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

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.