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A Method for Eliminating the Contribution of Agrobacterium tumefaciens to Studies of Nucleic Acid Biosynthesis in Crown Gall Tumors

Department of Biological Sciences, Youngstown State University, Youngstown, Ohio 44555

A procedure is described that permits the use of radiotracer methods for examining crown gall tumors in the presence of the initiating agent Agrobacterium tumefaciens. On the basis of sensitivity testing using the disc diffusion technique and the minimum inhibitory concentration procedure and incorporation of radioactive phosphorus into nucleic acids, it was determined that A. tumefaciens was most sensitive to tetracycline, oxytetracycline, and methacycline. It was further demonstrated that nucleic acid synthesis in tumor tissue from four representative plants including tomato (Lycopersicum esculentum L.), Bryophyllum daigremontiana (Hamet and Perrier), pinto beans (Phaseolus vulgaris L.), and carrot (Daucus carota L.) was unaffected by a concentration of tetracycline (50 µg/ml) which completely inhibited nucleic acid synthesis in the bacterium. On this basis a procedure is described which first eliminates surface contamination and then uses tetracycline to nullify the contribution of A. tumefaciens in radioactive studies using tumors initiated by this bacterium.