Fundamentals of Cytodifferentiation and Totipotency

In the strict sense, totipotency (from Latin totus — whole, entire) is the ability of a single cell to give rise to a complete organism, including all its tissues, organs, and extra‑embryonic structures (in plants, the endosperm) (Su et al., 2021). In multicellular animals, only the zygote and, in some groups, the cells of the very early embryo possess true totipotency (Fehér, 2019). For plants, however, this definition requires an important caveat.

In classical textbooks and even in modern articles, one often encounters the statement: “all plant cells are totipotent”. This is not entirely accurate but is a widely used simplification (Fehér, 2019). The reality is:

Thus, totipotency as applied to a plant somatic cell is its proven ability, under external influence, to reorganise its developmental program and form a fully fertile organism.

In contrast to totipotency, cytodifferentiation (or simply differentiation) is a process that is irreversible (under normal conditions), during which a cell acquires specific morphological, biochemical, and functional traits, losing its apparent universality (Sugiyama, 2015).

It is the path from a meristem stem cell to a highly specialised structure. For example:

A crucial feature of cytodifferentiation in plants is its reversibility under experimental conditions. Although in nature a phloem cell will not become a cortex cell, when excised from the tissue and placed in a specific medium it can undergo dedifferentiation — the reverse of differentiation — returning to a “young”, proliferating state (Fehér, 2019).

The key point that an agronomy student must grasp is: totipotency and differentiation are not mutually exclusive concepts; rather, they are the poles of a continuum of cellular states.

This distinguishes plants from animals, where differentiation is almost always accompanied by irreversible loss of potency (e.g., mammalian neurons do not divide).

In the next chapter, we will examine what dedifferentiation is and how the dormant totipotency of a plant cell is awakened.

As already noted, totipotency is the ability of a cell to form a complete organism, including all its tissues and extra‑embryonic structures (in flowering plants, the endosperm). In the strict sense, in plant ontogeny the truly totipotent cell is only the zygote (fertilised egg cell) and, possibly, the first few cells of the embryo at the 2–4 cell stage (Fehér, 2019).

What is unique to plants is that many somatic cells can regain this state upon induction (see Section 3). However, in normal development, totipotency is the prerogative of the beginnings.

Pluripotency (from Latin plures — several) is the ability of a cell to give rise to most cell types of the organism, but not all (typically unable to form extra‑embryonic tissues). In plants, the classic examples of pluripotent cells are the cells of apical meristems (shoot and root apical meristems) (Sablowski and Gutierrez, 2022).

A stem cell in the quiescent centre of the root or in the organising centre of the shoot is not totipotent: it cannot form a suspensor or endosperm. However, all tissues of the shoot (leaves, stem, flowers) or root originate from it. Pluripotency is the working state of cells that ensures continuous growth and renewal of organs.

Notably, when cultured in vitro on specific media, pluripotent meristem cells or “pericycle‑like” cells (see below) can give rise to both shoots and roots, and sometimes to somatic embryos — that is, they exhibit enhanced plasticity (Fehér, 2019; Chowdhury et al., 2026).

Multipotency — the ability of a cell to differentiate into several cell types, usually within one tissue lineage.

A classic example in plants is the cambium (lateral meristem). The cambium is a thin layer of dividing cells between xylem and phloem. Its initial cells are multipotent: they can give rise to only two cell types:

No other tissues (epidermis, leaf parenchyma, root cap) arise from the cambium. This is a strictly defined dichotomy.

Unipotent cells can give rise to only one cell type, but they themselves retain the ability to divide. In plants, clear examples are rare because meristem cells often retain a broader potential. Nevertheless, some initial cells in trichomes (hairs) or in the stomatal lineage can be considered unipotent (Chowdhury et al., 2026).

Finally, terminally differentiated cells are cells that have completed their development and lost the ability to divide. These include, for example, mature phloem sieve tube elements (enucleate), sclereids (stone cells), xylem cells, mature leaf chlorenchyma cells. Under normal conditions they do not divide and do not change their type. However — and this is a key difference from animals — even such cells retain latent totipotency: when excised from the tissue and treated with hormones, they can enter dedifferentiation and start dividing (Fehér, 2019).

A comparison with animals is very instructive here. In mammals, as differentiation proceeds, cell potencies decline rapidly and, as a rule, irreversibly. A neuron will never become a muscle cell. In plants, this loss of potencies is not final.

The hierarchy of plant cells can be imagined not as a ladder, but as a stretched spring: the higher the specialisation, the stronger the “epigenetic pressure” on the genome, but under certain conditions (wounding, hormones, stress) the spring can “uncoil” — the cell undergoes dedifferentiation and returns to a higher level of potency (up to totipotency) (Fehér, 2015).

In the next chapter, we will analyse in detail the mechanisms of this astonishing return.

The most important thing to understand about differentiation is: before the cell changes outwardly, it has already “decided” to change inwardly. This invisible “decision” is called determination (from Latin determinare — to limit, to define).

Determination is a stable, heritable change in the pattern of gene expression that predetermines the future fate of the cell, even if outwardly it still does not differ from its neighbours. It is a kind of “molecular blueprint” for future specialisation.

After the cell has passed the determination stage — made an invisible “decision” about its fate — the next stage arrives: realisation of that decision. It is at this stage that visible morphological and biochemical changes occur: the cell changes shape, its vacuole shrinks or expands, the cell wall thickens and becomes impregnated with lignin or suberin, plastids transform into chloroplasts, chromoplasts, or amyloplasts.

What serves as the immediate trigger for all these transformations? A key role here belongs to special transcription factors often called “master regulators” in the literature. These proteins stand at the top of hierarchical cascades: they turn on (or off) entire groups of genes required to form a specific cell type.

If determination and the work of master regulators are the invisible “molecular blueprint”, then cytodifferentiation proper is the implementation of that blueprint into real cellular architecture. For agronomy students, it is important to learn to recognise differentiated cells under the microscope and to understand what structural changes have taken place. Let us consider the main cytological features of differentiation.

The reader familiar with the basics of animal biology may rightly ask: do animal cells not also differentiate? Of course they do. But there are fundamental differences between plant and animal cell differentiation, and it is these differences that make plants unique objects for biotechnology. Understanding these distinctions is critically important for the agronomist working with in vitro cultures.

Let us compare the key aspects.

Let us examine these differences in more detail.

The term “dedifferentiation” appeared in botanical literature in the first half of the 20th century. It was understood as “the process by which mature or specialised cells lose their differentiated character and rejuvenate” (Bloch, 1941, cited in Sugiyama, 2015). For a long time, it was believed that for a somatic cell to give rise to callus or a somatic embryo, it must first return to a state resembling a meristematic (undifferentiated) cell.

However, in the last decade, this classical view has been substantially revised (Fehér, 2019; Sugiyama, 2015). Studies in Arabidopsis have shown that when callus is induced on a medium with high auxin, cells do not completely lose their identity. Instead, they transdifferentiate: leaf or stem cells acquire traits of …​ the root! It turned out that auxin‑induced callus expresses genes characteristic of lateral root primordia, and its cells originate from “pericycle‑like” stem cells that are scattered throughout the plant near vascular bundles (Fehér, 2019).

Thus, strictly speaking, classical dedifferentiation (a return to “ground zero”) does not always occur. More often, what takes place is transdifferentiation — a transition from one differentiated state to another, bypassing the stage of a totipotent meristematic cell (Sugiyama, 2015).

Nevertheless, in a broad sense, in biotechnological practice and in this article, the term “dedifferentiation” continues to be used to denote the process by which a specialised somatic cell (or group of cells) loses visible signs of its specialisation, activates the cell cycle, and acquires the ability to proliferate and be reprogrammed (Fehér, 2019).

Callus (from Latin callus — hard skin, callosity) is an unorganised mass of dividing cells that forms on the plant surface at a wound site or when an explant is cultured in vitro on a hormone‑containing medium (Sugiyama, 2015).

It is important to understand that callus is not a homogeneous mass of identical cells. On the contrary, it is heterogeneous: one can find cells at various degrees of differentiation (from nearly meristematic to partially specialised). Moreover, different types of callus have different fates.

For an agronomist‑biotechnologist, the ability to distinguish callus types is critically important: clonal micropropagation via organogenesis requires one type of callus, somatic embryogenesis another, and genetic transformation most often uses embryogenic or compact organogenic callus.

What makes a cell enter a dedifferentiated state and form callus? The main signals are hormones and stress.

If we compare a differentiated cell (e.g., a leaf mesophyll cell) with a callus cell derived from it, the differences are striking (Sablowski and Gutierrez, 2022; Fehér, 2015):

Thus, the dedifferentiated callus cell morphologically resembles a meristematic cell — small, dense, with intense metabolism.

A principled clarification is needed here. Dedifferentiation by itself is not equal to totipotency. Dedifferentiation is the process of losing specialised traits and returning to proliferation. Totipotency is the ability to realise the full genetic program of development.

Not all dedifferentiated callus cells are totipotent. In a typical callus, only a small fraction of cells (those expressing markers like SERK1 or WOX5) indeed retain the ability for somatic embryogenesis (Fehér, 2019; Su et al., 2021). Most callus cells are simply proliferating cells that are already determined toward root or parenchyma formation.

This is precisely why successful plant regeneration in vitro requires proper selection of genotype, explant type, and hormonal conditions: one must not merely induce dedifferentiation, but create conditions in which cells retain or re‑acquire totipotency.

In the next section (5), we will move on to the applied significance of these phenomena — how exactly totipotency, dedifferentiation, and cytodifferentiation work for the benefit of agriculture.

Essence of the method: From a small fragment of the mother plant (explant) — an apical or axillary meristem, a stem segment, a leaf, a root — on an artificial nutrient medium, many genetically identical regenerated plants (clones) are obtained. The process is based on the induction of dedifferentiation (callus formation) or direct activation of axillary meristems, followed by shoot and root regeneration through modification of the hormone balance (auxin/cytokinin) (Sugiyama, 2015; Malabadi et al., 2025).

Agronomic significance:

Essence of the method: Somatic cells of callus or directly of the explant, under certain conditions (often after removal of auxin or addition of abscisic acid), switch to an embryonic developmental pathway, forming somatic embryos — structures morphologically similar to zygotic embryos (Fehér, 2015; Su et al., 2021). Somatic embryos do not require fertilisation and can develop into fully functional plants.

Agronomic significance:

Essence of the method: Callus cultures, somatic embryos, or apical meristems can be stored for long periods under conditions of slow growth (reduced temperature, osmotic stress) or frozen in liquid nitrogen (−196 °C) — cryopreserved. Viability is maintained thanks to the latent totipotency of the cells (Fehér, 2019; Malabadi et al., 2025).

Agronomic significance:

Essence of the method: To obtain a transgenic plant, a foreign gene must be introduced into a plant cell, and then from a single transformed cell a whole plant must be regenerated. It is the totipotency of somatic cells that makes this possible. The two main approaches:

Agronomic significance:

Essence of the method: Immature anthers (microspores) or isolated microspores are cultured in vitro on a stress medium (often elevated temperature or osmotic shock). Instead of developing normally into a pollen grain, the microspore switches to an embryogenic pathway and forms a haploid somatic embryo or callus. Haploid plants are then used to produce doubled haploids (spontaneously or with colchicine), which are completely homozygous (Malabadi et al., 2025).

Agronomic significance:

Essence of the method: Callus or suspension cell cultures (cell suspensions in liquid medium) are grown in bioreactors. These cells retain the ability to synthesise secondary metabolites characteristic of the original plant: alkaloids, flavonoids, terpenes, glycosides, etc. (Malabadi et al., 2025).

Agronomic significance:

Essence of the method: Apical meristems 0.1–0.5 mm in size (with 1–2 leaf primordia) are excised and cultured in vitro. Meristems are usually free of viruses, bacteria, and fungi because these pathogens do not colonise the actively dividing initial cells (Malabadi et al., 2025).

Agronomic significance:

Essence of the method: During prolonged callus culture, mutations and epigenetic changes inevitably arise — somaclonal variability (Fehér, 2015; Malabadi et al., 2025). These changes can be stable and lead to the appearance of new economically valuable traits. Moreover, by fusing protoplasts (cells without walls) from different species, somatic hybrids can be obtained that do not cross sexually.

Agronomic significance: