Spurr s epoxy resin

Fully expanded leaf blades of 4-week-old seedlings were collected in the morning. For each line, three seedlings were selected and the middle parts of the leaf blades were cut into small segments and used immediately for microscopic observation. The extent of starch accumulation was determined by iodine staining of the rest (tips and bottom parts) of the sampled leaves. The small segments of leaf blades were fixed in 4% (w/v) paraformaldehyde and 2% (w/v) glutaraldehyde in 50 mM phosphate buffer (pH 7.2) for 24 h and post-fixed with 2% (w/v) osmium tetroxide aqueous solution in the same buffer for 2 h at 4°C. Dehydration and resin infiltration were performed in a graded series of acetone solution and propylene oxide. The samples were embedded in Spurr’s epoxy resin (Sigma-Aldrich, St. Louis, MO, USA). Ultrathin transverse sections were prepared with an ultramicrotome (Ultracut UTC, Leica, Germany) and stained with uranyl acetate and lead citrate, and observed using a transmission electron microscope (JEM-1010, JEOL Ltd., Tokyo, Japan) operated at 100 kV. Semithin transverse sections (1 μm thickness) were subjected to periodic acid–Schiff (PAS) staining and observed using a light microscope (Eclipse Ti-S, Nikon, Tokyo, Japan).

For leaf tissue preparation and electron microscopy, small leaf discs (4.0 mm × 1.2 mm) from within the gas exchange chamber were removed and infiltrated in a syringe with the fixative 2.5% glutaric aldehyde in 0.1 m phosphate buffer (pH = 7.6) at 4 °C, and post-fixed in 2% buffered osmium tetroxide at 20 °C for 2 h. The samples were embedded in Spurr’s epoxy resin (Sigma-Aldrich, St. Louis, USA). For light microscopy, semi thin leaf cross sections were stained with toluidine blue, and observed at 200× magnification with an Olympus IX71 light microscope (Olympus Optical, Tokyo, Japan). Ultrathin leaf cross sections were stained with 4% (w/v) uranyl acetate followed by 2% (w/v) lead citrate. Transmission electron microscope (H-7650; Hitachi – Science &Technology, Tokyo, Japan) and Soft Imaging System software (H-7650; Hitachi –Science & Technology, Tokyo, Japan) were used for observation and photography. The width and length of chloroplast was measured by software Image J, a free, Java-based image-processing package.

Anatomical analysis was down in 2017. Leaf segments (approximately 1 mm× 4 mm) were cut from the middle of the leaves (the midvein was avoided) using a blade at stem elongation (65 days after sowing), fixed immediately in 2.5% glutaraldehyde (v/v) in 0.1 mol L⁻¹ phosphate buffer (pH 7.4), and then post fixed with 2% osmium tetroxide for 2 h. Leaf segments were embedded in Spurr's epoxy resin (Sigma-Aldrich, St. Louis, USA). Semithin leaf cross sections were stained with toluidine blue and observed at 200× magnification with a light microscope (IX71, Olympus Optical, Tokyo, Japan). Soft Imaging System Software (version 1.15.3, Panoramic Viewer, 3DHISTECH, Budapest, Hungary) was used for the observation and photography. Leaf thickness was measured using Image-Pro Plus (version 6.0, Media Cybernetics, Maryland, USA).
Ultrathin leaf sections (90 nm thick) were examined for the ultrastructural observation of chloroplasts using a transmission electron microscope (H-7650, Hitachi, Japan) after staining the leaf sections with 2% uranyl acetate (w/v) and lead citrate. Chloroplast number was counted, and the chloroplast length and thickness were measured using Image-Pro Plus. The chloroplast surface area was calculated according to the Cesaro formula:
where L = 0.5× chloroplast length, and T = 0.5× chloroplast thickness.