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Read this article to learn about the prospects of drug production in plant tissue culture.
Plant cell cultures have great potential for the production of secondary metabolites. In recent years, considerable success has been achieved in increasing the secondary metabolites using cell suspension cultures in several plant species.
Plant cells grown in culture have potential to produce and accumulate chemicals similar to the parent plant from which they were derived.
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There are numerous reports describing the production of diverse secondary metabolites, viz., anthocyanin’s, alkaloids, carotenoids, flavones, coumarins, naphthaquinones, saponins, sesquiterpenes, steroidal alkaloids, sterols, tannins, terpenoids and several others.
Why Tissue Culture ?
Several pharmacologically active novel compounds are extracted from plants (Table 29.2). In plant systems, they accumulate in leaves (nicotine in Nicotiana), roots (ajmalicine in Catharanthus roseus), bark (quinine in Cinchona) or in the whole plant (ephedrine in Ephedra). Sometimes these products are produced in specialized differentiated tissues such as resin in resin ducts and latex in laticifers.
Except the herbaceous cultivated plants (e.g., Papaver somniferum), most of the secondary metabolites are accumulated after a certain age or maturity of the plant. In the case of tree or shrub species, e.g., Cinchona, Rauwolfia, Caniptotheca, Ochrosia, etc., plants attain maturity in a few years before they accumulate the active principle in high amounts. It is difficult to increase the area under plantation for a particular species and growth of plants takes it own time. To meet the ever-increasing demand (e.g., vincristine) the natural resources are not sufficient. The world political scenario may also affect the supply of a particular raw material.
To overcome all these hurdles, the industry requires alternative methods of assured supply of uniform material throughout the year. Harvesting of plants (except cultivated species) from natural forest resources is not only difficult, but also makes them endangered species; e.g., Ephedra gerardiana and several other Himalayan plants.
When plant material is not available throughout the year in a quantity sufficient for industrial production and chemical synthesis is not possible, particularly for large complex molecules, biotechnological methods offer an excellent alternative. But before implementing this approach, cost of the product and its demand should justify production by biotechnological means.
Production of various alkaloids in cultures, their yield and approximate price are given in Table 29.3. Demand and cost of colchicine is not very high, but both criteria are very high for vincristine. Alkaloids such as ephedrine are obtained by chemical synthesis, a cheap method, while nicotine is obtained from field-grown plants (again a cheap source); therefore, their production through plant tissue culture is not commercially viable.
Optimization:
Optimization of alkaloid production is done using physical factors (light, temperature, vessel type), nutrients (carbon source, nitrogen source – nitrate versus ammonium nitrogen, organic (reduced) nitrogen, precursor molecules, phosphate, etc.), plant growth regulators (auxin type and concentration, cytokinin) and perhaps complex organic supplements (casein hydro-lysate, coconut milk, yeast extract, etc.).
It has been established from a large number of reports that fast-growing cultures accumulate alkaloids in low amounts during exponential phase of growth and in high amounts during the stationary phase. During this phase nutrients are exhausted and primary metabolism comes to a halt and the stored pool of primary metabolites is diverted to the synthesis of secondary products. Alkaloids production in various plant species is presented in Table 29.4.
Zenk et. al. (1977) used a two step culture system: medium for optimal growth (growth or maintenance medium) and medium for production of alkaloids (production medium). In the latter medium, growth is practically arrested by manipulating the nutrients, viz., high sucrose (4-10%) and low phosphate, 2,4-D and nitrogen. On transfer of growing cells to the production medium, product yield is increased several-fold.
Various factors affecting the production of secondary metabolites in plant tissues grown in culture are presented in Figure 29.4. Initially, growth and production of secondary metabolites are optimized by manipulating the physico-chemical factors followed by selection of high-productive cells. Afterwards, use of the production medium and specific techniques such as elicitation, hairy roots and immobilization are applied. When cultures are considered suitable for commercial production, cultures are grown in bioreactors of increasing capacity to develop technology.
There are two approaches to enhance product synthesis and accumulation. The most widely applied empirical approach is optimization wherein medium and environmental factors of the cultures are manipulated. The second approach is to select suitable cells/tissues under normal or selective conditions. The most recent method is to combine both the approaches, i.e., selection of cells/tissues on the optimal medium condition for high product yield. For large scale production of a compound, stable high-producing cell lines of plants of interest have to be obtained.
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The conditions for high growth and conditions for high productions are different. High growth is associated with low yield of secondary metabolites and slow growth is associated with high yield of secondary metabolites. Therefore, all those nutrients and environmental conditions which support high growth are not suitable for the production of secondary metabolites such as low sucrose, high auxin (particularly 2, 4-dichlorophenoxyacetic acid) and high phosphate.
Therefore, growth medium and a medium for production re used for the production of secondary metabolites. Cells are grown for a period in growth medium then transferred in to production medium where high production is obtained. Increased synthesis of secondary products occurs during the stationary phase of cultures when primary metabolism and cell proliferation come to a halt. The production medium contains low or no auxin, moderate or high cytokinin, low phosphate and high to very high concentration of sucrose. In this medium, cells grow slowly and accumulate more secondary metabolites as nutrients in high concentrations are always available.
Selection of Cells:
An in vitro growing cell system has two components: the cell and its environment. In the above paragraphs, the details of manipulating physical and chemical environment of the cell are presented. Here, we discuss how selection procedures are helpful in increasing the yield of cultures. Before producing secondary metabolites at the industrial/commercial level, it is a prerequisite to achieve optimal yield of secondary metabolites through optimization and select the cells for maximal yield.
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Though the production of a secondary metabolite is a genetically controlled phenomenon, cells can be selected for high yield of secondary metabolites from a heterogeneous population to improve the overall quality of the cultures, in relation to the production of active principle. Before starting the selection procedures, it is necessary that heterogeneous nature of the cultures be established and a sensitive method of analysis is available to analyze a large number of clones.
The ‘stability’ of such selected clones is of paramount importance for developing further method to achieve industrial production. Cell suspensions are plated on Petri dishes, growing colonies are subcultures and clones are selected as described in plating technique.
Variability in secondary metabolite content is determined and high productive clones are selected. These clones may not be stable in production of secondary metabolites so, plating and selection is performed repetitively to obtain stable clones. This is presented in simplified form in the Figure 29.5. Repetitive selection yields high secondary metabolite productive clones.
