Advancing root architecture analysis: 3D neutron imaging of plants grown in slab rhizotrons

Abstract Background and aims Root system architecture (RSA) shapes biogeochemical concentration patterns in the rhizosphere. Root-soil studies are often conducted on plants cultivated in rectangular rhizotrons, including when using 2D hydrochemical analysis methods. However, roots naturally expand in three dimensions, with the rhizosphere extending accordingly. Three-dimensional neutron imaging can enhance interpretation of such studies, yet imaging flat, slab-shaped rhizotrons is technically challenging. This study presents a methodological comparison between conventional neutron tomography (NT) and neutron computed laminography (NCL) to assess whether NT under high-flux conditions can achieve image quality sufficient for 3D root segmentation, comparable to NCL, without requiring tilting of the rotation axis. Methods NT and NCL were applied to maize plants grown in rectangular rhizotrons. Imaging artifacts and their impact on root segmentation were assessed for two plants representing low and high soil moisture conditions suitable for neutron imaging. Results Both methods produced 3D tomograms of comparable quality across the tested moisture range, enabling effective segmentation of primary and seminal roots. Lateral root detection was more challenging and depended on soil moisture. NCL captured a greater number of horizontally oriented lateral roots while NT was more effective in resolving vertically oriented roots. Conclusions NCL is not required to resolve 3D RSA of maize plants in flat rhizotrons. Under high-flux neutron beam conditions, NT is preferable as it simplifies sample handling, reduces plant stress, avoids soil water redistribution and enables direct integration with timeseries of 2D chemical and neutron radiographic imaging.