4.7. Analysis of GUS Reporter Lines

Different developmental stages of at least four independent transgenic pSlTPS2::GUS and pSlTPS8::GUS lines were investigated for tissue-specific localization patterns using histochemical staining according to Jefferson et al. [73]. In brief, plant samples were submerged for 20 min in ice-cold 90% acetone. The acetone was then removed by replacing it with 2 mL of buffer A (50 mM Na₂HPO₄, 10 mM K₄[Fe(CN)₆], 10 mM K₃[Fe(CN)₆], 0.2% Triton X-100, pH 7.0) and vacuum infiltrated with GUS staining solution (buffer A plus 2 mM (f.c.) µL X-Gluc (5-bromo-4-chloro-3-indolyl-β-glucuronic acid) solution in DMSO) three times. Subsequently, the samples were incubated at 37 °C overnight. On the next day, the GUS staining buffer was carefully removed and replaced with 70% ethanol to bleach the green chlorophyll. Several changes of ethanol were performed until the tissue turned white. A stereo microscope imaging system (Discovery V20; Zeiss) was used to collect the images. The same settings were used to image a particular tissue from different plants. The entire experiment was carried out twice with independent batches of plants.