Callus culture involves growing unorganized plant cell masses (callus) in vitro. Key steps include selecting explant material from plant tissues, sterilizing and culturing explants on nutrient media supplemented with hormones like auxin and cytokinin. Callus forms through cell proliferation at cut surfaces and develops in three stages - induction, cell division, and differentiation. Callus cultures are significant for studying plant cell metabolism and regeneration, and can be used to rapidly determine nucleic acid content or trace metabolic processes.

1. Callus
Culture
M.PhooL Badshah

2. Callus culture
• Formed by the proliferation of parent tissue.
• Cells are parenchymatous, amorphous and unorganized. Callus
is formed as a result of injury at the cut ends of stem or a root.
• When tissue on culture produce unorganized mass of callus
with no regular form called callus culture.
• Rechinger, 1893 was firstly observed.
• Production and maintenance of callus on a solidified nutrient
medium under aseptic conditions.

3. Steps of Callus Culture:
• Selection and Collection of explant material.
• Selection and preparation of culture media.
• Surface sterilization of explant material.
• Preparation of explant.
• Inoculation (transfer) of explant in culture medium.
• Incubation of culture.
• Sub-culturing of culture.
• Regeneration of plant from callus (Organogenesis and
embryogenesis method).

4. Explant Material:
• Callus may initiate from explant s from any multicellular
plants.
• Explant from stem, root, leaf, flower, fruit or seed etc. may be
taken for culture.
• Examples of callus formation are:
 Storage parenchyma, Pericyclic cells of root, Cambial cells of
vascular bundles, Perivascular cells, Secondary phloem, Pith
cells, mesophyll cells and Cotyledons.

5. Reason of sub-culturing cells:
Callus
culture
Nutrient may be
exhausted
Callus
culture
Agar may be
desiccated.
Callus
culture
Cell metabolites
may accumulate
and cause
toxicity.

6. Callus Culture medium:
• Murashige and Skoog medium (MS).
• Kinetin used for initiation and maintaining callus.
• Usually required an exogenous supply of hormone for
• Initiation of callus.
• According to hormone requirement callus culture are:
 Auxin
 Cytokinins.
 Culture required both Auxin and Cytokinins
 Gibberellins.

8. Stages of Callus Culture development:
• Callus formation from an explants occurs in three stages:
 Induction stage:
Metabolism is stimulated and the cells prepare to divide. Cell
size remains unchanged.
 Cell division stage:
Cells divide actively and the cell size decreases. Cell division
is mainly periclinal and occurs towards the periphery giving
rise to wound cambial cells.

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 Differentiation stage:
 Cells differentiate by expansion and maturation.
 Rapidly growing calluses are more or less alike but as the
growth rate decrease the calluses show their characteristic
structures and forms.
 But all calluses have some similarities.
 Contain nodular or sheet meristems in group or scattered
through the tissue.

10. Significance of Callus Culture:
• Determine the total amount of nucleic acid (DNA or
RNA) rapidly from callus culture.
• By using tracer elements in callus culture the pith of
various metabolic processes has been determined.
• Friable callus is suitable for cell suspension culture.