Design of a comprehensive microfluidic and microscopic toolbox for the ultra-wide spatio-temporal study of plant protoplasts development and physiology

Plant protoplasts are basic plant cells units in which the pecto-cellulosic cell wall has been removed, but the plasma membrane is intact. One of the main features of plant cells is their strong plasticity, and their propensity to regenerate an organism from a single cell. Methods and differentiation protocols used in plant physiology and biology usually involve macroscopic vessels and containers that make difficult, for example, to follow the fate of the same protoplast all along its full development cycle, but also to perform continuous studies of the influence of various gradients in this context. These limits have hampered the precise study of regeneration processes.Herein, we present the design of a comprehensive, physiologically relevant, easy-to-use and low-cost microfluidic and microscopic setup for the monitoring of Physcomitrella patens (P. patens) growth and development on a long-term basis. The experimental solution we developed is made of two parts (i) a microfluidic chip composed of a single layer of about a hundred flow-through microfluidic traps for the immobilization of protoplasts, and (ii) a low-cost, light-controlled, custom-made microscope allowing the continuous recording of the moss development in physiological conditions. We validated the experimental setup with three proofs of concepts: (i) the kinetic monitoring of first division steps and cell wall regeneration, (ii) the influence of the photoperiod on growth of the protonemata, and (iii) finally the induction of leafy buds using a phytohormone, cytokinin.We developed the design of a comprehensive, physiologically relevant, easy-to-use and low-cost experimental setup for the study of P. patens development in a microfluidic environment. This setup allows imaging of P. patens development at high resolution and over long time periods.