Tissue Culture - Micropropagation



Through the use of biotechnology, desirable genetic traits can be transferred from one organism to another by transfer of DNA. In plants, the DNA of interest is transferred into the new plant by using Agrobacterium tumefaciens, a bacterium that can infect plant tissues and incorporate part of its DNA into the DNA of the host plant. Alternatively, a particle gun can be used to directly "shoot" the DNA into plant cells.
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In both methods, the target for the foreign DNA is a small piece of plant tissue or a small mass of plant cells. Once the DNA has been transferred, new plants must be regenerated from the small pieces of transformed plant tissue using micropropagation (tissue culture) techniques.

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Micropropagation is the aseptic culture of cells, pieces of tissue, or organs. It is possible to regenerate new plants from small pieces of plant tissue because each cell of a given plant has the same genetic makeup and is totipotent, that is, capable of developing along a "programmed" pathway leading to the formation of an entire plant that is genetically identical to the plant from which it was derived (a clone). In addition to its biotechnological applications, micropropagation is used commercially to asexually propagate plants. Using micropropagation, millions of new plants can be derived from a single plant. This rapid multiplication allows breeders and growers to introduce new cultivars much earlier than they could by using conventional propagation techniques, such as cuttings. Micropropagation also can be used to establish and maintain virus-free plant stock. This is done by culturing the plant's apical meristem, which typically is not virus-infected, even though the remainder of the plant may be. Once new plants are developed from the apical meristem, they can be maintained and sold as virus-free plants.

Micropropagation differs from all other conventional propagation methods in that aseptic conditions are essential to achieve success. The process of micropropagation can be divided into four stages:
1.    Initiation stage. A piece of plant tissue (called an explant) is (a) cut from the plant, (b) disinfested (removal of surface contaminants), and (c) placed on a medium. A medium typically contains mineral salts, sucrose, and a solidifying agent such as agar. The objective of this stage is to achieve an aseptic culture. An aseptic culture is one without contaminating bacteria or fungi.
2.    Multiplication stage. A growing explant can be induced to produce vegetative shoots by including a cytokinin in the medium. A cytokinin is a plant growth regulator that promotes shoot formation from growing plant cells.
3.    Rooting or preplant stage. Growing shoots can be induced to produce adventitious roots by including an auxin in the medium. Auxins are plant growth regulators that promote root formation. For easily rooted plants, an auxin is usually not necessary and many commercial labs will skip this step.
4.    Acclimatization. A growing, rooted shoot can be removed from tissue culture and placed in soil. When this is done, the humidity must be gradually reduced over time because tissue-cultured plants are extremely susceptible to wilting.