Skip to main content
SpringerLink
Log in

Genetic transformation of Bacopa monnieri by wild type strains of Agrobacterium rhizogenes stimulates production of bacopa saponins in transformed calli and plants

  • Original Paper
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

We have developed an efficient transformation system for Bacopa monnieri, an important Indian medicinal plant, using Agrobacterium rhizogenes strains LBA 9402 and A4. Transformed roots induced by strain LBA 9402 spontaneously dedifferentiated to callus while excised roots induced by strain A4 spontaneously showed induction of shoot buds within 10 days. PCR and RT-PCR analysis revealed the presence and expression of the rolAB and rolC genes at the transcription level in pRi A4 transformed cultures indicating that the TL-DNA was integrated retained and expressed in the A4-Ri transformed shoots. Transformed calli showed the presence of rolAB or rol A, TR and ags genes. Transformed plants showed morphological features typically seen in transgenic plants produced by A. rhizogenes. Growth and biomass accumulation was significantly higher in the transformed shoots (twofold) and roots (fourfold) than in the non-transformed (WT) plants. In pRi A4-transformed plants, the content of bacopasaponin D, bacopasaponin F, bacopaside II and bacopaside V was enhanced significantly as compared to WT plants of similar age while bacoside A3 and bacopasaponin C content was comparable with that of WT plants. Significant increase in content of five bacopa saponins could be detected in pRi 9402-transformed callus cultures. There is an overall stimulatory effect on accumulation of bacopa saponins in transformed plants and cells of B. monnieri establishing the role of endogenous elicitation by Ri T-DNA of A. rhizogenes.

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

Similar content being viewed by others

References

  • Agostini E, de Forchetti SM, Tigier HA (1997) Production of peroxidases by hairy roots of Brassica napus. Plant Cell Tiss Organ Cult 47:177–182

    Article  Google Scholar 

  • Agrawal A (1993) A comparative study of psychotropic drugs and bio-feedback therapy in the prevention and management of psychosomatic disorders. Ph.D. Thesis, Banaras Hindu University, Varanasi, India

  • Ambros PF, Matzke MA, Matzke JM (1986) Detection of 17 kb unique sequence (T-DNA) in plant chromosomes by in situ hybridization. Chromosoma 94:11–18

    Article  CAS  Google Scholar 

  • Anonymous (1997) Indian medicinal plants: a sector study. Occasional paper No. 54. Export-Import Bank of India. Quest publication, Bombay

    Google Scholar 

  • Bandyopadhyay M, Jha S, Tepfer D (2007) Changes in morphological phenotypes and withanolide composition of Ri-transformed roots of Withania somnifera. Plant Cell Rep 26:599–609

    Article  PubMed  CAS  Google Scholar 

  • 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  PubMed  CAS  Google Scholar 

  • Ben-Hayyim G, Martin-Tanguy J, Tepfer D (1996) Changing root and shoot architecture with the rolA gene from Agrobacterium rhizogenes: interactions with gibberellic acid and polyamine metabolism. Physiol Plant 96:237–243

    Article  CAS  Google Scholar 

  • Bhandari P, Kumar N, Singh B, Kaul VK (2006) Bacosterol glycoside, a new 13, 14-seco-steroid glycoside from Bacopa monnieri. Chem Pharm Bull 54:240–241

    Article  PubMed  CAS  Google Scholar 

  • Bouchez D, Tourneur J (1991) Organization of the agropine synthesis region of the T-DNA of the Ri plasmid from Agrobacterium rhizogenes. Plasmid 25:27–39

    Article  PubMed  CAS  Google Scholar 

  • Bulgakov VP (2008) Functions of rol genes in plant secondary metabolism. Biotechnol Adv 26:318–324

    Article  PubMed  CAS  Google Scholar 

  • Bulgakov VP, Tchernoded GK, Mischenko NP, Shkryl YN, Fedoreyev SA, Zhuravlev YN (2004) The rolB and rolC genes activate synthesis of anthraquinones in Rubia cordifolia cells by mechanism independent of octadecanoid signaling pathway. Plant Sci 166:1069–1075

    Article  CAS  Google Scholar 

  • Cardarelli M, Spano L, Mariotti D, Mauro ML, Van Sluys MA, Constantino P (1985) Identification of the genetic locus responsible for non polar root induction by Agrobacterium rhizogenes Ri plasmid. Plant Mol Bio 5:385–391

    Article  CAS  Google Scholar 

  • Cardarelli M, Spanó L, Mariotti D, Mauro ML, Constantino P (1987) The role of auxin in hairy root induction. Mol Gen Genet 208:457–480

    Article  CAS  Google Scholar 

  • Casanova E, Zuker A, Trillas MI, Moysset L, Vainstein A (2003) The rolC gene in carnation exhibits cytokinin- and auxin-like activities. Sci Hortic 97:321–331

    Article  CAS  Google Scholar 

  • Chakravarty AK, Sarkar T, Masuda K, Shiojima K, Nakane T, Kawahara N (2001) Bacopaside I and II: two pseudojujubogenin glycosides from Bacopa monniera. Phytochemistry 58(4):553–556

    Article  PubMed  CAS  Google Scholar 

  • Chakravarty AK, Garai S, Masuda K, Nakane T, Kawahara N (2003) Bacopasides III–V: three new triterpenoid glycosides from Bacopa monniera. Chem Pharm Bull 51:215–217

    Article  PubMed  CAS  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  PubMed  CAS  Google Scholar 

  • Chaudhuri KN, Ghosh B, Tepfer D, Jha S (2006) Spontaneous plant regeneration in transformed roots and calli from Tylophora indica: changes in morphological phenotype and tylophorine accumulation associated with transformation by Agrobacterium rhizogenes. Plant Cell Rep 25:1059–1066

    Article  PubMed  CAS  Google Scholar 

  • Chaudhuri KN, Das S, Bandyopadhyay M, Zalar A, Kollmann A, Jha S, Tepfer D (2009) Transgenic mimicry of pathogen attack stimulates growth and secondary metabolite accumulation. Transgenic Res 18:121–134

    Article  PubMed  CAS  Google Scholar 

  • Choi PS, Kim YD, Choi KM, Chung HJ, Choi DW, Liu JR (2004) Plant regeneration from hairy-root cultures transformed by infection with Agrobacterium rhizogenes in Catharanthus roseus. Plant Cell Rep 22:828–831

    Article  PubMed  CAS  Google Scholar 

  • Chomcznski P, Sacchi N (1987) Single step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction. Anal Biochem 162:156–159

    Google Scholar 

  • Chopra RN (1958) Indigenous drugs of India, 2nd edn. UN Dhur and sons, Calcutta, p 341

    Google Scholar 

  • Christey MC (1997) Transgenic crop plants using Agrobacterium rhizogenes-mediated transformation. In: Doran PM (ed) Hairy roots: culture and applications. Harwood Academic Publishers, Amsterdam, pp 99–110

    Google Scholar 

  • Delbarre A, Muller P, Imhoff V, Barbier-Brygoo H, Maurel C, Leblanc N, Perrot-Rechenmann C, Guern J (1994) The rolB gene of Agrobacterium rhizogenes does not increase the auxin sensitivity of tobacco protoplasts by modifying the intracellular auxin concentration. Plant Physiol 105:563–569

    PubMed  CAS  Google Scholar 

  • Elangovan V, Govindaswamy S, Ramamoorthy N, Balasubramanian K (1995) In vitro studies on the anticancer activity of Bacopa monnieri Fitoterapia 66(3):211–215

    Google Scholar 

  • Flores H, Hoy M, Pickard J (1987) Secondary metabolites from root cultures. Trends Biotechnol 5:64–69

    Article  CAS  Google Scholar 

  • Garai S, Mahato SB, Ohtani K, Yamasaki K (1996a) Dammarane-type triterpenoid saponins from Bacopa monniera. Phytochemistry 42:815–820

    Article  PubMed  CAS  Google Scholar 

  • Garai S, Mahato SB, Ohtani K, Yamaski K (1996b) Bacosaponin D-a pseudojujubogenin glycoside from Bacopa monniera. Phytochemistry 43:447–449

    Article  PubMed  CAS  Google Scholar 

  • Geissman TA (1972) A specialist periodical report. In: Biosynthesis, vol 1. The Chemical Society, London

  • Guillon S, Tremouillaux-Guiller J, Pati PK, Rideau M, Gantet P (2006) Hairy root research: recent scenario and exciting prospects. Curr Opin Plant Biol 9:341–346

    Article  PubMed  CAS  Google Scholar 

  • He YK, Wang JY, Gong ZH (1994) Root development initiated by exogenous auxin synthesis genes in Brassica sp. crops. Plant Physiol Biochem 32:493–500

    CAS  Google Scholar 

  • Hooykass PJJ, Klapwjik PM, Nuti MP, Schilperoort RA, Rorsch A (1977) Transfer of the A. tumefaciens Ti plasmid to avirulent Agrobacteria and Rhizobium ex planta. J Gen Microbiol 98:477–484

    Google Scholar 

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

    Article  CAS  Google Scholar 

  • Hughes EH, Shanks JV (2002) Metabolic engineering of plants for alkaloid production. Metabol Eng 4:41–48

    Article  CAS  Google Scholar 

  • Ionkova I, Witte L, Alfermann HA (1994) Spectrum of tropane alkaloids in transformed roots of Datura innoxin and Hyoscyamus xgyorffyi cultivated in vitro. Planta Med 60:382–384

  • Jain P, Kulshreshtha DK (1993) Bacoside A1, a minor saponin from B. monniera. Phytochemistry 33:449–451

    Article  CAS  Google Scholar 

  • Jaziri M, Homes J, Shimomura K (1994) An unusual root tip formation in hairy root tip culture of Hyoscyamus muticus. Plant Cell Rep 13:349–352

    Article  CAS  Google Scholar 

  • Jia H, Zhao B, Wang X, Wang Y (2008) Agrobacterium rhizogenes-mediated transformation and regeneration of the Apocynum venetum. Chin J Biotechnol 24(10):1723–1728

    Article  CAS  Google Scholar 

  • Jouanin L (1984) Restriction map of an agropine Ri plasmid and its homologies with Ti plasmids. Plasmid 12:91–102

    Article  PubMed  CAS  Google Scholar 

  • Jouanin L, Guerche D, Pamboukdjian N, Tourneur C, Casse-Delbart F, Tourneur J (1987) Structure of T-DNA in plants regenerated from roots transformed by Agrobacterium rhizogenes strain A4. Mol Gen Genet 206:387–392

    Article  CAS  Google Scholar 

  • Jung G, Tepfer D (1987) Use of genetic transformation by the Ri T-DNA of Agrobacterium rhizogenes to stimulate biomass and tropane alkaloid production in Atropa belladonna and Calystegia sepium roots. Plant Sci 50:145–151

    Article  CAS  Google Scholar 

  • Kirtikar KR, Basu BD (1981) Indian medicinal plants, Part II. Indian Press, Allahabad, pp 930–931

    Google Scholar 

  • Kittipongpatana N, Hock RS, Porter JR (1998) Production of solasodine by hairy root, callus, and cell suspension cultures of Solanum aviculare Forst. Plant Cell Tiss Org Cult 52:133–143

    Article  CAS  Google Scholar 

  • Koike Y, Hoshino Y, Mii M, Nakano M (2003) Horticultural characterization of Angelonia salicariifolia plants transformed with wild-type strains of Agrobacterium rhizogenes. Plant Cell Rep 21:981–987

    Article  PubMed  CAS  Google Scholar 

  • Kumar V, Satyanarayana KV, Itty S, Indu EP, Giridhar P, Chandrashekar A, Ravishankar GA (2006) Stable transformation and direct regeneration in Coffea canephora P ex. Fr. By Agrobacterium rhizogenes mediated transformation without hairy root phenotype. Plant Cell Rep 25:214–222

    Article  PubMed  CAS  Google Scholar 

  • Limami MA, Sun LY, Douat C, Helgeson J, Tepfer D (1998) Natural genetic transformation by Agrobacterium rhizogenes: annual flowering in two biennials, Belgian endive and carrot. Plant Physiol 118:543–550

    Article  PubMed  CAS  Google Scholar 

  • Mahato SB, Garai S, Chakravarty AK (2000) Bacosaponins E and F: two jujubogenin bisdesmosides from Bacopa monnieri. Phytochemistry 53:711–714

    Article  PubMed  CAS  Google Scholar 

  • Mano Y, Nabeshima S, Matsui C, Ohkawa H (1986) Production of tropane alkaloids by hairy root cultures of Scopolia japonica. Agric Biol Chem 50:2715–2722

    CAS  Google Scholar 

  • Mano Y, Ohkawa H, Yamada Y (1989) Production of tropane alkaloids by hairy root cultures of Duboisia leichhardtii by Agrobacterium rhizogenes. Plant Sci 59:191–201

    Article  CAS  Google Scholar 

  • Moyano E, Fornalè S, Palazòn J, Cusidò RM, Bonfill M, Morales C, Piñol MT (1999) Effect of Agrobacterium rhizogenes T-DNA on alkaloid production of Solanaceae plants. Phytochemistry 52:1287–1292

    Article  CAS  Google Scholar 

  • Moyano E, Jouhikainen K, Tammela P, Palazòn J, Cusidò RM, Piñol MT, Teeri TH, Oksman-Caldentey KM (2003) Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus. J Exp Bot 54(381):203–211

    Article  PubMed  CAS  Google Scholar 

  • Mukherjee DG, Dey CD (1966) Clinical trial on Brahmi. Int J Exp Med Sci 10:5–11

    CAS  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 

  • Nadkarni KM (1988) The Indian material medica. South Asia Books, Columbia, pp 624–625

    Google Scholar 

  • Nisha KK, Seetha K, Rajmohan K, Purushothama MG (2003) Agrobacterium tumefaciens-mediated transformation of Brahmi [Bacopa monniera (L.) Wettst.], a popular medicinal herb of India. Curr Sci 85:85–89

    CAS  Google Scholar 

  • Oksman-Caldentey KM, Kivelfi O, Hiltunen R (1991) Spontaneous shoot organogenesis and plant regeneration from hairy root cultures of Hyoscyamus muticus. Plant Sci 78:129–136

    Article  CAS  Google Scholar 

  • Palazón J, Cusidó RM, Gonzalo J, Bonfill M, Morales C, Piñol MT (1998) Relation between the amount of rolC gene product and indole alkaloid accumulation in Catharanthus roseus transformed root cultures. J Plant Physiol 153:712–718

    Google Scholar 

  • Petit A, David C, Dahl G, Ellis J, Guyon P, Casse-Delbart F, Tempé J (1983) Further extension of the opine concepts: plasmids in Agrobacterium rhizogenes co-operate for opine degradation. Mol Gen Genet 207:245–250

    Article  Google Scholar 

  • Pitta-Alverez SI, Giulietti AM (1995) Advantages and limitations in the use of hairy root cultures for the production of tropane alkaloids: use of anti auxins in the maintenance of normal root morphology. In Vitro Cell Dev Biol Plant 31:215–220

    Google Scholar 

  • Rahman LU, Verma PC, Singh D, Gupta MM, Banerjee S (2002) Bacoside production by suspension cultures of Bacopa monnieri (L.) Pennell. Biotechnol Lett 24:1427–1429

    Article  CAS  Google Scholar 

  • Rastogi S, Kulshreshtha DK (1998) Bacoside A2—a triterpenoid saponin from Bacopa monniera. Indian J Chem 38B:353–356

    Google Scholar 

  • Rastogi S, Pal R, Kulshreshtha DK (1994) Bacoside A3—a triterpenoid saponin from Bacopa monniera. Phytochemistry 36:133–137

    Article  PubMed  CAS  Google Scholar 

  • Saitou T, Kamada H, Harada H (1991) Isoperoxidase in hairy roots and regenerated plants of horseradish (Armoracia lapathifolia). Plant Sci 75:195–201

    Article  CAS  Google Scholar 

  • Schmülling T, Schell J, Spena A (1988) Single genes from Agrobacterium rhizogenes influence plant development. EMBO J 7:2621–2629

    PubMed  Google Scholar 

  • Sevón N, Dräger B, Hiltunen R, Oksman-Caldentey K-M (1997) Characterization of transgenic plants derived from hairy roots of Hyoscyamus muticus. Plant Cell Rep 16:605–611

    Article  Google Scholar 

  • Shkryl YN, Veremeichik GN, Bulgakov VP, Tchernoded GK, Mischenko NP, Fedoreyev SA, Zhuravlev YN (2008) Individual and combined effects of the rolA, B and C genes on anthraquinone production in Rubia cordifolia transformed calli. Biotechnol Bioeng 100:118–125

    Article  PubMed  CAS  Google Scholar 

  • Shrivastava N, Rajani M (1999) Multiple shoot regeneration and tissue culture studies on Bacopa monnieri (L.) Pennell. Plant Cell Rep 18:919–923

    Article  CAS  Google Scholar 

  • Slightom JL, Durand-Tardif M, Jouanin L, Tepfer D (1986) Nucleotide sequence analysis of TL-DNA of Agrobacterium rhizogenes agropine type plasmid. Identification of open reading frames. J Biol Chem 261:108–121

    PubMed  CAS  Google Scholar 

  • Sokal RR, Rohlf FJ (1987) Introduction to biostatistics. WH Freeman, New York

    Google Scholar 

  • Spéna A, Schmülling T, Koncz C, Schell J (1987) Independent and synergystic activity of rolA, B and C loci in stimulating abnormal growth in plants. EMBO J 6:3891–3899

    PubMed  Google Scholar 

  • Tanaka N, Takao M, Matsumoto T (1995) Vincamine production in multiple shoot culture derived from hairy roots of Vinca minor. Plant Cell Tiss Org Cult 41:61–64

    Article  CAS  Google Scholar 

  • Tepfer D (1984) Genetic transformation of several species of higher plants by Agrobacterium rhizogenes: phenotypic consequences and sexual transmission of the transformed genotype and phenotype. Cell 37:959–967

    Article  PubMed  CAS  Google Scholar 

  • Tiwari V, Singh BD, Tiwari KN (1998) Shoot regeneration and somatic embryogenesis from different explants of Brahmi [Bacopa monniera (L.) Wettst]. Plant Cell Rep 17:538–543

    Article  CAS  Google Scholar 

  • Tiwari V, Tiwari KN, Singh BD (2001) Comparative studies of cytokinins on in vitro propagation of Bacopa monniera. Plant Cell Tiss Org Cult 66:9–16

    Google Scholar 

  • Toivonen L, Rosenqvist H (1995) Establishment and growth characteristics of Glycyrrhiza glabra hairy root cultures. Plant Cell Tiss Org Cult 41:249–258

    Article  Google Scholar 

  • Verpoorte R, van der Heijden R, Schripsema J, Hoge JHC, ten Hoopen HJG (1993) Plant cell biotechnology for the production of alkaloids: present status and prospects. J Nat Prod 56:186–207

    Article  CAS  Google Scholar 

  • Vijayakumar M, Vijayakumar R, Stephen R (2010) In vitro propagation of Bacopa monnieri L.—a multipurpose medicinal plant. Indian J Sci Technol 3(7):781–786

    CAS  Google Scholar 

  • Vohora SB, Khanna T, Athar M (1997) Analgesic activity of bacosine, a new triterpene isolated from Bacopa monnieri. Fitoterapia 68:361–365

    CAS  Google Scholar 

  • Yang D-C, Choi Y-E (2000) Production of transgenic plants via Agrobacterium rhizogenes-mediated transformation of Panax ginseng. Plant Cell Rep 19:491–496

    Article  CAS  Google Scholar 

  • Zambriski P, Tempé J, Schell J (1989) Transfer and function of T-DNA genes from Agrobacterium Ti and Ri plasmids in plants. Cell 56:193–201

    Article  Google Scholar 

Download references

Acknowledgments

Sukanya Majumdar is indebted to the University Grants Commission for award of Junior and Senior Research Fellowship, to Dr. B. Chaubey, Reader Department of Botany for help in molecular analysis and to the Programme Coordinator, CAS, Dept of Botany, CU & Coordinator, BC Guha Centre for Biotechnology, CU, for facilities provided.

Author information

Authors and Affiliations

  1. Centre of Advanced Study, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, India

    Sukanya Majumdar & Sumita Jha

  2. Indian Institute of Chemical Biology, 4 Raja SC Mullick Road, Jadavpur, Kolkata, 700032, India

    Saraswati Garai

Authors
  1. Sukanya Majumdar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Saraswati Garai
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sumita Jha
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sumita Jha.

Additional information

Communicated by J. Register.

A contribution to the Special Issue: Plant Biotechnology in Support of the Millennium Development Goals.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Majumdar, S., Garai, S. & Jha, S. Genetic transformation of Bacopa monnieri by wild type strains of Agrobacterium rhizogenes stimulates production of bacopa saponins in transformed calli and plants. Plant Cell Rep 30, 941–954 (2011). https://doi.org/10.1007/s00299-011-1035-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-011-1035-9

Keywords

Search

Navigation