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The below mentioned article provides a study note on suspension culture.
A cell suspension culture consists of cell aggregates dispersed and growing in moving liquid media. It is normally initiated by transferring pieces of undifferentiated and friable calli to a liquid medium agitated by a suitable device (Fig. 17.3A-C).
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Agitation in the medium helps in two ways, it exerts a mild pressure on cell aggregates, breaking them into smaller clumps and single cells; secondly agitation maintains uniform distribution of cell and cell clumps in the medium. It also helps in good gaseous exchange between culture medium and air.
Suspension culture can also be achieved by using mechanical method i.e., sterile explants like stem, leaf, root or any kind of soft tissue can be gently grinded by glass homogenizer and then the homogenate containing intact cells or small tissue masses can be used for initiation of suspension culture.
Another method is enzymatic method which may also be applied for isolation of single cells by the use of pectinases to digest the pectin wall. Orbital shakers are widely used for initiation and serial propagation of plant suspension culture.
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The time period required for establishment of suspension culture from its callus tissue is called the initiation phase. During this phase the callus tissue breaks up, the cells grow and divide until the depletion of some nutrient in the medium. To know the time period for sub-culturing of a particular species the growth measurement in suspension culture is very much needed.
The growth measurement can be done by counting the cell number under simple microscope after staining and macerating the small aggregates. The growth can also be measured by collecting the cell mass and by taking the fresh weight or dry weight of the cell mass i.e., the packed cell volume (PCV) measurement.
During this period, five phases of growth can be observed:
(i) Lag phase: Where the cells prepare to divide.
(ii) Exponential phase: Where the rate of cell division is highest.
(iii) Linear phase: Where the cell divisional rate slows down but the cell expansion takes place.
(iv) Deceleration phase: Where the rate of cell division and cell elongation both decreases.
(v) Stationary phase: Where the number of cells and their size remain constant.
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After the initiation phase, the suspension culture can be passed through a nylon mesh to remove the larger clump and allowing the single cells or smaller cell aggregates to transfer into new medium for further culture. In the subsequent passages, the cell suspension is sub-cultured by taking a small aliquot and transferring into new medium containing flasks.
The concentration of growth hormones like auxin and cytokinin both play a critical role for the growth of cell suspension. The cells in suspension culture may vary in shapes and sizes. Those may be oval, round, elongated, etc. and mainly of thin walled.
Frequent sub-culturing in a suitable medium ensures to achieve rapid growth rate, uniform and viable cells. For sub-culturing the initial inoculum density is very critical for starting of the cell division. Very low density of cells is unable to start growing and also very high density of cells is inhibitory, i.e., after attaining certain density the sub-culturing is needed.
