Saponin extraction and thin layer chromatography analysis of ′CWS5095′

Saponins were extracted from the hypocotyls of mature dry seeds from each accession used in this study. Saponin extractions were kept for 24 h in ten-fold volumes of (v/w) of 80% (v/v) methanol (aqueous) at room temperature. The extracts were directly used for TLC analysis or stored at 4°C. The TLC was performed according to Krishnamurthy et al. [66], with slight modifications. Briefly, 5 μL of saponin extracts from each sample were directly loaded on pre-silica gel plates (TLC silica gel 60 F₂₅₄, Merck, Darmstadt, Germany) with a micropipette and air dried. The dried plates were developed in a developing chamber, which was saturated with the lower phase of a chloroform:methanol:water (65:35:10, v/v/v) mix for 50 min. The plates were dried at 90°C for 5 min and then developed by freshly prepared H₂SO₄ for 8 min in a closed chamber. The saponins were visualized by heating at 100–110°C for 10 min.