PLANT BIOTECHNOLOGY AND BIOSAFETY - A. P. Ermishin - 2015

CHAPTER 4. THE USE OF PLANT CELL CULTURES FOR THE PRODUCTION OF CHEMICAL COMPOUNDS

4.1. Secondary Metabolites, Their Role in Plant Life, and Value for Humans

A characteristic feature of higher plants is their ability to synthesize a wide range of relatively low-molecular-weight biologically active compounds, such as saponins, Coumarins, sterols, carotenoids, polyphenols, Alkaloids, Vitamins, Quinones, and others. Because their presence in The plant Cell is not strictly essential for maintaining viability, they are referred to as "secondary metabolites" (hereinafter referred to as SMs).

Plants that produce energy-rich seeds typically accumulate significant amounts of SMs with anti-nutritional properties. For instance, many legumes, along with a high protein content, contain Lectins, protease inhibitors, cyanogenic Glycosides, Tannins, isoflavonoids, and Oligosaccharides. The ability to synthesize these substances evolved and was preserved as a defense mechanism protecting the plant against herbivores, microorganisms, and Viruses.

Many plant SMs possess medicinal properties and have been used since ancient times to treat human diseases as well as in cosmetics. Examples include triterpene glycosides (steroidal sapogenins and saponins from ginseng, licorice, and aralia), yew Isoprenoids (anti-tumor taxanes), cardiac glycosides (cardenolides from foxglove, strophanthus, and lily of the valley), and alkaloids from Madagascar periwinkle, belladonna, colchicum, erythroxylum, opium poppy, periwinkle, rauwolfia, cinchona, and tobacco (atropine, hyoscyamine, scopolamine, colchicine, cocaine, codeine, morphine, papaverine, physostigmine, pilocarpine, quinine, quinidine, reserpine, D-tubocurarine, etc.). Notably, research aimed at discovering new, efficient natural SM producers has intensified sharply in recent years. For example, paclitaxel (taxol) from the yew tree Taxus brevifolia, identified as a cytotoxic agent against leukemia Cancer Cells, was discovered through a special US screening program for anti-carcinogenic substances, which tested approximately 110,000 compounds from 35,000 plant species samples.

Pyrethrins found in the flowers of pyrethrums, chrysanthemums, and tansy, along with rotenone from legume roots, are used as environmentally friendly

insecticides. Most spices, condiments, and beverages such as tea, coffee, and cocoa owe their distinct taste and aroma to the SMs present in the source plants.

For Structure/179.html">Practical Applications, SMs are extracted from natural raw Materials or produced via Chemical synthesis of the target compounds or their analogs (which are often more active than natural SMs). However, the concentration of valuable SMs in plants is frequently extremely low, while their chemical synthesis remains expensive or overly complex. For instance, meeting the annual market demand for taxol would require stripping the bark of several hundred thousand mature yew trees. Natural populations of many SM-producing plants are scarce, cultivation is often difficult (as in ginseng), and producer plants frequently exhibit slow growth rates and long life cycles (such as trees). In some cases, growing these producers is hindered by unfavorable climatic conditions or a lack of sufficient arable land. Consequently, producing SMs via plant cell biotechnology using in vitro cultures appears to be economically advantageous. Furthermore, these technologies offer the potential to generate novel SMs through biotransformation—the chemical conversion of SMs during cell culture via Hydrolysis, oxidation, reduction, hydroxylation, or hydrogenation.

4.2. Features of Secondary Metabolite Biosynthesis in Plant Cell Cultures

The biosynthesis of SMs in in vitro plant cell cultures differs in several ways from that in intact plants. In a whole plant, SM synthesis, transport, and storage are tightly regulated by the Organism as a whole, guided by its developmental program. Specialized cells, Tissues, and Organs exist where SMs accumulate. A critical prerequisite for establishing a PLANT CELL CULTURE is dedifferentiation, during which specialized explant cells may lose traits such as the ability to synthesize SMs. Even when this capacity is retained, SM biosynthesis in cell culture is generally less intense than in intact plants, and THE SPECTRUM OF synthesized SMs is significantly simpler.

A characteristic feature of cultured cells is the temporal Separation between Cell Division (which increases biomass) and SM synthesis. SM accumulation typically rises during the deceleration phase of cell population growth (at the end of the Cell Cycle) and peaks in the stationary phase. Although certain alkaloids are actively synthesized during maximum mitotic activity—that is, during exponential growth—this is an exception to the rule.

A positive correlation exists between SM accumulation and the degree of cellular differentiation in culture. Studies have clearly demonstrated that compact, slow-growing cultures contain higher levels of target SMs than loose, rapidly proliferating cultures. Presumably, the mechanisms (genetic and epigenetic) and conditions that slow down cell growth and proliferation simultaneously activate Secondary METABOLISM Enzymes. Consequently, commercial production of certain SMs via in vitro cell culture often relies on slower-growing callus cultures rather than cell Suspensions, which otherwise allow for rapid biomass accumulation. Nevertheless, large-scale industrial production of SMs via plant cell culture typically focuses on cell suspensions. Suspension cultures represent a more homogeneous, less differentiated cell population that grows much faster than the initial callus culture. Crucially, cell suspensions are suitable for continuous cultivation in large-scale bioreactors, which is essential for reducing production costs.

Despite the efforts of many biotechnology companies, some SMs have yet to be successfully produced in either callus or suspension cell cultures. This applies to high-value compounds such as the alkaloids of the opium poppy Papaver somniferum, tropane alkaloids from various Solanaceae species, and dimeric indole alkaloids from the Madagascar periwinkle Catharanthus roseus. Their biosynthesis in culture begins only after plant organ regeneration from callus or within organ-specific cell suspensions. Therefore, in vitro organ cultures (such as SHOOT or ROOT cultures) are utilized in such cases.

Shoot cultures are established by regenerating multiple shoots from callus or directly from explants containing apical Meristems. Cultivation is carried out on solidified or liquid nutrient media. In some cases, growing cultures under light helps enhance the yield of the final product. Shoot cultures are applied when target SMs are produced in the aerial organs of plants. For instance, monoterpene Essential Oils cannot be synthesized in undifferentiated callus or suspension cultures because these lack oil-secreting tissues, whereas shoot cultures accumulate them quite successfully. Prominent examples include the successful synthesis of artemisinin, an antimalarial compound from Artemisia annua, in shoot cultures. Similarly, anhydrovinblastine—a dimeric indole alkaloid serving as a precursor to anti-leukemic periwinkle alkaloids—accumulates in in vitro shoot cultures at levels comparable to those found in the leaves of intact plants. Another variation is the embryogenic culture, which consists of a cell suspension capable of forming somatic embryos at high frequencies under specific culture conditions.

Because many plants naturally accumulate SMs in their roots, utilizing in vitro root cultures to increase yield is the most straightforward and natural approach. There are two MAIN TYPES OF root cultures: normal root cultures and hairy root cultures (Fig. 4.1). The latter are obtained through plant cell transformation mediated by the bacterium Agrobacterium rhizogenes (The Mechanism of agrobacterial transformation is discussed in Chapter 9). Hairy root cultures are generally more efficient: they grow significantly faster than normal root cultures and do not require exogenous synthetic growth regulators in the nutrient medium because transformed cells synthesize them autonomously (whereas normal root cultures require Auxins). Consequently, the nutrient media for hairy root cultures are simpler, cheaper, and do not require optimization of growth regulator composition and concentrations. Avoiding synthetic growth regulators simplifies the regulatory approval process for pharmaceutical SM products. The primary challenge in utilizing hairy root cultures for SM production lies in the difficulty of transforming certain key SM-producing species with A. rhizogenes. Nevertheless, root cultures have proven highly effective for yielding valuable compounds such as tropane alkaloids from solanaceous plants and ginseng ginsenosides, among others.

Class="center">Fig. 4.1. Hairy root culture grown on solid nutrient medium (a) and in a bioreactor (b)

In intact plants, the biosynthesis of many SMs requires the coordinated action of both roots and aerial organs. For example, a precursor may be synthesized in the roots and subsequently transported to the stems or leaves, where it is converted into the final product. In such cases, single-organ in vitro cultures are ineffective. Solving this problem requires the co-cultivation of both root and shoot tissues from the same or different species. Under this setup, the precursor can be secreted into the nutrient medium and taken up by the other organ, where enzymatic conversion to the final product takes place. Mechanical wounding of the precursor-synthesizing organ can be applied to enhance this process. A successful example of this approach is the co-cultivation of hairy root and shoot cultures of belladonna (Atropa belladonna) or hybrid corkwood (Duboisia). Belladonna roots synthesize hyoscyamine, which requires the enzyme hyoscyamine 6$eta$-hydroxylase (H6H) for conversion into the valuable alkaloid scopolamine. Pure hairy root cultures of belladonna produce virtually no scopolamine due to low H6H levels, and pure shoot cultures fail to produce it as well. Only through the co-cultivation of root and shoot cultures has substantial scopolamine biosynthesis been successfully achieved.

The absence or insufficient activity of specific biosynthetic enzymes is often the reason why many SMs fail to be produced—or are produced in extremely low amounts—in plant cell cultures. However, supplementing the nutrient medium with metabolic intermediates can facilitate their conversion into the final product via endogenous cellular enzymes. This phenomenon of chemical transformation of exogenous substances by cultured plant cells or their extracts is known as "biotransformation." The substrate for biotransformation can be natural or synthetic, and the resulting product may either match compounds known from intact plants or represent a novel molecule specific to cultured cells. Biotransformation can be mediated by a single enzyme or a multi-enzyme cascade. As a rule, single-step biotransformation is more efficient, whereas the final product yield decreases as the number of sequential cellular enzymes involved in the process increases.

Several successful cases of SM production via plant cell biotransformation have been described in the literature, with production costs comparable to or lower than those of chemical synthesis. For instance, the biosynthesis of arbutin (used as a Skin-lightening agent) was achieved in cell cultures of Madagascar periwinkle (Catharanthus roseus) via the biotransformation of an inexpensive precursor, hydroquinone. Periwinkle cell cultures are also employed to produce valuable anti-cancer agents vinblastine and vincristine through the biotransformation of their precursor vindoline. Furthermore, cultured foxglove cells (Digitalis lanata and D. purpurea) can accumulate substantial amounts of the cardiac glycosides digoxin and/or digitoxin upon progesterone supplementation and, most importantly, can synthesize the medically valuable digoxin directly from digitoxin.

4.3. Selection and Preservation of Secondary Metabolite-Producing Cell Lines

The selection of high-yielding SM-producing cell lines begins with identifying plant species with the highest natural content of the target compound. Next, superior genotypes within that species are selected as an explant source to establish callus cultures, which are subsequently used to initiate suspension cultures. Finally, during suspension cultivation, the most productive and stable cell clones (lines) are isolated.

A characteristic feature of in vitro cultured cell populations is their heterogeneity (see Chapter 3). Significant cell-to-cell variations also exist regarding SM biosynthesis. For example, only about 10% of cultured cells of Catharanthus roseus are capable of actively accumulating anthocyanins. This indicates that isolating individual cell aggregates allows for the selection of stable lines with enhanced target SM production. The selection of productive lines is simplest when SM levels can be assessed visually—for instance, based on cell coloration intensity corresponding to pigment accumulation. In such cases, selection can be performed directly at the callus culture stage by sequentially subculturing and isolating the most intensely colored sectors. When visual selection is unfeasible, standard histochemical and quantitative Analytical Methods are applied to squash preparations of individual cell lines.

The efficiency of selecting productive cell lines can be enhanced using mutagens and selection on selective media (methods discussed in Chapter 6). Introduction/32.html">Genetic Engineering holds great promise for these purposes as well. The yield of SMs in cell culture can be substantially improved if the genetic and biochemical pathways of their biosynthesis are understood. This allows researchers to identify rate-limiting bottlenecks and selectively target individual pathway steps. Currently, the biosynthetic pathways for several valuable SMs have been elucidated, and individual genes encoding Key Enzymes have been cloned, enabling direct modulation of metabolic flux through genetic modification. Agrobacterial transformation using A. rhizogenes is particularly convenient here, as it simultaneously fulfills two objectives: introducing specific biosynthetic genes to enhance SM production and generating hairy root cultures, which further boost SM yields.

The selection of both productive and stable cell clones, along with the optimization of cultivation conditions, generally ensures a reliable level of secondary metabolite (SM) production over a reasonably long period. However, due to the inherent cytogenetic instability of cell cultures, a gradual decline in cell line productivity is a common occurrence. Consequently, The Challenge of preserving these lines arises, which can be addressed at various levels. First, it is essential to preserve the donor plants that served as explants for cell culture initiation, allowing the selection process for productive cell lines to be replicated using newly established cultures. To maintain such plants, it is convenient to employ in vitro plant genetic resource management methods, which will be discussed in the next chapter. Second, long-term preservation Methods for the most productive cell lines are available via cryopreservation in liquid nitrogen. It should also be noted that plant organ cultures, unlike callus and suspension cultures, exhibit significantly higher genotypic and phenotypic stability and are easier to regenerate from the explant donor plant. Therefore, whenever feasible, such cultures should be given preference.

4.4. Approaches to Enhancing Secondary Metabolite Yield in Plant Cell Cultures

Current approaches to increasing in vitro SM yields through the physiological regulation of plant cell metabolism include optimizing nutrient media (composition and concentrations of growth regulators, minerals, vitamins, sugars, and pH), lighting regimes, aeration (the CO₂/O₂ ratio), Temperature, and the choice of bioreactor type and dimensions. Optimal cultivation conditions can vary significantly depending on the specific case (plant species and SM) and therefore must be tailored individually.

Elicitation is a highly effective strategy for stimulating SM synthesis in cultured plant cells. In the context of plant cell culture production, elicitors are substances or other factors capable of inducing the biosynthesis of SMs. The application of elicitors is based on the fact that many SMs function as plant defense chemicals against biotic or environmental stresses. Accordingly, elicitors of microbial or plant origin (such as extracts from bacterial cells, Yeast, plants, Chitin, and chitosan) are classified as biotic elicitors, whereas physical (e.g., UV irradiation, heat or cold Shock) and chemical (e.g., heavy metals, Ca²⁺ ions) stressors are defined as abiotic elicitors. Elicitors have been shown not only to induce phytoalexin production but also to activate the biosynthetic potential for various constitutive metabolites in cultured plant cells. Their effectiveness can be remarkable; for instance, the yield of the sesquiterpene gossypol in tree cotton (Gossypium arboreum) cell culture was increased 100-fold using an elicitor derived from Verticillium dahliae. Similarly, adding elicitors to the nutrient medium of opium poppy (Papaver somniferum) cell cultures enhanced the biosynthesis of the alkaloid sanguinarine by 27-fold.

Investigations into plant Immunity mechanisms revealed that the exogenous signal Transduction system—which triggers cellular defense responses alongside reactive oxygen species, Ca²⁺ ions, and phosphoinositides—relies heavily on jasmonic acid. It was discovered that elicitor-induced biosynthesis of SMs in plant cell cultures is accompanied by a rapid, transient accumulation of jasmonic acid and its methyl ester, methyl jasmonate (H. Gundlach et al., 1992). This finding made it possible to use these compounds directly, rather than the elicitors themselves, to induce SM biosynthesis in cultured plant cells. Their high efficacy has been demonstrated in boosting SM yields across a wide range of plant species.

Research involving plant organ cultures has demonstrated that normal cellular metabolism requires tissue Organization. To enhance the organization of cultured plant cells in vitro and promote their differentiation, various cell immobilization techniques—namely, fixing cells in or on a carrier—are of considerable interest for increasing SM yields. Several methods exist for immobilizing plant cells:

✵ immobilization in an inert matrix: entrapment of cells within alginate, Agar, Collagen, or polyacrylamide;

✵ adsorption of cells onto an inert substrate: cells adhere to charged beads made of alginate, polystyrene, or polyacrylamide. Cultivating cells within a nylon mesh, polyurethane foam matrix, or hollow fibers (Fig. 4.2) also exemplifies this approach;

✵ covalent binding of cells to an inert carrier such as carboxymethyl Cellulose.

The most widely adopted methods for immobilizing plant cells involve embedding them within gel beads composed of naturally occurring Polysaccharides, such as gelatin, pectin, chitosan, agarose, calcium alginate, and related substances.

Fig. 4.2. Example of the immobilization of a madder (Rubia tinctorum L.) suspension culture on an inert substrate. Natural fibrous materials served as the substrate: (a) loofah fibers; (b) sisal fibers; (c) jute fibers. According to the authors, all three substrates had no adverse effect on biomass accumulation in the suspension culture or the in vitro production of the secondary metabolites alizarin and purpurin (after P. Nartop et al., 2013).

Cell immobilization substantially alters cellular metabolism. Immobilized cells exhibit a prolonged stationary growth phase. As growth processes slow down, primary metabolites become increasingly channeled into secondary metabolic pathways. Consequently, immobilized culture systems maintain stable SM biosynthesis over extended periods. It is believed that the immobilization process triggers a stress response in plant cells, leading to enhanced SM synthesis. The microenvironment within the matrix itself can act as a stress factor. Furthermore, polysaccharides (such as pectin, chitosan, and alginate) used as immobilization matrices can function as elicitors. By shielding maturing cells from external stressors, these matrices also help prevent apoptosis and the concurrent release of

proteases into the culture medium by dying cells. The accumulation of proteases typically leads to the degradation and loss of a portion of the SMs secreted by cells into the medium. Immobilizing cell cultures within a matrix helps preserve cell viability and maximizes the ultimate SM yield (Fig. 4.3).

Fig. 4.3. Patterns of intracellular SM accumulation and release into the nutrient medium in conventional suspension cultures versus immobilized cultures, specifically utilizing cell encapsulation within specialized polymer materials.

It should be noted, however, that the efficiency of cell immobilization is largely determined by the cells' ability to secrete the target product into the nutrient medium. While immobilization can enhance excretion, this trait is not characteristic of most plant cells, unlike microorganisms.

4.5. Industrial Production of Secondary Metabolites in PLANT CELL AND Organ Cultures

Industrial-scale production of SMs using cell cultures differs substantially from laboratory-scale Procedures. Most importantly, industrial manufacturing relies on bioreactors that not only exceed laboratory vessels in scale but also incorporate specialized mechanisms to ensure a continuous technological cycle, uniform cell distribution, proper aeration, and precise control over temperature, nutrient media composition, pH, and other parameters capable of influencing final product yield. Although A wide variety of bioreactors have been engineered and manufactured for the microbiological industry, only a fraction of them are suitable for cultivating Different types of plant cells.

Plant suspension cells (40–200 µm) are tens to hundreds of times larger than bacterial and fungal cells, and their dimensions change throughout ontogeny. While small and dense at the beginning of the exponential growth phase, they enlarge significantly and become heavily vacuolated in the stationary phase. As cells grow larger, the risk of mechanical damage during agitation increases markedly, with stem and root cultures being particularly vulnerable. Moreover, plant cells are denser than microbial cells and require efficient mixing. Sedimentation leads to The formation of "dead zones" within vessels, where rapid cell crowding and senescence occur.

Unlike microorganisms, which rarely form aggregates, plant cell suspensions typically consist of aggregates of varying sizes, considerably increasing culture density and mixing power requirements. Furthermore, Microbial growth rates are vastly higher (with doubling times measured in hours) compared to plant cells (doubling times measured in days). While microbial Fermentation can be initiated using heavily diluted inocula, plant cell culture requires an inoculum density of at least 5–10%. In vitro plant cells also exhibit a lower Respiration rate than microorganisms, meaning their aeration systems may differ significantly. Finally, plant cell cultures require far more complex and costly nutrient media than microbial cultures.

Two primary modes of cell cultivation are distinguished: closed and open systems. Closed systems operate in a batch (cumulative) mode. An inoculum of cells is placed into a defined volume of medium, and the bioreactor system remains entirely closed—with the exception of gas exchange—until the cultivation cycle is complete. In its simplest form, a conventional flask serves as a closed bioreactor. Open (continuous) plant cell cultivation systems involve the continuous supply of fresh nutrient medium alongside the harvest of both spent medium and a portion of The Cell biomass. Nutrient medium is fed progressively while old medium is removed, allowing cells to remain within the system throughout the entire production cycle.

Batch (cumulative) cultivation is the simplest and most traditional method for growing cells, employed primarily in laboratory settings. This approach relies on orbital shakers, roller bottles, or magnetic stirrers to aerate and agitate the cultures (Fig. 4.4, e). When scaling up from small flask cultures to multi-liter bioreactors, aeration and mixing are most commonly achieved using various impellers (propellers) rotating inside the vessels (Fig. 4.4, a). Drum-type bioreactors (cylindrical vats that slowly rotate around a horizontal axis) (Fig. 4.4, b) and pneumatic Column bioreactors—such as airlift systems, where mixing and aeration are driven by rising air bubbles (Fig. 4.4, c)—are also utilized for this purpose.

Fig. 4.4. Bioreactor types (a–d) for plant cell and organ cultures and examples of various bioreactors (e–h): (a) mechanically agitated bioreactors utilizing various impellers; (b) drum-type bioreactors; (c) pneumatically agitated (airlift) bioreactors; (d) single-use hydrodynamic mixing bioreactors; (e) a basic bioreactor setup consisting of suspension culture flasks on a programmable orbital shaker; (f and g) laboratory-scale automated Glass bioreactors; (h) an automated, programmable bioreactor system housed at the National Institute for Bioprocessing Research and Training (NIBRT) in Dublin, Ireland.

(after O. Yesil-Celiktas et al., 2010)

Culture vats are most commonly made of stainless steel. While durable, they are also quite expensive (Fig. 4.4, h). Consequently, The Use of inexpensive, single-use bioreactors made of flexible plastic holds the greatest promise. In these systems, culture mixing and aeration can be achieved hydrodynamically (Fig. 4.4, g). For embryogenic and shoot cultures, glass bioreactors can be employed, as cultivation requires external illumination from lamps positioned outside the vessel (Fig. 4.4, e, f). Alternatively, cultures can be illuminated from within the bioreactor using tubular or hollow-fiber lamps.

While bioreactors designed for microorganism cultivation are generally suitable for cell suspension cultures and embryogenic cultures, root cultures require substantial modification of such systems. This is due to the significant mechanical damage inflicted on roots by culture agitation. Therefore, bioreactors for root cultivation utilize various baskets, mesh partitions, and other devices to immobilize root cultures and prevent their contact with impellers. A promising approach involves cultivating roots in bioreactors equipped with "beds" (substrates supporting the root cultures) that are moistened with nutrient media via micro-irrigation devices or misting systems.

Attempts at large-scale plant cell cultivation date back to the late 1950s. As early as 1960, the feasibility of growing tobacco biomass in stainless steel vats was demonstrated. In 1977 in Japan, biomass of the tobacco plant Nicotiana tabacum was cultivated in 20 m³ bioreactors equipped with a mechanical agitation system. Currently, the volume of bioreactors used for growing tobacco biomass has reached 75 m³. This biomass is utilized to manufacture low-nicotine cigarettes.

In 1974, M. Tabata and coworkers demonstrated for the first time that shikonin derivatives could be synthesized in callus cultures of the gromwell plant Lithospermum erythrorhizon. Later, through targeted selection, a cell line was isolated that accumulated shikonin at levels 20 times higher than the initial culture, reaching 1.2 mg/g of fresh cell mass. Intensive research aimed at optimizing industrial cultivation conditions for this line culminated in the commercial production of shikonin by the Japanese company Mitsui Petrochemical Industry in 1983. Shikonin is a red naphthoquinone pigment accumulated in the root bark of many plants belonging to the Boraginaceae family. It exhibits antimicrobial, anti-inflammatory, wound-healing, and antitumor activities.

In Japan in 1988, commercial production of ginseng biomass began, which is used predominantly in the cosmetics industry

and as a dietary Supplement. Currently, cell cultures of about one hundred plant species are utilized across various countries in the biosynthetic industry to obtain substances of pharmacological importance. These include, for instance, various cardiac glycosides (cardenolides); anticancer agents such as paclitaxel (taxol), podophyllotoxin, vincristine, vinblastine, and camptothecin; secondary metabolites (SMs) with antibacterial and anti-inflammatory properties, such as protoberberines, shikonin, rosmarinic acid, and Echinacea polysaccharides (the latter also possessing immunostimulatory activity). Geraniol serves as a valuable source of Essential Fatty acids. Scopolamine exhibits pronounced anticholinergic activity and is used as a sedative and antiemetic agent (it is a component of motion sickness pills such as "Aeron"). A number of SMs are used as pigments in cosmetics (arbutin, carthamin, shikonin) and in the food industry (betacyanin, various anthocyanins), as well as Dyes in the textile industry (anthocyanin from Euphorbia). The currently existing avenues of industrial production of SMs using plant cell culture are illustrated in Fig. 4.5.

Fig. 4.5. Avenues of industrial production of secondary metabolites using plant cell culture

However, truly large-scale production of SMs via plant cell culture methods can only be discussed with respect to a small number of products: shikonin, taxol, berberine, and ginseng biomass (with companies in Japan and the USA acting as leaders). The primary limiting factor for the economically viable production of any substances using plant cell cultures is their insufficiently high productivity. Research conducted in many countries aimed at enhancing the efficiency of SM production through plant cell cultures gives grounds to expect significant progress in this field. Particularly great Prospects are associated with elucidating the biosynthetic pathways of SMs and applying genetic engineering approaches for targeted interventions at specific stages of SM biosynthesis.

Conclusion

Higher plants possess the ability to synthesize a diverse array of biologically active compounds whose presence in the plant cell is not strictly essential for maintaining life—namely, secondary metabolites (SMs). Many SMs are of considerable interest for pharmacology, cosmetics, agriculture, and the food and textile industries.

The biosynthesis of SMs in in vitro plant cell cultures exhibits distinct features compared to intact plants. In the plant organism, SM synthesis, transport, and storage are under the strict control of the whole organism and its programmed development. There are specialized cells, tissues, and organs in which SMs accumulate. A crucial prerequisite for establishing a plant cell culture is dedifferentiation, As a result of which specialized explant cells may lose traits such as the capacity for SM synthesis. When this capacity is retained, SM biosynthesis in cell culture generally occurs less intensively than in the intact plant, and the spectrum of synthesized SMs is significantly narrower.

A distinctive feature of cultured cells is that cell divisions leading to an increase in biomass and the synthesis of SMs are temporally uncoupled. SM accumulation increases during the deceleration phase of the cell population growth (at the end of the cell cycle) and reaches a maximum in the stationary phase. Certain alkaloids are actively synthesized during the phase of maximum mitotic activity—that is, during exponential growth—but this is an exception to the rule.

Cell suspension cultures represent the most homogeneous and least differentiated cell population, growing much faster than the initial callus culture. Cell suspensions are well-suited for continuous cultivation in large-volume bioreactors, which is a prerequisite for reducing production costs. However, some SMs have not yet been successfully produced in either callus or cell suspension cultures, despite prolonged and concentrated efforts by numerous biotechnology companies. Their synthesis in cell culture commences only following regeneration from callus or within a suspension of cells corresponding to the specific plant organ (secondary differentiation). Therefore, in such cases, in vitro organ cultures (shoots or roots) or embryogenic cultures are utilized.

Hairy root cultures, obtained by transforming plant cells with Agrobacterium rhizogenes, are the most efficient. They grow significantly faster than conventional root cultures and do not require The addition of synthetic growth regulators to the nutrient medium, as transformed cells are capable of synthesizing these compounds themselves. The nutrient medium for hairy root cultures is simpler and cheaper, requiring no optimization of composition or growth regulator concentrations.

A characteristic feature of in vitro cultured cell populations is their heterogeneity. Significant differences among cells also exist regarding SM biosynthesis parameters. This means that by cultivating individual cell aggregates, one can select strains capable of stably producing elevated yields of the target SM. The efficiency of selecting productive cell lines can be enhanced using mutagens and selection on selective media. The application of Genetic engineering METHODS holds great promise for these purposes. Agrobacterial transformation using A. rhizogenes is particularly convenient, as it simultaneously addresses two objectives: transferring individual enzyme genes that enhance SM biosynthesis and generating hairy root cultures, which also serve to increase SM production.

Current approaches to increasing SM yields in plant cell cultures through the physiological Regulation of cellular metabolism include optimizing nutrient media (composition and concentrations of growth regulators, mineral nutrients, vitamins, sugars, pH), lighting regimes, aeration (the CO₂/O₂ ratio), temperature, and selecting the appropriate bioreactor type and size.

An effective strategy for stimulating SM synthesis in cultured plant cells is elicitation. In the context of SM production via plant cell culture, elicitors are substances or other factors capable of inducing SM biosynthesis in plant cell cultures. The use of elicitors is based on the fact that many SMs function as plant defense responses to biotic or abiotic stresses. Accordingly, elicitors of microbial or plant origin (e.g., extracts of bacterial cells, yeast, plants, chitin, chitosan) are classified as biotic elicitors, whereas physical (e.g., UV irradiation, heat or cold shock) and chemical (e.g., heavy metals, Ca²⁺ ions) stress factors are defined as abiotic elicitors. In many cases, rather than using the elicitors themselves, it proves effective to induce SM biosynthesis in cultured plant cells by applying compounds involved in the intracellular signal transduction pathway—namely, jasmonic acid and methyl jasmonate. The use of biotechnologies based on cell immobilization also holds significant promise.

A wide variety of bioreactors have been developed and manufactured for the microbiological industry. However, far from all of them are suitable for various types of plant cell cultures. Plant cells in suspension are tens to hundreds of times larger than bacterial and fungal cells, correspondingly increasing the risk of mechanical damage during agitation. Shoot and root cultures are particularly susceptible to mechanical damage. Plant cells are heavier than microbial cells and require efficient agitation. Plant cell suspension cultures largely consist of aggregates of varying sizes, which significantly increases culture density and agitation costs. Compared to microorganisms, in vitro plant cells exhibit a lower respiration rate; consequently, their aeration systems may differ substantially from those used for microorganisms. Effective mixing and aeration are achieved in plant cell bioreactors equipped with impellers (mechanical agitation), as well as in drum-type bioreactors and pneumatic column bioreactors.

To prevent mechanical damage in bioreactors designed for root cultivation, various baskets, mesh partitions, and other devices are employed to immobilize root cultures and prevent their contact with impellers. A promising approach involves cultivating roots in bioreactors equipped with "beds" moistened with nutrient media via micro-irrigation devices or misting systems.

Currently, cell cultures of approximately one hundred plant species are utilized in the biosynthetic industry across various countries to obtain pharmacologically important substances. These include, for example, various cardiac glycosides (cardenolides); anticancer agents such as paclitaxel (taxol), podophyllotoxin, vincristine, vinblastine, and camptothecin; and SMs possessing antibacterial and anti-inflammatory properties, such as protoberberines, shikonin, rosmarinic acid, and Echinacea polysaccharides (the latter also exhibiting immunostimulatory activity). A number of SMs are used as pigments in cosmetics (arbutin, carthamin, shikonin) and in the food industry (betacyanin, various anthocyanins), as well as dyes in the textile industry (anthocyanin from Euphorbia). However, truly large-scale production of SMs using plant cell culture methods can only be discussed with respect to a relatively small number of products: shikonin, taxol, berberine, and ginseng biomass.

Review Questions

1. How do plant secondary metabolites differ from primary metabolites?

2. In which cases is it more advantageous to produce SMs via cell culture rather than using natural plant raw materials?

3. What are the differences in the biosynthesis of secondary metabolites in cell culture versus an intact plant?

4. Why do organ cultures accumulate secondary metabolites more efficiently compared to suspension or callus cultures?

5. What are the advantages of hairy root culture over cell suspension culture and conventional root culture?

6. How is the selection of high-yielding plant cell lines carried out?

7. What methods are used to preserve productive plant cell lines?

8. What is elicitation in plant cell cultures? Which elicitors are used to increase the yield of secondary metabolites in cell culture?

9. How are immobilized plant cell cultures obtained?

10. What are the differences between the industrial CULTIVATION OF MICROORGANISMS and that of plant cells?

11. What bioreactors are suitable for growing plant cell suspension cultures and root cultures?

12. In what year was large-scale industrial production of secondary metabolites using plant cell cultures initiated? What substance was it?

13. Name the main secondary metabolites produced on an industrial scale.