The classic experimental system for studying plant regeneration is the two-step tissue culture protocol. Explants, small pieces of plant tissue, are first placed on a callus induction medium containing a balanced ratio of the plant hormones auxin and cytokinin. Under these conditions, a subset of cells re-enters the cell cycle and proliferates into callus, a disorganized mass of cells that resembles lateral root primordia more than any mature tissue. When the callus is then transferred to a shoot induction medium rich in cytokinin, some cells adopt shoot identity and form a shoot apical meristem, the self-renewing stem cell hub that generates leaves, stems, and eventually flowers. Understanding the molecular switches that govern each of these transitions has become one of the central questions in plant developmental biology.

At the heart of the process lies hormonal control. Auxin, arguably the most influential player, drives dedifferentiation and callus formation, and its signaling operates through transport inhibitor response 1 receptors and auxin response factors that directly regulate reprogramming genes. Work in Arabidopsis thaliana has shown that auxin upregulates LEAFY COTYLEDON 1 and 2, transcription factors central to somatic embryogenesis. Studies in rice further revealed that local auxin gradients determine whether regenerating tissues form roots or shoots: high auxin with low cytokinin favors roots, while the opposite combination promotes shoots. PIN-FORMED transport proteins channel auxin directionally to specific sites, initiating de novo root organogenesis with remarkable spatial precision.

Cytokinin acts as auxin’s counterweight. It sustains cell division and, at high concentrations, promotes shoot primordia formation, while lower levels favor roots or somatic embryos. Cytokinin signaling flows through the Arabidopsis response regulator family, and type-B regulators such as ARR1, ARR2, ARR10, and ARR12 bind directly to the promoter of WUSCHEL, the master gene that maintains stem cell activity in the shoot apical meristem. Cytokinin also suppresses auxin accumulation through the YUCCA biosynthetic pathway, and remarkably, it reaches into the epigenetic machinery by modulating histone acetyltransferases, thereby making meristem maintenance genes more accessible for transcription. Mutations in type-B response regulators impair shoot regeneration, whereas overexpressing ARR12 enhances it, and WUSCHEL overexpression can rescue defective regeneration in double mutants, underscoring the centrality of this regulatory module.

Other hormones fine-tune the process. Gibberellins do not initiate reprogramming but sustain proliferation and maturation once cells have been redirected, promoting the elongation of somatic embryos into functional plantlets. Brassinosteroids, acting through the BRI1 receptor, synergize with auxin and cytokinin to boost cell division in callus and support organ formation. Abscisic acid, the stress hormone, plays the antagonist: elevated levels suppress cell division and organogenesis, inducing a dormancy-like state that prevents cells from reverting to totipotency under unfavorable conditions. ABA and cytokinin act antagonistically in shoot regeneration, illustrating how plants calibrate their regenerative responses against environmental risk.

Beneath the hormonal layer sits an elaborate gene regulatory network. Callus formation begins with the division of xylem pole pericycle cells, a process sharing molecular logic with lateral root initiation. The ALF4 protein initiates pericycle division, and root meristem genes including WOX5, WOX11, SCARECROW, PLETHORA 1 and 2, and SHORT ROOT become strongly upregulated. Mutant analyses confirm their importance: plt3plt5plt7 mutants lose shoot progenitor regeneration, and lbd16 mutants show reduced expression of WOX5 and PLETHORA genes with diminished shoot regeneration. On shoot induction medium, CUC1 and CUC2 transcription factors mark pre-meristematic zones, recruit PIN1 polarization, and activate SHOOT MERISTEMLESS to define future shoot progenitors, while ESR1 and ESR2 act upstream to promote the process. A transient root stem cell niche, marked by WOX5 expression, appears to be a prerequisite for the subsequent switch to shoot identity.

Small RNAs add another layer of temporal and spatial control. The miR156-SPL module explains why regeneration efficiency declines with plant age: juvenile plants with high miR156 repress SPL transcripts, keeping cytokinin responses active, whereas older plants accumulate SPL proteins that suppress type-B response regulators and reduce regeneration. miR165/166 promotes shoot formation, and the ARGONAUTE10 protein sequesters these microRNAs, with ago10 mutants showing elevated miR165/166 and enhanced regeneration. Meanwhile, a miR160-ARF10-ARR15 cascade fine-tunes the early balance between auxin and cytokinin signaling during callus formation, demonstrating how microRNA circuits integrate hormonal cues across developmental stages.

Perhaps the most striking insight from the review is that reprogramming is governed less by genetic change than by epigenetic rewiring. Global DNA hypomethylation is a hallmark of callus formation across species. In Arabidopsis, MET1 maintains CG methylation, and its loss increases WUSCHEL transcription and accelerates shoot formation; met1 mutants produce more shoots. Yet local hypermethylation also matters, silencing organ identity genes such as GSTU10, MAPK12, and BXL1 to help establish an undifferentiated cell mass. Histone marks are equally dynamic: H3K9ac and global H3 acetylation rise during callus formation, activating sugar metabolism, peptide signaling, and hormone transport genes while repressing photosynthesis programs. The demethylase JMJ30 removes repressive H3K9me3 marks from LBD16 and LBD29 promoters to enable callus formation, while ATX4 deposits activating H3K4me3 at shoot identity genes. Loss of the deacetylase HDA19 suppresses shoot regeneration, showing that both writing and erasing these marks is essential.

Proteomic studies, though still limited in number, reveal consistent patterns. Embryogenic cells accumulate heat-shock proteins as molecular chaperones, elevated glutathione-S-transferases that detoxify auxin, and cytoskeletal components such as tubulin and actin that establish cell polarity during division. Carbohydrate metabolism enzymes, photosynthetic proteins that appear as embryos mature, and nuclear import factors like importin all shift in abundance, reflecting the massive metabolic and architectural reorganization that accompanies the acquisition of pluripotency. Reactive oxygen species homeostasis intersects with auxin signaling during dedifferentiation, linking stress physiology directly to developmental potential.

New technologies are transforming the field’s resolution. Single-cell RNA sequencing has mapped the cellular composition of Arabidopsis and rice roots, revealed hormone signaling variation across cell clusters, and, when combined with ATAC-seq, identified WUSCHEL and DORNROESCHEN as crucial factors for regenerating mesophyll cells. CRISPR-based activation has been used to enhance somatic embryogenesis in tomato by targeting the SlWRKY29 gene, and CRISPR-dCas9 systems now allow precise transcriptional tuning of master regulators. Even artificial intelligence has entered the tissue culture lab: machine learning models, including neural networks and gradient boosting algorithms, have successfully predicted optimal media compositions for callus induction in carrot, micropropagation of lavender, and callogenesis in saffron, cutting both cost and experimental time. Together, these tools point toward a future in which regeneration efficiency can be engineered rather than stumbled upon, with implications for crop improvement, the propagation of recalcitrant and endangered species, and a deeper understanding of how any cell, given the right molecular instructions, can be persuaded to start over.

Subject of Research: Molecular mechanisms of cell fate reprogramming during de novo organogenesis in plants

Article Title: Understanding the molecular landscape of de novo organogenesis: insights into cell fate reprogramming

Article References: Nath, J., Kumari, A., Joshi, S., Gusain, S., Kumari, K., Yadav, S. K., & Joshi, R. (2025). Understanding the molecular landscape of de novo organogenesis: insights into cell fate reprogramming. Discover Biotechnology, 2(1), Article 27. https://doi.org/10.1007/s44340-025-00035-w

Image Credits: AI Generated

DOI: 10.1007/s44340-025-00035-w

Keywords: de novo organogenesis, cell fate reprogramming, auxin, cytokinin, WUSCHEL, epigenetics, DNA methylation, histone modification, callus, single-cell RNA sequencing, CRISPR, plant regeneration