Organogenesis

Organogenesis is the phase of embryonic development that starts at the end of gastrulation and continues until birth. During organogenesis, the three germ layers formed from gastrulation (the ectoderm, endoderm, and mesoderm) form the internal organs of the organism.

The endoderm of vertebrates produces tissue within the lungs, thyroid, and pancreas. The mesoderm aids in the production of cardiac muscle, skeletal muscle, smooth muscle, tissues within the kidneys, and red blood cells. The ectoderm produces tissues within the epidermis and aids in the formation of neurons within the brain, and melanocytes.

The cells of each of the three germ layers undergo differentiation, a process where less-specialized cells become more-specialized through the expression of a specific set of genes. Cell differentiation is driven by cell signaling cascades. Differentiation is influenced by extracellular signals such as growth factors that are exchanged to adjacent cells which is called juxtracrine signaling or to neighboring cells over short distances which is called paracrine signaling. Intracellular signals – a cell signaling itself (autocrine signaling) – also play a role in organ formation. These signaling pathways allow for cell rearrangement and ensure that organs form at specific sites within the organism. The organogenesis process can be studied using embryos and organoids.

Organs produced by the germ layers

Neural precursor cells fold and elongate to form the neural tube. Mesoderm cells condense to form a rod which will send out signals to redirect the ectoderm cells above. This fold along the neural tube sets up the vertebrate central nervous system.

The endoderm is the inner most germ layer of the embryo which gives rise to gastrointestinal and respiratory organs by forming epithelial linings and organs such as the liver, lungs, and pancreas. The mesoderm or middle germ layer of the embryo will form the blood, heart, kidney, muscles, and connective tissues. The ectoderm or outermost germ layer of the developing embryo forms epidermis, the brain, and the nervous system.

Mechanism of organ formation

While each germ layer forms specific organs, in the 1820s, embryologist Heinz Christian Pander discovered that the germ layers cannot form their respective organs without the cellular interactions from other tissues. In humans, internal organs begin to develop within 3–8 weeks after fertilization. The germ layers form organs by three processes: folds, splits, and condensation. Folds form in the germinal sheet of cells and usually form an enclosed tube which you can see in the development of vertebrates neural tube. Splits or pockets may form in the germinal sheet of cells forming vesicles or elongations. The lungs and glands of the organism may develop this way.

A primary step in organogenesis for chordates is the development of the notochord, which induces the formation of the neural plate, and ultimately the neural tube in vertebrate development. The development of the neural tube will give rise to the brain and spinal cord. Vertebrates develop a neural crest that differentiates into many structures, including bones, muscles, and components of the central nervous system. Differentiation of the ectoderm into the neural crest, neural tube, and surface ectoderm is sometimes referred to as neurulation and the embryo in this phase is the neurula. The coelom of the body forms from a split of the mesoderm along the somite axis

Plant organogenesis

In plants, organogenesis occurs continuously and only stops when the plant dies. In the shoot, the shoot apical meristems regularly produce new lateral organs (leaves or flowers) and lateral branches. In the root, new lateral roots form from weakly differentiated internal tissue (e.g. the xylem-pole pericycle in the model plant Arabidopsis thaliana). In vitro and in response to specific cocktails of hormones (mainly auxins and cytokinins), most plant tissues can de-differentiate and form a mass of dividing totipotent stem cells called a callus. Organogenesis can then occur from those cells. The type of organ that is formed depends on the relative concentrations of the hormones in the medium. Plant organogenesis can be induced in tissue culture and used to regenerate plants.

Categories

Direct organogenesis

Direct organogenesis is a method of plant tissue culture in which organs like roots and shoots develop directly from meristematic or non-meristematic cells, bypassing the callus formation stage. This process takes place through the activation of shoot and root apical meristems or axillary buds, influenced by internal or externally applied plant growth regulators. As a result, specific cell types differentiate to form plant structures that can grow into whole plants. This technique is commonly used for propagating various plant species, including vegetables, fruits, woody plants, and medicinal plants. Shoot tips and nodal segments are typically used as explants in this process. In some cases, adventitious structures arise from somatic tissues under specific conditions, allowing for the regeneration of shoots or roots in areas where they would not naturally develop. This approach is particularly effective in herbaceous species, and while adventitious regeneration can lead to a higher rate of shoot formation, axillary shoot proliferation remains the most widely used method in micropropagation due to its efficiency and practicality. The general sequence of organ development in this process follows the pattern: Primary Explant → Meristemoid → Organ Primordium.

Indirect organogenesis

Indirect organogenesis is a developmental process in which plant cells undergo dedifferentiation, allowing them to revert from their specialized state and transition into a new developmental pathway. This process is characterized by an intermediate callus stage, where cells lose their original identity and become morphologically adaptable, serving as the foundation for organ formation. The progression of indirect organogenesis involves several key phases, beginning with dedifferentiation, which enables the cells to attain competence, followed by an induction stage that leads to a fully determined state. Once determination is achieved, the cells undergo morphological changes, ultimately giving rise to functional shoots or roots. This process follows a structured developmental sequence: Primary Explant → Callus → Meristemoid → Organ Primordium, ensuring the organized formation of plant organs.

Factors affecting organogenesis

Explant

The ability to regenerate plants successfully depends on selecting the right explant, which varies among species and plant varieties. In direct organogenesis, explants sourced from meristematic tissues, such as shoot tips, lateral buds, leaves, petioles, roots, and floral structures, are often preferred due to their ability to rapidly develop into new organs. These tissues have high survival rates, fast growth, and strong regenerative potential in vitro. Meristems, shoot tips, axillary buds, immature leaves, and embryos are particularly effective in promoting regeneration across a wide range of plant species. Additionally, mature plant parts, including leaves, stems, roots, petioles, and flower segments, can also serve as viable explants for organ formation under suitable conditions. Plant regeneration occurs through the formation of callus, an undifferentiated mass of cells that later gives rise to new organs. Callus formation can be induced from various explants, such as cotyledons, hypocotyls, stems, leaves, shoot apices, roots, inflorescences, and floral structures, when cultured under controlled conditions. Generally, explants containing actively dividing cells are more effective for callus initiation, as they have a higher capacity for cellular reprogramming. Immature tissues tend to be more adaptable for regeneration compared to mature tissues due to their increased developmental plasticity. The size and shape of the explant also influence the success of culture establishment, as larger or more structurally favorable explants may enhance the chances of survival and growth. Callus development is primarily triggered by wounding and the presence of plant hormones, which may be naturally present in the tissue or supplemented in the growth medium to stimulate cellular activity and organ formation.

Culture medium, plant growth regulators, and gelling agent

Culture media compositions vary significantly in their mineral elements and vitamin content to accommodate diverse plant species requirements. Murashige and Skoog (MS) medium is distinguished by its high nitrogen content in ammonium form, a characteristic not found in other formulations. Sucrose typically serves as the primary carbohydrate source across various media types.

The interaction between auxins and cytokinins in regulating organogenesis is well-established, though responses vary by species. Some plants, such as tobacco, can spontaneously form shoot buds without exogenous growth regulators, while others like Scurrula pulverulenta, Lactuca sativa, and Brassica juncea strictly require hormonal supplementation. In B. juncea cotyledon cultures, benzylaminopurine (BAP) alone induces shoot formation from petiole tissue, similar to radiata pine where cytokinin alone suffices for shoot induction.

Research indicates that endogenous hormone concentrations, rather than exogenous application levels, ultimately determine organogenic differentiation. Among the various cytokinins (2iP, BAP, thidiazuron, kinetin, and zeatin) used for shoot induction, BAP has demonstrated superior efficacy and widespread application. Auxins similarly influence organogenic pathways, with 2,4-D commonly used for callus induction in cereals, though organogenesis typically requires transfer to media containing IAA or NAA or lacking 2,4-D entirely. The auxin-to-cytokinin ratio largely determines which organs develop.

Gibberellic acid (GA3) contributes to cell elongation and meristemoid formation, while unconventional compounds like tri-iodobenzoic acid (TIBA), abscisic acid (ABA), kanamycin, and auxin inhibitors have proven effective for recalcitrant species. Natural additives like ginseng powder can enhance regeneration frequency in certain cultures. Since ethylene typically suppresses shoot differentiation, inhibitors of ethylene synthesis such as aminoethoxyvinylglycine (AVG) and silver nitrate (AgNO3) are often employed to promote organogenesis, with documented success in wheat, tobacco, and sunflower cultures.

Agar is not an essential component of the culture medium, but quality and quantity of agar is an important factor that may determine a role in organogenesis. Commercially available agar may contain impurities. With a high concentration of agar, the nutrient medium becomes hard and does not allow the diffusion of nutrients to the growing tissue. It influences the organogenesis process by producing adventitious roots, unwanted callus at the base, or senescence of the foliage. The pH is another important factor that may affect organogenesis route. The pH of the culture medium is adjusted to between 5.6 and 5.8 before sterilization. Medium pH facilitates or inhibits nutrient availability in the medium; for example, ammonium uptake in vitro occurs at a stable pH of 5.5 (Thorpe et al., 2008).

Other factors

Season of the year

The timing of explant collection significantly impacts regenerative capacity in tissue culture systems, with seasonal variations playing a crucial role in organ formation success. This phenomenon is clearly demonstrated in Lilium speciosum, where bulb scales exhibit differential regenerative responses based on collection season. Explants harvested during spring and autumn periods readily form bulblets in vitro, while those collected during summer or winter months fail to produce bulblets despite identical culture conditions.

Similar seasonal dependency is observed in Chlorophytum borivillianum, a medicinally valuable species that shows markedly enhanced in vitro tuber formation during monsoon seasons compared to other times of year. This seasonal variation in morphogenic potential likely reflects differences in the physiological state of the source plant, including endogenous hormone levels, carbohydrate reserves, and metabolic activity that fluctuate throughout the annual growth cycle.

Oxygen gradient

Oxygen has a key role in tissue culture, which influences the organ formation. In some cultures, shoot bud formation takes place when the gradient of available oxygen inside the culture vessel is reduced, while induction of roots requires a high oxygen gradient.

Light

Light conditions, including both intensity and spectral quality, function as significant morphogenic signals in plant tissue culture systems. Spectral composition research has revealed distinct wavelength-dependent responses, with blue light generally promoting shoot organogenesis while red light wavelengths typically favor root induction. Sequential photoperiod exposure—blue light followed by red light—has been documented to effectively stimulate specific organogenetic pathways in certain species.

The regulatory effect of different wavelengths demonstrates how light quality can selectively control morphogenic outcomes. Artificial fluorescent lighting produces variable responses depending on the species, promoting root formation in some cultures while inhibiting it in others. Some species exhibit specialized light requirements, as observed in Pisum sativum (garden pea), where shoot bud initiation occurs optimally in darkness before exposure to light stimulates further development.

For most tissue culture applications, standard lighting protocols typically recommend illumination of approximately 2,000-3,000 lux intensity with a 16-hour photoperiod. However, certain species demonstrate exceptional light intensity requirements, exemplified by Nicotiana tabacum (tobacco) callus cultures, which require substantially higher light intensities of 10,000-15,000 lux to induce shoot bud formation or somatic embryogenesis.

Temperature

Temperature serves as a critical environmental factor in plant tissue culture systems, with optimal incubation temperatures varying significantly among species based on their natural habitat requirements. While 25°C represents the standard incubation temperature suitable for many plant species in vitro, species-specific temperature adaptations should be considered to maximize organogenic potential.

Geophytic species from temperate regions typically require lower temperature regimes than the standard protocol. Notable examples include bulbous plants such as Galanthus (snowdrop) which exhibits optimal growth at approximately 15°C, while certain cultivars of Narcissus (daffodil) and Allium (ornamental onion) demonstrate enhanced regeneration efficiency at around 18°C.

Conversely, species of tropical origin generally require elevated temperatures for optimal growth and organogenesis in culture. Date palm cultures thrive at 27°C, while Monstera deliciosa (Swiss cheese plant) exhibits peak regenerative performance at 30°C. These temperature requirements reflect evolutionary adaptations to the plants' native environmental conditions.

Ploidy level

Variation in chromosome number, that is, aneuploidy, polyploidy, etc., in plant cell culture has been well documented in the past. Chromosome instability of the cells results in gradual decline of morphogenetic potentiality of the callus tissue. Therefore, to maintain organogenic potential of the callus tissue and the chromosome stability, it is suggested that the time and frequency of subculture should be regularly followed.

Age of culture

Age of culture is often the key to successful organogenesis. A young culture/freshly subcultured material may produce organs more frequently than the aged ones. The probable reason for this is the reduction or loss of the organogenic potential in old cultures. However, in some plants, the plant regeneration capacity may retain indefinitely for many years

Developmental process

Dedifferentiation

The ability of cells to undergo organogenesis largely depends on the application of plant growth regulators (PGRs), which influence the developmental direction of the tissue. The balance between auxins and cytokinins plays a critical role in determining whether shoots or roots will form. A lower auxin-to-cytokinin ratio favors shoot regeneration, whereas a higher auxin concentration promotes root formation. For example, in Medicago sativa (alfalfa) cultures, an elevated level of kinetin combined with a low concentration of 2,4-D (a synthetic auxin) leads to shoot development, whereas increasing 2,4-D while reducing kinetin concentration encourages root formation. However, successful organogenesis is not solely dependent on PGR treatment. The physical size of the callus or developing tissue must reach a certain threshold to support proper organ formation, highlighting the importance of intercellular signaling in coordinating developmental processes.

Induction

The induction phase in organogenesis represents the transition period between a tissue achieving competence and becoming fully determined to initiate primordia formation. During this stage, an integrated genetic pathway directs the developmental process before morphological differentiation occurs. Research suggests that certain chemical and physical factors can interfere with genetically programmed developmental pathways, altering morphogenic outcomes. In the case of Convolvulus arvensis, these external influences were found to inhibit shoot formation, leading instead to callus development.

The conclusion of the induction phase is marked by a cell or group of cells committing to either shoot or root formation. This determination is tested by transferring the tissue from a growth regulator-supplemented medium to a basal medium containing essential minerals, vitamins, and a carbon source but no plant growth regulators. At this stage, the tissue completes the induction process and becomes fully determined to its developmental fate.

A key concept in this process is canalization, which refers to the ability of a developmental pathway to consistently produce a standard phenotype despite potential genetic or environmental variations. If explants are removed from a shoot-inducing medium before full canalization occurs, shoot formation is significantly reduced, and root development becomes the dominant outcome. This phenomenon highlights the morphogenic plasticity of plant tissues in vitro, demonstrating their ability to adjust to external conditions and developmental cues.

Differentiation

During this phase, the process of morphological differentiation begins, leading to the formation and development of the nascent organ. The initiation of organogenesis is characterized by a distinct shift in polarity, followed by the establishment of radial symmetry and subsequent growth along the newly defined axis, ultimately forming the structural bulge that marks organ initiation.

The sequential development of organogenesis can be observed in species such as Pinus oocarpa Schiede, where shoot buds are regenerated directly from cotyledons through direct organogenesis. However, the specific developmental patterns may vary across different plant species grown in vitro. The progression of organ formation includes distinct morphological changes, beginning with alterations in surface texture, the emergence of meristemoids, and the expansion of the meristematic region either vertically or horizontally. This is followed by the protrusion of the meristematic region beyond the epidermal layer, the formation of a structured meristem with visible leaf primordia, and eventually, the full development of an adventitious bud.

A notable characteristic of in vitro organogenic cultures is the simultaneous formation of multiple meristemoids on a single explant, with varying degrees of differentiation. Within the same explant, buds may exist in different developmental stages, ranging from early initiation to fully developed structures. Once the elongated shoots surpass a length of 1 cm, they are transferred to either in vitro or ex vitro rooting substrates, allowing for the completion of plantlet regeneration and the establishment of a fully formed plant.

Advantages and limitations

In the process of direct organogenesis, axillary shoots are generated directly from pre-existing meristems located at the shoot tips and nodes, offering a high rate of multiplication. One of the key advantages of this method is the low likelihood of mutations occurring in the organized shoot meristems, ensuring that the resulting plants maintain genetic consistency. This technique is particularly valuable for the production and conservation of economically and environmentally significant plants, as it allows for the efficient generation of multiple shoots from a single explant, maintaining uniformity across the propagated plants. Furthermore, all plants produced via direct organogenesis are true-to-type, meaning they are genetic clones of the original plant.

However, there are some limitations to organogenesis. Somaclonal variation, which can result in unwanted genetic diversity, is a potential issue, particularly in the indirect organogenesis process. Additionally, this technique may not be suitable for recalcitrant plant species, which are those that do not respond well to in vitro culture or regeneration protocols. These limitations highlight the need for ongoing research and optimization of methods for different plant species to overcome these challenges in plant propagation and conservation.

See also

• Ectoderm
• Embryogenesis
• Endoderm
• Eye development
• Gastrulation
• Germ layer
• Germ line development
• Gonadogenesis
• Heart development
• Histogenesis
• Limb development
• List of human cell types derived from the germ layers
• Mesoderm
• Morphogenesis
• Organoid

External links

• The dictionary definition of organogenesis at Wiktionary