Seventeen samples consisting of puried compounds and various ethanol extracts from plant sources were tested for actiity on the hdtiation of crown gall tumors on potato discs. The results demonstrated definite correlation between the ability of these samples to inhibit the formation of crown gail tumors and their activity on the P388 leukemia system in mice. Samples showing only cytotoxic effects in KB ceil cultures dW not affect tumor initiation in our system. The active materials had no effects on bacterial viability or on the ability of the bacteria to attach to a tumorbinding site.

The infectivity of Agrobacterium tumefaciens strain B6 was inhibited about 50% when these bacteria were inoculated on potato discs with equal viable cell counts of a weakly virulent strain of A. tumefaciens (B-48) The fact that crown gall host systems differ with respect to the degree of bacterial virulence shown by a given strain, the time it takes for tumor formation, and the size and number of tumors formed has been well established (1, 6, 7).The necessity for bacterial attachment to a specific site as a prerequisite for the formation of crown gall tumors on the primary leaves of Pinto beans and the time it takes for such attachment have been shown (5). Studies as to whether a similar type of attachment is necessary in other crown gall host systems have been limited due to the lack of another suitable quantitative bioassay for tumor formation, although the necessity for site attachment has been established for some nonquantitative host systems (8,10). With the recent development of the potato disc bioassay by Anand and Heberlein (1). we have undertaken studies which show that attachment to a specific site, which occurs in a short period of time, is a necessary prerequisite for tumor formation on potato discs.With the recent discovery that infectivity is due to the presence of a plasmid (11) found within tumor-inducing strains of Agrobacterium tumefaciens, experiments to determine the biochemical nature of the transformation process should increase in number. The need to understand more completely the nature of specific host systems for studying tumor formation takes on great importance. 2To whom reprint requests should be sent. MATERIALS AND METHODSof Northwestern University. The bacterium and yeast (which was obtained from the culture collection at the USDA Lab in Peoria) were grown for 48 hr in a medium containing 0.8% (w/v) nutrient broth, 0.1% (w/v) yeast extract, and 0.5% (w/v) sucrose on a New Brunswick Scientific Shaker at 27 C.Tumors were induced on potato discs by the method first described by Anand and Heberlein (1), and modified slightly in our laboratory (3). Red potatoes (Solanum tuberosum var. Red Russett) were purchased from the local market and soaked in 20%o Clorox (v/v) for 20 min. Potato discs were then obtained by placing a sterilized cork borer (20-mm diameter) through the potato and cutting 5-mm discs from the resulting plug. Five potato discs were then placed on Petri dishes containing 1.5% (w/v) agar and inoculated with 0.5 ml of bacterial suspension containing a known quantity of viable cells. The Petri dishes were placed at 27 C and exposed to 15 hr of room light and 9 hr of darkness daily. Twelve days following inoculation the tumors were counted with the aid of a dissecting scope. The tumors at this time, however, were clearly visible with the human eye. Each experimental sample consisted of three Petri dishes which contained five potato discs. Sterile techniques were employed throughout the above procedure.In order to insure that a maximum competition for attachment site...

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Incubation of embryoless barley (Hordeum vulgare) (4), the seeds were then washed 10 times with about 4 volumes of sterile distilled water in a sterile test tube (25 x 150 mm) covered with a Morton stainless steel closure. After the final wash they were covered with distilled water and soaked overnight at 4 C. Using aseptic technique, the seeds were cut in half perpendicular to their long axis, and the embryoless halves were used for the bioassays. Triplicates of two-half-seeds incubated in 1 ml total volume of medium in a 20-ml beaker were tested for each treatment. The medium contained 12.5 ,ug of chloromycetin per ml and, except as indicated in the text, was adjusted to pH 4.8 with either HCl or NaOH. The various substances incorporated in this medium were sterilized by filtration through 0.22 A pore diameter Millipore filters.After incubation at 30 C for various periods, the medium was removed, and the seeds and beaker were washed with 2 ml of sterile distilled water. The combined wash and incubation medium from each beaker was assayed for a-amylase by a slight modification of the procedure of Shuster and Gifford (15). The reaction mixtures consisted of 0.5 ml of enzyme plus 0.5 ml of soluble starch reagent. The starch reagent was prepared by dissolving soluble starch (1.5 mg/ml), KH.PO, (6 mg/ml), and CaCl, (0.22 mg/ml) in 0.1 M sodium acetate buffer at pH 4.8 and heating the solution until it started to boil. The enzyme-substrate mixtures were allowed to react for 1 to 10 min at 30 C, and the reaction was stopped by adding 2 mi of iodine reagent. The iodine reagent was prepared by diluting a stock I.-KI solution (60 mg of KI plus 6 mg of I, per ml of water) 100-fold with 0.05 N HCI. The tubes were held in an ice bath until diluted for absorbancy measurements by adding 5 ml of distilled water. The absorbancy of the blue starch-iodine complex was measured at 620 nm in a Gilford spectrophotometer. Dilutions of the enzyme were made so that no more than half of the starch in the reaction mixtures was hydrolyzed and under these conditions the kinetics of the reaction were zero order with respect to starch. The extent of hydrolysis was estimated from a standard curve for the starch-iodine complex, and the results are expressed as units of a-amylase released per half-seed. A unit is that amount of enzyme which hydrolyzes 100 ,ug of starch per min at 30 C. The values reported in individual experiments are the mean of the triplicate determinations.

The effect of the polyamine spermidine on the growth of crown gall tumors was determined using the potato disc bioassay. Addition of lmM spermidine resulted in a 30-50% increase in tumor growth. The spermidine effect was found to be biphasic, with lmM being optimal. Closely related polyamines including spermine, as well as other nitrogen containing compounds such as arginine and alanine, failed to promote tumor growth or inhibited the growth of these tumors. Endogenous levels of spermidine in crown gall tumor tissue were consistently greater than those of corresponding normal potato tissue. Rapidly dividing normal potato tissue derived from buds also contained elevated spermidine levels.