Dissection of regeneration response by tissue-specific induction and single-cell profiling

Regeneration, the development of plantlets from single cells, is generally required for in vitro propagation and transformation of plants. Usually, regeneration is effectuated by the exogenous addition of plant hormones based on trial-and-error application protocols. However, many economically important crops and cultivars are recalcitrant to hormone-mediated regeneration, which hugely impacts breeding and horticulture. We recently developed a new regeneration method employing activation of root stem-cell- related transcription factors without need for hormone application. This method allows dissection of regeneration in an exquisitely controlled manner by inducing the process cell-type specifically. Here, we advance our regeneration system to deduce cell-specific key aspects of regeneration using two crucial novel approaches: • First, we will use single-cell RNA-sequencing (scRNA-seq) to identify cell-specific transcriptional responses that convey regeneration competence, related to the fate and developmental stage of that cell. scRNA-seq is also used to characterize transitioning cell types during designated stages of regenerant development. Our induction system furthermore allows us to assess the cell-autonomy of the regeneration process by fluorescently tagging specifically the activated cells and their progeny. • Second, the induced regeneration system will be combined with a genetic scarring-based lineage tracing system to confidently infer lineage relationships between reprogrammed cell types. The combination of our cell-specific regeneration induction system, single-cell transcriptomics and lineage tracing as the regeneration program unfolds offers a unique opportunity to pinpoint cell-specific responses during plant regeneration. These we will validate during regeneration in other tissues and tomato. Together, our results will greatly benefit the rational design for improved crop regeneration methods.