L5 filter cube

Membrane integrity and cell viability were evaluated as previously described (Lecomte et al., 2014 (link)) 2 and 7 days after treatment. Briefly, 100 μL of the cell suspensions were stained with 10 μL fluorescein diacetate (0.1 mg.mL^-1) and 20 μL propidium iodide (1.5 mg.mL^-1), and incubated in the dark for 5 min. Stained cells were then observed under a fluorescence microscope (Leica DMR HC) equipped for illumination with a 100 W halogen bulb and a Leica L5 filter cube in order to detect green and red fluorescence simultaneously. The microscope was fitted with a digital camera (Qimaging, Retiga 2000R) and monitored using Image Pro Express 6.0 software. Images were acquired at 3 s and 500 ms exposure, a gain of 1 with 1 × 1 binning (1600 pixels × 1200 pixels, 300 pixels per inch), and the images were saved in 24 bit-color TIFF format. For each condition, three microscope slides were prepared from three different cell culture wells and three images were taken per slide. Green and red fluorescence indicated living and dead cells with damaged membranes, respectively. The ability of cells to differentiate and develop somatic embryos was then monitored 3 weeks post-treatment. Proembryogenic masses and somatic embryo formation were visually checked under a stereomicroscope (Olympus SZ61TR).

Fluorescent in situ hybridization (FISH) was performed based on methods described previously (Haroon et al., 2013 ; Batani et al., 2019 (link)) with minor modifications. Briefly, stored ilea scrapings were pelleted at 13,000 x g for 5 min and washed 2 times with PBS. After the second wash, pellets were resuspended in 50 mL transformation buffer (100 mM CaCl₂, 30 mM MnCl₂, 20 mM MgCl₂, 100 mM potassium acetate, and 10% glycerol V/V). Ilea scrapings were then supplemented with 2,000 ng/mL of the SFB-specific probe (GGG TAC TTA TTG CGT TTG CGA CGG CAC) corresponding to position 801–827 in the universal 16S rRNA gene sequence of SFB (Accession number: X87244) (Liao et al., 2012 (link)). The SFB-specific probe was 6-Carboxyfluorescein (6-FAM) labeled (Thermo Fischer, Waltham Massachusetts, United States). After hybridization and washing, FISH-tagged SFB were resuspended in PBS and stored at 4°C for microscopy (Batani et al., 2019 (link)). Fluorescence microscopy was performed utilizing a Leica DM4 B fluorescent microscope with L5 filter cube (excitation 480/40 nm) for the detection of the SFB probe (Leica, Wetzlar, Germany).