Quantitative Live Confocal Imaging in Aquilegia Floral Meristems

Background

Meristems are groups of pluripotent stem cells, typically located at the tips of shoots (Steeves and Sussex, 1989). Many fundamental features of meristems are shared across all vascular plants, e.g., the maintenance of a pool of undifferentiated cells, regulated cell proliferation and expansion, and control of post-embryonic organogenesis. However, there remains a great deal of unexplored variation in meristem structure and behavior across land plants. Exploring this diversity is hampered by the reliance on common developmental techniques, such as fixed tissue sectioning and imaging, that do not allow processes such as spatial and temporal patterns of cell division and expansion to be directly observed. In model systems such as Arabidopsis thaliana, genetic and molecular tools have been coupled with advancements in live imaging techniques to allow analyses of both cell behaviors and gene expression in real time. These tools have provided considerable progress in our understanding of meristem development (e.g., Prunet et al., 2016; Geng and Zhou, 2019; Shi et al., 2020; Caggiano et al., 2021; Silveira et al., 2022). However, these advancements are currently limited to a small number of model species, and there is a pressing need to develop quantitative live imaging techniques in non-model systems, and specifically, approaches that may be broadly practical across a range of plant taxa. Here, we present a detailed protocol for live imaging and analysis of floral meristems in Aquilegia coerulea, a member of the buttercup family (Ranunculaceae). This protocol provides a powerful tool to study the development of the meristem and initiation of floral organs, and should be easily adaptable to many plant lineages, including other emerging model systems. This protocol will allow researchers to explore questions outside the scope of common model systems.