Cytokinin-supplemented medium drives de novo shoot regeneration in petunia (Petunia hybrida) by bypassing the root-meristem program

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In vitro shoot regeneration is critical for plant propagation and serves as a model for cellular reprogramming. The classic Arabidopsis thaliana two-step system requires an auxin-rich callus-inducing medium to generate pluripotent callus with lateral root primordium (LRP) identity. However, whether this LRP-dependent pathway is universally required in one-step protocols using cytokinin-supplemented medium remains unclear. Here, we investigated one-step shoot regeneration from petunia ( Petunia hybrida ) leaf explants on cytokinin-supplemented medium using time-course RNA-seq. Clustering analysis of 7,357 differentially expressed genes identified six temporal expression patterns associated with wound response, metabolic activation, cell division, and shoot meristem formation. Strikingly, key LRP-associated pluripotency markers— WUSCHEL-RELATED HOMEOBOX 5 , PLETHORA 1a/b , and LATERAL ORGAN BOUNDARIES DOMAIN 16/29 —were not activated on cytokinin-supplemented medium, although they were readily induced on auxin-containing medium. These findings indicate that shoot regeneration proceeds via a root-meristem-independent pathway. Instead, on cytokinin-supplemented medium, shoot apical meristem (SAM) regulators were activated, including ENHANCER OF SHOOT REGENERATION 1a ( ESR1a , day 4), WUSCHEL (day 4), SHOOT MERISTEMLESS and NO APICAL MERISTEM (day 7). The auxin pathway was transiently suppressed but was subsequently reconfigured, with specific upregulation of AUXIN RESPONSE FACTOR 5 ( ARF5 ), the sole induced ARF member. Overexpression of petunia WOUND INDUCED DEDIFFERENTIATION 1a ( WIND1a ), a key wound-induced regulator in Arabidopsis , did not enhance regeneration, suggesting the canonical WIND-ESR1 axis is bypassed. We propose a model in which wounding and cytokinin coordinately drive callus proliferation, induce SAM genes, and upregulate PhARF5 without establishing an LRP-like state. This study reveals a streamlined, root-meristem-independent route to shoot regeneration, underscoring the plasticity of plant regenerative programs.

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