Ds red filter

Images were taken using a dissecting Leica M165 FC stereo‐epifluorescence microscope, with RFP and Ds‐RED filters for the detection of fluorescence, using the PLANAPO 1.0× objective, 0.63× zoom and Leica Application Suite V4.7 acquisition software. The autofluorescence of the wild‐type regenerated cowpea was evaluated using the same system. For testing transformation on the eight additional cowpea germplasm lines from NPGS, transgenic shoots expressing TdTomato were monitored with a Stemi SVII dissection stereoscope equipped with an HBO illuminator (Carl Zeiss, Thornwood, NY) and a Ds‐RED filter (excitation: 545/25 nm, emission: 605/70 nm, Chroma Technology, Bellows Falls, VT). Images were taken with an AxioCam camera (Carl Zeiss, Oberkochen, Germany) and AxioVision LE64 software and composed using Photoshop CC (Adobe, San Jose, CA).

Expression of fluorescent marker genes (i.e., ZsGreen, DsRed, and tdTomato) in plant tissues was observed using a Stemi SVII stereomicroscope equipped with an HBO illuminator (Zeiss, Thornwood, NY, USA), a FITC filter set (λ_excitation = 480 nm, and λ_emission = 515 nm; Chroma Technology, Bellows Falls, VT, USA), and a DsRED filter (λ_excitation = 545/25 nm, dichroic 565LP, λ_emission = 605/70 nm; Chroma Technology). Expression of ZsGreen in isolated embryos was observed under a Zeiss Axioskop 2 plus fluorescence microscope equipped with an 89 North^® PhotoFluor LM-75 illumination system (Chroma Technology) and a FITC filter set. Images were taken using an AxioCam camera (Carl Zeiss, Oberkochen, Germany) and the AxioVision LE64 software.

Capsules were harvested 9 through 17 days after pollination. Ovules were isolated and macerated in an enzymatic solution [27 ] composed of 12% (w/v) mannitol, 3 mM MES, 1% (w/v) cellulose R-10 (Research Products International), and 0.8% (w/v) Rhizopus sp. pectinase, with pH adjusted to 5.7, for 30 min with agitation at 60 rpm. The ovules were rinsed in a washing buffer composed of 12% (w/v) mannitol and 3 mM MES (pH 5.7) at least 3 times and then gently ground with a small pestle to release the developing embryos. Without removing integument debris, isolated embryos were mounted on a glass slide in the washing buffer and observed under a microscope (Zeiss, Thornwood, NY, USA) equipped with a PhotoFluor LM-75 illuminator (89 North, Williston, VT, USA) and a Ds-RED filter (excitation: 545/25 nm, emission: 605/70 nm, Chroma Technology, Bellows Falls, VT, USA). Images were taken using an AxioCam camera (Carl Zeiss, Oberkochen, Germany) and the AxioVision LE64 software. Embryos without visible damage were counted. Unless otherwise noted, all chemicals were obtained from Sigma-Aldrich.