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

Abstract

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.