Background

Silica deposition is central to grasses. Grasses deposit up to 10% of their dry weight as silica. The major sites of silica deposition in plants are root endodermal cells, abaxial epidermal cells of inflorescence bracts and silica cells in leaves (Kumar et al., 2017b). Almost the entire volume of silica cells is filled with solid, amorphous silica. Silica deposition in silica cells is physiologically controlled and takes place around the middle length of young leaves (Kumar et al., 2017a), which we named leaf-2 (Figure 1). Silica cell silicification is also a fast process completed within hours (Kumar and Elbaum, 2017), hence in the same leaf there are areas of high and low silicification intensity depending upon which area of leaf we are examining. To study the deposition process, we need a way to quantify the silica cell silicification level in different parts of the same leaf. In situ charring or spodogram preparation of plant material is an easy and cheap way to study silica deposition in plants. The plant material is burnt at temperatures typically above 500 °C for 3 h to overnight that oxidises all of the organic material. The ash remaining after the charring process contains silica and other minerals. The non-silicate minerals from the ash are dissolved by 1 N HCl and the remaining insoluble substance is silica. The in situ charring method can also be used to quantify the silica cell silicification level (Kumar et al., 2017a). The leaf piece is kept in between two glass slides to keep the leaf specimen flat (Figure 2), and then the specimen is burnt, ash washed with HCl (Figure 3) and subsequently with double distilled water to remove the mineral salts. The slide is then taken to a light microscope to count the number of silica cells silicified per unit length. Figure 4 shows a spodogram prepared from the middle of a young leaf. This part was analyzed to quantify silica deposition levels. Our method can be extended to quantify the silica cell silicification level in any leaf piece, but best results are obtained with young and still silicifying leaves.