Clones from a shooty tobacco crown gall tumor II: irregular T-DNA structures and organization, T-DNA methylation and conditional expression of opine genes

Abstract

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.