# Monitoring the stability of transgene expression in lettuce using the RUBY reporter

## TL;DR

This study identifies the LsUBI promoter/terminator as a reliable option for stable transgene expression in lettuce over multiple generations.

## Contribution

The study evaluates and compares the stability of transgene expression constructs in lettuce using the RUBY reporter system.

## Key findings

- The LsUBI promoter/terminator combination provides strong and stable transgene expression in lettuce across generations.
- The 35S promoter/tHSP combination shows frequent silencing despite initial strong expression.
- The AtUBI promoter/tRBCS combination offers intermediate expression levels with less silencing.

## Abstract

The RUBY reporter enabled the evaluation of different transgene expression constructs in lettuce, revealing that the lettuce ubiquitin promoter and terminator had strong expression that was stable over multiple generations.

Nearly four decades after the first transgenic lettuce was reported, constructs for stable transgene expression remain limited. Notably, the 35S promoter from the Cauliflower Mosaic Virus (35S), which drives strong expression of transgenes in several plant species, has often shown silencing and instability in lettuce. Other promoter/terminator combinations that are commonly used in plant expression vectors have not been extensively studied in lettuce. In this study, we evaluated three different expression constructs in two different horticultural types of lettuce using the non-invasive RUBY reporter, which allowed for the monitoring of transgene expression throughout the process of regeneration during tissue culture, throughout development of the primary transgenics, and in two subsequent sexual generations. The LsUBI promoter/terminator combination resulted in strong, uniform expression throughout regeneration, during growth of the primary transgenics, and in both subsequent generations. The AtUBI promoter/tRBCS combination showed slightly lower levels of expression and intermediate levels of silencing, while the 35S promoter/tHSP combination showed both initial strong expression and frequent silencing. Therefore, our data show that the LsUBI promoter/terminator combination provides strong, uniform expression that is unlikely to result in silencing and that the AtUBI promoter/tRBCS combination is an additional option for stable expression of transgenes in lettuce, especially if an intermediate expression level is desired.

The online version contains supplementary material available at 10.1007/s00299-025-03665-w.