Cytokinin/auxin balance in crown specific loci in the T-DNA (Agrobacterium tumefaciens/immunoaffinity chromatography/Tn5 n

Insertion of the transposon Tn5 into the T-region of the octopine Ti plasmid of Agrobacterium tumefaciens gives rise to crown gall tumors having altered morphology. Three loci within the T-DNA that control tumor morphology have been detected (Garfinkel, D. J., Simpson, R. B., Ream, L. W., White, F. F., Gordon, M. P. & Nester, E. W. (1981) Cell 27, 143-153). They influence tumor size (tml), production of roots (tmr), or production of shoots (tms). Cytokinin and auxin. levels in such mutant tumors were examined by HPLC/radioimmunoassay and HPLC/flu- orescence assay, respectively. Free indoleacetic acid levels (in pmol/g) were: uninfected tobacco stem tissues, 128; wild-type A348 tumors, 295; tml mutant tumors, 307; tmr mutant tumors, 129; and.tms mutant tumors, 70. Average trans-ribosylzeatin levels were correspondingly: 0.97, 48, 40, 0.54, and 1,400 pmol/g. trans- Ribosylzeatin/indoleacetic acid ratios were as high as 24 in shoot- producing tumors and as low as 0.003 in root-producing tumors.. The evidence strongly suggests that tumor phytohormone levels are determined by genes in the T-DNA. It is known from the work of several laboratories that a segment of DNA, the transferred (T) region of a large plasmid (the Ti plasmid) harbored by the inciting bacterium, Agrobacterium tumefaciens, is integrated into the host- genome during crown gall tumorigenesis (1). Although seven T-DNA transcripts have been reported (2, 3) in octopine crown gall tumors, no T-DNA- coded gene product has been identified with certainty other than octopine synthase. Other transcripts are presumably de- terminative in tumor growth and maintenance, but their func- tions are unknown (4-6). However, crown gall tumors do grow in culture in the absence of added auxin or cytokinin (7), and recent studies (8-10) indicate that cytokinin levels are increased in many, but not in all, tumor lines. The morphology of crown gall tumors is determined by at least three factors: (i) the strain of A, tumefaciens used to incite the tumor, (ii) the plant host, and (iii) the site of inoculation on the host (11). The work of Skoog and Miller (12) established that the morphology of normal tobacco tissue in culture is deter- mined by the cytokinin/auxin ratio in the medium. High and low exogenous cytokinin/auxin ratios lead respectively to shoot and root production. It seems very plausible, therefore, that hormone overproduction and imbalance may be general char- acteristics also of crown gall tumors.