In plant tissue culture, callus forms from detached explants in response to a high-auxin-to-low-cytokinin ratio on callus-inducing medium. Callus is a group of pluripotent cells because it can regenerate either roots or shoots in response to a low level of auxin on root-inducing medium or a high-cytokinin-to-low-auxin ratio on shoot-inducing medium, respectively¹. However, our knowledge of the mechanism of pluripotency acquisition during callus formation is limited. On the basis of analyses at the single-cell level, we show that the tissue structure of Arabidopsis thaliana callus on callus-inducing medium is similar to that of the root primordium or root apical meristem, and the middle cell layer with quiescent centre-like transcriptional identity exhibits the ability to regenerate organs. In the middle cell layer, WUSCHEL-RELATED HOMEOBOX5 (WOX5) directly interacts with PLETHORA1 and 2 to promote TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 expression for endogenous auxin production. WOX5 also interacts with the B-type ARABIDOPSIS RESPONSE REGULATOR12 (ARR12) and represses A-type ARRs to break the negative feedback loop in cytokinin signalling. Overall, the promotion of auxin production and the enhancement of cytokinin sensitivity are both required for pluripotency acquisition in the middle cell layer of callus for organ regeneration.

在植物组织培养中，愈伤组织从分离的外植体中形成，以响应愈伤组织诱导培养基上高生长素与低细胞分裂素的比例。愈伤组织是一组多能细胞，因为它可以再生根或芽，以响应根诱导培养基上的低水平生长素或芽诱导培养基上的高细胞分裂素与低生长素比率，分别¹。然而，我们对愈伤组织形成过程中多能性获得机制的了解是有限的。在单细胞水平分析的基础上，我们表明拟南芥的组织结构愈伤组织诱导培养基上的愈伤组织类似于根原基或根尖分生组织，具有静止中心样转录特性的中间细胞层表现出器官再生能力。在中间细胞层，WUSCHEL-RELATED HOMEOBOX5 (WOX5) 直接与 PLETHORA1 和 2 相互作用，促进拟南芥 1 的色氨酸氨基转移酶表达，从而产生内源性生长素。WOX5 还与 B 型拟南芥响应调节器 12 (ARR12) 相互作用并抑制 A 型ARR以打破细胞分裂素信号传导中的负反馈回路。总的来说，促进生长素的产生和细胞分裂素敏感性的增强都是愈伤组织中层细胞层获得多能性以实现器官再生所必需的。