Establishment of Suspension Cultures

Background
Suspension cultures are suspensions of individual plant cells and small cell clusters (microcalli)
grown in liquid media. Suspension cultures are established by transferring small pieces of callus to
liquid medium which is subsequently placed on a gyratory shaker. Within a few days individual
plant cells and microcalli should be detached from the original inoculum and growing in the
constantly agitated medium. Suspension cultures grow best if the larger pieces of callus are
removed after the culture has been initiated (Bottino, 1981).

Methods
1. Break up tobacco callus into small pieces and transfer to liquid medium (Tobacco Callus
Initiation Medium, without the agar). Use 25–50 ml medium in 250-ml flask for adequate aeration
for the swirling culture.
2. A thick suspension of callus should continue to proliferate within the next week on a gyratory
shaker.
3. Remove large pieces of callus from suspension culture by passing through a sterile sieve. Large
pieces of callus are usually detrimental to the maintenance of a suspension culture.
4. Suspension cultures can be multiplied by diluting 5 ml of the original strained suspension
culture to 50 ml total volume (1:10 dilution, vol:vol) of fresh medium. The growth rate and cell
generation time can be determined.

Usefulness of Suspension Cultures
Suspension cultures are used in a number of biotechnological procedures such as inoculum for
plant bioreactors, which resemble biofermentors. Valuable plant products can be extracted from
bioreactors during the growth of plant cells as cell suspensions. This type of biotechnology is still in
the initial stages of development and industrial scale-up. Suspension cultures treated with enzymes
that degrade cell walls produce protoplast cultures which can be used in DNA transformation
experiments as well as in protoplast fusion (somatic hybridization) experiments.