Cellulase onozuka r 10

Mitotic chromosome preparations were made according to a previously described procedure (Jenkins and Hasterok, 2007 (link)). In brief, the seeds were grown on filter paper moistened with tap water for 72 h at room temperature in the dark. Seedlings with two to three-cm-long roots were treated in ice-cold water for 24 h, fixed in a 3:1 (v/v) methanol:glacial acetic acid at 4°C overnight and stored at −20°C until use. After washing in a 0.01 mmol/L citric acid-sodium citrate buffer (pH 4.8), the roots were digested enzymatically for 1.5 h at 37°C in a mixture of 20% (v/v) pectinase (Sigma-Aldrich) and 2% (w/v) cellulase “Onozuka R-10” (Serva). After digestion, the meristems were dissected from the root tips and squashed in 45% acetic acid. After freezing on dry ice, the cover slips were removed and the preparations were air dried.

Actively growing root tips approximately 1.5 – 2.0 cm long were harvested separately from young hemp seedlings and immediately pre-treated with a 2 mM aqueous solution of 8-hydroxyquinoline for 2 h at room temperature (RT) and then for 2 h at 4°C in the dark. A 3:1 ethanol/glacial acetic acid (v/v) mix was used for fixation. Meristems 2 mm long were cut from fixed root tips and digested in a 10 μl enzyme solution (0.5% cellulase Onozuka R-10 (Serva, Germany) and 0.5% pectolyase Y-23 (Seishin Corp., Japan)) in 10 mM citrate buffer (pH = 4.9) for 1.5 h at +37°C. Suspended cells were used for chromosome preparation as described by Henegariu [49] (link) and Kato et al. [50] (link).

The CWRs obtained from dry stems were subjected to a saccharification assay. About 30mg of stem CWR was put into a 5ml disposable plastic tube together with 4ml of 5mM acetate buffer (pH 4.7) containing 4mg ml^–1 of a commercial cellulase preparation (cellulase Onozuka-R-10 from Trichoderma viride, Serva) and 0.1mg ml^–1 NaN₃. A blank tube was prepared containing the enzyme solution without any CWR. The reaction tubes (biological triplicates for each genotype plus the blank tube) were placed at 45 °C for 72h and on a carousel. After centrifugation (1500 g, 10min), the supernatant was subjected to glucose determination as follows. In a disposable 1ml spectrophotometric cuvette, 50 μl of the supernatant were mixed together with 1ml of Biomerieux reagent (Biomerieux Glucose RTU kit, Biomerieux, Lyon, France) and the colorimetric reaction was allowed to proceed for 30min. The absorbance was read at 505nm (against a cuvette containing 50 μl of H₂O and 1ml of reagent) and corrected for the glucose from the blank assay. The amount of glucose released by enzymatic hydrolysis of the stem CWR was calculated after appropriate calibration with standard glucose solutions. The pellets obtained from the CWR subjected to cellulase treatment were washed twice with water (with centrifugation for 10min at 1500 g between each wash) before freeze-drying and weight determination.

Cytogenetic preparations containing both mitotic chromosomes and interphase nuclei were made using a previously described procedure [29 (link)]. Briefly, the roots were cut off from the seedlings and washed in a 10 mM citric acid-sodium citrate buffer (pH 4.8), then digested enzymatically for 1.5 hours in a mixture containing 6% (v/v) pectinase (Sigma-Aldrich), 1% (w/v) cellulase ‘Onozuka R-10’ (Serva) and 1% (w/v) cellulase (Sigma-Aldrich). Root tips were squashed in a drop of 45% acetic acid. After freezing on dry ice, the cover slips were removed and the preparations were air dried and stored at 4°C until use.

Leaf protoplasts were extracted from 10 leaves of 4-week-old rosette of A. thaliana plants in WT and #236 background (n = 5 from each genotype) using the “Tape-Arabidopsis Sandwich method” described in Wu et�al., 2009 (link). For cell wall digestion, 1.25% of Cellulase Onozuka R-10 (Serva) and 0.3% of Macerozyme R-10 (Serva) were used. Protoplasts were washed with W5 solution [5 mM Glucose, 154 mM NaCl, 125 mM CaCl₂, 5mM KCl, 5 mM MES (Sigma-Aldrich), and imaged using an Olympus BX61]. Thirty-seven and forty-five pictures containing approximately 10,000 protoplasts were analyzed using ImageJ (https://imagej.nih.gov/ij/) to calculate cell size. The images were segmented using the "find edges" process and a color threshold. Touching cells were separated from the segmented binary images using the watershed process. Cell size was then measured using the analyze particles option.

Actively growing root tips approximately 1.5–2.0 cm long were harvested separately from young hemp seedlings and immediately pretreated with an aqueous solution of 2 mM 8-hydroxyquinoline for 2 h at room temperature (RT), and then for 2 h at 4 °C in the dark. An ethanol/glacial acetic acid (3:1 v/v) mixture was used for fixation. Meristems 2 mm long were cut from the fixed root tips and digested in a 10 mL enzyme solution (0.5% cellulase Onozuka R-10 (Serva, Germany) and 0.5% pectolyase Y-23 (Seishin Corp., Kobe, Japan) in a 10 mM citrate buffer (pH = 4.9)) for 1.5 h at +37 °C. Suspended cells were used for mitotic chromosome preparation as described by Kirov et al. [66 (link)]. Meiotic chromosome preparations were made from young flower buds as described by Divashuk et al., 2014 [15 (link)].