2012 Volume 29 Issue 3 Pages 285-291
Disease resistance potential of a synthetic fungal chitinase (NIC) gene was evaluated in transgenic Petunia hybrid employing Agrobacterium tumefaciens-mediated genetic transformation. The NIC gene (1271-bp in length) was synthesized to encode the same amino acid sequence (except 25Ser to 25Arg) as that of chitinase 1 gene (chi1) from Rhizopus oligosporus. As 18% of codons in the NIC gene were changed from fungal type (AT-rich) to plant type (GC-rich), the nucleotide sequence of NIC had 82% homology with the chi1 gene. Petunia hybrida ‘Danty Lady’ was transformed with A. tumefaciens EHA 101 harboring a binary vector plasmid containing NIC (gene of interest) and nptII (selection marker) genes. Putative transgenic plants were produced on MS medium containing kanamycin monosulphate as a selective chemical. PCR analysis revealed that the NIC and nptII genes are integrated into the genome of transgenic plants. Integrations of the transgenes were indicated by Southern blot analysis of genomic DNA from the transgenic plants. Expression of NIC gene at the mRNA level was confirmed by reverse-transcription-polymerase chain reaction (RT-PCR). Western blot analysis detected the accumulation of NIC protein in the leaves of transgenic plants. The transgenic plants exhibited enhanced resistance against Botrytis cinerea (grey mold) as indicated by inhibition of the fungal growth in detached leaves and by the total protein extract. From these results it could be concluded that the NIC gene was successfully integrated into the genome of transgenic petunia plants and produced the NIC protein. Expression of the synthetic chitinase, NIC gene, probably, conferred enhance resistance to B. cinerea in transgenic petunia.