RootReader2D (RR2D, Clark et al., 2013) was used to analyse images. First, a ‘batch process’ was carried out which automatically ‘thresholds’, ‘skeletonises’ and ‘builds segments’ of all images in bulk. Then, the the root system of individual images was measured by placing a marker at the ase and tip of the primary root. From these markers, RR2D automatically calculates primary root length (PRL), lateral root length(LRL) of all laterals and lateral root number (LRN).