Name: a simple protocol for mapping the plant root system architecture traits
Uploaded: 2023-02-10T13:10:00
Description: Watch this Scientific Journal Video about A Simple Protocol for Mapping the Plant Root System Architecture Traits at JoVE.com

We acknowledge the U.S. Department of Agriculture (Grant 58-6406-1-017) for supporting this research. We also acknowledge the WKU Biotechnology Centre, Western Kentucky University, Bowling Green, KY, USA, and the Director, CSIR Central Institute of Medicinal and Aromatic Plants, Lucknow, India, for providing the instrument facilities and support (CSIR CIMAP manuscript communication no. CIMAP/PUB/2022/103).&#160;SS acknowledges the financial support from Saint Joseph&#39;s University, Philadelphia, USA.

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
<ol>
	<li>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 c...

<ol>
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B., Sahi, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+deficiency+and+excess+of+zinc+on+morphophysiological+traits+and+spatiotemporal+regulation+of+zinc-responsive+genes+reveal+incidence+of+cross+talk+between+micro-+and+macronutrients.">Effects of deficiency and excess of zinc on morphophysiological traits and spatiotemporal regulation of zinc-responsive genes reveal incidence of cross talk between micro- and macronutrients.</a> Environmental Science and Technology. 47 (10), 5327-5335 (2013).</li><li>Jain, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Fe-responsive+genes+in+bioreduction+and+transport+of+ionic+gold+to+roots+of+Arabidopsis+thaliana+during+synthesis+of+gold+nanoparticles.">Role of Fe-responsive genes in bioreduction and transport of ionic gold to roots of Arabidopsis thaliana during synthesis of gold nanoparticles.</a> Plant Physiology and Biochemistry. 84, 189-196 (2014).</li><li>Williamson, L. C., Ribrioux, S. P., Fitter, A. H., Leyser, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphate+availability+regulates+root+system+architecture+in+Arabidopsis.">Phosphate availability regulates root system architecture in Arabidopsis.</a> Plant Physiology. 126 (2), 875-882 (2001).</li><li>Yang, T. J. W., Lin, W. D., Schmidt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profiling+of+the+Arabidopsis+iron+deficiency+response+reveals+conserved+transition+metal+homeostasis+networks.">Transcriptional profiling of the Arabidopsis iron deficiency response reveals conserved transition metal homeostasis networks.</a> Plant Physiology. 152 (4), 2130 (2010).</li><li>Kobae, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+transporter+of+Arabidopsis+thaliana+AtMTP1+is+localized+to+vacuolar+membranes+and+implicated+in+zinc+homeostasis.">Zinc transporter of Arabidopsis thaliana AtMTP1 is localized to vacuolar membranes and implicated in zinc homeostasis.</a> Plant Cell and Physiology. 45 (12), (2004).</li><li>Al-Ghazi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+responses+of+Arabidopsis+root+architecture+to+phosphate+starvation:+evidence+for+the+involvement+of+auxin+signalling.">Temporal responses of Arabidopsis root architecture to phosphate starvation: evidence for the involvement of auxin signalling.</a> Plant, Cell and Environment. 26 (7), 1053-1066 (2003).</li><li>S, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> National Institutes of Health. , 1997-2007 (1997).</li><li>Dubrovsky, J. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+of+Arabidopsis+under+gold+exposure+to+identify+putative+genes+involved+in+the+synthesis+of+gold+nanoparticles+(AuNPs).Genomics+Data.">Microarray analysis of Arabidopsis under gold exposure to identify putative genes involved in the synthesis of gold nanoparticles (AuNPs).Genomics Data.</a> 3, 100-102 (2015).</li></ol>

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 u...

The root system architecture (RSA), which is underground, is a vital organ for plant growth and productivity1,2,3. 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 nutrients1,2,3,4. The RSA exhibits a high degree of developmental plasticity, which means that the environment is always used to make decisions about development2,5. 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 environments2,5.
The RSA senses varying nutrient concentrations and renders phenotypic alterations4,6,7,8,9,10,11,12. Studies suggest that root morphology/RSA is highly plastic compared to shoot morphology1,3. RSA trait mapping is highly effective in recording the effect of changing the surrounding soil environment1,11,12.
In general, discrepancies in the effect of various nutrient deficiencies on the root phenotype have been reported in many earlier studies3,11,13,14,15. 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 condition6,8. In contrast, a decrease in LR density under Pi deficient conditions has also been reported by other authors3,13,16. 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 medium11,14,15. Numerous nutrients are frequently present in significant amounts in the agar media, including P, Zn, Fe, and many more11,14,15. 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 wedges1,10,11.
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&#39;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 roots1,10,11,12. 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&#39;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Arabidospsis thaliana (Col 0)</td>
			<td>Lehle Seeds</td>
			<td>WT-02</td>
			<td>Columbia (Col-0**, no markers)*</td>
		</tr>
		<tr>
			<td>Art brushes</td>
			<td>Amazon or any other vendor</td>
			<td></td>
			<td>Water color round brush size no. 14 (8 mm), 16 (9.5 mm), 18 (12 mm), and 20 (14.2 mm)</td>
		</tr>
		<tr>
			<td>Automated Microscope with digital camera</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>LAS version 4.12.0, Leica Microsystems</td>
		</tr>
		<tr>
			<td>Imaging Software</td>
			<td>ImageJ</td>
			<td></td>
			<td>ImageJ V 
			&#160;1.8.0</td>
		</tr>
		<tr>
			<td>Magenta box GA-7</td>
			<td>Fisher Scientific&#160;</td>
			<td>50-255-176</td>
			<td></td>
		</tr>
		<tr>
			<td>Medicago sativa</td>
			<td>Johnny&#39;s Seeds</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri-plate (150 mm x 15 mm)</td>
			<td>USA Scientific</td>
			<td>8609-0215</td>
			<td>150 mm x 15 mm PS Petri Dish (https://www.usascientific.com)</td>
		</tr>
		<tr>
			<td>Photo camera</td>
			<td>Cannon or Nikon</td>
			<td></td>
			<td>Any high mega pixel (atleast 12 mega pixel per inch) camera on macro mode</td>
		</tr>
		<tr>
			<td>Plant-Agar</td>
			<td>Sigma-Aldrich</td>
			<td>A3301</td>
			<td>Agargel&#160; Suitable for plant tissue culture</td>
		</tr>
		<tr>
			<td>Polycarbonate Sheets</td>
			<td>Amazon</td>
			<td></td>
			<td>1 mm&#160; thick</td>
		</tr>
		<tr>
			<td>Polypropylene Mesh</td>
			<td>Amazon</td>
			<td></td>
			<td>Pore size 250 &#181;m, 500 &#181;m and 1000 &#181;m</td>
		</tr>
		<tr>
			<td>Scanner</td>
			<td>Epson</td>
			<td></td>
			<td>Epson Perfection V700 Photo (Scan at 600 dpi)</td>
		</tr>
	</tbody>
</table>

a simple protocol for mapping the plant root system architecture traits

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 &#176;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.

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&#39;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<sup cl...

Watch this Scientific Journal Video about A Simple Protocol for Mapping the Plant Root System Architecture Traits at JoVE.com

A Simple Protocol for Mapping the Plant Root System Architecture Traits

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.

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar ...

This is a non-invasive hydroponic method to measure the root system architecture using routine lab equipments. This protocol allows the complete visualization of the plant's entire root system by manually spreading it. One of the main advantages of the RSA analysis is that it allows for studying plant root system without the need of which can introduce unwanted elemental contamination.This method can record the direct environmental interaction including hormonal, nutrients, and climactic conditions with the RSA of plant systems. Begin the surface sterilization of Arabidopsis seeds by soaking a tiny scoop of approximately 100 seeds in distilled water at room temperature. After 30 minutes, briefly centrifuge the seeds at 500 G for five seconds using a tabletop centrifuge.Next, decant the water, and add 700 microliters of 70%ethanol before vortexing the tube for a few seconds. Centrifuge the seeds again. If required, repeat vortexing and centrifugation, ensuring that 70%ethanol treatment does not exceed three minutes.After three minutes, immediately rinse the seeds with sterile water. Next, treat the seeds using diluted commercial bleach containing a drop of TWEEN 20 for seven minutes. Mix the seeds with bleach solution by inverting the tubes rapidly eight to 12 times.After a brief centrifugation, decant the supernatant using a one milliliter pipette, and rinse the seeds at least five times with sterile water, following the same vortexing procedure. Leave the surface sterilized seeds in water, and incubate them for two to three days at four degrees Celsius for stratification. Autoclave the standard magenta box half filled with distilled water.Cut the autoclaved polycarbonate sheet into four by eight centimeter rectangles with a midpoint notched more than halfway through the rectangle so that two rectangles may slot together to form an X shape. Use this setup to hold the 250 micrometer pore size polypropylene mesh cut into six by six centimeter squares. In the laminar airflow cabinet, add sterile half MS basal media with vitamins plus 1.5%sucrose to each box to reach the bottom edge of the polypropylene mesh.Sow the surface sterilized seeds on the mesh hydroponically, and grow them for three days. After three days, transfer the seedlings onto a 500 micrometer pore size mesh, and allow them to grow for two days. Next, transfer the seedlings onto the control media and to the experimental media, and let the seeds grow for seven days.Add 10 to 20 milliliters of autoclaved filtered tap water to the Petri plate. Gently pull out the seedlings from the 500 micrometer mesh. With the help of a round art brush, spread plant-like roots in the water-filled plate and submerge them in water.Tilt the plate slightly to remove the water. Scan or photograph these Petri plates appropriately. Measure the root system architecture, or RSA traits, using freely available ImageJ software, then open the file to be analyzed.After setting the scale, 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. To measure the length of the known scale bar in pixels, click on analyze, followed by measure on the toolbar.Make a note of the pixel length. Open the set scale dialogue box by clicking the set scale tab in the analyze tab. Check pixel length in the distance in pixels field.Next, enter the scale bar value in the known distance field, and set the unit of length as millimeters. Lock the scale for this particular image by clicking on okay. Use the segmented line tool for a line selection that outlines the root length.After finishing the outlining, adjust the line selection by clicking and dragging the small black and white handles along the outline. Under the analyze tab of ImageJ, select the measure command and quantify the root length. Transfer the measured data to a spreadsheet by right clicking on the results window, selecting copy all from the popup menu, switching to the spreadsheet, and pasting the data.For RSA traits measurement and calculation, measure the primary root length between the hypocotyl junction to the root tips end. Then measure the first order lateral roots, or one degree LR length, and second order lateral roots, or two degree LR length. Once done, copy and paste all the measurements into the spreadsheet.Measure and record the branching zone of the primary root, or BZPR, that spans the first lateral root emerging point to the last lateral root emerging point. Similarly, record the number of lateral roots originating within the boundary of the BZPR. Next, measure the average length of the first and higher order lateral roots.Derive the average length of the primary lateral roots by dividing the total length of the primary lateral roots by the total number of primary lateral roots. Then to measure the average length of the second order lateral roots, calculate the average length of the secondary lateral roots by dividing the total length of the secondary lateral roots by the total number of secondary lateral roots. Measure the primary lateral root density by dividing the number of primary lateral roots by the length of the BZPR.Next, measure the branching zone of individual lateral roots and calculate the secondary lateral root density by dividing the number of secondary lateral roots by the length of the branching zones of second order lateral root lengths. Once done, measure the total root length, or TRL. This is the aggregate of primary roots and primary and secondary lateral root lengths.The hydroponic system used in this experiment worked well, reflecting the apparent contrasting phenotype under inorganic phosphate deficient and sufficient conditions. Various RSA traits were analyzed under contrasting inorganic phosphate regimes in hydroponic conditions. The inorganic phosphate deficient treatment invoked a root phenotype exhibiting a shorter, shallower, and less branched RSA, compared to the inorganic phosphate sufficient condition.Primary root length was significantly attenuated under the inorganic phosphate deficient condition. Significant and rapid gain in the primary root length in the presence of 1.25 millimolar inorganic phosphate or control media indicated the efficiency of the hydroponic system, and reflected the physiomorphological changes adequately. The branching zone was significantly reduced under the inorganic phosphate deficient condition.The average length of the primary lateral root was significantly reduced in the pi deficient condition. The average secondary lateral root length was similarly reduced due to the phosphate deficient condition, however, it was lower in quantity than the average primary lateral root length. The number of primary and secondary lateral roots heavily decreased under the phosphate deficient condition, compared to the control, 1.25 millimolar inorganic phosphate condition.The primary lateral root density was not changed under the phosphate deficient condition, relative to the control, 1.25 millimolar inorganic phosphate condition. The secondary lateral root density did not show any significant change, indicating the importance of lateral root density for gaining insight into the RSA plasticity. I gently removed seedlings from mesh using tweezers.I spread roots in a water filled plate using a round brush, all the primary roots, and spread it straight, spread lateral roots symmetrically, spread second order lateral roots linked to first order lateral roots. A similar method can be used to calculate the soot area of Arabidopsis plant tips. The Arabidopsis leaves can be spread on the other plate followed by image capture and analysis using freely available ImageJ software to determine the soot area.This protocol can be used to answer any questions about the environment's influence on root system plasticity.

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