cell and tissue culture; biochemical conversions; clonal propagation

Chapter 13 Plant cell and tissue culture; biochemical conversions; clonal propagation

One of the rapidly expanding areas of pharmacognosy has involved the application of the artificial culture of plant cells, tissues and organs to the study of medicinal plants. Principal topics include the development of commercial production of expensive biomedicaments, the discovery of new metabolites, the selection of superior strains of medicinal plants, the elucidation of biosynthetic pathways of secondary metabolites with isolation of corresponding enzymes, and the improvement of medicinal plant species by genetic engineering.

INDUSTRIAL SIGNIFICANCE

A number of factors militate against dependence by the pharmaceutical industry on the use of botanical sources of drugs, and these have been, to some extent, responsible for the reluctance of industry to invest in the exploitation of the plant kingdom. These factors include the following.

1 Availability of raw material. Some plants, although highly desirable as sources of biochemicals, just cannot be produced in an economically sufficient quantity to satisfy demand. An example is Strophanthus sarmentosus seeds, which, early in the search for corticosteroid precursors (1950s), were known to contain a very suitable compound, sarmentogenin, conveniently substituted in the steroid C ring (see Chapter 23). But the plants, tropical lianes existing as different chemical races (Chapter 14), are not particularly abundant and are difficult to cultivate. More recent examples include the very limited supply of the Pacific yew (Taxus brevifolia) the principal source of taxol, a diterpene with considerable potential as a starting material for the semisynthesis of promising anticancer drugs and also Coleus forskohlii, an Indian species now listed as vulnerable to extinction in the wild as a result of indiscriminate collection for the isolation of forskolin, a diterpenoid used in the treatment of glaucoma and heart disease.

2 Fluctuation of supplies and quality. The production of crude drugs is subject to the vagaries of the climate, to crop disease, to varying methods of collection and drying which influence quality, and to the inherent variation of active constituents arising from plants of the same species having different genetic characteristics.

3 Political considerations. New compounds of promising medicinal value are increasingly reported in previously uninvestigated tropical or subtropical species situated in countries of uncertain political persuasion. Industry is not prepared to undertake the prolonged and expensive development of such material.

4 Patent rights. Generally, it is not possible to patent a naturally occurring plant metabolite as such, only a novel method for its extraction and isolation. Hence, there is little incentive for a pharmaceutical company to spend many years and vast sums of money on launching a new natural product over which it has no patent rights.

Following from the above, it is not surprising that industry world-wide takes a close interest in the commercial possibilities of cultivating particular species of plant cells, under conditions analogous to the production of antibiotics, that will yield biomedicinals. By this means, production could at all times be geared to demand, and a product of standard quality assured. Furthermore, a highly sophisticated and specific method of production can be patented.

Shikonin, a dye and antibacterial, is commercially produced by the cultivation of Lithospermum plant cells and the production of the ginsenosides and antitumour alkaloids of Catharanthus roseus are currently being developed; Japanese patents exist concerning the manufacture of many secondary metabolites including the purple pigment from Melissa officinalis, the production of catharanthine and ajmalicine by cell cultures derived from C. roseus anthers, the manufacture of an analogue of taxol (q.v.) by callus cultures of Taxus spp., and tropane alkaloids from Duboisia tissue cultures. A vast amount of work has been reported during the last decade and the majority of common medicinal plants, and many less common ones, have been subjected to cell culture investigation. Nevertheless, in the majority of cases, yields of metabolites have been commercially disappointing. Staba, a pioneer in the investigation of medicinal plant cell culture, stated (J. Nat. Prod., 1985, 48, 203) ‘there are arguably as many gravestones as milestones along the way in developing plant tissue culture systems for the production of secondary metabolites’. Ultimately, of course, as Fowler pointed out over 25 years ago (Chem. Ind., 1981, 229), ‘no matter how elegant the science, the fundamental criterion has to be price comparability coupled with profitability’, a fact clearly obviated by subsequent events.

CULTIVATION OF PLANT CELLS

Although the feasibility of artificially cultivating plant cells had long been recognized, and White had propagated isolated tomato roots for periods of over 30 years, it was only some few decades ago that modern developments in the cultivation of cells of higher plants as a callus, or as a suspension liquid culture, really began. In this connection the publication of P. R. White’s Cultivation of Plant and Animal Cells in 1954 and H. E. Street’s developmental work at Leicester University deserve mention.

Cultures of single cells growing under controlled conditions in a liquid medium, or callus cultures consisting of undifferentiated masses of cells developing on a semi-solid medium, can be initiated from parenchymatous tissues of shoots, roots and other plant structures (see Fig. 13.1). The maintenance of such cultures depends on an adequate supply of nutrients, including growth factors, and a controlled sterile environment. The cells, although undifferentiated, contain all the genetic information present in the normal plant. By suitable manipulation of the hormone content of the medium, it is possible to initiate the development of roots, shoots, and complete plants from the callus cell culture and to encourage the division of cells in a suspension culture.

Fig. 13.1 Origin of plant cell cultures.

Several forms of suspension culture are commonly utilized, as follows.

1 Batch suspension cultures. In this technique the cells multiply in a liquid medium which is being continuously agitated to break up any cell aggregates. Except for the circulation of air, the system is ‘closed’ with respect to additions or subtractions from the culture. Typically, the original inoculation of cells into the medium is followed by a lag period and then, after increasing in mass, the cells undergo a period of exponential growth and division. Finally, a stationary growth phase is reached, at which point some component of the medium, essential for growth, has probably been exhausted. Growth will recommence when cells are transferred to a fresh medium or when more medium is added to the original culture.

2 Semicontinuous cultures. In this instance the system, an ‘open’ one, is designed for the periodic removal of culture and the addition of fresh medium, by which means the growth of the culture is continuously maintained.

3 Continuous cultures. Two forms of this ‘open’ system are the chemostat and turbostat systems, in which the volume of culture remains constant and fresh medium and culture are, respectively, continuously added and withdrawn. The essential feature of these two systems is that cell proliferation takes place under constant conditions. In the chemostat arrangement, a steady state is achieved by adding medium in which a single nutrient has been adjusted so as to be growth-limiting; this contrasts with the batch culture method, in which the transient conditions in which the cells find themselves lead to continuous changes in their growth rate and metabolism.

The problems associated with plant-cell culture are not completely identical with those encountered in the fermentation of microorganisms and fungi. With the former there is much slower cell proliferation and it takes about 3–6 weeks to progress from the shake-flask level (300 ml) to production capacity (20 000 litres). Also in plant cell cultures, large aggregates of cells may form, which then exist under different environmental conditions from the suspended cells. Dispersal of aggregates by the use of the usual fermenter paddles was originally considered too vigorous for fragile plant cells, resulting in their rupture. To overcome this, various designs of low-shear fermenters including the air-lift or drum types were designed. However, such refinements are not always necessary and the shikonin production mentioned above is, in fact, carried out in conventional stirred-tank vessels. As R. Verpoorte et al. have pointed out (J. Nat. Prod., 1993, 56, 186), for the industrial application of plant cell cultures, the recognition that shear- tolerant plant cell cultures exist is important because the fermentation industry at present uses stirred tanks almost exclusively. Investment in new ingenious bioreactors, as reported for experimental cell cultures in recent years, would place a major constraint on the commercialization of plant cell biotechnology.

To maximize on the cell mass produced, the cell suspension culture eventually becomes very dense and this presents problems of even aeration.

Most pilot studies have utilized fermenters of 5–15 litres capacity and the reported scaling up to 20 000 and more recently 75 000 litres in West Germany and Japan presents chemical engineering problems of considerable complexity.

PRODUCTION OF SECONDARY METABOLITES

The genetic information required for the manufacture of secondary metabolites is also present in the undifferentiated cells of the species concerned, and when activated should lead to the production of these materials. Much interest has been aroused by this aspect of cell culture with the aim of growing particular plant cells on a commercial scale for the production of valuable metabolites.

A pioneer in the cell culture of medicinal plants was E. J. Staba of Minnesota University, and his group was the first to demonstrate that many medicinal plants did produce in cell culture their characteristic secondary metabolites, albeit often in low yield. Notable advances were made by Zenk and colleagues who in 1975 demonstrated a 10% (dry weight) production of anthraquinone derivatives in a Morinda citrifolia culture—at that date the highest yield of secondary metabolite achieved by cell culture. By 1991 (M. H. Zenk, Phytochemistry, 1991, 30, 3861) almost 1000 species of callus were deposited in the collection at Braunschweig. Commercially orientated research has concentrated on those species that produce high-value speciality phytochemicals. Obvious examples are Catharanthus roseus (dimeric antitumour alkaloids), ginseng (ginsenosides) and Taxus species (taxol).

Apart from the general problem of low yield of product other factors which need to be addressed with cell cultures as a source of phytopharmaceuticals are: instability of cell lines, compartmentalization and isolation of the products, and the nature of the metabolites produced. Some points concerning these problems are given below.

Low production of desired metabolites

Knowledge of the enzymology of secondary metabolite formation, although rapidly expanding, is still incomplete. Secondary metabolic processes compete with primary metabolism for precursors and potential bottlenecks for the former may involve those enzymes linking the primary and secondary pathways, for example, tryptophan decarboxylase converting tryptophan to tryptamine in the formation of indole alkaloids and cyclase enzymes involved in the synthesis of cyclohexanoid monoterpenes from geranylpyrophosphate. With cell cultures, as distinct from whole plants, particular genes may be repressed and need to be activated by suitable elicitors, a technique which is currently an important area of research and is discussed below.

The compositions of the media in which culture cells are grown have been extensively investigated with a view to increasing both the biomass and secondary metabolites. Often, as reported with Dioscorea deltoidea for instance, rapidly dividing cells produce little or no metabolites of interest and a change from a growth medium (high biomass) to a production medium is required to effect the necessary biosynthesis. In this connection Zenk’s ‘alkaloid production medium’ for ajmalicine in C. roseus may be noted, together with the effects of long-term starvation of phosphate on levels of purine nucleotides and related compounds (F. Shimano and H. Ashihara, Phytochemistry, 2006, 67, 132).

Variations in the relative hormonal contents of the growth medium can also affect metabolism. It has been reported that reduced concentrations of 2,4-D increased alkaloid formation in C. roseus cultures and that abscisic acid and antigibberellin compounds have similar effects. With Thalictrum minus, ethylene has been shown to activate the production of berberine in cell cultures from the key intermediate (S)-reticuline and the ethylene-producing reagent 2-chloroethylphosphoric acid stimulates anthraquinone production in callus cultures of Rheum palmatum. Conversely, cardenolide accumulation in Digitalis lanata tissue cultures is decreased by ethylene. Cytokinins have been found to enhance secondary metabolite accumulation in a number of tissue culture studies—indole alkaloids (C. roseus), condensed tannin (Onobrychis sp.), coumarins (Nicotiana sp.), rhodozanthin (Ricinus sp.), berberine (Thalictrum minus). Rhodes et al. found a five-fold increase in alkaloid content of a culture of Cinchona ledgeriana occurs when cells are transferred from a 2,4-D, benzyladenine medium to one containing IAA and zeatin riboside. For information on plant hormones, see Chapter 12.

Although alkaloids from a wide range of medicinal plants have been produced satisfactorily by cell culture in the laboratory, a singular lack of success has been experienced in obtaining quinine and quinidine from Cinchona cultures and morphine and codeine from those of Papaver somniferum although, in both cases, other alkaloids are formed. To some extent the problems with the former are being overcome by the use of transformed roots (see below) but the growth rate is very slow. With morphine biosynthesis it appears that lack of developed laticiferous tissue in the unorganized cell culture may be responsible because cytodifferentiation leading to latificer-type cells leads to morphinan alkaloid production. One problem with Catharanthus cell cultures has been their inability to dimerize the requisite indole monomers to form the medicinally important anticancer alkaloids vinblastine and vincristine. In a similar way the accumulation of monoterpenes in cell cultures of some volatile oil-producing plants is severely limited, probably because of the absence of such storage structures as glands, ducts and trichomes. Thus, in Rosa damascena callus and suspension cultures, negligible amounts of monoterpenes are accumulated, although enzymes with high activity for the conversion of mevalonate and IPP into geraniol and nerol (see Chapter 18) are extractable from the apparently inactive callus. In this case, non-compartmentalization of the metabolites probably leads to their further metabolism. This is supported by the finding that, when added to cultures of Lavandula angustifolia, the monoterpenoid aldehydes geranial, neral and citronellal are reduced to their corresponding alcohols, geraniol, nerol and cirtronellol which, once formed, disappear from the cultures over about 15 h.

In studies on the phenolic antioxidant compounds produced by in vitro cultures of rosemary, A. Kuhlmann and C. Rohl (Pharm. Biol., 2006, 44, 401) find the content of carnosic acid, carnosol and rosmaric acid to be dependent on the differentiation grade of the cell culture type. Higher concentrations of rosmaric acid were measured in suspension cultures than in shoot and callus cultures, whereas the former on average produced three-fold less carnosic acid than the two latter cultures. Carnesol could not be detected in suspension cultures.

With Ginkgo biloba although a satisfactory biomass of undifferentiated cells could be produced on a manufacturing scale, the poor level of ginkgolides produced renders it of scant importance.

It has been observed that the origin (stem, root, etc.) of the callus can play an important part in determining the biochemistry of the subsequent culture.

Improved metabolite production may sometimes be achieved by the addition of precursors to the culture medium. Thus, addition of coniferin (a phenylpropane) to cell suspension cultures of Podophyllum hexandrum improved podophyllotoxin production 12.8-fold and an increase in quinoline alkaloids was obtained with Cinchona ledgeriana cultures fed with L-tryptophan. With transformed root cultures of Catharanthus roseus, the addition of the precursor loganin to the culture medium has been shown to increase the production of both ajmalicine and serpentine at the early stationary phase of growth, although it produced no increases during the early and late exponential growth phases. Catharanthine production was unaffected but was increased, together with the other alkaloids, by multiple feedings of loganin (see E. N. Gaviraj and C. Veeresham, Pharm. Biol., 2006, 44, 371 and references cited therein).

Light intensity

and selective wavelengths of light have been shown to have a stimulating effect on the production of some secondary metabolites in various tissue cultures. Thus, in one report (1990) blue light enhanced, whereas red light decreased, diosgenin production in Dioscorea deltoidea callus cultures. A recent example of the stimulant effect of UV-B radiation on secondary metabolism in callus cultures is the research of F. Antognoni et al. (Fitoterapia, 2007, 78, 345) on Passiflora quadrangularis. Daily doses of UV-B radiation (12.6, 25.3, 37.9 KJ m⁻²) produced increases in the flavonoid production of orientin, isoorientin, vitexin and isovitexin. Isoorientin accumulation in the callus after 7 days reached levels comparable to those found in the fresh leaves of greenhouse-raised plants. However, such beneficial treatments are difficult to accommodate with conventional stirred-tank fermentors.

The selection of high-yielding cell lines has been a major factor in countering low productivity. Such selection, perhaps involving a few plants from several thousand, has been greatly facilitated by the use of modern immunoassays (q.v.). In the case of Catharanthus roseus cultures, for example, recent research has concentrated on the production of the dimeric alkaloids vinblastine and vincristine (q.v.), the important anticancer drugs. The alkaloids are produced at the end of a complex biogenetic pathway in which the monomers are first produced. The latter, as corynanthe-, strychnos- and aspidosperma-type alkaloids can all be produced (0.1–1.5%) in culture using Zenk’s alkaloid production medium. Different cell cultures derived from any one species of plant may vary enormously in their synthetic capacities, so that, in the above case, distinct high ajmalicine-producing and high serpentine-producing strains are possible.

Examples of other plants for which somaclonal variation has been exploited include Nicotiana rustica (nicotine) (of no commercial interest), Coptis japonica (berberine), Anchusa officinalis (rosmarinic acid), Lithospermum erythrorhizon (shikonin) and Hyoscyamus muticus (hyoscine). For Thalictrum minus (berberine) a strain giving a 350-fold increase in alkaloid production has been reported.

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Jul 18, 2016 | Posted by admin in PHARMACY | Comments Off

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