Summary
Transformed clones from a shooty tobacco crown gall tumor, induced byAgrobacterium tumefaciens strain LBA1501, having the auxin locus of the TL-region inactivated by a Tn1831 insertion, were investigated for their T-DNA structure and expression. It has been described previously (28) that in addition to clones with an expected phenotype (phytohormone independent growth in tissue culture (Aut+), shoot regeneration (Reg+) and octopine synthesis (Ocs+)), clones were obtained with an aberrant phenotype. One of these clones, TSO38, is Aut+Reg+ but shows little or no octopine synthesis activity (Ocs-). Subclones of TSO38, however, are either Ocs- or Ocs+. Ocs- shoots become Ocs+ under certain states of differentiation, indicating that the octopine synthase gene is present. The fact that in the Ocs- subclones the octopine synthase gene is not expressed, is probably due to DNA methylation (29). The present paper describes that shoots derived from both an Ocs+ and an Ocs- subclone of TSO38, which were negative for the presence of mannopine (Mas-) and agropine (Ags-), became Mas+Ags+ after culturing on medium containing the hypomethylating agent 5-azacytidine. This means that both in the Ocs- line and in the Ocs+ line expression of TR-DNA opine genes most likely was hampered by DNA methylation. The T-DNA structures of an Ocs- and an Ocs+ TSO38 subclone proved to be identical and surprisingly complex. No intact copy of Tn1831 was present. TL-DNA and TR-DNA segments, present in high copy numbers, were truncated; several T-DNA segments existed in tandem arrangements. When DNA from an Ocs+ and an Ocs- subclone of TSO38 were compared for cleavability by the methylation sensitive restriction enzymes HpaII and MspII, differences were detected, but it became also clear that both lines contained methylated T-DNA segments. This indicates that the Ocs- and the Ocs+ TSO38 subclones differ only quantitatively in respect to degree of T-DNA methylation.
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Peerbolte, R., Leenhouts, K., Hooykaas-van Slogteren, G.M.S. et al. Clones from a shooty tobacco crown gall tumor II: irregular T-DNA structures and organization, T-DNA methylation and conditional expression of opine genes. Plant Mol Biol 7, 285–299 (1986). https://doi.org/10.1007/BF00752901
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DOI: https://doi.org/10.1007/BF00752901