Cuticle Analysis of Plant Seeds

Toluidine blue (TBO) staining is known to examine the cuticular properties of plant^{85 (link)}. Experiments were performed as described earlier^{86 ,87 (link)} using seeds^{88 (link)}. TBO solution was prepared with 10 mM sodium citrate, pH 4.4; 0.05w/v toluidine blue and 0.4 v/v tween 20. Ten seeds each of wild type and rga1 were immersed in the solution in microcentrifuge tubes and incubated at room temperature for not more than 3 min. They were gently rinsed with water with a pipette for 3 min to remove unbound stain. They were treated with 80% ethanol and incubated at 37 ºC for 2 h and absorbance readings were taken at 626 and 430 nm using Agilent Cary 60 spectrophotometer.