Isolation and Characterization of Agrobacterium Tumefaciens from Crown Gall and Soil Samples

Agrobacterium tumefaciens is popularly known as nature’s genetic engineer. It is a Gram-negative pathogenic bacterium which is generally found in soil. It causes tumour formation in plants due to its ability to perform inter-kingdom DNA transfer. Due to this characteristic of A. tumefaciens to act as a gene vector it is used as a gene jockeying tool. Its virulence is due to presence of Ti plasmid, T-DNA and vir regions of the plasmid. This paper focuses on the isolation and characterization of Agrobacterium tumefaciens isolated from two samples- crown gall present on Azadirachta indica (neem tree) and soil present around Phaseolus vulgaris (bean) plant. The tumour sample was surface sterilized using Tween-20, 70% ethanol and mercuric chloride solution while the soil sample was serially diluted. Isolation was done by streaking the samples onto MacConkey agar plates that is selective for Agrobacterium. On performing Gram staining it was determined that bacterium was Gram-negative. Antibiotic Sensitivity test performed using Kirby-Bauer method revealed that the isolate was sensitive to kanamycin (1.5 cm) and tetracycline (2.4 cm), visualized as zone of inhibition, while being resistant to cefuroxime and rifampin (0 cm). Biochemical tests revealed that the isolate was positive for motility test (highly motile), oxidase test (disc turns blue), catalase test (effervescence produced), citrate utilization test (Simmon’s citrate agar turned blue) and H2S production test (Kligler’s iron agar turned black). Positive result was obtained for the pathogenicity test in carrot, which confirmed tumour forming ability of the isolate. Leaves of brinjal plant which were infected with the isolate were then tested for GUS activity and blue coloured spots were observed which are positive for GUS assay. From the obtained results, it was concluded that the organism isolated was indeed Agrobacterium tumefaciens. However, the strain can be confirmed only by using advanced techniques such as DNA sequencing and polymerase chain reaction (PCR).