Stability and expression of transferred DNA in F1 tobacco transformants studied at various states of differentiation

Abstract

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.

Tobacco genes encoding acidic and basic isoforms of pathogenesis-related proteins display different expression patterns

“…To investigate whether other TMV-inducible tobacco genes are also induced as a result of an elevated cytokinin concentration, the levels of mRNAs A -I in the shoot line SNT1005 were analysed. This line contains part of the octopine TL-DNA of Agrobacterium tumefaciens including an active cytokinin gene (T-cyt gene; [20]). Due to T-cyt-directed overproduction ofcytokinins SNT1005 shoots are unable to develop a root system and form numerous stunted side-sprouts.…”

“…Petit Havanna SR1 lines SNTI005 (T-DNA-transformed) and SNT1016 (untransformed) [20] were subcultured axenically at 26 °C and 2000 lux (12-h light cycle) on hormone-free LS medium at monthly intervals. Etiolated SNT1016 plants were obtained by growing subcultured plants for 3 weeks in the dark.…”

T-DNA rearrangements due to tissue culture: somaclonal variation in crown gall tissues

“…1D). Northern blots revealed that TL-DNA genes 5, 7 and 2 are not expressed in NT1 due to the deletion, whereas TL-DNA transcripts 1, 4, 6a/b and 3 were detected [14,20]. The absence of gene 2 implies that the TL-DNA auxin locus is not intact in NT1 (Aux-).…”

“…C: Extent of the T-region clone pOTY8 [6]. D: Extent of TL-DNA segment present in NT1 and NTl-like seedlings (including NT1.013) [14,20]. E: Open reading frames of genes on TL-DNA and TR-DNA according to Barker et al [2]; genes are numbered according to Willmitzer et al [26] (TL) and Salomon et al [23] (TR); genes which have a clear phenotypic effect, are hatched.…”

“…Germination of the seeds showed that half of the progeny phenotypically resembled the female parent (NTl-like seedlings and the other half resembled the male parent (SRl-like seedlings). Twenty-two NTl-like seedlings and thirteen SRl-like have been kept in tissue culture for four years and were studied for phenotypic stability, T-DNA content and T-DNA expression [20].…”