Cellulase

Cell nuclei on nanofiber randomly oriented PVDF-TrFE scaffolds were imaged after staining for 10 min with DAPI (1 μg/ml), then placed in a 1.7-ml microcentrifuge tube with 1 ml of BY-2 medium containing either 0.05% trypsin (Sigma-Aldrich, T2601), 0.8% cellulase (Duchefa Biochemie, C8001), 0.2% Macerozyme R-10 (Duchefa Biochemie, M8002), or 1.5% pectate lyase (Megazyme, E-PLYCJ), and shaken at room temperature for 1 hour. Mock treatments used BY-2 medium alone. Scaffolds were washed in 25 ml of fresh medium three times and then stained for 10 min with DAPI for imaging.

Pisum sativum plants were grown in a climatized chamber at 25 °C with a light/dark photoperiod of 16:8 hours. Intra-veining strips were cut out of leaves from 2-week-old plants (1.5 g) and incubated for 210 min at 30 °C with gentle shaking (70 rpm) in 10 mL of 1 M mannitol, 100 mM MES-KOH, pH 5.6, 0.37% w/v macerozyme, and 1.5 % w/v cellulase (Duchefa Biochemie, Haarlem, The Netherlands). The digestion reaction was stopped by adding 2 mL of 2 mM MES, 154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, pH 5.7, and the cell suspension was filtered through a polypropylene mesh (pore diameter: 105 µm; tissue thickness: 121 µm—polyester Woven filter, Spectrum Labs, Rancho Dominguez, CA, USA). Digested cells were washed twice by resuspending them in 2 mL of 4 mM MES, 0.4 M mannitol, and 15 mM MgCl₂, pH 5.7. Finally, they were pelleted by gentle centrifugation (3 min at 100× g), resuspended in 1 mL of the same buffer, counted, and stored at 4 °C after having confirmed their integrity at the microscope.

Chromosome counting was carried out according to Schwarzacher [53 (link)]. Briefly, thallus tips from freshly started R. fluitans 001TC and R. fluitans BoGa cultures ensuring high cell division rates, were collected and transferred to ice water for 24 h and afterwards incubated in saturated 8-hydroxyquinoline solution for 3 h. Thallus pieces were then fixed overnight in Carnoy’s solution. The next day, thalli were washed five times with 10 mM citric acid buffer (pH 4.6) to remove the fixative. Thalli were then digested with 2.5% pectolyase (Duchefa, Haarlem, The Netherlands), 2.5% pectinase (Sigma-Aldrich, Darmstadt, Germany), and 2.5% cellulase (Duchefa, Haarlem, The Netherlands) for 30 min and washed with ddH₂O. DAPI (10 µg/mL) staining was performed on object slides, tissue was then squashed with a cover slip, and staining results were immediately observed using a Leica DM5000 B microscope.