Application of Immunological Principles (Vaccines, Diagnostics), Tissue and Cell Culture Methods for Plants and Animals

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The technique of growing plant cells, tissues, organs in an artificial prepared nutrient medium static or liquid under septic conditions.

Haberlandt (1898) successfully cultured somatic cells of higher plants in simple nutrient solution.

Plant tissue cultures are generally classified according to the type of in vitro growth viz. callus and suspension cultures or the explant used for culture initiation e.g., embryo culture, anther culture etc.

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Explant:

The plant part which is excised from the original and is used for initiating a culture. Explant is disinfected with antimicrobial chemicals in procedure called surface sterilisation. Common disinfectants are dilute hypochlorite, methiolate or clorox water.

Sterilisation:

The vessels, media and instruments are also suitably treated with steam, dry heat or alcohol, or subjected to filtration to make them free from microbes.

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Culture medium:

It is required for the desired growth and development of the explants. It contains inorganic salts, certain vitamins, sucrose and the desired growth regulators (e.g., auxins like 2, 4-D and cytokinins like BAP). The cultures are generally kept in a culture room at about 24°C with some illumination.

Callus and Suspension Cultures

Callus culture:

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Callus is an unorganised mass of loosely arranged parenchymatous cells which develop from parent tissue due to proliferation of cells. Darkness and solid medium gelled by agar stimulates callus formation. The medium generally possess auxin 2, 4-D (2, 4- dichlorophenoxy acetic acid) and often a cytokinin like BAP (benzyl aminopurine). A callus mass is generally obtained in 2-3 weeks.

Suspension culture:

Consists of single cells and small groups of cells suspended in a liquid medium. Generally medium possesses the auxin (2,4-D). Suspension culture must be constantly agitated at 100 – 250 rpm. Agitation helps in aeration of culture, mixing of the medium and breakage of cell aggregates into smaller cell groups. This culture grows much faster than callus culture.

i. With the passage of time, cell or tissue dry matter (biomass) increases, the level of nutrients in the medium decreases and medium volume declines due to evaporation.

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ii. Subculturing-Cultures are regularly divided and transferred to containers having fresh medium. During subculture, only a part of the culture from a vessel is transferred in the new culture vessel.

iii. The callus and suspension cultures can be used to achieve cell biomass production, regeneration of plantlets, production of transgenic plants and isolation of protoplasts.

Regeneration of plantlets

Plantlets can be obtained from cultured cells by two different routes:

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(a) Short regeneration followed by roots of the shoots

(b) Regeneration of somatic embryos followed by their germination.

Regeneration:

Formation of organised structures like shoot, root, somatic embryo (embryoid) or plantlet from callus/ suspension and other forms of tissue culture. Regeneration is possible because plant cells are totipotent.

1. Shoot Regeneration:

Shoot regeneration is promoted by a cytokinin (e.g., BAP) while root regeneration is promoted by an auxin like NAA (napthalene acetic acid). Callus culture first kept on a BAP containing medium, differentiates into shoots. When the shoot become 2-3 cm long they are excised and transferred to an auxin containing medium. Roots develop at the lower ends to shoot to form plantlets.

2. Somatic embryo regeneration:

It is induced by a relatively high concentration of an auxin e.g., 2, 4-D. These young embryo develop into mature embryos either on the same medium or on another medium. Mature somatic embryos germinate to yield complete plantlets.

Establishment in the field:

Plantlets are transferred to small earthen pots in a green house under reduced light and high humidity for 3-4 weeks. It brings about hardening, which helps the plantlets to develop root system and leaf structure capable of withstanding field conditions. These hardened plants are then established in the field.

Applications

1. Rapid clonal propagation:

All the cells in a callus or suspension culture are derived from a single explant by mitotic division so all plantlets regenerated from a callus/suspension culture generally have the same genotype and constitute a clone. The technique used is rapid propagation of superior and rare lines e.g., oil palm.

2. Somaclonal variation:

There are genetic variations present among plant cells of a culture. This variation have been incorporated in plant breeding programmes, e.g., rust resistance and high temperature tolerance in wheat, resistance to Tongo virus and leaf hopper in rice etc.

3. Transgenic plants:

Transgene is a gene that is transferred into an organism by genetic engineering. Transgenic organism is such organism which contains and expresses a transgene. Transgenics are cells or organisms with transgenes. Transgenic cells are multiplied in tissue culture. Plantlets and plant are then regenerated from them.

4. Resistance to weedicides:

Weedicides are added to obtain desired level of resistance in culture. The developed resistant cells are then regenerated to form plantlets and plants.

5. Induction and selection of mutations:

Mutagens are added to single cell liquid cultures for induction of mutations. Useful mutants are selected for further breeding.

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