Fig. 5

Agrobacterium-mediated transformation of pBIF-egfp in P. funiculosum NCIM1228. a Hygromycin-resistant transformants on LMP hygromycin selection plate with Chlorpromazine and Triton X-100. b PCR confirmation of transformants. Left panel shows the amplification of 500 bp fragment of hygromycin cassette and right panel shows amplification of reporter gene EGFP from genomic DNA of hygromycin-resistant transformants, with no amplification in lane with NCIM1228 genomic DNA (-ve control). + ve lane showed amplification from pBIF-egfp construct. c Fluorescence microscopy of pBIF-egfp transformants showing GFP fluorescence, NCIM1228 was taken as negative control. d Southern blot of pBIF-egfp transformants showing integration of T-DNA having egfp gene. E-GFP PCR amplified fragment was used as probe. NCIM1228 (WT) was taken as negative control. e PCR-based detection of two copies of cbh1 gene, one native and second recombinant, in hygromycin-resistant pBIF-CBH1 transformants. Left panel shows 1870 bp amplification of acquired cbh1 gene under GPD promoter using hygromycin cassette internal primer and cbh1 gene internal reverse primer. NCIM1228 was taken as negative control and pBIF-EGFP was taken as positive control. Right panel shows 1560 bp amplification of innate copy of cbh1 gene under native promoter. pBIF-egfp was taken as negative control showing no amplification