A hit-and-run strategy for protoplast reprogramming and regeneration into transgene-free plants

The ability of protoplasts to regenerate into whole plants underpins advances in crop engineering and pluripotency research. However, most protoplasts exhibit poor division and limited shoot regeneration, restricting their broader utility. Here, we present EppTec ( E fficient regeneration of transgene-free whole P lants from P rotoplasts reprogrammed by T ransiently E xpressed C ombinatorial factors), a transient expression platform using defined combinatorial factors (CFs) to unlock protoplast pluripotency and enable transgene-free whole-plant regeneration. Among 24 CFs tested, co-transfection of SEPW (SCR, ESR1, PSK5, and WOX5) markedly enhanced regeneration across diverse plant species in an evolutionarily conserved manner. We demonstrate that SEPW co-transfection induces epigenomic reprogramming, formation of a distinct cell cluster undergoing reprogramming and cell-cycle re-entry, and long-term reprogramming into pluripotent cells. These findings establish EppTec as a robust strategy to restore the regenerative capacity of plant cells from diverse species. EppTec may serve as a platform to revolutionize regeneration-based plant biotechnologies and conserve endangered plant species.