Incipient stem cell niche conversion in tissue culture: using a systems approach to probe early events inWUSCHEL-dependent conversion of lateral root primordia into shoot meristems

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Adventitious shoot organogenesis contributes to the fitness of diverse plant species, and control of this process is a vital step in plant transformation andin vitropropagation. New shoot meristems (SMs) can be induced by the conversion of lateral root primorida/meristems (LRP/LRMs) or callus expressing markers for this identity. To study this important and fascinating process we developed a high-throughput methodology for the synchronous initiation of LRP by auxin, and subsequent cytokinin-induced conversion of these LRP to SMs. Cytokinin treatment induces the expression of the shoot meristematic geneWUSCHEL(WUS) in converting LRP (cLRP) within 24–30 h, andWUSis required for LRP → SM conversion. Subsequently, a transcriptional reporter forCLAVATA3(CLV3) appeared 32–48 h after transfer to cytokinin, marking presumptive shoot stem cells at the apex of cLRP. Thus the spatial expression of these two components (WUSandCLV3) of a regulatory network maintaining SM stem cells already resembles that seen in a vegetative shoot apical meristem (SAM), suggesting the very rapid initiation and establishment of the new SMs. Our high-throughput methodology enabled us to successfully apply a systems approach to the study of plant regeneration. Herein we characterize transcriptional reporter expression and global gene expression changes during LRP → SM conversion, elaborate the role ofWUSandWUS-responsive genes in the conversion process, identify and test putative functional targets, perform a comparative analysis of domain-specific expression in cLRP and SM tissue, and develop a bioinformatic tool for examining gene expression in diverse regeneration systems.

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