Trace Metal Detection by X-ray Microanalysis in Vascular Cells of Roots and Leaves from Field-Grown Sugar Maple Seedlings

Abstract

The low survival of sugar maple (Acer saccharum) seedlings growing in unglaciated acidic forest soils (pH<4.0) in north-central Pennsylvania may be due in part to aluminum (Al) or manganese (Mn) phytotoxicity. Although foliar analysis has shown high concentrations (>2,000 ppm) of Mn, the location of potentially toxic elements in leaves or absorptive fine roots has not been studied in field-grown seedlings. This study of vascular (xylem and phloem) cells was conducted to determine by energy-dispersive X-ray microanalysis (EDX) whether Mn and/or Al could be detected in fine root or leaf midvein vascular cells. A secondary objective was to determine if vascular cells prepared by chemical fixation (CF) for conventional TEM or by freeze-substitution (FS) for EDX showed similar ultrastructural characteristics.

Four sugar maple seedlings from an unglaciated field site (Potter County PA) were excavated, and roots were washed carefully. Two mm-long segments of fine roots and leaf midveins from each seedling were immersed in 3% glutaraldehyde for 6 hours, transferred to 0.1 M phosphate buffer, and transported to the Delaware Research Lab where tissues were post-fixed in OsO₄ (in buffer), dehydrated in ethanol, and embedded in PolyBed-Araldite epoxy resin (CF method).