Fig. 2
Fluorescence detection of GFP expression in hyphae regenerated from transformed protoplast of V. dahliae. a GFP expression after 18 h of incubation. Protoplasts (200 μl of 1 × 106 /ml) were transformed with 12 μg of either GFP plasmid or linear GFP cassette and cultured. Fluorescence was observed in hyphae regenerated from transformed protoplast of V. dahliae after 18 h incubation in TB3. b Transformation efficiency of protoplasts using electroporation (300-500 V) or PEG-mediated transformation (PEG-4000, 6000 and 8000). After transformation, the protoplasts were cultured in TB3 broth for 18 h. Number of transformants was calculated per microgram DNA by counting the number of hyphae with GFP fluorescence. c Silencing of GFP expression with siRNA. Vd-GFP protoplasts were transformed with 10 μM of 4 different siRNAs (siRNA-gfp1, siRNA-gfp2, siRNA-gfp3 and siRNA-gfp4) separately by PEG-mediated transformation. The regenerated mycelia from the transformed protoplasts were observed for GFP fluorescence. d Assay for siRNA inhibition of GFP. Inhibition of GFP expression by siRNA-gfp1, siRNA-gfp2, siRNA-gfp3 and siRNA-gfp4 was compared after in the regenerated hyphae from the transformed protoplasts