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.
