An efficient method for organic acetylation and use of DL-phosphinothricin as a negative selection agent in argE transgenic rice

Abstract

We present an efficient method for the production of N-acetyl-L-phosphinothricin (N-AcPt) from commercial DL-phosphinothricin (DL-PPT) by organic acetylation for use as a negative selection agent (NSA) that induces cell death in argE transgenic rice. DL-PPT was efficiently converted into N-AcPt with tetrahydrofuran (THF) and acetic anhydride (Ac2O). Chemical changes were confirmed using NMR and ATR-FTIR analyses. DL-PPT was toxic but N-AcPt did not show cytotoxic effects on leaf discs or seed germination of wild-type rice. Conversely, in argE-hpt transgenic rice, non-toxic N-AcPt showed the negative selection (NS) effect by inducing cell destruction in leaf discs and restricting seed germination. For inducing NS, ≥0.1 mg ml(-1) and ≥0.5 mg ml(-1) of N-AcPt were effective in leaf and seed assays, respectively. Further, the NS effect occurred faster in the leaf assay compared with the seed germination assay, again indicating the leaf assay was a more sensitive indicator of N-AcPt as an NSA to argE transgenic rice than the seed germination assay. This negative selection approach could be useful for the development of selectable marker free transgenic plants in the economically important monocot species and its commercialization for multiple gene transformation.

Keywords: Acetylation; N-AcPt; N-Acetyl-l-phosphinothricin; N-acetyl-l-phosphinothricin; NS; NSA; NSM; Negative selection agent; PPT; PS; PSA; PSM; Phosphinothricin; SM; SMF; Transgenic rice; argE Gene; negative selectable marker; negative selection; negative selection agent; phosphinothricin; positive selectable marker; positive selection; positive selection agent; selectable marker; selectable marker free.