Measuring Inorganic Orthophosphate in Oilseed Rape Using Reflectoquant Technology

Abstract

Reflectoquant analytical technology was assessed for measuring inorganic orthophosphate (Pi‐mainly H2PO4 − but also HPO4 2 −) in oilseed rape plants (Brassica napus). The Reflectoquant system consists of a hand‐held reflectometer (RQflex2) combined with phosphate sensitive test‐strips, which use molybdate‐blue chemistry. Reflectoquant slightly under‐estimated Pi concentrations in standard potassium phosphate solutions, and had a linear measurement range between 5–100 mg PO4 L− 1 (0.05–1.05 mM Pi). Variability in phosphate test‐strip performance was large with five test‐strips per sample being required for 95% confidence in the mean value. Potentially interfering ions and molecules at concentrations commonly found in plant sap—chloride, nitrate, citrate, ATP, fructose‐6‐phosphate, and NADP—had no significant effect on the Pi assays. The Reflectoquant method gave similar results to a typical laboratory assay for Pi in rape leaf blades. In practice, plant sap or tissue water samples would need to be diluted up to 40 times to bring Pi into the linear range of the Reflectoquant system and this also ensures minimal interference from other cell constituents. Five methods of extracting Pi from oilseed rape leaves were compared in the laboratory‐fresh, freeze‐thaw, freeze‐dry, microwave‐dry and oven‐dry. Tissue water was pressed from leaves in the first two methods, whilst Pi in the (milled) material from the three drying methods was extracted by shaking with 2% acetic acid. Tissue water and acid extracts were analyzed for Pi by the molybdate‐blue method using either a laboratory spectrometer or Reflectoquant technology. Extraction method, but not analytical method, had a significant effect on Pi concentrations which increased in the order oven‐dry > microwave‐dry > freeze‐thaw = freeze‐dry > fresh. The freeze‐thaw method coupled with Reflectoquant technology was deemed most suitable for the on‐farm measurement of Pi in oilseed rape leaves.