Shrivastava S.

Plant tissue culture, the growth of plant cells outside an intact plant, is a technique essential in many areas of the plant sciences. It relies on maintaining plant cells in aseptic conditions on a suitable nutrient medium. The culture can be sustained as a mass of undifferentiated cells for an extended period of time or regenerated into whole plants.

Because plant cell culture is not affected by changes in environmental conditions, improved production may be available in any place or season. Therefore, studies on the production of useful metabolite by plant cell culture have been carried out on an increasing scale since the end of the 1950's. Their results stimulated more recent studies on the industrial application of this technology in many countries. However, there are still a few barriers that must be overcome before commercialization of many other products can occur. To overcome barriers hindering industrial application of plant cell cultures, however, it is required to conduct more fundamental research, including elucidation of biosynthetic pathways of many useful secondary metabolites in plants and mechanisms for their biosynthesis, collaboration with a number of researchers in other scientific fields is also very helpful. In this review, the background of research on plant cell culture, various approaches to improve the productivity of secondary metabolites and different techniques are discussed.

Introduction

From centuries, mankind is totally dependent on plants as a source of carbohydrates, proteins and fats for their food & shelter. In addition, plants are valuable source of a wide range of secondary metabolites, which are used as pharmaceuticals, agrochemicals, flavors, fragrances, colors, biopesticides and food additives. Over 80% of the approximately 30,000 known natural products are of plant origin ^1,2 . In 1985, 3500 new chemical structures have been identified and 2600 derived from the higher plants. Worldwide, 121 clinically useful prescription drugs are derived from plants ³ . Even today, 75% of the world’s population relies on plants for traditional medicine. In the US , where chemical synthesis dominates the pharmaceutical industry, 25% of the pharmaceuticals are based on plant-derived chemicals ^4,5 .

Plants will continue to provide novel products as well as chemical models for new drugs in the coming centuries ⁶ . The advent of chemical analysis and the characterization of molecular structures have helped in precisely identifying these plants and correlating them with their activity under controlled experimentation. Despite advancements in synthetic chemistry, there is need of biological sources for a number of secondary metabolites including pharmaceuticals ⁷ . Elaborative pathways from basic primary metabolites, which are synthesized immediately as a result of photosynthetic activity, produce secondary metabolites. Other techniques like hairy root culture, biotransformation, immobilization and elicitations are used for the increased production of secondary metabolites. Through plant tissue culture the totipotent characteristics of plant can be used for the in vitro regeneration of plant.

Tissue culture is an experimental technique through which mass of cell is produced from the explants tissue. The callus produced can be utilized directly to regenerate plantlets or to extract or to manipulate some primary and secondary metabolites. Callus culture and suspension culture are the basic technique used to produce the desired metabolites of plants ⁸ . The plant and tissue cultures have been enabled to increase the knowledge in many areas including differentiation, cell division, cell nutrition and cell preservation. But now cells are cultivated in-vitro in bulk or as clone from single cells to grow whole plants from isolated meristem, then induce callus and develop complete plantlets by organogenesis or by embryogenesis. The research needs are based on the elements of scientific progress and development of new techniques, which either enables more critical experiment to be undertaken, or rendering easy accessibility to complicated problems through experimental studies ^9,10 .

1. Need for a biotechnological approach

Biotechnology offers an opportunity to exploit the cell, tissue, organ or entire organism by growing them in-vitro and to genetically manipulate them to get desired compounds. Since the world population is increasing rapidly, there is extreme pressure on the available cultivable land to produce food and fulfill the needs. Therefore, for other uses such as production of pharmaceuticals and chemicals from plants, the available land should be used effectively. The development of micropropagation methods for a number of medicinal plant species has been already reported and needs to be adopted ¹¹ .

Chemical engineers are currently developing improved and appropriate bioreactors for the improvement of production systems by adopting techniques of growth and metabolite production coupled with downstream processing of the products. The improvements in molecular biological research have given a new dimension to in-vitro culture as well as for plant improvement, enhancing the yields of the product and resulting in multiple products or producing novel products from genetically engineered plants. Moreover, the need for safer drugs without side effects has led to the use of natural ingredients with proven safety. These factors have laid emphasis on the use of biotechnological methods to enhance the production of pharmaceuticals and food additives in quality and quantity.

2. History in plant tissue culture

Haberlandt (1902) first envisioned the concept of plant tissue culture when he cultivated cells of various species on a medium containing glucose and peptone. Although he did not observe cell division, he described an approach whereby cellular physiology could be followed without the complexity of the whole level intervening. Haberlandt's pioneering research provided the groundwork for the first instances of callus induction and sustainable cell growth nearly forty years after his original experiments. Three independent and nearly simultaneous reports described callus initiation from carrots ¹² and tobacco ¹³ in-vitro cultures.

Callus induction from other dicotyledonous species soon followed (Gautheret, 1983) and callus from the first monocot was reported by LaRue (1947) in Zea mays endosperm cultures. One of the reasons for their success in initiating and maintaining these cultures was the use of the plant growth regulators. The discovery of indole acetic acid (IAA) by Went (1926) provided a means to initiate callus from nontumorous tissues. Furthermore, the positive influence of coconut milk on cell proliferation was attributed to new class of growth regulator, cytokinins ¹⁴ (i.e. kinetin as described by Miller et al., 1955). The combined use of IAA and kinetin by Skoog and Miller ¹⁵ (1958) illustrated the synergism association with these compounds regarding the development of synchronized shoot-root plant regeneration. It should be pointed out the phrase 'plant tissue culture' is a really a misnomer in that the only tissue of plant origin that can be cultured is wound callus. In other instance of in-vitro culture where differentiation has occurred into shoots or roots, mixed tissues are involved in the final organs produced.

In a historical perspective, Cocking (1983) described problems and frustrations associated for the initial attempt to isolate and culture protoplasts. However, there have been several advances in this technology since the early 1970s. This includes the first reports of regeneration from protoplasts of Nicotiana tobaccum . The work of Sjepared and Totten (1977) with Solanum tuberosum was also a milestone since it represented the first case of protoplast-derived plant regeneration from a major agronomic crop. Advances are occurring in several monocotyledonous species such as Dactylis glomerata , Oryza sativa , Saccharum species, Triticum aestivum and Zea mays , where plant regeneration is now possible. A survey of historical milestone in plant tissue culture is shown in Table 1.

3. Strategies to increase secondary metabolite production                                   

During the past decade, a considerable progress has been made to stimulate formation and accumulation of secondary metabolites using plant cell cultures ¹⁶ . The adopted strategies for enhancing the secondary metabolites are obtaining efficient cell lines for growth, screening of high-growth cell line to produce metabolites of interest, immobilization of cells to enhance yields of extra-cellular metabolites and to facilitate biotransformation, use of elicitors to enhance productivity in a short period of time, permeation of metabolites to facilitate downstream processing, adsorption of the metabolites to partition the products from the medium and to overcome feedback inhibition and scale-up of cell cultures in suitable bioreactors.

4. Types of nutrient media

The number of nutrient media is uses from time to time, their names and composition are given below in Table 2.

5. Plant cell cultures

Plant tissue culture provides an alternative way for the production of phytochemicals of therapeutic importance. Callus culture and suspension culture are the basic techniques used to produce the desired metabolites. Now a day techniques like hairy root culture, immobilization and elicitation are used to enhance the production of secondary metabolites. The plant tissue culture have some salient feature which includes, the clone generated through tissue culture which are identical in size, development stage and rate of metabolic activities and are capable of performing the transformative activity which involves biotransformation to produce primary and secondary metabolites in culture medium. Through plant tissue culture, the totipotent characteristic of plant cell lend them amenable to in-vitro regeneration into whole plant via embryogenesis, organogenesis or through micropropagation. Growing research in tissue culture technology provides the opportunities of biosynthesis of a variety of natural products using tissue culture. Different techniques of plant tissue culture are given below;

5.1. Organ cultures as a source of pharmaceuticals

Since production of secondary metabolites is generally higher in differentiated tissues, there are attempts to cultivate shoot cultures and root cultures for the production of medicinally important compounds, these organ cultures are relatively more stable ¹⁷ . There are a number of medicinal plants whose shoot cultures have been studied for metabolites (Table 3). Similarly, root cultures are valuable sources of medicinal compounds, root systems of higher plants generally exhibit slower growth and are difficult to harvest.

5.2. Hairy root cultures for pharmaceuticals

The ability of Agrobacterium rhizogenes to induce hairy roots in a range of host plants has lead to studies on it as a source of root-derived pharmaceuticals ¹⁸ . Tepfer ¹⁹ (1990) summarized 116 plants belonging to 30 dicotyledonous families wherein hairy roots have been induced. Hairy roots are induced by transfer of T-DNA from the plasmid of A. rhizogenes ²⁰ to host tissue, resulting in root formation. A number of examples are given in Table 4.

5.3. Immobilization of plant cells for the production of secondary metabolites

Improvement in the secondary metabolite production of cell cultures is often associated with the organization and differentiation of plant cells. The concept of organization and differentiation led to the use of immobilization technology, which has long been used for microbes and enzymes. Immobilization is defined as a technique, which confines a catalytically active enzyme or cells on a fixed support and prevents its entry into liquid phase ²¹ . Immobilized plant cells have been used for single and multistep biotransformation of precursors to desired products as well as for the de-novo biosynthesis of secondary metabolites. Immobilization can have a dramatic impact on cellular physiology and secondary product formation. The more dramatic responses are summarized in Table 5.

5.4. Plant tissue culture by Micropropagation

Micropropagation is a combination of the arts and sciences of plant multiplication in-vitro and plant acclimatization. It is the true-to-type propagation of a genotype that comprises many steps-stock plant care, explants selection and sterilization, media manipulation to obtain proliferation, rooting, acclimation, and growing on of liners and is usually associated with commercial production. The purpose of micropropagation is to produce carbon copies of original unique plants or more simply put to grow clones in quantity. The micropropagation process is the culmination of all the inventions, theories, and discoveries man has made regarding the anatomy and physiology of the living world. When the long histories of other approaches to deliberate plant propagation, it is shortest but it is the most promising technique, today about 150 plants are commercially micropropagated. Many of these have reached the limits of their improvement by traditional methods. The emphasis on sustainable agriculture, increasing world population and the loss of prime land to housing and industry make this method of propagation indispensable. The history of micropropagation is shown in Table 6.

5.5. Plant tissue culture by Biotransformation

Biotransformations are chemical reactions catalyzed by cells, organs or enzymes. Biotransformations explore the unique properties of biocatalysts, namely their stereo-and regiospecificity and their ability to carry out reactions at no extreme pH values and temperatures. The biotransformation is another technique of tissue culture in which chemical conversion of an exogenously supplied substance can be done by the living cell culture. It is for commercial exploitation of secondary metabolites. Biotransformation is defined as chemical transformations that are catalyzed by microorganism or their enzymes ⁸ . The unlimited enzymatic potential of plant cell culture can in principle is used for bioconversion purpose. Biotransformation is an area of biotechnology that has achieved a considerable attention. It is the process in which pharmaceutically less important precursor is converted into the compound of interest. Biotransformation of digitoxin to gitoxin purpurea glycoside-A & purpurea glycoside-B from suspension culture of D. Purpurea ²² . Such modification can takes place at several positions in the molecules. Rakson & Baker ²³ (1988) excellently summarized the advantages & disadvantages of biocatalyst. Cell suspension cultures, immobilized cells, enzyme preparations and hairy root cultures can be applied for the production of food additives or pharmaceuticals by biotransformation process (Table 7).

Summary and Conclusion

Since last two decades there have been considerable efforts was made in the use of plant cell cultures in bioproduction, bioconversion or biotransformation and biosynthetic studies. The potential commercial production of pharmaceuticals by cell culture techniques depends upon detailed investigations into the biosynthetic sequence. The great enthusiasms of biotechnologists have been seen in the potential use of cell culture in the production of valuable secondary products. Plant tissue culture is a noble approach to obtain their substances in large scale. Many companies in India and abroad are showing interest in this direction. Tissue culture is an alternative way for the production of phytochemical of therapeutic importance. Other techniques like hairy root culture, biotransformation, immobilization and elicitations are used for the increased production of secondary metabolites. By this approach an increase production of secondary metabolites are obtained. In present scenario it is an effective and efficient procedure for converting less medicinally important plant metabolites to a valuable product.

References

1. Phillipson, J.D., (1990), Plants as source of valuable products. In: Charlwood, B.V., Rhodes, M.J.C., editors. Secondary products from plant tissue culture. Oxford : Clarendon Press, 1–21.

2. Balandrin, M.J., Klocke, J.A., (1988), Medicinal, aromatic and industrial materials from plants. In: Bajaj YPJ, editor. Biotechnology in agriculture and forestry. Medicinal and aromatic plant , 4, 1–36.

3. Payne, G.F., Bringi, V., Prince, C., Shuler, M.L., (1991), Plant cell and tissue culture in liquid systems. Munich : Hanser Publ ., 1–10.

4. Glaser, V., 1999, Billion-dollar market blossoms as botanicals take root, Nat Biotechnol , 17, 17–8.

5. Farnsworth, N.R., 1985, The role of medicinal plants in drug development . In: Kroogsgard-Larsen P, Brogger Christenses S, editors, Natural products and drug development , Denmark: Munsgaard Copenhagen, 17–30.

6. Cox, P.A., Balick, M.J., 1994, The ethanobotanical approach to drug discovery, Sci Am , 82–7.

7. Pezzuto, J.M., 1995, Natural product cancer chemoprotective agents . In: Arnason JT, Mata R, Romeo JT, and editors. Recent advances in phytochemistry, Phytochemistry of medicinal plants , 29, 19–45.

8. Vyas, S.P., Dixit VK, (1999), Pharmaceutical biotechnology , 298, 299.

9. Mantell, S.H. and Smith, H., (1983), Plant Biotechnology , Cambridge University Press, Cambridge , 3.

10. Evans, D.A., Sharp, W.R., Ammirato, P.V. and Yamada, Y., (1983), Handbook of plant cell culture ”, Macmillan Publishing Co., New York , 1, 4.

11. Naik, G.R., (1998), Micropropagation studies in medicinal and aromatic plants. In: Khan IA , Khanun A, editors, Role of biotechnology in medicinal and aromatic plants. Hyderabad : Ukaz Publications , 50–6.

12. Gautheret, R. J., (1939), C.R. Hebd. Seances Acad. Sc ., 208, 118-120.

13. White, N.J., (1939), Am. J. Bot ., 26, 59-64.

14. Miller, C.O., Skoog, F., Von Saltza, M.H. and Strong, F.M., (1955), J . Am. Chem. Soc., 77, 1392.

15. Skoog, F. and Miller, C.O., (1958), Symp. Soc.m Exptl., 11:118-130

16. Ravishankar, G.A., Rao, R.S., (2000), Biotechnological production of phyto-pharmaceuticals, J Biochem, Mol Biol Biophys , 4, 73 –102.

17. Roja, G, (1994), Biotechnology of indigenous medicinal plants. PhD Thesis, Bombay University , Bombay .

18. Flores , H.E., Vivanco, J.M., Loyola-Vorgas, M., (1999), Trends Plant Sci., 4, 220–6.

19. Tepfer, D., (1990), Physiol. Plant , 79, 14–6.

20. Ambros, P.F., Matzke, A.J.M. and Matzyke, M.A., (1986), Embo J ., 5, 2073–7.

21. Lindsey, K., Yeoman, M.M., (1983), Novel experimental systems for studying the production of secondary metabolites by plant tissue cultures. In: Mantell SH, Smith H, editors. Plant biotechnology . London : Cambridge Univ. Press, 39–66.

22. Karow, E.O. and Passives, (1956), P.M ., Ind. Eng. Chem , 48, 2213.

23. Rakson, I.G. and Baker, P., (1988), Spec. Chem. , 239-240.

24. Purohit, S.S. and Mathur, S.K., (1990), Fundamentals of Biotechnology , A.B.P. Publication, 123-142.

Table 1: A survey of historical milestones in plant cell and tissue culture techniques24