Culture of Plant Materials in Plant Tissue Culture

Culture of Plant Materials in Plant Tissue Culture:

Explant Culture:

There are a variety of forms of seed plants, such as trees, herbs grass, which exhibit the basic morphological units, i.e., root, stem and leaves, versatile of all types of tissues. They are capable of division and growth.

Development of a tissue is characterized:

(i) Cell division,

(ii) Cell elongation, and

(iii) Cell differentiation.

For this reason, the explants from healthy and young part of the plant are used. Presence of parenchyma is first consideration in a particular species, parenchyma from stems, rhizomes, tubers; root is easily accessible and will generally respond quickly to culture conditions in vitro.

Callus Formation and its Culture:

In nature, callus develops by infection of microorganisms from wounds due to stimulation by endogenous growth hormones, the auxins and cytokinins. However, it has been artificially developed by adopting tissue culture techniques.

A callus is an amorphous mass of loosely arranged thin walled parenchyma cells developing from proliferating cells of the parent tissue. The unique feature of callus is that the abnormal growth has logical potential to develop normal root, shoots and embryoids ultimately forming a plant.

In callus culture, cell division in the explant forms a callus, an un-organised mass of cells. It is maintained on a medium gelled usually with agar. The medium ordinarily contains the auxin 2, 4 – D (2, 4 – dichlorophenoxyacetic acid), and often a cytokinin BAP (benzylaminopurine).

When an explants is placed on such a medium, many of the cells become meristematic and begin to divide. In about 2 to 3 weeks, a callus mass is obtained.

Cell (Suspension) Culture:

A suspension culture consists of single cells and small groups of cells suspended in liquid medium. Cell suspension is prepared by transferring a fragment of callus (about 500 mg.) to the liquid medium (500 ml.) and agitating them aseptically to make the cells free in medium. The medium ordinarily contains the auxin 2, 4 – D. Suspension cultures must be constantly agitated at 100 – 250 rpm (revolutions per minute).

Agitation serves three important purposes.

They are as follows:

(i) Aeration of culture;

(ii) Constant mixing of the medium and

(iii) Breakage of cell aggregates into smaller cell groups. Suspension cultures grow much faster than callus cultures.

It is difficult to have suspension of single cell. However, the suspension includes single cell, cell aggregates (varied number of cells), residual inoculum and dead cells. King (1980) has described that a good suspension consists of a high proportion of single cells than small cluster of cells.

Cell suspension cultures have many advantages over the callus cultures.

They are as follows:

(i) The suspension can be pipetted.

(ii) They are less heterogeneous and cell differentiation is less pronounced.

(iii) They can be cultured in volumes up-to 1500 litres.

(iv) They can be subjected to more stringent environmental controls.

(v) They can be manipulated for production of natural products by feeding precursors.

Sub-Culturing:

After some time, the under-mentioned three things happen in all types of plant tissue cultures:

(i) Cell/tissue dry matter known as biomass increases,

(ii) The level of nutrients in the medium decreases, and

(iii) The medium volume declines due to evaporation.

Hence, if tissue cultures were kept in the same culture vessel, they will die in due course of time. Due to this reason, cells/tissues are regularly transferred into new culture vessels containing fresh media. This process is called sub-culturing.

Precaution is taken that during sub-culture, only a part of the culture from a vessel is transferred into the new culture vessel.

Difference between callus and suspension cultures:

Callus Culture:

a. Her cell division in the explant forms a callus, which is an unorganised mass of cells.

b. It is maintained on a medium gelled usually with agar.

c. The medium ordinarily contain the auxin 2, 4-D, and often a cytokinin like BAP.

d. There is no need of agitation.

e. In about 2-3 weeks, a callus mass is obtained.

Suspension Culture:

a. Suspension culture consists of single cells and small groups of cells.

b. Here, cells are suspended in liquid medium.

c. Usually the medium contains the auxin 2, 4-D.

d. They must be constantly agitated at 100-250 rpm (revolutions per minute).

e. suspension culture grow much faster than callus cultures.

 Uses of Callus and Suspension Cultures:

The callus and suspension cultures can be used to achieve cell biomass production which may be used for biochemical isolation.

Also used for regeneration of plantlets, i.e., newly regenerated plants through tissue culture.

Production of transgenic plants and isolation of protoplasts