Hayta S, Bayraktar M, Baykan erel S, Gurel A Direct plant regeneration from different explants through micropropagation and determination of secondary metabolites in the critically endangered endemic Rhaponticoides mykalea.
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Tasty superfood from plant cell cultures
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Plant cell cultures for the production of recombinant proteins | Nature Biotechnology
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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  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.
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.
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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 common 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.