Lsm 510 meta confocal scanning laser inverted microscope

To analyze the regulation of AHP6 expression in response to BOL activity, one drop of β-estradiol or mock solution was applied per inflorescence in DRNL-ER AHP6::GFP plants. The β-estradiol solution contained 10 μM β-estradiol (Sigma–Aldrich) with 0.01% Silwet L-77 (Lehle Seeds). A solution containing DMSO and Silwet L-77 in the same concentration as in the β-estradiol solution was used for the mock treatment.
Transverse sections of the gynoecia were made 48 h after the treatments, according to Reyes-Olalde et al. (2013) (link). The sections were visualized and images were captured using a LSM 510 META confocal scanning laser inverted microscope (Carl Zeiss). Propidium Iodide (PI; at 0.01 mg/mL) was used as a counterstain. PI was excited using a 514-nm line and GFP was excited using a 488-nm line of an Argon laser. PI emission was filtered with a 575-nm long pass (LP) filter and GFP emission was filtered with a 500–550-nm bandpass (BP) filter.

The coding sequence of ZmRAP2.7 was cloned into the BamHI and EcoRI sites of the pEZS-NL transient expression vector under the control of the 35S promoter to generate pEZS-35S:ZmRAP2.7-EGFP construct (Zuo et al., 2015 (link)). Maize protoplasts were isolated from etiolated maize seedlings of inbred B73 for transformation as described by Yoo et al. (2007) (link). After the incubation at 24°C for 12 h in the dark, GFP fluorescence in the transformed protoplasts was visualized using a LSM510 META confocal scanning laser inverted microscope (Carl Zeiss, Jena, Germany).

For light pictures and phenotype analysis the plant material was dissected and observed using a Leica EZ4 D stereomicroscope (Leica, Wetzlar, Germany). Scanning electron microscopy images were captured using a Zeiss EVO40 environmental scanning electron microscope (Carl Zeiss, Oberkochen, Germany) with a 20 kV beam, and the signal was collected using the BSD detector, for which plant tissue was collected and directly observed in the microscope. For fluorescent microscopy, the images were captured using a LSM 510 META confocal scanning laser inverted microscope (Carl Zeiss, Oberkochen, Germany). Propidium iodide (PI) was excited using a 514-nm line and GFP was excited using a 488-nm line of an Argon laser. PI emission was filtered with a 575-nm longpass (LP) filter and GFP emission was filtered with a 500–550-nm bandpass (BP) filter.