Preparation of Barley Root Samples for SEM Analysis

Samples for the SEM analysis were prepared according to the procedure that had been developed for barley roots^{61 (link)}. In brief, barley roots from seven-day-old seedlings that had been grown in a hydroponic culture in a 1/16 Hoagland medium (control plants) and in a 5 nm AuNPs solution (50 µg/ml concentration) were cut into 1 cm segments and fixed in 3% glutaraldehyde in a 0.1 M sodium phosphate buffer, pH 7.2 (24 h at RT). Next, the samples were washed three times for 15 min each with a phosphate buffer followed by post-fixation in 2% osmium tetraoxide for 2 h at RT. The material was subsequently washed three times in the same buffer and dehydrated in a graded ethanol/water series of 50%, 60%, 70%, 80%, 90%, 95% and 100% (10 min each step). Next, the samples were critical-point dried using carbon dioxide in a Pelco CPD2 apparatus (Ted Pella Inc., Redding, CA, USA) and then mounted on aluminium stubs with double-sided carbon tape and sputter coated with a thin film of gold in a Pelco SC-6 sputter coater (Ted Pella Inc., Redding, CA, USA). The samples were imaged using a Hitachi SU8010 field emission scanning electron microscope FE-SEM (Hitachi High-Technologies Corporation, Tokyo, Japan).