Preparation of Intact Tissue for Microscopic Analysis of the Endosperm Cell Layer in Developing and Mature Arabidopsis Seeds

Keisuke Seta; Daiki Shinozaki; Kohki Yoshimoto

doi:10.3791/68217

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Method Article

Preparation of Intact Tissue for Microscopic Analysis of the Endosperm Cell Layer in Developing and Mature Arabidopsis Seeds

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DOI:

10.3791/68217

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May 16th, 2025

Keisuke Seta¹ , Daiki Shinozaki¹^,²^,³ , Kohki Yoshimoto¹

¹Department of Life Sciences, School of Agriculture, Meiji University, ²Organization for the Strategic Coordination of Research and Intellectual Properties, Meiji University, ³Plant Functional Biotechnology, Agro-Biotechnology Research Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo

Summary

This protocol describes the preparation of intact samples of the endosperm cell layer in Arabidopsis thaliana seeds. The method requires only common laboratory equipment, such as an injection needle and precision forceps, and enables high-resolution fluorescent live-cell imaging of endosperm cells in both developing and mature seeds.

Abstract

In Arabidopsis seeds, the endosperm, a single layer of living cells located between the embryo and the testa, plays a critical role in regulating seed maturation, dormancy, and germination. Microscopic analysis of intact endosperm cells is essential for understanding the physiological functions of the endosperm at cellular and molecular levels. However, sample preparation has been challenging due to the small size of Arabidopsis seeds and the location of the endosperm cell layer beneath the testa. This article details the preparation of intact endosperm cell layer samples suitable for microscopic observation and analysis in both developing and mature seeds. This method enables the observation of large areas and numerous intact endosperm cells without requiring fixation or sectioning. Additionally, the protocol utilizes only standard laboratory equipment, such as injection needles, precision forceps, and stereo microscopes. This approach successfully enables high-resolution live-cell imaging of fluorescent signals, such as green fluorescent protein (GFP), in intact endosperm cells. This method allows for the observation of intracellular localization and movement of various proteins, as well as the morphology of organelles, in the endosperm cells of different Arabidopsis mutants. This protocol contributes to the elucidation of novel endosperm functions and expands the potential for cellular and molecular studies of this essential tissue.

Introduction

Because plants are sessile organisms, seed germination is a crucial event that determines their fate. The decision to germinate is strictly regulated by both internal and environmental factors, such as primary seed dormancy levels, temperature, light intensity and wavelength, and nitrogen concentration¹^,²^,³^,⁴^,⁵^,⁶. Seeds have complex structures consisting of multiple tissue types⁷. In Arabidopsis dry seeds, the embryo, which develops into a ....