Application of secondary metabolites
Secondary metabolites are widely used in many industrial products, some of them are

1. Ø Nuetraceuticals
2. Ø Textile Industry
3. Ø Cosmoceuticals
4. Ø Pharmaceuticals
5. Ø Perfume Industry, etc.

Terpenoids, Steroids and steroids are used in 25% of the prescribed drugs, and almost all cancer drugs are sourced from plants. Colours and fragrances are used in textile and food industry. Some of the plant species that produce the secondary metabolites of great industrial importance. Like Taxas burgata which produce Taxol is used in cancer treatment, all parts of the plant has the capacity to produce this compound.Coleus forskohli which produces the compound Forskohlin is used in treatment for Glaucoma, only roots are capable of producing the secondary metabolite in this plant. Lithospermum erythrorhizon which produce Shinkonin.

Production of secondary metabolites

The process of invitro culture of cells for the large scale production of secondary metabolite is complex, and involves the following aspects

1. Ø Developing Mother Culture
2. Ø Selection of cell lines for high yield of secondary metabolites.
3. Ø Large scale cultivation of plant cells
4. Ø Medium composition and effect of nutrients
5. Ø Elicitor induced production of secondary metabolites
6. Ø Effect of environmental factors
7. Ø Biotransformation using plant cell culture
8. Ø Secondary metabolites and analysis.

Developing Mother Culture

The culture, which is produced, first is called mother culture. Different cultures has been developed like callus, multiple shoots, roots, etc among these which of the culture can be produced easily, will be selected for further culturing.
The callus can be developed from explant or from a mother culture, which is already developed. Different types of callus can be obtained like Nodular Callus, Friable Callus Pigment Callus, Embryonic Callus, etc. among those friable callus (loosely packed callus) is preferred. If friable callus is not producing it can be induced by certain treatments. When the friable callus is put in liquid medium and kept in shaker the cells of the callus will get dissociated. Usually 75ml of the media is taken in 250ml flask, the optimum inoculating density is 5mg / 75ml, the pH of the medium should be 5.6.the flask is kept in a Gyratory Shaker at 120 rpm, which is for the aeration of the medium. The speed varies depending on the plant species.

Sub Culturing:

Sub culturing can be done when the cells are in the exponential stage, the stage of development can be identified by growth pattern studies. The culturing can be done by batch culture or by continous culture. One of the method is 5ml of inoculum from the suspension [75ml] is transferred 70ml of fresh medium. Like this way 15 or more flasks of culture can be produced. Another method is that, divide the media equally into two parts and add the fresh media to make-up to 75ml and this process is continued.
For identification of the stage and analysis of the product growth pattern studies are very much important. Growth study is done for 25days form the starting. For studying growth different methods (Optical Density, Dry weight, Cell counting) can be used. By this we can know the Packed Cell Volume (PCV) along with that content analysis also can be done so we will get a clear idea that on particular day at a particular stage of growth the cell is producing the secondary metabolite. Moreover the target of the product also can be known,ie. Whether it is produced Intracellular or Extracellular.

Callus Culture

Selection of cell lines for high yield of secondary metabolites 
The purpose of tissue culture is to produce high amount of secondary metabolite, but in general majority of the callus and suspension cultures produce less quantity of secondary metabolites. The reason for this is mainly due to the lack of fully differentiated cells in the cultures. There are some techniques ultimately useful for the separation of producers and non-producers. The techniques employed for this are cell cloning, Visual and Chemical analysis and selection for resistance.

This is a simple procedure and involves the growth of single cells (taken from suspension culture) in a suitable medium. Each cell population is then screened for secondary metabolite formation. The cells with high ability are selected and maintained by subcloning.

Aggregate Cell Cloning:

Visual identification of cell lines producing coloured secondary metabolites will help in the selection of high-yielding cells. This method is simple but the limitation is that the desired metabolite should be coloured.The chemical analysis can be done in some colonies derived from single cell cultures.Radioimmunoassay is the most commonly used, Microspectrophotometry and fluorescent antibody techniques are also been used.

Immobilized Cell Cultures


Plant cells can be made immobile or immovable and used in culture systems. The cells are physically immobilized by entrapment. Besides individual cells it is possible to immobilize aggregate cells or even calluses. Homogenous cell suspensions are more suitable for immobilization.

Fluidized bed or fixed bed bioreactors are employed to use immobilized cells for large scale cultivation. In fluidized bed bioreactors, the immobilized cells are agitated by a flow of air or by pumping the medium. In contrast, in the fixed bed bioreactor, the immobilized cells re kept stationary (not agitated) and perfused at a slow rate with an aerated culture medium. The list of plant species which immobilized cells employed for the production of secondary metabolites

Elicitor-induced production of secondary metabolites

Elicitors produced within the plant cells are called as endogenous elicitors (Eg: Pectin, pectic acid and other polysaccharides) when the elicitors are produced by microorganisms they are called as exogenous elicitors (Eg: Chitins, Glucans). All the elicitors of biological orgin are biotic elicitors. Physical (Cold, Heat, UV light, etc.) and chemical agents (ethylene, fungicides, antibiotics) can also increase product formation such elicitors are called as abiotic elicitors.



Factors affecting Secondary Metabolite production


There are various factors affecting Secondary metabolite production, some of the major factors includes

1. Light
2. Incubation Temperature
3. pH of the medium
4. Aeration of the culture, etc

Biotransformation using plant cell cultures


The conversion of one chemical into another (ie, a substrate into a final product) by using biological systems (ie, cell suspensions) as biocatalysts is regarded as Biotransformation or bioconversion the biocatalyst may be free or immobilized, and the process of Biotransformation involve one or more enzymes.