The core challenge the team faced is one that has stymied progress across many orphan crops: without an efficient tissue culture system, scientists cannot easily insert genes, edit genomes, or study gene function in a living plant. For most well-studied cereals such as rice, maize, and wheat, decades of optimization have produced robust protocols in which immature embryos—the young, undifferentiated plant tissue harvested from developing seeds—are coaxed into forming callus, a mass of regenerable cells, and then into whole plants. But immature embryos come with a heavy logistical price. They must be collected during a narrow window of seed development, which means researchers need a constant supply of flowering plants, careful timing, and considerable labor. For a crop like Taiwan oil millet, whose growth habits and flowering patterns are not well characterized in laboratory settings, this dependency creates a bottleneck that can stall research for years.

The Taiwanese team’s solution was elegant in its simplicity: use seedlings instead. Rather than harvesting immature embryos at a precise developmental moment, the researchers germinated mature seeds under controlled conditions and used the resulting young seedlings as the starting material for callus induction. This shift removes the strict developmental-stage constraints that plague embryo-based systems, allowing experiments to proceed year-round. Seeds can be stored, germinated on demand, and processed in batches, making the entire workflow scalable and accessible even to laboratories without specialized greenhouse facilities for maintaining large populations of flowering plants. The approach also sidesteps the seasonal limitations that would otherwise dictate when experiments could begin.

Importantly, the team did not start from scratch. They drew on insights from previously reported tissue culture systems in sorghum, a close C4 relative with a more established research infrastructure. By adapting sterilization procedures, callus induction media, and regeneration conditions that had proven successful in sorghum, the researchers gave themselves a rational starting point rather than an exhaustive trial-and-error search. This cross-species knowledge transfer is a strategy increasingly favored in orphan crop research, where funding and time are limited and lessons learned in better-studied relatives can dramatically shorten the path to a working protocol.

The optimization process touched every stage of the regeneration pipeline. Seed sterilization protocols had to be tuned to eliminate microbial contamination without damaging the delicate seedling tissue. Callus induction conditions were refined so that efficient callus formation occurred within just four weeks of culture initiation. The researchers also incorporated plant growth regulators, the hormone-like signaling molecules that steer plant cells between growth, differentiation, and organ formation, and found that their judicious use further improved regeneration efficiency. Once formed, the calli could be reduced in size and maintained their proliferative capacity through repeated subculturing, meaning that a single batch of starting material could sustain extended lines of experimentation rather than being exhausted after one round.

The headline numbers from the study are striking. The optimized protocol achieved a regeneration efficiency of 97.9 percent, meaning that nearly every callus that entered the regeneration phase gave rise to a complete plant. From the initial induction of callus to mature plants that had been acclimatized to soil, the entire process took between twelve and fourteen weeks. That timeline is competitive with established cereal systems and represents a practical pace for research programs that need to generate, evaluate, and propagate genetically modified or edited lines. Equally significant, the calli displayed stable regenerative capacity over extended culture periods, a property that guards against the gradual loss of regenerability that often undermines long-term tissue culture work in other species.

Why does all this matter beyond the laboratory bench? Taiwan oil millet is what agricultural scientists call an orphan crop: a species of local importance that has been largely ignored by commercial breeding and international research. Orphan crops are frequently adapted to marginal environments—poor soils, drought, high elevations—precisely because they were never selected for maximum yield under ideal conditions. As climate change intensifies pressure on global agriculture, there is growing interest in mining these resilient species for traits that could fortify food systems. Taiwan oil millet, cultivated traditionally by Indigenous peoples in Taiwan’s mountains, embodies that resilience. A working regeneration system is the first prerequisite for identifying the genetic basis of its hardiness and, if desired, introducing those traits into other crops or improving the millet itself through modern breeding.

The second major motivation is scientific rather than agricultural: C4 biology. Most plants, including rice and wheat, use the C3 photosynthetic pathway, in which a key enzyme often wastes energy by binding oxygen instead of carbon dioxide. C4 plants, which include maize, sorghum, sugarcane, and Taiwan oil millet, have evolved a biochemical and anatomical specialization that concentrates carbon dioxide around the carbon-fixing machinery, making photosynthesis dramatically more efficient in hot, bright conditions. Engineers and plant biologists have long dreamed of installing C4 traits into C3 staple crops to boost yields, but the underlying biology remains incompletely understood. A C4 grass that is small, tractable, and now amenable to tissue culture and, prospectively, genetic manipulation could serve as a model organism for dissecting C4 photosynthesis in ways that towering maize plants or slow-growing sugarcane cannot. Taiwan oil millet’s compact growth habit and newly established regenerability position it as exactly such a model.

The authors are explicit that the regeneration system is a foundation rather than a finished toolkit. Stable transformation and genome editing in plants depend on the ability to regenerate whole plants from cells that have taken up foreign DNA or undergone targeted edits. With a 97.9 percent regeneration rate and sustained callus proliferation now demonstrated, the path to developing those downstream systems in Taiwan oil millet is open. The team acknowledges the contributions of collaborators who provided and maintained the plant materials, including researchers at National Chiayi University and the Hualien District Agricultural Research and Extension Station, underscoring how much of this work rests on the careful stewardship of a crop that exists largely outside mainstream seed banks and commercial channels. The research was supported by Taiwan’s National Science and Technology Council.

The broader lesson of the study resonates across plant science. As genome editing tools such as CRISPR have become fast and inexpensive, the limiting step in improving neglected species is often not the editing itself but the plant biology surrounding it: getting cells to take up DNA, and getting edited cells to become plants. By demonstrating that a simple, scalable seedling-based approach can deliver near-complete regeneration in a previously intractable orphan grass, the Taiwanese team has offered a template that other researchers working on underutilized crops may follow. For Taiwan oil millet, a species that fed mountain communities for generations before fading from the fields, the work represents a chance at a second life—first as a window into one of nature’s most efficient photosynthetic machines, and perhaps eventually as a resilient grain returned to cultivation in a warming world.

Subject of Research: Development of a seedling-based tissue culture regeneration system for the orphan crop Taiwan oil millet to enable genetic studies, crop domestication, and C4 photosynthesis research

Article Title: Establishment of a seedling-based regeneration system for Taiwan oil millet (Eccoilopus formosanus) to support crop domestication and C4 biology

Article References: Hsieh, K.-T., Chuang, H.-Y., Hsu, C.-H., Chen, Y.-F., Li, W.-H., Lin, H.-C., Hsing, Y.-I. C., Hong, C.-Y., & Chang, M.-C. (2026). Establishment of a seedling-based regeneration system for Taiwan oil millet (Eccoilopus formosanus) to support crop domestication and C4 biology. Plant Methods. https://doi.org/10.1186/s13007-026-01599-2

Image Credits: AI Generated

DOI: 10.1186/s13007-026-01599-2

Keywords: Taiwan oil millet, Eccoilopus formosanus, tissue culture, plant regeneration, orphan crops, C4 photosynthesis, callus induction, plant growth regulators, genome editing, crop domestication, seedling-based culture, Plant Methods

The histone in question is H3, one of the core proteins around which DNA is wound in every plant cell. At lysine 27, the twenty-seventh amino acid of that protein, the cell faces a fundamental choice. Acetylating this residue, essentially attaching a small chemical acetyl group to it, generally loosens the grip of chromatin and switches genes on. Deacetylating the same residue does the reverse, tightening the packaging and silencing the underlying DNA. For decades, textbooks have treated these two processes as opposing forces in a tug of war, with activating marks on one set of genes and repressive marks on another. The work highlighted by the Silesian team, originally reported by Iwase and colleagues in Molecular Plant, reveals something far more surprising: WIND1 appears to promote both sides of this chemical contest at the same time, and the cell is all the more responsive for it.

WIND1 is an AP2-family transcription factor first identified in Arabidopsis thaliana, the thale cress that serves as botany’s favorite laboratory workhorse. Its name betrays its origin story. When plant tissue is wounded, WIND1 springs into action, driving dedifferentiation, the process by which mature cells shed their specialized character and regain a stem-like, pluripotent state. This is the biological foundation of callus formation, the mass of undifferentiated cells that sprouts at wound sites and in tissue culture, and from which entire plants can ultimately be regenerated. Earlier studies had shown that WIND1 could induce callus not only in Arabidopsis but also in rapeseed, tomato and tobacco, hinting that its reprogramming power is broadly conserved among flowering plants.

What makes the new findings so compelling is the mechanism behind that power. According to the research summarized in the commentary, WIND1 simultaneously promotes histone H3 acetylation at lysine 27, known to chromatin biologists as H3K27ac, and induces the expression of genes associated with the embryogenic response. At the same time, the factor activates deacetylation of that very same histone mark and represses genes tied to differentiation. In other words, WIND1 does not simply flip a global switch from off to on. Instead, it acts like a master electrician rewiring a building, cutting power to the rooms that keep the cell locked in its old identity while flooding the rooms that define an embryo with light, all in one coordinated operation.

The context in which this reprogramming unfolds is somatic embryogenesis, the remarkable process by which vegetative cells, with no connection to flowers, ovules or seeds, are coaxed into forming embryos. Understanding the triggers of this embryogenic program remains, as the commentary’s authors put it, a major focus of modern molecular biology, and for good practical reasons. Somatic embryogenesis is widely used for clonal propagation of elite crops and forest trees, for cryopreservation of embryogenic material, and for genetic transformation, the insertion of new genes into plant genomes. A bibliometric analysis cited in the commentary counts more than nine thousand articles on somatic embryogenesis spanning fifty-five years, a testament to both the scientific fascination and the commercial stakes involved.

The economic dimension is hard to overstate. In conifers, where embryogenic tissue induction and maintenance remain stubborn bottlenecks, researchers are exploring small molecules to overcome the barriers. In citrus, recent work has shown that peptide signals can significantly enhance transformation efficiency even as they inhibit shoot regeneration, underscoring how finely balanced these developmental pathways are. In Europe, the micropropagation of economically important fruit species continues to face challenges that better mechanistic understanding could help solve. Woody species in particular depend on cryopreservation of embryogenic material for long-term conservation of genetic resources. Every insight into how cells are pushed into an embryogenic state therefore translates directly into faster, cheaper and more reliable propagation and breeding pipelines.

Against this backdrop, WIND1’s dual role in histone modification takes on real biotechnological significance. If the factor coordinates both acetylation and deacetylation to drive the embryogenic transition, then manipulating WIND1 or the enzyme complexes it recruits could make recalcitrant crop species far more amenable to regeneration and transformation. The commentary situates the finding within a broader landscape of research on developmental regulators that boost transformation efficiency in maize and other plants, and on the chromatin accessibility dynamics that underpin somatic embryogenesis. A hierarchical transcriptional regulatory network has been mapped for the process, and LEAFY COTYLEDON genes, especially LEC2, a B3-domain transcription factor famous for inducing embryo development when ectopically expressed, have long been recognized as essential players. WIND1 now emerges as a regulator that sits upstream of the epigenetic machinery itself, commanding the very chemical marks that open and close the genome.

The deeper conceptual payoff of the work may lie in how it reframes the biology of pluripotency. In animal systems, histone acetyltransferases and histone deacetylases are often studied as antagonists, and inhibitors of histone deacetylases are already in clinical use as cancer therapies, reflecting the power of tipping the acetylation balance. Plants appear to have evolved a more integrated solution: rather than relying on a global shift in one direction, the WIND1 pathway deploys both modifications in a targeted, gene-specific manner, activating embryogenic genes while silencing differentiation genes in the same cells at the same time. The commentary’s authors, whose work is supported by the National Science Centre of Poland under the OPUS 26 plus LAP call in the Weave program, emphasize that this coordinated integration of opposing marks is what enables the cell fate transition during somatic embryogenesis. It is a reminder that epigenetic regulation is less a switch than a symphony, with a single conductor drawing both crescendo and silence from the same orchestra.

There are also intriguing parallels with wound signaling beyond WIND1 itself. The peptide REF1 has recently been identified as a local wound signal promoting plant regeneration, and related peptides have been shown to enhance transformation in citrus. Wound-induced signals and chromatin remodeling evidently form a connected circuit: injury triggers molecular messengers, which activate transcription factors such as WIND1, which in turn remodel histone marks to unlock the embryogenic program. Even close relatives of WIND1 in other species, such as the AP2 transcription factor ThWIND1-L from the salt-tolerance relative Thellungiella halophila, suggest that this regulatory module is ancient and adaptable. As reviews of epigenetic regulation in plant regeneration make clear, the field is converging on a picture in which transcription factors and chromatin modifiers operate as an inseparable unit, with factors like WIND1 serving as the bridge between external signals and the epigenetic code.

For now, the commentary by Falińska, Nowak and Wójcikowska serves as both a synthesis and a provocation. It distills the message that the embryogenic transition in plants hinges on a transcription factor capable of directing opposing H3K27 modifications, and it challenges researchers to think of acetylation and deacetylation not as rivals but as partners in reprogramming. If that principle holds across species, the implications stretch from the tissue culture bench to the forest nursery and the crop field, wherever scientists seek to coax a mature cell back to the beginning. The question posed in the original title, to acetylate or to deacetylate, turns out to have a surprising answer: for WIND1, the answer is both, and that is precisely what gives the factor its power to rewrite a cell’s destiny.

Subject of Research: Epigenetic control of plant somatic cell reprogramming by the WIND1 transcription factor

Article Title: To acetylate or deacetylate? WIND1 directs opposing H3K27 modifications in plant somatic cell reprogramming

Article References: Falińska, J., Nowak, K., & Wójcikowska, B. (2026). To acetylate or deacetylate? WIND1 directs opposing H3K27 modifications in plant somatic cell reprogramming. Plant Molecular Biology, 116(5), Article 100. https://doi.org/10.1007/s11103-026-01765-z

Image Credits: AI Generated

DOI: 10.1007/s11103-026-01765-z

Keywords: WIND1, histone acetylation, H3K27, somatic embryogenesis, plant regeneration, epigenetics, transcription factor, pluripotency, chromatin remodeling, callus formation, Arabidopsis, plant biotechnology

Protoplasts are plant cells stripped of their rigid cellulose walls, usually digested free from leaf tissue by cocktails of commercial enzymes. Freed from the wall, they behave like single cells in culture: they can be fused with cells of other species to sidestep sexual incompatibility, transformed with gene-editing machinery such as CRISPR components, and pushed to regenerate entire plants through somatic embryogenesis. Because protoplasts can develop into plantlets faster and at larger scale than conventional callus-based micropropagation, they have long been attractive to breeders of crops where open-pollinated cultivars produce inconsistent yields, as is the case for carrot and many other members of the Apiaceae family. Carrot itself is a historic model in this field; the first protoplast isolation from carrot root slices was reported in 1972, and the species has since served as a workhorse for studies of somatic hybridization, cytoplasmic male sterility, and transformation.

The new study compared two carrot varieties, the hybrid Champion F1 and the male-sterile Naigara, using leaves from aseptically grown seedlings as the starting material. Leaf tissue avoids the laborious cell suspension cultures traditionally used for carrot protoplasts, which can take three to eight months to establish and have historically delivered plating efficiencies of twenty percent or less. Leaves of three- to four-week-old seedlings were finely chopped, plasmolyzed in mannitol solution, and digested overnight with enzyme mixtures built around Cellulase Onozuka R-10, Macerozyme R-10, and Pectolyase Y-23. Released protoplasts, roughly 22 micrometers in diameter and green with randomly distributed chloroplasts, were sieved, pelleted by gentle centrifugation, and purified on a sucrose gradient before counting in a hemocytometer.

The enzyme recipes mattered, and in a variety-dependent way. For Naigara, the mixture of one percent Cellulase Onozuka R-10 with 0.1 percent Pectolyase Y-23 yielded 2.29 million protoplasts per gram of fresh leaf, while a stronger mixture of two percent cellulase with 0.2 percent Macerozyme R-10 raised the yield to 3.74 million per gram. Champion F1 showed the opposite trend: the pectolyase-based mixture delivered the best result, 4.9 million protoplasts per gram, nearly double the 2.42 million obtained with the macerozyme recipe. Viability, assessed by fluorescein diacetate staining under a fluorescence microscope, exceeded 70 percent in all cases, peaking at 87.5 percent for Champion F1 with the pectolyase mixture. The authors attribute the different responses of the two varieties primarily to variations in cell wall structure, a reminder that even within a single species, protoplast work remains a bespoke craft. The yields reported are comparable to those in prior Daucus studies, which have ranged from roughly 1.3 to 5.9 million protoplasts per gram across species and varieties.

The bitter gourd result is the study’s most novel contribution. Bitter gourd is valued across Asia for both nutrition and medicinal compounds, but its long juvenile phase, resistance to transformation, and variety-dependent regeneration protocols have hampered conventional breeding. The team tested ten different enzyme combinations on leaves of the open-pollinated variety KSP 1522, and only one recipe, combining 1.5 percent Cellulase Onozuka R-10 with 0.2 percent Macerozyme R-10, worked. The protoplasts proved highly sensitive: most other enzyme treatments either failed to liberate cells or produced protoplasts that burst during centrifugation. The successful combination yielded 1.18 million protoplasts per gram, about 24 micrometers in diameter, with viability of just over 80 percent that declined only modestly, by around eight percent, after 24 hours. Because viability above roughly 65 percent is generally considered sufficient for regeneration, the authors present this as a foundational protocol, while cautioning that it is optimized for this specific variety and explant, and that yields remain sub-optimal pending further refinement.

For carrot, the team went beyond isolation and demonstrated the full pipeline from naked cell to field-ready root. Protoplasts were embedded in thin layers of calcium alginate, a technique that protects the fragile cells, allows undisturbed cell walls to regenerate, prevents clumping, and improves mass transfer of nutrients because the matrix is thin. Within four to six weeks, visible macrocolonies emerged, developed into proembryonic masses, and then differentiated into globular, torpedo, and cotyledonary-stage somatic embryos over one to two months. Embryos were released from the alginate with a mannitol and sodium citrate solution and transferred to regeneration medium containing zeatin and naphthaleneacetic acid. Plantlets appeared within weeks, and germination frequency was so high that thousands of plantlets, too many to count individually, were produced. Of 332 plantlets hardened off in cocopeat under gradually reduced humidity, 319 survived acclimatization, a 96 percent success rate with no morphological abnormalities.

The cold storage experiments are arguably the most practically significant. Plant genetic resources are conventionally conserved as seeds, field collections, or in vitro cultures, with cryopreservation in liquid nitrogen as the gold standard for recalcitrant material. But cryopreservation is expensive, technically demanding, and, for carrot protoplasts, previously documented only once, with a colony-forming capability of just 19 percent after six months in liquid nitrogen. The Indian team instead stored Champion F1 protoplasts in carrot petiole protoplast medium at ordinary refrigerator temperatures of 2 to 8 degrees Celsius. After five months, the protoplasts were still viable by FDA staining, although exact counts were complicated by clumping, and they showed partial cell division upon rewarming. When embedded in alginate and cultured, the cold-stored protoplasts regenerated into plantlets within a week of transfer to regeneration medium.

The outcome of that regeneration pipeline was remarkable: from 253 plants hardened after regeneration from five-month-old cold-stored protoplasts, 216 survived acclimatization, an 85 percent efficiency. The regenerated plants completed a three-month growth cycle and produced healthy orange carrots, confirming that the entire pathway, from wall-less cell stored in a refrigerator to harvestable storage root, can function without cryogenic equipment. The authors are careful to note the limitations: the study rests on only two data points, and systematic work across longer storage durations with multiple time points will be needed to establish how far this simple approach can be pushed.

Taken together, the results offer breeding programs a practical toolkit. Reliable leaf-based protoplast isolation eliminates the need for long-lived suspension cultures; demonstrated cold storage at 2 to 8 degrees Celsius gives labs a low-cost buffer for scheduling fusions, transformations, and regeneration experiments weeks or months apart; and the first protoplast protocol for bitter gourd opens a route toward transgene-free genome editing in a crop whose medicinal value and breeding difficulties have long made it a difficult target. With CRISPR components already shown to be deliverable to protoplasts, and gene-edited carrots previously produced via protoplast transformation, the ability to isolate, bank, and regenerate these cells on demand brings somatic hybridization and precision editing for both crops a step closer to routine practice.

Subject of Research: Protoplast isolation, cold storage and plant regeneration in carrot and bitter gourd

Article Title: Protoplast isolation in Daucus carota and Momordica charantia: regeneration and cold storage studies in carrot

Article References: Ranaware, A. S., Kushwaha, S. B., Kunchge, N., Prakash, G., & Lele, S. S. (2025). Protoplast isolation in Daucus carota and Momordica charantia: regeneration and cold storage studies in carrot. Discover Biotechnology, 2(1), Article 24. https://doi.org/10.1007/s44340-025-00034-x

Image Credits: AI Generated

DOI: 10.1007/s44340-025-00034-x

Keywords: protoplasts, carrot, bitter gourd, Daucus carota, Momordica charantia, plant regeneration, somatic embryogenesis, cold storage, tissue culture, cellulase, plant breeding, CRISPR

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

The story begins with a decades-old foundation. Since Martin Skoog and Folke Miller demonstrated in 1957 that the balance of two plant hormones, auxin and cytokinin, dictates whether cultured tissue forms roots or shoots, tissue culture has been largely an empirical craft. Laboratories fine-tune media recipes, hormone concentrations, and explant choices, but comparatively little attention has gone to the genetic determinants of regeneration competence. This gap matters because regeneration ability varies dramatically between species and even between cultivars of the same crop. Tomato is a striking example. Some lines transform and regenerate with ease while others remain stubbornly recalcitrant, and earlier work had already mapped quantitative trait loci and identified a handful of major genes influencing the trait. What was missing was a clear molecular culprit, a gene whose loss could be shown to shut regeneration down completely, and whose restoration could switch it back on. The Spanish team, screening a collection of more than 4,000 tomato T-DNA insertion lines generated with an enhancer trap construct, found exactly such a mutant.

The mutant, named dor, looked deceptively ordinary at first glance. When cotyledon and hypocotyl explants from the mutant seedlings were placed on callus-inducing medium, they dedifferentiated normally, proliferating into callus tissue just as wild-type explants did. But the process stopped there. On shoot-inducing medium, no adventitious buds ever emerged from any explant. On root-inducing medium, no adventitious roots formed either. The tissue remained trapped in an undifferentiated state, unable to take the decisive step of organizing itself into meristems, the specialized structures that generate new organs. The defects extended into ordinary development as well. Twenty-day-old dor seedlings had embryonic root systems roughly half the weight and total length of their wild-type counterparts, with a normal average root diameter but far fewer root tips, indicating that the formation of lateral roots was specifically impaired. Adventitious roots arising from stem tissue showed the same stunted, sparsely branched character. By sixty days, both the roots and the aerial parts of the mutant were visibly underdeveloped, although the plant could eventually complete its life cycle, producing normal flowers and fruits, only more slowly than usual.

Grafting experiments delivered the most telling clue about how the mutant’s peculiar phenotype fits together. When a wild-type scion was grafted onto a dor rootstock, the mutant root system remained as abnormal as ever. But when a dor scion was grafted onto wild-type roots, the shoot development of the mutant recovered dramatically, becoming nearly indistinguishable from a normal plant. In other words, the poor aerial growth of dor plants was not an independent shoot defect but a downstream consequence of a defective root system. The root genotype was calling the shots. This pointed the researchers toward a fundamental cellular process rather than an organ-specific one. Hormone profiling deepened the puzzle further. Measurements of auxins, cytokinins, gibberellins, salicylic acid, and jasmonic acid in cotyledon explants revealed no significant differences between mutant and wild type, with abscisic acid the only hormone showing a statistically significant difference, and even that vanished when measured in leaves. Supplementing the culture media with various auxins, or doubling hormone concentrations, failed to rescue the mutant. Whatever DOR does, it apparently operates beyond the classical hormone-driven pathways that dominate regeneration biology textbooks.

The genetic detective work took an unexpected turn. Segregation analysis in the T1 progeny showed the mutation behaved as a single recessive gene, but when the researchers tested whether the visible T-DNA insertion cosegregated with the phenotype, it did not. A few kanamycin-sensitive seedlings carried the mutant phenotype despite lacking the insert, revealing that the dor mutation had arisen not from the inserted DNA but from somaclonal variation, a spontaneous genetic change occurring during the tissue culture process used to generate the lines. An allelic mutant, dor-MM, was subsequently identified in an independent Money Maker T-DNA line, and a complementation cross between the two produced entirely mutant F1 offspring, proving both mutations disrupted the same gene. To find it, the team turned to mapping-by-sequencing, crossing the mutant to a wild tomato accession, sequencing pooled DNA from wild-type and mutant F2 plants, and scanning the genome for the region where allele frequencies diverged. The signal converged on the distal end of chromosome 12, where variant analysis uncovered a single thymine insertion in exon 14 of a gene called Solyc12g098670, causing a frameshift and a premature stop codon that truncated the predicted 532-amino-acid protein at position 511. The Money Maker allele carried its own frameshift, a thymine deletion in exon 10. All 40 mutant F2 plants were homozygous for the insertion, while the 158 wild-type plants were either heterozygous or free of it.

The identity of the gene came as a genuine surprise. Solyc12g098670 encodes a signal peptide peptidase-like protease, a member of the SPPL family of intramembrane aspartyl proteases, most closely related to the Arabidopsis proteins AtSPPL3 and AtSPPL5, sharing 54.8 and 57.5 percent sequence identity respectively. These are unusual enzymes. Rather than cutting proteins in watery cellular compartments, they cleave within the oily interior of biological membranes, and their best-known relatives in humans play crucial roles in the immune response, residing in the endoplasmic reticulum, the Golgi apparatus, lysosomes, and the plasma membrane. In Arabidopsis, one SPP and five SPPL-like genes are known, and the canonical SPP protein is essential for pollen function, cleaving signal peptides and failing catastrophically when disrupted, with mutant alleles transmissible through pollen at less than two percent the normal rate. In rice, OsSPPL1 and OsSPPL2 participate in endoplasmic reticulum-associated protein degradation and stress tolerance. But no plant SPPL protease had ever been connected to rooting or regeneration. Expression analysis showed that DOR is nearly ubiquitous in tomato, active in roots, stems, leaves, and across every stage of reproductive development from tiny floral buds to ripe fruit, peaking in breaker-stage fruit, a pattern closely mirroring AtSPPL3 in Arabidopsis.

Two independent lines of evidence confirmed that Solyc12g098670 really is DOR, and they came with an accidental experiment built in. When the team silenced the gene with RNA interference constructs, transformation efficiency collapsed to 0.42 percent, compared with the laboratory’s usual rate of around 15 percent, and no silenced lines could be recovered at all in the P73 background. The two silenced plants that were eventually obtained, one in Money Maker and one in the wild relative Solanum pennellii, displayed exactly the dor phenotype: delayed rooting, short sparsely branched roots, failure to form adventitious roots, and impaired shoot regeneration. In the T1 progeny of the silenced line, only the kanamycin-resistant plantlets, which retained the silencing construct, failed to regenerate. CRISPR/Cas9 knockout told the same story, with an editing efficiency of 5.82 percent, also well below normal. Two edited Money Maker lines carried loss-of-function alleles predicted to produce truncated proteins, and their T1 progeny all resembled the dor mutant, with reduced shoot development and slow, sparsely branched adventitious roots. The very difficulty of generating these lines was itself evidence: reducing DOR activity in the genome cripples the regeneration machinery that gene transformation itself depends upon.

The clincher came from the opposite direction. Introducing the DOR gene under a strong constitutive promoter into the mutant plants rescued the phenotype completely, restoring the capacity to form roots and yielding a transgenic plant with vegetative development indistinguishable from wild type, despite having no functional native copy. Overexpression in healthy plants produced no visible abnormalities, although a curious ceiling emerged: none of the 30 overexpression lines exceeded roughly twice wild-type expression levels, hinting that the plant may not tolerate much higher doses of the protease. Intriguingly, the Arabidopsis homologs did not behave the same way. T-DNA mutants disrupting AtSPPL3 showed only mildly stunted growth with perfectly normal roots and intact regeneration capacity, while AtSPPL5 disruption produced no phenotype at all, consistent with its barely detectable expression. Functional redundancy among Arabidopsis SPPL genes might partly explain the difference, as recently demonstrated in rice where only double mutants show stress sensitivity, but the severe defects caused by DOR loss in both cultivated tomato and its wild relative S. pennellii indicate that the closest tomato paralog cannot compensate. SPP/SPPL proteases, it seems, have functionally diversified across plant lineages, with rapeseed SPPL4 governing pollen fertility, Arabidopsis SPP governing gametophyte development, and tomato DOR governing the regeneration of entire organs.

The practical implications could be substantial. Regeneration efficiency is a major bottleneck for transforming recalcitrant crops, and DOR now offers a defined genetic target for manipulating morphogenetic competence. The researchers suggest that introducing DOR into poorly regenerating tomato cultivars, or even into notoriously difficult species such as woody perennials and legumes, might enhance their tissue culture responsiveness, with the complementation experiment serving as proof of principle. A caution accompanies the promise, however, in the apparent upper limit on DOR expression levels. Beyond the application, the finding reframes the biology of totipotency itself. Because dor callus dedifferentiates normally but fails at the determination phase, when cells commit to becoming organs, DOR likely enables some core cellular process, perhaps the proteolytic processing of membrane-associated signaling substrates, that allows cells to perceive or execute organogenic instructions. The endogenous substrates of the protease remain unknown, and whether plant SPPL proteins activate signaling peptides during developmental reprogramming is now an open and tantalizing question. What is certain is that a protein family once studied mainly for its role in human immunology has, in a tomato mutant that could not make roots, revealed one of the hidden gatekeepers of plant regeneration.

Subject of Research: Identification of the tomato DOR gene encoding a signal peptide peptidase-like protease required for adventitious organogenesis and rooting

Article Title: Tomato DOR encodes a signal peptide peptidase-like protein required for adventitious organogenesis and rooting

Article References: Jáquez-Gutiérrez, M., Bretones, S., Martin-Vásquez, C., Fonseca, R., Aguiar, A., Pineda, B., Lozano, R., Moreno, V., Yuste-Lisbona, F. J., & Atarés, A. (2026). Tomato DOR encodes a signal peptide peptidase-like protein required for adventitious organogenesis and rooting. Plant Cell Reports, 45(10), Article 297. https://doi.org/10.1007/s00299-026-03979-3

Image Credits: AI Generated

DOI: 10.1007/s00299-026-03979-3

Keywords: tomato, DOR gene, adventitious organogenesis, rooting, root development, signal peptide peptidase-like, SPPL family, Solanum lycopersicum, plant tissue culture, CRISPR/Cas9, mapping-by-sequencing, plant regeneration

Plant tissue culture begins with a biological reset. Under carefully controlled conditions, specialized cells can lose aspects of their original identity and begin dividing as an undifferentiated tissue known as callus. With the right hormonal signals, some callus can later form roots, shoots or embryos and eventually develop into a complete plant. This ability, called cellular totipotency, underpins several crop technologies, including Agrobacterium-mediated transformation, particle bombardment, doubled-haploid production and CRISPR-based genome editing. Maize, however, is notably difficult to culture, particularly when researchers work with tropical and subtropical genotypes. Immature zygotic embryos have historically been favored because they can produce embryogenic callus, but they are available only during a narrow developmental window and generally require controlled pollination, seasonal planning and suitable greenhouse facilities. Seedling-derived explants could offer a more accessible alternative because they can be produced from mature seeds and prepared under laboratory conditions throughout the year.

Danielle Christelle Tinak Ekom and Abba Haïcha Diko evaluated the approach using the local varieties ATP, CHABA, CHH and KASSAI. The seeds were surface-sterilized and germinated on Murashige and Skoog basal medium supplemented with sucrose and plant growth regulators. After two weeks, the researchers cut the seedlings into two types of explants. Internodes were split longitudinally to expose tissue near the shoot meristem, while young leaves were cut into pieces approximately half a centimeter long. The explants were then placed on five callus-induction media, each based on the same mineral salts and vitamins but containing different combinations of the synthetic auxin 2,4-dichlorophenoxyacetic acid, or 2,4-D, and the cytokinins benzylaminopurine, known as BAP, or kinetin. The formulations also included casein hydrolysate, silver nitrate and spermidine, compounds used to support culture performance and, in the case of silver nitrate, reduce ethylene accumulation that can inhibit maize callus growth.

The researchers maintained the cultures in complete darkness at approximately 25 degrees Celsius for six weeks, transferring them to fresh medium after 21 days. They recorded the percentage of explants that formed callus and calculated relative fresh weight growth rate, a measure based on the increase between initial and final tissue weight. Statistical analysis used a randomized complete block design covering four varieties, two explant types and five media, with two-way analysis of variance followed by Duncan’s multiple range test at a significance threshold of 0.05. Callus became visible after about one week on all media and from both types of explant. By the end of the culture period, the tissues displayed several forms, including soft, watery white or cream callus; cream-to-brown callus; and more friable, granular tissue that showed a tendency toward early root formation. The authors emphasize that these appearances are preliminary indicators, not proof that a callus is embryogenic.

The clearest pattern was the strong influence of genotype. Split internodes from CHABA produced the highest reported induction response, reaching 76.2 percent on one medium, while ATP and CHH generally performed better than KASSAI. Media designated M1 and M3 were broadly effective for internode-derived callus, with induction above 50 percent in several variety combinations. M2 produced the weakest responses, indicating that its balance of auxin and cytokinin was poorly suited to internode callogenesis in these materials. The leaf explants told a slightly different story. ATP reached the highest leaf-based induction rate, 80.25 percent on M3, followed by CHABA and KASSAI, whereas CHH was the least responsive leaf source. Even so, every variety formed callus from leaf pieces under the tested conditions, showing that the tissue could provide a useful secondary route when internodes are unsuitable.

Growth rate revealed another important distinction. Split internodes consistently generated more rapidly expanding callus than leaves. CHABA showed the strongest proliferation, with relative fresh weight growth rates exceeding 5,000 percent on M1 and M3. Leaf-derived callus from the same variety also grew vigorously, surpassing 3,000 percent on those media, but remained less proliferative overall. The researchers caution that such striking percentages should not be interpreted as equivalent to a five-thousand-fold increase in useful biological material or as evidence of superior regeneration potential. Fresh weight can rise sharply when callus absorbs water, becomes highly vacuolated and develops a loose, watery structure. In other words, rapid tissue expansion may reflect hydration as much as the production of dense, developmentally competent cells. This distinction is crucial for laboratories choosing material for transformation, because abundant callus is not necessarily embryogenic callus.

The hormone results fit the basic biology of plant regeneration. Auxins such as 2,4-D can promote dedifferentiation and stimulate the formation of early callus, while cytokinins help regulate cell division and influence whether tissue continues proliferating or begins differentiating. The most favorable responses generally came from media containing 2 to 2.5 milligrams per liter of 2,4-D together with BAP or kinetin, corresponding to the M1–M3 group. The outcome was not universal, however: the same medium could produce very different results in different varieties, and the interaction between genotype and medium was statistically significant. That variability reflects the fact that tissue culture is governed not only by the recipe in the vessel but also by the genetic and physiological state of the plant. Differences in hormone signaling, cell-cycle control, stress responses and tissue organization can determine whether an explant remains inactive, produces watery callus or enters a pathway capable of regeneration.

The study therefore represents a foundation rather than a finished transformation platform. The authors did not test whether the induced calli could produce shoots, roots and fertile plants, nor did they use histological or molecular markers to confirm embryogenic competence. Further experiments must identify which callus types can regenerate, determine whether the tissues remain genetically stable during prolonged culture and optimize the transition from induction media to regeneration media. Those steps will be especially important for CHABA and ATP, the varieties that displayed the most promising combinations of induction and proliferation. If subsequent work succeeds, seedling-derived split internodes and leaves could make tropical maize biotechnology less dependent on immature embryos and specialized facilities. That would give researchers a practical starting point for improving locally adapted varieties threatened by climate change, pests, diseases and declining soil quality, while preserving the genetic resources that farmers already rely on.

One practical strength of the protocol is that it begins with mature caryopses rather than relying on a precisely timed reproductive tissue. The seedlings were generated under defined laboratory conditions, and the explants were prepared at a common two-week developmental stage. That standardization can reduce one source of experimental variation: the physiological differences associated with embryo age. It does not eliminate variation altogether, because seed quality, germination behavior and the exact position of an internode or leaf segment may still affect the cells that respond. For this reason, a useful next step would be to define explant sampling landmarks and seedling size criteria in enough detail for independent laboratories to reproduce the comparison.

The treatment design also illustrates why tissue-culture optimization is usually empirical rather than transferable as a single universal recipe. The five media varied the relative influence of 2,4-D, BAP and kinetin, allowing the researchers to examine hormonal combinations rather than testing an auxin alone. Such comparisons can reveal a response window in which cells divide without immediately differentiating, but the best induction medium may not be the best medium for later plant development. Regeneration commonly requires a change in hormonal conditions, and the study’s results should therefore be used to select candidate combinations for subsequent experiments, not as a complete culture system.

Several components of the medium are particularly relevant to interpreting the results. Casein hydrolysate supplies a complex mixture of nitrogenous and other organic compounds, while spermidine is associated with processes involved in cell proliferation and stress responses. Silver nitrate was included because ethylene can accumulate in sealed culture vessels and suppress callus proliferation. These additives may have contributed to the observed responses, but their effects were not separated experimentally from those of the growth regulators. A future factorial comparison could determine whether each component is necessary for every genotype or whether some varieties would respond equally well to a simpler and less costly formulation.

The statistical structure provides a framework for identifying interactions that would be missed by comparing averages alone. With varieties, explant sources and media combined in a randomized complete block design, the researchers could assess whether a medium’s effect depended on genetic background or tissue type. Nevertheless, callus induction percentage and fresh-weight growth rate describe quantity more directly than developmental quality. Confirmation of embryogenic potential will require regeneration tests, characterization of shoot and root formation, and evaluation of plants recovered from culture. Testing regenerated plants for fertility and phenotypic or genetic stability would then determine whether the method can support breeding and transformation rather than merely produce proliferating tissue.

That distinction is important for tropical maize improvement. A protocol that works across several locally maintained varieties can serve as a screening platform, helping researchers compare transformation or editing conditions without first obtaining immature embryos from every genotype. It may also support experiments on varieties whose agronomic value is local but whose tissue-culture behavior has not been extensively documented. The immediate contribution of this work is thus methodological: it expands the set of accessible starting tissues and identifies genotype-specific responses that can guide the more demanding stages of regeneration and genetic improvement.

Subject of Research: Callus induction from seedling-derived explants in tropical maize

Article Title: Seedling-derived split internode and leaf explants as efficient alternatives for callus induction in tropical maize

Article References: Tinak Ekom, D. C., & Diko, A. H. (2026). Seedling-derived split internode and leaf explants as efficient alternatives for callus induction in tropical maize. BMC Agriculture, 2(1), Article 28. https://doi.org/10.1186/s44399-026-00051-z

Image Credits: AI Generated

DOI: 10.1186/s44399-026-00051-z

Keywords: Tropical maize, Callus induction, Plant tissue culture, Split internodes, Leaf explants, 2,4-D, Plant regeneration, Crop biotechnology, Seedling-derived, split, internode, leaf