Abstract: Highlights•MyROOT 2.0 is an image analysis tool for high-throughput root length measurement.•MyROOT 2.0 reduces user intervention to a minimum.•Presents previously unreported experiments to evaluate several constituting modules of MyROOT 2.0.•Using Arabidopsis thaliana seedlings grown on agar plates as a case study.•Obtaining measurements four times more accurate than its competitors.
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