Endogenous Auxin Quantification by HPLC

Endogenous auxin extraction and quantification from tip cuttings of plants with 30 and 60 days after sowing were performed according to Kim et al. (2006), with modifications as described by De Almeida et al. (2015). Briefly, chromatography was carried out on a Shimadzu C18 reverse-phase column (250 mm × 4.6 mm), with corresponding guard column, in a Shimadzu SPD-20A HPLC equipment using a gradient system of three mobile phases: Solvent A: 10% methanol, 0.3% acetic acid; Solvent B: 90% methanol, 0.3% acetic acid; Solvent C: 100% acetonitrile. All solutions were previously filtered through 0.45 μm Millipore^® membranes and degassed. Flow rate was 1.0 ml min^-1 and detection was done with a Shimadzu RF-10A XL fluorescence detector (Emission at 360 nm, Excitation at 282 nm). To quantify IAA, 20 μl of each sample was injected, and an external standard curve was generated using IAA (Acros Organics, Geel, Belgium). The identification of IAA content from samples of E. globulus was based on retention time and co-chromatography with authentic IAA standard. The contents of IAA in samples were expressed as nmol of IAA per gram of extracted fresh weight.