Abstract: Twenty six-mer forward and 23-mer reverse primers were designed using the published nucleotide sequence data of cellular T-DNA of Nicotiana glauca. Primers annealing temperature and Mg+2 concentration were optimized for a polymerase chain reaction. At target sequence of about 1050 bp overlapping the rolB and rolC genes were successfully amplified from the genomic DNA of N. glauca. The amplified sequence was cloned into pUC18 vector. The cloned rol B-C fragment showed a high base sequence homology to the rol B and C genes located on the T-DNA of Ti and Ri plasmids of Agrobacterium tumefaciens and Agrobacterium rhizogenes respectively.