Determination of copper concentration in rice by ICP-OES

This protocol outlines the steps for detecting the copper content in rice plants using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES).

1. Sample Collection and Preparation
   • Harvest the shoots and roots of rice plants (4-6 weeks), separately for both mock-inoculated and virus-infected samples.
   • Wash the harvested tissues three times with water
   • Rinse three times with washing buffer containing 10 mM Na2-EDTA and 5 mM CaSO4.
2. Drying and Grinding
   • Dry the washed samples at 70°C for 7-10 days until completely desiccated.
   • Grind the dried samples into a fine powder using a suitable grinder.
3. Digestion
   • Digest 100 mg of the powdered sample in 2 ml of 70% concentrated HNO₃ (Sigma-Aldrich, ACS reagent) at a temperature of up to 130°C for 2-3 hours until fully dissolved.
4. Dilution and Analysis
   • Dilute the digested sample with deionized water (ddH₂O) to achieve a final concentration of  approximately 5% HNO3.
   • Centrifuge the diluted sample at 4000 rpm for 15-20 minutes, take the  supernatant for the analysis.
   • Determine the copper concentration in the solution using an ICP-OES (Prodigy 7, Leeman, USA) based on the characteristic electromagnetic radiation emitted by each atom or ion. This analysis can be conducted at the Analytical Instrumentation Center of Peking University (PKUAIC, Beijing, China).
5. Controls and Replicates
   • Use diluted 5% HNO₃ as a blank control to account for background readings.
   • Each sample should have at least three to four biological replicates, with each biological replicate consisting of pooled tissues from at least 10 rice plants.
6. Quantification
   • The ICP-OES will provide the exact concentration of different elements. To obtain the copper content in different rice tissues, subtract the background element content of HNO₃ from the readings.

Note:  This protocol can be adapted to detect the concentration of other elements in various plant tissues.