18.The growth stages in suspension culture

The system of growing single cells and small cell aggregates in a liquid growth medium that is kept agitated by means of bubbling, shaking, or stirring so the cells do not settle out. Microorganisms and cells derived from callus tissues may be grown in this way. Growth is maintained by providing continuous aeration and by either transferring portions of the suspension to fresh medium or replacing a part of the culture with fresh medium. Suspension cultures of plant cells derived from friable masses of callus show a similar growth curve to cultures of microorganisms in that there is a lag phase and a logarithmic phase of growth. Cells of many plant species can be induced to form embryoids in suspension culture, which, if removed from the culture and given appropriate conditions, will develop into complete plants.

Cell suspension is prepared by transferring a fragment of callus (about 500 mg) to the liquid medium (500 ml) and agitating them aseptically to make the cells free in medium. It is difficult to have suspension of single cell. However, the suspension includes single cell, cell aggregates (varied number of cells), residual inoculum and dead cells (Dodds and Roberts, 1985). King (1980) has described that a good suspension consists of a high proportion of single cells than small cluster of cells. It is more difficult to have a good suspension than to find optimum environmental factors for cell separation. King and Street (1977) described the techniques of cell separation by changing the nutritional composition of medium. No standard technique for separation of cells from callus has been recommended. When cells are transferred into a suitable medium they divide after lag phase (no cell division) and linearly increases their population. After some time, based on nutrient level, the rate of cell division decelerates until it comes to stationary phase (see Fig. 14.2) At this stage, to keep the cells viable, it is essential to subculture the cells. By using plate technique, cell lines can be raised where a mass of cells is spread over medium. Further, growth of plated cells depends on cell density. Street (1977) suggested that cell density should be determined before subculturing. Growth of culture depends on a critical cell density below which culture will not grow, for instance, for a clone of Acer pseudoplatanus 9-15 x 103 cells/ ml are required. Generally cultures are agitated on orbital shaker or magnetic stirer for good result. Thus different types of works can be carried out by using cell suspensions. The cultured cells of a higher plant is inherently dualistic. On one hand it possesses necessary genetic informations for existence on cell level (reproduction, growth, mature state, programmed death). On the other hand, the cultured cells retainsupplementary informations that determine the production of substances which are important for integrative functions and biocoenotic interactions of the plants. The informations determining the progression to a programme for the development of a whole plant are also redundant for existence at cell level (Butenko, 1985). Cell culture systems have been employed in numerous morphological analyses by varying the origin of cells and physicochemical factors (White, 1963; Street, 1973). Kurz and Constabel (1979) have described the properties of cultured plant cell suspensions in common with culture of microorganisms as (i) they grow in sterile environment, (ii) they are homogeneous in size, (iii) they have a doubling time which is longer than that of microorganisms but considerably shorter than cells in situ, and (iv) they can be grown on a large scale.

19 The immobilized plant cells. The adventage of immobilized cells culture. Cell immobilization is a technique to fix plant cells in a suitable matrix. Cell immobilization is different from cell entrapment in that immobilized cells can be entrapped cells but also the cells are absorbed onto support materials. Plant cells grow slowly, they produce targetted compounds slowly, they are more easily disrupted by physical stress and their behaviour (growth and synthesis) is influenced by chemical signals by neigbouring. Then by immobilization, the plant cells are protected from liquid shear forces. Moreover, immobilization facilitates the importance of cellular cross talk, which can establish inter-cellular communication by the action of signalling molecules. This should enhance the biosynthetic of plant cells. Freely suspended plant cells mostly accumulate their secondary metabolistes in the stationary phase of their growth cycle, at the point of time their growth stop. Entrapment of plant cells is one the means to create non-growth condition under which the production of secondary metabolites may be improved. Advantages: retention of biomass enables its continuous reutilization as a production system. The high cell density allows a reduction in contact in packed bed catalyst leading to an increased volumetric productivity; separates cells from medium and the product is extra cellular, which will simplify downstream processing compared to extract from tissue; allows a continuous process, which increase volumetric productivity; compatible with non-growth associated product formation; reduces some problems associated with the cultivation of plant cells such as the formation of aggregates, and susceptibility to mechanical damage (shear stress) are problems which do not affect immobilized system compared to cell culture.