In vitro transcripts were synthesized from linearized pWSMV-GFP (pSP6-WSMV-GFP-6K1/CI) [38 (link)], pWSMV-GFP-P1-W303A, and pWSMV-GFP-ΔP1 [44 (link)] and mechanically inoculated onto fully expanded wheat seedlings at the single leaf stage as described in Tatineni et al. [38 (link)]. Inoculated wheat plants were incubated in a greenhouse at 22 to 27 °C with a 16 h photoperiod. Inoculated leaves at 10 days post-inoculation (dpi) and upper non-inoculated leaves at 21 dpi were collected for GFP fluorescence observation using a Zeiss Stereo Discovery V12 Fluorescence Microscope equipped with a narrow band GFP filter set 38 with 400–450 nm excitation and 450–490 nm emission. Fluorescent micrographs were captured using an AxioCam MRc5 camera attached to the fluorescent microscope. One µg of linearized plasmid DNA of pP2C2S-PVX [39 (link)], pPVX-WSMV-P1, or pPVX-WSMV-P1-W303A was transcribed in vitro and mechanically inoculated onto fully expanded N. benthamiana leaves at the six-leaf stage and incubated in a growth chamber at 25 °C with a 16 h photoperiod.
Stereo discovery v12 fluorescence microscope
Manufactured by Zeiss
The Stereo Discovery V12 Fluorescence Microscope is a high-performance instrument designed for a wide range of applications. It features a zoom system that provides a magnification range of 6.3x to 80x, enabling detailed observation and analysis of specimens. The microscope is equipped with a fluorescence illumination system, allowing for the visualization of fluorescently labeled samples. The Stereo Discovery V12 is a versatile tool suitable for various research and industrial applications.
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2 protocols using stereo discovery v12 fluorescence microscope
1
Wheat Seedling Inoculation and GFP Visualization
2
Inoculation and Fluorescence Imaging of PVX and WSMV Mutants
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Linearized pPVX (pP2C2S-PVX; Chapman et al., 1992) , pPVX-P7, pPVX-P7-W219A were transcribed in vitro and mechanically inoculated onto fully expanded N. benthamiana leaves at the 6-leaf stage. The inoculated plants were incubated at 18 °C in a growth chamber with a 16 h photoperiod. Total RNA was extracted using the Tripure isolation reagent (Roche) method from upper noninoculated symptomatic leaves at 21 and 28 days postinoculation (dpi). In vitro transcripts from pWSMV-GFP (pSP6-WSMV-GFP-6KI/CI; Tatineni et al., 2011) , pWSMV-GFP-ΔP1, pWSMV-GFP-ΔP1-P7 (Gupta et al., 2018) , or pWSMV-GFP-ΔP1-P7-W219A were mechanically inoculated onto wheat seedlings at the single-leaf stage, and incubated in a greenhouse at 22-27 °C with a photoperiod of 16 h. Inoculated leaves and upper noninoculated leaves were examined under a Zeiss Stereo Discovery V12 Fluorescence Microscope by using a narrow-band GFP filter set 38 for green fluorescence at excitation and emission ranges of 400-450 nm and 450-490 nm, respectively. Fluorescent micrographs were taken using an AxioCam MRc5 camera mounted on the fluorescent microscope.
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