3.2.1. Callus cultures
Callus is a coherent and amorphous tissue, formed when plant cells multiply in a disorganised way. It is often induced in or upon parts of an intact plant by wounding, by the presence of insects or microorganisms, or as a result of stress. Callus can be initiated in vitro by placing small pieces of the whole plant (explants) onto a growth-supporting medium under sterile conditions. Under the stimulus of endogenous growth regulators or growth regulating chemicals added to the medium, the metabolism of cells, which were in a quiescent state, is changed, and they begin active division. During this process, cell differentiation and specialisation, which may have been occurring in the intact plant, are reversed, and the explant gives rise to new tissue, which is composed of meristematic and unspecialised cell types.
During dedifferentiation, storage products typically found in resting cells tend to disappear. New meristems are formed in the tissue and these give rise to undifferentiated parenchymatous cells without any of the structural order that was characteristic of the organ or tissue from which they were derived. Although callus remains unorganised, as growth proceeds, some kinds of specialised cells may again be formed. Such differentiation can appear to take place at random, but may be associated with centres of morphogenesis, which can give rise to organs such as roots, shoots and embryos. The de novo production of plants from unorganised cultures is often referred to as plant regeneration.
Although most experiments have been conducted with the tissues of higher plants, callus cultures can be established from gymnosperms, ferns, mosses and thallophytes. Many parts of a whole plant may have an ultimate potential to proliferate in vitro, but it is frequently found that callus cultures are more easily established from some organs than others. Young meristematic tissues are most suitable, but meristematic areas in older parts of a plant, such as the cambium, can give rise to callus. The choice of tissues from which cultures can be started is greatest in dicotyledonous species. A difference in the capacity of tissue to give rise to callus is particularly apparent in monocotyledons. In most cereals, for example, callus growth can only be obtained from organs such as zygotic embryos, germinating seeds, seed endosperm or the seedling mesocotyl, and very young leaves or leaf sheaths, but so far never from mature leaf tissue (e.g. Green and Phillips, 1975; Dunstan et al., 1978). In sugar cane, callus cultures can only be started from young leaves or leaf bases, not from semi-mature or mature leaf blades.
Even closely associated tissues within one organ may have different potentials for callus origination. Thus when embryos of Hordeum distichum at an early stage of differentiation are removed from developing seeds and placed in culture, callus proliferation originates from meristematic mesocotyl cells rather than from the closely adjacent cells of the scutellum and coleorhiza (Granatek and Cockerline, 1979).
The callus formed on an original explant is called 'primary callus'. Secondary callus cultures are initiated from pieces of tissue dissected from primary callus (Fig. 1.8.). Subculture can then often be continued over many years, but the longer callus is maintained, the greater is the risk that the cells thereof will suffer genetic change (see Chapter 10).
Callus tissue is not of one single kind. Strains of callus differing in appearance, colour, degree of compaction and morphogenetic potential commonly arise from a single explant. Sometimes the type of callus obtained, its degree of cellular differentiation and its capacity to regenerate new plants, depend upon the origin and age of the tissue chosen as an explant. Loosely packed or 'friable' callus is usually selected for initiating suspension cultures (see below).
Some of the differences between one strain of callus tissue and another can depend on which genetic programme is functioning within the cells (epigenetic differences). Variability is more likely when callus is derived from an explant composed of more than one kind of cell. For this reason there is often merit in selecting small explants from only morphologically uniform tissue, bearing in mind that a minimum size of explant is normally required to obtain callus formation.
The genetic make up of cells is very commonly altered in unorganised callus and suspension cultures. Therefore another reason for cell strains having different characteristics, is that they have become composed of populations of cells with slightly different genotypes. Genetic and epigenetic changes occurring in cultures are described in greater detail in Chapters 10 and Volume 2. The growth, structure, organisation and cytology of callus are discussed in various chapters of the book edited by Street (1977a), and also in the review by Yeoman and Forche (1980).
Unorganised plant cells can be grown as callus in aggregated tissue masses, or they can be freely dispersed in agitated liquid media. Techniques are similar to those used for the large-scale culture of bacteria. Cell or suspension cultures, as they are called, are usually started by placing an inoculum of friable callus in a liquid medium (Fig. 1.8). Under agitation, single cells break off and, by division, form cell chains and clumps which fracture again to give individual cells and other small cell groups. It is not always necessary to have a previous callus phase before initiating suspension cultures. For example, leaf sections of Chenopodium rubrum floated on Murashige and Skoog (1962) medium in the light, show rapid growth and cell division in the mesophyll, and after 4 days on a rotary shaker they can be disintegrated completely to release a great number of cells into suspension (Geile and Wagner, 1980).
Because the walls of plant cells have a natural tendency to adhere, it is not possible to obtain suspensions that consist only of dispersed single cells. Some progress has been made in selecting cell lines with increased cell separation, but cultures of completely isolated cells have yet to be obtained. The proportion and size of small cell aggregates varies according to plant variety and the medium in which the culture is grown. As cells tend to divide more frequently in aggregates than in isolation, the size of cell clusters increases during the phase of rapid cell division. Because agitation causes single cells, and small groups of cells, to be detached, the size of cell clusters decreases in batch cultures as they approach a stationary growth phase (see below).
The degree of cell dispersion in suspension cultures is particularly influenced by the concentration of growth regulators in the culture medium. Auxinic growth regulators increase the specific activity of enzymes, which bring about the dissolution of the middle lamella of plant cell walls (Torrey and Reinert, 1961). Thus by using a relatively high concentration of an auxin and a low concentration of a cytokinin growth regulator in the culture medium, it is usually possible to increase cell dispersion (Narayanaswamy, 1977). However, the use of high auxin levels to obtain maximum cell dispersion will ensure that the cultured cells remain undifferentiated. This may be a disadvantage if a suspension is being used to produce secondary metabolites. Well-dispersed suspension cultures consist of thin-walled undifferentiated cells, but these are never uniform in size and shape. Cells with more differentiated structure, possessing, for example, thicker walls and even tracheid-like elements, usually only occur in large cell aggregates.
Many different methods of suspension culture have been developed. They fall into two main types: batch cultures in which cells are nurtured in a fixed volume of medium until growth ceases, and continuous cultures in which cell growth is maintained by continuous replenishment of sterile nutrient media. All techniques utilise some method of agitating the culture medium to ensure necessary cell dispersion and an adequate gas exchange.
Batch cultures. Batch cultures are initiated by inoculating cells into a fixed volume of nutrient medium. As growth proceeds, the amount of cell material increases until nutrients in the medium are depleted or there is the accumulation of an inhibitory metabolite. Batch cultures have a number of disadvantages that restrict their suitability for extended studies of growth and metabolism, or for the industrial production of plant cells, but they are nevertheless widely used for many laboratory investigations. Small cultures are frequently agitated on orbital shakers onto which are fixed suitable containers, which range in volume from 100 ml (Erlenmeyer conical flasks) to 1000 ml (spherical flasks); the quantity of medium being approximately the same as the flask volume. The shakers are usually operated at speeds from 30-180 rpm with an orbital motion of about 3 cm. Alternatively, stirred systems can be used.
Continuous cultures. Using batch cultures, it is difficult to obtain a steady rate of production of new cells having constant size and composition. Attempts to do so necessitate frequent sub-culturing, at intervals equivalent to the doubling time of the cell population. Satisfactorily balanced growth can only be produced in continuous culture, a method, which is especially important when plant cells are to be used for the large-scale production of a primary or secondary metabolite. Continuous culture techniques require fairly complicated apparatus. Agitation of larger cultures in bio-reactors is usually achieved by stirring with a turbine and/or by passing sterile air (or a controlled gaseous mixture) into the culture from below and releasing it through plugged vents. Mechanically stirred reactors damage plant cells by shearing. This is minimised in air-lift reactors. Different bioreactor designs are illustrated in Fig. 1.9.
The use of suspension cultures in plant propagation. The growth of plant cells is more rapid in suspension than in callus culture and is also more readily controlled because the culture medium can be easily amended or changed. Organs can be induced to develop in cell suspensions: root and shoot initiation usually commences in cell aggregates. Somatic embryos may arise from single cells. Cells from suspensions can also be plated onto solid media where single cells and/or cell aggregates grow into callus colonies from which plants can often be regenerated. For these reasons suspension cultures might be expected to provide a means of very rapid plant multiplication. There are two methods: • plants may be obtained from somatic embryos formed in suspensions. Once embryos have been produced, they are normally grown into plantlets on solid media, although other methods are potentially available (Chapter 2);
• cells from suspensions are plated onto solid media where single cells and/or cell aggregates grow into callus colonies from which plants can often be regenerated.
In practice neither of these techniques has been sufficiently reliable for use in plant propagation.
Immobilised cell cultures. Plant cells can be captured and immobilised by being cultured in a gel which is afterwards solidified (see Chapter 4). This technique has only limited application to plant micropropagation, but is now employed quite widely when plant cells are grown for the production of their secondary products or for the bio-transformation of chemical compounds (Lindsey and Yeoman, 1983).
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