Callus culture-Initiation and maintenance of callus culture. Initiation of suspension culture.

Assessment of cells in suspension-mitotic index, EC, cell viability and growth curve.
A callus consists of an amorphous mass of loosely arranged thin-walled parenchyma cells
arising from the proliferating cells of the parent tissue.
Freshly cut pieces of surface sterilized plant tissues are grown in agar medium with
appropriate nutrients with suitable proportion of auxin and cytokinin exhibit callusing at cut ends,
which gradually extends to the entire surface of the tissue. For callus initiation, aseptically transfer
explants to the required semi solid medium and gently press them into the agar so that good contact
is made. Radical tips will callus well if laid horizontally on the agar whereas stem section may
produce more callus if placed vertically with one cut end in the agar.
Within 2-3 weeks, explants should show new growth as pustules or protuberances or as a fine
mat across the surface depending on the distribution and mitotic activity of the parenchyma
residing in the excised tissues. Continued growth may leave the core of the explants fairly
undisturbed or the explants may disintegrate as the callus grows. Some callus growths are heavily
lignified and hard in texture, whereas others break easily into small fragments. Fragile growths that
readily separate are termed "friable" cultures. Callus may appear yellowish, white, green or
pigmented with anthocyanin. Pigmentation may be uniform throughout the callus or some regions
may remain un pigmented. Callus tissue is not uniform. Assuming a suitable medium has been
selected, most explants should have produced sufficient callus to allow for subculture within 3-7
weeks in view of cell growth , nutrient depletion and medium drying. Remove newly formed callus
from the initial explant at this stage by cutting with the sterile scalpel. Once well established, most
callus cultures will require regular subculture at approximately 4-6 weekly intervals. Repeated
subculture on an agar medium improves the friability of the callus
Callus maintenance
When cultured for several weeks, any callus will show signs of ageing, noted as deceleration
of growth, necrotic or browning and finally desiccation. Aging usually is the result of one or several of
the following factors:
1.

Exhaustion of nutrients.

2.

Inhibition of nutrient diffusion.

3.

Evaporation accompanied by an increase in the concentration of some constituents of the

medium and

4.

Accumulation of metabolites, some of which may be toxic.

So, transfer of healthy, vigorous callus pieces about 5mm in diameter to 30 ml of fresh medium
(subculture) at intervals of 4-6 weeks will maintain the callus line
Applications of callus culture
1. To study nutrition requirement of plants
2. To study cell and organ differentiation and morphogenesis
3. Somaclonal variations and its exploitation
4. Developing cell suspension cultures and protoplasts cultures
5. Genetic transformation using ballistic particle gun technology
6. In the production of secondary metabolism and regulation
7. The development of shoots, roots or somatic embryos from which whole plants can
subsequently be produced
Suspension cultures
Cultures of single cells (minority) and small cell aggregates (majority) that proliferate and complete a
growth cycle while suspended in liquid medium. It is also referred to cell culture or cell suspension
culture. Cell suspension is aggregates of cells suspended in liquid medium.
Initiation
1. Cell suspensions typically are initiated from friable callus by inoculating into liquid medium.
2. Individual cells and/or cell aggregates are maintained in suspension by agitation or aeration,
which also minimizes hypoxia.
3. After the initial passage, culture is typically filtered to eliminate large tissue masses

Growth phases

1. Lag phase -cells activate metabolism for cell proliferation, conditioning is occurring
2. Exponential phase-cell mass gain is exponential due to primarily to cell division
3. Linear/progressive/deceleration phase -Linear mass gain due primarily to cell expansion
4. Stationary phase -growth ceases
Assessment of cells in suspension-Growth parameters
1. Cell count
a. Two volumes 8 % Chromium trioxide, heated at 70C (2-15 min.) ,cooled, agiated
for 10 min., centrifuged and pellet is suspended in 8% saline. After 10-15 min., free
cells are counted in haemocytometer or
b. 0.1% pectinase
2. Packed cell volume (PCV)
1. Graduated centrifuge tube -100 x g for 5min.
2. Expressed as ml of pellet/ml of sample
3. Cell fresh weight (gm / ml)
1. Pre weighed filter paper,
2. Washed in distilled water under vacuum
4. Cell dry weight (gm / ml)
1. Filter discs are dried in an oven for 12h at 60C
5.

Viability of cell
Cytoplasmic streaming(phase contrast),TTC reduction(respiratory efficiency-TTC to
formazon), Fluorescein Di Acetate (FDA, 0.01%), Evans blue(0.025%, dead cells)

6. Electrical conductivity
7. Mitotic Index -

Total No. cells in active mitosis

------------------------------------Total observed cells

X 100

Broadly there are three types of suspension cultures

1. Batch cultures -Cells are nurtured in a fixed volume of medium until growth ceases.
2. Continuous cultures - Cell growth is maintained by continuous replenishment of
sterile nutrient medium. There are two types of continuous culture types
a. Closed continuous culture
b. Open continuous culture
3. Plant cells and groups may be encapsulated in a suitable material or entrapped in
membranes or stainless screens.
Applications of cell culture
1.

Useful in the isolation of protoplasts

2. Useful in cell cloning by the plating technique with or without specific treatment like
mutagens,
3. Development of cell lines for various types of resistance like salt or drought tolerance, toxin
resistant lines
4. Suspensions from medicinally important plants for the production of secondary metabolites
such as alkaloid
5. Produce valuable products including secondary metabolites through bio transformation

Questions
1.
2.
3.
4.
5.

How will you define callus?

How will you establish and maintain the callus?
Write down the applications of callus culture.
What are suspensions?
Describe some growth parameters used for monitoring the
growth of cell suspensions?
6. What are batch, continuous and immobilized cultures?
7. List the applications of suspension culture.