ClearDepth Method for Evaluations of Root Depth in Soil-Filled Pots

Despite its significant relevance to drought adaptation, optimization of nutrient acquisition, and carbon sequestration in soil, genetic factors determining root depth remain poorly explored, mostly due to the limitations of the methods currently available to estimate it. Although several such methods have been developed for crops, their applicability to large-scale studies and those involving smaller, more fragile root systems is severely limited. To address this, we have developed ClearDepth, a simple, non-destructive, low-cost method. In ClearDepth, the root system develops naturally inside the soil in clear pots. As it expands, secondary roots reach the transparent walls of the pot ("wall roots"), becoming visible. The shallowness of each wall root is then measured (wall root shallowness, WRS), and the depth of the root system is expressed as the average of all single WRS measurements. We demonstrated the suitability of ClearDepth for root depth studies using <i>Arabidopsis thaliana</i> and <i>Oryza sativa</i> (rice), two species with contrasting root system architecture (RSA) and root size. The robustness and sensitivity of the WRS trait allow us not only to reproducibly discriminate between shallow and deep root systems but also to detect smaller yet significant differences in depth determined by the influence of environmental factors, such as light. Here, we present a comprehensive protocol for utilizing this method. Key features • ClearDepth measures the depth of a minimum number of secondary roots, set by the user, to estimate the depth of the root system. • The method captures differences of root depth at a spatio-developmental stage rather than at one specific time point after planting. • ClearDepth captures differences in root depth independently of differences in total root biomass.