Callus is defined as an unorganized tissue mass growing on solid substrate. Callus forms
naturally on plants in response to wounding, infestations, or at graft unions (Bottino, 1981). Since
extensive callus formation can be induced by elevated hormone levels, tissue culture media
designed to produce callus contain pharmacological additions of cytokinins and auxins.
Callus formation is central to many investigative and applied tissue culture procedures. Callus
can be multiplied and later used to clone numerous whole plants. Additionally, various genetic
engineering protocols employ callus initiation procedures after DNA has been inserted into cells;
transgenic plants are then regenerated from transformed callus. In other protocols callus is
generated for use in biotechnological procedures such as the formation of suspension cultures from
which valuable plant products can be harvested.

Callus Formation
Explants from several parts of large intact plants can be used to form callus. The most
successful explants are often young tissues of one or a few cell types. Pith cells of young stem are
usually a good source of explant material. Initially, callus cells proliferate without differentiating,
but eventually differentiation occurs within the tissue mass. Actively dividing cells are those
uppermost and peripheral in the callus. The extent of overall differentiation usually depends on the
hormone balance of the support medium and the physiological state of the tissue.

Callus Multiplication
Actively growing callus can be initiated on culture media with an even physiological balance of
cytokinin and auxin (Tobacco Callus Initiation Medium; Appendix E). After callus biomass
increases two to four times (after 2–4 weeks of growth), callus can be divided and placed on fresh
Tobacco Callus Initiation Medium for callus multiplication. Multiplication procedures can be
repeated several times (up to eight sequential transfers) before gross chromosome instability (or
contamination) occurs.

Differentiation and Plant Regeneration
Multiplied callus can be stimulated to form shoots by increasing the cytokinin concentration and
decreasing auxin content of culture media (Tobacco Shoot Development Medium; Appendix E).
Shoot masses can be cut apart and transferred to rooting medium. Once rooted, regenerated plants
can be acclimatized to natural rather than "in vitro" growth conditions. Regenerated plants are
especially valuable if the parent plant was itself unique or if the plants were genetically engineered.
If, for example, multiplied callus was first used to form suspension cultures on which genetic
engineering or cell selection was accomplished, resultant regenerated plants via tissue culture could
possess special traits or capabilities.

Materials and Methods
Callus Formation (Bottino, 1981)
1. Obtain a 5-cm section of tobacco stem.
2. Cut off all leaves.
3. Immerse it in a beaker of 95% ethanol for 15 seconds.
4. In the laminar flow hood, expose the pith by cutting away epidermis, cortex, and vascular
tissue with a sterile scalpel (Figure 9.5).
5. Slice the exposed length of pith into a sterile petri dish.
6. Cover the dish to keep pith sterile.
7. Aseptically slice 5-mm cross-sections of pith.
8. Transfer one cross-section to each plate of Tobacco Callus Initiation Medium.
9. Cover the dishes, seal with parafilm, and place in an incubator at 22–25°C.

Callus Multiplication
1. Obtain a plate of tobacco callus.
2. Aseptically divide the callus into smaller pieces.
3. Transfer divided callus pieces to fresh Tobacco Callus Initiation Medium.

When cultured in an appropriate medium having auxin and cytokinin, explants will give rise to an unorganized, growing and dividing mass of cells called callus. Callus cultures are initiated from a small part of an organ or tissue segment called the explants on a growth supporting solidified nutrient medium under sterile conditions. Any part of the plant organ or tissues may be used as the explants. At the time of callus formation, there is some degree of dedifferentiation happens both in morphology and metabolism. One of the major consequences of this dedifferentiation is that most plant cultures lose their ability to perform photosynthesis. The necessitates of the addition of other components such as carbon and vitamins source to the culture media, in addition to the unusual mineral nutrients.

Morphology of callus:

Callus varies considerably in appearance and texture, ranging from hard nodular cell masses to friable soft ones. They maybe white or creamish, orange, green either in whole or part as a result of chloroplast development. The shape of individual cells within the callus mass ranges from the near spherical or markedly elongated.

A typical unorganized plant callus initiated from a new explants or piece of previously initiated calli has three stages of development.

The induction of cell division.
A period of active cell division during which differentiated cells lose specialized features they may have