Site-Directed Mutagenesis and GFP Imaging

Site-directed mutagenesis was performed using the plasmid pENTR^TM1A-CmPT2 constructed in our previous studies [13 (link)]. All mutated constructs were recombined into the C-terminus GFP fusion vector pMDC43 by the LR reaction (as described in Gateway^® Technology with Clonase^® II) [35 (link)]. Plasmid DNA was bombarded into onion (Allium cepa) epidermal cells using a helium-driven gene gun (PDS-1000; Bio-Rad, Hercules; CA, USA) according to the manufacturer’s instructions [36 (link)]. The onion epidermal cells were incubated on Murashige and Skoog (MS) solid medium plates in the dark for 16–20 h. GFP expression was monitored using confocal laser scanning microscopy at 488 nm (Zeiss, Oberkochen, Germany) [37 (link)].