Gene silencing in transgenic tobacco hybrids: frequency of the event and visualization of somatic inactivation pattern

Thomas Schmülling¹ * & Horst Röhrig²

Mol Gen Genet 249: 375-390.

¹ Universität Tübingen, Lehrstuhl fⁿr Allgemeine Genetik, Auf der Morgenstelle 28, D-72076 Tübingen, Germany
² Max-Planck-Institut fⁿr Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany

Summary

We have investigated the stability of the expression of different T-DNA located genes in hybrid tobacco lines. These lines were obtained to restore rolC induced male sterility in kanamycin resistant P35S- rolC transgenic tobacco plants by rolC antisense genes. We observed for five different tester lines in a total of 158 hybrids in 20% of the F1 progeny and in 35% of the backcrossed F2 progeny inactivation of transgene expression, as indicated by the loss of kanamycin resistance. In 3% of all crosses a complete loss of antibiotic resistance was noted, while in most affected hybrid progeny only part of the population became kanamycin sensitive. On T-DNAs harboring several genes, single genes could be selectively inactivated. Gene inactivation was not restricted to one of the two T-DNAs. Somatic silencing, as visualized by a cell-specific 35SGUSINT marker gene, occurred in a random fashion or exhibited an inherited specific pattern. The type of somatic silencing pattern indicated developmental control of the process. Two phenotypic classes could be distinguished with respect to frequency and timing of the inactivation process. Rapid gene inactivation occurring within few weeks after germination of hybrid seedlings was characterized by a complete methylation of restriction sites in the promoter of the silenced gene, resetting of gene expression during meiosis, heridity of the developmentally controlled program of gene silencing in subsequent generations and a rapid reactivation of gene expression after genetic separation of the different T-DNAs. In contrast, a slow type of gene inactivation was of a more stochastic nature and recognized only in hybrids of the backcrossed F2 generation. In this case the degree of promoter methylation, which could extent beyond the T-DNA borders, was not correlated with the reduction of steady state poly(A)⁺ mRNA level, the silenced state was transmitted through meiosis and reactivation lasted several generations. The implications of the observations for our understanding of the gene inactivation process are discussed.