Skip to main content
SpringerLink
Log in

Gentiana dinarica Beck. hairy root cultures and evaluation of factors affecting growth and xanthone production

  • Original Paper
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

The induction and establishment of hairy root cultures of Gentiana dinarica using two strains of Agrobacterium rhizogenes (A4M70GUS and 15834/PI) is reported for the first time. Strain 15834/PI had higher induction rate of hairy roots (32.15 %) than strain A4M70GUS (6.12 %). Transgenic nature of the roots was confirmed by GUS assay and PCR analysis. Two clones per strain (A4M70GUS-D and -I, and 15834/PI-2 and -3) with marked differences in general morphology and growth rate were further studied. The methanol extracts of hairy root clones were analyzed for xanthones content using HPLC method. The effects of the type of carbohydrate source (sucrose, fructose or glucose) at different concentrations on the growth parameters (growth index, dry weight, fresh/dry weight ratio), phenolic and xanthone production, and free radical scavenging activity of the transgenic clones were evaluated. Statistical two level factorial design was used to define optimal conditions for growth and successful secondary metabolite production in G. dinarica hairy root clones. The results showed that clones A4M70GUS-D and 15834/PI-3 were the superior ones. These two clones had the highest dry weight on 116.8 mM sucrose, producing up to threefold higher amounts of total phenolics and norswertianin-1-O-primeveroside than other clones, untransformed roots and roots of wild-grown plants.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

MS:

Murashige and Skoog (1962)

DW:

Dry weight

FW:

Fresh weight

BA:

6-Benzyladenine

NAA:

α-Naphthaleneacetic acid

PCR:

Polymerase chain reaction

DPPH:

1,1-Diphenyl-2-picrylhydrazyl

References

  • Batra J, Dutta A, Singh D, Kumar S, Sen J (2004) Growth and terpenoid indole alkaloid production in Catharanthus roseus hairy root clones in relation to left- and right-termini-linked Ri T-DNA gene integration. Plant Cell Rep 23:148–154

    Article  CAS  PubMed  Google Scholar 

  • Brand-Williams W, Cuvelier ME, Berset C (1995) Use a free radical method to evaluate antioxidative activity. LWT Food Sci Technol 28:25–30

    Article  CAS  Google Scholar 

  • Cao TW, Geng CA, Ma YB, He K, Wang HL, Zhou NJ, Zhang XM, Tao YD, Chen JJ (2013) Xanthones with anti-hepatitis B virus activity from Swertia mussotii. Planta Med 79:697–700

    Article  CAS  PubMed  Google Scholar 

  • Chaudhuri KN, Ghosh B, Tepfer D, Jha S (2005) Genetic transformation of Tylophora indica with Agrobacterium rhizogenes A4: growth and tylophorine productivity in different transformed root clones. Plant Cell Rep 24:25–35

    Article  CAS  PubMed  Google Scholar 

  • Chericoni S, Testai L, Calderone V, Flamini G, Nieri P, Morelli I, Martinotti E (2003) The xanthones gentiacaulein and gentiakochianin are responsible for the vasodilator action of the roots of Gentiana kochiana. Planta Med 69:767–770

    Article  Google Scholar 

  • Cho HJ, Widholm JM, Tanaka NY, Nakanishi Y, Merooka Y (1998) Agrobacterium rhizogenes-mediated transformation and regeneration of the legume Astragalus sinicus (Chinese milk vetch). Plant Sci 138:53–65

    Article  CAS  Google Scholar 

  • Fotie J, Bohle S (2006) Pharmacological and biological activities of xanthones. AntiInfect Agents Med Chem 5:15–31

    Article  CAS  Google Scholar 

  • Georgiev M, Pavlov A, Bley T (2007) Hairy root type plant in vitro systems as sources of bioactive substances. Appl Microbiol Biotechnol 74:1175–1185

    Article  CAS  PubMed  Google Scholar 

  • Hayta S, Gurel A, Aagun IH, Altan F, Ganzera M, Tanyolac B, Bedir E (2011) Induction of Gentiana cruciata, hairy roots and their secondary metabolites. Biologia 66:618–625

    Article  CAS  Google Scholar 

  • Hirakawa K, Yoshida M, Nagatsu A, Mizukami H, Rana V, Rawat MSM, Oikawa S, Kawanishi S (2005) Chemopreventive action of xanthone derivatives on photosensitized DNA demage. Photochem Photobiol 81:314–319

    Article  CAS  PubMed  Google Scholar 

  • Hosokawa K, Matsuki RM, Oikawa Y, Yamamura S (1997) Genetic transformation of gentian using wild-type Agrobacterium rhizogenes. Plant Cell Tissue Organ Cult 51:137–140

    Article  Google Scholar 

  • Hostettmann-Kaldas M, Hostettmann K, Sticher O (1981) Xanthones, flavones and secoiridoids of American Gentiana species. Phytochemistry 20:443–446

    Article  CAS  Google Scholar 

  • Hu ZB, Du M (2006) Hairy root and its application in plant genetic engineering. J Integr Plant Biol 48:121–127

    Article  CAS  Google Scholar 

  • Janković T, Krstić D, Šavikin-Fodulović K, Menković N, Grubišić D (2002) Xanthones and secoiridoids from hairy root cultures of Centaurium erythraea and C. pulchellum. Planta Med 68:944–946

    Article  PubMed  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: b-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    PubMed Central  CAS  PubMed  Google Scholar 

  • Jensen SR, Schripsema J (2002) Chemotaxonomy and pharmacology of Gentianaceae. In: Struwe L, Albert VA (eds) Gentianaceae: systematics and natural history. Cambridge University Press, Cambridge, pp 573–631

    Google Scholar 

  • Jeong G-T, Park D-H, Ryu H-W, Lee W-T, Park K, Kang C-H, Hwang B, Woo J-C (2002) Optimum conditions for transformed Panax ginseng hairy roots in flask culture. Appl Biochem Biotech 98–100:1129–1139

    Article  Google Scholar 

  • Jovanović-Dunjić R. (1973) Gentiana L. In: Josifović M (ed) Flore de la Republique Socialiste de Serbie V. Academie Serbe des Sciences et des Artes, Belgrade, pp 412–425

  • Keil M, Härtle B, Guillaume A, Psiorz M (2000) Production of amarogentin in root cultures of Swertia chirata. Planta Med 66:452–457

    Article  CAS  PubMed  Google Scholar 

  • Krstić D, Janković T, Aljančić I, Šavikin-Fodulović K, Menković N, Milosavljević S (2004) Phytochemical investigation of Gentiana dinarica. Biochem Syst Ecol 32:937–941

    Article  Google Scholar 

  • Krstić-Milošević D, Janković T, Vinterhalter B, Menković N, Aljančić I, Vinterhalter D (2013) Influence of carbohydrate source on xanthone content in root cultures of Gentiana dinarica Beck. Plant Growth Regul 71:147–155

    Article  Google Scholar 

  • Larcher G, Morel C, Tronchin G, Landreau A, Seraphin D, Richomme P, Bouchara JP (2004) Investigation of the antifungal activity of caledonixanthone E and other xanthones against Aspergillus fumigatus. Planta Med 70:569–571

    Article  CAS  PubMed  Google Scholar 

  • Luo C-T, Mao S-S, Liu F-L, M-x Yang, Chen H, Kurihara H, Li Y (2013) Antioxidant xanthones from Swertia mussoti, a high altitude plant. Fitoterapia 91:140–147

    Article  CAS  PubMed  Google Scholar 

  • Mahendran G, Manoj M, Rajendra Prasad KJ, Narmatha Bai V (2013) Evaluation of anti-inflammatory and antinoceceptive activity of xanthones from Swertia corymbosa (Griseb.) Wight ex C.B. Clarke. Int J Pharm Pharm Sci 5:523–529

    CAS  Google Scholar 

  • Menković N, Šavikin Fodulović K, Vinterhalter B, Vinterhalter D, Janković T, Krstić D (2000a) Secoiridoid content in hairy roots of Gentiana punctata. Pharm Pharmacol Lett 2:73–75

    Google Scholar 

  • Menković N, Šavikin-Fodulović K, Momčilović I, Grubišić D (2000b) Quantitative determination of secoiridoid and γ-pyrone compounds in Gentiana lutea cultured in vitro. Planta Med 66:96–98

    Article  PubMed  Google Scholar 

  • Mishiba K, Nishihara M, Abe Y, Nakatsu T, Kawamura H, Kodama K, Takesawa T, Abe J (2006) Production of dwarf potted gentian using wild-type Agrobacterium rhizogenes. Plant Biotechnol NAR 23:33–38

    Article  CAS  Google Scholar 

  • Momčilović I, Grubišić D, Kojić M, Nešković M (1997) Agrobacterium rhizogenes-mediated transformation and plant regeneration of four Gentiana species. Plant Cell Tissue Organ Cult 50:1–6

    Article  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Ninković S, Djordjević T, Vinterhalter B, Uzelac B, Cingel A, Savić J, Radović S (2010) Embryogenic responses of Beta vulgaris L. callus induced from transgenic hairy roots. Plant Cell Tissue Organ Cult 103:81–91

    Article  Google Scholar 

  • Nishihara M, Nakatsuka T, Hosokawa K, Yokoi T, Abe Y, Mishiba K, Yamamura S (2006) Dominant inheritance of white-flowered and herbicide-resistant traits in transgenic gentian plants. Plant Biotechnol 23:25–31

    Article  CAS  Google Scholar 

  • Peres V, Nagem TJ, de Oliviera FF (2000) Tetraoxygenated naturally occurring xanthones. Phytochemistry 55:683–710

    Article  CAS  PubMed  Google Scholar 

  • Pontus S, Michael AP, Chaim I (2006) Use of Gentiana lutea extracts as an antimicrobial agent. European Patent EP1663271

  • Porter J (1991) Host range and implications of plant infection by Agrobacterium rhizogenes. Crit Rev Plant Sci 10:387–421

    Article  Google Scholar 

  • Praveen N, Murthy HN (2012) Synthesis of withanolide A depends on carbon source and medium pH in hairy roots of Withania somnifera. Ind Crops Prod 35:241–243

    Article  CAS  Google Scholar 

  • Singleton VL, Rossi JA (1965) Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic 16:144–153

    CAS  Google Scholar 

  • Smigocki AC, Puthoff DP, Zuzga S, Ivic-Haymes SD (2009) Low efficiency processing of an insecticidal Nicotiana proteinase inhibitor precursor in Beta vulgaris hairy roots. Plant Cell Tissue Organ Cult 97:167–174

    Article  CAS  Google Scholar 

  • Suginuma C, Akihama T (1995) Transformation of Gentiana with Agrobacterium rhizogenes. Acta Hortic 392:153–160

    CAS  Google Scholar 

  • Tepfer M, Casse-Delbart F (1987) Agrobacterium rhizogenes as a vector for transforming higher plants. Microbiol Sci 4:24–28

    CAS  PubMed  Google Scholar 

  • Tiwari RK, Trivedi M, Guang ZC, Guo GQ, Zheng G-C (2007) Genetic transformation of Gentiana macrophylla with Agrobacterium rhizogenes: growth and production of secoiridoid glucoside gentiopicroside in transformed hairy root cultures. Plant Cell Rep 26:199–210

    Article  CAS  PubMed  Google Scholar 

  • Van Larebake N, Genetello CH, Hernalsteens JP, De Picker A, Zaenen I, Messens E, Van Montagu M, Schell J (1977) Transfer of Ti plasmids between Agrobacterium strains by mobilization with the conjugative plasmid RP4. Mol Gen Genet 152:1119–1124

    Google Scholar 

  • Verma PC, Rahman LU, Negi AS, Jain DC, Khanuja SPS, Banerjee S (2007) Agrobacterium rhizogenes-mediated transformation of Picrorhiza kurroa Royle ex Benth.: establishment and selection of superior hairy. Plant Biotechnol Rep 1:169–174

    Article  Google Scholar 

  • Vinterhalter B, Orbović V, Vinterhalter D (1999) Transgenic root cultures of Gentiana punctata L. Acta Soc Bot Pol 68:275–280

    Article  Google Scholar 

  • Vinterhalter B, Momčilović I, Vinterhalter D (2000) High biomass producing root cultures of Gentiana punctata L. transformed with Agrobacterium tumefaciens C58C1 (pArA4b). Arch Biol Sci 52:85–90

    Google Scholar 

  • Vinterhalter B, Krstić-Milošević D, Janković T, Milojević J, Vinterhalter D (2012) In vitro propagation of Gentiana dinarica Beck. Cent Eur J Biol 7:690–697

    Article  CAS  Google Scholar 

  • Vinterhalter B, Krstić-Milošević D, Janković T, Zdravković-Korać S, Vinterhalter D (2013) Quantitative determination of secoiridoid and xanthone glycosides of Gentiana dinarica Beck cultured in vitro. Acta Physiol Plant 35:567–574

    Article  Google Scholar 

  • Weathers PJ, Hemmavanh DD, Walcerz DB, Cheetham RD, Smith TC (1997) Interactive effects of nitrate and phosphate salts, sucrose, and inoculum culture age on growth and sesquiterpene production in Artemisia annua hairy root cultures. In Vitro Cell Dev Biol Plant 33:306–312

    Article  CAS  Google Scholar 

  • Wu CFJ, Hamada M (2009) Experiments: planning, analysis and parameter design optimization, 2nd edn. John Wiley, New York

    Google Scholar 

  • Yimdjo MC, Azebaze AG, Nikengfack AE, Meyer AM, Bodo B, Fomum ZT (2004) Antimicrobial and cytotoxic agents from Calophyllum inophyllum. Phytochemistry 65:2789–2795

    Article  CAS  PubMed  Google Scholar 

  • Zhang HL, Xue SH, Pu F, Tiwari RK, Wang XY (2010) Establishment of hairy root lines and analysis of gentiopicroside in the medicinal plant Gentiana macrophylla. Russ J Plant Physiol 57:110–117

    Article  CAS  Google Scholar 

  • Zheng H-H, Luo C-T, Chen H, Lin J-N, Ye C-L, Mao S-S, Li Y-L (2014) Xanthones from Swertia mussoti as multitarget-directed antidiabetic agents. Chem Med Chem 9:1374–1377

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Bacterial strain A4M70GUS was obtained by courtesy of Dr. Landre, Univ. Pierre and Marie Curie, Paris, France. The present work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Grant No. 173015.

Conflict of interest

The authors have no conflicts of interest to declare.

Author information

Authors and Affiliations

  1. Institute for Biological Research “Siniša Stanković”, University of Belgrade, Bulevar despota Stefana 142, 11060, Belgrade, Serbia

    Branka Vinterhalter, Dijana Krstić-Milošević, Slavica Ninković & Dragan Vinterhalter

  2. Institute for Medicinal Plant Research “Dr Josif Pančić”, Tadeuša Košćuška 1, 11000, Belgrade, Serbia

    Teodora Janković & Dejan Pljevljakušić

  3. Molecular Plant Pathology Laboratory, USDA-ARS, Beltsville, MD, 20705, USA

    Ann Smigocki

Authors
  1. Branka Vinterhalter
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dijana Krstić-Milošević
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Teodora Janković
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dejan Pljevljakušić
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Slavica Ninković
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ann Smigocki
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Dragan Vinterhalter
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Teodora Janković.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Vinterhalter, B., Krstić-Milošević, D., Janković, T. et al. Gentiana dinarica Beck. hairy root cultures and evaluation of factors affecting growth and xanthone production. Plant Cell Tiss Organ Cult 121, 667–679 (2015). https://doi.org/10.1007/s11240-015-0737-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-015-0737-z

Keywords

Search

Navigation