Chara braunii is an emerging model species for studying plant evolution and development. Various members of the Chara genus have been used for pioneering studies of electrophysiology, cytoplasmic streaming, and cell biology. However, many studies have been limited by challenges, such as overgrowth with epiphytes or non-continuous growth throughout the year. Here, we present protocols for the cultivation, germination, and in vivo staining of C. braunii NIES-1604 to overcome these limitations. We constructed the custom-made cultivation chamber equipped with the illumination unit that allows for the precise simulation of natural conditions and continuous cultivation of C. braunii NIES-1604 throughout the year. By determining the optimal stratification period for C. braunii NIES-1604 oospores, we achieved a germination rate of 26%. Further, we present fluorescence-based in vivo staining protocols for routine in vivo staining of cellular structures including the plasma membrane, cell wall, and nuclei. Altogether, our affordable method of controlled cultivation of C. braunii NIES-1604 and other procedures serve as a valuable resource for establishing Chara cultures in laboratory settings and advancing its use as a model organism. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Construction of a cultivation chamber for C. braunii NIES-1604 growth Basic Protocol 2: Vegetative propagation of C. braunii NIES-1604 Basic Protocol 3: Germination of C. braunii NIES-1604 oospores Basic Protocol 4: In vivo staining of C. braunii NIES-1604 with fluorescent dyes.

Department of Experimental Plant Biology Faculty of Science Charles University Prague Czech Republic

Institute of Experimental Botany Czech Academy of Sciences Prague Czech Republic

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