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

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Publikace nespadá pod Ústav výpočetní techniky, ale pod Středoevropský technologický institut. Oficiální stránka publikace je na webu muni.cz.
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ATTULURI Venkata Pardha S. SANCHEZ LOPEZ Juan Francisco MAIER Lukáš PARUCH Kamil ROBERT BOISIVON Helene

Rok publikování 2022
Druh Článek v odborném periodiku
Časopis / Zdroj Plant Reproduction
Fakulta / Pracoviště MU

Středoevropský technologický institut

Citace
www open access published article
Doi http://dx.doi.org/10.1007/s00497-022-00453-4
Klíčová slova Arabidopsis seed clearing; Microscopy imaging; Fluorescence preservation; ClearSee alpha; FAST9; Iohexol
Přiložené soubory
Popis 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.
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