A plant callus is a mass of undifferentiated or loosely organized cells that forms when plant tissue is wounded or when cut pieces of a plant are cultured on nutrient media containing the right balance of hormones. It looks like a lumpy, often pale or translucent growth, and it serves as something between a biological bandage and a cellular blank slate. Callus formation sits at the intersection of wound healing and developmental reprogramming, and it has become one of the most important tools in modern plant biotechnology.
How Callus Forms in Nature
When a plant is injured, certain cells near the wound quickly re-enter the cell cycle and begin dividing. In a study using Arabidopsis hypocotyls (the stem-like segment of a seedling), researchers found that new cell divisions are restricted to the vasculature and pericycle cells, the tissue layers that surround the plant’s internal plumbing. Surrounding cell types like the cortex and epidermis did not start dividing, which means the callus does not arise from just any cell at the wound site. Instead, specific inner cell layers are the ones that respond.1PubMed Central. Wounding Triggers Callus Formation via Dynamic Hormonal and Transcriptional Changes
This wound response is fast. Within hours, transcription factors called WIND proteins are switched on at the injury site. WIND1, the best-studied member of this family, drives cells to lose their specialized identity and start proliferating, forming the mass of loosely organized cells we call a callus. When WIND1 is artificially overexpressed, it can push cells into this dedifferentiated state even without the hormones normally required, suggesting that WIND1 is a powerful master switch for the process.2PubMed. The AP2/ERF transcription factor WIND1 controls cell dedifferentiation in Arabidopsis Follow-up work showed that the WIND family of transcription factors also orchestrates vascular reconnection and defense responses, so wound callus is not just filler tissue. It is actively involved in healing and protecting the plant.3PubMed Central. WIND transcription factors orchestrate wound-induced callus formation, vascular reconnection and defense response in Arabidopsis
The Hormone Balancing Act
Two plant hormones dominate the story of callus induction: auxin and cytokinin. Researchers discovered decades ago that when these two hormones are supplied at a roughly equal, intermediate ratio, they promote the formation of undifferentiated callus. Tip the ratio toward more auxin, and the tissue grows roots instead. Tip it toward more cytokinin, and shoots emerge.4PubMed Central. Plant Callus: Mechanisms of Induction and Repression – Section: CALLUS FORMATION IN VITRO AND IN NATURE This insight, first demonstrated by Folke Skoog and his collaborators at the University of Wisconsin using tobacco tissue, remains the foundational principle behind laboratory callus culture. They showed that by adjusting the relative concentrations of auxin and kinetin (a synthetic cytokinin) in the growth medium, it was possible to steer the tissue toward shoots, roots, or continued undifferentiated callus growth.5PubMed Central. The Scientific Roots of Modern Plant Biotechnology
In nature, a wounded plant ramps up its own hormone production at the injury site, which is why callus forms without any laboratory intervention when you see a knobby growth on a pruned branch or a graft union. In the lab, researchers supply these hormones externally through the culture medium, giving them precise control over what the tissue does next.
Not a Simple Reset Button
For years, plant biologists assumed that callus formation was a kind of rewind, pushing specialized cells backward to some generic, embryonic-like state. The reality turned out to be more surprising. Research on Arabidopsis showed that callus tissue, no matter what organ it comes from, closely resembles the tip of a root meristem. Even callus derived from petals, which are as far from a root as you can get, shares this root-like identity. The study also found that mutant plants unable to initiate lateral roots could not form callus from any organ, whether roots, leaves, or petals. This means that callus formation essentially hijacks a root-development program rather than reverting cells to a truly blank slate.6PubMed Central. Arabidopsis regeneration from multiple tissues occurs via a root development pathway
This finding reshapes how researchers think about totipotency, the ability of a single plant cell to regenerate an entire organism. The cell is not returning to “zero” but rather adopting a specific developmental program that happens to be flexible enough to serve as a launching pad for further regeneration.
Epigenetic Switches Behind the Transition
The shift from a differentiated leaf cell to a callus cell is not just about which genes are turned on or off. It involves changes to the chemical tags on DNA and the proteins that package it. In peach leaves cultured to produce callus, researchers observed widespread loss of DNA methylation and removal of a repressive histone mark called H3K27me3. These epigenetic changes loosened the chromatin structure around genes involved in auxin and cytokinin signaling, effectively unlocking them for expression. When the researchers applied a chemical that blocks the enzyme responsible for removing H3K27me3, callus formation dropped dramatically. Conversely, adding a drug that strips away DNA methylation boosted callus development.7PubMed Central. Epigenetic reprogramming of H3K27me3 and DNA methylation during leaf-to-callus transition in peach
In cannabis callus, a separate transcriptomic study found that hundreds of epigenetic-related genes were differentially expressed between embryogenic callus (the type capable of forming embryos) and non-embryogenic callus (the type that just sits there and grows). Genes encoding histone deacetylases and chromatin remodeling factors were notably upregulated in the embryogenic type, pointing to active epigenetic reshuffling as the tissue acquires the potential to regenerate a whole plant.8PubMed Central. Transcriptomic Profiling of Embryogenic and Non-Embryogenic Callus Provides New Insight into the Nature of Recalcitrance in Cannabis
Embryogenic Versus Non-Embryogenic Callus
Not all callus is created equal. Under a microscope, the two main types look strikingly different. Embryogenic callus, the type that can eventually give rise to whole plants through somatic embryogenesis, tends to form compact, nodule-like structures made up of small, tightly packed cells. Some of this tissue is coated with a fibrillar extracellular matrix. In contrast, non-embryogenic callus is covered with rounder, looser cells that often have large fluid-filled vacuoles and more developed internal membrane systems.9PubMed. Histological and ultrastructural observation reveals significant cellular differences between Agrobacterium transformed embryogenic and non-embryogenic calli of cotton
Getting a plant species to produce embryogenic callus rather than the non-embryogenic kind is one of the biggest practical challenges in tissue culture. Many economically important plants are considered “recalcitrant,” meaning they stubbornly refuse to form the right type of callus under standard conditions. Researchers have been working to crack this by identifying the genes and transcription factors that distinguish the two types. One key player is BABY BOOM, a transcription factor that activates a network of embryogenesis-related genes. Overexpressing it can push callus toward the embryogenic pathway in species that normally resist it.10PubMed. Role of BABY BOOM Transcription Factor in Promoting Somatic Embryogenesis and Genetic Transformation in a Woody Magnoliid Liriodendron11Plant Physiology. The BABY BOOM Transcription Factor Activates the LEC1-ABI3-FUS3-LEC2 Network to Induce Somatic Embryogenesis
From Callus to Whole Plant
Once you have a mass of callus, the next step in tissue culture is often to regenerate a shoot. This requires shifting the hormone balance in the culture medium toward cytokinin. At the molecular level, cytokinin triggers a two-step activation of a gene called WUSCHEL, which establishes the shoot stem cell niche. First, the cytokinin-rich environment removes repressive epigenetic marks from the WUSCHEL locus. Then, cytokinin-responsive transcription factors called type-B ARRs directly bind and activate WUSCHEL expression, while simultaneously suppressing auxin accumulation to reinforce shoot identity.12PubMed Central. A Two-Step Model for de Novo Activation of WUSCHEL during Plant Shoot Regeneration13The Plant Cell. Type-B ARABIDOPSIS RESPONSE REGULATORs Specify the Shoot Stem Cell Niche by Dual Regulation of WUSCHEL
The full journey from a single protoplast (a plant cell with its wall removed) to a whole plant passes through several stages: cell wall recovery, re-entry into the cell cycle, callus proliferation, shoot regeneration, root regeneration, and finally establishment of a new plantlet.14Biology Methods and Protocols. Efficient regeneration of protoplasts from Solanum lycopersicum cultivar Micro-Tom Each step requires different conditions, and losing the tissue at any point is common. That fragility is part of why tissue culture remains as much an art as a science for many species.
Environmental Factors That Shape Callus Growth
Beyond hormones, other conditions influence how well callus forms and proliferates. Light intensity is a major factor. In a study on the ornamental succulent Haworthia, low light levels produced callus proliferation rates above 80%, while higher light intensity dropped those rates to below 30% for the same hormone combinations.15PubMed Central. Effects of light intensity and plant growth regulators on callus proliferation and shoot regeneration in the ornamental succulent Haworthia This is consistent with the general practice of culturing callus in dim light or even total darkness, since strong light can trigger unwanted differentiation or stress responses.
Sugar supply matters too. Callus cells, unlike green leaves, are often not photosynthesizing efficiently, so they rely on sugar in the growth medium as their energy source. In experiments on Justicia gendarussa, callus grown without any sucrose did not accumulate any biomass at all. The best growth came with moderate sucrose concentrations around two to three percent, with diminishing returns at higher levels.16Biotechnology Reports. Effects of light, sucrose concentration and repetitive subculture on callus growth and medically important production in Justicia gendarussa Burm.f. Metabolomic studies on tomato callus confirm that the process of callus induction is energetically expensive, with increased activity in the TCA cycle and pentose phosphate pathway to fuel all that cell division.17PubMed Central. Metabolomic homeostasis shifts after callus formation and shoot regeneration in tomato
Callus in Grafting
Grafting, the practice of joining two plant parts so they grow as one, depends heavily on callus to seal the union. When a scion is attached to a rootstock, callus proliferates at the junction and fills gaps between the cut surfaces. This newly formed tissue then differentiates to reconnect the vascular systems of the two partners. In Arabidopsis, WIND1 mutants that produce less wound callus also show reduced grafting success in leaf grafting experiments. The correlation between callus production and grafting efficiency appears broadly across the grafting literature.18Molecular Plant. What Is a Plant Callus and How Does It Form? – Section: Tissue adhesion and plasmodesmata formation
That said, the relationship is not absolute. When a skilled grafter achieves tight alignment between the scion and rootstock, the two pieces can fuse even when callus formation is genetically reduced. In those cases, callus seems less critical for the initial attachment and more important for reinforcing it afterward and filling in where the alignment is imperfect.
Biotechnology Uses
Callus culture underpins several major branches of plant biotechnology. One of the most commercially significant is the production of secondary metabolites, the complex chemicals plants make for defense, signaling, and other purposes. Many of these molecules have pharmaceutical value, including compounds used against cancer, cardiovascular disease, and infections. Growing callus or cell suspension cultures in bioreactors allows researchers to harvest these chemicals without destroying entire plants, an advantage when the source species is rare or slow-growing.19Engineering. Biotechnology Applications of Plant Callus Cultures – Section: 4. Production of secondary metabolites for therapeutic purposes20PubMed Central. Production of secondary metabolites using tissue culture-based biotechnological applications
Callus also serves as the entry point for genetic transformation. In a common workflow, pieces of callus are infected with Agrobacterium tumefaciens, a soil bacterium that naturally inserts its DNA into plant cells. The transformed callus is then screened and regenerated into whole plants carrying the new gene. In Liriodendron (tulip tree) hybrids, this approach achieved a transformation efficiency above 60% for regenerated callus clumps, and PCR screening confirmed that nearly all plants from resistant lines carried the transgene.21PubMed Central. Agrobacterium-Mediated Genetic Transformation of Embryogenic Callus in a Liriodendron Hybrid (L. Chinense × L. Tulipifera) Similar pipelines using embryogenic callus have been adapted for genome editing in fruit trees like litchi, where CRISPR/Cas9 components are delivered through the same Agrobacterium-mediated route.22Horticultural Plant Journal. Agrobacterium tumefaciens-mediated transformation of embryogenic callus and CRISPR/Cas9-mediated genome editing in ‘Feizixiao’ litchi
Micropropagation, the mass production of genetically identical plants, also relies on callus as an intermediate stage. This is how many ornamental plants, fruit tree rootstocks, and plantation crops are multiplied commercially.23ScienceDirect. Biotechnology Applications of Plant Callus Cultures – Section: 1. Introduction
The Problem of Somaclonal Variation
One trade-off with callus-based propagation is that the process is not always genetically stable. Cells that go through tissue culture can accumulate mutations or shifts in epigenetic marks, a phenomenon called somaclonal variation. This can show up as unexpected differences in growth habit, flower color, disease resistance, or other traits among plants regenerated from the same callus line.24PubMed Central. Somaclonal variations and their applications in horticultural crops improvement
The variation is driven primarily by mutations that arise during culture, and stress conditions magnify the problem. Hormonal imbalances in the medium, chemical exposure during sterilization, and even the wounding involved in preparing the explants all contribute. Oxidative stress has been identified as a particularly common driver of these culture-induced mutations.25OBM Genetics. Somaclonal Variation in Callus Cultures of Rose Periwinkle, Catharanthus Roseus L. Under Induced Salt and Osmotic Stresses For applications that demand genetic uniformity, like commercial clonal propagation, this is a headache that requires careful monitoring. But breeders sometimes exploit somaclonal variation deliberately as a source of novel traits, screening regenerated plants for useful mutations that would be difficult to produce any other way.
Why Some Species Resist Callus Culture
Despite the universality of the underlying biology, many plant species are notoriously difficult to culture. Conifers, most monocots, and many woody perennials either refuse to form callus efficiently, form only non-embryogenic callus, or fail to regenerate shoots from callus that does form. The cannabis study mentioned earlier is a case in point: the researchers profiled nearly 200 hormone-related genes to understand why some cannabis callus lines become embryogenic while others do not, and found that the difference comes down to very specific patterns of gene expression and epigenetic regulation that are difficult to reproduce reliably.8PubMed Central. Transcriptomic Profiling of Embryogenic and Non-Embryogenic Callus Provides New Insight into the Nature of Recalcitrance in Cannabis
Overcoming recalcitrance is one of the active frontiers in plant science. Strategies include engineering the expression of morphogenic genes like BABY BOOM and WUSCHEL, optimizing culture conditions for individual genotypes, and screening for the right combination of hormone types and concentrations. Progress is real but incremental, and the gap between model plants like Arabidopsis and economically important crops remains wide for many species. The fact that callus formation hijacks a lateral-root program helps explain part of the challenge: species with different root biology may not respond the same way to the signals researchers use to trigger callus in lab-friendly plants.