Comparing the efficiency of six clearing methods in developing seeds of Arabidopsis thaliana.

key messageClearSee alpha and FAST9 were optimized for imaging Arabidopsis seeds up to the torpedo stages. The methods preserve the fluorescence of reporter proteins and seed shape, allowing phenotyping embryos in intact seeds. Tissue clearing methods eliminate the need for sectioning, thereby helping better understand the 3D organization of tissues and organs. In the past fifteen years, clearing methods have been developed to preserve endogenous fluorescent protein tags. Some of these methods (ClearSee, TDE, PEA-Clarity, etc.) were adapted to clear various plant species, with the focus on roots, leaves, shoot apical meristems, and floral parts. However, these methods have not been used in developing seeds beyond the early globular stage. Tissue clearing is problematic in post-globular seeds due to various apoplastic barriers and secondary metabolites. In this study, we compared six methods for their efficiency in clearing Arabidopsis thaliana seeds at post-globular embryonic stages. Three methods (TDE, ClearSee, and ClearSee alpha) have already been reported in plants, whereas the others (fsDISCO, FAST9, and CHAPS clear) are used in this context for the first time. These methods were assessed for seed morphological changes, clearing capacity, removal of tannins, and spectral properties. We tested each method in seeds from globular to mature stages. The pros and cons of each method are listed herein. ClearSee alpha appears to be the method of choice as it preserves seed morphology and prevents tannin oxidation. However, FAST9 with 60% iohexol as a mounting medium is faster, clears better, and appears suitable for embryonic shape imaging. Our results may guide plant researchers to choose a suitable method for imaging fluorescent protein-labeled embryos in intact Arabidopsis seeds.