Sandhu, Jagdeep SinghGumber, Hardeep Kaur2016-10-242016-10-242010http://krishikosh.egranth.ac.in/handle/1/81571In the present study two anti-fungal gene constructs were generated comprising CaMV35SChitinase- NOS and CaMV35S-Glucanase-NOS using 905 bp and 1764bp Open Reading Frames (ORFs) respectively from the indigenously cloned chitinase (Accession no. GU187942) and glucanase (Accession no. GU300766) genes. ORFs were PCR amplified using specifically designed primers containing overhanging restriction sites and ligated into pBI121 cleaved by corresponding restriction enzymes. The gene constructs were further subcloned into high copy number plasmids pUC18 and pGEM 9Zf(-) respectively for particle bombardment of citrus cells for transient expression analysis. The gene constructs were bombarded individually as well as in combination. PCR analysis demonstrated insertion of gene construct in 78% of calli bombarded with chitinase gene construct, in 28% of the calli bombarded with glucanase gene construct and in 9.3% of the calli co-bombarded with both the gene constructs. Keywords: Sub-cloning, chitinase, glucanaseenbiotechnologyglucanaseThesis