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Sunday, February 22, 2009

Secondary Metabolite Production In Plants


Secondary Metabolite Production in Plants-Applications

Secondary Metabolite Production

Secondary metabolites are chemical compounds and the chemical compounds produced by plants are collectively called as phytochemicals.

Secondary metabolites are those chemical compounds that do not participate in metabolism of plants. But these compounds are involved in disease resistance (from fungus, bacteria, viral, and pests), for pollination and to compact in extreme conditions of stress. The stress may be biotic or abiotic [Drought, Cold, Temparature, etc.].some of the secondary metabolites are Alkaloids, Steroids, terpenoids, essential oils, flavours, fragrance, colours & pigments.




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
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.

Cell Cloning:

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:

A high yielding plant of desired metabolite is selected and its explants are cultured on a solid medium. After establishing callus cultures, high metabolite producing calluses are identified, and they are grown in suspension cultures. Cell aggregates from these cultures are grown on solid medium. The freshly developed cell aggregates are divided into two parts. One half is grown further, while the other half is used for the quantitative analysis of the desired metabolite produced. The cell lines with high yield of secondary metabolites are selected and are used for scale-up in suspension cultures. This is followed large-scale tissue culture in a bioreactor.

Visual or Chemical Analysis:
A direct measurement of some of the secondary metabolites produced by cell lines can be done either by visual or chemical analysis.
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.

Selection for Resistance:
Certain cells resistant to toxic compounds may lead to the formation of mutant cells, which can overproduce a primary metabolite, and then a secondary metabolite. Such mutants can be selected and used to produce the desired metabolite in the large quantities.

Large Scale Cultivation of Plant Cells
To achieve industrial production of the desired metabolite, large-scale cultivation of the plant cell is required. Plant cells when cultured exhibit changes in volumes and thus variable shapes and sizes. Further, cultured cells have low growth rate and genetic instability. All these aspects have to be considered for the mass cultivation of cells. the following four different culture systems are widely used:

1. The Free-Cell Suspension Culture
2. Immobilized Cell culture
3. Two Phase System culture
4. Hairy root culture

The Free-Cell Suspension Culture:
Mass cultivation of plant cells is most frequently carried out by cell suspension cultures. Care should be taken to achieve good growth rate of cells and efficient formation of the desired secondary metabolite. Many specially designed bioreactors are in use for free-cell suspension cultures.

Bioreactors for the Production of Secondary metabolites
  • Batch Bioreactors
  • Continuous Bioreactors
  • Multistage Bioreactors
  • Airlift Bioreactors
  • Stirred Tank Bioreactors
Two important aspects should be considered for good success of suspension cultures
  1. Adequate and Continous Oxygen Supply
  2. Minimal generation of hydrodynamic stresses due to aeration and agitation.
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.

Surface immobilized plant cell (SIPC) technique efficiently retains the cells and allow them to grow at a higher rate. When the cells are immobilized there is a better cell-cell contact, and the cells are protected from high shear and stresses. All these helps in the maximal production of secondary metabolite.
The common methods used for entrapment are

Entrapment of cells in gels: The cells / protoplast can be immobilized in several gels.Eg: Alginate,agarose,carrageenin.The gels may be either used individually or in combination.The techniques involved for immobilization of plant cells are comparable to those used for the immobilization of microorganism or other cells 

Entrapment of cells in nets or foams: Polyurethane or nets with various pore sizes are used.The actively growing plant cells in suspension can be immobilized on these foams.The cells divide within the compartments of foam and foam aggregates.
Entrapment of cells in hollow-fibre membranes: Tubular hollow fibres composed of cellulose acetate silicone polycarbonate and organized into parallel bundles are used for immobilization of cells.It is possible to entrap cells between the fibres

Bioreactors for use of immobilized cells

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



Two Phase System Culture
Plant cells can be cultivated in an aqueous two phase system for production of secondary metabolites. In this technique cells are kept apart from the product by separation in the bioreactor. This is advantageous since the product can be removed continuously. Certain polymers are used for the separation of the phases.Various products have been used for phase separation. Hooker and Lee cultured tobacco cells in aqueous two phase compraising of Dextran & PEG for the production of phenolic compounds.Silicon fluid has been used for the phase separation of Eschscholtzia californica cell cultures.This fluid enhanced benzophenan-thridine alkaloids three fold compared to single phase culture.

Hairy Root Culture
Hairy root cultures are for the production of root-associated metabolites. In general, these cultures have high growth rate and genetic stability.
For the production of hairy root cultures, the explant material is inoculated with pathogenic bacterium, Agrobacterium rhizogenes. The organism contains root-inducing (Ri) plasmid that causes genetic transformation of plant tissues, which finally results in the hairy root cultures. Hairy roots produced by plant tissues have metabolite features similar to that of normal tissues.
Elicitor-induced production of secondary metabolites
The production of secondary metabolites in plant cultures is generally low and does not meet the commercial demands. the synthesis of majority of secondary metabolites involves multistep reactions and many enzymes. It is possible to stimulate any step to increase product formation.
Elicitors are the compounds of biological orgin, which stimulate the production of secondary metabolites, and the phenomenon of such stimulations are called as elicitation.
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
All these factors will have determinal effect on the production of secondary metabolite.

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.
The biotechnological application of plant cell cultures in Biotransformation reactions involves the conversion of some less important substances to valuable medicinal or commercially important products. In Biotransformation, it is necessary to select such cell lines that possess the enzymes for catalyzing the desired reactions. Eg: hydroxylation, reduction, and glycosylation.

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