Ds 5mc

Mature stage plants and flowers at anthesis were photographed with an Olympus C-770 digital camera (Tokyo, Japan). Mature anthers were examined using a Nikon SMZ1500 stereoscope and photographed with a Nikon DS-5Mc digital camera (Tokyo, Japan). The wild-type and osabcg15 mutant anthers were separately crushed and stained in 1 % I₂–KI solution for 3–5 s to dye starch then photographed with a Nikon E600 microscope. Observation of anther development by semi-thin sections and transmission electron microscopy (TEM) was performed as described by Li et al. (2006 (link)) using an Hitachi H-7500 transmission electron microscope (Tokyo, Japan). Bright-field photographs of anther cross-sections were taken under a Nikon E600 microscope using a Nikon DS-5Mc digital camera, and the TEM results were photographed using a Gatan 832 CCD camera (Pleasanton, CA, USA). For scanning electron microscopy (SEM) observations, anthers were collected and processed essentially as described by Keijzer et al. (1996 (link)) and observed with a Quanta 200 scanning electron microscope (FEI, Hillsboro, OR, USA) under a strong vacuum. Anthers from different developmental stages, as defined by Zhang et al. (2011 (link)), were collected based on spikelet length and lemma/palea morphology.

EGFP expression was observed under an epifluorescence inverted microscope (NikonT300, Japan) on D7. Briefly, different stages of implantation embryos were exposed to blue light (excitation wavelength 488 nm, emission wavelength 530 nm), the EGFP expression signal was observed and fluorescent photos acquired with a CCD camera (DS-5Mc, Nikon, Japan).

For light microscopy observations, flowers were fixed for 4 h at room temperature in a mixture of 2.5% (v/v) glutaraldehyde and 2.5% (v/v) formaldehyde (obtained from paraformaldehyde) in 0.05 M cacodylate buffer pH 7.0. After fixation, the plant material was rinsed with cacodylate buffer, then dehydrated in a graded acetone series, and finally embedded in Spurr’s resin. Semi-thin (0.5–1.5 μm) transverse sections were prepared using a Sorvall MT 2B ultramicrotome and glass knives, and mounted on glass microscope slides.
For histochemical analyses, semi-thin control sections for light microscopy were stained with 0.05% (w/v) alcoholic Toluidine Blue O (TBO). Detection of protein and water-insoluble polysaccharides was undertaken using Aniline Blue Black (ABB) and the periodic acid-Schiff reaction (PAS), respectively [25 ]. Moreover, sections of plant material prepared for TEM were treated with Sudan Black B (SBB) to detect the presence of lipids [26 (link)].
Observations and photographic documentation were undertaken using a Nikon Eclipse E 800 light microscope equipped with a Nikon DS-5Mc camera with Lucia Image software (University of Gdańsk, Gdańsk, Poland).