Materials and Reagents

Glass slides
Paper towel
Aluminium foil
Plastic disposable droppers
Scalpel
Ceramic tile
Young Sorghum bicolor plants
Double distilled water
Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148 )
1 N Hydrochloric acid (HCl) (see Recipes)

Equipment

Scalpel
Forceps
Muffle furnace (Alfi Laboratory supplies)
Binocular microscope (Motic, model: SMZ168 Series )
Light microscope (Nikon Instruments, model: Eclipse 80i )

Procedure

Sampling
1. Young plants (up to one month old) where silica cell silicification level needs to be estimated are brought to the lab. The length of youngest leaf visible from outside is recorded.
  Note: We name this youngest, visibly emerging leaf as leaf-2 (Kumar et al., 2017a) (Figure 1).
2. Under a binocular microscope and using a scalpel and forceps, the older leaves are carefully removed from leaf-2. Leaf-2 is then cut into 10 equal segments, along the length of the leaf.
  
  Figure 1. Sketch showing a young sorghum plant. Leaf-2 is indicated with the arrow. Figure was adapted from Kumar et al. (2017a) with permission from the John Wiley and Sons publications. Original authors of the article and the publisher retain the copyright of this figure.
Spodogram preparation
1. Segments of leaf-2 are laid flat on a glass slide. Segments from the base of leaf-2 are always rolled; hence 1-2 drops of double distilled water are put on the glass slide and the segment is pressed against the drop. This way, the segment lies flat and sticks to the slide. A small piece of aluminium foil is folded 2-3 times and is kept near the two shorter sides of the slide (Figure 2). Another fresh slide is kept on top of the slide holding the sample in place. This step is repeated until all the segments are put on slides. All the slides are kept on a clean ceramic tile, put in a muffle furnace and burnt at 550 °C between 3 h to overnight. The furnace is turned off and allowed to cool down to room temperature which usually takes more than 12 h.
  Note: The aluminium foil in between the two slides makes sure that the two slides do not touch each other when the tissue sample is burnt. We do not go above 580 °C, as the glass slides may crack, distort, melt or stick together. We use the ceramic tile that is used in flooring in houses. The ceramic tile functions as a solid base where all the slides can be put together on one base. We do not write anything on the slides with a marker pen as burning erases all the marks. Rather, before putting the samples for burning, we note down the sample names and their orientation with respect to one of the sides of the tile. Burning for three hours is sufficient for quantitative estimation of silica cell silicification in leaves.
  
  Figure 2. Image showing a leaf-2 segment in between two glass slides for spodogram preparation
2. Slides with burnt leaf material are put on paper towel without disturbing them. Using a plastic dropper, the ash is very carefully and gently washed with about 1-1.5 ml of 1 N HCl from one of the sides of the slides (Figure 3). The slides are then washed with about 1-1.5 ml of double distilled water to remove the HCl solution and solutes. The two slides are then separated and dried at room temperature.
  Note: Sample names can be written on dried slides.
  
  Figure 3. A burnt leaf-2 segment being washed with 1 N HCl
Data acquisition
1. After the slides dried, the bottom slides (without cover slip) are taken to the transmitted light microscope and visually scanned under low magnification (4x) to look for areas having silicified cells. The area on the slide with the longest, unbroken stretch of silica cells is identified and chosen for data acquisition. Ten sequential images are acquired following the rows of silica cells in one direction.
  Note: All the images for data analysis must be acquired using a common high magnification objective; otherwise the data will not be uniform. Images must be taken in such a way that the rows of silica cells are parallel to a side of the screen.
2. We use 20x objective for all our images and the dimensions of all our images are 690 x 506 µm.

Data analysis

Numbers of silicified silica cells are counted in each row of an image and only the row with the highest number of silicified silica cells is considered for data analysis. In the beginning of silica cell silicification process, only the boundaries of silica cells look silicified and in the spodogram only the cell boundary is lightly visible. Those cells are also counted as silicified cells. Sometimes, in the more mature segment of leaf-2, many silica cells join together and form of polylobate bodies. In this case, three lobes are regarded as one silica cell. We show our method with an example (Figure 4) below. In this spodogram image, there are three rows of silica cells, the top row (row-1) with 8 silica cells, the row in the middle (row-2) with 7 silica cells, whereas the row in the bottom (row-3) has 13 silica cells. Hence, the silicification score of this image is 13. The silicification score from each of the ten sequential images of a sample was averaged. The average number becomes the silica cells silicification status of that particular segment. Silicification scores of different segments are calculated and compared.

Figure 4. A spodogram image under 20x magnification. Silica cells are encircled with dotted ovals. Scale bar = 100 µm.

Recipes

1 N HCl
The strength of commercially available HCl is 12.1 N. We added 8.26 ml of commercially available HCl to 91.74 ml of double distilled water to make the final concentration of HCl to 1 N

Acknowledgments

SK is thankful to the Planning and Budgeting Committee, Council of Higher Education, Israel for a post-doctoral fellowship. The research was supported by Israel Science Foundation grant 534/14. The authors declare that any conflict of interest or competing interests do not exist.

References

Kumar, S. and Elbaum, R. (2017). Interplay between silica deposition and viability during the life span of sorghum silica cells. New Phytol 217(3):1137-1145.
Kumar, S., Milstein, Y., Brami, Y., Elbaum, M. and Elbaum, R. (2017a). Mechanism of silica deposition in sorghum silica cells. New Phytol 213(2): 791-798.
Kumar, S., Soukup, M. and Elbaum, R. (2017b). Silicification in grasses: Variation between different cell types. Front Plant Sci 8: 438.