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Enhanced production of asiaticoside from hairy root cultures of Centella asiatica (L.) Urban elicited by methyl jasmonate

  • Genetic Transformation and Hybridization
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Abstract

Transformed root (“hairy root”) cultures have been shown to be a good model for the study of many secondary metabolites. However, economically important compounds such as asiaticoside and madecassoside are produced in insignificant amounts in the root of Centella asiatica (L.) Urban. To overcome this problem, C. asiatica was transformed using Agrobacterium rhizogenes strain R1000 that harbors pCAMBIA1302 encoding the hygromycin phosphotransferase (hpt) and green fluorescence protein (mgfp5) genes and the hairy culture was coupled with elicitation technique. Hairy roots were obtained at a frequency of up to 14.1% from a tissue junction between the leaf and petiole. Abundant hairy roots were observed when co-cultivation of the plant with A. rhizogenes was done for 7 days (36.1%). Transformation was confirmed by PCR and Southern blot analyses. Five weeks after inoculation, no asiaticoside was detected in the hairy root samples. However, when 0.1 mM methyl jasmonate (MJ) was applied as an elicitor to the culture medium for 3 weeks, a large quantity of asiaticoside was generated (7.12 mg/g, dry wt). In the case of gene expression, 12 h after MJ treatment the expression of the CabAS (C. asiatica putative β-amyrin synthase) gene in the hairy roots is significantly different from that of the control and this level of transcripts was maintained for 14 days. Our results showed that production of C. asiatica hairy roots could be optimized and the resulting cultures could be elicited with MJ treatment for enhanced production of asiaticoside.

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Abbreviations

CabAS :

Centella asiatica putative beta-amyrin synthase

GFP:

Green fluorescence protein

HPLC:

High-performance liquid chromatography

hpt :

Hygromycin phosphotransferase

MJ:

Methyl jasmonate

RT-PCR:

Reverse transcriptase-polymerase chain reaction

References

  • Alpizar E, Dechamp E, Espeout S, Royer M, Lecouls AC, Nicole M, Bertrand B, Lashermes P, Etienne H (2006) Efficient production of Agrobacterium rhizogenes-transformed roots and composite plants for studying gene expression in coffee roots. Plant Cell Rep 25:959–967

    Article  PubMed  CAS  Google Scholar 

  • Aoyagi H, Kobayashi Y, Yamada K, Yokoyama K, Kusakari K, Tanaka H (2001) Efficient production of saikosaponins in Bupleurum falcatum root fragments combined with signal transducers. Appl Microbiol Biotechnol 57:482–488

    Article  PubMed  CAS  Google Scholar 

  • Aziz ZA, Davey MR, Power JB, Anthony P, Smith RM, Lowe KC (2007) Production of asiaticoside and madecassoside in Centella asiatica in vitro and in vivo. Biol Plant 51:34–42

    Article  CAS  Google Scholar 

  • Baek YW (1997) Micropropagation of Centella asiatica (L.) Urban by in vitro cultures and production of triterpene glycosides. PhD thesis, Chonnam University, Gwangju

  • Bercetche J, Chriqui D, Adam S, David C (1987) Morphogenetic and cellular reorientation induced by Agrobacterium rhizogenes (strains 1855, 2659 and 8196) on carrot, pea and tobacco. Plant Sci 52:195–210

    Article  Google Scholar 

  • Bonfill M, Mangas S, Cusidó RM, Osuna L, Piñol MT, Palazón J (2006) Identification of triterpenoid compounds of Centella asiatica by thin-layer chromatography and mass spectrometry. Biomed Chromatogr 20:151–153

    Article  PubMed  CAS  Google Scholar 

  • Chen DT, Ye HC, Li GF (2000) Expression of a chimeric farnesyl diphosphate synthase gene in Artemisia annua L. transgenic plants via Agrobacterium tumefaciens-mediated transformation. Plant Sci 155:179–185

    Article  PubMed  CAS  Google Scholar 

  • Damgaard O, Rasmussen O (1991) Direct regeneration of transformed shoots in Brassica napus from hypocotyls infections with Agrobacterium rhizogenes. Plant Mol Biol 17:1–8

    Article  PubMed  CAS  Google Scholar 

  • Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15

    Google Scholar 

  • Flores HE, Vivanco JM, Loyola-Vargas VM (1999) Radicle biochemistry: the biology of root-specific metabolism. Trends Plant Sci 4:220–226

    Article  PubMed  Google Scholar 

  • Hamill JD, Rounsley S, Spencer A, Todd G, Rhodes MJC (1991) The use of the polymerase chain reaction in plant transformation studies. Plant Cell Rep 10:221–224

    Article  CAS  Google Scholar 

  • Hayashi H, Huang PY, Inoue K (2003) Up-regulation of soyasaponin biosynthesis by methyl jasmonate in cultured cells of Glycyrrhiza glabra. Plant Cell Physiol 44:404–411

    Article  PubMed  CAS  Google Scholar 

  • Haralampidis K, Bryan G, Qi X, Papadopoulou K, Bakht S, Melton R, Osbourn A (2001) A new class of oxidosqualene cyclases directs synthesis of antimicrobial phytoprotectants in monocots. Proc Natl Acad Sci USA 98:13431–13436

    Article  PubMed  CAS  Google Scholar 

  • Kang HJ, Anbazhagan VR, You XL, Moon HK, Yi JS, Choi YE (2006) Production of transgenic Aralia elata regenerated from Agrobacterium rhizogenes-mediated transformed roots. Plant Cell Tissue Organ Cult 85:187–196

    Article  CAS  Google Scholar 

  • Kartnig T, Hoffmann-Bohm K (1992) Centella. In: Hänsel R, Keller K, Rimpler H, Schneider G (eds) Hager’s handbuch der pharmazeutischen praxis. Springer, Berlin

    Google Scholar 

  • Kim OT, Kim MY, Hong MH, Ahn JC, Hwang B (2004) Stimulation of asiaticoside production from Centella asiatica whole plant cultures by elicitors. Plant Cell Rep 23:339–344

    Article  PubMed  CAS  Google Scholar 

  • Kim OT, Kim MY, Huh SM, Bai DG, Ahn JC, Hwang B (2005) Cloning of a cDNA probably encoding oxidosqualene cyclase associated with asiaticoside biosynthesis from Centella asiatica (L.) Urban. Plant Cell Rep 24:304–311

    Article  PubMed  CAS  Google Scholar 

  • Komari T, Ishida Y, Hiei Y (2004) Plant transformation technology: Agrobacterium-mediated transformation. In: Christou P, Klee H (eds) Handbook of plant biotechnology. Wiley, England

    Google Scholar 

  • Lee MH, Jeong JH, Seo JW, Shin CG, Kim YS, In JG, Yang DC, Yi JS, Choi YE (2004) Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squalene synthase gene. Plant Cell Physiol 45:976–984

    Article  PubMed  CAS  Google Scholar 

  • Lu MB, Wong HL, Teng WL (2001) Effects of elicitation on the production of saponin in cell culture of Panax ginseng. Plant Cell Rep 20:647–677

    Article  CAS  Google Scholar 

  • Mangas S, Bonfill M, Osuna L, Moyano E, Tortoriello J, Cusido RM, Piñol MT, Palazón (2006) The effect of methyl jasmonate on triterpene and sterol metabolisms of Centella asiatica, Ruscus aculeatus and Galphimia glauca cultured plants. Phytochemistry 67:2041–2049

    Article  PubMed  CAS  Google Scholar 

  • Matsuda H, Morikawa T, Ueda H, Yoshikawa M (2001) Medicinal foodstuffs. XXVII. Saponin constituents Gotu Kola (2): Structures of new ursane- and oleanane-type triterpene oligoglycosides, centellasaponin B, C, and D, from Centella asiatica cultivated in Sri Lanka. Chem Pharm Bull 49:1368–1371

    Article  PubMed  CAS  Google Scholar 

  • Mihaljevic S, Stipkovic S, Jelaska S (1996) Increase of root induction in Pinus nigra explants using agrobacteria. Plant Cell Rep 15:610–614

    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 

  • Nath S, Buragohain AK (2005) Establishment of callus and cell suspension cultures of Centella asiatica. Biol Plant 49:411–413

    Article  Google Scholar 

  • Niu X, Li P, Veronese P, Bressan RA, Weller SC, Hasegawa PM (2000) Factors affecting Agrobacterium tumefaciens-mediated transformation of peppermint. Plant Cell Rep 19:304–310

    Article  CAS  Google Scholar 

  • Ottani MP, Schel JHN, Hänisch ten Cate Ch H (1990) Variation in structure and plant regeneration of Agrobacterium rhizogenes transformed and control roots of the potato cv. Bintje. Plant Cell Tissue Organ Cult 20:25–34

    Article  Google Scholar 

  • Paramageetham Ch, Prasad Babu G, Rao JVS (2004) Somatic embryogenesis in Centella asiatica L. an important medicinal and neutraceutical plant of India. Plant Cell Tissue Organ Cult 79:19–24

    Article  Google Scholar 

  • Patra A, Rai B, Rout GR, Das P (1998) Successful plant regeneration from callus culture of Centella asiatica (L.) Urban. Plant Growth Regul 24:13–16

    Article  CAS  Google Scholar 

  • Pointel JP, Boccalon H, Cloarec M, Ledebehat C, Joubert M (1987) Titrated extract of Centella asiatica (TECA) in the treatment of venous insufficiency of the lower limbs. Angiology 38:46–50

    PubMed  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloing: a laboratory manual, 2ndedn. Cold Spring Harbor Laboratory Press, Plainview

    Google Scholar 

  • Shi HP, Kintzios S (2003) Genetic transformation of Pueraria phaseoloides with Agrobacterium rhizogenes and puerarin production in hairy roots. Plant Cell Rep 21:1103–1107

    Article  PubMed  CAS  Google Scholar 

  • Srivastava S, Srivastava AK (2007) Hairy root culture for mass-production of high-value secondary metabolites. Crit Rev Biotechnol 27:29–43

    Article  PubMed  CAS  Google Scholar 

  • Suzuki H, Achnine L, Xu R, Matsuda SPT, Dixon RA (2002) A genomic approach to the early stages of triterpene saponin biosynthesis in Medicago truncatula. Plant J 32:1033–1048

    Article  PubMed  CAS  Google Scholar 

  • Tao J, Li L (2006) Genetic transformation of Torenia fournieri L. mediated by Agrobacterium rhizogenes. S Afr J Bot 72:211–216

    Article  Google Scholar 

  • Tiwari KN, Sharma NC, Tiwari V, Singh BD (2000) Micropropagation of Centella asiatica (L.), a valuable medicinal herb. Plant Cell Tissue Organ Cult 63:179–185

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Prof. Lourdes B. Cardenas, Institute of Biological Sciences, University of the Philippines Los Baños, for critical review of this manuscript.

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Authors and Affiliations

  1. Division of Ginseng and Medicinal Crop, National Institute of Crop Sciences, RDA, Suwon, 441-857, South Korea

    Ok-Tae Kim, Kyong-Hwan Bang, Yu-Su Shin, Su-Jin Jung, Dong-Yun Hyun, Young-Chang Kim, Nak-Sul Seong & Seon-Woo Cha

  2. College of Agriculture, Life and Environment Science, Chungbuk National University, Cheongju, 361-763, South Korea

    Min-Jeong Lee

  3. Department of Biology and Institute of Plant Resources, Chonnam National University, Gwangju, 500-757, South Korea

    Baik Hwang

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  1. Ok-Tae Kim
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  2. Kyong-Hwan Bang
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Correspondence to Baik Hwang.

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Communicated by J. R. Liu.

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Kim, OT., Bang, KH., Shin, YS. et al. Enhanced production of asiaticoside from hairy root cultures of Centella asiatica (L.) Urban elicited by methyl jasmonate. Plant Cell Rep 26, 1941–1949 (2007). https://doi.org/10.1007/s00299-007-0400-1

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  • DOI: https://doi.org/10.1007/s00299-007-0400-1

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