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Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues, or organs under sterile conditions on a nutrient culture medium of known composition. It is widely used to produce clones of a plant in a method known as micropropagation. Different techniques in plant tissue culture may offer certain advantages over traditional methods of propagation, including:
Plant tissue culture relies on the fact that many plant parts have the ability to regenerate into a whole plant (cells of those regenerative plant parts are called totipotent cells which can differentiate into various specialized cells). Single cells, plant cells without cell walls (protoplasts), pieces of leaves, stems or roots can often be used to generate a new plant on culture media given the required nutrients and plant hormones.
Preparation of plant tissue for tissue culture is performed under aseptic conditions under HEPA filtered air provided by a laminar flow cabinet. Thereafter, the tissue is grown in sterile containers, such as Petri dishes or flasks in a growth room with controlled temperature and light intensity. Living plant materials from the environment are naturally contaminated on their surfaces (and sometimes interiors) with microorganisms, so their surfaces are sterilized in chemical solutions (usually alcohol and sodium or calcium hypochlorite) [1] before suitable samples (known as explants) are taken. The sterile explants are then usually placed on the surface of a sterile solid culture medium but are sometimes placed directly into a sterile liquid medium, particularly when cell suspension cultures are desired. Solid and liquid media are generally composed of inorganic salts plus a few organic nutrients, vitamins, and plant hormones. Solid media are prepared from liquid media with the addition of a gelling agent, usually purified agar.
The composition of the medium, particularly the plant hormones and the nitrogen source (nitrate versus ammonium salts or amino acids) have profound effects on the morphology of the tissues that grow from the initial explant. For example, an excess of auxin will often result in a proliferation of roots, while an excess of cytokinin may yield shoots. A balance of both auxin and cytokinin will often produce an unorganised growth of cells, or callus, but the morphology of the outgrowth will depend on the plant species as well as the medium composition. As cultures grow, pieces are typically sliced off and subcultured onto new media to allow for growth or to alter the morphology of the culture. The skill and experience of the tissue culturist are important in judging which pieces to culture and which to discard.
As shoots emerge from a culture, they may be sliced off and rooted with auxin to produce plantlets which, when mature, can be transferred to potting soil for further growth in the greenhouse as normal plants. [2]
The specific differences in the regeneration potential of different organs and explants have various explanations. The significant factors include differences in the stage of the cells in the cell cycle, the availability of or ability to transport endogenous growth regulators, and the metabolic capabilities of the cells. The most commonly used tissue explants are the meristematic ends of the plants like the stem tip, axillary bud tip, and root tip. [3] These tissues have high rates of cell division and either concentrate or produce required growth-regulating substances including auxins and cytokinins.
Shoot regeneration efficiency in tissue culture is usually a quantitative trait that often varies between plant species and within a plant species among subspecies, varieties, cultivars, or ecotypes. Therefore, tissue culture regeneration can become complicated especially when many regeneration procedures have to be developed for different genotypes within the same species.
The three common pathways of plant tissue culture regeneration are propagation from preexisting meristems (shoot culture or nodal culture), organogenesis, and non-zygotic embryogenesis.
The propagation of shoots or nodal segments is usually performed in four stages for mass production of plantlets through in vitro vegetative multiplication but organogenesis is a standard method of micropropagation that involves tissue regeneration of adventitious organs or axillary buds directly or indirectly from the explants. Non-zygotic embryogenesis is a noteworthy developmental pathway that is highly comparable to that of zygotic embryos and it is an important pathway for producing somaclonal variants, developing artificial seeds, and synthesizing metabolites. Due to the single-cell origin of non-zygotic embryos, they are preferred in several regeneration systems for micropropagation, ploidy manipulation, gene transfer, and synthetic seed production. Nonetheless, tissue regeneration via organogenesis has also proved to be advantageous for studying regulatory mechanisms of plant development.
The tissue obtained from a plant to be cultured is called an explant.
Explants can be taken from many different parts of a plant, including portions of shoots, leaves, stems, flowers, roots, single undifferentiated cells, and from many types of mature cells provided they still contain living cytoplasm and nuclei and are able to de-differentiate and resume cell division. This has given rise to the concept of totipotency of plant cells. [4] [5] However, this is not true for all cells or for all plants. [6] In many species explants of various organs vary in their rates of growth and regeneration, while some do not grow at all. The choice of explant material also determines if the plantlets developed via tissue culture are haploid or diploid. Also, the risk of microbial contamination is increased with inappropriate explants.
The first method involving the meristems and induction of multiple shoots is the preferred method for the micropropagation industry since the risks of somaclonal variation (genetic variation induced in tissue culture) are minimal when compared to the other two methods. Somatic embryogenesis is a method that has the potential to be several times higher in multiplication rates and is amenable to handling in liquid culture systems like bioreactors.
Some explants, like the root tip, are hard to isolate and are contaminated with soil microflora that becomes problematic during the tissue culture process. Certain soil microflora can form tight associations with the root systems, or even grow within the root. Soil particles bound to roots are difficult to remove without injury to the roots that then allows a microbial attack. These associated microflora will generally overgrow the tissue culture medium before there is significant growth of plant tissue.
Some cultured tissues are slow in their growth. For them there would be two options: (i) Optimizing the culture medium; (ii) Culturing highly responsive tissues or varieties. [7] Necrosis can spoil cultured tissues. Generally, plant varieties differ in susceptibility to tissue culture necrosis. Thus, by culturing highly responsive varieties (or tissues) it can be managed. [7]
Aerial (above soil) explants are also rich in undesirable microflora. However, they are more easily removed from the explant by gentle rinsing, and the remainder usually can be killed by surface sterilization. Most of the surface microflora do not form tight associations with the plant tissue. Such associations can usually be found by visual inspection as a mosaic, de-colorization, or localized necrosis on the surface of the explant.
An alternative for obtaining uncontaminated explants is to take explants from seedlings which are aseptically grown from surface-sterilized seeds. The hard surface of the seed is less permeable to the penetration of harsh surface sterilizing agents, such as hypochlorite, so the acceptable conditions of sterilization used for seeds can be much more stringent than for vegetative tissues.
Tissue-cultured plants are clones. If the original mother plant used to produce the first explants is susceptible to a pathogen or environmental condition, the entire crop would be susceptible to the same problem. Conversely, any positive traits would remain within the line also.
Plant tissue culture is used widely in the plant sciences, forestry, and horticulture. Applications include:
Developing Somaclonal variation
Plant | Somaclonal variant | Traits |
Sugarcane | 'Ono' | resistance to Fiji disease. |
Citronella java | 'Bio-13' (by CIMAP , Lucknow) | 37% more oil |
Climate resilience
- As in Kaveri Vaman (by NRCB , Tamil Nadu) , a Tissue Culture Banana Mutant to withstand heavy rains. [18]
Secondary metabolites production
- Such as Caffeine from coffea arabica, Nicotine from Nicotiana rustica or phenolic acids from Echinacea purpurea . [19]
Induction of flowering
- In trees with delay in flowering or Bamboo - where some species flower once in their life but may live longer than 50 years. [20]
In vascular plants, the roots are the organs of a plant that are modified to provide anchorage for the plant and take in water and nutrients into the plant body, which allows plants to grow taller and faster. They are most often below the surface of the soil, but roots can also be aerial or aerating, that is, growing up above the ground or especially above water.
The vascular cambium is the main growth tissue in the stems and roots of many plants, specifically in dicots such as buttercups and oak trees, gymnosperms such as pine trees, as well as in certain other vascular plants. It produces secondary xylem inwards, towards the pith, and secondary phloem outwards, towards the bark.
Plant hormones are signal molecules, produced within plants, that occur in extremely low concentrations. Plant hormones control all aspects of plant growth and development, including embryogenesis, the regulation of organ size, pathogen defense, stress tolerance and reproductive development. Unlike in animals each plant cell is capable of producing hormones. Went and Thimann coined the term "phytohormone" and used it in the title of their 1937 book.
Vegetative reproduction is a form of asexual reproduction occurring in plants in which a new plant grows from a fragment or cutting of the parent plant or specialized reproductive structures, which are sometimes called vegetative propagules.
Tissue culture is the growth of tissues or cells in an artificial medium separate from the parent organism. This technique is also called micropropagation. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar. Tissue culture commonly refers to the culture of animal cells and tissues, with the more specific term plant tissue culture being used for plants. The term "tissue culture" was coined by American pathologist Montrose Thomas Burrows. This is possible only in certain conditions. It also requires more attention. It can be done only in genetic labs with various chemicals.
Plant callus is a growing mass of unorganized plant parenchyma cells. In living plants, callus cells are those cells that cover a plant wound. In biological research and biotechnology callus formation is induced from plant tissue samples (explants) after surface sterilization and plating onto tissue culture medium in vitro. The culture medium is supplemented with plant growth regulators, such as auxin, cytokinin, and gibberellin, to initiate callus formation or somatic embryogenesis. Callus initiation has been described for all major groups of land plants.
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 form the internal organs of the organism.
Plant embryonic development, also plant embryogenesis, is a process that occurs after the fertilization of an ovule to produce a fully developed plant embryo. This is a pertinent stage in the plant life cycle that is followed by dormancy and germination. The zygote produced after fertilization must undergo various cellular divisions and differentiations to become a mature embryo. An end stage embryo has five major components including the shoot apical meristem, hypocotyl, root meristem, root cap, and cotyledons. Unlike the embryonic development in animals, and specifically in humans, plant embryonic development results in an immature form of the plant, lacking most structures like leaves, stems, and reproductive structures. However, both plants and animals including humans, pass through a phylotypic stage that evolved independently and that causes a developmental constraint limiting morphological diversification.
Somaclonal variation is the variation seen in plants that have been produced by plant tissue culture. Chromosomal rearrangements are an important source of this variation. The term somaclonal variation is a phenomenon of broad taxonomic occurrence, reported for species of different ploidy levels, and for outcrossing and inbreeding, vegetatively and seed propagated, and cultivated and non-cultivated plants. Characters affected include both qualitative and quantitative traits.
Kinetin (/'kaɪnɪtɪn/) is a cytokinin-like synthetic plant hormone that promotes cell division in plants. Kinetin was originally isolated by Carlos O. Miller and Skoog et al. as a compound from autoclaved herring sperm DNA that had cell division-promoting activity. It was given the name kinetin because of its ability to induce cell division, provided that auxin was present in the medium. Kinetin is often used in plant tissue culture to induce callus formation and regenerate shoot tissues from callus.
Micropropagation or tissue culture is the practice of rapidly multiplying plant stock material to produce many progeny plants, using modern plant tissue culture methods.
Indole-3-butyric acid (1H-indole-3-butanoic acid, IBA) is a white to light-yellow crystalline solid, with the molecular formula C12H13NO2. It melts at 125°C in atmospheric pressure and decomposes before boiling. IBA is a plant hormone in the auxin family and is an ingredient in many commercial horticultural plant rooting products.
A seedless fruit is a fruit developed to possess no mature seeds. Since eating seedless fruits is generally easier and more convenient, they are considered commercially valuable.
In biology, explant culture is a technique to organotypically culture cells from a piece or pieces of tissue or organ removed from a plant or animal. The term explant can be applied to samples obtained from any part of the organism. The extraction process is extensively sterilized, and the culture can be typically used for two to three weeks.
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.
Embryo rescue is one of the earliest and successful forms of in-vitro culture techniques that is used to assist in the development of plant embryos that might not survive to become viable plants. Embryo rescue plays an important role in modern plant breeding, allowing the development of many interspecific and intergeneric food and ornamental plant crop hybrids. This technique nurtures the immature or weak embryo, thus allowing it the chance to survive. Plant embryos are multicellular structures that have the potential to develop into a new plant. The most widely used embryo rescue procedure is referred to as embryo culture, and involves excising plant embryos and placing them onto media culture. Embryo rescue is most often used to create interspecific and intergeneric crosses that would normally produce seeds which are aborted. Interspecific incompatibility in plants can occur for many reasons, but most often embryo abortion occurs In plant breeding, wide hybridization crosses can result in small shrunken seeds which indicate that fertilization has occurred, however the seed fails to develop. Many times, remote hybridizations will fail to undergo normal sexual reproduction, thus embryo rescue can assist in circumventing this problem.
Hyperhydricity is a physiological malformation that results in excessive hydration, low lignification, impaired stomatal function and reduced mechanical strength of tissue culture-generated plants. The consequence is poor regeneration of such plants without intensive greenhouse acclimation for outdoor growth. Additionally, it may also lead to leaf-tip and bud necrosis in some cases, which often leads to loss of apical dominance in the shoots. In general, the main symptom of hyperhydricity is translucent characteristics signified by a shortage of chlorophyll and high water content. Specifically, the presence of a thin or absent cuticular layer, reduced number of palisade cells, irregular stomata, less developed cell wall and large intracellular spaces in the mesophyll cell layer have been described as some of the anatomic changes associated with hyperhydricity.
Somatic embryogenesis is an artificial process in which a plant or embryo is derived from a single somatic cell. Somatic embryos are formed from plant cells that are not normally involved in the development of embryos, i.e. ordinary plant tissue. No endosperm or seed coat is formed around a somatic embryo.
Maryam Jafarkhani Kermani is an Associate Professor in the Department of Tissue and Cell Culture at the Administration of Agriculture and Biotechnology Research Institute of Iran (ABRII). She is an Iranian scientist whose main research area is agricultural tissue culture and mainly studies plants in the Rosaceous family.
Plant cryopreservation is a genetic resource conservation strategy that allows plant material, such as seeds, pollen, shoot tips or dormant buds to be stored indefinitely in liquid nitrogen. After thawing, these genetic resources can be regenerated into plants and used on the field. While this cryopreservation conservation strategy can be used on all plants, it is often only used under certain circumstances: 1) crops with recalcitrant seeds e.g. avocado, coconut 2) seedless crops such as cultivated banana and plantains or 3) crops that are clonally propagated such as cassava, potato, garlic and sweet potato.
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