Catharanthus Roseus Alkaloids: Application of Biotechnology for Improving Yield

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Catharanthus roseus alkaloids: Application of biotechnology for improving yield

Data in Plant Growth Regulation · May 2012 DOI: 10.1007/s10725-012-9704-4

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Catharanthus roseus alkaloids: application of biotechnology for improving yield

Abdul Mujib • Abdul Ilah • Junaid Aslam • Samar Fatima • Zahid Hameed Siddiqui • Mehpara Maqsood

Received: 6 September 2011 / Accepted: 17 April 2012 Ó Springer Science+Business Media B.V. 2012

Abstract Catharanthus roseus (L.) G. Don. is a well SE Somatic embryos known medicinal plant. It produces several phyto- TDC Tryptophan decarboxylase compounds many of which show anticancerous properties. SSS Strictosidine synthetase The yields of these compounds are however, very low. In this present article, the current development of secondary metabolite synthesis in C. roseus involving biotechnology has been reviewed keeping in mind the various basic fac- Introduction tors that inﬂuence yield. The importance of cell culture, the role of culturing conditions and other approaches aiming at Catharanthus roseus (L.) G. Don. belonging to the family higher production of alkaloids have been discussed. The Apocynaceae, is an important dicotyledonous medicinal genes encoding important enzymes, proteomics, expressed plant. It has a pantropic distribution, naturalized in conti- sequence tag and transcription factors in relation to alka- nental Africa, America, Asia, Australia, Southern Europe loid yield have also been summarized in order to under- and in some islands of the Paciﬁc Ocean. The plant is stand the regulatory mechanisms of C. roseus. cultivated as an ornamental, used particularly for beddings, borders and for mass effect. It blooms throughout the year Keywords Catharanthus roseus Indole alkaloids and is propagated by seeds or by cuttings. Biotechnology In vitro culture conditions C. roseus has been studied extensively for its remarkable anticancer constituents. Vincristine and vinblastine are two Abbreviations very powerful anticancerous compounds of terpenoid- 2, 4-D 2, 4-Dichlorophenoxyaceticacid indole alkaloids class found in C. roseus. Among other BA/BAP 6-Benzyladenine components, (Table 1) ajmalicine and serpentine are used DMSO Dimethylsulfoxide in relieving hypertension and related cardiac disorders. The

GA3 Gibberellic acid yield of these two compounds (vincristine and vinblastine, HPLC High performance liquid chromatography Fig. 1) in particular, is very low, therefore is expensive. KN Kinetin This poses a serious challenge to all including biotech- MS Murashige and Skoog nologists as to improve the yield and to reduce the unit NAA a-Naphthalene acetic acid cost. In biotechnology based alkaloid enrichment program, PGR Plant growth regulator the following two approaches have currently been adopted: (1) use of superior cell lines from various sources and optimization of cultural conditions/factors that control A. Mujib (&) A. Ilah J. Aslam S. Fatima synthesis and (2) metabolic engineering and/or over Z. H. Siddiqui M. Maqsood expression of key enzymes (Van der Heijden et al. 2004). Cellular Differentiation and Molecular Genetics Section, In recent years, various aspects of C. roseus and its Department of Botany, Hamdard University, New Delhi 110062, India alkaloids were discussed (Van der Heijden et al. 1989; e-mail: [email protected] Moreno et al. 1995). In this present article, we reviewed 123 Plant Growth Regul

Table 1 Some important anticancerous alkaloids/derivatives isolated study and improve the yield where several plant parts such from cell cultures of C. roseus as stem-node, root, leaf, ﬂower, anther and seed have been Alkaloid Sources of isolation cultured as primary explants sources (Moreno et al. 1995).

Ajmalicine Callus, suspension, shoot, hairy root Alsotonine Callus Anthirine Suspension Culture of callus Cathindine Suspension Serpentine Callus, suspension, shoot, hairy root Callus is an undifferentiated tissue which is a good source Akuammicine Callus, suspension, shoot of secondary metabolites and other natural products. It is Lochneridine Callus, suspension, hairy root also used to obtain suspension of cells, a key source to get embryos in masses and help in isolating protoplasts at ease. Horhammericine Suspension, shoot In C. roseus, various explants have been tested on several Tabersonine Callus, suspension basal media with or without plant growth regulators Vindoline Suspension, shoot, crown gall (PGRs) for obtaining callus (Akcam and Yurekli 1995; Vindolinine Suspension, shoot Mujib et al. 1995; Moreno et al. 1995; Van der Heijden Catharanthine Suspension, shoot, root et al. 2004). The agar-solidiﬁed medium was found 3,4 anhydro vinblastine Shoot more responsive compared to liquid, in which 2, 4-D Leurosine Shoot (0.5–2.0 mg/L) induced fast growing callus from nearly all Catharine Shoot the explant sources. It becomes necessary to transfer cal- Vinblastine Callus, crown gall, shoot, somatic embryo luses on fresh nutrient medium for further growth. The Vincristine Shoot, somatic embryo alteration of media, its components and manipulation of Sources Van der Heijden et al. (1989, 2004); Moreno et al. (1995) PGRs are also often essential to restore growth of declining callus. afresh the current biotechnological developments that aim to enrich alkaloids in C. roseus.

Cell suspension Explant and other primary cultures Since early 1980s it has been noted that the plant cell’s Various undifferentiated and differentiated tissues such as growth is fast in agitated-suspension compared to solid callus, suspension, shoot, hairy root have been induced to medium because of easier uptake of nutrients by the

Fig. 1 Chemical structure of vinblastine and vincristine

Vinblastine

Vincristine

123 Plant Growth Regul cultured tissues. The biomass growth could easily be An optimized hairy root culture protocol was established improved by using a variety of bioreactors (Schlatmann on MS with sucrose as carbon and lactalbumin as nitrogen et al. 1994). The process has generally been accomplished source, in which, enhanced level of total alkaloids was by transferring friable (1.0 g) callus to the liquid medium noticed compared to other explants and calli (Sun and Zeng on gyratory shaker (120 rpm). At this suspension period of 2005). Treated with antagonists (verapamil and CdCl2) that growth, early sub culturing (10–15 days interval) promotes blocked Ca?2 fluxes across the plasma membrane over faster growth by forming free cells and cell aggregates as 25 % increase of total alkaloid content in hairy root cul- new cultures. The cell suspension has often been used for tures of C. roseus was observed by Moreno-Valenzuela alkaloid synthesis studies in C. roseus as the extraction of et al. (2003). Higher concentration of ajmalicine and alkaloids is far more simple, it also uptake precursors and catharanthine was found in genetically transformed Cath- other nutrients more rapidly that favours enhanced accu- aranthus hairy root cultures (Vazquez-Flota et al. 1994). It mulation of alkaloids (Junaid et al. 2009; Zhao et al. 2009). was also noted that vindoline and catharanthine producing hairy roots were thin and slow in growth (Palazoń et al. 1998). The study also indicated that early sub culturing Shoot and root culture induced fast growth of hairy tissues that accumulated enhanced level of lochnericine. Genetically transformed The cultivation of plant parts, i.e., shoot and root has also (with Agrobacterium rhizogenes) roots grew well in simple been practised for in vitro biosynthesis of secondary hormone-free medium and high accumulation of secondary metabolites (Pietrosiuk et al. 2007). A high accumulation metabolite was noticed in Catharanthus root culture. of ajmalicine in multiple shoot cultures of C. roseus was Various bioreactors and complex fermenters were tried to reported in Murashige and Skoog medium on high con- improve the biomass of hairy-roots that accumulated better centration of IAA (11.42 lM) and a low (2.22 lM) con- production of secondary metabolites (Nuutila 1994). centration of BA (Satdive et al. 2003). Vindoline synthesis Efforts to engineer C. roseus hairy roots to produce com- in multiple shoot culture of C. roseus was investigated in mercially significant amounts of valuable compounds like various (continuous light, 16 h) photoperiod regimes and vinblastine and vincristine may require the development of the authors observed that the vindoline biosynthesis was tools to study the effects of over expressing key metabolic very related with shoot morphogenesis (Campos-Tamayo and regulatory genes. In this direction, a glucocorticoid- et al. 2008). Similar protocol was earlier described where inducible and an alcohol-inducible promoter system were shoots were maintained for over a year without any tissue used to study the effects of gene overexpression within the vitrification, disaggregation or callus formation (Hernań- terpenoid indole alkaloid pathway on metabolite produc- dez-Domıńguez et al. 2006). The content of vindoline was tion (Peebles et al. 2007). The effect of the engineered quantified that reached values similar to those found in indole pathway on accumulation of phenolic compounds in leaves from field-grown plants. Leaves and flowers of two response to ectopic overexpression of tryptophan feedback- different phenotypes of C. roseus (pink flower and white resistant anthranilate synthase holoenzyme (AS alpha and flower) were examined by flow-injection electrospray beta) was analyzed in C. roseus hairy roots and it was noted ionization mass spectrometry in which variable amounts of that the uninduced and induced AS alpha and beta hairy vincristine and vinblastine were detected (Favretto et al. roots accumulated up to 1.2 and 4.5 mg/g DW respectively 2001). The agitated liquid medium was very productive for over a period of 72 h (Chung et al. 2007). multiple-shoot regeneration, growth processes and high The genetic engineering and expression of the terminal incidences of root formation were obtained on the same step of vindoline biosynthesis, deacetylvindoline-4-O- medium which is rich sources of several compounds acetyltransferase (DAT) in C. roseus hairy root cultures (Mujib et al. 1995). Yield variation was also observed in was also described (Magnotta et al. 2007). The conducted callus originated from white and rosy periwinkle. Root biochemical analyses showed that several hairy root lines callus was always found highly rich in alkaloids for the two expressed high levels of DAT enzyme activity compared to varieties compared to other sources (Marfori and Alejar control hairy root cultures. High performance liquid chro- 1993). matography (HPLC) of hairy root extracts also revealed an altered alkaloid profile with respect to ho¨rhammericine accumulation in DAT expressing lines. The growth and Hairy root culture alkaloid synthesis in C. roseus hairy root clones after Ri T-DNA gene insertion was made recently (Batra et al. Roots often contain a variety of secondary metabolites, 2004). The accumulation of considerable amounts of root- thus cultivation of hairy roots has been practised in vitro specific alkaloids ajmalicine and serpentine was observed as an alternative approach to produce these products. in the presence of both the T(L)-DNA and T(R)-DNA 123 Plant Growth Regul genes (C1) and the T(L)-DNA gene (C3) alone. The be used as raw material for secondary metabolite produc- possible role of the T(L)-DNA and T(R)-DNA genes on tion. The same technique is also used to generate trans- growth and alkaloid accumulation in these root clones was genics en masse as the embryo originates from single also described. The expression profiles of one early and embryo mother cell. In C. roseus, however, the incidence two late TIA biosynthetic pathway genes, namely, strict- of induction of embryogenic callus has been relatively new osidine synthase, desacetoxyvindoline 4-hydroxylase and (Junaid et al. 2006). It was quite different from non- deacetylvindoline 4-O-acetyl transferase were attempted in embryogenic callus and was induced from hypocotyl of in C. roseus in which authors noted differentially regulated vitro germinated seeds. In Catharanthus, in vitro embryo- terpenoid indole alkaloid (TIA) biosynthetic pathway in genesis was earlier categorized into three distinct stages: response to different abiotic stresses (Dutta et al. 2005, (1) initiation and proliferation of SEs (2) maturation and 2007). Semiquantitative RT-PCR analysis of TIA and (3) germination or plantlet conversion. The SEs were related primary pathway genes in response to dehydration, induced on BAP (0.5 mg/L) ? 2, 4-D (0.5 mg/L) supple- low temperature, salinity, UV-light and wounding revealed mented medium where the embryos took the usual distinct their negative regulation in response to low temperature. (globular and heart-shaped) appearances. Advanced, coty- ledonary embryos (5–6 mm length) matured fast on opti-

mized 2.60 lMGA3 added solid MS medium. Later, green In vitro embryogenesis and alkaloid synthesis mature SEs were germinated and produced emblings (Fig. 2a–d) on medium with optimized concentration of The development of somatic embryos (SEs) on vegetative 2.24 lM BA and 7.38 lM IBA. The same research group tissues of cultivated plants is called in vitro embryogenesis studied and identiﬁed various factors (pH, gelrite, light, or somatic embryogenesis. This process of in vitro sugar alcohols, polyethylene glycol and amino acid) that embryogeny has been reported in a number of plant genera inﬂuenced in vitro embryogeny in C. roseus (Junaid et al. (Thorpe 1995; Mujib and Samaj 2006). It offers the pos- 2008). In an early event, callus was obtained from anther sibility of obtaining unlimited production of somaclones to on 1.0 mg/L NAA and 0.1 mg/L kinetin containing

Fig. 2 a Embryogenic callus (a) (b) with developing embryos; b somatic embryo (arrowheads) at proliferated stage; c germinating embryos (arrowheads); d regenerated plantlet.(a, c, 1 mm; b, 1 cm; and d, 0.5 cm)

123 Plant Growth Regul medium, subsequently, embryos were produced in liquid of sugars. It was earlier reviewed that the presence of C/N medium on same hormonal combinations, and later plant- in carbohydrate is also an important factor in regulating lets were regenerated (Kim et al. 1994). alkaloid yield. The caulogenesis rate decreased with the Recently, quantitative estimation of vincristine was overall ion concentration along with the ammonium ions carried out using HPLC from in vitro grown tissues and a presence in the medium, nitrogen supplied in the form of comparison was made from ex vitro raised plantlets. Vin- nitrate and excess amount of Ca?? (compared to K?) ions cristine content was noted to be tissue speciﬁc as leaf callus produced double biomass with tripled yield of alkaloids and germinated embryos produced maximum vincristine (Moreno et al. 1995). In hairy-root culture, maximum (Junaid et al. 2009). growth was observed when the medium contained 0.56 lM

In an effort, protein proﬁles of different embryonic tis- NaH2PO4 as phosphate and 12.97 lM KNO3 as nitrate. sues in C. roseus were analyzed and compared by using However, the alkaloid production was maximum at SDS-PAGE (Ilah et al. 2009); a 38 and a 33 kDa protein lower levels of phosphate, nitrate and ammonia. A higher were noted to be present in torpedo embryos but these two ajmalicine production at low levels of phosphate and protein bands were absent in callus and heart embryos. It ammonia in the medium was reported previously (Schlat- was also noted that most of the important bands are com- mann et al. 1992). mon at three different stages of development; as the Sucrose, glucose, maltose, galactose and lactose were embryo progressed towards maturity (torpedo stage) a few frequently used in cultured media as source of energy, distinct low intensity bands were also appeared. It was also sucrose, however, has been the unanimous choice in predicted that these two proteins might be responsible for Catharanthus as increased alkaloid yield was consistently in vitro embryogenesis in C. roseus. noticed at enhanced levels. In hairy-root culture, sucrose only improved biomass growth without any increase of alkaloid yield. The glucose as energy source was effective Culture conditions for increased ajmalicine production in hairy-root culture (Moreno et al. 1995). Vitamins and organic components Cultural conditions of plant cell culture laboratory affect had a marginal effect on the production of indole alkaloids. biomass growth and production of alkaloids in C. roseus. These are several basic nutrient and cultural factors that inﬂuence synthesis, these are summarized below: Effect of plant growth regulators (PGRs)

PGRs are unique component of plant culture media that Medium composition regulate in vitro morphogenesis processes such as callus formation and shoots differentiation. The secondary metab- The composition of the medium significantly influences olite synthesis has also been influenced by PGRs. The auxin cultural growth and secondary metabolite synthesis. In and cytokinin are added in culture media either alone or in general, tissue culture media contain inorganic and organic combinations routinely which regulate alkaloids biosynthesis components, vitamins and carbohydrates, but the number pathway (Moreno et al. 1995). An antagonistic effect of of inorganic and organic salts and their levels are different. gibberellins and cytokinins on monoterpenoid indole alkaloid Although MS medium is most widely used, other media are (MIA) biosynthesis and their possible impact on elements of also used during cell culture studies. The effect of different the signal transduction was reported very recently (Amini media component on indole alkaloid generation was pre- et al. 2009). It was also observed that the inhibitory effect of viously reviewed (Van der Heijden et al. 1989; Moreno gibberellin was correlated with the silencing of two genes, et al. 1995). The ammonium and nitrate ions are common encoding enzymes of the MIA biosynthetic pathway, which source of nitrogen, with few exceptions these compounds was normally up regulated by cytokinins. Exogenously promoted growth and differentiation but inhibited alkaloid applied cytokinin considerably increased the synthesis of production (Van Gulik et al. 1993). A structured nutritional ajmalicine and serpentine in untransformed cotyledon callus model was recently proposed to describe growth and while the use of 2, 4-D reduced the accumulation of sec- nutritional behaviour of C. roseus hairy roots (Cloutier ondary metabolic products; BAP and in some cases NAA et al. 2008); it demonstrated the cell specific growth rate stimulated the accumulation of vindoline and catharanthine from concentration of intracellular nutrients such as inor- in Catharanthus (Garnier et al. 1996). - ? ganic phosphate (Pi), nitrogen sources (NO3 ) and NH4 In Catharanthus, alkaloid synthesis is a two-stage pro- and sugars. Two-level Michaelis-Menten kinetics was used cess; in the first stage, a rapid accumulation of biomass - to describe Pi and NO3 uptake while simple Michaelis- occurs while alkaloid synthesis takes place in the second Menten kinetics was exploited for the description of uptake stage (Moreno et al. 1995). The medium compositions at 123 Plant Growth Regul two stages are also entirely different, in the second stage BA photoperiod, 16 h light proved to be very effective for was added into the medium that induced enhanced level of faster embryo proliferation while 12 h light improved alkaloids. At hormonal regulation time, cytokinin removed embryo maturation and germination process (Junaid et al. the effects of auxin (Decendit et al. 1992); and in some cases 2007). In previous studies, the light or dark photoperiod it favoured calcium influx which along with calmodulin induced somatic embryogenesis was reported (Fiore et al. stimulated the accumulation of alkaloids (Liu et al. 1990). 1997; Torne et al. 2001) but the molecular mechanisms by A higher transcription of enzyme tryptophan decarboxylase, which it influences embryogenesis are however, limited. strictosidine synthetase was reported to be induced by IAA, Analysis of wheat has, however, indicated that the light Kinetin and BA in Catharanthus. Although the higher TDC induced expression of some proteins were involved where activity and protein accumulation by overexpression of phytochrome acted as important key molecule (Nato et al. cDNA was reported in crown gall callus but the yield of 2000; Kevei and Nagy 2003). The intensity and the periods indole alkaloid was not significantly improved. of light exposure influenced secondary metabolism (Zhao et al. 2001b). The ajmalicine and serpentine syntheses were controlled by light intensity, so was vindoline. The catha- Temperature ranthine production was completely inhibited by the absence of light. It was observed that monochromatic light The temperature is an important element that influences in such as blue (450 nm) and red (670 nm) wavelengths did vitro cultural growth and alkaloid metabolism in C. roseus. not influence growth and alkaloids content significantly in Various temperatures were earlier tested for their ability to C. roseus (Hirata et al. 1992). The dark periods longer than grow and synthesize alkaloids (Bailey and Nicholson 1990). 12 h stimulated shoot elongation but it reduced alkaloid The biomass growth was found maximum at 35 °C and synthesis. It was also noticed that vindoline and catharan- highest production of serpentine was noticed at 25 °C. At the thine were enhanced under intense light (20–30 w/m2). same time, temperature-independent alkaloid biosynthesis Higher serpentine accumulation was reported in light and even inhibition were also reported (Toivonen et al. 1992). induced chloroplast containing increased levels of chloro- phyll (Loyola-Vargas et al. 1992). The information of light on alkaloid synthesis is, however, less in C. roseus. pH

The alkaloids biosynthesis has been influenced by the pH Oxygenation of the medium. The used ranges of pH in plant culture media do not seem to have much influence on production, In culture vessels, different types of gases particularly CO2 however, this set pH value at times fluctuates and affecting and ethylene are produced. These gases and gradual loss of secondary metabolism. The influence of buffered media O2 are reported to be detrimental for normal growth and upon the growth and alkaloid productivity was studied in metabolism of cultured tissues (Moreno et al. 1995). The

C. roseus hairy root as the buffers minimized the shifts of aeration which generates O2, help to restore normal growth media pH (Morgan et al. 2000). It was found that the processes and thereby regularizes alkaloid production. The specific yield of lochnericine was significantly lower; tab- traditional shake flask and various modern stirring devices ersonine yield was significantly higher, while the specific have been regularly employed (Moreno et al. 1995). High yields of ajmalicine, serpentine, and horhammericine dissolved oxygen and improved gaseous permeability at remained unchanged in addition to buffers. It was known different aerated conditions modify secondary metabolism that the cell-culture itself acidified the medium rapidly and to a large extent. This situation was noticed at higher caused differential response (Sakano et al. 1997). It was oxygen level linked ajmalicine production compared to low observed that 5.5 initial pH was very effective for ser- dissolved oxygen (Schlatmann et al. 1994). Various design- pentine biosynthesis (Moreno et al. 1995). In a recent types of fermentors have been used for providing enhanced report, maximum embryogenesis percentage, embryo pro- oxygen level. Carboy system, roller flask, V-shaped reac- liferation, maturation and germination in C. roseus were tors, stirred-jar fermentors, airlift bioreactor were com- noted on 5.8–6.0 pH adjusted medium (Junaid et al. 2006). monly employed (Moreno et al. 1995; Van der Heijden et al. 2004). An investigation on bioreactor consideration for secondary metabolite production from plant cell culture Influence of light in Catharanthus was made in which the impeller speed of 100 rpm was suggested as most appropriate for the accu- Catharanthus embryogeny, an important method to obtain mulation of alkaloids, which increased culture growth rates higher amount of alkaloid, was influenced by the cultural (Paynee et al. 1988). Ajmalicine production was monitored 123 Plant Growth Regul by using various types of vessels and it was noted that Alkaloids biosynthesis, metabolic engineering biomass was not affected much by the use of various cul- and target site regulation ture vessels and the yield was also variable, even lower level of ajmalicine was noted on increasing level of bio- The biosynthesis of secondary metabolites particularly the mass (Hoopen et al. 1994). synthesis of vincristine and vinblastine in C. roseus is quite complex. The schematic pathway (Fig. 3) clearly indicates the participation of several enzymes, proteins genes Bioreactor application including regulatory genes and compartments (Table 2). The process of synthesis starts with the amino acid trypto- In vitro alkaloids production has mainly been conducted on phan and monoterpenoid geraniol in two different path- cell suspension culture in which a rotary shaker is desir- ways. There are about 35 intermediates, 30 different able. For mass scale production larger size cultural vessels, enzymes, regulatory genes, and 7 intra- and inter-cellular fermenters and bioreactors are essential (Schlatmann et al. compartments are associated for the synthesis of vinblastine 1994). However, a bench-top bioreactor allowing contin- (Van der Heijden et al. 2004; Ziegler and Facchini 2008). uous extraction of secondary metabolites was also designed The knowledge of substrates, total enzyme-systems, prod- in C. roseus cell suspensions (Valluri 2009). The simple uct formation (excretion and storage) is also necessary for fermentors and the complex vessels are provided with secondary metabolite synthesis regulation. The information stirring devices for improved aeration. There are other of several biosynthetic routes of Catharanthus, the required vessels too, in which filtered air is provided by a com- precursors and the necessary enzyme-complex are known pressor. The bioreactors with low shear stress have been partially. The enzymes that have major regulation on sec- found more useful for culture growth and alkaloid pro- ondary metabolites production were isolated and purified, duction. The bubble-column and airlift reactors have also and in some cases, the nucleotide structures of concerned been recommended, though other designs with improved genes were also sequenced (Van der Heijden et al. 2004). mechanical configurations are being regularly introduced The tryptophan decarboxylase (TDC) and strictosidine in commercial bioreactor markets. Impellers with different synthetase (SSS) are the two most important enzymes. designs and speed controls have also been developed to The former helps in tryptamine formation from tryptophan reduce shear-agitation. It was also noticed that at high while the other catalyses the coupling of tryptamine and impeller speeds (1,000 rpm), culture maintained its steady secologanin to produce strictosidine. Recently, the identi- growth and alkaloid formation ability (Moreno et al. 1995). fication and characterization of a C. roseus cDNA encoding The immobilization of plant cell has been suggested for an S-adenosyl-L-methionine-dependent N methyltransfer- better accumulation of secondary metabolites in Catha- ase (NMT) that catalyzes a nitrogen methylation involved ranthus sp. An immobilization process can maintains in vindoline biosynthesis has been reported suggesting cells for longer period of time and generates extracellular that NMT is a good candidate to over-express in tissue and accumulation of secondary metabolites that could cut down cell culture systems (Liscombe et al. 2010). Several other cost considerably through increased production (Moreno enzymes, i.e., geraniol-10-hydroxylase (g10H), NADPH- et al. 1995). Considering other potential applications, var- cytochrome P-450 reductase, anthranilate synthetase ious immobilized systems such as alginate, agarose, agar, (AS)—the enzyme with similar TDC activity, are known in carrageenan, polyurethane foam etc. were developed (Van the biosynthesis of indole alkaloids, the TDC enzyme was der Heijden et al. 1989). Agar and agarose were found to be purified from cell-suspension cultures and cDNA was iso- effective for long term maintenance of cell in C. roseus. lated. The identification and functional characterization of The alginate mediated immobilized cells retained respira- a cytochrome P450 gene in a new subfamily of CYP71, tory and biosynthetic activity for extended period of time CYP71BJ1, involved in alkaloid biosynthesis was cur- resulting in enhanced tryptamide, ajmalicine and serpen- rently established in C. roseus (Giddings et al. 2011). tine accumulations. During the last few years, surface Levac et al. (2008) purified 16-hydroxytabersonine-16-O- immobilization has been proposed using different types of methyltransferase (16OMT) proteins from leaf epidermis, matrices for large scale production of alkaloids (Moreno which catalyses the second of six steps in the conversion of et al. 1995). Unfortunately, immobilized cells accumulated tabersonine into vindoline, and the gene was cloned. The reduced level of tryptamide, ajmalicine and catharanthine information about the enzyme SSS, the gene sequence and compared to suspended cells. Cells immobilized with gel, other related properties were also known (Moreno et al. matrice entrapment in polysaccharide beads, polyacryl- 1995). amide sheet, polyester fiber mat with porous layer of SiO2 Mevalonate biosynthesis is also considered to be an were used in Catharanthus in which better accumulation of integrated part of the indole alkaloid production in Cath- alkaloids was induced (Carturan et al. 1998). aranthus. Several enzymes catalyse at different stages were 123 Plant Growth Regul

Fig. 3 Schematic steps of Shikimate Pathway Mevalonate Pathway Methyl-erythritol phosphate (MEP) pathway indole alkaloid synthesis pathway Acetyl-CoA Pyruvate + glyceraldehyde 3- phosphate

3-Hydroxy-3 methylglutaryl-CoA 1-Deoxy-D-xylulose-5-phosphate

Mevalonate 2-C-methyl-D-erythritol-4-phosphate

Isopentenyl diphosphate Dimethyl-allyl diphosphate DXS

Chorismate Geranyl-diphosphate

G10H AS Geraniol

Anthranilate 10-Hydroxy geraniol

Tryptophan Loganin

TDC SLS STR Tryptamine Secologanin (Indole pathway) (Monoterpenoid pathway) SGD

Strictoisidine

Cathenamine

Ajmalicine Tabersonine Catharanthine T16H

Serpentine 16 Hydroxytabersonine Lochnericine D4H Deacetylvindoline Vinblastine,Vincristine Horhammericine DAT Vindoline studied, of which the 3-hydroxy-3-methylglutaryl coen- maximum being in root (Meijer et al. 1993). Even the zyme A reductase and the gene encoding this enzyme amount of these alkaloids vindoline, catharanthine, 304 were studied extensively (Van der Heijden et al. 1994). anhydrovinblastine varied at different ontological ages. Besides, many other enzymes have also been identiﬁed that The enzyme SSS was known to be present in vacuole while metabolize strictosidine and after several biosynthetic the substrate on which it is active is located in the cytosol, intermediates cathenamine and ajmalicine were produced so the enzyme needs to be transported from vacuole to (Moreno et al. 1995). Later, it was also noted that the cytoplasm. It was reported that the subcellular compart- ontological development regulated alkaloid metabolism as mentation is an important site of regulation of secondary differential mRNA level for TDC, SSS and cytochrome metabolism where the enzyme recognized and separated P-150 was transcribed at various phases of development, the substrates from their end products (Moreno et al. 1995).

123 Plant Growth Regul

Table 2 Enzymes/proteins and their localization in alkaloid synthesis pathway of C. roseus Enzyme/protein Location

Tryptophan decarboxylase Cytosol, plastid, vacuole, several enzymes are membrane bound Anthranylate synthetase Chorismate mutase Isochorismate synthase Strictosidine synthetase Strictosidine b glucosidase Desacetoxyvindoline 4-hydroxylase Geraniol 10-hydroxylase Glutamine synthatase Peroxidase Tabersonine 11-hydroxylase Secologanin synthase Acetyl CoA:deacylvindoline 17 O-acetyltransferase Acetyl CoA: minovincinine-O-acetyltransferase NADPH-cytochrome P-450 reductase SAM: methoxy2,16-dihydro, 16 hydrotabersonine N-methyl transferase Mevalonate kinase Phosphomevalonate kinase 1-deoxy D xylulose 5 phosphate synthase 2C methyl D-erythritol 2,4 cyclodiphosphate synthase 1-deoxyD-xylulose 5-phosphate reductoisomerase S-adenosyl-L methionine: 16-hydroxyltabersonine O methyltransferase S-adenosyl-L methionine: 2,3-dihydro-3 hydroxytabersonine N-methyltransferase S-adenosyl-L methionine: loganic acid methyl transferase Cathenamiine synthase Geissoschizine synthase Tetrahydroalstonine synthase Catharanthus AP2 domain protein 2 Catharanthus AP2 domain protein 3 Box P Binding Factor a S-adenosyl-L-methionine-dependent N methyltransferase 16-methoxy-2,3-dihydro-3-hydroxytabersonine-N-methyltransferase (NMT)a Cytochrome P450a 16-hydroxytabersonine-16-O-methyltransferase (16OMT)a Sources Van der Heijden et al. (1989, 2004); Moreno et al. (1995) a Newly isolated protein or enzyme: Levac et al. (2008); Liscombe et al. (2010); Giddings et al. (2011)

Three important cellular components vacuole, cytosol and expressed in C. roseus cell cultures, which showed and plastid are known to be the important cellular regions enhanced levels of ajmalicine and tetrahydroalstonine in in which biosynthetic machinery lies within membranes comparison with control lines (Pomahacova´ et al. 2009). for alkaloid metabolism (Pomahacova´ et al. 2009). ATP-binding cassette (ABC) transporters, present in the cell membrane are known to export various monoterpenoid Precursor feeding alkaloids in C. roseus. Thus, over-expression of an ABC transporter may think to affect the regulation of the alka- The addition of various precursors enhanced secondary loid biosynthetic pathway. CjMDR1, an ABC transporter metabolites synthesis and a number of compounds have so gene originally isolated from Coptis japonica, was inserted far been identiﬁed. The compounds directly used as

123 Plant Growth Regul precursors were L-tryptophan, loganin, loganic acid, seco- was also produced from catharanthine and vindoline from loganin while the other groups of compounds interfere in extracted enzymes of immobilized cell suspension culture. secondary metabolism, are ferulic acid, phenyl ether and The AVLB was consequently biotransformed to pro- dimethylpiperidines. The amino acids have been added to duce vincristine, an important compound with antitumour culture media for enhanced production of different indole- activity (Moreno et al. 1995). template based alkaloids. The L-tryptophan has stimulative The production of many new compounds by mid-pro- effect on Catharanthus alkaloids. It was noted that compact cess biotransformation method has also been obtained on callus cluster (CCC) of C. roseus showed cohesive callus cultural cells of C. roseus and biotransformation products, aggregates with certain levels of cellular or tissue differ- capsaicin 4-O-beta-D-glucopyranoside, capsaicin 4-O-(6- entiation that produced about two-fold more indole alka- O-beta-D-xylopyranosyl)-beta-D-glucopyranoside (116 lg/ loids than normal dispersed-cell cultures (Zhao et al. g fresh wt of cells) and capsaicin 4-O-(6-O-alpha-L-arabi- 2001a). The studies also indicated that the addition of KCl, nopyranosyl)-beta-D-glucopyranoside (83 lg/g fresh wt of mannitol, and a variety of synthetic precursors and bi- cells) were isolated from the cell suspension after 3 days of oregulators markedly improved indole alkaloid production. incubation with capsaicin (Shimoda et al. 2007). Four new Treatment with 250 mM mannitol and 4 g/L KCl yielded compounds after biotransformation of alpha-santonin was 42.3 and 33.6 mg/L of ajmalicine, respectively; the yields obtained by cell suspension of C. roseus (Yang et al. 2005). were about four-fold higher than the control. Succinic acid, The same Catharanthus cell suspension was earlier used tryptamine, and tryptophan feedings also significantly and two biotransformed products triptriolide and 12 beta, increased ajmalicine (41.5, 36.9, and 31.8 mg/L respec- 13 alpha-dihydroxytriptonide were synthesized from tri- tively) and catharanthine contents (21.1, 17.2, and 18 mg/L ptolide and triptonide respectively. More significantly, cell respectively). Recently, chemically modified artificial suspension enzymes involved in specific reactions also substrates have also been added as feeding study in order to catalysed several other biochemical reactions in producing semi-synthesis of novel compounds (Runguphan et al. arbutin and glycerrhetic acid constituents in C. roseus 2010). (Ning et al. 2004). It is believed that the biotransformation The mechanism by which these compounds regulate and the use of appropriate precursors could be one of the biosynthetic pathway is still not well understood. The realistic methods for secondary alkaloid preparations. compounds from glycosides class e.g., secologanin and loganin also influenced alkaloid production and a nearly two fold increase of alkaloid synthesis was noted in cul- Transformation tures, fed with secologanin at their growth phase (Nauda- scher et al. 1990). A high accumulation of ajmalicine and Catharanthus produces commercially important anti- strictosidine was noted in secologanin, loganin and loganic cancer indole alkaloids that are used in pharmaceutical acid added media (Moreno et al. 1993). After feeding with industry. During biosynthesis, various precursor molecules geraniol, 10-hydroxygerariol, or loganin a marked increase are produced as intermediates from different pathways in tabersonine accumulation was also noted recently which help to synthesize desired indole alkaloids by active (Morgan and Shanks 2000). Terpenoid indole alkaloid participation of several enzymes. In order to control the (TIA) accumulation has recently been observed to be synthesis of these alkaloids, Agrobacterium mediated influenced by the availability of precursors. Loganin genetic modification has been initiated in Catharanthus. It feeding in transgenic C. roseus TDC and/or STR cell line was noticed that the transformed calluses of Catharanthus efficiently improved alkaloid accumulation (1,200 lmol/L produced higher levels of tryptophan decarboxylase and TIA), compared to wild type cell accumulated of about tryptamine but the increase of indole alkaloids production 580 lmol/L (Whitmer et al. 2000). Precursor feeding was not that significant (Goddijn et al. 1995). The trypto- of tryptamine and loganin improved accumulation of phan decarboxylase enzyme catalyses one of the key steps ajmalicine in C. roseus transgenic cells (Whitmer et al. in the biosynthesis of indole alkaloids by converting 1998). tryptophan into tryptamide. The light inducible ipt gene introduction has been attempted in Catharanthus as this gene regulates endogenous cytokinin accumulation. The Biotransformation cytokinin in turn regulates many aspects of plant growth including secondary metabolism. The transformed cotyle- In Catharanthus, biotransformation has been used for the donary callus that contained the same ipt gene accumulated synthesis of vinblastine using two precursor compounds higher level of cytokinin but no improved alkaloid- catharathine and vindoline (Datta and Srivastava 1997). synthesis was, however, observed. Thus, it was believed Another related compound, 3040-anhydrovinblastine (AVLB) that the endogenous level did not mimic the effects of 123 Plant Growth Regul exogenously applied cytokinin (Garnier et al. 1996). active compound lies in extracts was found to be protein- Recently, increased vincristine production from Agrobac- aceous in nature (Menke et al. 1999). Methyl jasmonate terium tumefaciens C58 induced shooty teratomas of (MeJA) is widely used elicitor, which after elicitation of C. roseus was observed (Begum et al. 2009). Authors noted C. roseus hairy roots changes in the accumulation of alka- that the dimeric alkaloid vincristine in the transformed loids such as ajmalicine, serpentine, ajmaline and catha- cultures was present at a concentration of 0.011 that was ten- ranthine (Lee-Parsons et al. 2004; Ruiz-May et al. 2009; fold higher compared to untransformed control cultures. Va´zquez-Flota et al. 2009); this compound is believed to act Transformation of an alkaloid biosynthetic gene with reen- as secondary messenger in regulating alkaloid synthesis. gineered substrate specificity into C. roseus was conducted Endogeneous elicitors can also be released from plant cells recently (Runguphan and O’Connor 2009). This transgenic on treatment with macerozyme which induced TDC and plant cell produced a variety of unnatural alkaloid com- PAL activity during alkaloid synthesis (Seitz et al. 1989; pounds when co-cultured with simple, achiral precursors Garnier et al. 1996; Moreno-Valenzuela et al. 1999). that the reengineered enzyme was designed to accept. Elicitors of abiotic nature include light, UV, several stress agents and all play a key role in producing alkaloids in C. roseus. Several studies showed that light significantly Elicitor’s role on alkaloid accumulation influenced the synthesis of alkaloids as the level of vindoline and serpentine was high in cell cultures of C. roseus An elicitor is defined as a substance that induces the syn- (Zhao et al. 2001a, b). thesis of compounds, used in defense responses (Ebel and Many stress factors like heavy metals influenced alka- Scheel 1997). The production of secondary metabolites has loid synthesis in C. roseus as well. The indole alkaloid been enhanced by several such compounds, which are accumulation was reported to be high on copper and cad- biotic and abiotic in nature. In the biotic class the microbial mium added media. In another report, enhanced alkaloids extracts like Yeast, Pythium, Botrytis, Phytophthora, yield under water-stressed conditions was observed Pseudomonas have been very effective in a variety of cell (Mustafa et al. 2009). Increased level of nicotinamide and cultures including Catharanthus (Valluri 2009; Mustafa trigolline was noted after treated with 2, 2 azobis dehy- et al. 2009). After exposing periwinkle cell cultures to drochloride (AAPH), an oxidative stress agent, and in biotic—(Aspergillus niger, crude chitin) and abiotic elici- cultures irradiated with UV (Bergland et al. 1996). tors (mannitol, methyl jasmonate) it was noted that most of The effects of UV-B stress on the production of terpenoid the biotic elicitors were effective at day 15, while the indole alkaloids in C. roseus hairy roots was studied and it abiotic elicitors were effective at day 20 for alkaloid syn- was observed that the UV-B light enhanced significant thesis (Valluri 2009). The culture filtrate of P. aphanider- increases in the production of TIAs, including precursors to matum stimulated alkaloid synthesis was observed by vinblastine and vincristine (Binder et al. 2009). Cell sus- inducing over accumulation of phenylalanine ammonia pension cultures of C. roseus in late exponential phase and lysate (PAL) that catalyzed the synthesis of phenolic stationary phase were irradiated with UV-B. The stationary compounds in the medium (Mustafa et al. 2009). phase cultures were more responsive to UV-B irradiation Elicitor prepared from Penicillium citrinum cell wall compared to late exponential phase in which catharanthine evoked several responses, including rapid generation of and vindoline increased threefold and 12-fold, respectively, nitric oxide (NO), which was essential for triggering on treatment with a 5-min UV-B irradiation (Ramani and catharanthine in C. roseus cells (Xu and Dong 2005). Jayabaskaran 2008). UV-B-induced signaling events lead- Acetylsalicylic, a derivative of salicyclic acid was used in ing to enhanced-production of catharanthine was studied in A. tumefaciens transformed cell lines which showed enri- C. roseus cell suspension and a model for signal transduction ched levels of alkaloids. Similarly, elicited with pectinase cascade was proposed (Ramani and Chelliah 2007). The and jasmonate (JA) the in vitro cultures induced improved study demonstrated that the cell surface receptor(s), Ca2? level of alkaloids, the maximum improvement being in influx, medium alkalinization, calcium dependent protein horhammericine (500 %) whilst minimum improvement kinase (CDPK), H2O2 and mitogen activated protein kinase (60 %) was noted in serpentine when JA was used in hairy (MAPK) play significant roles in UV-B signaling leading to root culture (Rijhwani and Shanks 1998). stimulation of Tdc and Str genes and the accumulation of These biotic elicitors in particular contain compounds catharanthine in C. roseus. Previous study suggested that like oligosaccharides or glycopeptides which facilitate the UV induced stress influenced the synthesis of alkaloids elicitation effect, the active principle is however yet to be in C. roseus as both the content of vinblastine and leurosine illustrated (Van der Heijden et al. 2004). Yeast extracts were high in irradiated cultures (Zhao et al. 2001a). enhanced transcription of strictosidine synthase, one of the The combination of two or more stress factors that key enzymes in alkaloid metabolism in C. roseus, and the synergistically influenced secondary metabolite synthesis 123 Plant Growth Regul have been reported (Saenz et al. 1993; Zhao et al. 2001c). ajmalicine production when added to C. roseus culture; An increased alkaloid accumulation was noted by using later it was established that the carboxyl groups, present in fungal elicitor vanadyl sulphate and potassium chloride, the alginate have an important role in elicitation reaction higher concentrations however, reduced cell viability (Akimoto et al. 1999). Recently, mixed alginate elici- (Kargi and Potts 1991). Similarly, a two-fold increase in tors were prepared with exogenous elicitor (autoclaved alkaloid production was reported when tryptophan, fungal A. macleodii), endogenous elicitor (alginate oligomers), elicitor and vanadyl sulphate were used together as elicitors and trans-4,5-dihydroxy-2-cyclopenten-1-one (Aoyagi (Kargi and Ganapathi 1991). et al. 2006). The prepared mixed alginate elicitors signifi- In addition, various chemical compounds (organic and cantly promoted 50-phosphodiesterase (PDase) production inorganic) increased the accumulation of catharanthine, (2.67 U/mL) by C. roseus, and the productivity was serpentine and tryptamine in culture. The substances like increased 120-fold compared to the control without cell vanadyl sulphate, sodium chloride, potassium chloride and growth inhibition. The use of trans-cinnamic acid, an sorbitol have also been used as elicitors. A 90 % increase inhibitor of phenylalanine ammonia lyase (PAL) activity, in catharanthine production over control was noticed in results in significant increase in the alkaloid production in sodium chloride added (1.7 g/L) medium while potassium C. roseus. Mannitol-induced osmotic stress induced chloride addition resulted in about 200 % increase in marked increment in the total alkaloid. catharanthine. The effect of NaCl on the growth and Molecular studies suggested that elicitors activate signal alkaloid content was studied in C. roseus seedlings and it transduction pathways which in turn stimulate transcrip- was noted that the vindoline, catharanthine, vincristine, and tional control of several defense genes including genes that vinblastine contents were maximum at 50 mmol 9 L(-1) encode enzymes participating in alkaloid synthesis path- NaCl stress, which was significantly higher than the non ways. Elicitor-based signaling model has been proposed treated and other cultural treatments (Wang et al. 2008). recently for enhanced activation of gene expression in Alginate, acted as an endogenous elicitor and promoted C. roseus (Fig. 4) where yeast elicitor controlled alkalization

Elicitor Medium alkalinization

Cl- + - K O2 O2

ROS (H2O2)

Plasma membrane

NADPH Receptor CDPK oxidase

H2O2 H+ Ca+ Catharanthine Octadecanoid Protein bisindole Pathway MAPK phosphatases alkaloids

Jasmonate Vindoline

Protein kinase Vacuole Vindoline

GT-1? ORCA deacyl Vindoline CrBPF TDC/STR

Nucleus Plastid

Fig. 4 Model for the elicitor mediated signal transduction leading to activation of genes in indole alkaloid synthesis pathways (modified after Memelink et al. 2001) 123 Plant Growth Regul of the medium by influxing proton molecule, particularly Cryopreservation of cell lines the calcium. It activates the octadecanoid pathway leading to the synthesis of jasmonic acid (JA). JA behaves as The superior cell-lines capable of high production of sec- secondary messenger and influences the synthesis of ondary metabolites require screening and preserving, since nuclear proteins ORCA2 and ORCA3. These proteins the efficiency of cell-lines in culture often deteriorated with interact with TDC- and ‘JA-responsive STR’ promoter of time. In order to maintain secondary metabolite synthesis several biosynthetic genes and activate their gene expres- for long term, several cryo-preservation protocols have sion (Memelink et al. 2001). As discussed above, a number been employed using different pre-treatments, cryo-pro- of key gene’s activities such as tryptophan decarboxylase tectants, cooling and thawing processes. C. roseus LD50/ (tdc), strictosidine synthetase (sss), geraniol 10 dehydro- h13 cell lines were preserved for over 6 months in the genase (gh), anthranilate synthetase (as) increased as an presence of sorbitol and 5 % DMSO, and using a two-step effect of ‘elicitor-receptor’ mediated signaling (Van der freezing process, the cell lines was noted to continue sec- Fits and Memelink 2000). ondary metabolites synthesis, even following cryopreservation (Mannonen et al. 1990). The storage under mineral oil has also been very useful for the pre-cells for in vitro High cell density culture preservation which resumed growth in renewed suspensions on fresh liquid media (Bachiri et al. 1995). Recently, Secondary metabolites production by high cell density cul- an efficient cryopreservation protocol was established for ture feeding has been attempted with or without much suc- embryogenic cell suspension cultures of C. roseus (Samar cess. It was noted that the high-inoculum-density cultures et al. 2009). It was a vitrification-based cryopreservation produced higher ajmalicine compared to low density culture, method wherein embryogenic cells were exposed to a but catharanthine content was nearly the same between the preculture/pretreatment medium prior to their immersion two inoculum density culture systems (Lee and Shuler in liquid nitrogen. During preculture, different sucrose 2000). However, the ammonium, nitrate, and sugars uptake (0.09–0.6 M) and sorbitol (0.2–0.6 M) levels were used, was high in low-inoculum-density cultures (50 g FW/L) com- among pretreatments Dimethyl sulphoxide (DMSO) (5 or pared to the high-inoculum density cultures (100 g FW/L). 10 %) and glycerol (5 or 10 %) at six different levels either In contrast, current study indicated that the alkaloid pro- alone or in combinations (PT 1–PT 6) were tested. The duction was remarkably very low when inoculum potential cryopreservation treatment combinations of either 0.4 M was increased (Moreno-Valenzuela et al. 2003). The low sucrose, 5 % DMSO, and 5 % glycerol (PT-5) or 0.4 M oxygen levels and inadequate nutrient uptake are among the sucrose and 5 % DMSO (PT-1) resulted in the highest possible causes for low metabolite accumulation at high cell frequency of viability of embryogenic cultures. density culture (Schlatmann et al. 1992).

Association of proteomics, expressed sequence tag Superior cultivars/cell lines in C. roseus and transcription factor in alkaloid biosynthesis

HPLC-based metabolic profiling was made in 50 different Two cDNA libraries from RNA were generated and iso- cultivars of C. roseus, which showed nearly equal amount lated from the base part of young leaves and from root tips of alkaloids; one of the cultivars however, produced low and 9,824 random clones were selected for unidirectional level of vindoline, suggesting thereby a possibility of sequencing, to yield 3,327 related sequences and 1,696 existence of altered biosynthetic pathway (Magnotta et al. singletons by cluster analysis (Murata et al. 2006). Putative 2006). Recently, after induced mutagenesis, two mutants, functions of 3,663 clones were also assigned from 5,023 one with small, pollen-less anthers (OR-EA) and another non-redundant ESTs to establish a resource for transcrip- with ‘‘pin’’ flowers (EMS 13-2) were developed in tome analysis and gene discovery in this medicinal plant. C. roseus strain OR and cultivar ‘‘Dhawal,’’ respectively, A proteomic approach was also conducted to study the the yield of alkaloids, however, has not been checked yet identification of novel proteins associated with alkaloid (Kulkarni and Baskaran 2008). It was noted that some of biosynthesis in C. roseus (Jacobs et al. 2005). Mass spec- the cell lines of C. roseus produced higher levels of trometry (MALDI-MS/MS) analysis identified 58 protein ajmalicine and serpentine (Moreno et al. 1995). Even in spots, including two isoforms of strictosidine synthase cases significantly variable synthesis and accumulation of (EC 4.3.3.2), which catalyzes the synthesis of strictosidine alkaloids was noted in Catharanthus cell cultures, initiated in the alkaloid biosynthesis. It also recognized tryptophan from different explant sources i.e., anther, leaf and meri- synthase (EC 4.1.1.28), which is required for alkaloid stem (Benjamin et al. 1990). precursor tryptamine; and 12-oxophytodienoate reductase, 123 Plant Growth Regul which catalyzes the last step in the biosynthesis of the which is underway, may offer more number of untapped regulator jasmonic acid. genes, which may unravel some of the unexplored regu- The influence of MADS-box transcription factor latory mechanisms, controlling alkaloid synthesis in Agamous-like 12 (Agl12) from Arabidopsis thaliana on the C. roseus. differentiation of suspension cells was studied in C. roseus, as in vitro undifferentiated suspension tissues failed to Acknowledgments The first author is highly thankful to Central produce indole alkaloids (Montiel et al. 2007). The Agl12 Council for Research in Unani Medicine (CCRUM) and Department of Botany, Hamdard University (Jamia Hamdard), New Delhi, for expression in Catharanthus suspension induced globular providing financial assistance and other facilities. The help rendered parenchyma-like organization but failed to form complete by present and past research students is also acknowledged. We wish root differentiation. The study also revealed that the to thank anonymous reviewers for their comments on manuscript and transgenic cell lines expressing Agl12 selectively enhanced critical reading. the expression of genes encoding enzymes that control early biosynthesis steps of indole alkaloids and eventually References produced significant amounts of ajmalicine, an antihyper- tensive TIA. 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