Analysis of hairy root culture of Rauvolfia serpentina ... - CIMAP Staff

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Analysis of hairy root culture of Rauvolfia serpentina ORIGINAL RESEARCH 597to screen and select the best performer amongst a wider,independently generated, heterogeneous background,as reported in earlier analogous studies (Christey andBraun, 2005). Chemical profiling of a large number ofindependently generated hairy root clones through theapplication of conventional complex solvent extractionprocess is undeniably a herculean task.In order to profile the hairy root clones of Rauvolfiaserpentina quickly and with great accuracy for the characterizationof the indole alkaloids expressed in them,we have utilized a new mass spectrometric ionizationtechnique, DART (direct analysis in real time). DARTis coupled to a time-of-flight mass analyzer that providesselectivity and accurate elemental composition assignmentthrough exact mass measurement. The DARTtechnique has been found to be extremely useful fordirect detection of chemicals on surfaces withoutrequiring sample preparation such as solvent extraction.In this communication, we report the identificationof two alkaloids, vomilenine and reserpine, that wereexpressed in the hairy root cultures of R. serpentina bydirectly analyzing the hairy roots using the DARTspectrometric method. The structures of the two alkoidswere confirmed as vomilenine (1) and reserpine (2) byexact measurement of their molecular formulae. Tothe best of our knowledge, this is the first report ofchemical profiling of the hairy roots of plant tissueculture origin using DART technique. Moreover, thisis also the first report of expression of reserpine in thehairy root culture of R. serpentina.EXPERIMENTALInduction and establishment of hairy roots. Rauvolfiaserpentina plants (8–10 weeks), maintained under in-vitroconditions on semisolid MS (Murashige and Skoog, 1962)medium supplemented with 2.0 mg/L 6-benzylaminopurine(BAP) and 0.1 mg/L α-naphthalene acetic acid (NAA), wereused as the explant source. The young leaves were inoculatedthrough pricking with a 48-h-old suspension cultureof A. rhizogenes strain A 4 (a kind gift from Professor D.Tepfer, INRA, Versailles Cedex, France), grown in liquidYMB (Hooykass et al., 1977) medium (O.D. 600 = 0.9–1.0) andco-cultured on hormone-free, semi-solid MS medium. After48 h of co-cultivation with the bacterial strain, the explantswere transferred on to the same medium containing 1.0 g/L ofCephalaxin (Ranbaxy, India) under dark conditions. Similartypes of explants, pricked with a sterile needle devoid of thebacterial suspension, were cultured under uniform conditionsas controls. The emerging hairy roots were subsequentlytransferred to the half and full strengths of the MS mediumcontaining 3% (w/v) sucrose for their further proliferation.Once established, the individual hairy root clones were transferredto liquid MS medium with the same concentrationof antibiotic and incubated on a rotary shaker in the dark at25 ± 1°C under constant agitation (80 rpm). The antibioticconcentration was progressively lowered and finally completelyomitted after 4 months. Roots excised from in-vitrogrown complete plantlets were cultured under identical conditionsin liquid MS medium supplemented with 1.0 mg/Lindole-3-butyric acid (IBA) to serve as control roots.Characterization of hairy roots. Polymerase chain reactionanalysis was carried out to prove the transformed nature ofthe independently generated hairy root clones following apublished procedure (Rahman et al., 2004).DART mass spectrometryThe mass spectrometer used was a JMS-100 TLC (AccuTof)atmospheric pressure ionization time-of-flight mass spectrometer(Jeol, Tokyo, Japan) fitted with a DART ion source.The mass spectrometer was operated in positive-ion modewith a resolving power of 6000 (full-width at half-maximum).The orifice 1 potential was set to 28 V, resulting in minimalfragmentation. The ring lens and orifice 2 potentials were setto 13 and 5 V, respectively. Orifice 1 was set to a temperatureof 100°C. The RF ion guide potential was 300 V. Data acquisitionwas from m/z 10 to 1050. The DART ion source wasoperated with helium gas flowing at approximately 4 L/min.The gas heater was set to 300°C. The potential on the dischargeneedle electrode of the DART source was set to 3000 V, electrode1 to 100 V and the grid to 250 V. The sample (hairyroot) was positioned in the gap between the DART sourceand mass spectrometer for measurements. Exact mass calibrationwas accomplished by including a mass spectrum of neatpolyethylene glycol (1:1 mixture PEG 200 and PEG 600)in the data file. The mass calibration was accurate to within± 0.003 u. Using the Mass Center software, the elementalcomposition could be determined on selected peaks.RESULTS AND DISCUSSIONEmergence of hairy roots could be seen after four weeksof bacterial inoculation of the leaf explants with the A.rhizogenes strain A 4 at 81.25% relative transformationfrequency. In accordance with an earlier report (Benjaminet al., 1993), most of the emerging roots of the presentstudy tend to form calli in the half-strength, hormonefree,liquid basal MS medium during further sub-culturing.However, on the basis of potential to grow without callusingalong with maximum elongation and branchingbehavior, one particular hairy root clone could be selectedfor further studies out of 30 independent hairyroot lines. The selected hairy root clone exhibited anRol-A-positive result, which indicated its transformednature. The biosynthetic potential of this particularhairy root clone has been evaluated through applicationof the newly developed DART technique.Mass spectrometry is a very important tool for thedetermination of molecular weight and structure determinationof organic molecules. It has also been utilizedin the field of biotechnology as one of the core methodsfor characterization of different compounds. TheCopyright © 2008 John Wiley & Sons, Ltd. Biomed. Chromatogr. 22: 596–600 (2008)DOI: 10.1002/bmc
598 ORIGINAL RESEARCHK. P. Madhusudanan et al.different ion sources that have been used in massspectrometry include electron ionization, chemical ionization,fast atom bombardment, field desorption/fieldionization, electrospray ionization and matrix-assistedlaser desorption ionization.DART, a new ionization source, has recently beendeveloped by Cody and Laramee (Cody et al., 2005). InDART, the sample is not directly exposed to highvoltages, laser beams, radiation or plasma. DART iscoupled with a time-of-flight mass spectrometer. DARTis based on the atmospheric pressure interaction oflong-lived electronic excited state atoms or vibronicexcited state molecules with atmospheric gases and thesample. Helium or nitrogen is used for producingthe ionizing plasma. The gas flows through a chamberwhen an electric discharge (corona discharge) producesions, electrons and excited state metastable atoms andmolecules. These metastable species, in turn, generateprotonated gaseous water clusters in ambient air. Thewater clusters on proton exchange with the sample giverise to [M + H] + ions of the sample. Thus, proton transferis the dominant mechanism in positive ionization. Innegative ionization mode, oxygen water clusters arereported to be responsible for ionizing the sample. TheDART gas flow is towards the mass spectrometer inletorifice across a gap of 5–25 mm. This gap is opened tothe laboratory room and the material to be analyzed isheld in this gap in the stream of warm gas. The DARTion source has been used to analyze an extremely widerange of analytes including drugs (tablets, formulationsetc.), metabolites in body fluid, explosives, forensic substances,synthetic-organic and organo-metallic compounds,food, spices, beverages, fatty acids in bacteria andalso materials on surfaces such as paper or currency.Recently, we have utilized the DART technique forthe detection and identification of taxoid molecules thatwere expressed in the cell culture of Taxus wallichiana(Banerjee et al., 2007).The hairy root cultures of different Rauvolfia specieshave been explored in the past in the search for analternative source of reserpine. However, in the firstreport, 40-day-old hairy root cultures of R. serpentinaproduced only trace amounts of serpentina, while allthe other targeted indole alkaloids, i.e. reserpine, ajmalineand raucaffricine, remained absent (Benjamin et al.,1993). The same research group subsequently revealedthat pure hairy root cultures without the problemof callusing could never be obtained in R. serpentinaand the mixed hairy root + callus cultures produced onlyajmaline (0.045% dry weight [DW]) and serpentine(0.007% DW; Benjamin et al., 1994). Klyushnichenkoet al. (1995) later reported yohimbine as the majoralkaloid on a backdrop of small unidentified impuritiesin the hairy root cultures of R. serpentina and R.vomitoria. Sheludko et al. (2002a,b), on the other hand,have reported the synthesis indole alkaloid of raumaclinegroup and a new alkaloid of the sarpagine group in thehairy root cultures of R. serpentina. Recently Sudhaet al. (2003) have also reported the production ofajmalicine and ajmaline in the hairy root cultures ofR. micrantha.As DART is a new ionization technique and itssensitivity is quite high, we have decided to analyze thehairy roots of R. serpentina by this technique as it hasnot yet been applied for this purpose. The intact hairyroots of R. serpentina were held in front of the DARTsource. A mass spectrum was instantly obtained (Fig. 2).The peaks at m/z 351 and 609 were assigned to the [M+ H] + of two nitrogen-containing compounds. The twonitrogen containing compounds (alkaloids) were characterizedas vomilenine (1) and reserpine (2) (Fig. 1) onthe basis of their exact mass measurement which gaveFigure 1. Chemical structures of vomilenine (1) and reserpine (2).Copyright © 2008 John Wiley & Sons, Ltd. Biomed. Chromatogr. 22: 596–600 (2008)DOI: 10.1002/bmc
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Page 5: 600 ORIGINAL RESEARCHK. P. Madhusud

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