Group A6Methods Used for the

Cultivation of Plant Cells

Tan Siew Yen

Mun Hui SinNuraini Binti

AbdullahSiti Rabaah Binti Hamzah

Callus Culture

Method 1 - Callus Culture

Callus is explants that cultured on the appropriate medium, with auxin and a cytokinin, give rise to an unorganised, growing and dividing mass of cells.

Any plant tissue that have dividing cells can be used as an explant.

Exercised plant material is placed on solidified medium containing nutrients and hormones that promote rapid cell differentiation.

Callus forms can be quite large (>1cm) (Shuler and Kargi, 2002).

Method 1 - Callus culture

During callus formation there is some degree of dedifferentiation or changes in morphology and metabolism that can cause plant cultures to lose the ability to photosynthesise.

Overcome by the addition of other components such as vitamins and carbon source to the culture medium.

Auxin – Promote root initiation. Cytokinin – Induce growth of shoots

Figure 1: Function of Auxin and Cytokinin

Method 1 - Callus culture

Suspension

Culture

Method 2 - Suspension Culture

There are two categories of callus cultures: compact or friable. In compact callus, the cells are densely aggregated while in friable callus the cells are only loosely associated with each other and the callus becomes soft and breaks apart easily.

Friable callus provides the inoculum to form cell-suspension cultures.

The friability of callus is improved by manipulating the medium components, repeated subculturing or culturing it on medium with a low concentration of gelling agent.

Method 2 - Suspension Culture

Callus culture is then transferred into liquid medium to establish suspension culture, which is place on shaker to supply the cells with sufficient oxygen.

With moderate agitation, cells or small aggregates of cells will slough off.

A platform shaker is used to give a circular motion in a viable speed control (30-150 rpm) (Shuler and Kargi, 2002).

Callus culture is performed in the dark (27°C & pH 5.5) as light can encourage differentiation of the callus (Shuler and Kargi, 2002).

Method 2 - Suspension Culture

During long-term culture, ‘habituation’ occurs. The culture may lose the requirement for auxin and/or cytokinin.

Under the correct conditions, these released cells continue to grow and divide, eventually producing a cell-suspension culture.

The inoculum should not be too large as toxic products released from damaged or stressed cells can build up to lethal levels.

Cell suspensions can be maintained by batch cultures in conical flasks. They are continually cultured by repeated subculturing into fresh medium.

Method 2 - Suspension Culture

This results in dilution of the suspension and the initiation of another batch growth cycle. The degree of dilution during subculture should be determined empirically for each culture. Too great a degree of dilution will result in a greatly extended lag period or, in extreme cases, death of the transferred cells.

After subculture, the cells divide and the biomass of the culture increases, until nutrients in the medium are exhausted and/or toxic by-products build up to inhibitory (stationary phase).

If cells are left in the stationary phase for too long (~2-3 weeks), they will die and the culture will be lost. Therefore, cells should be transferred to fresh medium as they enter the stationary phase (Shuler and Kargi, 2002).

Figure 2: Steps in Plant Cell Culture (Dodds & Roberts, 1995).

Figure 3: Steps in Plant Cell Culture (Shuler and Kargi, 2002).

Figure 4: Callus Culture (right) and Suspension Culture (left) of Coleus Blumei (Dodds & Roberts, 1995).

Thank You

References

Dodds, J. H. and Roberts, L. W. (1995). Experiments in plant tissue culture. Cambridge University Press, Cambridge.

Shuler, M. L. and Kargi, F. (2002). Bioprocess Engineering: Basic Concept. 2nd Ed. Upper Saddle River, NJ: Prentice Hall PTR