A. Plant Growth Regulators
Effects of plant growth regulators, especially auxin and cytokinin, on secondary metabolism in cell cultures have been extensively investigated. It is well known that auxin is essential and cytokinin is preferable to induce cell dedifferentiation and to maintain cell proliferation in vitro. In has also been widely recognized that the concentration and balance of auxin and cytokinin affect organ regeneration from cultured cells. These growth regulators regulate secondary metabolism in in vitro-cultured cells probably through controlling cell differentiation. However, the effects of auxin and cytokinin are variable from species to species and from product to product, and the mechanism by which the plant growth regulator up- or down-regulates the particular secondary metabolism is not clear in most cases.
Gibberellin is usually not added to culture medium, and only a few reports describe its effect on natural product biosynthesis. Production of berberine in Coptis japonica cell cultures was increased by gibberellin (15). In contrast, gibberellin inhibited shikonin biosynthesis in Lithospermum erythrorhizon cell cultures (16).
For practical application of plant cell cultures to secondary metabolite production, it is desirable to culture the cells without phytohormones, especially when the product is used as a crude extract, for example, in the case of anthocyanin, because contamination of the phytohormones from the culture medium may influence human health. In mammalian cell cultures, hybridomas produced by fusion of antibody-producing cells having no proliferation activity with highly proliferative myeloma cells are used to produce monoclonal antibodies. A similar approach could be possible in plant cell cultures. Actually, protoplasts from petals of Petunia hybrida were fused with protoplasts from cultured crown gall tumor cells, and microcalli thus produced grew vigorously on hormone-free medium and formed anthocyanin characteristic of parent petals (17).
Optimization of medium nutrients is important to increase the productivity of the particular secondary metabolites. There are a number of reports describing the effects of medium nutrients on secondary metabolism in plant cell cultures. Many of these investigations seem to indicate a negative correlation between cell proliferation and secondary metabolism. It might be possible that any manipulation for inhibiting cell growth leads to an increase in the productivity of secondary metabolites, leading to establishment of a two-stage culture system for production of phytochemicals where the cells are first cultured in the medium appropriate for maximum biomass production and then transferred to the growth-limiting medium for maximum productivity of secondary metabolites as established for shikonin production in Lithospermum erythrorhizon cell cultures (18).
One of the most important nutritional factors is the phosphate level in the medium. Since Nettlership and Slator (19) first reported in 1974 that use of a phosphate-free medium increased the alkaloid productivity of Peganum harmala cells, it has been recognized that reducing the phosphate concentration results in growth limitation and a concomitant increase in the level of secondary products.
Nitrogen is essential to support cell growth as a source of protein and nucleic acid synthesis, thus affecting secondary metabolism. Generally, culture medium contains N sources as NH4f and NO^~, and both the concentration of total nitrogen and the ratio of NH4 to NOJ regulate cell growth and secondary metabolism. In many cases, reducing the total nitrogen concentration in the medium leads to lower cell growth and higher product formation as typically reported for anthocyanin production by Vit is vinifera (20) cell cultures. However, cell growth and production of betacyanin in Phytolacca americana cell cultures increased with an elevated nitrogen supply
(21). If used as a sole nitrogen source, NHÎ" is often toxic to cell growth. Shikonin production in L. erythrorhizon cell suspension cultures was completely inhibited when the cells were cultured in NH^-containing medium
(22). It is important to find an optimum ratio of NH4f to N03 for attaining maximum production of secondary metabolites.
Sucrose is utilized most as a carbon source. In contrast to phosphate and nitrogen, an increase in the initial sucrose concentration in culture medium leads to an increase in secondary metabolite production. The enhancing effect of sucrose was most impressively shown in the case of rosmarinic acid formation in Coleus blumei cell suspension cultures, where the rosmarinic content increased sixfold in a medium containing 5% sucrose compared with that in the control medium (2% sucrose), reaching 12% of dry weight (23). This effect was not due to the higher osmotic pressure because addition of mannitol to low-sucrose medium did not increase rosmarinic acid production. In contrast, the stimulatory effect of sucrose on anthocyanin production in Vitis vinifera cell cultures was shown to be due to osmotic stress (24). The carbon-to-nitrogen ratio is also an important factor in secondary metabolism as shown by anthocyanin production in Vitis cell cultures (25).
Although less investigated compared with macronutrients, micronutri-ents are also expected to affect secondary metabolism. In fact, the shikonin content in L. erythrorhizon cell cultures increased drastically with an increasing Cu2+ level in the medium (22).
Elicitors are the active components in extracts of microbial and plant origin that induce defense responses when applied to plant tissues. The elicitors produced by microorganisms and plants are referred as biotic elicitors, while physical and chemical stresses such as ultraviolet (UV) irradiation, heat or cold shock, and heavy metals also induce a wide range of defense responses and are defined as abiotic elicitors. Abiotic elicitors are thought to induce the release of biotic elicitors from plant cell walls. It has been shown that elicitors are capable of not only inducing de novo formation of phytoalexins but also activating biosynthetic potentials of various constitutive metabolites in cultured plant cells. Production of sequiterpene gossypol in Gossypium arboretum was increased over 100-fold by elicitors prepared from Verticil-lum dabliae elicitors (26). Elicitor treatment increased the biosynthesis of the benzophenanthridine alkaloid sanguinarine 26-fold in Papaver som-niferum cell cultures (27). Induction of isoflavonoid biosynthesis in Pueraria lobata cell cultures by either a biotic elicitor yeast extract or the abiotic elicitor CuCl2 has also been extensively investigated, especially at the molecular level (28).
Elicitors provide important clues to understanding the molecular basis of the transducing pathway through which exogenous signals lead to secondary product biosynthesis, involving various signal compounds such as reactive oxygen species, jasmonic acid, Ca2+, and phosphoinositides. Induction of secondary metabolism by elicitors in cell suspension cultures of various plant species was correlated with earlier rapid and transient accumulation of jasmonic acid and its methyl ester methyl jasmonate, and jas-monic acid was proposed to be a key signal compound in the cellular process of elicitation leading to the accumulation of various secondary metabolites in the cultured plant cells (29). Production of various phytochemicals including rosmarinic acid (30), alkannin (31), taxol (32), shikonin (33), and stilbene (34) has been reported to be induced by jasmonic acid or methyl jasmonate. A cDNA encoding geranylgeranyl diphosphate synthase, which catalyzes an important biosynthetic step leading to taxol, was cloned from Taxus canadensis cell cultures pretreated with methyl jasmonate to induce taxol biosynthesis (35). This suggests that jasmonic acid (or its methyl ester) may be used as an inducer of secondary metabolism in cultured plant cells not only for practical application but also for basic research.
Physical factors controlling secondary metabolite production synthesis in cultured plant cells include light, temperature, medium pH, aeration, cell density, etc. The effect of light on natural product biosynthesis is quite varied. Light illumination usually induces chloroplast differentiation, which sometimes leads to elevation of secondary metabolism. A lupine alkaloid lupanine was produced only in the green callus of Thermopsis lupinoides cultured under light illumination (36). Light illumination is often essential to induce anthocyanin biosynthesis, although its biosynthesis is not localized in chloroplasts. In contrast, biosynthesis of nicotine in tobacco cells (37) and shikonin in Lithospermum erythrorhizon cell cultures is inhibited by light illumination (38). From a biotechnological viewpoint, a light requirement for secondary metabolite formation is problematic because it is difficult to provide adequate illumination without affecting temperature.
The optimal culture temperature and medium pH are usually between 20 and 25°C and between 5.6 and 6.0, respectively. Although these factors are expected to affect secondary metabolism in cultured cells, they have not received much attention so far. Aeration is also an important factor to regulate both cell growth and product yield, especially in bioreactor cultures, and is discussed later in this chapter and in a separate chapter. Cell density, which is mostly determined by cell inoculum size, is also an important factor affecting product yield. High-density cultures were established for berberine production by Coptis japónica cultures (39) and anthocyanin production by Perilla frutescens cultures (40).
One of the most important factors controlling secondary metabolite production is cell-to-cell variation. Within a population of cultured cells there is a difference in metabolic behavior, especially in ability to synthesize particular metabolites even if the cell population was induced from the same piece of explant and cultured in the same physical and chemical environments. Although a molecular biological basis for such cellular variation in secondary metabolism has not yet been clarified, it has been well recognized that selective subculture of cell aggregates whose content of the secondary metabolite is higher than others will eventually result in the isolation of so-called high-producing cell lines for a particular secondary metabolite. Such selection is easy to perform when the target compound is a pigment (41) but still possible for colorless compounds by using a convenient cell-squash method (42) or a semiautomated immunoassay (43).
Another important biological factor is stability of the biosynthetic capability of cultured plant cells. Alkaloid-producing cell lines of Catharan-thus roseus that were established by repeated selection lost their biosynthetic ability during subcultures; the indole alkaloid content decreased 70-fold over 8 years of subculture (44). A similar kind of biochemical instability was also reported for nicotine in Nicotiana rustica callus (45), cardenolides in Digitalis purpurea callus (46), and cinnamic acid in Capsicum frutescens cell suspension (47). These results indicate that it is important to subculture the cells under selection pressure or with occasional reselection.
Anthocyanin constitutes a major flavonoid pigment. It is ubiquitous in the plant kingdom and provides scarlet to blue colors in flowers, fruits, leaves, and storage organs. Chemically, they are based on a single aromatic molecule, that of delphinidin, and all are derived from this pigment by hydrox-ylation, methylation, or glycosylation (Fig. 1). Interest in anthocyanins as food colorants has been increasing not only because they are less toxic than synthetic red dyes but also because they exhibit significant antioxidant activity, which might protect against cardiovascular diseases and certain cancers (48).
There have been a number of publications describing anthocyanin production by plant cell cultures; some of the more recent ones are summarized in Table 1. Progress in sophisticated spectroscopic technology such as fast atom bombardment mass spectrometry (FAB-MS) and two-dimensional nuclear magnetic resonance (2D-NMR) together with high-performance liquid chromatography (HPLC) separation has led to elucidation of complex structures of anthocyanin as reported in cell suspension cultures of Perilla sp. (49), Daucus carota (50), Ajuga reptans (51), and Ajuga pyramidalis (52).
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