An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

This method can help answer key questions in the field of Root and Rhizosphere Science, such as how roots respond to localized patches of nutrients. The main advantages of this technique are that it allows repeated, non-invasive measurements of a single root system in soil, but it doesn't require specialized equipment and it's relatively inexpensive. Gather the parts for the Rhizobox for assembly.

These include front and back panels made of clear acrylic about 40 centimeters by 61 centimeters. At this point, there are side and bottom spacers on top of the back panel. The spacers are aligned with previously drilled holes in the panels.

Cut a length of polyester padding to go along the bottom of the Rhizobox. Lay the padding on the back panel directly above the bottom spacer to help prevent leaking. Put the top panel in position on the spacers to hold the padding in place.

Now get hardware to assemble the Rhizobox. Use a screw, two washers, and hex nut for each of the drilled holes. Place a washer on the front panel and tighten the screw through it to the back panel.

Another washer and the hex nut. To avoid soil loss, ensure that all of the screws are very tight. Also, create two spacers to form the treatment and control patches.

Make them with high density polyethylene sheets and secure a screw at the top to allow only partial insertion. For light deprivation and heat reduction, prepare a protective case that can enclose the box. Finally, build a frame to encourage roots to grow against the back panel.

This PVC frame holds the box at an angle of approximately 55 degrees. Have ready a homogenized mixture of field soil and sand. Begin with a large bag containing the substrate mixture for each Rhizobox.

Label two small ziptop bags per box for the treatment and control patches. Use a scale and weigh 30 grams of the soil sand substrate. Transfer the substrate into the control bag.

In the same way, add 30 grams of substrate to the treatment bag. Then weigh one gram of nitrogen 15 labeled nitrogen source and add it to the treatment bag. Mix the substrate and nitrogen source thoroughly.

Work with one Rhizobox and it's patch spacers. Insert the two spacers into the Rhizobox until the screw prevents further motion. Mark the depth of the bottom edge of the spacers on the side of the Rhizobox.

Then remove the spacers for the next steps. Using a funnel, fill the Rhizobox from the large bag of substrate to the marked depth. Move the funnel back and forth slowly and evenly to fill the Rhizobox uniformly.

When the substrate is at the marked depth, put the spacers into the box. They should be five centimeters from the left and right sides of the box. Continue filling the box until the substrate is about five centimeters from the top of the box.

Next, arrange for slow irrigation of the substrate in the box with 150 milliliters of water with drip emitters or by hand. Let it rest several hours or overnight so that the water diffuses and the area around each spacer is thoroughly wet. At this point, remove the spacers to leave an empty cavity for the patches.

Now, tape transparency film to the outside of the box. Use the transparency to label one patch as treatment and the other as control. Then use the funnel and the prepared control substrate to fill the control patch.

This is the labeled control patch after it has been filled. Move on to fill the treatment patch with the prepared treatment substrate in the same manner. When done, use a permanent marker to trace the boundary of each patch on the transparency film.

Next, fill the Rhizobox evenly with the remaining substrate from the large bag. Finish by tracing the top of the substrate on the transparency. Once the box is ready, transplant a germinated seed.

First, use a narrow spatula to dig a hole at the center of the Rhizobox. The hole should be 2.5 centimeters deep. Place the germinated seed inside ensuring the radical is oriented directly downward.

Trace the location of the seed on the transparency attached to the box. Cover the seed, then water it with up to 50 milliliters of deionized water. Next, enclose the box in its case to prevent light from interacting with the roots during growth.

Take the box to the frame prepared for it. Place the Rhizobox on the frame to encourage growth toward the back frame. Retrieve the box after every three to four days of plant growth.

With the case removed, trace the plants visible roots by using a different color pen each time. As in this example, be consistent and systematic for this step. Return the covered box to its stand and ensuring it is in its original orientation.

Here, the total root lengths of the traced roots on the back of 24 Rhizoboxes are plotted against root lengths measured using a scanner and computer software. A similar plot for traced root patterns at the front of the box demonstrates the roots grew preferentially against the back of the box. The consistent slopes in this plot of the logarithm of the total root length as a function of days of growth suggests the growth rates were similar between the different boxes.

Here are data for root length density for one maize genotype. The density for the control patch is smaller than the density for the nitrogen treated patch. The experiment compared root length density for six separate maize genotypes.

In all cases, the root length density was greater in the nitrogen 15 labeled treatment patch versus the control patch. This protocol can be combined with other methods such as sphygmography or florescence in situ hybridization in order to visualize the spacial distribution of enzyme activity or microbes.