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Background Root system architecture and especially its plasticity in acclimation to variable environments play a crucial role in the ability of plants to explore and acquire efficiently soil resources and ensure plant productivity. Non-destructive measurement methods are indispensable to quantify dynamic growth traits. For closing the phenotyping gap, we have developed an automated phenotyping platform, GrowScreen-Agar, for non-destructive characterization of root and shoot traits of plants grown in transparent agar medium. Results The phenotyping system is capable to phenotype root systems and correlate them to whole plant development of up to 280 Arabidopsis plants within 15 min. The potential of the platform has been demonstrated by quantifying phenotypic differences within 78 Arabidopsis accessions from the 1001 genomes project. The chosen concept ‘plant-to-sensor’ is based on transporting plants to the imaging position, which allows for flexible experimental size and design. As transporting causes mechanical vibrations of plants, we have validated that daily imaging, and consequently, moving plants has negligible influence on plant development. Plants are cultivated in square Petri dishes modified to allow the shoot to grow in the ambient air while the roots grow inside the Petri dish filled with agar. Because it is common practice in the scientific community to grow Arabidopsis plants completely enclosed in Petri dishes, we compared development of plants that had the shoot inside with that of plants that had the shoot outside the plate. Roots of plants grown completely inside the Petri dish grew 58% slower, produced a 1.8 times higher lateral root density and showed an etiolated shoot whereas plants whose shoot grew outside the plate formed a rosette. In addition, the setup with the shoot growing outside the plate offers the unique option to accurately measure both, leaf and root traits, non-destructively, and treat roots and shoots separately. Conclusions Because the GrowScreen-Agar system can be moved from one growth chamber to another, plants can be phenotyped under a wide range of environmental conditions including future climate scenarios. In combination with a measurement throughput enabling phenotyping a large set of mutants or accessions, the platform will contribute to the identification of key genes., Plant Methods, 16 (1), ISSN:1746-4811

Additional file 1.GrowScreen-Agar mechanical setup parts list and technical drawings, list of phenotyped germplasm, and description of modified Hoagland solution.