Leaves and seeds were collected starting at 42 DAP with 14-day intervals until seed harvest. The 3rd fully expanded young leaf on the main-stem from terminal were collected. To observe the anatomical structure of the mesophyll cells, a leaf sample was cut into 5-mm segments along the midrib and fixed in FAA (formalin: alcohol: glacial acetic acid was 90:5:5, V). The fixed material was then dehydrated using an ethanol series, cleared in xylene, embedded in paraffin wax, and then cut into 4–6-μm-thick sections using a rotary microtome (RM2016, Leica, Germany). The sections were stained with both safranine and fast green and fixed in neutral balata. They were subsequently observed and photographed with a microscope (Nikon Eclipse E100, Nikon digital sight DS-U3, Japan).
Tree peony seeds were fixed in FAA and 2.5% glutaraldehyde respectively. The sections fixed in FAA were stained with PAS (Periodic Acid Schiff) and naphthol sulfonic acid (Servicebio, Wuhan, China). The starch granules were stained to purplish red, the cell wall was also dyed purplish red, and the protein presented yellow. The seeds fixed in 2.5% glutaraldehyde were immersed in 1% citric acid and then embedded in paraffin sections. The sections were stained with Nile red (Servicebio, Wuhan, China) in the dark, and the lipids appeared orange-red or red under a fluorescent microscope.
Tree peony seeds were fixed in FAA and 2.5% glutaraldehyde respectively. The sections fixed in FAA were stained with PAS (Periodic Acid Schiff) and naphthol sulfonic acid (Servicebio, Wuhan, China). The starch granules were stained to purplish red, the cell wall was also dyed purplish red, and the protein presented yellow. The seeds fixed in 2.5% glutaraldehyde were immersed in 1% citric acid and then embedded in paraffin sections. The sections were stained with Nile red (Servicebio, Wuhan, China) in the dark, and the lipids appeared orange-red or red under a fluorescent microscope.