callus culture -tissue culture

Callus Culture

Dr. Amit Kumar Dutta, Ph.D

Callus Culture

Callus is basically a more or less non-organized tumor tissue, which is usually arises on wounds of differentiated tissues & organs.

Callus

An unorganised, growing and dividing mass of cells. Callus is usually composed of unspecialised parenchyma cells Manipulation of the auxin to cytokinin ratio in the medium can lead to the development of shoots, roots or somatic embryos from which whole plants can subsequently be produced.

Culture Types

Cultures are generally initiated from sterile pieces of a whole plant.

Pieces are termed ‘explants’, and may consist of pieces of organs, such as leaves or roots, or may be specific cell types, such as pollen or endosperm.

Many features of the explant are known to affect the efficiency of culture initiation.

Generally, younger, more rapidly growing tissue (or tissue at an early stage of development/meristematic) is most effective.

Culture Types

• Modes of Culture

The plant cells if cultured on a solid surface will grow as friable, pale brown, unorganized mass of cells called callus.

Tissues and cells of plant cultured in a liquid medium aerated by agitation grow as suspension of single cells and cell clumps.

For growth, the cells need to divide, whereas, the cells breaking up from explant are mature, nondividing.

Modes of Culture

Differentiated tissue undergoes modifications to become meristematic.

This phenomenon of a mature cell reverting back to meristematic state to form undifferentiated callus tissue is called dedifferentiation.

Callus culture:

The culture of undifferentiated mass of cell on agar media produced from an explant of a seedling or other plant part is called callus culture.

For callus formation, auxin and cytokinins, both are required.

Callus can be subcultured indefinitely by transferring a small piece of the same to fresh agar medium.

Subculturing needs to be done every 3-5 weeks in view of cell growth, nutrient depletion and medium drying.

Callus culture

The rate of growth of callus grown on solid agar medium is relatively slow. The new cells are formed on the periphery of existing callus mass.

Consequently, callus consists of cells which vary considerably in age. Since nutrients are gradually depleted from the agar, a vertical nutrient gradient is formed.

Cell Culture

Callus is placed into a liquid medium (usually the same composition as the solid medium used for the callus culture) and then agitated, Single cells and/or small clumps of cells (aggregates) are released into the medium.

Under the correct conditions, these released cells continue to grow and divide, eventually producing a cell-suspension culture.

After subculture, the cells divide and the biomass of the culture increases in a characteristic fashion, until nutrients in the medium are exhausted and/or toxic by-products build up to inhibitory levels—this is called the ‘stationary phase’.

Cell Culture

If cells are left in the stationary phase for too long, they will die and the culture will be lost. Therefore, cells should be transferred before the stationary phase.

Cell Culture

Two Categories:

Compact

Friable

Categories

Compact callus, the cells are densely aggregated,

Friable callus, the cells are only loosely associated with each other and the callus becomes soft and breaks apart easily. Friable callus provides the inoculum to form cell-suspension

Usually made up of two types of tissue:-

Differentiated

Non-Differentiated

Plant Tissue is a differentiated tissue (Roots, Stem, Leaves, Flowers, etc) which is used as a starting material for callus induction.

Dedifferentiation plays a very important role, enabling mature cells in an explant isolated from an adult plant to be redetermined.

Callus formation takes place under the influence of exogenously supplied growth regulators present in the nutrient medium.

Formation

Requirements can be put into three categories:-

Auxin alone(In Monocotyledons)Cytokinin alone

Cytokinin and Auxin(Carrot)

Other Important factors are:-

Genotype

Composition of the Nutrient Medium

Physical Growth Factor

Temperature Light

Murashige & Skoog (1962)

Modify, Sucrose & Glucose (2-4%) is usually employed as the sugar source.

Effect of light on callus formation is dependent on the plant species.

Light may be required in some cases and darkness in other cases.

Temperature of 22-280C is normally required for C/Formation.

Murashige & Skoog (1962)

Callus tissue from different plant species may be different in structure and growth habit.

Murashige & Skoog (1962)

Callus growth within a plant species may also depend on factors.

as the original position of the explant within the plant, and the growth conditions

Murashige & Skoog (1962)

After Callus Induction,

Grown further on a new medium (Subculturing).

Exhibit an S-shaped or Sigmoid Pattern of growth during each passage.

The main use of callus culture is for purposes of maintaining cell lines and for morphogenesis.

Use/ Purpose

Low degree of uniformity among cells in callus, slower growth rate and development of nutrient gradients, the usefulness of callus in experimental system is limited

Use/ Purpose

Callus cultures can also be used to initiate cell suspensions, which are used in a variety of ways in plant transformation studies.

Use/ Purpose

Phases

Five Phases of callus growth

Lag Phase:-

Cells prepare to divide.

Exponential Phase:-

Rate of Cell Division is highest.

Linear Phase:-

Cell Division slows, but rate of cell expansion increases.

Deceleration Phase:-

Rate of Cell Division & elongation decreases.

Stationary Phase:-

The number & Size of cells remain constant.

Linear

Log

Time (days)

Lag

Exponential

Linear/progressive deceleration

Stationary

Callus Growth Measurement

Monitored by fresh weight measurements(Convenient for observing the growth of cultures).

Dry weight measurements are more accurate(requires sacrifice the samples)

Mitotic Index measurement of Cell Division rates require extensive sampling to reduce sample error.

Not easy to perform.

Mitotic Index

Thank You

Any Difficulties Contact to Respective Teacher