Bx51 pd72 ix71 microscopic imaging system

Fresh latitudinal sections of the receptacles of “Red Zaosu” and “Zaosu” were manually cut and temporarily preserved in a 5% (w/v) ascorbic acid solution during microscopic observation. Paraffin sectioning of the “Red Zaosu” receptacle was performed^{26 (link),27 (link)}. Fresh “Red Zaosu” flowers were immediately fixed in a formaldehyde–acetic acid–alcohol solution. The receptacles were separated, dehydrated, embedded in paraffin, cut into 10-μm slices, and stained with Fast green and safranin. The safranin-labeled xylem was imaged using a 450-nm excitation filter in combination with a 520–550-nm emission filter. Histological analysis was carried out using a BX51 + PD72 + IX71 microscopic imaging system (Olympus, Tokyo).

The full-length PbMYB12b CDS was amplified and fused into pCambia 2300 to generate the transgene construct p35S::GFP-PbMYB12b using a ClonExpress One Step Cloning Kit (Vazyme) based on homologous recombination technology. The construct was transiently introduced into onion epidermal cells by injection, and control cells were transformed in the same way with p35S::GFP. After their transformation, the onion epidermal samples were held for 16 h at 25 °C in the dark, after which GFP fluorescence was imaged using a BX51 + PD72 + IX71 microscopic imaging system (OLYMPUS).

The artificially shaded young leaves of “Red Zaosu” explants, artificially bagged carpopodia of “Red Zaosu”, naturally growing young leaves of “Zaosu” explants, and naturally growing carpopodia of “Zaosu” were incubated with fluorescence dye (40 μM Fluo-3/AM ester, 0.2 mM CaCl₂, and 50 mM sorbitol) at 4 °C for 5 min in the dark. The calcium signal was assessed using a 450-nm excitation filter in combination with a 520–550-nm emission filter on a BX51 + PD72 + IX71 microscopic imaging system (Olympus). The total calcium concentration was quantified by flame atomic absorption spectrophotometry.