Figure 14.24B Some specific examples of specialized stems that are easily propagated asexually. Left: A tulip bulb. Center. Crocus corms. Right: A ginger rhizome.
antibiotic we can administer as a cure. The best way to eliminate the effects of plant disease is to prevent exposure to pathogens. This is nearly impossible in greenhouse and field environments. However, it is possible to maintain plants in a disease-free status if we grow them in sterile test tubes through micropropagation. Other advantages of micropropagation include the capacity to grow large numbers of plants in a small area, minimal maintenance required for established plants (they do not need to be watered), and rapid multiplication.
Propagating plants through micropropagation is similar to growing them as cuttings. The major difference is that the plants are grown in vitro in a sterile medium and maintained in special controlled environment rooms. The medium includes a support matrix composed of agar, a gelatinous material extracted from red algae. Inorganic salts are added to the medium to provide macro- and micronutri-ents, such as nitrogen, phosphorous, calcium, and iron. Sucrose is added to supplement the sugars produced by the plant. In addition, vitamins such as thiamine, nicotinic acid, and inositol are generally included in the growth medium. Commonly, growth regulators are also added. After the ingredients are combined and pH is adjusted, the mixture is poured into test tubes. The tubes are then capped and put in an autoclave (a large form of pressure cooker) to sterilize them (Fig. 14. 25). When the medium cools, it solidifies like Jell-O™. This acts as the "soil" in the micropropagation system, providing plants with support, nutrients, and water.
Micropropagation, like other forms of asexual propagation, relies on the property of totipotency (capacity of a cell to give rise to any structure of a mature organism) of plant cells. Each living cell has the genetic information and, therefore, the capacity to develop into any cell type. Micropropagation usually begins with an excised piece of leaf or stem tissue, or explant, and carries it through three steps.
The first step is establishment of explants in tissue culture. Micropropagation requires sterile plant material as well as growth media. Plant parts must be disinfested to remove surface contaminants without killing the plant tissue.
Common disinfestants include bleach and ethanol. This procedure does not remove internal contaminants, including pathogens, so it is important to begin with disease-free plants. After plant parts are disinfested, they are inserted into the growth medium in test tubes under sterile conditions. Often, a special reach-in chamber, called a laminar flow hood, is used for this step (Fig. 14.26). Filtered air is blown across the work surface in the chamber to prevent the introduction of contaminants. Test tubes containing sterile plants are then placed in a clean room with artificial lighting and temperature control. The goal of this first step is to obtain sterile, viable plant tissue cultures (Fig. 14.27).
After cultures are established, they typically will be induced to develop multiple shoots in a multiplication medium. These microshoots can be separated and placed in a new medium by a process called subculturing (Fig. 14.28).This step is similar to propagation by cuttings, except it is carried out under sterile conditions. It is not unusual to subculture plants every four weeks, making approximately four new plants from every one in a test tube. At this rate, it is theoretically possible to produce a million plants from one plant in just 10 months. Although these multiplication rates
are not realized in commercial systems, many tissue culture laboratories have the capacity to produce millions of plants per year.
The third step in micropropagation is root formation. Some explants, such as those from African violets, will spontaneously produce roots in multiplication medium. In other
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cases, plants are induced to form roots in vitro by transferring them to a rooting medium. Compared to the multiplication medium the rooting medium usually contains reduced levels of cytokinins and increased auxin levels. When possible, the most economical approach is an ex vitro one, in which microshoots are rooted in potting mix and treated as cuttings.
The last step in micropropagation is the transfer of plants back to an outdoor environment. This is often the most difficult step. Because the humidity is high in test tubes, plants grown in vitro do not produce as much wax on their cuticles as do those grown outdoors. In addition, their stomata do not close as readily in response to water stress. Therefore, tissue culture plantlets must be acclimatized to an outdoor environment. First, the humidity in the growth chamber is reduced for a few weeks before the plants are brought out of their test tubes. Then, when they are transferred to soil in pots, they are maintained in a high-humidity environment for several weeks.
Commercial micropropagation has become a successful venture with a number of plants. Some plants that propagate slowly by other asexual means may be rapidly increased with micropropagation. These include orchids, Boston fern, African violet, and Hosta. In some cases, with the use of tissue culture techniques, as many plants can be produced in a month as would be produced in a year with other techniques. New cultivars can also be rapidly multiplied to meet high market demand. For example, new apple rootstocks are often propagated in vitro because conventional asexual reproduction methods cannot adequately supply market needs. Tissue culture protocols, using various combinations of nutrients and growth regulators, have been developed for a number of woody plants that are otherwise difficult to root.
Tissue culture is now being used to propagate endangered plant species. Small pieces of just a few plants can be used to establish tissue cultures. Then, nearly unlimited numbers of plantlets can be produced and returned to their natural habitat.
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