Fluorescence Microscopy Analysis of Transgenic Plants

The analysis of transgenic calli, cells or plants were performed as previously described [62 (link)]. Fluorescence microscopy was carried out using the tdTomato filter set: 554-nm excitation and 581-nm emission wavelength with an Olympus stereo microscope model SZX12 (Olympus America, Center Valley, PA, USA) (for callus imaging) and an Olympus BX51 epifluorescence (for cell imaging). Confocal microscopy images were produced using a confocal Leica TCS SP8 microscope. The samples were excited with a 543 nm HeNe laser and fluorescence emission was collected from 590 to 610 nm for pporRFP. Fluorescence intensity was measured using a spectrofluorometry according to methods described by Millwood [69 (link)] with a Fluorolog^®-3 system (Jobin-Yvon and Glen Spectra, Edison, NJ, USA). Triplicate spectra/peak emission absorbance was adjusted by removing the background signal from corresponding controls used for each sample. For each sample, the youngest fully expanded leaf from T₀ lines was chosen to measure the intensity of fluorescence in non-transgenic control and putatively transgenic plants.