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Attempts to Detect Agrobacterium tumefaciens DNA in Crown-Gall Tumor Tissue ¹

^a Plant Disease Resistance Research Unit, Agricultural Research Service, United States Department of Agriculture, and Department of Plant Pathology, University of Wisconsin, Madison, Wisconsin 53706

Primary and secondary crown gall tissue cultures were established from sunflower plants (Helianthus annuus, variety Mammoth Russian) wound-inoculated with Agrobacterium tumefaciens (Smith and Townsend) Conn strain B₆. Growth rates of tumor tissues and habituated healthy sunflower stem section tissues on basal medium lacking auxin and cytokinin were compared to those of healthy sunflower stem section tissue grown on the same medium with added phytohormones. No difference was detected in the thermal denaturation midpoints (74.8 C) and melting profiles in 25 mM sodium phosphate (pH 6.8), or the buoyant densities in cesium chloride equilibrium centrifugation (1.687 g cm^–3), between deoxyribonucleic acids (DNAs) isolated from crude nuclear preparations of the four tissue types. No satellite DNA was observed in equilibrium centrifugation of unsheared plant DNAs.

Heterologous DNA renaturation kinetic analyses were performed in 0.14 M sodium phosphate (pH 6.8) at 70 C. Thermal stability measurements of reassociated DNA revealed less than 1% of mismatched base pairs. Reannealing of sheared, denatured, radioactive A. tumefaciens B₆ DNA (molecular weight, 325,000 daltons) in the presence of a 5400-fold excess of sheared calf thymus, healthy tissue, or secondary sunflower crown gall DNA obeyed second order kinetics, with a Cot of 2.8, identical to that observed when B₆ DNA was reannealed in the absence of foreign DNA.

Reannealing rates of B₆ DNA in the presence of 5400-fold excesses of DNA from two lines of primary sunflower crown gall were increased 2.24- or 1.47-fold. Digestion of the tumor DNA preparations with pancreatic deoxyribonuclease I until no detectable DNA remained, followed by restoration of solution viscosity by added calf thymus DNA, failed to remove the acceleration effect of the tumor DNA preparations. Reisolation of the reannealed nucleic acid formed in this experiment, and digestion with ribonuclease A or deoxyribonuclease I revealed that the double-stranded fraction was composed entirely of DNA-DNA duplexes, with no detectable DNA-RNA hybrids.

The data indicate that tumor, but not healthy tissue DNA preparations contain some factor or factors (not DNA) which accelerate the reannealing of bacterial DNA. Sunflower tumor tissue DNAs, therefore, do not contain integrated A. tumefaciens DNA sequences in amounts greater than a random of the bacterial genome per diploid amount of plant DNA, or a complete bacterial genome per five diploid plant cell DNA equivalents. Further, the possibility of the presence of many copies of a specific portion greater than 5% of the bacterial genome is excluded.

¹ Research cooperative with the College of Agricultural and Life Sciences, University of Wisconsin, Madison, and the Agricultural Research Service, United States Department of Agriculture.