Quantification of Heat Treatment-Induced Transgene Silencing Using Single Cells of Nicotiana tabacum

Abstract

The growth of transgenic phosphinothricin-resistant single cell cultures under unusually high temperatures (37 °C) led to the loss of herbicide resistance in more than 90% of the cells (Walter et al. 1992). A strong reduction of herbicide resistance could be observed in transgenic tobacco plants after an incubation for 1 to 10 days at 37 °C (Neumann et al. 1996). In this study we tried to quantify the phenomenon analysing the reduction of the transgene encoded phenotype in single cells of different tobacco lines. The transgenic lines used carried transgenes with different promoters, coding regions and terminator sequences. In the single cell system, a reversible loss of the transgene encoded phenotype was detectable during the heat treatment. After the reduction of the growth temperature to 24 °C, the number of cells which lost the transgene encoded phenotype decreased, reaching the initial level after approximately 6 days. On the single cell level, evidence for DNA methylation associated with the phenomenon could not be found by in vivo incubation of heat-treated plants with 5-azacytidine, a compound preventing DNA methylation. Transient expression of a gus A gene transferred to protoplasts of transgenic plants was not disturbed by a heat treatment of cells, while the integrated transgenes were still inactivated in the same experiment.