Polyamine Extraction and Quantification

Fresh material (200 mg) from selected transgenic HRs grown on solid MS20 medium was transferred to 2-mL reaction tubes (Sarstedt, Nümbrecht, Germany) and powdered by means of metal balls in liquid nitrogen in a Mixer Mill MM 400 (Retsch, Haan, Germany) at a frequency of 30/s for 1 min. The powder was extracted in 1 mL perchloric acid (5%, v/v) containing 2 nmol 1,7-diaminoheptane as an internal standard under freeze-thaw cycles according to Minocha et al. [87 (link)]. Subsequently, the pH was raised to pH 9–10 with 10 M NaOH (approx. 50 μL per 1000 μL extract). Precolumn derivatization and chromatography of polyamines were carried out according to Kaltenegger et al. [88 (link)] using a fluorescence detector (Figure A1).