Grafts from the SR1 tobacco crown-gall lines NT1 (having a deletion eliminating part of the transferred (TL)-DNA auxin locus) and NT2 (having an IS60 insertion in gene 2 of this auxin locus) were cross-pollinated with pollen from nontransformed SR1 tobacco plants. One half of the resulting F1 progeny resembled the female parent ("transformed" NT1-like and NT2-like seedlings respectively) and one half resembled the male parent ("non-transformed" SR1-like seedlings). For three states of differentiation (callus, shoot, graft) all phenotypic markers of the transformed seedlings studied were identical to those of the transformed female parent. Most phenotypic markers of non-transformed seedlings corresponded with markers of the male parent. Unlike the SR1 male parent, however, the SR1-like seedlings showed the maternal traits hyperstyly and male sterility. These two traits were inherited by 100% of the F1 seedlings studied. Ninety percent of the non-transformed F2 seedlings were still male-sterile whereas in as much as 50-100% of the non-transformed F3 progeny, male fertility had been restored. The SR1-like F1 seedlings did not contain any T-DNA. At the level of restriction-fragment analysis the T-DNA structures of all 22 NT1-like seedlings examined were identical to the T-DNA structure of their female parent NT1. The steady-state level of transcripts 4 (cytokinin locus) and 6a/6b relative to transcript 3 (octopine-synthase locus) was less in shoots and grafts than in callus. Observed variation in shoot morphology among the twenty-two NT1-like seedlings was not correlated with T-DNA structure, organization and expression at the level of steady-state mRNA. The T-DNA structure of NT2 and its transformed seedlings deviated from regular border-to-border TL-DNA, in that it extended beyond the left border repeat.

After three years of apparent stability in tissue culture, the single cell derived shooty crown gall line sNT1.013 produced a revertant shoot which had switched from non-rooting (Rod(+)) and octopine synthesizing (Ocs(+)) to Rod(-) Ocs(-), indicating that in this revertant TL-DNA genes 4 (causing the Rod(+) trait) and gene 3 (causing the Ocs(+) trait) had been inactivated. Southern blots revealed that the inactivation of these T-DNA genes was the result of a considerable rearrangement of DNA sequences, accompanied by deletions and possibly also by DNA amplifications. This study for the first time unambiguously proves that foreign genes which have been introduced via Agrobacterium tumefaciens can, at a low frequency, be inactivated after T-DNA integration because of reorganization of T-DNA sequences during tissue culture. This can be considered as an event of somaclonal variation.