Fig. 2

Analyses of viability, quality and transformation of cassava protoplasts. Viability of freshly isolated protoplasts was determined by Evans’ Blue Dye staining and visualisation under bright field microscopy. Analysis of protoplast quality was done by flow cytometric density measurement where events are discriminated by size and granularity, represented in log scale density plots. The size and shape of cassava protoplasts are measured by their effect on the forward scatter (FSC-A) and side scatter (SSC-A) of the laser. Stable transformation with the CRISPR construct was determined by fluorescence microscopy visualisation of eGFP fluorescence through the GFP filter and bright field. a Protoplasts from model M. esculenta cv.60444; b Protoplasts from susceptible M. esculenta T200 (c); Protoplasts from tolerant M. esculenta TME3. Non-viable cells are stained blue. d Plot of model M. esculentac v.60444 protoplast density (e) Plot of susceptible M. esculenta T200 protoplast density (f) Plot of tolerant M. esculenta TME3 protoplast density. Circled regions correspond to desirable protoplasts. g M. esculenta T200 protoplasts visualised through the GFP filter (h) M. esculenta T200 protoplasts visualised through both the bright field and GFP filters