TITLE: An improved digestion and analysis procedure for silicon in plant tissue
AUTHORS: Noah Langenfeld, Bruce Bugbee

[DESCRIPTION]
Silicon (Si) in plant tissues reduces abiotic and biotic stress, but it is incorporated as silica (SiO2), which is difficult to solubilize for analysis. We modified an oven-induced tissue-digestion method to improve Si solubilization and validated the accuracy by quantifying the mass-balance recovery of Si from hydroponic solution and in plant tissues. Leaf, stem, and root tissues were dried, finely-ground, and digested in 12.5 molar sodium hydroxide at 95 °C. The solutions were then acidified with hydrochloric acid to achieve a pH of 2 for measurement of Si using the molybdate blue colorimetric method. Interference of phosphorus (P) in the analysis was minimized by increasing the addition of oxalic acid from 0.6 to 1.1 molar. We recovered 101 ± 13% of the Si in leaf, stem, and root tissues across 15 digestions. This Si recovery was fourteen-fold higher than the standard acid-extraction method and similar to a USDA-ARS alkaline-extraction method. Our procedure offers a low-cost, accurate method for extraction and analysis of Si in plant tissues.

[BEFORE_START]
Preheat the oven to 95 °C.


[STEPS]
SECTION: Sample preparation
4. Triple rinse a 50-mL polyethylene screw-cap centrifuge tube with 100 millimolar (mM) sodium hydroxide.

SECTION: Sample preparation
5. Triple rinse the 50-mL polyethylene screw-cap centrifuge tube with distilled water.
SECTION: Sample preparation
6. Dry the tube and cap with a clean paper towel.
SECTION: Sample preparation
7. Add about 100 mg of dry and ground plant tissue to the tube. Record the exact mass. Ensure all ground tissue is transferred to the bottom of the tube and not stuck on the side.

SECTION: First digestion
8. Add 5 drops of octyl-alcohol to the ground tissue in the bottom of the tube to reduce foaming.
SECTION: First digestion
9. Add 2 mL of 30 % (v/v) hydrogen peroxide to the bottom of the tube. Wash the inside of the tube free from the tissue sample with the hydrogen peroxide as it is added.

SECTION: First digestion
10. Tighten the screw cap and place the tube upright (standing inside a 250 mL glass beaker works well)  into a 95 °C oven for 30 min.

SECTION: Sample preparation
3. Preheat an oven to 95 °C.

SECTION: Second digestion
11. After 30 min, remove the tube from the oven using heat-safe gloves.

SECTION: Second digestion
12. Inside a fume hood, add 4 mL of12.5 Molarity (m) sodium hydroxide to the tube. Add the sodium hydroxide slowly to avoid excess foaming.

SECTION: Sample fixing
14. After 4 hours, remove the tube from the oven using heat-safe gloves.
SECTION: Second digestion
13. Gently vortex the tube, replace the cap, and return to the 95 °C oven for an additional 240 min.

SECTION: Sample fixing
15. Add 1 mL of 5 mM ammonium fluoride to the tube to facilitate the formation of monosilicic acid.

SECTION: Sample fixing
16. Add 9 mL of 6 Molarity (m) hydrochloric acid to neutralize the sample. Add the hydrochloric acid slowly to avoid foaming. The solution should turn clear after addition of the acid.

SECTION: Sample fixing
17. Add distilled water to the tube up to 50 mL.
SECTION: Sample analysis
18. Use deionized water to prepare a 1:25 dilution of the sample with a final volume of 10 mL . Place sample into a 10 mL glass vial or test tube.

SECTION: Sample drying
1. Dry fresh plant tissue at 80 °C for at least 2880 min. Water can remain in tissue below  80 °C, which increases dry mass, and volatile compounds can be driven off above  80 °C, which reduces dry mass.
SECTION: Sample grinding
2. Grind dry plant tissue in a mortar and pestle to a uniform, fine powder. Particle sizes should be less than about 0.1 mm in diameter (consistency of fine sand).
SECTION: Sample analysis
19. Add 6 drops of 6 Molarity (m) hydrochloric acid to the sample vial. Cap the vial and invert to mix.

SECTION: Sample analysis
20. Add 12 drops of 81 millimolar (mM) ammonium molybdate. Cap the vial and invert to mix. Wait 5 min .

SECTION: Sample analysis
21. Add 8 drops of 1.1 Molarity (m) oxalic acid. Cap the vial and invert to mix. Wait 2 min .

SECTION: Sample analysis
22. Add 100 mg of 90 Mass Percent sucrose and 10 Mass Percent isoascorbic acid. Cap the vial and invert to mix until solids have dissolved. Wait 5 min .

SECTION: Sample analysis
23. If using a LaMotte Smart3 colorimeter, select the Silica - Low Range method. Insert vial into colorimeter to obtain measurement of silica in the sample. If using a spectrophotometer, prepare a calibration curve from 0 to 4 ppm silica and analyze all samples at 650 and 815 nm.