Linsmaier skoog media

Histochemical staining of GUS activity was performed as described (Kim et al. 2006 ). Prior to GUS staining, etiolated Arabidopsis (Col-0) seedlings were grown vertically for 5 days in darkness on agar plates. Light-grown seedlings were grown vertically on agar plates for 14 days at 22 °C under a 16 h-light/8 h-dark cycle. The agar plates contained 0.8 % (w/v) Gellan Gum (Sigma-Aldrich), 0.5× Linsmaier & Skoog media (Caisson Laboratories) and 1.5 % (w/v) sucrose. Shoot inflorescences were obtained from 6-week-old plants grown on soil (Farfard Super Fine Germinating Mix, American Clay Works & Supply Company, USA) under a 16 h-light/8 h-dark cycle at 25 °C. To determine the effect of indole-3-acetic-acid (IAA) treatment on GUS expression in roots, 6-d-old seedlings grown vertically on 0.5× LS agar plates were incubated in 0.5× LS liquid media containing either 0 M (negative control) or 1 µM IAA for 24 h prior to GUS staining. Root samples were mounted on glass slides in 50 % glycerol, and images were acquired on an Olympus BX-51 upright microscope. All other images were taken on an Olympus SZX-12 stereomicroscope, with samples submerged in 70 % ethanol in a petri dish.

PDI8_pro:GUS seedlings were grown vertically on 1/2× LS agar plates [0.8 % (w/v) Gellan Gum (Sigma-Aldrich, St. Louis, MO), 1/2x Linsmaier & Skoog media (Caisson Laboratories, Smithfield, UT) and 1.5 % (w/v) sucrose] for 7 or 14 days at 22 °C under a 16 h-light/8 h-dark cycle. Shoot inflorescences were obtained from 6-week-old PDI8_pro:GUS plants grown on Farfard Super-Fine Germinating Mix (Sun Gro Horticulture, Agawam, MA) under a 16 h-light/8 h-dark cycle at 25 °C. GUS staining was performed as described [17 ]. Briefly, the tissue samples were fixed in 90 % ice-cold acetone for 20 min at 25 °C, then washed with staining buffer (50 mM sodium phosphate buffer (pH 7.0), 0.2 % Triton X-100, 2 mM potassium ferrocyanide, and 2 mM potassium ferricyanide) three times on ice, then submerged in staining buffer containing 1 mM 5-bromo-4-chloro-3-indoxyl-β-D-glucuronide cyclohexylammonium salt (X-gluc). The tissues were vacuum infiltrated briefly, then incubated O/N at 37 °C. After staining, the samples were incubated in 70 % ethanol to extract soluble pigments, repeating with fresh 70 % ethanol as necessary. Images of GUS staining in roots and stomata were acquired on an Olympus BX-51 upright microscope, with the samples mounted on glass slides in 50 % glycerol. All other images were taken on an Olympus SZX-12 stereomicroscope, with samples submerged in 70 % ethanol in a petri dish.