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

Summary

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.

Abstract

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing ¹⁵N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine ¹⁵N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Introduction

Although often overlooked compared to their aboveground counterparts, roots play a critical role in plant nutrient acquisition. Given the substantial carbon cost of root construction and maintenance, plants have evolved mechanisms to develop roots only where foraging is worth the investment. Root systems can thus efficiently and dynamically mine resource patches by proliferating in hotspots, upregulating rates of uptake, and rapidly translocating nutrients to the phloem for further transport¹. Plasticity responses can vary widely among plant species or genotypes^2,3 and depending on the ....

Protocol

1. Preparation of the Front and Back Panels, and Spacers

1. Prepare the front and back panels.
   1. Cut two pieces of clear 0.635 cm thick acrylic to 40.5 cm wide by 61 cm long per box or purchase pre-cut pieces (see Table of Materials).
   2. Using a drill bit designed for acrylic, drill holes 0.635 cm in diameter 1.3 cm from the side edges at 2.5, 19, 38, and 53.3 cm from the top. Drill holes 1.3 cm from the bottom edge at 2.5, 20.3, and 38 cm from the left side (Figure 1).
      NOTE: It is most efficient to use a drill press for a stack of six to ten sheets at a time, but a hand drill can also be ....

Results

Roots grew preferentially against the back of the box, as anticipated. Total traced root length on the back of the box ranged from 400 to 1,956 cm, as compared to 93-758 cm on the front of the box. Pairwise Pearson correlation coefficients were calculated between scanned root length and traced root length on the front of the box, back of the box, and the sum of front and back was used to determine whether tracing accurately reflected total root length (n = 23, as the plant in one box died.......

Discussion

The rhizoboxes described in this protocol can be used to answer varied questions in root and rhizosphere science, and have found diverse uses elsewhere^{10,20,21,22,23,24,25}. Other researchers have captured time-lapse images of rhizoboxes^21,.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.27 cm diameter PVC pipe	JM Eagle	530048	305 cm per box, cut into lengths as specified in the protocol
PVC side elbows	Lasco	315498	2 per box
PVC 90-degree elbows	Charlotte	PVC 02300 0600	4 per box
PVC T joints	Charlotte	PVC 02402 0600	4 per box
Extruded acrylic panes	TAP Plastics	N/A	2 per box, 0.64 cm thick x 40.5 cm wide x 61 cm long
HDPE spacers (sides)	TAP Plastics	N/A	2 per box, 0.64 cm thick x 2.5 cm wide x 57 cm long
HDPE spacers (bottom)	TAP Plastics	N/A	1 per box, 0.64 cm thick x 2.5 cm wide x 40.5 cm long
HDPE spacers (patch)	TAP Plastics	N/A	2 per box, 0.64 cm thick x 3.8 cm wide x 28 cm long
Polyester batting	Fairfield	#A-X90	2.5 cm x 40.5 cm strip per box
20-thread screws	N/A	N/A	3.2 cm long, 0.64 cm diameter
Washers	N/A	N/A	0.64 cm internal diameter
Hex nuts	N/A	N/A	sized to fit the screws
Light deprivation fabric	Americover, Inc.	Bold 8WB26.5	1 piece 95 cm wide and 69 cm long per box
Sand	Quikrete	No. 1113
Field soil	N/A	N/A
Transparencies for tracing	FXN	FXNT1319100S	One per side of the box to be traced