4. Strategies for enhanced production of secondary metabolites in plant cell cultures

4.1. Proper selection of cell lines

The heterogeneity within the cell population can be screened by selecting cell lines capable of accumulating higher level of metabolites.

4.2. Manipulation of medium

The constituents of culture medium, like nutrients, phytohormones and also the culture conditions, like temperature, light etc. influence the production of secondary metabolites. For e.g., if sucrose concentration is increased from 3% to 5%, production of rosamarinic acid is increased by five times. In case of shikonin production, IAA enhances the yield whereas 2,4-D and NAA are inhibitory.

4.3. Addition of Elicitors

Elicitors are the compounds which induce the production and accumulation of secondary metabolites in plant cells. Elicitors produced within the plant cells include cell wall derived polysaccharides, like pectin, pectic acid, cellulose etc. Product accumulation also occurs under stress conditions caused by physical or chemical agents like UV, low or high temperature, antibiotics, salts of heavy metals, high salt concentrations which are grouped under abiotic elicitors. Addition of these elicitors to the medium in low concentration enhances the production of secondary metabolites.

4.4. Addition of precursors

Precursors are the compounds, whether exogenous or endogenous, that can be converted by living system into useful compounds or secondary metabolites. It has been possible to enhance the biosynthesis of specific secondary metabolites by feeding precursors to cell cultures. For example, amino acids have been added to suspension culture media for production of tropane alkaloids, indole alkaloids. The amount of precursors is usually lower in callus and cell cultures than in differentiated tissues. Phenylalanine acts as a precursor of rosmarinic acid; addition of phenylalanine to Salvia officinalis suspension cultures stimulated the production of rosmarinic acid and decreased the production time as well. Phenylalanine also acts as precursor of the N-benzoylphenylisoserine side chain of taxol; supplementation of Taxus cuspidata cultures with phenylalanine resulted in increased yields of taxol. The timing of precursor addition is critical for an optimum effect. The effects of feedback inhibition must surely be considered when adding products of a metabolic pathway to cultured cells.

4.5. Permeabilisation

Secondary metabolites produced in cells are often blocked in the vacuole. By manipulating the permeability of cell membrane, they can be secreted out to the media. Permeabilisation can be achieved by electric pulse, UV, pressure, sonication, heat, etc. Even charcoal can be added to medium to absorb secondary metabolites.

4.6. Immobilisation

Cell cultures encapsulated in agarose and calcium alginate gels or entrapped in membranes are called immobilised plant cell cultures. Immobilization of plant cells allows better cell to cell contact and the cells are also protected from high shear stresses. These immobilized systems can effectively increase the productivity of secondary metabolites in a number of species. Elicitors can also be added to these systems to stimulate secondary metabolism.

4.7. Limitations

• Production cost is often very high.

• Lack of information of the biosynthetic pathways of many compounds is a major drawback in the improvement of their production.

• Trained technical manpower is required to operate bioreactors.