X-ray computed tomography (CT) allows us to visualize root system architecture (RSA) beneath the soil, non-destructively and in a three-dimensional (3-D) form. However, CT scanning, reconstruction processes, and root isolation from X-ray CT volumes, take considerable time. For genetic analyses, such as quantitative trait locus mapping, which require a large population size, a high-throughput RSA visualization method is required. We have developed a high-throughput process flow for the 3-D visualization of rice (Oryza sativa) RSA (consisting of radicle and crown roots), using X-ray CT. The process flow includes use of a uniform particle size, calcined clay to reduce the possibility of visualizing non-root segments, use of a higher tube voltage and current in the X-ray CT scanning to increase root-to-soil contrast, and use of a 3-D median filter and edge detection algorithm to isolate root segments. Using high-performance computing technology, this analysis flow requires only 10?min (33?s, if a rough image is acceptable) for CT scanning and reconstruction, and 2?min for image processing, to visualize rice RSA. This reduced time allowed us to conduct the genetic analysis associated with 3-D RSA phenotyping. In 2-week-old seedlings, 85% and 100% of radicle and crown roots were detected, when 16?cm and 20?cm diameter pots were used, respectively. The X-ray dose per scan was estimated at??0.09?Gy, which did not impede rice growth. Using the developed process flow, we were able to follow daily RSA development, i.e., 4-D RSA development, of an upland rice variety, over 3?weeks. We developed a high-throughput process flow for 3-D rice RSA visualization by X-ray CT. The X-ray dose assay on plant growth has shown that this methodology could be applicable for 4-D RSA phenotyping. We named the RSA visualization method ‘RSAvis3D’ and are confident that it represents a potentially efficient application for 3-D RSA phenotyping of various plant species.
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