Tm4000plus scanning electron microscope

Computer-Aided Design (CAD) models of hollow cylinders and gyroidal columns were created on Fusion 360 (Autodesk, USA), exported as Standard Tessellation Language (STL) files, and sliced using Netfabb 2017 (Autodesk, USA). A Solflex 350 (W2P Engineering, Austria) DLP printer was employed to fabricate all parts. Post-printing, the parts were washed three times in isopropyl alcohol in an ultrasonic bath (Allendale Ultrasonics, UK) and then fully cured in water with a xenon Otoflash G171 unit (NK-Optik, Germany). The parts were stored in sterile 0.1 M phosphate buffer until use. A TM4000Plus scanning electron microscope (SEM, Hitachi, Japan) and a Zeiss Crossbeam 550 focused ion beam (FIB) SEM (Jena, Germany) were used for imaging, with samples prepared by freeze fracturing with liquid nitrogen and drying in ethanol, followed by a final wash in hexamethyldisilazane before sputter coating using an Emscope SC500 (Bio-Rad, UK). Mean pore sizes and distributions were evaluated from the SEM images (see section 2 in the Additional file 1 for additional details).

The incorporation of PVA into α-cellulose and BEKP was qualitatively confirmed by ATR-FTIR spectroscopy using a Varian Cary 630 spectrometer. Spectra were recorded using oven-dried samples as they were obtained.
Thermogravimetric analysis (TGA) was carried out by means of a TG209 F3 Tarsus thermogravimetric analyzer (NETZSCH Instruments). Samples (ca. 5 mg) were heated at 10 ºC min⁻¹ from 25 to 600 ºC, under nitrogen, at a flow rate of 20 mL min⁻¹.
Backscattered electron imaging was performed using a TM4000Plus scanning electron microscope (SEM) with EDS (Hitachi), operating at an acceleration voltage of 15 kV.
Cross-linking of α-cellulose and BEKP with PVA was also assessed by the ability of the materials to sorb and desorb water. Thus, the water uptake was measured by following a methodology adapted from the DIN standard 53,814. Briefly, the method consists of immersing a sample in water which is then left to swell overnight. Afterward, the samples were centrifuged at 3000 rpm, for 30 min, in an IEC Centra-3C centrifuge. The excess water was removed, the samples were weighed, and the mass was recorded, m_eq. The samples were then dried in an oven, at 60 °C, until constant weight, m₀. In each of the experiments, which were carried out in quadruplicate, the water uptake (%) was calculated using Eq. 1: $$Wateruptake\left(\%\right)=\left(m_{\mathit{eq}}-m_0\right)\times 100/m_0$$

For scanning confocal microscopy examination, the samples, including the abscission layers with pedicels and spikelets of weedy rice, were collected 1–2 days before or at flowering. These samples included the SH4 (S5), qSH1 (Q5), and SH4/qSH1 double gene edited (SQ1) lines, in addition to the weedy rice parent (“C9”). The samples were longitudinally cut and stained with 0.1% acridine orange solution for 15 min. The prepared samples were fixed on glass slides and then observed by the FV1000 laser scanning confocal microscope (Olympus Corp., Tokyo, Japan) under laser sources of 488 and 559 nm.
For scanning electronic microscopy examination, the pedicel samples were collected after maturity of the weedy rice seeds. The pedicel samples, including the abscission layers, were cut from the spikelet, soaked in the fixative solution containing 2.5% (pH 7.2–7.4) glutaraldehyde for more than 4 h, and rinsed with 0.1 m phosphate buffer. The prepared samples were dehydrated with ethanol, soaked in tertiary butanol for more than 15 min, and dried with a carbon dioxide critical point dryer. After removing the spikelet, the pedicel samples were conducted with vacuum ion sputtering coating and observed (the fractures surface facing up) using the TM4000 Plus scanning electron microscope (Hitachi Ltd., Tokyo, Japan).