Abstract
Centella asiatica (L), a herbaceous plant belonging to the family Apiaceae, possesses great medicinal value owing to the presence of important and characteristic triterpenoids as secondary metabolites. These triterpenoid secondary metabolites are found in leaves in substantial quantities whereas negligible amounts may be detected sometimes in root tissues. In the resent study direct rhizogenesis was induced from C. asiatica leaf explants using different concentrations and combinations of auxins (IBA/IAA/NAA) leading to the production of distinct root morphotypes. A number of culture conditions such as pH, nature of carbon sources (glucose, fructose, mannitol and maltose) as well as concentrations of sucrose exhibited their strong influence in terms of induction of root morphotypes, accumulation of total secondary metabolites and expression of key pathway genes. Phytochemical profiling using HPLC revealed that all root morphotypes accumulated enhanced amounts of triterpenoids. The enhanced phytochemical accumulation was further validated by the coherent pattern of expression of key genes related to their biosynthetic pathway in root morphotypes. The results have revealed that the hormonal combinations in the culture media not only mediated differential morphogenic responses but also regulated secondary metabolites accumulation in non-transgenic rhizogenic roots. The results of the study are promising for the utilization of such in vitro root morphotypes. The root morphotypes may act as alternative bioresources for the production of industrially important and leaf associated asiaticosides and other important triterpenoids for the commercial purposes.
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Introduction
Centella asiatica L. has been used as a medicinal herb in India, China, Srilanka, Nepal, Madagascar and other countries. The plant belongs to the family Apiaceae and is commonly known as Indian pennywort, madukparni or gotu kola. It is one of the most important herbs for medicinal benefits like treating skin problems, healing wounds, revitalizing nerves and brain cells (Aziz et al. 2007). The medicinal properties are because of the presence of pentacyclic triterpenoid saponins collectively known as centellosides and asiaticosides. These bioactive pentacyclic C30 phytochemicals are asiatic acid, madecassic acid and their glycosides asiaticoside and madecassoside putatively synthesized via mevalonic acid pathway (Fig. 1). Leaves of the plant are chiefly valued for their medicinal properties because of high concentrations of the triterpenoids in the tissue (Aziz et al. 2007). Recently, our group has sequenced and analyzed C. asiatica leaf transcriptome, and identified transcripts associated with regulatory and structural function as well as biosynthetic pathways with special relevance to secondary metabolism (Sangwan et al. 2013). Earlier studies have reported that the triterpenoid content was higher in callus and suspension cultures of C. asiatica than in leaves and it varied with different varieties and collections (Randriamampionona et al. 2007). Several methods have been employed to enhance the secondary metabolite production such as using elicitors like methyl jasmonate, salicylic acid etc. in the culture conditions. Moreover, genotypes of C. asiatica have also shown considerable influence on the formation of callus, adventitious roots and secondary metabolite accumulation (Mercy et al. 2011, 2012; Chaurasiya et al. 2007). Native roots of C. asiatica are reported to possess trace amounts of triterpenoids (Aziz et al. 2007). Previous studies on medicinal plants have shown expression of pathway genes and accumulation of secondary metabolites could be well co-related (Sangwan et al. 2007, 2008). In the present study we targeted to induce generation of diverse root morphotypes of C. asiatica with enhanced ability to produce secondary metabolites. For molecular validation of altered secondary metabolite synthesis by root morphotypes, key genes of biosynthetic pathway were also analyzed for their expression levels. The aim of this study was to explore an alternative strategy to produce enhanced levels of asiaticosides and other related triterpenoidal phytochemicals in C. asiatica in a non-transgenic and direct rhizogenesis manner. The investigation could reveal hormone induced enhancement up to several folds for the production of commercially important triterpenoid secondary metabolites in rhizogenic roots in C. asiatica.
Materials and methods
Plant material
The nodal segments of Centella asiatica were collected from the plants maintained in the glass house of CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow (India) and used as explants for in vitro shoot generation.
Induction and proliferation of multiple shoots
Nodal segments were thoroughly washed under running tap water for 2 h, followed by 10 % (v/v) detergent (Teepol™) for 5 min, surface-sterilized with 0.1 % (w/v) mercuric chloride for 7 min, rinsed three times with sterile distilled water and then inoculated on Murashige and Skoog (MS) medium supplemented with different cytokinins such as BAP (6-benzyl aminopurine at 0.5, 1, 2 and 4 mg l−1 concentrations) alone and in combination with kinetin (0.5, 1, and 2 mg l−1).
Direct rhizogenesis from leaves
Leaf explants from in vitro generated shoot culture of C. asiatica were used for direct rhizogenesis. Leaves were inoculated on MS media at half and full strength supplemented with four different auxins, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and α-naphthalene acetic acid (NAA) as presented in Table 1. IBA was used alone or in combination with IAA and NAA. Each leaf was placed on medium with their abaxial surface in contact with the medium.
Optimization of culture conditions for secondary metabolite production
Following physical factors of culture conditions were modified to evaluate their effect on secondary metabolite production in adventitious roots directly induced on leaves of C. asiatica. Media of different pH (4.0, 4.5, 5.0, 5.3, 5.6, 5.9, 6.2, 6.5 and 7.0) were prepared and used for inducing direct rhizogenesis. Various concentrations (1, 2, 3, 4 and 5 %) of sucrose were used as carbon source in the MS media to observe their effect on secondary metabolite production. Also, other carbon sources (3 % each of sucrose, glucose, fructose, maltose and mannitol) were used for evaluating their affect on triterpenoid accumulation in different root morphotypes.
All the culture media contained 3 % (w/v) sucrose (except the experiments where the effect of different concentration of sucrose and of various carbon sources was planned to be monitored) and solidified with 0.8 % (w/v) agar. The pH of media was adjusted to 5.6 (except where the experiment involved studying the effect of pH) prior to autoclaving at 121 °C and 15 lb/inch2 pressure. Each set of combinations consisted of six replicates and were performed as three independent experiments. The cultures were evaluated after 45 days in terms of numbers and height of shoots/roots per explant. All the cultures were maintained at 25 ± 1 °C under 14 h photoperiod (30 µmol m−2 s−2) supplied from cool white fluorescent tube.
Extraction and analysis of secondary metabolites
For extraction of triterpenoidal secondary metabolites such as centellosides, asiaticosides etc. from the root morphotypes, 1.0 g of the dried tissue were harvested and crushed in liquid nitrogen to fine powder and soaked in 5 ml methanol for 2 h with shaking on a Rocker platform. Methanolic extract was filtered and the filtrate was saved and residue was subjected to re-extraction for 2 h as above using the same volume of the solvent. The filtrates were pooled and partitioned with equal volume of n-hexane and n-hexane layer was discarded to remove fatty substances. The methanolic phase was retained and evaporated till dryness, collected in 1.5 ml eppendorf tube and dissolved in HPLC grade methanol for HPLC analysis. The standard methanolic stock solutions of asiaticoside, asiatic acid, madecassoside and madecassic acid were prepared at the concentration of 1 mg ml−1. Three concentrations (each in triplicate) 5, 10 and 15 µl of the standard phytochemicals were subjected to HPLC under optimized conditions of resolution to generate calibration curves for quantitative estimations. HPLC analyses were carried out on a Waters HPLC system that comprised of a high pressure constant flow pump (600E), auto sampler injector (20 µL) and Waters 2996 dual λ absorbance detector and chromatographic separation was performed with a Waters C18 column with pore size of 4 μm and UV detection at 216 nm. Samples were run in triplicate, using modified protocol as described earlier (Rafamantananaa et al. 2009).
Semi-quantitative and quantitative real time expression analysis
Gene expression analysis was carried out for four key triterpenoid pathway genes viz. hydroxyl methyl glutaryl Co-A reductase (CaHMGR), farnesyl pyrophosphate synthase (CaFS), squalene synthase (CaSS) and squalene epoxidase (CaSQE) by semi-quantitative and quantitative real time PCR (Step one™ Real time PCR system, Applied Biosystems) in selected C. asiatica root morphotypes induced under various culture conditions e.g. concentration of auxins, sucrose, different pH and carbon source. Total RNA was extracted from tissues by TRI reagent (InVitrogen) according to manufacturer’s instructions. RNA was quantified by NanoDrop ND-1000 (Nanodrop Technologies) and verified for integrity by electrophoresis on a 1.2 % (w/v) agarose gel. Equal amounts of DNase I (Fermentas) treated total RNA (5 μg) were used to synthesize first strand cDNA by Revert Aid™ cDNA synthesis kit (Fermentas) as per the manufacturer’s instructions. cDNA was further diluted and used as template in quantitative real time PCR (qRT-PCR) and semi quantitative PCR using gene specific primers. Primers were designed using sequences available from transcriptome by Beacon designer™ 8.0 software. All the reactions were carried out in triplicates with two biological repeats. Actin was used as an endogenous control for normalization of the relative quantitative estimation. Real time PCR amplification was performed using SYBR Green ROX mix (Applied Biosystems) using 5 pmol of each primer as described earlier (Sangwan et al. 2013).
Results
Effect of plant growth regulators on direct rhizogenesis and generation of root morphotypes
All the tested hormonal combinations were found to induce direct rhizogenesis on the leaves of C. asiatica. Half strength MS medium was found to be more suitable in terms of growth responses (data not shown). The ratio and concentrations of different auxins and their effect root morphology and their triterpenoid content are presented in Table 1. The roots, finest in appearance were produced in the media containing different concentrations of IBA alone. Increase in the IBA concentration in the medium resulted in decrease in the number as well as length of the roots (Fig. 2A). The roots induced at higher IBA concentrations appeared harder in texture and were bushy in shape. When the rhizogenesis was induced using combinations of IBA + IAA at lower concentrations, finer, longer and green roots were produced. However, with increase in concentrations of IBA and IAA roots become harder and bushy (Fig. 2A). The combination of IBA + NAA in the medium for rhizogeneis exhibited entirely different morphology. IBA in combination with NAA, showed no significant effect on number of induced roots but root color and texture exhibited interesting observations. The color of roots induced with IBA and NAA combinations was brownish white or white. On lower concentrations of IAA and NAA, the roots induced were curved and on increment of hormone concentration, roots turned bushy. Similarly, root morphoforms differeing in the growth, appearence and textures were induced under treatment with pH, sucrose concentrations and different carbon sources in the media (Fig. 2B–D).
Effect of plant growth regulators on secondary metabolite accumulation in induced rhizogenic root morphotypes
To understand the characteristics of roots produced on supplementation of the medium with different concentrations and combinations of different auxins, production of four major triterpenoids viz. asiaticoside, asiatic acid, madecassoside and madecassic acid. The data obtained from phytochemical analysis of root morphotypes indicated that the levels of secondary metabolite were highly influenced by plant growth regulators. Control roots grown without any growth regulators in the medium accumulated asiatic acid at low concentration (1.33 ± 0.003) which was enhanced (3.46 ± 0.041) in the presence of IBA (2 mg l−1). The maximum accumulation of the secondary metabolites was found in the medium containing IBA (1 mg l−1) followed by IBA + IAA (1 mg l−1 each) i.e. 11.93 ± 0.106 and 11.47 ± 0.044 mg g−1 DW, which was about eightfold higher than control roots. All other combinations also exerted marked effect on enhanced triterpenoids accumulation as compared to the roots grown on medium devoid of any growth hormones (Fig. 3A). The different combinations of auxins induced accumulation of variable amounts of individual bioactive compounds. On increasing the IBA concentration from 1 to 3 mg l−1, asiaticoside content increased but it decreased at further higher concentrations (4–5 mg l−1). Quantitatively, the amount of asiatic acid remained either higher or equal to asiaticoside, indicating that this glycon-aglycon pair of pentacyclic triterpenoid was dynamically inter- converted with flux governed by balance of glycosyltransferase and glycosidase activities. At this range of auxin concentrations, IBA was the most favorable for production of the highest amount of madecassic acid. Madecassic acid and madecassoside also appeared inter-convertible as high amount of madecassic acid resulted in low accumulation of its glycoside (madecassoside). It could be inferred that madecassic acid glycosides were less in their usual steady state levels as only limited proportion of it was converted to its glycoside, probably due to limiting level/efficiency of the relevant glycosyltransferase. The ratios of glycosides to aglycons appeared to have a particular pattern throughout the alterations in the media compositions for rhizogenesis. When glycosides were noted to be present in lower amounts, their aglycons were found to be present in higher amounts and vice versa. This suggested that the hormonal combinations regulated bioactive compounds by controlling flux movement from active to storage form through modulations in the catalytic activities of the relevant enzymes by mediating the effect at transcriptional and/or translational level.
Effect of various physical factors on rhizogenesis and secondary metabolite accumulation
IBA concentration at 1 mg l−1 was observed to be the best for enhancing levels of total secondary metabolites. Therefore, this concentration was used to evaluate the effect of various physical factors on rhizogenesis and secondary metabolite production.
Experiments related to effect of pH of the medium revealed that the texture of the roots was affected by pH (Fig. 3B). At lower pH (4.0 to 5.0), the roots were fine in texture with small length and bushy growth morphoform. Whereas, at the pH range of 5.3–6.2, the roots were fine in texture and longer in size. Further, higher pH (6.5 to 7.0) of the medium led to induction of bushy, small and hardened roots. Total metabolite accumulation was found at highest concentration in the tissue (11.93 ± 0.106 mg g−1 DW) at routinely used pH (pH 5.6). Higher pH (5.9 to 7.0) of the medium resulted in gradual decline in metabolite accumulation (7.86 ± 0.101 to 1.78 ± 0.015 mg g−1 DW) in the root tissues. Whereas, lower pH (acidic range) of the medium triggered decrease of accumulation of secondary metabolites (3.28 ± 0.036 mg g−1DW). However, when individual metabolite accumulation was analyzed, an interesting pattern appeared, indicating that accumulation of certain metabolites was also controlled by pH.
An interesting pattern of changes in the secondary metabolite accumulation was observed with the change in the nature of carbon sources used in the medium. Glucose appeared to be more suitable carbon source than sucrose for accumulation of the total secondary metabolites, the levels in the tissue noted to be 17.92 ± 0.058 and 11.93 ± 0.106 mg g−1 DW, respectively. Fructose and maltose induced far lower level of accumulation of the triterpenoid secondary metabolites whilst mannitol failed to induce rhizogenesis at all. Roots generated in response to supplementation of the medium with sucrose and glucose were long, green and fine in texture, whereas shorter length roots were induced with fructose and maltose as carbon source. Sucrose is the most common carbon source used for plant tissue and cell cultures, serving as the principal energy source as well as serving as a carbon source (as intermediary metabolites of its catabolic pathway) for secondary metabolite biosynthesis. Sucrose at 4 % concentration was found to be the most suitable for the highest accumulation (17.01 mg g−1 DW) of total content of triterpenoids (Fig. 3C). Whereas, sucrose at lower and higher concentration in the medium decreased accumulation of triterpenoid secondary metabolites (Fig. 3).
Expression analysis of key pathway genes
Initially, gross pattern of expression of the selected key genes of metabolic pathway related to the triterpenoid biosynthesis was investigated in different morphotypes through semi-quantitative PCR using gene specific primers (Fig. 4A–D). The quantitative assessment of these changes in the levels of transcripts of these genes was obtained by qRT-PCR in relation to their association with changes in the secondary metabolites accumulation. Expression of all the four pathway genes (CaHMGR, CaSQE, CaSS and CaFS) were maximal in the root morphotypes induced using IBA (1 mg l−1) followed by roots obtained with IBA/IAA (1 mg l−1 each), IBA (3 mg l−1), IBA/IAA (4 mg l−1) and IBA/NAA (2 mg l−1) as compared to control in vivo roots (Fig. 5A–D). Relatively lower (two–five folds) expression of the pathway genes was observed in root morphotypes grown at different pH (Fig. 5E–H). Maximal gene expression was observed in root morphotypes obtained with media pH of pH 5 and 6.2 and minimal in those with medium having pH 4 and 7. For control conditions, the culture medium of pH 5.6 was used and it was noted to have maximum level of the secondary metabolites. Expression profiles of the pathway genes displayed smaller differences in response to increasing concentration of sucrose. At 4 % sucrose concentration, the gene expression levels were higher but the elevation was not more than five folds in any of the genes (Fig. 5I–L). Abundance of transcripts of these selected genes was higher when glucose was used as carbon source in the medium than with sucrose and fructose. It was least with the use of maltose as carbon source (Fig. 5M–P). In general, the in vitro roots grown on glucose exhibited two–ten fold higher expression of the genes. When sucrose was used as a carbon source, CaHMGR and CaFS genes were expressed at almost same level as in case of glucose, however expression of CaSQE and CaSS dropped by about 2.5 fold when sucrose replaced glucose as the carbon source in the medium.
Discussion
Auxins are responsible for maintaining plant cell and tissue culture systems, and are associated with the promotion of growth, callus proliferation, rooting and tissue morphological diversity. Among auxins (IAA, IBA and NAA) studied in this investigation with respect to their effect on induction of rhizogenesis in Centella asiatica leaf, and on morphological and metabolic features of these in vitro roots, IBA was found to be the most effective for the root induction. IBA in combination with IAA was found to be the best combination with respect to induction of maximal number of roots with substantial root length. Several studies have shown that IBA may remain stable for longer time than NAA. Thus IBA may be more efficient auxin for proper functioning in the cells by staying stable in the medium for longer durations. Also, there are several reports that suggest that lower concentrations of IBA are more suitable to induce larger number of roots by increased cytosolic streaming than that at higher concentration of IBA (Tominaga et al. 1998). Higher levels of auxins have also been reported to induce higher levels of degraded metabolites that inhibit the root formation. Lower concentrations of plant growth regulators are known to induce longer roots with their finer texture whilst higher concentrations trigger formation of shorter roots with hardened texture in Hypericum perforatum (Cui et al. 2010). Reports also suggest that non-transformed tissues are more sensitive to exogenous auxin applications and eventually leading to generation of their diverse morphotypes than transformed tissues (Koperdakova et al. 2009). Half strength of MS salts was better than full strength for achieving rhizogenesis in the leaves of C. asiatica similar to our observations in case of W. somnifera (Sabir et al. 2013). The lower salt concentrations may enhance the nutrient availability and mobilization, whilst higher salt concentrations may cause lowering of water potential to the extent that it may in turn result in poor water and nutrient absorption leading to restricted root growth The thresholds of these promoting and limiting effects of salt concentrations may also vary with the species and tissues (Cui et al. 2010; Sabir et al. 2011; Sabir et al. 2013).
Phytochemical analysis of leaf rhizogenesis derived root morphotypes of Centella asiatica clearly establishes the strong effect of plant growth regulators on secondary metabolite accumulation. Although, reports are available on the effect of plant growth regulators on secondary metabolite content in C. asiatica (Yoo et al. 2011) but the studies so far have remained limited to in vitro shoots. IBA has been shown to stimulate accumulation of phenolics and flavonoids in the roots of H. perforatum (Cui et al. 2010). Similarly, studies on hairy root cultures of Nepeta cataria also suggest that lower concentrations of IBA amongst the auxins (IBA, IAA and NAA) induced better root growth and rosmarinic acid accumulation (Yang et al. 2010). Addition of higher concentration of BAP in presence of lower concentration of IBA has been shown to be beneficial for secondary metabolite production in Thymus vulgaris (Karalija and Parić 2011). Contrarily, enhanced accumulation of total phenolics at high concentration of IBA has been noted hairy roots of Panax ginseng, although growth of the roots was inhibited. While IAA has been shown to induce better root growth but it was not accompanied by a significant increase in secondary metabolite synthesis (Jeong et al. 2007). Similarly, high concentrations of IBA have been demonstrated to be able to enhance ginsenoside production in P. ginseng suspension cultures (Lian et al. 2002).
Our results also demonstrate that presence of NAA had a negative effect on biomass and secondary metabolite production in in vitro generated roots of C. asiatica similar to the reports on Rauwolfia serpentina and H. perforatum (Pandey et al. 2010; Cui et al. 2010). Interestingly, it has been reported that, in planta, exogenous auxins did not affect overall asiaticoside content (Kim et al. 2004). This detrimental effect of auxins on secondary metabolite production in C. asiatica in planta may have been due to decreased asiaticoside accumulation in storage organs or to obstructed formation of shoots that normally contained abundant asiaticoside. We can correlate our findings such that, auxins inhibited the growth of shoots and eventually secondary metabolite accumulation declined in shoots but at the same time if auxin triggers the root formation without shoots (site of abundant asiaticoside production), it induces secondary metabolite accumulation in the roots. Previous reports suggest that pH of the medium can influence direct root formation (rhizogenesis) on Nautilocalyx leaf segment (Venverloo 1976). A slightly acidic pH seems to be preferred by most species because acidity is necessary for auxin action. The analysis of effect of lower medium pH (4.0 to 7.0) on secondary metabolite accumulation in C. asiatica revealed that highest accumulation occurred at pH 5.6 of the medium. In W. Somnifera cell suspension cultures maintained at pH 6.0 have proved to be best for high withanolide accumulation (Sabir et al. 2011) whereas, in Bacopa monnieri shoot cultures, initial medium pH set at 4.5 has been reported to be most suitable for biomass accumulation and bacoside A production (Naik et al. 2010). These differential effects may be manifested due to variations in the catalytic properties of the enzymes with respect to pH of the medium as also through the effects originating at the level of the expression of genes involved in biosynthesis and/or regulation of secondary metabolites (Deduke et al. 2012).
Sucrose is the most routine carbohydrate used as carbon source in cell and tissue culture, considering as the primary and transportable energy source and primary source for secondary metabolite biosynthesis (Singh and Luthra 1988). Therefore, changing the sucrose concentration may influence the secondary metabolite accumulation. Among various concentrations tried, 4 % sucrose in the medium resulted in maximum secondary metabolite accumulation. While other studies reported use of 3 % sucrose as optimum concentration for secondary metabolite accumulation in W. Somnifera (Sabir et al. 2013) and P. ginseng (Jeong et al. 2007). Contrarily, no requirement of sucrose was observed for high accumulation of secondary metabolite in B. monnieri (Naik et al. 2010). The carbon sources serve as energy and osmotic agents to support the growth of plant tissues. In addition, growth and root initiation are highly energy requiring processes that can occur at the expense of available substrates metabolizable through energy yielding pathways, which are mainly carbohydrates. Incidentally, intermediary metabolites of energy yielding carbohydrate oxidation pathways also serve as precursors (e.g. acetyl coenzyme A, phosphoenol puruvate, erythrose-4-phosphate, pyruvate, glyceraldehyde-3-phosphate, amino acids) of secondary metabolites. The most general use of sucrose as carbon source in tissue culture studies is due to its efficient uptake across the plasma membrane and its ease of transportability through phloem of most plants. Glucose has also been reported to have various effects on the in vitro growth of plants. In case of Pinus pinea glucose proved better carbon source for root induction than sucrose (Zavattieri et al. 2009). In the present study, glucose was found to be more suitable than other carbon sources tested (sucrose, glucose, fructose, maltose and mannitol) for the secondary metabolite accumulation in C. asiatica. Artemisinin accumulation in Artemisia annua hairy roots and seedling cultures has been shown to be influenced by the type of carbon source (Wang and Weathers 2007). Different accumulation pattern in the presence of individual sugars might be due to differential signaling levels of sugars.
Auxins play a crucial role in growth and development of a plant by regulating and influencing several diverse functions such as apical dominance, root initiation, cell extension, division and differentiation. Many auxin response factors (ARFs) which on interacting with auxin regulate genes related to several physiological and biochemical processes of higher plants (Chapman and Estelle 2009). These include various transcription factors involved in the signal pathways responsible for root formation. Carbohydrates play important roles, not only by providing energy and carbon chains for biosynthetic processes in new meristems and roots, but also by affecting gene expression, in co-action with auxin. Utilization of carbon source is also metabolically linked to the biosynthesis of secondary metabolites (Singh and Luthra 1988; Luthra et al. 1993). Growth and differentiation of tissues can be modulated by carbohydrate signals through alterations in metabolic fluxes and carbohydrate concentrations during development which may regulate gene expression (Dacosta et al. 2013; Yadav et al. 2014). This study recognizes glucose as the best carbon source for the enhanced production of secondary metabolites in C. asiatica. Similarly, silymarin production in Silybum marianum has been shown to be enhanced with the use of glucose as carbon source (Al-Hawamdeh et al. 2013). However, mannitol was not effective in inducing root formation in C. asiatica, similar to the case with Chrysanthemum. Mannitol, the sugar alcohol, does not metabolize through core primary carbon metabolism yielding energy and biosynthetic precursors (Da Silva 2004). Sugar alcohols are perceived by cells as chemical signals, with very high in vitro concentrations acting as chemical stress agents (Steinitz 1999). Contrary to our report, Kagmi (1999) reported that the shoots of Japanese persimmon were able to grow better when cultured on autoclaved half strength Murashige and Skoog medium containing 0.2 M fructose. Roots have been shown to be induced in 94 % of the shoots after 45 days of culture period whereas only 30 % of roots were obtained on media containing the same concentration of sucrose or glucose (Kagmi 1999). In case of A. annua as well, artemisinin biosynthesis has been demonstrated to be regulated differentially under different carbon levels (Wang and Weathers 2007). Influence of carbon sources such as fructose, sucrose, glucose, sorbitol and maltose at various concentrations on rooting in apple rootstock MM106 is also available (Bahmani et al. 2009).
Elevated accumulation of asiaticoside might also be associated with larger biomass of roots. To verify the raison de etre of of metabolite level enhancements under various treatments, semi-quantitative and quantitative analysis of expression of genes relevant to asiaticoside biosynthesis pathway was performed. The expression profiles of all the key genes of the central pathway genes were in accordance with the levels of secondary metabolite accumulation under the respective treatments as also shown in recent reports (Bose et al. 2013; Yadav et al. 2014; Narnoliya et al. 2014). The root morphotypes generated under IBA (1 mg l−1) supplementation in the medium exhibited high levels of expression of the selected pathway genes. CaHMGR showed less variation in fold expression which suggested that either its expression was not be a first degree of rate limiting step. Even the first degree of limitation, if existent at this step, it may be obviated by cross-talk assisted sharing of IPPs generated by plastid operated DOXP/MEP pathway of isoprenogenesis, instead of all IPPs contributed by mevalonate pathway alone (Chaurasiya et al. 2012). The observation that slightly acidic medium (pH 5.6) was optimum for maximal triterpenoids accumulation whilst neutral pH (7.0) resulted in lower root growth and metabolites production may be because acidic medium could be required for auxins action.
Our results on in vitro roots showed that the optimum concentration of IBA (1 mg l−1) found for secondary metabolite accumulation was different from the optimum concentration (IBA + NAA at 4 and 2 mg l−1) required for biomass production. Therefore, high metabolite accumulation/yield may not directly be linked with high biomass, rather both of these parameters are regulated by hormonal combinations. The observed concordance of gene expression patterns (as obtained through both semi-quantitative PCR and quantitative real time PCR) with the patterns of secondary metabolite content in various treatments, implied that regulation/stimulation of triterpenoid biosynthesis was under hormonal combination. Thus, our results reveal the possibility of substantial stimulation of biosynthesis of triterpenoids and their saponins by various physical factors such as plant growth regulators, pH of medium, and type and concentration of carbon sources. This entails possibility of maneuver in production of asiaticoside-the secondary metabolites for commercial purposes in better yields from non-transgenic roots from native non-/poor producer tissue of C. asiatica.
Conclusions
The studies presented here exhibited that the hormonal and other growth conditions generated diverse root morphotypes as a process of rhizogenesis induction in C. asiatica leaf. The hormonal and other culture conditions of in vitro roots could be maneuvered to regulate triterpenoid biosynthesis and for production of the same secondary metabolites as usually produced by the shoots in planta and in vitro cultures of C. asiatica. The secondary metabolite production profiles were biosynthetically corroborated to the observed matching patterns of the expression levels of the selected genes related to the metabolic pathway for generation of the metabolites. Thus, the study provides promising opportunity of an alternative platform to produce enhanced levels of asiaticosides and other related triterpenoidal phytochemicals in C. asiatica in a non-transgenic and direct rhizogenesis manner. The study also revealed that triterpenoid biosynthesis and accumulation was directly under hormonal regulation and modulation in C. asiatica.
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Acknowledgments
JS and FS are thankful to CSIR, New Delhi and UGC, New Delhi for financial assistance in the form of senior research fellowship. The authors wish to express their sincere thanks to the director, CSIR-CIMAP, for constant encouragement and providing necessary facilities. The financial Grant from DBT, New Delhi to carry out above studies is gratefully acknowledged.
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Jyoti Singh and Farzana Sabir have equally contributed to this work.
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10725_2014_9931_MOESM1_ESM.ppt
Supplementary material HPLC profiles of (a-b) with respect to the four major bioactive authentic compounds resolved under standard conditions; (c), representative HPLC chromatogram of extract from the C. asiatica roots produced by induction of rhizogenesis by media supplementation with auxins (1mgl−1) IBA (PPT 277 kb)
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Singh, J., Sabir, F., Sangwan, R.S. et al. Enhanced secondary metabolite production and pathway gene expression by leaf explants-induced direct root morphotypes are regulated by combination of growth regulators and culture conditions in Centella asiatica (L.) urban. Plant Growth Regul 75, 55–66 (2015). https://doi.org/10.1007/s10725-014-9931-y
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DOI: https://doi.org/10.1007/s10725-014-9931-y