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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.{{cn|date=June 2025}}
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'', ''[[Lettuce|Lactuca sativa]]'', and ''[[Brassica juncea]]'' strictly require hormonal supplementation. In ''B. juncea'' cotyledon cultures, [[6-Benzylaminopurine|benzylaminopurine]] (BAP) alone induces shoot formation from petiole tissue, similar to [[Pinus radiata|radiata pine]] where cytokinin alone suffices for shoot induction.{{cn|date=June 2025}}
Research indicates that endogenous hormone concentrations, rather than exogenous application levels, ultimately determine organogenic differentiation. Among the various [[Cytokinin|cytokinins]] (2iP, BAP, thidiazuron, kinetin, and zeatin) used for shoot induction, BAP has demonstrated superior efficacy and widespread application. [[Auxin|Auxins]] similarly influence organogenic pathways, with 2,4-D commonly used for callus induction in cereals, though organogenesis typically requires transfer to media containing [[Indole-3-acetic acid|IAA]] or [[1-Naphthaleneacetic acid|NAA]] or lacking 2,4-D entirely. The auxin-to-cytokinin ratio largely determines which organs develop.{{cn|date=June 2025}}
[[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]], [[Nicotiana|tobacco]], and [[Common sunflower|sunflower]] cultures.{{cn|date=June 2025}}
[[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 Root|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.{{cn|date=June 2025}}
Similar seasonal dependency is observed in ''[[Chlorophytum borivilianum|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.{{cn|date=June 2025}}
==== Oxygen gradient ====
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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.{{cn|date=June 2025}}
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 ''[[Pea|Pisum sativum]]'' (garden pea), where shoot bud initiation occurs optimally in darkness before exposure to light stimulates further development.{{cn|date=June 2025}}
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.{{cn|date=June 2025}}
==== 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.{{cn|date=June 2025}}
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 (plant)|Narcissus]]'' (daffodil) and ''[[Allium]]'' (ornamental onion) demonstrate enhanced regeneration efficiency at around 18°C.{{cn|date=June 2025}}
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.{{cn|date=June 2025}}
==== Ploidy level ====
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=== 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.
=== 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.{{cn|date=June 2025}}
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.{{cn|date=June 2025}}
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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.{{cn|date=June 2025}}
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 [[Meristem|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.{{cn|date=June 2025}}
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.{{cn|date=June 2025}}
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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.{{cn|date=June 2025}}
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.{{cn|date=June 2025}}
===Cell elongation===
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[[File:Growth of a Plant.svg|thumb|This image shows the development of a normal plant. It resembles the different growth processes for a leaf, a stem, etc. On top of the gradual growth of the plant, the image reveals the true meaning of phototropism and cell elongation, meaning the light energy from the sun is causing the growing plant to bend towards the light aka elongate.]]
Plant growth and development are mediated by specific [[plant hormone]]s and plant growth regulators (PGRs) (Ross et al. 1983).<ref name="ross">Ross, S.D.; Pharis, R.P.; Binder, W.D. 1983. Growth regulators and conifers: their physiology and potential uses in forestry. p. 35–78 ''in'' Nickell, L.G. (Ed.), Plant growth regulating chemicals. Vol. 2, CRC Press, Boca Raton FL.</ref> Endogenous hormone levels are influenced by plant age, cold hardiness, dormancy, and other metabolic conditions; [[Photoperiodism|photoperiod]], drought, temperature, and other external environmental conditions; and exogenous sources of PGRs, e.g., externally applied and of [[Rhizosphere|rhizospheric]] origin.{{cn|date=June 2025}}
=== Morphological variation during growth ===
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===Adventitious structures===
Plant structures, including, [[Root|roots]], [[Bud|buds]], and [[Shoot (botany)|shoots]], that develop in unusual locations are called ''adventitious''.{{cn|date=June 2025}}
Adventitious roots and buds usually develop near the existing vascular tissues so that they can connect to the [[xylem]] and [[phloem]]. However, the exact ___location varies greatly. In young stems, adventitious roots often form from [[Ground tissue#Parenchyma|parenchyma]] between the [[vascular bundle]]s. In stems with secondary growth, adventitious roots often originate in phloem parenchyma near the [[vascular cambium]]. In stem cuttings, adventitious roots sometimes also originate in the [[Callus (cell biology)|callus]] cells that form at the cut surface. Leaf cuttings of the ''[[Crassula]]'' form adventitious roots in the epidermis.<ref>McVeigh, I. 1938. Regeneration in ''Crassula multicava''. ''American Journal of Botany'' 25: 7-11. [https://www.jstor.org/stable/2436624]</ref>
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Some leaves develop adventitious buds, which then form adventitious roots, as part of [[vegetative reproduction]]; e.g. piggyback plant (''[[Tolmiea menziesii]]'') and mother-of-thousands (''[[Kalanchoe daigremontiana]]''). The adventitious plantlets then drop off the parent plant and develop as separate [[Cloning|clone]]s of the parent.{{cn|date=June 2024}}
[[Coppicing]] is the practice of cutting [[tree]] stems to the ground to promote rapid growth of adventitious shoots. It is traditionally used to produce poles, fence material or firewood. It is also practiced for [[biomass]] crops grown for fuel, such as [[Populus|poplar]] or [[willow]].
{{anchor |adventitious root}}
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[[File:Adventitious roots on Odontonema aka Firespike.jpg|thumb|Roots forming above ground on a cutting of ''[[Odontonema]]'', also known as firespike]]
Adventitious rooting may be a stress-avoidance acclimation for some species, driven by such inputs as [[hypoxia (environmental)|hypoxia]]<ref>{{cite journal | url=https://doi.org/10.1007%2FBF00384595 | doi=10.1007/BF00384595 | title=Ethylene-promoted adventitious rooting and development of cortical air spaces (Aerenchyma) in roots may be adaptive responses to flooding in Zea mays L | year=1979 | last1=Drew | first1=M. C. | last2=Jackson | first2=M. B. | last3=Giffard | first3=S. | journal=Planta | volume=147 | issue=1 | pages=83–88 | pmid=24310899 | s2cid=7232582 | url-access=subscription }}</ref> or nutrient deficiency. Another ecologically important function of adventitious rooting is the vegetative reproduction of tree species such as ''[[Willow|Salix]]'' and ''[[Sequoia sempervirens|Sequoia]]'' in [[riparian]] settings.<ref>{{cite journal | url=https://www.jstor.org/pss/2952507 | jstor=2952507 | title=The Ecology of Interfaces: Riparian Zones | last1=Naiman | first1=Robert J. | last2=Decamps | first2=Henri | journal=Annual Review of Ecology and Systematics | year=1997 | volume=28 | pages=621–658 | doi=10.1146/annurev.ecolsys.28.1.621 | s2cid=86570563 | url-access=subscription }}</ref>
The ability of plant stems to form adventitious roots is utilised in commercial propagation by [[Cutting (plant)|cutting]]s. Understanding of the physiological mechanisms behind adventitious rooting has allowed some progress to be made in improving the rooting of cuttings by the application of synthetic auxins as rooting powders and by the use of selective basal wounding.<ref>{{cite journal | url=https://doi.org/10.1007%2Fs11627-999-0076-z | doi=10.1007/s11627-999-0076-z | title=Review the formation of adventitious roots: New concepts, new possibilities | year=1999 | last1=De Klerk | first1=Geert-Jan | last2=Van Der Krieken | first2=Wim | last3=De Jong | first3=Joke C. | journal=In Vitro Cellular & Developmental Biology - Plant | volume=35 | issue=3 | pages=189–199 | s2cid=44027145 | url-access=subscription }}</ref> Further progress can be made in future years by applying research into other regulatory mechanisms to commercial propagation and by the comparative analysis of molecular and ecophysiological control of adventitious rooting in 'hard to root' vs. 'easy to root' species.{{cn|date=June 2024}}
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==Leaf development==
The genetics behind leaf shape development in ''[[Arabidopsis thaliana]]'' has been broken down into three stages: The initiation of the [[leaf primordium]], the establishment of [[dorsiventrality]], and the development of a marginal [[meristem]]. Leaf primordium is initiated by the suppression of the genes and proteins of the class I ''[[Evolutionary history of plants|KNOX]]'' family (such as ''SHOOT APICAL MERISTEMLESS''). These class I KNOX proteins directly suppress [[gibberellin]] biosynthesis in the leaf primodium. Many genetic factors were found to be involved in the suppression of these genes in leaf primordia (such as ''ASYMMETRIC LEAVES1,'' ''BLADE-ON-PETIOLE1'', ''SAWTOOTH1'', etc.). Thus, with this suppression, the levels of gibberellin increase and leaf primorium initiates growth.{{cn|date=June 2024}}
==Flower development==
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