Clonal analysis of heterogeneous crown gall tumor tissues induced by wild-type and shooter mutant strains ofAgrobacterium tumefaciens-expression of T-DNA genes.

Tumors were induced by anAgrobacterium tumefaciens strain with a wild-type octopine Ti plasmid and by shooter mutants with a transposon insertion in the auxin-locus of the T-region. Cloning of isolated axenic tumor tissues revealed that in all cases they consisted of tumor cells (10-26%) next to a majority of normal cells. The tumor clones that had been induced by the strain with the wild-type Ti plasmid all grew as amorphous calli. Tumor, clones induced by a shooter mutant were of two different types. One type of clone grew well on phytohormone-free medium. this type invariably regenerated tumorous shoots abundantly on this medium. The other type of clone only grew after the addition of auxin and cytokinin to the culture medium, but slow growth also took place in the presence of only auxin. This type never regenerated shoots spontaneously. After stimulation by a high level of kinetin, however, a few shoots were also obtained from these clones. One of these shoots, like other tumorous shoots, contained the tumor-specific enzyme octopinesynthase (Ocs), but in contrast to other tumorous shoots formed a root-system.The expression of T-DNA genes in shoots proliferating from the cloned tumor tissues induced by a mutant with an insertion in the region for transcript tr. 2 was studied by northern blot hybridization. Except for tr.2 the T-DNA transcripts were detected in the tumorous shoots analysed, including the transcript, tr.1 from the auxin-locus and tr.4 from the cytokinin-locus. This shows that the presence of these transcripts, which are assumed to be responsible for the tumorigenic character of tumor cells, does not interfere with the differentiation of shoot cells.One of the shooty tumor clones (TSO38) showed an unstable character with regard to octopine synthase activity (Ocs±). For, TSO38 and some of its subclones, it was found that only 4% of the regenerated shoots were Ocs(+). Northern blot hybridization revealed that the mRNA for octopine synthase was present in extremely low quantity in the population of TSO38 derived shoots.The finding that it was possible to force shoots from clone TSO38 and from subclone TSO38-23(-) that were Ocs(-) to become Ocs(+), proved that the gene for octopine synthase was present in the Ocs(-) shoots and that this gene showed unstable expression due to regulation at the level of transcription.