A dynamic WUSCHEL / Layer 1 interplay directs shoot apical meristem formation during regeneration

De novo shoot apical meristem (SAM) organogenesis during regeneration in tissue culture has been investigated for several decades, but the precise mechanisms governing early-stage cell fate specification remain elusive. In contrast to SAM establishment during embryogenesis, in vitro SAM formation occurs without positional cues, and is characterized by spontaneous cellular patterning. Here, we have elucidated the initial stages of SAM organogenesis and the molecular mechanisms that orchestrate gene patterning to establish SAM homeostasis. We found that SAM organogenesis in tobacco calli initiates with protuberance formation followed by the formation of an intact L1 layer covering the nascent protuberance. Acquisition of L1 cellular identity is indispensable for de novo SAM formation, which also requires WUSCHEL (WUS) and the cellular capacity to direct anticlinal cell divisions. An intriguing finding is that TONNEAU1 silencing prevents the exclusive occurrence of anticlinal divisions in the outermost layer of the protuberances and suppresses the acquisition of L1 cellular identity, ultimately impeding regeneration. This study exposes an intricate interplay between L1 and WUS expression and that any disruption in this interplay compromises shoot formation. It further provides a novel molecular framework for the characterization of WUS/L1 interplay-mediated shoot apical meristem formation during regeneration.