A Microscopy Toolkit for Root Studies in B. distachyon.

The study of plant organogenesis presents significant challenges due to the small number of cells involved in its initial stages. Observing the initial cell divisions becomes increasingly challenging when transitioning from Arabidopsis thaliana (Arabidopsis) to other species. Lateral root (LR) initiation is an essential process for enhancing a plant's ability to access water and nutrients. In most cases, LR formation starts in the pericycle of the parent root, giving rise to a new meristem. Understanding the mechanisms coordinating LR development is important for improving plant resilience to biotic and abiotic stresses. The prediction of the precise timing and location of LR initiation along the root axis remains challenging, even in Arabidopsis. This is magnified when attempting to observe LR development in crops. Brachypodium distachyon (Brachypodium) has emerged as a versatile model for cereal crops. However, studying Brachypodium LR development requires a revision of protocols and methodologies to be applied from seedling growth to root imaging. Here, we present protocols for seed preparation, in vitro growth, and tissue clearing for Brachypodium. Whereas ClearSee appeared to be unsuitable for rendering Brachypodium root tissues transparent after a reasonable incubation period, our modified DEEP-Clear method resulted in improved efficiency in tissue clearing and is compatible with major fluorescent proteins and dyes. Finally, we introduce a simple and straightforward approach to locally synchronize LR development. These tools and methodologies are crucial for advancing our knowledge of plant root system architecture, transitioning from a model plant to agronomically important species.