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Yield improvement strategies for the production of secondary metabolites in plant tissue culture: silymarin from Silybum marianum tissue culture S. AbouZid
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Department of Pharmacognosy, Faculty of Pharmacy, University of Beni-Suef, Beni-Suef 62111, Egypt Published online: 20 Jun 2014.
To cite this article: S. AbouZid (2014) Yield improvement strategies for the production of secondary metabolites in plant tissue culture: silymarin from Silybum marianum tissue culture, Natural Product Research: Formerly Natural Product Letters, 28:23, 2102-2110, DOI: 10.1080/14786419.2014.927465 To link to this article: http://dx.doi.org/10.1080/14786419.2014.927465
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Natural Product Research, 2014 Vol. 28, No. 23, 2102–2110, http://dx.doi.org/10.1080/14786419.2014.927465
REVIEW Yield improvement strategies for the production of secondary metabolites in plant tissue culture: silymarin from Silybum marianum tissue culture S. AbouZid*
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Department of Pharmacognosy, Faculty of Pharmacy, University of Beni-Suef, Beni-Suef 62111, Egypt Plant cell culture can be a potential source for the production of important secondary metabolites. This technology bears many advantages over conventional agricultural methods. The main problem to arrive at a cost-effective process is the low productivity. This is mainly due to lack of differentiation in the cultured cells. Many approaches have been used to maximise the yield of secondary metabolites produced by cultured plant cells. Among these approaches: choosing a plant with a high biosynthetic capacity, obtaining efficient cell line for growth and production of metabolite of interest, manipulating culture conditions, elicitation, metabolic engineering and organ culture. This article gives an overview of the various approaches used to maximise the production of pharmaceutically important secondary metabolites in plant cell cultures. Examples of using these different approaches are shown for the production of silymarin from Silybum marianum tissue culture. Keywords: plant tissue culture; secondary metabolites; silymarin; yield improvement
Plant tissue culture systems are established using part of the plant (explant) that is sterilised with a detergent solution, cut to approximately 1 cm in length, and transferred to a solid medium. The plant material is incubated aseptically at appropriate temperature for several weeks until callus is produced. The callus is a mass of undifferentiated tissue that develops on or around an injured or cut plant surface or in tissue culture. The callus is sub-cultured by transferring a small piece to a fresh solid medium. After several subsequent transfers, the callus is ready to be transferred to a liquid medium to form a cell suspension culture. A piece of the callus is transferred to a liquid medium in an Erlenmeyer flask and the vessel placed on a rotary shaker. The culturing conditions (speed of shaker, light intensity, pH of the medium and so on) depend on the plant species under investigation. By sub-culturing for several generations, a fine cell suspension culture containing small cell aggregates and single cells is established. The cells in suspension are used for a large-scale culture with jar-fermenters and tanks (Kyte & Kleyn 1996). Plant cell culture can be an alternative for the production of pharmaceutically important secondary metabolites. This technology depends on using plant cell cultures in a similar manner to microbial fermentation for factory-type production of target metabolites. There are many advantages in using plant cell culture as a source for secondary metabolites. For example, production would be independent of variation in crop quality or failure; yield of target compounds would be constant and geared to demand; there is no difficulty in applying good manufacturing practice to the early stages of production; production would be possible anywhere under strictly controlled conditions; independency of political problems; free from risk of contamination with pesticides, herbicides, agrochemicals or fertilisers; and new methods of
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production can be patented. Few examples for commercial application are known until now (Kayser & Warzecha 2012). Paclitaxel was produced by Phyton Biotech, USA and Samyang Genex, Korea using plant cell culture established from Taxus species. Scopolamine from tissue culture derived from Dubosia species was commercialised by Sumitomo Chemical Industries, Japan. Dried ginseng cells were commercialised by Nitto Denko Corporation, Japan. Nevertheless, many problems hinder the commercialisation of this technology. For example, technical problems related to culture of plant cells compared with the easier and much wellstudied fermentation of microbial cells. Another problem is the instability of cell lines so that the product may disappear on prolonged cultures. A major problem is the low productivity to reach a cost-effective process due to lack of differentiation and/or organisation. In this article, different approaches to enhance the production of secondary metabolite in plant tissue culture are surveyed. Application of these approaches to enhance the production of silymarin, a well-known hepatoprotective agent from Silybum marianum (L.) Gaertn. (Asteraceae), is discussed as an example. Silymarin is one of the most investigated plant extracts with known mechanism of action for the oral treatment of toxic liver damage (Polyak et al. 2010). Its annual world consumption exceeds 500 million USD. This constitutive natural product is mainly found in the fruits of S. marianum, and it consists of a number of flavonolignans such as silybin, isosilybin, silychristin and silydianin (Figure 1). Cell cultures of this plant species were able to produce silychristin as the major flavonolignan (Sa´nchezSampedro et al. 2005; Elwekeel et al. 2012). Application of different yield improvement strategies to these cell cultures changed the profile of the produced flavonolignans qualitatively and quantitatively. Yield improvement strategies Preliminary considerations On beginning plant tissue culture experiments for the production of secondary metabolites, we have to choose the plant species, variety, and cultivar producing the required quantitative and qualitative profile of the target metabolites (Ananga et al. 2013). This is usually underestimated in many of the published work in this field. Firouzi et al. (2013) examined the effect of using four ecotypes of S. marianum on growth pattern and flavonolignan production in cell culture. The different ecotypes showed significant differences in the studied parameters. Second and of equal importance is the selection of appropriate explant for the initiation of a callus culture. Typically, a good explant should be small, healthy, taken from middle part of the plant, and contain meristematic tissues. It was reported that cell growth and dry weight were higher in S. marianum cell suspension culture derived from cotyledon explants compared with hypocotyl explants (Firouzi et al. 2013). In addition, silymarin production level showed significant difference in cell cultures derived from different explants. Screening cell lines On selecting the cell line that will be used for the production of the desired metabolite, researchers should optimise many factors such as initial high level of the secondary metabolites, growth rate, culture stability, localisation of the metabolite and responsiveness of the culture to the planned yield enhancement strategy (Shuler 1999). An important term ‘clonal selection’ refers to the production of a population of cells all possessing the same trait. The emphasis is always given to the level of the product; however, the growth rate of the culture is very important for an economic process. Instability of the culture may result from genetic or epigenetic factors. Epigenetic factors are caused by alterations in the environment and do not result in permanent change in cell genome.
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HO
O
O
CH2OH HO
OCH3
O OH
O
CH2OH OCH3
O OH
OH
OH
OH O
OH O Silybin A
Silybin B OCH3
OCH3 O
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HO
O
OH HO
O
O
O
O
CH2OH
CH2OH
OH
OH OH O
OH O Isosilybin A
Isosilybin B OH
OH
O
O HO
OH
OH
O OH
HO
OH
O
CH2OH OCH3
OH
CH2OH OCH3
OH O
OH O
Isosilychristin
Silychristin
OH OCH3
O O HO
O OH OH OH O Silydianin
Figure 1. Chemical structures of silymarin flavonolignan components.
Manipulation of the components of the culture medium Plant tissue culture media include some or all of the following components: macronutrients, micronutrients, vitamins, amino acids, carbon source, growth regulators, solidifying agent and undefined organic supplements (Saad & Elshahed 2012). The most frequently used media are Murashige and Skoog (MS) medium (Murashige & Skoog 1962), Linsmaier and Skoog medium (Linsmaier & Skoog 1965), Gamborg medium (Gamborg et al. 1968) and Nitsch and Nitsch medium (Nitsch & Nitsch 1969).
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The concentration of some of the media components is usually manipulated to provide requirements for optimal growth and secondary metabolite production. In case of S. marianum cell culture, production of silymarin in cell cultures of S. marianum was studied using different concentrations of KNO3, KH2PO4, Feþ2 and Caþ2 (Cacho et al. 1999). An increase in the concentration of CaCl2 reduced the content of silymarin. Removal of CaCl2 induced significant increase in the accumulation of silymarin and its precursors but reduced the growth of the cells. Removal of other salts such as KNO3 and KH2PO4 also reduced the growth. Elimination of iron from the medium led to a total loss of cell viability. The effect of calcium deprivation on cell growth and silymarin production by S. marianum cell cultures was studied (Cacho et al. 2013). It was concluded that the removal of calcium from the medium advanced the onset of stationary phase of growth. In addition, growth inhibition was suggested to be responsible for maximum silymarin accumulation. Precursor feeding Addition of inexpensive precursors or metabolic intermediates to the culture media may increase the yield of the target metabolites. This requires a thorough knowledge of the biosynthetic pathways of the target metabolite. Phenylalanine and coniferyl alcohol, main precursors involved in silymarin flavonolignan biosynthesis (AbouZid & Ahmed 2013), were added to S. marianum cell cultures to increase silymarin production. Addition of coniferyl alcohol (46 mM) resulted in increase in silydianin level after 72 h (Tumova et al. 2006). Treating S. marianum cell cultures with phenylalanine enhanced silymarin production (Firouzi et al. 2013). Elicitation Elicitation is defined as the induction of secondary metabolite production by molecules or treatments called elicitors. Elicitors can be classified according to their origin and chemical structures as either biotic or abiotic. Biotic elicitors can be either of defined chemical composition, e.g. methyl jasmonate, or of complex nature, e.g. yeast extract, polysaccharides and glycoproteins. Abiotic elicitors can be either physical – e.g. ultraviolet irradiation, osmotic stress – or chemical – e.g. salts of heavy metals. Figure 2 shows a classification system for
Elicitors
Biotic
Defined composition
Abiotic
Complex composition
Chemical
Chitosan
Fungal homogenate
Heavy metal salts
Pectin
Yeast extract
Physical
Thermal stress Osmotic stress
Chitin
UV radiation
Methyl jasmonate
Wounding
Figure 2. Classification of elicitors used to improve secondary metabolite production in plant tissue culture according to their origin and chemical structure.
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different types of elicitors. Elicitation has been applied successfully to cultures of various plant species in the experimental-scale and large-scale reactors (Krzyzanowska et al. 2012; Rahimi et al. 2012). Secondary metabolites responsive to elicitation are usually involved in resistance against a variety of stimuli such as heat treatment, osmotic stress, wounding and fungal infection. Some of these metabolites are constitutively expressed, phytoanticipins, while others are synthesised after a pathogen attack, phytoalexins. Many factors affect the elicitation process such as elicitor specificity, elicitor concentration and culture conditions. The use of elicitor, which is not specific to the species, can lead to ineffective elicitation. There is an optimum concentration of the elicitor that can lead to maximum production of secondary metabolites. Growth stage at which the elicitor is added and medium composition can significantly affect the success of the elicitation process. Therefore, application of an effective elicitation strategy requires extensive trial and error procedures. Elicitors from different origin have been successfully used to increase secondary metabolite yield in plant tissue culture (Cai et al. 2012; Saw et al. 2012; Veerashree et al. 2012). Jasmonic acid and its methyl ester are the most popular elicitors. The mechanism of action of jasmonate was studied in Catharanthus roseus (Van der Fits & Memelink 2000). It acts by the induction of gene expression through transcription factor with a conserved jasmonate-response domain. The differential effects of several elicitors were evaluated to increase silymarin production in S. marianum cell culture (Sa´nchez-Sampedro et al. 2005). The elicitors tested were chitosan, chitin, yeast extract, salicylic acid and methyl jasmonate. Salicylic acid, chitin and chitosan used at different concentrations did not stimulate the accumulation of silymarin. This emphasises the importance of selecting specific elicitor for each plant species. Yeast extract at an optimum concentration of 50 mg/mL added to 3-days old S. marianum cell cultures for 48 h was able to slightly increase silymarin content in the cells and threefold higher level accumulated in the medium. Methyl jasmonate was ineffective at 10 mM concentration. When the elicitor was added at a concentration of 100 mM to 3-days old cell cultures, it produced significant accumulation of silymarin both in the cultured cells and in the medium. Silymarin production peaked between 48 and 72 h after the addition of methyl jasmonate. Cell growth and silymarin production was increased by the addition of 0.2, 0.4, 0.8 and 1 mM Agþ to S. marianum cell suspension culture (Ashtiani et al. 2010). Treatment of the cells with 0.8 mM Agþ resulted in increase in silymarin production 30-fold higher than the control. Organ culture Organ cultures are used to avoid the main limitations of instability and low product yield associated with cell cultures due to lack of differentiation and organisation. Production of secondary metabolites in tissue cultures is usually higher when plant cells are organised into tissues/organs (Misawa 1994). The expression of secondary metabolic pathways in organised cultures is not surprising because it mimics exactly what the plant does. Shoot culture S. marianum multiple shoot culture was established and maintained on MS media containing 2isopentenyl adenine as cytokinin. The cultures were able to accumulate silymarin (0.437% DW), of which silybin A and silybin B were the main components (El Sherif et al. 2013). Root culture These are the roots excised from sterile plantlets and continuously cultured on hormonal media. Root cultures are typical examples of organ cultures that can be used for the production of
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phytochemicals. Root cultures have been used as standard experimental system in studies of inorganic nutrition, nitrogen metabolism, plant growth regulation and root development. The slow growth of the intact plant root remains the main disadvantage of this system (LoyolaVargas & Miranda-Ham 1995). S. marianum roots were cultured in liquid MS media on a rotary shaker at 100 rpm (Elwekeel et al. 2012). The major flavonolignans produced by the cultured roots were silychristin and silydianin. Neither silybin nor isosilybin were detected in the cultured roots. Elicitation of the root culture with methyl jasmonate (100 mM) increased the content of the produced flavonolignans to approximately 300% of the control culture.
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Hairy root culture Hairy roots are obtained by genetically transforming plant tissues with the natural genetic engineer Agrobacterium rhizogenes. A part of the bacterial DNA (T-DNA) is integrated and expressed into the plant genome. This T-DNA is present in the bacterial Ri (root-inducing) plasmid. Hairy root cultures have been known for more than three decades and developed from more than 100 plant species (Georviev et al. 2007). It offers many advantages for secondary metabolite production compared with the low yield level obtained with plant cell cultures. Among these advantages are fast growth, ability to grow from low inoculum, genetic and biochemical stability, and ability to synthesise secondary metabolites at levels similar to or higher than that produced by the mother plant. Recent advances in bioreactor design allowed hairy root technology to be scaled up. Hairy roots can be cultured under optimal conditions using mist bioreactors developed by RooTecw (Witterswil, Switzerland). Hairy root culture of S. marianum was established to be used as an alternative source for the production of silymarin flavonolignans (Alikaridis et al. 2000; Rahnama et al. 2008). Eight hairy root lines were cultured in liquid MS medium. These root lines showed significant differences in biomass and silymarin flavonolignan production. Surprisingly, the production of silymarin from these organised cultures was comparable or lower than that from cell cultures (Table 1). Metabolic engineering The accumulation of secondary metabolites can be enhanced by increasing the flux through the biosynthetic pathway to the desired product, increasing the number of producing cells and decreasing the catabolism of the desired product (Verpoorte et al. 1999). Increasing the flux to Table 1. Silymarin production in S. marianum tissue culture using different yield improvement strategies. Plant/culture Fruits Cell culture Cell culture Cell culture Cell culture Shoot culture Intact roots Hairy roots Hairy roots Hairy roots Transgenic HR Hairy roots a
Silymarin content (mg/g DW ^ SE) 23.40 ^ 3.3 (1.5– 3%) 0.31 ^ 0.0 0.71 ^ 0.0 6.34 ^ 0.07 11.50 ^ 0.07 0.437a 0.05 ^ 0.00 0.11 ^ 0.00 1.89 ^ 0.02 1.20 ^ 0.00 0.87 ^ 0.05 0.22 ^ 0.00
Yield improvement approach
Reference
Precursor feeding Change in media (- Caþ2) Elicitation (100 mM MeJ) Organ culture Organ culture Organ culture Organ culture þ SA Organ culture þ Agþ Metabolic engineering Scale up þ MeJ
Cacho et al. (1999) Firouzi et al. (2013) Firouzi et al. (2013) Cacho et al. (1999) Sa´nchez-Sampedro et al. (2005) El Sherif et al. (2013) Elwekeel et al. (2012) Rahnama et al. (2008) Khalili et al. (2009) Khalili et al. (2010) Rahnama et al. (2013) Rahimi et al. (2012)
Calculations were made as % DW. SEs are available for individual silymarin components in the referenced report.
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the desired product can be achieved by genetic engineering of the biosynthetic pathway leading to the desired product. This requires thorough knowledge of the enzymatic and genetic constitution of the pathway. The enzymes catalysing the rate-limiting step can be expressed from a stronger promoter or a heterologous gene encoding a similar step can be introduced into the plant cells. An example to the use of this approach is the over-expression of chalcone synthase in S. marianum hairy root culture (Rahnama et al. 2013). This enzyme catalyses a key step in the flavonoid biosynthesis pathway. It is involved in the biosynthesis of the flavonoid taxifolin, the immediate precursor of silymarin flavonolignans (Figure 3) (AbouZid & Ahmed 2013). Taxifolin is converted into silymarin flavonolignans by oxidative coupling to coniferyl alcohol. Hairy root culture harbouring chalcone synthase transgene was established on liquid MS media. COOH COOH
COOH
NH2 O2
PAL
NADPH OH p-coumaric acid
Cinnamic acid
L-Phe
O2 CoA ligase
NADPH COOH
OH CoAS HO 3 x malonyl-CoA
O
OH Caffeic acid
Chalcone synthase
SAM
OH HO
COOH
OH
OH
O MeO OH
HO
OH Ferulic acid
O
OH
CH2OH
O OH
O2 HO
O OH MeO OH OH
O
Taxifolin
OH Coniferyl alcohol
Figure 3. Chalcone synthase, a key enzyme in the pathway leading to silymarin flavonolignans.
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Silymarin content in the hairy roots harbouring the transgene was seven-fold higher than the non-transgenic hairy roots. Multiple strategies Application of more than one of the previously mentioned strategies may result in increase in yield of secondary metabolites in plant tissue culture and bring the process to a commercial level. For example, salicylic acid was used to increase the content of silymarin in hairy root culture of S. marianum (Khalili et al. 2009). This elicitor was added to 28-days old hairy roots for 24 h at 6 mg/50 mL culture. There was 2.52-fold increase in the silymarin content compared with control culture. It was observed that the hairy roots treated with salicylic acid grew slower than the non-treated hairy roots. Effect of elicitation of S. marianum hairy roots with Agþ was reported (Khalili et al. 2010). Agþ was added to 28-days old hairy roots at different concentrations. Hairy roots treated with 2 mM Agþ for 96 h showed the highest silymarin production (1.2 mg/g DW). Scaling up cultivation of S. marianum hairy roots was carried out in a bioreactor (Rahimi et al. 2012). Accumulation of silymarin increased from 0.13 mg/g DW in control cultures to 0.22 mg/g DW in cultures treated with 100 mM methyl jasmonate for 72 h. Conclusion Plant tissue culture can be a promising source for a continuous, standardised and stable production of useful secondary metabolites. Several approaches have been applied to increase the yield of secondary metabolites produced by plant tissue culture. Elicitation and metabolic engineering of the biosynthetic pathways leading to the target metabolites in organised cultures has proven to produce the highest increase in yield. This was clearly shown in the production of flavonolignans from S. marianum tissue culture. Combining several approaches may achieve the improvement in yield required to apply an economical process on a commercial scale. In addition, understanding metabolic regulation of secondary metabolite biosynthesis on the molecular level will greatly help in such efforts. References AbouZid S, Ahmed O. 2013. Silymarin flavonolignans: structure–activity relationship and biosynthesis. In: Atta-urRahman, editor. Studies in natural products chemistry. Vol. 40. Oxford (UK): Elsevier; p. 469– 484. Alikaridis F, Papadakis D, Pantelia K, Kephalas T. 2000. Flavonolignan production from Silybum marianum transformed and untransformed root cultures. Fitoterapia. 71:379–384. Ananga A, Georgiev V, Ochieng J, Phills B, Tsolova V. 2013. Production of anthocyanins in grape cell cultures: a potential source of raw material for pharmaceutical, food, and cosmetic industries. Rijeka, Croatia: InTech. Ashtiani SR, Hasanloo T, Bihamta MR. 2010. Enhanced production of silymarin by Agþ elicitor in cell suspension cultures of Silybum marianum. Pharm Biol. 48:708–715. Cacho M, Mora´n M, Corchete P, Ferna´ndez-Ta´rrago J. 1999. Influence of medium composition on the accumulation of flavonolignans in cultured cells of Silybum marianum (L.) Gaertn. Plant Sci. 144:63–68. Cacho M, Torres Domı´nguez A, Elena-Rossello´ JA. 2013. Role of polyamines in regulating silymarin production in Silybum marianum (L.) Gaertn (Asteraceae) cell cultures under conditions of calcium deficiency. J Plant Physiol. 170:1344–1348. Cai Z, Kastell A, Mewis I, Knorr D, Smetanska I. 2012. Polysaccharide elicitors enhance anthocyanin and phenolic acid accumulation in cell suspension cultures of Vitis vinifera. Plant Cell Tissue Organ Cult. 108:401– 409. El Sherif F, Khattab S, Ibrahim AK, Ahmed SA. 2013. Improved silymarin content in elicited multiple shoot cultures of Silybum marianum. Physiol Mol Biol Plant. 19:127–136. Elwekeel A, AbouZid S, Sokkar N, Elfishway A. 2012. Studies on flavonolignans from cultured cells of Silybum marianum. Acta Physiol Plant. 34:1445–1449. Elwekeel A, Elfishway A, AbouZid S. 2012. Enhanced accumulation of flavonolignans in Silybum marianum cultured roots by methyl jasmonate. Phytochem Lett. 5:393–396.
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Yield improvement strategies for the production of secondary metabolites in plant tissue culture: silymarin from Silybum marianum tissue culture S. AbouZid
a
a
Department of Pharmacognosy, Faculty of Pharmacy, University of Beni-Suef, Beni-Suef 62111, Egypt Published online: 20 Jun 2014.
To cite this article: S. AbouZid (2014) Yield improvement strategies for the production of secondary metabolites in plant tissue culture: silymarin from Silybum marianum tissue culture, Natural Product Research: Formerly Natural Product Letters, 28:23, 2102-2110, DOI: 10.1080/14786419.2014.927465 To link to this article: http://dx.doi.org/10.1080/14786419.2014.927465
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Natural Product Research, 2014 Vol. 28, No. 23, 2102–2110, http://dx.doi.org/10.1080/14786419.2014.927465
REVIEW Yield improvement strategies for the production of secondary metabolites in plant tissue culture: silymarin from Silybum marianum tissue culture S. AbouZid*
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Department of Pharmacognosy, Faculty of Pharmacy, University of Beni-Suef, Beni-Suef 62111, Egypt Plant cell culture can be a potential source for the production of important secondary metabolites. This technology bears many advantages over conventional agricultural methods. The main problem to arrive at a cost-effective process is the low productivity. This is mainly due to lack of differentiation in the cultured cells. Many approaches have been used to maximise the yield of secondary metabolites produced by cultured plant cells. Among these approaches: choosing a plant with a high biosynthetic capacity, obtaining efficient cell line for growth and production of metabolite of interest, manipulating culture conditions, elicitation, metabolic engineering and organ culture. This article gives an overview of the various approaches used to maximise the production of pharmaceutically important secondary metabolites in plant cell cultures. Examples of using these different approaches are shown for the production of silymarin from Silybum marianum tissue culture. Keywords: plant tissue culture; secondary metabolites; silymarin; yield improvement
Plant tissue culture systems are established using part of the plant (explant) that is sterilised with a detergent solution, cut to approximately 1 cm in length, and transferred to a solid medium. The plant material is incubated aseptically at appropriate temperature for several weeks until callus is produced. The callus is a mass of undifferentiated tissue that develops on or around an injured or cut plant surface or in tissue culture. The callus is sub-cultured by transferring a small piece to a fresh solid medium. After several subsequent transfers, the callus is ready to be transferred to a liquid medium to form a cell suspension culture. A piece of the callus is transferred to a liquid medium in an Erlenmeyer flask and the vessel placed on a rotary shaker. The culturing conditions (speed of shaker, light intensity, pH of the medium and so on) depend on the plant species under investigation. By sub-culturing for several generations, a fine cell suspension culture containing small cell aggregates and single cells is established. The cells in suspension are used for a large-scale culture with jar-fermenters and tanks (Kyte & Kleyn 1996). Plant cell culture can be an alternative for the production of pharmaceutically important secondary metabolites. This technology depends on using plant cell cultures in a similar manner to microbial fermentation for factory-type production of target metabolites. There are many advantages in using plant cell culture as a source for secondary metabolites. For example, production would be independent of variation in crop quality or failure; yield of target compounds would be constant and geared to demand; there is no difficulty in applying good manufacturing practice to the early stages of production; production would be possible anywhere under strictly controlled conditions; independency of political problems; free from risk of contamination with pesticides, herbicides, agrochemicals or fertilisers; and new methods of
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production can be patented. Few examples for commercial application are known until now (Kayser & Warzecha 2012). Paclitaxel was produced by Phyton Biotech, USA and Samyang Genex, Korea using plant cell culture established from Taxus species. Scopolamine from tissue culture derived from Dubosia species was commercialised by Sumitomo Chemical Industries, Japan. Dried ginseng cells were commercialised by Nitto Denko Corporation, Japan. Nevertheless, many problems hinder the commercialisation of this technology. For example, technical problems related to culture of plant cells compared with the easier and much wellstudied fermentation of microbial cells. Another problem is the instability of cell lines so that the product may disappear on prolonged cultures. A major problem is the low productivity to reach a cost-effective process due to lack of differentiation and/or organisation. In this article, different approaches to enhance the production of secondary metabolite in plant tissue culture are surveyed. Application of these approaches to enhance the production of silymarin, a well-known hepatoprotective agent from Silybum marianum (L.) Gaertn. (Asteraceae), is discussed as an example. Silymarin is one of the most investigated plant extracts with known mechanism of action for the oral treatment of toxic liver damage (Polyak et al. 2010). Its annual world consumption exceeds 500 million USD. This constitutive natural product is mainly found in the fruits of S. marianum, and it consists of a number of flavonolignans such as silybin, isosilybin, silychristin and silydianin (Figure 1). Cell cultures of this plant species were able to produce silychristin as the major flavonolignan (Sa´nchezSampedro et al. 2005; Elwekeel et al. 2012). Application of different yield improvement strategies to these cell cultures changed the profile of the produced flavonolignans qualitatively and quantitatively. Yield improvement strategies Preliminary considerations On beginning plant tissue culture experiments for the production of secondary metabolites, we have to choose the plant species, variety, and cultivar producing the required quantitative and qualitative profile of the target metabolites (Ananga et al. 2013). This is usually underestimated in many of the published work in this field. Firouzi et al. (2013) examined the effect of using four ecotypes of S. marianum on growth pattern and flavonolignan production in cell culture. The different ecotypes showed significant differences in the studied parameters. Second and of equal importance is the selection of appropriate explant for the initiation of a callus culture. Typically, a good explant should be small, healthy, taken from middle part of the plant, and contain meristematic tissues. It was reported that cell growth and dry weight were higher in S. marianum cell suspension culture derived from cotyledon explants compared with hypocotyl explants (Firouzi et al. 2013). In addition, silymarin production level showed significant difference in cell cultures derived from different explants. Screening cell lines On selecting the cell line that will be used for the production of the desired metabolite, researchers should optimise many factors such as initial high level of the secondary metabolites, growth rate, culture stability, localisation of the metabolite and responsiveness of the culture to the planned yield enhancement strategy (Shuler 1999). An important term ‘clonal selection’ refers to the production of a population of cells all possessing the same trait. The emphasis is always given to the level of the product; however, the growth rate of the culture is very important for an economic process. Instability of the culture may result from genetic or epigenetic factors. Epigenetic factors are caused by alterations in the environment and do not result in permanent change in cell genome.
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HO
O
O
CH2OH HO
OCH3
O OH
O
CH2OH OCH3
O OH
OH
OH
OH O
OH O Silybin A
Silybin B OCH3
OCH3 O
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HO
O
OH HO
O
O
O
O
CH2OH
CH2OH
OH
OH OH O
OH O Isosilybin A
Isosilybin B OH
OH
O
O HO
OH
OH
O OH
HO
OH
O
CH2OH OCH3
OH
CH2OH OCH3
OH O
OH O
Isosilychristin
Silychristin
OH OCH3
O O HO
O OH OH OH O Silydianin
Figure 1. Chemical structures of silymarin flavonolignan components.
Manipulation of the components of the culture medium Plant tissue culture media include some or all of the following components: macronutrients, micronutrients, vitamins, amino acids, carbon source, growth regulators, solidifying agent and undefined organic supplements (Saad & Elshahed 2012). The most frequently used media are Murashige and Skoog (MS) medium (Murashige & Skoog 1962), Linsmaier and Skoog medium (Linsmaier & Skoog 1965), Gamborg medium (Gamborg et al. 1968) and Nitsch and Nitsch medium (Nitsch & Nitsch 1969).
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The concentration of some of the media components is usually manipulated to provide requirements for optimal growth and secondary metabolite production. In case of S. marianum cell culture, production of silymarin in cell cultures of S. marianum was studied using different concentrations of KNO3, KH2PO4, Feþ2 and Caþ2 (Cacho et al. 1999). An increase in the concentration of CaCl2 reduced the content of silymarin. Removal of CaCl2 induced significant increase in the accumulation of silymarin and its precursors but reduced the growth of the cells. Removal of other salts such as KNO3 and KH2PO4 also reduced the growth. Elimination of iron from the medium led to a total loss of cell viability. The effect of calcium deprivation on cell growth and silymarin production by S. marianum cell cultures was studied (Cacho et al. 2013). It was concluded that the removal of calcium from the medium advanced the onset of stationary phase of growth. In addition, growth inhibition was suggested to be responsible for maximum silymarin accumulation. Precursor feeding Addition of inexpensive precursors or metabolic intermediates to the culture media may increase the yield of the target metabolites. This requires a thorough knowledge of the biosynthetic pathways of the target metabolite. Phenylalanine and coniferyl alcohol, main precursors involved in silymarin flavonolignan biosynthesis (AbouZid & Ahmed 2013), were added to S. marianum cell cultures to increase silymarin production. Addition of coniferyl alcohol (46 mM) resulted in increase in silydianin level after 72 h (Tumova et al. 2006). Treating S. marianum cell cultures with phenylalanine enhanced silymarin production (Firouzi et al. 2013). Elicitation Elicitation is defined as the induction of secondary metabolite production by molecules or treatments called elicitors. Elicitors can be classified according to their origin and chemical structures as either biotic or abiotic. Biotic elicitors can be either of defined chemical composition, e.g. methyl jasmonate, or of complex nature, e.g. yeast extract, polysaccharides and glycoproteins. Abiotic elicitors can be either physical – e.g. ultraviolet irradiation, osmotic stress – or chemical – e.g. salts of heavy metals. Figure 2 shows a classification system for
Elicitors
Biotic
Defined composition
Abiotic
Complex composition
Chemical
Chitosan
Fungal homogenate
Heavy metal salts
Pectin
Yeast extract
Physical
Thermal stress Osmotic stress
Chitin
UV radiation
Methyl jasmonate
Wounding
Figure 2. Classification of elicitors used to improve secondary metabolite production in plant tissue culture according to their origin and chemical structure.
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different types of elicitors. Elicitation has been applied successfully to cultures of various plant species in the experimental-scale and large-scale reactors (Krzyzanowska et al. 2012; Rahimi et al. 2012). Secondary metabolites responsive to elicitation are usually involved in resistance against a variety of stimuli such as heat treatment, osmotic stress, wounding and fungal infection. Some of these metabolites are constitutively expressed, phytoanticipins, while others are synthesised after a pathogen attack, phytoalexins. Many factors affect the elicitation process such as elicitor specificity, elicitor concentration and culture conditions. The use of elicitor, which is not specific to the species, can lead to ineffective elicitation. There is an optimum concentration of the elicitor that can lead to maximum production of secondary metabolites. Growth stage at which the elicitor is added and medium composition can significantly affect the success of the elicitation process. Therefore, application of an effective elicitation strategy requires extensive trial and error procedures. Elicitors from different origin have been successfully used to increase secondary metabolite yield in plant tissue culture (Cai et al. 2012; Saw et al. 2012; Veerashree et al. 2012). Jasmonic acid and its methyl ester are the most popular elicitors. The mechanism of action of jasmonate was studied in Catharanthus roseus (Van der Fits & Memelink 2000). It acts by the induction of gene expression through transcription factor with a conserved jasmonate-response domain. The differential effects of several elicitors were evaluated to increase silymarin production in S. marianum cell culture (Sa´nchez-Sampedro et al. 2005). The elicitors tested were chitosan, chitin, yeast extract, salicylic acid and methyl jasmonate. Salicylic acid, chitin and chitosan used at different concentrations did not stimulate the accumulation of silymarin. This emphasises the importance of selecting specific elicitor for each plant species. Yeast extract at an optimum concentration of 50 mg/mL added to 3-days old S. marianum cell cultures for 48 h was able to slightly increase silymarin content in the cells and threefold higher level accumulated in the medium. Methyl jasmonate was ineffective at 10 mM concentration. When the elicitor was added at a concentration of 100 mM to 3-days old cell cultures, it produced significant accumulation of silymarin both in the cultured cells and in the medium. Silymarin production peaked between 48 and 72 h after the addition of methyl jasmonate. Cell growth and silymarin production was increased by the addition of 0.2, 0.4, 0.8 and 1 mM Agþ to S. marianum cell suspension culture (Ashtiani et al. 2010). Treatment of the cells with 0.8 mM Agþ resulted in increase in silymarin production 30-fold higher than the control. Organ culture Organ cultures are used to avoid the main limitations of instability and low product yield associated with cell cultures due to lack of differentiation and organisation. Production of secondary metabolites in tissue cultures is usually higher when plant cells are organised into tissues/organs (Misawa 1994). The expression of secondary metabolic pathways in organised cultures is not surprising because it mimics exactly what the plant does. Shoot culture S. marianum multiple shoot culture was established and maintained on MS media containing 2isopentenyl adenine as cytokinin. The cultures were able to accumulate silymarin (0.437% DW), of which silybin A and silybin B were the main components (El Sherif et al. 2013). Root culture These are the roots excised from sterile plantlets and continuously cultured on hormonal media. Root cultures are typical examples of organ cultures that can be used for the production of
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phytochemicals. Root cultures have been used as standard experimental system in studies of inorganic nutrition, nitrogen metabolism, plant growth regulation and root development. The slow growth of the intact plant root remains the main disadvantage of this system (LoyolaVargas & Miranda-Ham 1995). S. marianum roots were cultured in liquid MS media on a rotary shaker at 100 rpm (Elwekeel et al. 2012). The major flavonolignans produced by the cultured roots were silychristin and silydianin. Neither silybin nor isosilybin were detected in the cultured roots. Elicitation of the root culture with methyl jasmonate (100 mM) increased the content of the produced flavonolignans to approximately 300% of the control culture.
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Hairy root culture Hairy roots are obtained by genetically transforming plant tissues with the natural genetic engineer Agrobacterium rhizogenes. A part of the bacterial DNA (T-DNA) is integrated and expressed into the plant genome. This T-DNA is present in the bacterial Ri (root-inducing) plasmid. Hairy root cultures have been known for more than three decades and developed from more than 100 plant species (Georviev et al. 2007). It offers many advantages for secondary metabolite production compared with the low yield level obtained with plant cell cultures. Among these advantages are fast growth, ability to grow from low inoculum, genetic and biochemical stability, and ability to synthesise secondary metabolites at levels similar to or higher than that produced by the mother plant. Recent advances in bioreactor design allowed hairy root technology to be scaled up. Hairy roots can be cultured under optimal conditions using mist bioreactors developed by RooTecw (Witterswil, Switzerland). Hairy root culture of S. marianum was established to be used as an alternative source for the production of silymarin flavonolignans (Alikaridis et al. 2000; Rahnama et al. 2008). Eight hairy root lines were cultured in liquid MS medium. These root lines showed significant differences in biomass and silymarin flavonolignan production. Surprisingly, the production of silymarin from these organised cultures was comparable or lower than that from cell cultures (Table 1). Metabolic engineering The accumulation of secondary metabolites can be enhanced by increasing the flux through the biosynthetic pathway to the desired product, increasing the number of producing cells and decreasing the catabolism of the desired product (Verpoorte et al. 1999). Increasing the flux to Table 1. Silymarin production in S. marianum tissue culture using different yield improvement strategies. Plant/culture Fruits Cell culture Cell culture Cell culture Cell culture Shoot culture Intact roots Hairy roots Hairy roots Hairy roots Transgenic HR Hairy roots a
Silymarin content (mg/g DW ^ SE) 23.40 ^ 3.3 (1.5– 3%) 0.31 ^ 0.0 0.71 ^ 0.0 6.34 ^ 0.07 11.50 ^ 0.07 0.437a 0.05 ^ 0.00 0.11 ^ 0.00 1.89 ^ 0.02 1.20 ^ 0.00 0.87 ^ 0.05 0.22 ^ 0.00
Yield improvement approach
Reference
Precursor feeding Change in media (- Caþ2) Elicitation (100 mM MeJ) Organ culture Organ culture Organ culture Organ culture þ SA Organ culture þ Agþ Metabolic engineering Scale up þ MeJ
Cacho et al. (1999) Firouzi et al. (2013) Firouzi et al. (2013) Cacho et al. (1999) Sa´nchez-Sampedro et al. (2005) El Sherif et al. (2013) Elwekeel et al. (2012) Rahnama et al. (2008) Khalili et al. (2009) Khalili et al. (2010) Rahnama et al. (2013) Rahimi et al. (2012)
Calculations were made as % DW. SEs are available for individual silymarin components in the referenced report.
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the desired product can be achieved by genetic engineering of the biosynthetic pathway leading to the desired product. This requires thorough knowledge of the enzymatic and genetic constitution of the pathway. The enzymes catalysing the rate-limiting step can be expressed from a stronger promoter or a heterologous gene encoding a similar step can be introduced into the plant cells. An example to the use of this approach is the over-expression of chalcone synthase in S. marianum hairy root culture (Rahnama et al. 2013). This enzyme catalyses a key step in the flavonoid biosynthesis pathway. It is involved in the biosynthesis of the flavonoid taxifolin, the immediate precursor of silymarin flavonolignans (Figure 3) (AbouZid & Ahmed 2013). Taxifolin is converted into silymarin flavonolignans by oxidative coupling to coniferyl alcohol. Hairy root culture harbouring chalcone synthase transgene was established on liquid MS media. COOH COOH
COOH
NH2 O2
PAL
NADPH OH p-coumaric acid
Cinnamic acid
L-Phe
O2 CoA ligase
NADPH COOH
OH CoAS HO 3 x malonyl-CoA
O
OH Caffeic acid
Chalcone synthase
SAM
OH HO
COOH
OH
OH
O MeO OH
HO
OH Ferulic acid
O
OH
CH2OH
O OH
O2 HO
O OH MeO OH OH
O
Taxifolin
OH Coniferyl alcohol
Figure 3. Chalcone synthase, a key enzyme in the pathway leading to silymarin flavonolignans.
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Silymarin content in the hairy roots harbouring the transgene was seven-fold higher than the non-transgenic hairy roots. Multiple strategies Application of more than one of the previously mentioned strategies may result in increase in yield of secondary metabolites in plant tissue culture and bring the process to a commercial level. For example, salicylic acid was used to increase the content of silymarin in hairy root culture of S. marianum (Khalili et al. 2009). This elicitor was added to 28-days old hairy roots for 24 h at 6 mg/50 mL culture. There was 2.52-fold increase in the silymarin content compared with control culture. It was observed that the hairy roots treated with salicylic acid grew slower than the non-treated hairy roots. Effect of elicitation of S. marianum hairy roots with Agþ was reported (Khalili et al. 2010). Agþ was added to 28-days old hairy roots at different concentrations. Hairy roots treated with 2 mM Agþ for 96 h showed the highest silymarin production (1.2 mg/g DW). Scaling up cultivation of S. marianum hairy roots was carried out in a bioreactor (Rahimi et al. 2012). Accumulation of silymarin increased from 0.13 mg/g DW in control cultures to 0.22 mg/g DW in cultures treated with 100 mM methyl jasmonate for 72 h. Conclusion Plant tissue culture can be a promising source for a continuous, standardised and stable production of useful secondary metabolites. Several approaches have been applied to increase the yield of secondary metabolites produced by plant tissue culture. Elicitation and metabolic engineering of the biosynthetic pathways leading to the target metabolites in organised cultures has proven to produce the highest increase in yield. This was clearly shown in the production of flavonolignans from S. marianum tissue culture. Combining several approaches may achieve the improvement in yield required to apply an economical process on a commercial scale. In addition, understanding metabolic regulation of secondary metabolite biosynthesis on the molecular level will greatly help in such efforts. References AbouZid S, Ahmed O. 2013. Silymarin flavonolignans: structure–activity relationship and biosynthesis. In: Atta-urRahman, editor. Studies in natural products chemistry. Vol. 40. Oxford (UK): Elsevier; p. 469– 484. Alikaridis F, Papadakis D, Pantelia K, Kephalas T. 2000. Flavonolignan production from Silybum marianum transformed and untransformed root cultures. Fitoterapia. 71:379–384. Ananga A, Georgiev V, Ochieng J, Phills B, Tsolova V. 2013. Production of anthocyanins in grape cell cultures: a potential source of raw material for pharmaceutical, food, and cosmetic industries. Rijeka, Croatia: InTech. Ashtiani SR, Hasanloo T, Bihamta MR. 2010. Enhanced production of silymarin by Agþ elicitor in cell suspension cultures of Silybum marianum. Pharm Biol. 48:708–715. Cacho M, Mora´n M, Corchete P, Ferna´ndez-Ta´rrago J. 1999. Influence of medium composition on the accumulation of flavonolignans in cultured cells of Silybum marianum (L.) Gaertn. Plant Sci. 144:63–68. Cacho M, Torres Domı´nguez A, Elena-Rossello´ JA. 2013. Role of polyamines in regulating silymarin production in Silybum marianum (L.) Gaertn (Asteraceae) cell cultures under conditions of calcium deficiency. J Plant Physiol. 170:1344–1348. Cai Z, Kastell A, Mewis I, Knorr D, Smetanska I. 2012. Polysaccharide elicitors enhance anthocyanin and phenolic acid accumulation in cell suspension cultures of Vitis vinifera. Plant Cell Tissue Organ Cult. 108:401– 409. El Sherif F, Khattab S, Ibrahim AK, Ahmed SA. 2013. Improved silymarin content in elicited multiple shoot cultures of Silybum marianum. Physiol Mol Biol Plant. 19:127–136. Elwekeel A, AbouZid S, Sokkar N, Elfishway A. 2012. Studies on flavonolignans from cultured cells of Silybum marianum. Acta Physiol Plant. 34:1445–1449. Elwekeel A, Elfishway A, AbouZid S. 2012. Enhanced accumulation of flavonolignans in Silybum marianum cultured roots by methyl jasmonate. Phytochem Lett. 5:393–396.
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