Plant Accessible Tissue Clearing Solvent System (PATCSOS) for 3-D Imaging of Whole Plants

Tissue clearing is a technique to make the inner structure of opaque tissue visible to achieve 3-dimensional (3-D) tissue imaging by unifying the refractive indexes of most of the cell components. Tissue clearing is widely used in animal tissue imaging, where whole body 3-D imaging has been realized. However, it has not been widely used in plant research. Most plant tissue clearing protocols have their disadvantages, including low efficiency, not being fluorescence-friendly and poor transparency on tissues with a high degree of lignification. In this work, we developed a new plant tissue clearing method for whole plant imaging, named Plant Accessible Tissue Clearing Solvent System (PATCSOS), which was based on the Polyethylene Glycol-associated Solvent System (PEGASOS). The PATCSOS method realized extensive transparency of plant tissues, including the flower, leaf, stem, root, and seed of Arabidopsis thaliana, with high efficiency. The PATCSOS method consists of four main steps: fixation, decolorization/delipidation, dehydration, and clearing. Subsequently a rapid and efficient clearing of mature plant tissue can be achieved. With PATCSOS, we can image Arabidopsis seedling in their entirety in 3-D using endogenous cellulose autofluorescence. What’s more, the PATCSOS method is compatible with fluorescence protein imaging and GUS staining, which greatly expands the applicability of this method. We also imaged intact Nicotiana benthamiana leaf and Zea mays embryos. Our results showed that the PATCSOS clearing method is an excellent tool to study plant development and cell biology.