Filter set 38

Observation and recording of the fluorescence emission by GFP were carried out using an epifluorescence microscope (Zeiss Axioscope Lab A.1 equipped with Filter Set 09, BP 450–490 nm and Filter Set 38, BP 470/540 nm; Zeiss, Oberkochen, Germany). The light source was provided by a 470 nm LED lamp. Images were acquired with a Canon Rebel T3 camera using the software EOS Utility (Canon Inc., Tokyo, Japan).

Evaluation of GFP expression over time was achieved through the use of whole plant vacuum-agroinfiltration assays (as described by Chialva et al., 2018 (link)) along with gene transfer experiments in ‘Thompson Seedless’ somatic embryos. The GFP reporter gene expression was evaluated by epifluorescence microscopy in leaves at 1, 3, 5, 8, 13, 16, 19, and 23 days post-infiltration (dpi) (Figure 1A) and in embryogenic callus between 4 and 47 dpi (Figure 1B). Samples were observed using a Zeiss Axioscope Lab A.1 epifluorescence microscope equipped with Filter Set 09 (BP 450–490 nm) and Filter Set 38 (BP 470/540 nm; Zeiss, Oberkochen, Germany). The light source was a 470-nm LED lamp. Images were acquired with a Canon Rebel T3 camera using EOS Utility software (Canon Inc., Tokyo, Japan). The green channel was quantified via eight images per point using ImageJ software (National Institutes of Health, United States). Data was subjected to one-way ANOVA test with a 5% level of significance using Statgraphics Centurion XV (Manugistics Inc., Rockville, MD, United States).

Visualisation of fluorescent protein expression in slice was either performed directly at the setup described above or using a confocal laser-scanning microscope (LSM, 510 META, Axiovert 200, Zeiss, Oberkochen, Germany). At the LSM, EGFP emission (EM) was detected via a 505–530 nm bandpass-filter (BP) with 488 nm for excitation (EX) and tdTomato EM was recorded through a 560 nm longpass filter (543 nm for EX). At the electrophysiology setup, fluorescence could be recorded at the side port of the Examiner Z1 via a color camera (DFK41BU02, The Imaging Source Europe GmbH). EGFP was detected using a filter set (Zeiss, Filter set 38) consisting of a BP 450–490 nm for EX, a beam splitter (BS) 495 nm, and an EM filter BP 500–550 nm. For the separation of tdTomato-fluorescence a Texas red filter set (F20–309; AHF analysentechnik AG) was used (EX: BP 580–604 nm; BS 615 nm; EM BP 625–725 nm). For cell counting, composites of the EGFP and tdTomato images were generated in ImageJ (https://fiji.sc/) and assessed with the cell counter plugin by two investigators. Additionally, a dualband filter set (F56-019, AHF analysentechnik AG) was used to visualise EGFP and tdTomato at once.