Poplar Leaf Protoplast Isolation and Transformation

Protoplasts from 1-month-old poplar leaves were isolated following previously described methods [75 (link)]. Briefly, 1-month-old poplar leaves were cut into filaments and then placed into an enzyme solution configured with 2% cellulase and 0.5% pectinase and kept in at 28°C in darkness for 3 hours. The liquid was then subjected to centrifugation and 2 mM MES at pH 5.7, 154 mM NaCl, 125 mM CaCl2,5 mM KCl (W5) was added to dilute the poplar leaf protoplasts. Different combinations of plasmids (pRI101-GFP/ProGbFLS::LUC, ProGbFLS::LUC/35S::GbHY5, ProGbFLS::LUC/35S::GbMYB1, ProGbFLS::LUC/35S::GbHY5/35S::GbMYB1, 35S::nLUC/35S::GbMYB1-nLUC and 35S::GbMYB1-nLUC/35S::GbHY5-nLUC) were co-transformed into the protoplasts and cultured for 16 hours. LUC and REN activity was detected using a GLO-MAX20/20 Fluorescence Detector with a Dual Luciferase Reporter Assay Kit (Promega Corp., Madison, WI, USA).