Three types of differentiated cells are commonly found in callus and cell cultures; these are vessels and tracheids (the cells from which the water-conducting vascular xylem is constructed), and cells containing chloroplasts (organelles carrying the green photosynthetic pigment, chlorophyll). Phloem sieve tubes may be present but are difficult to distinguish from undifferentiated cells.
Callus cultures are more likely to contain tracheids than any other kind of differentiated cell. The proportion formed depends on the species from which the culture originated and especially upon the kind of sugar and growth regulators added to the medium. This is discussed further in Chapter 10.
Tracheid formation may represent or be associated with an early stage in the development of shoot meristems. Nodules containing xylem elements in callus of Pelargonium have, for example, been observed to develop into shoots when moved to an auxin-free medium (Chen and Galston, 1967; Cassells, 1979).
The rapid cell division initiated when tissue is transferred to a nutrient medium usually occurs in meristems formed around the periphery of the explant. Cell differentiation does not take place in callus cultures during this phase but begins when peripheral meristematic activity is replaced or supplemented by the formation of centres of cell division deeper in the tissue. These internal centres generally take the form of meristematic nodules that may produce further expanded and undifferentiated cells (so contributing to callus growth) or cells that differentiate into xylem or phloem elements. Nodules can form primitive vascular bundles, with the xylem occurring centrally and the phloem peripherally, separated from the xylem by a meristematic region.
The formation and maintenance of green chloroplasts in cultured plant cells represents another form of cellular differentiation which is easy to monitor, and which has been studied fairly extensively. When chloroplast-containing cells from an intact plant are transferred to a nutrient medium they begin to dedifferentiate. This process continues in the event of cell division and results in a loss of structure of the membranes containing chlorophyll (thylakoids) and the stacks (grana) into which they are arranged, and the accumulation of lipid-containing globules. The chloroplasts eventually change shape and degenerate.
Callus cells frequently do not contain chloroplasts but only plastids containing starch grains in which a slightly-developed lamellar system may be apparent. All the same, many calluses have been discovered that do turn green on continued exposure to light and are composed of a majority of chloroplast-containing cells. Chloroplast formation can also be connected with the capacity of callus to undergo morphogenesis. Green spots sometimes appear on some calluses and it is from these areas that new shoots arise. By subculturing areas with green spots, a highly morphogenic tissue can sometimes be obtained. The formation of chloroplasts and their continued integrity is also favoured by cell aggregation. When green callus tissue is used to initiate suspension cultures, the number of chloroplasts and their degree of differentiation are reduced. Nevertheless, there can be some increase in chlorophyll content during the stationary phase of batch cultures.
The level of chlorophyll so far obtained in tissue cultures is well below that found in mesophyll cells of whole plants of the same species, and the rate of chlorophyll formation on exposure of cultured cells to the light is extremely slow compared to the response of etiolated organised tissues. The greening of cultures also tends to be unpredictable and even within individual cells, a range in the degree of chloroplast development is often found. In the carbon dioxide concentrations found in culture vessels, green callus tissue is normally photomixotrophic (i.e. the chloroplasts are able to fix part of the carbon that the cells require) and growth is still partly dependent on the incorporation of sucrose into the medium (Vasil and Hildebrandt, 1966). However, green photoautotrophic callus cultures have been obtained from several different kinds of plants. When grown at high carbon dioxide concentrations (1-5%), without a carbon source in the medium, they are capable of increasing in dry weight by photosynthetic carbon assimilation alone (see Street, 1977a).
Photoautotrophic cell suspensions have also been obtained. They too normally require high carbon dioxide levels, but cell lines of some species have been isolated capable of growing in ambient CO2 concentration (Xu et al., 1988). Why cultured cells do not freely develop fully functional chloroplasts is not fully known. Some hypotheses have been summarised by Dalton (1980). The cytology of chloroplast formation is described in Yeoman and Street (1977). Photoautotrophic growth of shoots is described in Chapter 2.
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