A comprehensive rhizobox pipeline for analyzing pepper root system architecture under well-watered and water deficit conditions

Background Drought stress can significantly impede plant productivity, adversely impacting crop yields. The root system is an important plant organ contributing to drought resistance mechanisms. Therefore, assessing root systems under drought stress conditions can provide insights to identify root traits associated with enhanced drought resistance. When seeking dense and high-quality root data, root phenotyping can be complex, costly, and time-consuming. The objectives of this study were to establish a method to grow chili pepper plants in a soil-based rhizobox container under water deficit conditions and compare two methods for collecting two-dimensional root trait data from their roots. Method We grew two chile peppers (Capsicum annuum) accessions in soil-based rhizobox containers to analyze the responses of root architecture traits under well-watered and water-deficit conditions during the vegetative stage. The root traits were phenotyped using two different methods. The first method involved non-destructive in-box imaging of roots in situ through acrylic glass while the plant grew. The second method involved scanning destructively harvested and washed roots—the gold standard for root measurements. For the first method, we developed a pipeline for rhizobox studies to demonstrate the response of root system architecture to water deficit over time and assessed the quality of non-destructive in-box imaging methods as compared to scans of destructively harvested and washed roots. We used a relatively large rhizobox (53.34 cm in width x 78.73 cm in height) into which we established and maintained well-watered and water deficit conditions based on the field capacity and permanent wilting point of the soil (Bodner et al. 2017; Cassel & Nielsen 1986).Our in-box root imaging pipeline captures high-resolution root images with an affordable camera that can achieve a maximum resolution of 9152 x 6944 pixels, as well as high-quality root segmentation using a robust graphical user interface-based software called RootPainter (Smith et al. 2022). Results Root growth decreased under water deficit compared to well-watered conditions. There were strong positive relationships between total root length using the washed scanned method and the in-box imaging method. The same was observed for root perimeter and most of the total root length distinct root diameter classes, but not for average root diameter. Some of these relationships weakened under water deficit conditions. In addition, we also found a strong relationship between root biomass and total root length using both phenotyping methods. Conclusion Overall, we developed a rhizobox pipeline for phenotyping the root system architecture of chile pepper plants under both well-watered and water-deficit conditions. We showed that measurements taken via non-destructive in-box imaging strongly predict those taken directly on washed scanned roots, with the added benefit of allowing repeated measurements over time.