Cellulase rs

We tested several cell wall digestion enzymes from multiple manufacturers and found the performance of the enzymes varied between manufacturers, as well as between batches from the same manufacturers. We tested driselase (Sigma), cellulase (Sigma), cellulase R10 (Duchefa, Yakult), cellulase RS (Duchefa, Yakult), pectolyase (Duchefa; discontinued), pectinase (Sigma), macerozyme R10 (Duchefa) and hemicellulase (Sigma) for conventional whole-mount protocol. We chose cellulase RS (Duchefa), hemicellulase (Sigma) and pectinase (Sigma) or macerozyme R10 (Duchefa) for expansion microscopy based on their performance and availability.

Sporophytic metaphase chromosome slide preparations were performed according to the technique of Waminal et al. (2012 (link)) with some modifications. Briefly, meristematic tips (~2 mm) were digested in a 50 μl pectolytic enzyme solution (2% Cellulase RS [Duchefa, Haarlem, The Netherlands, C8003.001] and 1% Pectolyase Y-23 [Duchefa, P8004.0001] in 100 mM citrate buffer) for 2 h at 37°C, and washed with distilled water. Roots were then transferred into a microtube containing chilled Carnoy's solution and vortexed for 30 s at room temperature. After disposing of the supernatant, the pellet was resuspended in (9:1 v/v) aceto-ethanol. The cellular suspension was then pipetted onto clean glass slides prewarmed in a humid chamber. After air-drying, slides were fixed for 5 min in 2% formaldehyde (Vrana et al., 2012 (link)), quickly dipped into distilled water, and dehydrated using ascending concentrations of ethanol (70, 90, and 100%).