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Types of Plant tissue cultures
Based on the explants some other plant tissue culture types are
1. Organ culture
2. Meristem culture
3. Protoplast culture
4. Cell culture.
The culture of embryos, anthers, ovaries, roots, shoots or other organs of plants on culture media.
The culture of any plant meristematic tissue on culture media.
Protoplasts are cells without a cell wall, but bounded by a cell membrane or plasma membrane. Using protoplasts, it is possible to regenerate whole plants from single cells and also develop somatic hybrids. The steps involved in protoplast culture.
i. Isolation of protoplast: Small bits of plant tissue like leaf tissue are used for isolation of protoplast. The leaf tissue is immersed in 0.5% Macrozyme and 2% Onozuka cellulase enzymes dissolved in 13% sorbitol or mannitol at pH 5.4. It is then incubated over-night at 25°C. After a gentle teasing of cells, protoplasts are obtained, and these are then transferred to 20% sucrose solution to retain their viability. They are then centrifuged to get pure protoplasts as different from debris of cell walls.
ii. Fusion of protoplast: It is done through the use of a suitable fusogen. This is normally PEG (Polyethylene Glycol). The isolated protoplast are incubated in 25 to 30% concentration of PEG with Ca++ ions and the protoplast shows agglutination (the formation of clumps of cells) and fusion.
iii. Culture of protoplast: MS liquid medium is used with some modification in droplet, plating or micro-drop array techniques. Protoplast viability is tested with fluorescein diacetate before the culture. The cultures are incubated in continuous light 1000-2000 lux at 25°C. The cell wall formation occurs within 24-48 hours and the first division of new cells occurs between 2-7 days of culture.
iv. Selection of somatic hybrid cells: The fusion product of protoplasts without nucleus of different cells is called a cybrid. Following this nuclear fusion happen. This process is called somatic hybridization.
The growing of cells including the culture of single cells or small aggregates of cells in vitro in liquid medium is known as cell suspension culture. The cell suspension is prepared by transferring a portion of callus to the liquid medium and agitated using rotary shaker instrument. The cells are separated from the callus tissue and used for cell suspension culture.
Production of Secondary Metabolites
Cell suspension culture can be useful for the production of secondary metabolites like alkaloids, flavonoids, terpenoids, phenolic compounds and recombinant proteins. Secondary metabolites are chemical compounds that are not required by the plant for normal growth and development but are produced in the plant as ‘byproducts’ of cell metabolism. For Example: Biosynthesis and isolation of indole alkaloids from Catharanthus roseus plant cell culture.
The process of production of secondary metabolites can be scaled up and automated using bio-reactors for commercial production. Many strategies such as biotransformation, elicitation and immobilization have been used to make cell suspension cultures more efficient in the production of secondary metabolites. Few examples of industrially important plant secondary metabolites are listed below in the table:
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