhances Rice Root Morphology
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Sterilized deionized water-washed 7-day-old Malaysia MR-219 rice seedlings were grown in experimental and reclaimed soil. For the experimental group, homogenous seedlings were soaked with S32 strain suspensions (108 cells/mL) for 30 min. Then, three seedlings were sown in each plastic pot. The plants were grown under natural growth conditions, maintaining a 20% absolute water content. Rice seedlings were harvested 21 days after sowing. Approximately 5 × 108/mL of live washed bacterial S32 cells were used as inoculum in each bacterial treatment.
The root morphology of the MR-219 rice was determined using a root scanner (Expression 1680, Epson). Total root length (cm), total surface area (cm2) and total volume (cm3) were quantified using a scanner (Expression 1680, Epson) equipped with a 2 cm deep plexiglass tank (20.30 cm) filled with H2O (El Zemrany et al., 2007 (link)). The scanned data were processed by Win-Rhizo software (Regent Instruments Inc.).
The root morphology of the MR-219 rice was determined using a root scanner (Expression 1680, Epson). Total root length (cm), total surface area (cm2) and total volume (cm3) were quantified using a scanner (Expression 1680, Epson) equipped with a 2 cm deep plexiglass tank (20.30 cm) filled with H2O (El Zemrany et al., 2007 (link)). The scanned data were processed by Win-Rhizo software (Regent Instruments Inc.).
10
Root Morphology and Activity Measurements
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In this study, for each of the normal (unlabeled) groups of each treatment, three seedlings were selected at random for the root morphology measurements, and root total length (RL) and root surface area (RSA) were selected to evaluate the root morphology. After completing the root sample preparations as described by Xing et al. (2021) (link), the values of RL and RSA were measured using the WinRHIZO software (Regent Instruments Canada, Inc.). The samples were prepared according to Xu et al. (2020) (link). Subsequently, a 1/1,000 electronic balance was used to measure the dry weight. Meanwhile, the determination of root activity was also measured using the triphenyltetrazolium chloride (TTC) method of Chen et al. (2018) (link).
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