Callus Culture- Growth and Maintenance (Types of Tissue culture Part 3)

Callus tissue means an unorganised prolifer­ative mass of cells produced from isolated plant cells, tissues or organs when grown aseptically on artificial nutrient medium in glass vials un­der controlled experimental conditions. Induction of cell division in the permanent tissue is highly dependent on the high auxin content (e.g., 2, 4-D) in the medium. The hormone requirement for callus induction may be auxin alone, cytokinin alone or auxin and cytokinin in different ratios. The type of growth regulator requirement and its concentration in the medium depends strongly on the genotype and endogenous hormone content of an explant.
Initiation of Callus Culture:
Callus Culture: Initiation and Maintenance
1. Selection of explant material: Any kind of permanent but living tissue such as parenchyma, collenchyma, cortical tissue, pith cells, phloem tissue, cambium cells or meristematic tissue may take part in callus formation. The permanent tissue cells become rejuvenated and the influence of endogenous or exogenously applied growth substances leads to the stimu­lation of cell division.
2. Preparation of culture medium: Induction of cell division in the permanent tissue is highly dependent on the high auxin content (e.g., 2, 4-D) in the medium. The hormone requirement for callus induction may be auxin alone, cytokinin alone or auxin and cytokinin in different ratios. The type of growth regulator requirement and its concentration in the medium depends strongly on the genotype and endogenous hormone content of an explant.
2.Surface sterilization of Explant material
3.Preparation of Explant and inoculation of explant in culture medium
For the initiation of callus cultures, tissues from young seedling or from juvenile part of the mature plant either grown in vivo or in vitro are generally taken, either steri­lised (when grown in vivo) or cut aseptically (when grown in vitro) and inoculated aseptically on a nutrient medium provided with different combinations of exogenous growth substances.
4.Incubation of Culture
5. Maintenance of Callus Culture:
After callus induction, the callus is grown further on a new medium, which is referred to as sub-culturing. During sub-culturing the callus tissue shows sigmoid growth pattern on a particular media. The time interval for sub-culturing is calculated according to the growth pattern of the particular tissue type and genotype of the plant. When the callus growth reaches to the stationary phase of growth then it needs to be cultured on to a fresh medium. Callus tissue from different explants of different plant species may be different in structure and growth habit: white or coloured, soft (watery) or hard, friable (easy to sepa­rate the cells) or compact
4. Plantlets can be obtained from cultured cells by two different ways:
(i) Organogenetic method- Shoot regeneration followed by rooting of the shoots, and
(ii) Embryogenetic method- Regeneration of somatic embryos followed by their germination.
The development of an organized structure, like root, shoot or somatic embryo from cultured cells can be described as regeneration.

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