In this article we will discuss about the Cell Suspension Culture:- 1. Meaning of Cell Suspension Culture 2. Initiation of Cell Suspension Culture 3. Medium 4. Aeration 5. Significance.
Meaning of Cell Suspension Culture:
Cell suspension culture is the culture of cells or cell aggregates dispersed in a moving liquid medium. Batcher and Ingram (’76) noted that all suspension cultures contain cell aggregates in addition to free cells (Fig. 21).
Cell suspension cultures were grown successfully in several plants, such as, Nicotiana tabacum, Tagetes erecta, Daucus carota, Antirrhinum and Picea glauca. Suspension cultures from various plants show different proportions of free cells and cell aggregates.
In suspension culture of Antirrhinum cell aggregates are more frequent than free cells. In suspension culture of Paul’s Scarlet rose free cells occur at a very high frequency.
Cells of a suspension culture often possess large nuclei, dense cytoplasm and starch grains. Cells show hormone habituation, increase in ploidy level and loss in totipotency. Cell separation is high and cell doubling period is 24-78 hours. But cell suspensions from different sources vary considerably in the expression of these characteristics.
Suspension cultures are mainly of two types—batch culture and continuous culture.
(a) In batch culture cells are grown in a fixed volume of medium. It is a closed system culture.
(b) In continuous culture by pumping a constant flow of the culture medium is maintained in the culture vessel. So there is replacement of equal volume of culture medium. New cell formation is more or less equal to the amount of old washed out cells. Thus the culture is maintained in a steady condition.
Continuous culture may be closed or open type. In an open system fresh medium is added and spent medium is withdrawn and the cells are also harvested. In a closed culture all cells remain in the culture, thus the number of cells increases until the stationary phase is readied.
Initiation of Cell Suspension Culture:
Cell suspension cultures can be initiated by transferring fragments of friable callus tissue to a liquid medium, which is constantly agitated. Cells of friable callus tissue readily dissociate. Highly friable callus is most suitable for suspension culture. Friability depends upon certain minerals, such as, the ratio of potassium and magnesium, amount of calcium etc..
Non-friable callus is sometimes used for suspension culture. As the cells do not separate easily, so auxin concentration of the liquid medium is increased or enzymes, such as, cellulose and pectinase are added to help dissociation. High auxin and cytokinin ratio sometimes favours formation of friable culture.
Besides callus, soft tissues from hypocotyl or cotyledon of sterile seedlings or embryos can be taken for suspension culture. This tissue is broken in a hand operated glass homogenizer and then the cells transferred to the medium.
By subsequent subculture residual part of the inoculum can be excluded. The culture is sometimes allowed to settle for few seconds and then the cell suspension is taken only from the upper part of the culture. Critical initial density or minimum effective density is the smallest inoculum from which a new suspension culture can be successfully grown.
Medium of Cell Suspension Culture:
The culture medium is composed of mineral salts, sucrose, vitamins and hormones. In some cases higher concentration of cytokinin and auxin is needed.
In liquid medium considerable pH change may occur with the addition of the inoculum because the medium is usually lightly buffered. To stabilize the pH of the medium, solid buffer, such as, sparingly soluble compounds of calcium de-hydrogen orthophosphate, calcium phosphate and calcium carbonate are used.
Aeration of Cell Suspension Culture:
Cell suspension culture is constantly shaken by a platform shaker at a speed of 100-120 r.p.m. in a 250 c.c. Erlenmeyer flask. The volume of the flask and the amount of the culture medium are important.
The amount of the culture liquid should be about 20% of the total volume of the flask. Other methods for aeration are magnetic stirrer, roller culture and Steward’s auxophyton. In micro-culture no special method for aeration is necessary.
Growth of the Cells in the Liquid Medium:
After introducing the inoculum in the culture medium initially the cells fail to divide. This is the lag period. Then cell division starts and there is an increase in cell population. It reaches a maximum density per unit volume.
Gradually the rate of cell division decreases (deceleration phase) due to exhaustion of the medium or accumulation of toxic substances. Ultimately the cells reach a stationary phase (Fig. 22). Then the cells are sub-cultured to maintain viability.
Subculture:
Subculture should be done when the culture has reached the maximum cell density, otherwise the cells may die and lysis occurs. Subculture is usually done at early stationary phase or during progressive deceleration phase. Usually in a suspension culture maximum cell density is reached within 18-25 days.
But in some cases maximum cell density is reached within 6-9 days. Before first subculture, the culture is passed through a stainless steel filter or nylon net to remove the cell aggregates and residual inoculum. By repeated subcultures a cell suspension culture can be maintained for a long time.
Cell Suspension Culture of Carrot:
Young and freshly growing carrot callus is taken and is placed on a petridish lined with filter paper. The sides of the callus are trimmed and an inoculum of about 0.5—0.75 gm callus is transferred to each flask containing the culture medium.
Usually Murashige and Skoog’s basal medium supplemented with 1.0 mg/1 2-4D and sucrose is used. Presence of auxin in the medium increases the friability of the tissue.
The flask with the inoculum is kept in a shaker moving at a speed of 100 r.p.m. and at a temperature of 25-27°C.
After 7-10 days the culture is strained through a nylon mesh to remove cell aggregates and residual inoculum. Subculture is done on a fresh medium.
Cell counting can be done with a homocytometer or a Sedgwick-rafter slide.
Significance of Cell Suspension Culture:
(1) Cell culture may be used in future for the whole or partial synthesis of secondary plant products, such as, alkaloids, glycosides etc.
(2) By cell culture many problems in applied botany can be solved.