Leaves of V. faba, T. repens, C. annuum, N. tabacum and T. aestivum were freeze-dried for 36 hours in lyophilizer (MSA3.6P, Sartorius, Germany). The samples were then moved into a drying oven (Thermo, USA) and dried for 2 hours at 60 °C. After that, 10 mg of treated samples were weighed precisely by a high precision electronic balance (LL3000, Thermo, USA) at room temperature and transferred into 50 ml plastic tubes and covered. All organized samples were then used for L-DOPA and dopamine extraction and assay.
To analyze relative L-DOPA concentrations in plants phloem, 100 g of leaves of V. faba, T. repens, C. annuum, N. tabacum and T. aestivum were cut into 5 pieces and extracted in 1 ml EDTA solution (5 mM, pH = 7.0) for 24 hours (20 ± 1 °C). Extracted solutions were then filtered into a new tube; and 100 mg leaves of V. faba were extracted by grinding as a positive control47 48 (link)49 (link). Each treatment was replicated six times.
To analyze relative L-DOPA concentrations in plants phloem, 100 g of leaves of V. faba, T. repens, C. annuum, N. tabacum and T. aestivum were cut into 5 pieces and extracted in 1 ml EDTA solution (5 mM, pH = 7.0) for 24 hours (20 ± 1 °C). Extracted solutions were then filtered into a new tube; and 100 mg leaves of V. faba were extracted by grinding as a positive control47 48 (link)49 (link). Each treatment was replicated six times.