Resistance and Susceptibility of Carrot Roots to Crown-gall Tumor Formation.

Crown-gall tumor cells induced by Agrobacterium tumefaciens (Sm. and Town.) Conn develop from normal plant cells by the action on them, in sequence, of several causal factors.1 Inoculations of susceptible species frequently result in fairly uniform tumors in 100 per cent of the trials. Inoculations of other plant species are not always successful or may result in tumors showing considerable size variations. Smith et al.2 and Magnus3 noted that different carrots formed tumors showing a gradient in size. DeRopp,4 in this laboratory, found that carrot roots could be divided into three groups relative to the amount of tumor tissue formed on transverse or longitudinal slabs of carrot root. He termed these "strong," "moderate," or '6nonreactor" carrots, depending on whether the inoculated surface formed large, coalescing masses of tumor tissue, a moderate number of discrete or barely confluent tumors, or no tumor tissues. These reaction categories were independent of the strain of bacteria and were not ascribable to varietal differences or to growth conditions. During the development of a quantitative bio-assay for tumor formation,5 it was found that disks of young secondary phloem excised from radial cylinders of different carrot roots and inoculated on their cambial-adjacent face exhibited similar variations in tumor-forming capacity. The weight of tumor tissue per disk in 15 days ranged from less than 5 mg. to over 200 mg. Phloem tissue possessing any predetermined tumor-forming capacity continued to show this capacity within 10-15 per cent even after two to three months storage at 8° C. The differences in tumor formation between the strong reactor (SR) and the weak reactor (WR) carrots might be ascribed to the presence of an inhibitor in the WR carrots or to the absence from them of one or more substances necessary for optimal tumor formation. This report is concerned with an investigation of these alternatives. Methods.-Disks of young secondary phloem were cut from radial cylinders of carrot roots of predetermined tumor-forming capacity. WR disks were taken from roots which formed less than 20 mg. of tumor tissue in 15 days, and SR disks were excised from carrots forming more than 80 mg. of tumor tissue under the same conditions. Agrobacterium tumefaciens, strain B6, used throughout this study, was grown in nutrient broth in shake cultures for 18 hours at 260 C. A 5-mm. platinum loop delivered ca. 106 bacteria to each disk, and the suspension was spread over the cambial-adjacent surface of the disk. Carrot juice (wound juice) was prepared by grinding peeled carrots in a meat-chopper, pressing out the juice in a Carver press, clarifying for 30 minutes at 10,000 X g, and sterilizing by successive passage through 015, 02, and 03 Selas porcelain filter candles. Eight to 12 disks were used per variable in each experiment. All were incubated at 260 C. on moistened filter paper in Petri dishes. Results.--Disks of phloem tissue from known SR and WR carrots were placed in Petri dishes, and 0.1 ml. of wound juice from either SR or WR carrots was ap-