Modified Pseudo-Schiff Propidium Iodide for Staining the Shoot Apical Meristem in Arabidopsis

Background

Most of the above-ground plant organs are derived from the shoot apical meristem (SAM) (Steeves and Sussex, 1989; Dinneny and Benfey, 2008). As a key tissue involved in plant growth and development, it plays a role in maintaining the ability of stem cells to divide continuously (Fletcher, 2018; Hu et al., 2018), controlling the initiation of organs development (Spinelli et al., 2011; Balkunde et al., 2017), accurately regulating the spatio-temporal specific gene expression (Mayer et al., 1998; Long et al., 1996; Williams, 2021), and integrating the signals from various cell types (Zhao et al., 2010; Azizi et al., 2015; Chung et al., 2019). Thus, exploring the mechanism of complex regulation patterns in the meristem is inseparable from observation of meristem at the cellular level.

Unlike the inflorescence meristem, the SAM of Arabidopsis is tightly wrapped by the surrounding leaf primordia and leaves, which makes it difficult to observe its cellular structure in its deeper part (Moreno et al., 2006). Scientists usually observe the cell structure and morphogenesis of SAM by semithin sections or tissue clearing, but only one cell layer or the surface layer of the SAM is observed (Koi and Kato, 2007; Zhang et al., 2021; Wang et al., 2022). To visualize three-dimensional structure of plant tissues, scientists prefer to use fluorescent dyes, such as propidium iodide, to stain tissues for optical section for laser scanning confocal microscopy (Haseloff, 2003; Truernit et al., 2008; Prunet et al., 2016; Shi et al., 2016).

Previously, we observed the cell structure of SAM and inflorescence meristem in Arabidopsis by modifying the pseudo-Schiff propidium iodide staining method (Li et al., 2022). Compared with traditional methods, this method presents several advantages. First, it allows the observation of the different cell layers in the whole SAM, which is a continuous and complete structure. Second, the cellular structure of the initial leaf primordium is observable except for the SAM. Third, unlike the traditional method that takes seven days, this method is simple in terms of procedure and time saving, taking only three days to complete. It can be foreseen that this method will likely be applicable to the observation of other tissues or SAM of other plant species. The operation details are described in the following section.