Callus Culture

Callus is basically a more or less non-organized

tumor tissue which usually arises on wounds of

differentiated tissues and organs. Thus, in

principle, it is a non-organized and little-

differentiated tissue. The cells in callus are of a

parenchymatous nature. When critically

examined, callus culture is not homogeneous

mass of cells, because it is usually made up of

two types of tissue: differentiated and non-

differentiated. Explant tissue is a differentiated

tissue (roots, stem, leaves, flowers, etc.) which

is used as a starting material for callus induction.

These explant tissues generally show distinct

planes of cell division, cell proliferation and

organization into specialized structures such as

vascular systems. If there are only differentiated

cells present in an isolated explant, then

dedifferentiation must take place before cell

division can occur. Parenchyma cells present in

the explant usually undergo this differentiation.

If the explant already contains meristematic

tissue when isolated, then this can divide

immediately without dedifferentiation taking

place. Dedifferentiation plays a very important

role, enabling mature cells in an explant isolated

from an adult plant to be redetermined. In this

process, adult cells are temporarily able to revert

from the adult to the juvenile state. The

rejuvenated cells have a greater growth and

division potential and under special

circumstances are able to regenerate into organs

and/or embryos.

Callus formation takes place under the

influence of exogenously supplied growth

regulators present in the nutrient medium. The

type of growth regulator requirement and its

concentration in the medium depends strongly

on the genotype and endogenous hormone

content of an explant. These requirements can

be put into three categories:

1. Auxin alone (especially in monocotyledons)

2. Cytokinin alone

3. Both auxin and cytokinin (carrot)

If the callus is difficult to induce, or if juvenile

callus is needed, then immature embryos or

seedlings or parts of these are used. It should

be taken into account that the type of starting

material (juvenile or adult) and the original

position of the explant in the plant reflects the

endogenous growth regulator level which have

an important influence on processes such as cell

division and organ and embryo formation.

Many other factors are important for callus

formation: genotype, composition of the nutrient

medium, physical growth factors (light,

temperature, etc.). The Murashige and Skoog

(1962) mineral medium, or modifications of this

are often used. Sucrose or glucose (2–4%) is

usually employed as the sugar source. The effect

of light on callus formation is dependent on

the plant species; light may be required in

some cases and darkness in other cases. A

temperature of 22–28°C is normally advantageous

for callus formation Callus tissue induced from different plant

species may be different in structure and growth

habit: white or coloured, soft (watery) or hard,

friable (easy to separate into cells) or compact.

The callus growth within a plant species may also

depend on factors such as the original position of

the explant within the plant, and the growth

conditions.

After callus induction, the callus is grown

further on a new medium which is referred to as

subculturing. When subcultured regularly on

agar medium, callus cultures will exhibit an S-

shaped or sigmoid pattern of growth during each

passage (See Fig. 7.1). There are five phases

of callus growth:

1. Lag phase, where cells prepare to divide.

2. Exponential phase, where the rate of cell

division is highest.

3. Linear phase, where cell division slows but

the rate of cells expansion increases.

4. Deceleration phase, where the rates of cell

division and elongation decreases.

5. Stationary phase, where the number and

size of cells remain constant.

Callus growth can be monitored by fresh

weight measurements, which are convenient

for observing the growth of cultures over time

in a non-destructive manner. Dry weight

measurements are more accurate than fresh

weight, but this method requires sacrifice of the

samples. Mitotic index measurement of cell

division rates require extensive sampling to

reduce sample error and are not easy to perform.