Elementary plant Biochemistry and Biotechnology (2+1)

Cell suspension culture

• To obtain free cells, pieces of undifferentiated and friable calli are transferred to liquid medium in flask or some other suitable vial and the medium is continuously agitated by a suitable device. Such cultures is called ‘suspension culture’. Agitation of medium serves at least two functions. First, it exerts a mild pressure on cell aggregates, breaking them into smaller clumps and single cells. Second, the agitation maintains uniform distribution of cells and cell clumps in the medium. Movement of the medium also provides gaseous exchange between the culture medium and culture air.


• Most suspension cultures are obtained by transfer of friable callus clumps to agitated liquid medium of the same composition as that used for callus growth. Agitation rates on orbital shakers should be in the range of 100-150 rpm. The degree of cell separation of established cultures of high friability can be modified by changing the composition of the nutrient medium, particularly the concentration of growth regulators. When the plant material is first placed on the medium, there is an initial lag period prior to any sign of cell division (Figure).


• During the incubation period, the biomass of the suspension cultures increases due to cell division and cell enlargement. This continues for a limited period after which the growth stops due to the exhaustion of some factors or the accumulation of certain toxic metabolites in the culture medium. When a small aliquot of the cell suspension at this stage, is transferred to a fresh medium of the same composition, the cell growth is revived. This is an exponential rise in cell number and a linear increase in cell population.
  


• There is a gradual deceleration in the division rate. After three to four cell generations the growth declines and finally the culture enters the stationary phase. Finally the cells enter a stationery or non dividing stage. In order to maintain the viability of the culture, the cells should be sub cultured early during this stationery phase i.e., in exponential phase by frequent (every 2-3 days) subculture of the suspensions. Prolonged maintainance of cultures in the stationary phase may result in extensive death and lysis of cells.


• Subculture involves the aseptic transfer of a suitable volume of inoculum to fresh medium using either pipettes (or) autoclavable metal syringes and transfer by simply tipping culture into the new vessel up to a graduation mark to ensure approximately constant inoculum’s size. At the time of subculture, the flask is allowed to stand for a few seconds to allow the large colonies to settle down and suspension is taken from the upper part of the culture. Regular practice of this procedure should allow the build-up of a fine suspension.


• The texture of a callus is genetically controlled. Certain celli may not give good dispersion under normal media conditions. However, it has been possible to improve the tissue dissociation by manipulating the media composition and subculture routine. Addition of small amounts of 2, 4-D, hydrolytic enzymes such as cellulose and pectinase or substances like yeast extract had a promotory effect on cell dispersion.