Microscopic Observation of the Culm Wall

To observe the tissue morphology of the culm wall in cross section. The samples were first separated from the different positions of the standard bamboo culm, and then further cut into smaller bamboo strips with a size of about 3 × 1 × 1 cm. At least 3 samples were collected from each culm position of the two bamboo species, representing at least 3 biological replicates. These samples were fixed with the FAA solution (70% ethanol, formalin, and acetic acid, 18:1:1, v/v/v) for 3 days and then softened in 70% alcohol-glycerin (1:1, v/v) for 2 weeks. After embedding in PEG2000, the samples were cut into 20-micrometre cross sections using a Microtome 860 (American Optical Corporation, New York, USA). These sections were stained with Safranin O and aqueous Alcian Blue and then observed under a Leica DM2500 light microscope (Leica, Wetzlar, Germany) (Wang et al., 2016).

Based on these cross-sectional photographs of the bamboo culm, we measured the density of the vascular bundles, the size, and the area ratio of the individual wall tissue components of the two bamboo species using ImageJ version 1.48 software.

For the analysis of the longitudinal tissue morphology of the culm wall, samples from different culm positions of standard bamboo were divided into pieces of approximately 1.5 cm³. After a series of pre-treatments, including fixation of the samples (with FAA solution), and softening (70% alcohol and glycerin), which was similar to that of the pervious section. The longitudinal section of the samples was smoothed with a sharp blade. After dehydration and drying, these samples were sprayed with gold and observed under the JEOL JSM-6300 scanning electron microscope (JEOL, Tokyo, Japan). A detailed protocol can be found in a study by Wang et al. (2019).