Methodology of Tissue culture:
Explant culture:
Proper explant is excised from the plant which may be a
- cell,
- tissue
- a piece of plant organ.
Generally parenchyma tissue or meristem is used as explants.
It is sterilized properly and placed on solid nutrient medium.
The cells from explant absorb nutrients and start multiplying.
Callus formation and its culture:
The callus is unorganized mass of cells produced due to growth of the explant.
Generally it has thin walled living parenchyma cells.
It develops due to proliferation of cells from explants.
All the cells of callus are identical because they are produced by mitosis only.
Organogenesis:
Now the growth hormones like auxins and cytokinins in proper proportion are provided to the callus to induce formation of organs.
- If auxins are more, roots are formed (rhizogenesis)
- If the cytokinins are in more quantity then the shoot system begins to develop (caulogenesis).
Formation of cell or suspension culture:
For formation of cell or suspension culture the callus can be transferred to liquid nutrient medium and it is agitated.
Due to this the cells from callus get separated.
This cell culture is to be agitated constantly at 100-250 rpm.
The agitation serves the purpose of aeration, mixing of medium and prevents the aggregation of cells.
However, generally the suspension culture shows high proportion of single isolated cells and also small clumps of cells.
Suspenion culture grows much faster than callus culture.
They need to be sub-cultured every week.
The cell or suspension culture has some advantages as -
- cells can be cultured on larger scale.
- suspension culture is more or less homogenous and
- the cells present in the culture do not show much differentiation.
- It is easier to use for subculturing.
By the callus and suspension culture we can achieve cell biomass production which can be utilized for
- biochemical isolation,
- regeneration of new plantlets,
- formation of transgenic plants
- protoplast culture.
