4.9. Suberin Detection/Staining in the Roots Using Fluorol Yellow 088

In order to detect the suberin lamellae in the endodermis, periderm and other ectopic suberin in other cell walls (if present), whole roots from 21-day-old plants were cleared with 1% (w/v) NaOH overnight as previously described by Peterson and co-workers [56,57]. This method successfully removed cell wall polysaccharides and cleared the roots. The whole roots were then stained with lipophilic fluorochrome, fluorol yellow 088, for 1 h and viewed under an epifluorescence microscope using an ultraviolet filter set (excitation filter BP 365, dichroic mirror FT 395, barrier filter LP 397; Zeiss, Oberkochen, Germany). The aliphatic component of suberin in root endodermis (younger root zones with a primary growth) and periderms (mature, basal zones with a secondary growth) can be detected by yellow fluorescence under ultraviolet light as described by Brundrett et al. [58]. For comparison between genotypes, photographs were taken at 40 mm from the root tip to detect the suberin lamellae in the endodermis, and very base of the root to detect peridermal suberin. Since the root lengths of all genotypes were the same, the staining results were comparable. Three to four whole root systems from each genotype were used for suberin staining.