Synchronizaation of suspension culture, cellular totipotency, cytodifferentiation

1. BHARATHIDASAN UNIVERSITY
SYNCHRONIZATION OF SUSPENSION CULTURE,
CELLULAR TOTIPOTENCY AND CYTODIFFERENTIATION
Presented by:
Bhanu Kiran

2. Content
 Synchronization of suspension culture
Physical methods
Chemical methods
 Cellular totipotency
 Cytodifferentiation

3. SYNCHRONIZATION OF SUSPENSION
CULTURE
 Cells in suspension cultures vary greatly in size, shape DNA and nuclear content. Moreover, the
cell cycle time varies considerably within individual cells. Therefore, cell cultures are mostly
asynchromous.
 This variation complicates studies of biochemical, genetic physiological and other aspects of cell
metabolism. A synchronous culture is one in which the majority of cells proceed through each
cell cycle phase (G,S ,G2 and M) simultaneously.
 There are two ways of achieving synchronization in suspension culture:
1. Physical Method
2. Chemical method

4. Physical methods
1. Selection by Volume:
Synchronization may be achieved on the basis of selecting the size of cell aggregates present
even in the finest possible suspension cultures. Cell fractionation is employed for selection.
2. Temperature Shock:
Low temperature shocks combined with nutrient starvation are reported to induce
synchronization of suspension culture.

5. Chemical method
1. Starvation:
The principle of starvation is based on depriving suspension cultures of an essential growth
compound leading to a stationary growth phase.
Resupplying the missing compounds is expected to induce resumption of cell growth synchronously.
Growth hormone starvation is also reported to induce synchronization of cell cultures.
2. Inhibtion:
Synchronization is achieved by temporarily blocking the progression of events in the cell cycle and
accumulating cells in a specific stage using a biochemical inhibitor. On release the block cells with
synchronously enter the next stage.
Inhibitors of DNA synthesis ( 5-aminourail, 5-flurodexypurine, hydroxyurea or excess thymidine) in
cell cultures accumulate cells at the G1/S boundary.

6. 3. Mitotic Arrest:
Colchicine has been widely used to arrest cells at metaphase. Suspension cultures in
exponential growth are supplied with 0.02% (w/v) colchicine for 4-8 hr in order to inhibit
spindle formation.

7. Cellular Totipotency
 Cellular Totipotency is the ability of a single cell to produce all cell types and
to organise them into an entire organism when cultured in a suitable culture
medium at appropriate temperature and aeration conditions.
 Spores and Zygote are examples of totipotent cells.
 In more or less suitable medium, the totipotent cells of the callus tissue give
rise to meristematic nodules or meristemoids by repeated cell division.
 This may subsequently give rise to vascular differentiation or it may form a
primordium capable of giving rise to a shoot or root.
 Sometimes the totipotent cell may produce embryoids through sequential
stages of development such as globular stage, heart shaped stage and torpedo
stage etc.

8.  After prolonged culture, it has been observed that calluses in some species
(e.g. Ntcotiana tabacum, Citrus aurantifolia etc.) maybe- come habituated.
This means that they are now able to grow on a standard maintenance
medium which is devoid of growth hormones.
 The cells of habituated callus also remain totipotent and are capable to
regenerate a plant without any major manipulation.

10.  In some plant species, the crown gall bacterium (Agrobactenum tumefaciens)
induces a special type of tumour, called teratomas, the cells of which possess
the capacity to differentiate shoot buds and leaves when they are grown in
culture for unlimited periods. Thus it is clear that the mode of expression of
totipotency of plant cell in culture varies from plant to plant and also helps us
to understand the process of differentiation in vitro.

11. Cytodifferentiation
 During growth and maturation of callus tissues few dedifferentiated cells undergo
cytoquiescence and cytosenescence and these phenomenon's are associated with
differentiation of vascular tissues and this whole developmental process is called
cytodifferentiation.