Spore Preparation and Protoplast Isolation to Study Gravity Perception and Response in Ceratopteris richardii

Early studies revealed a highly predictable pattern of gravity-directed growth and development in Ceratopteris richardii spores. This makes the spore a valuable model system for the study of how a single-cell senses and responds to the force of gravity. Gravity regulates both the direction and magnitude of a trans-cell calcium current in germinating spores, and the orientation of this current predicts the polarization of spore development. In order to make Ceratopteris richardii cells easier to transform and image during this developmental process, a procedure for isolating protoplasts from Ceratopteris richardii gametophytes has been developed and optimized. These protoplasts follow the same developmental pattern as Ceratopteris richardii spores and can be used to monitor the molecular and developmental processes during single-cell polarization. Here, we describe this optimized procedure, along with protocols for sterilizing the spores, sowing them in solid or liquid growth media, and evaluating germination and polarization.