Stem Morphometry and Raman Analysis

Stem samples (1-cm-long) were collected from the middle region of the stem (10 cm above the cotyledon scar) from ten individual control and ten individual stressed plants. For morphometric analyses samples were fixed in EtOH (70% w/v) for a minimum of three days at 4°C before being dehydrated through a graded EtOH/Histoclear series and embedded in Paraplast Plus. Stem transversal sections (10 μm) were obtained with a Leica RM65 microtome equipped with an S65 metal blade. Sections were mounted on glass slides and stained with Toluidine Blue-O (TBO) prior to observation with an Olympus BH-2 light microscope. For Raman microspectroscopy, samples were embedded in PEG (polyethylene glycol) 1,500, as previously described (Gierlinger, 2014 (link)). Transversal sections (30 μm) were obtained with a Leica RM2065 microtome for Raman focusing. PEG was removed by multiple water baths and sections were air dried prior to mounting in bidistilled water with a coverslip sealed by nail polish to avoid desiccation. Standard glass slides (superfrost™) and glass coverslips were used.