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Abstract Studies on plant secondary metabolites have been increasing over the last 50 years. These molecules are known to play a major role in the adaptation of plants to their environment, but also represent an important source of active pharmaceuticals. Plant cell culture technologies were introduced at the end of the 1960s as a possible tool for both studying and producing plant secondary metabolites. Different strategies, using in vitro systems, have been extensively studied with the objective of improving the production of secondary plant compounds. Undifferentiated cell cultures have been mainly studied, but a large interest has also been shown in hairy roots and other organ cultures. Specic processes have been designed to meet the requirements of plant cell and organ cultures in bioreactors. Despite all of these efforts of the last 30 years, plant biotechnologies have led to very few commercial successes for the production of valuable secondary compounds. Compared to other biotechnological elds such as microorganisms or mammalian cell cultures, this can be explained by a lack of basic knowledge about biosynthetic pathways, or insufciently adapted reactor facilities. More recently, the emergence of recombinant DNA technology has opened a new eld with the possibility of directly modifying the expression of genes related to biosyntheses. It is now possible to manipulate the pathways that lead to secondary plant compounds. Many research projects are now currently being carried out and should give a promising future for plant metabolic engineering. 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Secondary metabolite; Metabolic engineering; Plant; Cell; Tissue; Culture; Hairy root; Shoot; Bioreactor
1. Introduction Two hundred years of modern chemistry and biology have described the role of primary metabolites in basic life functions such as cell division and growth, respiration, storage, and reproduction. In biology, the concept of secondary metabolite can be attributed to Kossel [1]. He was the rst to dene these metabolites as opposed to primary ones. Thirty years later an important step forward was made by Czapek [2] who dedicated an entire volume of his plant biochemistry series to what he named endproduckt. According to him, these products could well derive from nitrogen metabolism by what he called secondary modications such as deamination. Compared to the main molecules found in plants, these secondary metabolites were soon dened
* Corresponding author. E -mail address: bourgaud@ensaia.inpl-nancy.fr (F. Bourgaud).
by their low abundance, often less than 1% of the total carbon, or a storage usually occurring in dedicated cells or organs. In the middle of the 20th century, improvement of analytical techniques such as chromatography allowed the recovery of more and more of these molecules, and this was the basis for the establishment of the discipline of phytochemistry. Paper chromatography obviously revealed that some of these molecules were pigments, however, other possible implications of such secondary compounds in plant life were still largely mysterious as stated by Czapeks term end products. Thanks to the improvement of biochemical techniques and the rise of molecular biology, it has been clearly demonstrated that secondary products play a major role in the adaptation of plants to their environment. These molecules largely contribute to plant tness by interacting with the ecosystems. They have been described as being antibiotic, antifungal and an-
0168-9452/01/$ - see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 1 ) 0 0 4 9 0 - 3
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tiviral, and therefore able to protect plants from pathogens (phytoalexins), and also anti-germinative or toxic for other plants (allelopathy). Besides, they constitute important UV absorbing compounds, thus preventing serious leaf damage from the light [3]. They also act on animals, such as insects (anti-feeding properties) or even cattle for which forage grasses such as clover or alfalfa can express estrogenic properties and interact with fertility (see [4,5] for reviews on the chemical ecology of secondary compounds). Plant secondary compounds are usually classied according to their biosynthetic pathways [5]. Three large molecule families are generally considered: phenolics, terpenes and steroids, and alkaloids. A good example of a widespread metabolite family is given by phenolics: because these molecules are involved in lignin synthesis, they are common to all higher plants. However, other compounds such as alkaloids are sparsely distributed in the plant kingdom and are much more specic to dened plant genus and species. This narrower distribution of secondary compounds constitutes the basis for chemotaxonomy and chemical ecology. Due to their large biological activities, plant secondary metabolites have been used for centuries in traditional medicine. Nowadays, they correspond to valuable compounds such as pharmaceutics, cosmetics, ne chemicals, or more recently nutraceutics. Recent surveys have established that in western countries, where chemistry is the backbone of the pharmaceutical industry, 25% of the molecules used are of natural plant origin [6]. A good example could be aspirin (acetylsalicylate) which derives from salicylate. The genuine molecule can be isolated in large quantities from many plants (Spiraea ulmaria, Betula lenta ), but the chemical is synthesized as an acetyl-derivative in order to lower secondary effects (stomachaches). Chemical synthesis apart, the production of plant secondary metabolites has, for a long time, been achieved through the eld cultivation of medicinal plants. However, plants originating from particular biotopes can be hard to grow outside their local ecosystems. It also happens that common plants do not withstand large eld cultures due to pathogen sensitiveness (anthracnose on Hypericum perforatum or Arnica montana ). This has led scientists and biotechnologists to consider plant cell, tissue and organ cultures as an alternative way to produce the corresponding secondary metabolites.
time that undifferentiated cells, such as callus or cell suspension cultures were not able to produce secondary compounds, unlike differentiated cells or specialized organs [7]. Zenk and co-workers experimentally demonstrated that this theory was wrong, as they could observe dedifferentiated cell culture of Morinda citrifolia yielding 2.5 g of anthraquinones per liter of medium [8]. This nding opened the door to a large community of vitro culturists who extensively studied the possible use of plant cultures for the production of secondary compounds of industrial interest (mainly pharmaceutics and dyes).
2. Plant cell cultures Evidence that plant cell cultures are able to produce secondary metabolites came quite late in the history of in vitro techniques. It had been considered for a long
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Fig. 1. Guidelines for the production of secondary metabolites from plant cell.
erratic production of secondary metabolites with dedifferentiated cell cultures has been carried out with nonstabilized cell lines. This argument is generally poorly discussed in the literature (with the exception of specialized work on gene silencing [12]) because markers are missing to evaluate the state of genetic stability of a culture. In a recent work conducted in our laboratory, it was decided that a cell line could not be regarded as stabilized until growth parameters could be repeated during three consecutive subculture cycles in stable culture conditions. It is clear that this condition is necessary but not sufcient to conclude that there is genetic stability. However, because growth is a strong polygenic character it was considered as being better than nothing to evaluate the ending of erratic phenomena. Therefore, we closely monitored growth parameters such as length of lag, of log phases, and growth speed during the log phase, for each of the 217 different callus lines obtained from Psoralea plants (Legumi nosae ) and capable of producing isoavones [13]. Results showed that an increasing number of the 217 callus lines had stable growth parameters over time. Moreover, once considered as stable a cell line never went back to erratic growth parameters which strengthened the validity of our hypothesis. In our experiments 90% of the collection was growth stabilized after 16 subculture cycles (48 weeks). Very few authors indicate the time necessary to stabilize cell cultures. However, the period we determined is rather short compared to the 2 years reported by Fett Neto et al. [14].
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Table 1 Effect of environmental conditions and elicitor treatments on furocoumarin content in higher plants Environmental factor or elicitor Plant diseases Ceratocystis mbriata Sclerotinia sclerotiorum Unknown Erwinia caroto6ora Rhodotula rubra Phoma companata Pseudomonas cichorii Insect damages Effect of light UV UV UV Air quality Acidic fog Ozone Temperatures Cold (15 C, control 26 C) Hot (32 C, 21 C) control 21 C) Chemicals CuSO4 CuSO4 NaCl NaCl H2SO4 Ca(OCl)2 Plant species Effect on furocoumarin content Authors
Pastinaca sati6a (root apex) Apium gra6eolens (stalks) Daucus carota (roots) Apium gra6eolens (stalks) Ruta gra6eolens (hydroponic) Pastinaca sati6a (leaves) Glehnia littoralis (roots) Pastinaca sati6a (leaves) Apium gra6eolens (stalks) Ruta gra6eolens (leaves) Glehnia littoralis (roots) Apium gra6eolens (leaves) Petroselinum crispum (leaves) Apium gra6eolens (leaves) Psoralea cinerea (leaves) Apium gra6eolens (leaves) Psoralea cinerea (fruits) Ruta gra6eolens (leaves) Psoralea cinerea (fruits) Ruta gra6eolens (leaves) Psoralea cinerea (fruits)
20 (8-MOP) 235 (Psoralen) 24 (Total furocoumarins) 77 (8-MOP) 24 (Total furocoumarins) No modication 5 (Total furocoumarins) 9 (psoralen) 2.2 (8-MOP) 1.8 (Psoralen) 3.4 (Linear furocoumarin) 2.510 (Total furocoumarin) 2 (Psoralen)
[28] [29]
[26] [30]
2.2 (Linear furocoumarins) 2.8 (Psoralen) Decrease but higher percentage on leaf surface 2 (Psoralen) Decrease but higher percentage on leaf surface 1.5 (Psoralen)
Different molecules from this family were investigated. Linear furocoumarins refer to a mixture of various molecules derived from psoralen. 8-MOP is for 8-methoxypsoralen (also called xanthotoxin).
Cell suspensions constitute a good biological material for studying biosynthetic pathways. Indeed, compared to callus cultures they allow the recovery of large quantities of cells from which enzymes can be more easily isolated [16]. These biosynthetic studies enable
the researcher to spot limiting enzyme activities (or genes) in the production of valuable metabolites. These bottleneck steps can sometimes be overlapped by feeding the cell cultures with a precursor metabolite that corresponds to the product of a limiting enzymatic
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activity although there is always the risk of activating a feedback inhibition somewhere else on the pathway [17]. Many traditional strategies can be used to increase the production of secondary metabolites but elicitation is usually one of the most successful. This consists in applying chemical or physical stresses to the cell suspension cultures that will trigger the production of secondary metabolites that are normally not produced. This is currently done with biotic elicitors (chitosan, autoclaved mycelium of pathogenic fungi, various protein extracts) or abiotic factors (temperature, UV light, heavy metal salts, pH, etc.). Elicitation can be very efcient at increasing secondary metabolite production as shown in Table 1. This table gives the results that were obtained, in the literature with furanocoumarins, for various plant species and elicitor treatments. These molecules are well-known phytoalexins and their synthesis can be greatly multiplied, using adequate methods. Table 1 clearly shows that pathogen fungus or bacterial attacks are the most efcient for increasing the furanocoumarin content. This statement is also true for most other secondary metabolites, which explains that fungus or bacterial autoclaved preparations are frequently used for elicitation. Other methods than elicitation have been developed with cells in liquid systems such as immobilization [32]. In this case plant cells or micro aggregates are encapsulated in polymers (alginate, carraghenans, etc.), and this usually enhances the production of secondary metabolites [33]. The main explanations for this come from a possible matrix effect of the polymers around the cells which could mimic a tissue organization between them. This is supposed to give rise to the so-called biochemical differentiation which favors the synthesis of secondary products [34].
becoming older [38]. Many studies describe alternative agitation processes that tend to lower this lysis such as air-lift or bubble reactors instead of traditional propeller helixes [39]. This allows biomass production to increase to a level compatible with industrial processes. After successful optimization of the biomass production in a bioreactor, plant cell cultures must undergo well-adapted processes to achieve a good production of secondary metabolites. Traditional processes described for microorganisms can be used for plant cell cultures such as batch, fed-batch (semi-continuous), perfusion and continuous fermentations. Basically, the process design is governed by (1) the relationships between growth and secondary metabolite synthesis; and (2) the possibility for the secondary products to be excreted or not in the medium. (1) If secondary products are produced at the end of the growth phase, it is logical to consider a two-step process where a rst reactor is used for building up the biomass, and a second one for metabolite production [40]. On the other hand, when the production of a given metabolite is growth-associated, a single-step reactor is sufcient to grow the cells and recover the molecules at the same time [40]. (2) If the metabolites remain intracellular, it is usually necessary to kill the biomass, so that the chemicals can be extracted from the cells. This leads to a batch or fed-batch process. Conversely, extracellular production avoids destruction of the biomass for the extraction of the compounds as they can be directly recovered from the medium. This characteristic allows the building of a continuous system with an improved productivity, compared to a standard batch. When a compound tends to be intracellular, it is sometimes possible to force the excretion with permeabilization methods. This can be obtained following different treatments such as pH shock [41] or the addition of various chemicals (detergents, oligosaccharides, [42]) bearing in mind that they must preserve cell viability. Perfusion systems have also been designed with encapsulated cells [15]. In addition to the effect previously described on metabolite production, this treatment usually ensures protection for the cells against shear stress. Among the hundreds of secondary plant products that have been investigated with undifferentiated cell cultures, many works have been published on indole alkaloids [43], tropane alkaloids [44], and taxanes [45], for which the reader is invited to refer to the bibliographical reviews cited herein. However, the development of the shikonin production by Tabata and co-workers has a special place in the recent plant cell culture history ([46], and for a review, see [40]). Indeed, it was one of the rst and most successful stories of an industrial scale-up associating plant cell culture and bioreactor technologies. Another well-known plant cell culture success is represented by the production of ginseng at a very large scale (20 000 25 000 l, see [47]).
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Fig. 2. Guidelines for the production of secondary metabolites from plant organ cultures.
3. Organ cultures Plant organs represent an interesting alternative to cell cultures for the production of plant secondary products. Two types of organ are generally considered for this objective: hairy roots and shoot cultures (Fig. 2).
growth and tropane alkaloid production was stable over a period of 5 years. However, some experimental results have been reported that do not match the hypothesis of a straightforward genetic stability of transformed roots. Yukimune et al. [56] observed a decrease in growth rate during repeated selection cycles when trying to increase tropane alkaloid production of Duboisia hairy root lines. These changes can probably be attributed to the variation of the expression of Ri tDNA oncogenes in the transformed roots [57,58]. In addition to their growth capacities, hairy roots display interesting properties regarding the production of secondary compounds. The metabolite pattern found in hairy roots is similar, if not always identical to that of plant roots [59,60]. In most cases, the differences are only on trace compounds [61]. A major characteristic of hairy roots is that they are able to produce secondary metabolites concomitantly with growth. Hence it is possible to get a continuous source of secondary compounds from actively growing hairy roots [62], unlike the usual results obtained with cell suspension cultures. The same strategies, as developed for cell cultures, have been used to increase the metabolite production from hairy roots. Successful results have been obtained by modifying the nutrient composition of the medium [63], or applying elicitors [64]. It is also possible to cultivate roots under light, in order to get green tissues. These light-adapted hairy roots usually demonstrate different biosynthetic capacities, compared to dark-grown ones [65], due to the presence of chloroplastic enzymes. However, growth rates can be severely depressed with hairy roots under light exposure [66].
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heterogeneity is always possible along the growing organ. In the case on hairy roots, each root tip grows linearly, and the older part of the roots are still present in the culture system. As a consequence, the total root biomass is always composed of young and older tissues. These tissues present various possibilities for the synthesis of secondary compounds as stated by Bourgaud et al. [79] with avonoids. Young tips of Psoralea roots were demonstrated as more capable of synthesizing isoavones (daidzein) whereas old roots accumulated isoavone-derivatives like coumestrol. In most organ cultures, the production of secondary plant products is usually concomitant with growth. As a consequence, and unlike most secondary metabolites in cell suspension systems, it is possible to use a single-stage bioreactor for both growing the biomass and producing the compounds. However, like for cell cultures, most of the secondary metabolites tend to remain intracellular [62,79] especially when growth is still active [76]. Specic bioreactors have been designed for hairy root cultures in order to overcome the limiting factors existing for biomass and secondary metabolite production. Submerged cultures have successfully been replaced by dispersed liquid systems such as nutrient mist reactors [80] or drip-tube techniques [62]. Two-phase systems have also been used to facilitate the release and recovery of the secondary compounds in the medium [81,82]. This technology helps to continuously remove the compounds from the medium, and helps to prevent the feedback repression of the synthesis [62,83]. Despite all the improvements that have been made to reach a better understanding of plant organ cultures in bioreactors, this technology has led to even fewer commercial successes than cell suspension cultures for the production of secondary metabolites. Yet, plant organ cultures are still regarded as promising and many projects are under development [84,85].
4. Need for breakthroughs for the production of secondary compounds Industrial interest emerged quite early in the story of plant vitro cultures and secondary metabolites. Indeed, at least six large-scale industrial productions were engaged between 1976 and 1986 [86].1 However, since that time, this technology has led to only a few applications for the production of commercial compounds. This lack of success can be attributed to several bottlenecks. First of all, it is obvious that industrial processes were started at a time when basic knowledge was
1 Ubiquinone-10 (Japan Tobacco & Salt Pub. Co.), ginseng (Svoboda Co.; Nitto Denko Co.); shikonine (Mitsui Petrochemical Ind. Ltd), berberine (Yamaguchi Co.), rosmarinic acid (Natterdam Co.).
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severely lacking on both plant vitro cultures and secondary compounds. It is astonishing to realize that a key-problem like cell viability, and its assessment, has been studied only recently with plant cells [37,38], whereas this topic has been considered for a long time with animal cell cultures [87]. Also, secondary metabolites are produced following long biosynthetic pathways that can involve dozens of enzymes. This synthesis is much more complex than for recombinant proteins produced with mammalian or prokaryotic biotechnologies, which usually involve one or two genes. This can partially explain the lack of success of plant cell and tissue cultures compared to other expression systems. A second bottleneck has to do with the economic feasibility of plant cell and organ cultures. Indeed, this technology requires high-cost bioreactors associated with aseptic conditions that are expensive to maintain [88]. Besides, unlike mammalian cell cultures which produce high-value molecules, plant vitro cultures have also been considered for the production of low or moderate-value molecules such as food ingredients. As a consequence, economic feasibility is even more difcult to reach. There have been many attempts in the last decade to address the problem of cost-effectiveness of plant vitro culture technologies. Several new routes have been investigated. The rst one is the design of low-cost bioreactors associated with a reduction of online controls and probes, and a simplied sterilization process [88]. Another possible strategy could be the use of plastic bags as already in use for the cultivation of microalgae [89]. Such bags are available in varying volumes (20 1000 l) and an aeration system can be adapted. These plastic bags are obviously much cheaper to use than culture reactors. However, they do not address all the problems encountered with cell cultures such as cell autotrophy or sedimentation. Other authors have tried to change more radically the concept of plant cell tissue and organ cultures in bioreactors without the use of a traditional culture chamber. A new system has been proposed by Borisjuk and co-workers [90] although it was designed for the production of recombinant proteins from genetically modied plants. The basic concept is to grow entire plants in a system where roots are maintained in sterile conditions and separated from the aerial parts. The recombinant proteins are recovered in the nutrient solution that surrounds the roots with the help of the rhizospheric excretion from the plant. This concept is interesting because it uses whole plants. It addresses the problem of biomass production encountered with cell suspension cultures that are hard to develop. Another system has been recently proposed by Gontier and co-workers [91]. This last system has been designed specically for the production of secondary compounds. The basic principle is to get rid of sterility but to keep a culture medium that allows the
manipulation of secondary compound production (elicitation, addition of precursors, etc.). Sterility is essential because a traditional in vitro culture medium is constituted of organic compounds, especially sugars, that would be altered by microorganisms if maintained in non-sterile conditions. If we use whole plants that are photosynthetically active, it is possible to avoid the use of organic compounds in a mineral-based medium. In this case, the medium is less amenable to contamination in open conditions. The other basic concept of this device is to yield secondary compounds in a non-destructive manner for the plants, therefore allowing repeated and regular harvests from the same biomass. In practice, this is achieved by growing the plants in hydroponic or aeroponic non-sterile conditions and by treating the medium in order to elicit the synthesis and to allow the excretion of the desired compounds, out of the roots. The last step is the recovery of the compounds from the medium with the help of purication processes. As a conclusion, this system uses the plants for what they can do best in natural conditions: they grow quite quickly when they are photosynthetically active. It also uses the concept of culture medium for what it is most useful in reactor culture technology: it allows a close control of secondary compound production. This technology has been has been successfully used at pilot scale on three different model plants: Datura innoxia for tropane alkaloids, Ruta gra6eolens for furocoumarins, and Taxus baccata for paclitaxel. Many attempts have been made to invent new devices that could lead to breakthroughs for the production of secondary compounds. However, the major changes in the area of plant secondary metabolites have probably been achieved thanks to the rise of molecular genetics in recent years, through the so-called metabolic engineering approach.
5. Plant metabolic engineering The last 15 years have produced a large quantity of results regarding the biosynthetic pathways leading to secondary metabolites. Concomitantly, at the beginning of the 1990s, a new discipline called metabolic engineering appeared. According to Bailey [92], metabolic engineering is: the improvement of cellular activities by manipulation of enzymatic, transport, and regulatory functions of the cell with the use of recombinant DNA technology. In many cases this approach relies on the identication of limiting enzyme activities, after successful pathway elucidation and metabolite mapping (metabolomics). Such limiting steps can be improved with an appropriate use of genetic transformation. Most of the strategies developed so far have played on the introduction of genes isolated from more efcient organisms, promoters that enhance the expression of a
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target gene, or antisens and co-suppression techniques for the obtainment of knock-down plants for the desired traits. Besides their own biosynthetic capacities, plants can also be used as host organisms for the production of recombinant proteins. This corresponds to the so-called molecular pharming when the technology utilizes eldgrown transgenic plants. This last topic is outside the scope of this review as it is not typically focused on the production of secondary compounds. However, many research projects relevant to this category are currently being developed to which the reader is invited to refer [93,94].
antisens inhibition of the 4-coumarate:coA ligase activity. They demonstrated up to a 45% reduction of lignin, associated with a 15% cellulose increase in the transformants. This compensatory effect is particularly relevant for the paper industry as cellulose is the most valuable part of wood pulp. Other results of considerable interest have been reached with Arabidopsis lignication mutants that accumulate lignin syringyl monomers to the detriment of guaiacyl units [105]. This was obtained by the overexpression of ferulate-5-hydroxylase, a major P450 gene from the pathway, and was also veried in other model plants such as tobacco and poplar [106]. This strategy could well lead to an improved chemical degradability of lignin as syringyl-based lignin is more easily processed. More detailed results about lignin genetic engineering can be found in dedicated bibliographical reviews [107,108].
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F. Bourgaud et al. / Plant Science 161 (2001) 839 851 [2] F. Czapek, Spezielle Biochemie, Biochemie der Panzen, vol. 3, G. Fischer Jena, 1921, p. 369. [3] J. Li, T.M. Ou-Lee, R. Raba, R.G. Amundson, R.L. Last, Arabidopsis mutants are hypersensitive to UV-B radiation, Plant Cell 5 (1993) 171 179. [4] K.B.G. Torssell, Chemical ecology, in: K.B.G. Torssell (Ed.), Natural Product Chemistry, A Mechanistic Biosynthetic and Ecological Approach, Swedish Pharmaceutical Press, 1997, pp. 42 79. [5] J.B. Harborne, Classes and functions of secondary products, in: N.J. Walton, D.E. Brown (Eds.), Chemicals from Plants, Perspectives on Secondary Plant Products, Imperial College Press, 1999, pp. 1 25. [6] G.F. Payne, V. Bringi, C. Prince, M.L. Shuler, The quest for commercial production of chemicals from plant cell culture, in: G.F. Payne, V. Bringi, C. Prince, M.L. Shuler (Eds.), Plant Cell and Tissue Culture in Liquid Systems, Hanser, 1991, pp. 1 10. [7] A.D. Krikorian, F.C. Steward, Biochemical differentiation: the biosynthetic potentialities of growing and quiescent tissue, in: F.C. Steward (Ed.), Plant Physiology, A Treatise, Academic, Press, 1969, pp. 227 326. [8] M.H. Zenk, Chasing the enzymes of secondary metabolism: plant cultures as a pot of gold, Phytochemistry 30 (1991) 3861 3863. [9] M. Hamburger, K. Hostettmann, Bioactivity in plants: between phytochemistry and medicine, Phytochemistry 30 (1991) 3864 3874. [10] G.E.P. Box, The exploration and exploitation of response surfaces: some general considerations and examples, Biometrics 10 (1954) 16 60. [11] P.J. Larkin, W.R. Scowcroft, Somaclonal variation A novel source of variability from cell cultures for plant improvement, Theoretical and Applied Genetics 60 (1981) 197 214. [12] I.A. Senior, Uses of plant gene silencing, Biotechnology and General Engineering Review 15 (1998) 79 119. [13] V. Bouque, F. Bourgaud, C. Nguyen, A. Guckert, Production of daidzein by callus cultures of Psoralea species and comparison with plants, Plant Cell Tissue and Organ Culture 53 (1998) 35 40. [14] A.G. Fett Neto, W.Y. Zhang, F. DiCosmo, Kinetics of taxol production, growth and nutrient uptake in cell suspensions of Taxus cuspidate, Biotechnology and Bioengineering 44 (1994) 205 210. [15] G.F. Payne, V. Bringi, C. Prince, M.L. Shuler, Immobilized plant cells, in: G.F. Payne, V. Bringi, C. Prince, M.L. Shuler (Eds.), Plant Cell and Tissue Culture in Liquid Systems, Hanser, 1991, pp. 179 223. [16] D.K. Dougall, Tissue culture and the study of secondary (natural) products, in: P.K. Stumpf, E.E. Conn (Eds.), The Biochemistry of Plants, A Comprehensive Treatise. Secondary Plant Products, vol. 7, Academic Press, 1981, pp. 21 34. [17] G.P. Bolwell, C.L. Cramer, C.J. Lamb, W. Schuch, R.A. Dixon, L-Phenylalanine ammonia-lyase from Phaseolus 6ul garis : modulation of the levels of active enzymes by trans -cinnamic acid, Planta 169 (1986) 97 107. [18] C. Johnson, D.R. Brannon, J. Kuc, Xanthotoxin: a phytoalexin of Pastinaca sati6a root, Phytochemistry 12 (1973) 2961 2962. [19] S.K. Chaudhary, O. Ceska, P.J. Warrington, M.J. AshwoodSmith, Increased furocoumarin content of celery during storage, Journal of Agriculture and Food Chemistry 33 (1985) 1153 1157. [20] O. Ceska, S.K. Chaudhary, P.J. Warrington, M.T. AshwoodSmith, Furocoumarins in the cultivated carrot, Daucus carota, Phytochemistry 25 (1986) 81 83. [21] G. Surico, L. Varvaro, M. Solfrizzo, Linear furocoumarin accumulation in celery plants infected with Erwinia caroto6ora pv. carotovora, Journal of Agriculture and Food Chemistry 35 (1987) 406 409.
also be transformed into sanguinarine by a parallel pathway. SMT overexpression allowed an increase in the berberine content of Coptis transgenic cells (20%) to the detriment of the competitive pathway. The above results, obtained by the Yamada group, demonstrates the value of playing on a well-dened gene, for which the corresponding enzyme has been identied as a limiting step in a synthesis. However, multiple gene families are usually involved in the synthesis of a single secondary metabolite, and therefore it is important to think about multiple gene transformation and coordinate gene expression for the future. The rst encouraging results were described by Beck von Bodman et al. [111]. They succeeded in expressing a polygenic construction driven by a single promoter in tobacco plants. Two genes placed on the construct were co-ordinately expressed in the same secondary pathway (mannopine synthesis). Although this biosynthesis has no pharmaceutical relevance, this basic research demonstrates that it is possible to create a new metabolic channeling in the case of plant secondary compounds and is full of promise for research to come.
6. Prospects Many other examples could be presented with plant metabolic engineering as this research area is developing actively, but being comprehensive on this topic is outside the scope of this review. For further readings, a rst book entirely dedicated to this approach in the eld of plant secondary metabolism has been recently published [112], conrming that the discipline is healthy. Metabolic engineering is probably a large step forward but playing on the genes will not solve all the problems that have prevented the development of commercial success in the eld of plant secondary metabolites. To cite just a few non-scientic problems, the so-called molecular pharming has not gained public acceptance yet, due to environmental concerns about transgenic plants, especially in Europe. A possible alternative to eld grown plants could be to use transformed cells or organs in reactors, as they are by denition cultivated under closed conditions, and therefore do not exhibit any environmental risk. The long-awaited breakthrough to reach an industrial accomplishment is probably in our hands with the combination of plant cell, tissue, and organ culture technologies and metabolic engineering.
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