Agrobacterium tumefaciens Site Attachment as a Necessary Prerequisite for Crown Gall Tumor Formation on Potato Discs

Abstract

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...

Crown Gall Tumor Disc Bioassay

“…Red Russett) by the method first described by Anand and Heberlein (1), and modified slightly in our laboratory (7). Twelve The initial step in the formation of crown gall tumors involves the attachment of the bacterium to a tumor-binding site (9,11). This attachment on the potato disc system is complete within 15 min following inoculation (9).…”

“…Twelve The initial step in the formation of crown gall tumors involves the attachment of the bacterium to a tumor-binding site (9,11). This attachment on the potato disc system is complete within 15 min following inoculation (9). To test the possibility that any of the active test materials were in any way preventing bacterial attachment, they were added at various times after the inoculation of the bacteria to the potato discs (Table III).…”

Agrobacterium tumefaciens-mediated transformation of yeast.

“…Chromosomal virulence genes. A. tumefaciens cells with mutations in the chromosomal genes chvA, chvB, or exoC are defective in either the synthesis or transport of ,B 1,2-glucan which appears to be required for efficient attachment to and transformation of plant cells (34)(35)(36)(37)(38)(39)(40). To State of the Transferred T-DNA.…”

Inhibition of crown-gall tumorigenesis with plant extracts

“…The initial step in the formation of crown gall tumors involves the attachment of the bacterium to a tumorbinding site (Glogowski & Galsky, 1978;Lippincott & Lippincott, 1969). This attachment on the potato disc system is complete within 15 min following inoculation (Glogowski & Galsky, 1978).…”

“…This attachment on the potato disc system is complete within 15 min following inoculation (Glogowski & Galsky, 1978). To test the possibility that any of the active test materials were in any way preventing bacterial attachment, they were added at various times after the inoculation of the bacteria to the potato discs ( Table 2).…”