Staining localization of ferric reduction on roots

Abstract

Tomato plants (cv. ‘Rutgers') were grown in nutrient solutions without and with FeEDDHA or at low phosphorus levels to evaluate the formation of prussian blue (Fe₄[Fe(CN)₆]3‐nH₂O), a stain used in determining the site of ferric reduction on roots. The effect of Fe source, Fe concentration, solution pH, and time were evaluated. Prussian blue was compared with nitro‐BT, and agar systems with Fe‐chelates and ferrous color reagents. Ferric iron was supplied as a chelate, Fe‐EDDHA or Fe‐HEDTA; or as a salt, FeNH₄(SO₄)₂ or Fe(NO₃)₃. The staining procedure producing the most distinct and localized stain on the roots was the combination of FeNH₄(SO₄)₂ and ferricyanide both at 100 μM for approximately 1 hour. The initial pH was 3.0 to maintain ferric soluble. Greater salt and ferricyanide concentrations, lower solution pH's or lengthening staining times resulted in stains on most of the root with less differences in the extent of staining between Fe sufficient and Fe deficient plants. When roots were stained with nitro‐BT, only the young root hairs on lateral roots or near apical root tips were stained. Hairs on ‐P roots did not become stained with prussian blue while ‐Fe root hairs were extensively stained. These results indicate that root hairs on Fe‐stressed tomatoes reduce ferric to ferrous and that only root hairs grown under Fe deficiency were capable of high rates of ferric reduction.