Methods available for propagating plants in vitro are largely an extension of those already developed for conventional propagation. In vitro techniques have the following advantages over traditional methods:
• Cultures are started with very small pieces of plants (explants), and thereafter small shoots or embryos are propagated (hence the term 'micropropagation' to describe the in vitro methods). Only a small amount of space is required to maintain plants or to greatly increase their number. Propagation is ideally carried out in aseptic conditions (avoiding contaminations). The often used term "axenic" is not correct, because it means "free from any association with other living organisms". Once cultures have been started there should be no loss through disease, and the plantlets finally produced should be ideally free from bacteria, fungi and other micro-organisms. (Most often this is not the case, see Vol. 2).
• Methods are available to free plants from specific virus diseases. Providing these techniques are employed, or virus-tested material is used for initiating cultures, certified virus-tested plants can be produced in large numbers. Terminology such as virus-free and bacteria-free should not be used, as it is impossible to prove that a plant is free of all bacteria or viruses. One can only prove that a plant has been freed from a specific contaminant provided the appropriate diagnostic tools are available.
• A more flexible adjustment of factors influencing vegetative regeneration is possible such as nutrient and growth regulator levels, light and temperature. The rate of propagation is therefore much greater than in macropropagation and many more plants can be produced in a given time. This may enable newly selected varieties to be made available quickly and widely, and numerous plants to be produced in a short while. The technique is very suitable when high volume production is essential.
• It may be possible to produce clones of some kinds of plants that are otherwise slow and difficult (or even impossible) to propagate vegetatively.
• Plants may acquire a new temporary characteristic through micropropagation which makes them more desirable to the grower than conventionally-raised stock. A bushy habit (in ornamental pot plants) and increased runner formation (strawberries) are two examples.
• Production can be continued all the year round and is more independent of seasonal changes.
• Vegetatively-reproduced material can often be stored over long periods.
• Less energy and space are required for propagation purposes and for the maintenance of stock plants (ortets).
• Plant material needs little attention between subcultures and there is no labour or materials requirement for watering, weeding, spraying etc.; micropropagation is most advantageous when it costs less than traditional methods of multiplication; if this is not the case there must be some other important reason to make it worthwhile.
The chief disadvantages of in vitro methods are that advanced skills are required for their successful operation.
• A specialised and expensive production facility is needed; fairly specific methods may be necessary to obtain optimum results from each species and variety and, because present methods are labour intensive, the cost of propagules is usually relatively high (Vol. 2). Further consequences of using in vitro adaptations are although they may be produced in large numbers, the plantlets obtained are initially small and sometimes have undesirable characteristics.
• In order to survive in vitro, explants and cultures have to be grown on a medium containing sucrose or some other carbon source. The plants derived from these cultures are not initially able to produce their own requirement of organic matter by photosynthesis (i.e. they are not autotrophic) and have to undergo a transitional period before they are capable of independent growth. More recently techniques have been proposed which allow the production of photo-autotrophic plants in vitro (Kozai and Smith, 1995).
• As they are raised within glass or plastic vessels in a high relative humidity, and are not usually photosynthetically self-sufficient, the young plantlets are more susceptible to water loss in an external environment. They may therefore have to be hardened in an atmosphere of slowly decreasing humidity and increased light. The chances of producing genetically aberrant plants may be increased.
A more extended discussion of all these points will be found in other sections.
The methods that are theoretically available for the propagation of plants in vitro are illustrated in Fig 2.1 and described in the following sections of this Chapter. They are essentially:
• by the multiplication of shoots from axillary buds:
• by the formation of adventitious shoots, and/or adventitious somatic embryos, either a) directly on pieces of tissue or organs (explants) removed from the mother plant; or b) indirectly from unorganised cells (in suspension cultures) or tissues (in callus cultures) established by the proliferation of cells within explants; on semi-organised callus tissues or propagation bodies (such as protocorms or pseudo-
bulbils) that can be obtained from explants (particularly those from certain specialised whole plant organs).
The techniques that have been developed for micropropagation are described in greater detail in the following sections of this Chapter. In practice most micropropagated plants are produced at present by method (i), and those of only a few species (which will be instanced later) by method (ii). Shoots and/or plantlets do not always originate in a culture by a single method. For example, in shoot cultures, besides axillary shoots, there are sometimes adventitious shoots formed directly on existing leaves or stems, and/or shoots arising indirectly from callus at the base of the explant. The most suitable and economic method for propagating plants of a particular species could well change with time. There are still severe limitations on the extent to which some methods can be used. Improvements will come from a better understanding of the factors controlling morphogenesis and genetic stability in vitro.
Rooting Somatic embryos have both a root and a shoot meristem. Under ideal conditions they can grow into normal seedlings. The shoots procured from axillary or adventitious meristems are miniature cuttings. Sometimes these small cuttings form roots spontaneously, but usually they have to be assisted to do so (Fig 2.2). The small rooted shoots produced by micropropagation are often called plantlets.
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