Imaging the Cytoskeleton in Living Plant Roots

For the past two decades, genetically encoded fluorescent proteins have emerged as the most popular method to image the plant cytoskeleton. Because fluorescent protein technology involves handling living plant cells, it is important to implement protocols that enable these delicate plant specimens to maintain optimal growth for the entire duration of the imaging experiment. To this end, we rely on a system that consists of a large coverslip coated with nutrient-supplemented agar. This agar-coverslip system is planted with surface-sterilized Arabidopsis thaliana seeds expressing cytoskeletal fluorescent protein reporters. The agar-coverslip system with planted seeds is then maintained in an environmentally controlled growth chamber. The entire setup is transferred onto the stage of a confocal microscope for imaging when roots of germinated seedlings reach a desired length. For plants with larger roots such as Medicago truncatula, the polymerized nutrient-supplemented agar is gently lifted or cut and used to secure pre-germinated seeds on the coverslip prior to root imaging. The agar-coverslip system we use for imaging the cytoskeleton in living roots along with general methods for expressing green fluorescent protein (GFP)-based cytoskeletal reporters in hairy roots of Medicago truncatula is described here.