A Simple Protocol for Mapping the Plant Root System Architecture Traits

Summary

We use simple laboratory tools to examine the root system architecture (RSA) of Arabidopsis and Medicago. The plantlets are grown hydroponically over mesh and spread using an art brush to reveal the RSA. Images are taken using scanning or a high-resolution camera, then analyzed with ImageJ to map traits.

Abstract

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar tolerance to environmental challenges. An experimental protocol is presented for setting up the hydroponic system, plantlet growth, RSA spreading, and imaging. The approach used a magenta box-based hydroponic system containing polypropylene mesh supported by polycarbonate wedges. Experimental settings are exemplified by assessing the RSA of the plantlets under varying nutrient (phosphate [Pi]) supply. The system was established to examine the RSA of Arabidopsis, but it is readily adaptable to study other plants like Medicago sativa (Alfalfa). Arabidopsis thaliana (Col-0) plantlets are used in this investigation as an example to understand the plant RSA. Seeds are surface sterilized by treating ethanol and diluted commercial bleach, and kept at 4 °C for stratification. The seeds are germinated and grown on a liquid half-MS medium on a polypropylene mesh supported by polycarbonate wedges. The plantlets are grown under standard growth conditions for the desired number days, gently picked out from the mesh, and submersed in water-containing agar plates. Each root system of the plantlets is spread gently on the water-filled plate with the help of a round art brush. These Petri plates are photographed or scanned at high resolution to document the RSA traits. The root traits, such as primary root, lateral roots, and branching zone, are measured using the freely available ImageJ software. This study provides techniques for measuring plant root characteristics in controlled environmental settings. We discuss how to (1) grow the plantlets, and collect and spread root samples, (2) obtain pictures of spread RSA samples, (3) capture the images, and (4) use image analysis software to quantify root attributes. The advantage of the present method is the versatile, easy, and efficient measurement of the RSA traits.

Introduction

The root system architecture (RSA), which is underground, is a vital organ for plant growth and productivity^1,2,3. After the embryonic stage, plants undergo their most significant morphological changes. The way in which the roots grow in the soil greatly affects the growth of plant parts above ground. Root growth is the first step in germination. It is an informative trait as it uniquely responds to different available nutrients^1,2,3,4. The RSA exhibits a high degree of developmental plasticity, which means that the environment is always used to make decisions about development^2,5. Changes in the environment have made crop production more difficult in the present scenario. On a continuous basis, the RSA incorporates environmental signals into developmental choices⁵. As a result, a thorough understanding of the principles behind root development is essential for learning how plants respond to changing environments^2,5.

The RSA senses varying nutrient concentrations and renders phenotypic alterations^{4,6,7,8,9,10,11,12}. Studies suggest that root morphology/RSA is highly plastic compared to shoot morphology^1,3. RSA trait mapping is highly effective in recording the effect of changing the surrounding soil environment^1,11,12.

In general, discrepancies in the effect of various nutrient deficiencies on the root phenotype have been reported in many earlier studies^{3,11,13,14,15}. For example, there are several contrasting reports on phosphate (Pi) starvation-induced changes in the number, length, and density of lateral roots (LRs). An increase in LR density has been reported under the Pi deficient condition^6,8. In contrast, a decrease in LR density under Pi deficient conditions has also been reported by other authors^3,13,16. One of the prominent causes of these inconsistencies is the use of the elemental contamination-prone gelling medium, which agar often contains¹⁰. Researchers typically grow their experimental plants on an agar-based plate system and record the root traits. Numerous RSA traits are frequently concealed or entrenched within the agar material and cannot be documented. Experiments linked to inducing nutrient deficiency, in which users often exclude one component totally from the medium, cannot be performed in elemental contamination-prone gelling medium^11,14,15. Numerous nutrients are frequently present in significant amounts in the agar media, including P, Zn, Fe, and many more^11,14,15. Furthermore, RSA growth is slower in agar-based media than in non-agar-based liquid medium. As a result, there is a need to establish an alternate non-agar-based approach for quantifying and qualitatively recording the phenotype of RSA. Consequently, the current method has been developed, in which plantlets are raised in a magenta box-based hydroponic system atop a polypropylene mesh supported by polycarbonate wedges^1,10,11.

This study presents a detailed improvised version of the earlier method described by Jain et al.¹⁰. This strategy has been tuned for current demands in plant root biology and can also be used for plants like Alfalfa, other than model plants. The protocol is the primary way to measure the changes in RSA, and it only requires simple equipment. The present protocol illustrates how to phenotype several root features, such as primary and lateral roots in normal and modified medium (Pi deficient). Step-by-step directions and other helpful hints gleaned from the author's experiences are provided to help the researchers follow along with the methodologies offered in this method. The present study aims to provide a simple and effective method for revealing the entire root system of plants, including higher-order LRs. This method involves manually spreading the root system with a round watercolor art brush, allowing for precise control over the exposure of the roots^1,10,11,12. It does not require expensive equipment or complicated software. This method has improved nutrient uptake and growth rate; plants have a nutrient-rich solution easily absorbed by their roots. The present method is suitable for researchers who wish to map the traits of a plant's root system in detail, particularly during early development (10-15 days after germination). It is suitable for small root systems, model plants like Arabidopsis and tobacco, and non-conventional plants like Alfalfa until their root system fits in the magenta boxes.

The steps for phenotypic analysis of RSA development in Arabidopsis are outlined in this protocol as follows: (1) the method of seed surface sterilization for plants (Arabidopsis), (2) the steps to set up the hydroponic system, followed by seed sowing on a medium, (3) procedure for taking out the complete seedings and spreading on the Petri plate for RSA analysis, (4) how to record the images for RSA, and (5) calculate important RSA parameters using ImageJ software.

Protocol

The whole protocol is summarized schematically in Figure 1, showing all the essential steps involved in revealing the root system architecture (RSA) of plantlets. Protocol steps are given in detail below:

1. Arabidopsis seed surface sterilization

1. Transfer a tiny scoop (approximately 100 seeds = approximately 2.5 mg) of seeds to a microfuge tube, and soak for 30 min in distilled water at room temperature (RT). This entire procedure is carried out in the aseptic condition.
2. Briefly centrifuge the microfuge tube containing seeds at 500 x g for 5 s, using any tabletop centrifuge at RT to let the seeds settle down.
3. Decant the water, add 700 µL of 70% (v/v) ethanol, vortex for a few seconds, and spin. Repeat vortexing and spinning if required, but ensure the treatment time of 70% ethanol remains at 3 min.
4. After 3 min, immediately rinse once with sterile water. Keep the ethanol washing step as timely as possible, as prolonged ethanol exposure decreases germination.
5. Treat the seeds with diluted commercial bleach (4% v/v) with a drop of Tween-20 for 7 min. Mix the seeds with bleach solution by inverting the tubes rapidly 8-12 times, followed by a brief centrifuge (500 x g for 5 s at RT). Froth is seen appearing in the tube.
6. Decant the supernatant using a 1 mL pipette and rinse the seeds with at least five washes with sterile water, following the same vortexing procedure.
7. Leave the surface sterilized seeds in water and incubate for 2-3 days at 4 °C for stratification¹⁰.

2. Setting a hydroponic system for seed germination

1. Half-fill a standard magenta box with distilled water and autoclave it. Autoclave the polycarbonate sheet (clear color and smooth texture) and cut 4 cm x 8 cm rectangles, with a midpoint notched more than halfway through the rectangle so that two rectangles may slot together to form an X shape¹⁰. Use this setup to hold the polypropylene mesh (6 cm x 6 cm squares of 250 µm pore size, or depending upon the requirement) cut from 12x 24 inch sheets¹⁰.
   NOTE: Polypropylene is highly resistant to acids, alkalis, and other chemicals; therefore, it has been opted. Autoclaving tends to distort polypropylene mesh; hence, it is recommended to be carried separately wrapped in aluminum foil. Typical autoclaving conditions of 16 min, 121 °C, 15 psi, or 775 mm Hg are recommended.
2. Add sterile half-MS basal media with vitamins + 1.5% (w/v) sucrose, as described by Shukla et al.¹, to each box to reach the bottom edge of the polypropylene mesh in a laminar flow. All the procedures are carried out under aseptic conditions.
3. Sow the surface-sterilized seeds on the mesh (250 µm pore size) hydroponically and allow them to grow for 3 days.
4. After 3 days, transfer the seedlings onto a mesh (500 µm pore size) and allow them to grow for 2 days.
5. After 2 days (total of 5 days), transfer the seedlings onto the control media (i.e., modified MS nutrient media¹ containing 2.0 mM NH₄NO₃, 1.9 mM KNO₃, 0.15 mM MgSO₄·7H₂O, 0.1 mM MnSO₄·H₂O, 3.0 µM ZnSO₄·7H₂O, 0.1 µM CuSO₄·5H₂O, 0.3 mM CaCl₂·2H₂O, 5.0 µM KI, 0.1 µM CoCl₂·6H₂O, 0.1 mM FeSO₄·7H₂O, 0.1 mM Na₂EDTA·2H₂O, 1.25 mM KH₂PO₄, 100 µM H₃BO₃, 1 µM Na₂MoO₄·2H₂O, 1.5% sucrose, 1.25 mM MES, pH 5.7 adjusted with 0.1 M MES [pH 6.1]) and to the experimental media (e.g., P- [0 mM] treatment; KH₂PO₄ is replaced with 0.62 mM K₂SO₄ from the control media composition as mentioned above¹. For excess Pi treatments, the concentration of KH₂PO₄ is increased in modified MS medium [2.5, 5.0, 10.0, 20.0 mM]¹) and let the seeds grow for 7 days.
   ​NOTE: A larger mesh pore size (500 µm) facilitates the smooth picking of entire seedlings out without any damage or need of cutting at the hypocotyl. Plantlets grow under standard growth conditions (i.e., 16 h light/8 h dark photoperiod, 150 µmol·m^-2·s^-1 light intensity, 60%-70% humidity) at 23 °C.

3. Examination of RSA

1. Prepare agar (1.1%) plates for root spreading (Petri plate size: 150 mm x 15 mm).
2. Add 10-20 mL of autoclaved filtered tap water to the Petri plate, as mentioned above. Gently pull out the seedlings from the mesh (500 µm) and submerge them in water on the plates.
3. Gently spread each plantlets' root in the water-filled plate with the help of a round watercolor art brush (sizes: no. 14, 16, 18, and 20).
   NOTE: While carrying out the spreading of the root system, first get hold of the primary root and spread it into a straight line, as it serves as an axis. Then, spread the LRs symmetrically on each side of the primary root, wherever possible. After that, spread the second-order LR linked to the first-order LR. This spreading process is a kind of art; do it gently, slowly, like an artist drawing an image of the RSA.
4. Tilt the plate slightly to remove the water.
   ​NOTE: At this point, the procedure can be paused by putting these spread plates at 4 °C. Later, when image processing is required, take out the plates and place them at RT for a while. Wipe out the condensed water, and then the image can be processed conveniently.

4. Recording images for RSA

1. Scan or photograph these Petri plates appropriately.
   NOTE: For obtaining high-quality photographs, the 600 dpi resolution is recommended for scanning, and at least a 12 megapixel camera is recommended for photography.
2. Measure the root system architecture traits using freely available ImageJ software (https://imagej.nih.gov/ij/index.html). To quickly follow the steps to measure the root length using ImageJ software, please refer to the example "measuring DNA contour length"​¹⁷.
   NOTE: These steps are followed to measure the root lengths on pictures captured using a high-resolution scanner or camera.
   1. Use a given distance of length to set the scale. The known distance of the scale bar in Figure 3 is 2 cm. Select the Straight Line tool from the ImageJ toolbar (fifth tool from the left). Use the Straight-Line tool to create a line selection that outlines the scale bar. Finish outlining by right-clicking, double-clicking, or clicking in the box at the beginning.
   2. Measure the length of the known scale bar in pixels using the Analyze > Measure toolbar. Make a note of the pixel length.
   3. Open the Set Scale dialogue box by clicking the Set Scale tab in the Analyze tab. In the Distance in Pixels field, enter the pixel length (as noted above). Next, in the Known Distance field, enter the value, as shown by the scale bar (here, it is 20 mm). Set the Unit of Length as mm. The pixel-aspect ratio is 1.0. Now, the scale is specified by the x number of pixels per millimeter. To lock the scale for this particular image, click on OK.
   4. Create a line selection that outlines the root length using the Segmented Line tool. Finish outlining by right-clicking, double-clicking, or clicking in the box at the beginning. Click and drag the small black and white "handles" along the outline to adjust the line selection as needed.
   5. Use the Measure command under the Analyze tab of ImageJ to quantify the length of the root. To transfer the measured data to a spreadsheet, right-click on the Results window, select Copy All from the popup menu, switch to the spreadsheet, and then paste the data.
      NOTE: As described above, set the scale using the known distance of the scale bar in the ImageJ set scale option. This gives the number of pixels per unit length. It is required to freshly set the scale every time, whenever a new image is being analyzed.
3. Measurement and calculation of RSA traits
   1. Measure the primary root length between the hypocotyl junction to the root tip's end.
   2. Measure the first- and second-order LR length.
   3. Measure the branching zone (BZ) of the primary root. The branching zone of the primary root (BZ^PR) spans the first LR emerging point to the last LR emerging point.
   4. Record the number of LRs, which is the number of LRs originating within the boundary of the BZ^PR.
   5. Measure the average length of the first- and higher-order LRs. Derive the average length of the first-order LR (1° LR) (centimeter per root) by dividing the total length of the 1° LR by the total number of 1° LRs.
   6. Measure the average length of the second-order LR. Calculate the average length of the second-order LR (2° LR) by dividing the total length of the 2° LR by the total number of 2° LRs.
   7. Measure the 1° LR density. Calculate the 1° LR density (number of 1° LRs per unit length of BZ^PR) by dividing the number of 1° LRs by the length of the BZ^PR.
   8. Measure the 2° LR density. Calculate the 2° LR density by dividing the number of 2° LRs by the length of the BZ of 1° LRs (number of 2° LRs per unit length of the BZ of 1° lateral roots).
   9. Measure the total root length (TRL). This is the aggregate of the primary root, 1° LR, and 2° LR (and more if present) lengths.

5. Root hair measurement

NOTE: Although the hydroponic system is not good at promoting root hair growth and development, despite being as robust as it is in solid growth media, it is still important to study it in the present context. Follow the steps below to analyze root hair development in a 5 mm section from the tip of the primary root of the seedlings.

1. Chop off a 2 cm section of the primary root from the root tip.
2. Mount the root section on a slide using 10% glycerol as a mounting medium.
3. Place the slide under a stereo microscope.
4. Use the axial carrier to visualize and capture images of the root hairs.
5. Analyze the images to study the structure and characteristics of the root hairs using ImageJ software as earlier described.

Representative Results

The different morphometric traits of root system architecture (RSA) are measured using simple laboratory tools, and the steps are depicted schematically in Figure 1. The details of the hydroponic setup demonstrate the protocol's potential in measuring the RSA (Figure 1 and Figure 2).

Given the observed differences in gelling agents, we used a hydroponic growing system to conduct all the studies^3,10. As a proof of concept, this hydroponic system works well, reflecting the apparent contrasting phenotype under Pi deficient and sufficient conditions (Figure 3). Arabidopsis seeds were hydroponically cultivated for 5 days in a half-MS medium on a polypropylene mesh, as illustrated in Figure 2. The plantlets were transplanted after 5 days into Pi deficient and sufficient conditions, and allowed to grow for 7 days (Figure 3).

Demonstration of RSA traits under varied nutrient (Pi) supply
We have followed an established scheme to analyze and record the traits of the RSA³. Different RSA traits were analyzed under contrasting Pi regimes in hydroponic conditions (Figure 3). The P_i deficient treatment (0 mM Pi) invoked a typically reported root phenotype exhibiting a shorter, shallower, and less branched RSA compared to the Pi sufficient condition (Figure 3A). Primary root length was significantly attenuated under the Pi deficient condition (Figure 3A,B). Primary root length, rapidly gained in the presence of Pi (1.25 mM), shows the efficiency of the hydroponic system reflecting the physio-morphological changes adequately (Figure 3A,B). The TRL was significantly decreased under the Pi deficient condition (Figure 3B). The branching zone (BZ^PR), as shown in Figure 3A, was significantly attenuated under the Pi deficient condition (Figure 3B).

Of these traits, the most affected was the TRL, because of the high difference in LR number between the two Pi conditions. The vigorous growth of the RSA under the Pi sufficient condition (1.25 mM) was due to a significant increase in the number and length of 1° LRs. Thus, a rapid RSA change occurred, mainly due to the alteration in LR development. We measured the number of LRs originating within the boundary of the branching zone of the primary root, as they are considered more meaningful^1,3,18. The average length of the 1° LR (centimeter per root) was significantly reduced in the Pi deficient condition (Figure 3C). The average 2° LR length was similarly reduced, due to the P₀ condition; however, it was lower in quantity than the average 1° LR length (Figure 3C). The number of 1° LRs and 2° LRs heavily decreased under the P₀ condition compared to the P_1.25 condition (Figure 3D). The 1° LR density (number of 1° LRs per centimeter of BZ^PR length) was not changed under the P₀ condition relative to the P_1.25 condition (Figure 3E). The 2° LR density (number of 2° LRs per centimeter of the BZ of 1° LRs) also showed no significant change (Figure 3E). As a result, determining the density of LRs is essential for gaining helpful insight into the RSA plasticity.

Analysis of root hair development under varying phosphate (Pi) regimes
The effect of Pi supply on root hair development was studied in the primary root of seedlings. It was found that root hair length increased with increasing concentrations of Pi up to 2.5 mM, but decreased at 5 mM and 10 mM. However, at 20 mM, root hair length returned to near-peak levels (Supplementary Figure 1). The number of root hairs was significantly higher at 0 mM compared to all other Pi supplies, with the highest number observed at 20 mM (Supplementary Figure 1).

Application of the present method on plants other than Arabidopsis
We have examined the feasibility of the present method on plants other than Arabidopsis, taking Medicago sativa (Alfalfa) as a test plant. The protocol has been modified according to the requirement of two aspects: (1) the seed surface sterilization method, and (2) the pore size of the polypropylene mesh (now larger, 1,000 µm). The surface seed sterilization and stratification protocol always need to be optimized depending upon the selected plants to study. For Alfalfa, steps were followed as described by Weeks et al.¹⁹. After surface sterilization, the seeds were incubated at 4 °C for 7 days for stratification. After that, we followed the same procedure described in this protocol with a modification of mesh pore size. As shown in Figure 4A,B, the plantlets were well-grown, with an altered RSA under varying supplies of Pi. Figure 4C displays the root system plasticity under 1.25, 0, and 20 mM of Pi nutrient solution. Excess Pi (20 mM) and deficient Pi supply led to diminishing the development of the root system, as compared to the Pi sufficient (1.25 mM) condition (control) (Figure 4C). The RSA can be mapped for different traits using the ImageJ software described in the protocol. Hence, the protocol is simple, efficient, and can be easily modified according to the selected plant species. It offers the opportunity to study the RSA of diverse plant species under different nutrient conditions.

Figure 1: Schematic of the procedure. The schematic diagram outlines the major steps involved in the method protocol for mapping the RSA. Please click here to view a larger version of this figure.

Figure 2: Display of hydroponic setup in magenta boxes to grow plantlets. (As described by Jain et al.¹⁰, with modifications). (A) Two rectangular pieces (4 cm x 8 cm) with notches of polycarbonate wedges for assembling the setup. (B) The assembly of polycarbonate wedges into each other through notch turning into an X shape to support the mesh surface. (C) A 250 µm polypropylene mesh sheet (6 cm x 6 cm). (D) Top view of the assembly of polycarbonate wedges in the magenta box. (E) Assembly of the polypropylene mesh on the polycarbonate wedges in a magenta box filled with media. (F) A display of Arabidopsis plantlets germinated on the mesh. Please click here to view a larger version of this figure.

Figure 3: Demonstration of typical RSA modulation under varying nutrient conditions (Pi deficient [0 mM] and sufficient [1.25 mM]) using this phenotyping method. Arabidopsis (Col-0) seedlings are grown hydroponically in 0.5x MS media for 5 days, and thereafter subjected to Pi deficient and sufficient supplies (0 and 1.25 mM, respectively) and grown for 7 days, as shown in Figure 2. (A) Individual plantlets are pulled out from the polypropylene mesh (500 µm) and spread on the agar Petri plates with the help of a round art brush and water. Data of RSA traits are presented for (B) primary root (PR) length, total root length (TRL), branching zone (BZ), (C) average (Av.) first order lateral root length (1° LR length), average (Av.) second order lateral root length (2° LR length), (D) number of 1° LR and 2° LR, and (E) 1° LR and 2° LR density. Values are means ± SE; n = 21. This figure has been modified from Shukla et al.¹. Please click here to view a larger version of this figure.

Figure 4: Demonstration of Medicago sativa (Alfalfa) root system as an example to show the applicability of this method to other plants besides Arabidopsis. (A) Side view of the magenta box showing the growth of Alfalfa plantlets. (B) The top view of the magenta box shows the shoot's emergence on the polypropylene mesh (pore size of 1,000 µm). (C) The typical root system architecture (RSA) of Alfalfa modulation under varying nutrient conditions (Pi deficient [0 mM], excess Pi [20 mM], and sufficient or control [1.25 mM]) using this RSA phenotyping method. Alfalfa seedlings are grown hydroponically in 0.5x MS media for 5 days, subjected to Pi deficient, sufficient, and excess supplies (0, 1.25, and 20 mM, respectively), and grown for 7 days. Individual plantlets are pulled out from the polypropylene mesh (1,000 µm) and spread on the agar Petri plates, as shown in C, with the help of an art brush and water. Please click here to view a larger version of this figure.

Supplementary Figure 1: Different excess Pi concentrations modulate root hair development. WT seedlings were grown hydroponically, as described in the protocol. (A) A 1-2 cm section from the tip of the primary root was chopped and mounted on a slide with 10% glycerol, and a 5 mm region from the tip was documented for the root hair number and length. Data are presented for (B) the root hair length and (C) the number of root hairs in a 5 mm region of the primary root tip. Values are means ± SE; n = 10 (B and C). Bars with different alpha letters differ significantly (p ≤ 0.05) according to Student's paired t-test. Please click here to download this File.

Discussion

This work demonstrated mapping RSA utilizing simple laboratory equipment. Using this method, phenotypic alterations are recorded at the refined level. The benefit of this strategy is that the shoot portion never comes in contact with the media, so the phenotype of the plantlets is original. This method involves setting up a hydroponic system to grow plantlets as described in the protocol. Then, each plantlet is taken out intact and placed on an agar-filled Petri plate. The root system is then allowed to spread manually using an art brush, and photographs are taken to be analyzed with ImageJ software^1,10,11,12.

Seed germination requires seed surface sterilization to remove bacteria, fungi, and viruses. Alcohol - 70% ethanol - is used to sterilize seed surfaces. To disinfect seeds without destroying them, the treatment time of alcohol sterilization needs to be carefully followed. Seed germination decreases when alcohol sterilization is overdone. Treatment timings vary with different plant species (e.g., for Arabidopsis, the time limit is 3 min^{1,10,11,12,20} only, while for Medicago sativa, it is 5 min¹⁹. In order to facilitate smooth picking of the RSA for analysis, it is important to limit the number of seeds per mesh or magenta box, to prevent entanglement of the root system among itself^1,10,11,12. This can be achieved by using a smaller number of seeds per mesh. For example, using four seeds per mesh can help to reduce the risk of entanglement while allowing for robust growth and development of the root systems. It is important to note that the optimal number of seeds per mesh depends on the specific plant species and the goals of the RSA analysis. For example, if an experiment requires tissue for further downstream processing requirements like RNA isolation, in this case, bulk sowing is recommended (100 seeds per mesh in the case of Arabidopsis)^1,10,11,12. Picking plantlets from the mesh is a delicate process that requires utmost care and attention^1,10,11. It is important to approach this task slowly, gently, and carefully to avoid damaging the delicate plantlets. To pick plantlets from the mesh, it is recommended to use fine tweezers or forceps to grasp the plantlets gently but firmly. The plantlets should be carefully lifted out of the mesh to avoid disturbing the root systems or causing any damage to the plantlets. It is essential to be patient and take the time to carefully remove each plantlet from the mesh to ensure that they are not damaged during the process. It is a gradual process that should be carried out slowly, gently, and carefully to ensure the success of the experiment. In order to accurately measure the RSA of plantlets, it is crucial to mark a scale on the Petri plate to allow for precise measurement^1,10,11. This can be done by using a permanent marker to draw a line on the Petri plate at a known distance, such as 1 cm or 2 cm. The scale should be placed along the edge of the Petri plate in a visible location. Using a brush, it is also essential to take utmost care when spreading the RSA. The primary root should be carefully positioned in the center of the Petri plate, and the lateral roots should be spread out on both sides of the primary root. The root system should be partially submerged in water to facilitate spreading. To measure each new Petri plate, one must set the scale in the ImageJ software each time.

Few modifications can be employed to improve the efficiency, noninvasiveness, and effectiveness of RSA analysis¹. One such improvement is to alter the pore size of the polypropylene mesh used to hold the plantlets. The pore size of the mesh can be adjusted to suit the specific needs of the plant species being studied and to optimize the growth and development of the root systems^1,10,11,12. For example, a bigger mesh pore size (500 µm) facilitates the smooth picking of entire seedlings without cutting the hypocotyl, which was earlier practiced^10,11,12. Further, a larger pore size may be more suitable for larger plant species RSA, while a smaller pore size may be more appropriate for smaller plant species. Another modification that can be made is to wrap the polypropylene mesh in aluminum foil to prevent it from bending. This can help maintain the shape and integrity of the mesh, making it suitable to serve as a flat floor matrix. In addition to these modifications, other troubleshooting techniques can be employed to address any issues that may arise during RSA analysis. For example, if the plantlets are not growing or developing as expected, it may be necessary to adjust the environmental conditions, such as the temperature, humidity, and light levels. If the root systems are entangled, reducing the number of seeds per mesh may be necessary, as mentioned above.

One of the main advantages of RSA analysis is that it allows for studying plant root systems without the need for agar. Agar is commonly used as a solidifying agent in plant tissue culture and seed germination experiments. However, using agar can introduce elemental contamination that can potentially generate artifacts and affect the accuracy of the results¹⁰. By excluding the requirement for agar, RSA analysis eliminates the risk of agar-derived elemental contamination and the potential for artifacts. This makes RSA analysis a more reliable and accurate method for studying plant root systems^1,3,10,11,12. For example, the effects of Pi deprivation on lateral root density have been the subject of a number of contradictory reports. It has been reported that the LR density increases when Pi is low^6,8. In contrast, a drop in lateral root density has also been found in Pi deficient conditions^3,13,16. These discrepancies may be attributable to the agar-based growth media system, in which workers utilize different brands of agar to jellify media with varying degrees of Pi contamination¹⁰. Again, experiments seeking to illustrate the effect of Zn deficiency on RSA may not be conducted adequately utilizing the agar-based Petri-plate method, because the agar-based gelling medium also includes Zn contamination¹¹. Therefore, performing RSA analysis may not be appropriate to investigate nutrient deficiency using the agar-based gelling medium. Second, plantlets grown on agar-based gelling media do not develop as quickly as those cultivated hydroponically. Third, because the RSA is typically embedded in an agar medium, numerous root features are not correctly displayed. Fourth, removing the RSA from the medium often causes substantial damage to the RSA and renders it a destructive sampling technique.

The previous hydroponic technique, as published by Jain et al.¹⁰, used a polypropylene mesh size of 250 µm, which had narrower pores that did not enable pulling out the intact RSA. As a result, in that particular case, we had to cut the plantlets from the hypocotyl region to separate the RSA, turning it into a destructive sampling method^10,11,12. The existing method is improvised to turn it non-destructive by using a polypropylene mesh with a larger pore size (500 µm) that allows pulling out whole Arabidopsis plantlets intact without inflicting any damage to the RSA¹. It is worthwhile to note that we can always adjust the pore size of the polypropylene mesh, depending on the plantlet's type. For instance, Figure 4 illustrates how a similar approach may be used to map the RSA of different plants, such as Medicago sativa (Alfalfa). We have opted for the polypropylene pore size of 1 mm to accommodate the Medicago root system.

One disadvantage of this system might be the development of root hairs, which often do not flourish under hydroponic systems compared to agar plate systems. The primary cause is the easy availability of nutrients in liquid media rather than in solid media. Even though we observed the development of root hairs (not robust) using the same system and obtained the results (Supplementary Figure 1). The availability of Pi strongly affects root hair development^10,11. Herein, root hair length increased initially till 2.5 mM, and then declined.

Altogether, the key advantages of the system are: (1) it is a simple and precise method that does not require any sophisticated high-end equipment; (2) the method allows rapid growth of the plantlets due to its hydroponic nature; (3) it is a non-destructive method; (4) manual spreading of the RSA allows the proper display of each trait, revealing the hidden RSA, offering full control to the user; and (5) the method employs a freely available imaging software (i.e., ImageJ), which is also simple to use.

Materials

Name	Company	Catalog Number	Comments
Arabidospsis thaliana (Col 0)	Lehle Seeds	WT-02	Columbia (Col-0**, no markers)*
Art brushes	Amazon or any other vendor		Water color round brush size no. 14 (8 mm), 16 (9.5 mm), 18 (12 mm), and 20 (14.2 mm)
Automated Microscope with digital camera	Leica Microsystems		LAS version 4.12.0, Leica Microsystems
Imaging Software	ImageJ		ImageJ V  1.8.0
Magenta box GA-7	Fisher Scientific	50-255-176
Medicago sativa	Johnny's Seeds
Petri-plate (150 mm x 15 mm)	USA Scientific	8609-0215	150 mm x 15 mm PS Petri Dish (https://www.usascientific.com)
Photo camera	Cannon or Nikon		Any high mega pixel (atleast 12 mega pixel per inch) camera on macro mode
Plant-Agar	Sigma-Aldrich	A3301	Agargel  Suitable for plant tissue culture
Polycarbonate Sheets	Amazon		1 mm  thick
Polypropylene Mesh	Amazon		Pore size 250 µm, 500 µm and 1000 µm
Scanner	Epson		Epson Perfection V700 Photo (Scan at 600 dpi)