Plant development
Important structures in plant development are buds, shoots, roots, leaves, and flowers; plants produce these tissues and structures throughout their life from meristems located at the tips of organs, or between mature tissues. Thus, a living plant always has embryonic tissues. By contrast, an animal embryo will very early produce all of the body parts that it will ever have in its life. When the animal is born, it has all its body parts and from that point will only grow larger and more mature. However, both plants and animals pass through a phylotypic stage that evolved independently and that causes a developmental constraint limiting morphological diversification.
According to plant physiologist A. Carl Leopold, the properties of organization seen in a plant are emergent properties which are more than the sum of the individual parts. "The assembly of these tissues and functions into an integrated multicellular organism yields not only the characteristics of the separate parts and processes but also quite a new set of characteristics which would not have been predictable on the basis of examination of the separate parts."
Growth
A vascular plant begins from a single celled zygote, formed by fertilisation of an egg cell by a sperm cell. From that point, it begins to divide to form a plant embryo through the process of embryogenesis. As this happens, the resulting cells will organize so that one end becomes the first root while the other end forms the tip of the shoot. In seed plants, the embryo will develop one or more "seed leaves". By the end of embryogenesis, the young plant will have all the parts necessary to begin in its life.Plant organogenesis
Once the embryo germinates from its seed or parent plant, it begins to produce additional organs through the process of organogenesis. New roots grow from root meristems located at the tip of the root, and new stems and leaves grow from shoot meristems located at the tip of the shoot. Branching occurs when small clumps of cells left behind by the meristem, and which have not yet undergone cellular differentiation to form a specialized tissue, begin to grow as the tip of a new root or shoot. Growth from any such meristem at the tip of a root or shoot is termed primary growth and results in the lengthening of that root or shoot. Secondary growth results in widening of a root or shoot from divisions of cells in a cambium.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 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 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 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 contributes to cell elongation and meristemoid formation, while unconventional compounds like tri-iodobenzoic acid, abscisic acid, 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 and silver nitrate 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.
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.