Skip to main content
SpringerLink
Log in

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

  • Genetic Transformation and Hybridization
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Hairy root cultures of Catharanthus roseus var. Prabal were established by infecting the leaves with Agrobacterium rhizogenes agropine-type A4 strain. Two hundred and fifty independent root clones were evaluated for growth, morphology, number of integration of Ri T-DNA genes and alkaloid contents. On the basis of growth pattern, type of branching and number of lateral roots we were able to separate the hairy root clones into four categories. However based on the integration of the Ri TL-DNA and TR-DNA genes, there were only three different categories of independent hairy root clones—C1 (rolA&B +/ags +), C2 (rolA&B -/ags +) and C3 (rolA&B +/ags −). Southern hybridization analysis revealed both single and multiple copies of T-DNA integration in the root clones. The accumulation of considerable amounts of the root-specific alkaloids ajmalicine and serpentine was observed in the presence of both the TL-DNA and TR-DNA genes (C1) and the TL-DNA gene (C3) alone. Two rolA&B − but ags + clones (C2) accumulated much less or only very negligible amounts of ajmalicine. The possible role of the TL-DNA and TR-DNA genes on growth and alkaloid accumulation in these root clones is discussed.

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. 1a–d
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7a,b
Fig. 8

Similar content being viewed by others

Abbreviations

ags :

Agropine synthase

Ri :

Root-inducing

T L -DNA :

Left-terminus DNA

T R -DNA :

Right-terminus DNA

TIAs :

Terpenoid indole alkaloids

References

  • Aoki T, Matsumoto H, Asako Y, Matsunaga Y, Shimomura K (1997) Variation of alkaloid productivity among several clones of hairy roots and regenerated plants of Atropa belladonna transformed with Agrobacterium rhizogenes 15834. Plant Cell Rep 16:282–286

    Article  CAS  Google Scholar 

  • Bhadra R, Shanks JV (1997) Transient studies of nutrient uptake, growth and indole alkaloid accumulation in heterotrophic cultures of hairy roots of Catharanthus roseus. Biotechnol Bioeng 55:527–534

    Article  CAS  Google Scholar 

  • Brillanceau MH, David C, Tempe J (1989) Genetic transformation of Catharanthus roseus G. Don by Agrobacterium rhizogenes. Plant Cell Rep 8:63–66

    CAS  Google Scholar 

  • Cardarelli M, Mariotti D, Pomponi M, Spano L, Capone I, Costantino P (1987) Agrobacterium rhizogenes T-DNA genes capable of inducing hairy root phenotype. Mol Gen Genet 209:475–480

    CAS  Google Scholar 

  • Chilton MDM, Que Q (2003) Targeted integration of T-DNA into the Tobacco genome at double-stranded breaks: new insights on the mechanism of T-DNA integration. Plant Physiol 133:956–965

    Article  CAS  PubMed  Google Scholar 

  • Chilton MD, Tepfer DA, Petit A, David C, Casse-Delbart F, Tempe J (1982) Agrobacterium rhizogenes inserts T-DNA into the genomes of the host plant root cells. Nature 295:432–434

    CAS  Google Scholar 

  • Christey MC (2001) Use of Ri-mediated transformation for production of transgenic plants. In Vitro Cell Dev Biol Plant 37:687–700

    CAS  Google Scholar 

  • De Paolis A, Mauro ML, Pomponi M, Cardarelli M, Spano L, Costantino P (1985) Localization of agropine-synthesizing functions in the TR region of the root-inducing plasmid of Agrobacterium rhizogenes 1855. Plasmid 13:1–17

    PubMed  Google Scholar 

  • Durand-Tardif M, Broglie R, Slightom J, Tepfer D (1985) Structure and expression of Ri T-DNA from Agrobacterium rhizogenes in Nicotiana tabacum: organ and phenotypic specificity. J Mol Biol 186(3):557–564

    CAS  PubMed  Google Scholar 

  • Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158

    CAS  PubMed  Google Scholar 

  • Hanisch ten Cate CH, Loonen AE, Ottaviani MP, Ennik L, van Eldik G, Stiekema WJ (1990) Frequent spontaneous deletions of Ri T-DNA in Agrobacterium rhizogenes transformed potato roots and regenerated plants. Plant Mol Biol 14:735–741

    PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  • Jung KH, Kwak SS, Kim SW, Lee H, Choi CY, Liu JR (1992) Improvement of the catharanthine productivity in hairy root cultures of Catharanthus roseus by using monosaccharides as a carbon source. Biotechnol Lett 14:695–700

    CAS  Google Scholar 

  • Khanuja SPS, Shasany AK, Darokar MP, Kumar S (1999) Rapid isolation of DNA from dry and fresh samples of plants producing large amounts of secondary metabolites and essential oils. Plant Mol Bio Rep 17:1–7

    Article  Google Scholar 

  • Kumar S, Fladung M (2002) Transgene integration in aspen: structures of integration sites and mechanism of T-DNA integration. Plant J 31:543–551

    Article  CAS  PubMed  Google Scholar 

  • Lemcke K, Schmulling T (1998) A putative rolB gene homolog of the Agrobacterium rhizogenes TR-DNA has different morphogenetic activity in tobacco than rolB. Plant Mol Biol 36:803–808

    Article  CAS  PubMed  Google Scholar 

  • Limami MA, Sun LY, Douat C, Helgeson J, Tepfer D (1998) Natural genetic transformation by Agrobacterium rhizogenes. Plant Physiol 118:543–550

    Article  CAS  PubMed  Google Scholar 

  • Morgan JA, Barney CS, Penn AH, Shanks JV (2000) Effects of buffered media upon growth and alkaloid production of Catharanthus roseus hairy roots. Appl Microbiol Biotechnol 53:262–265

    Article  CAS  PubMed  Google Scholar 

  • Parr AJ, Peerless ACJ, Hamill JD, Walton NJ, Robins RJ, Rhodes MJC (1988) Alkaloid production by transformed root cultures of Catharanthus roseus. Plant Cell Rep 7:309–312

    CAS  Google Scholar 

  • Rijhwani SK, Shanks JV (1998) Effect of elicitor dosage and exposure time on biosynthesis of indole alkaloids by Catharanthus roseus hairy root cultures. Biotechnol Prog 14:442–449

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Shanks JV, Bhadra R (1997) Characteristics of selected hairy root lines of Catharanthus roseus. In: Doran PM (ed) Hairy roots-culture and applications. Harwood Academic, The Netherlands, pp 51–63

  • Singh DV, Maithy A, Verma RK, Gupta MM, Kumar S (2000) Simultaneous determination of Catharanthus alkaloids using reversed phase high performance liquid chromatography. J Liq Chromatogr 23:601–607

    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. J Biol Chem 261:108–121

    CAS  PubMed  Google Scholar 

  • Spena A, Schmulling T, Koncz C, Schell JS (1987) Independent and synergistic activity of rol A, B and C loci in stimulating abnormal growth in plants. EMBO J 6:3891–3899

    CAS  Google Scholar 

  • Taylor BH, Amasino RM, White FF, Nester EW, Gordon MP (1985) T-DNA analysis of plants regenerated from hairy root tumor. Mol Gen Genet 201:554–557

    CAS  Google Scholar 

  • Tikhomiroff C, Jolicoeur M (2002) Screening of Catharanthus roseus secondary metabolites by high-performance liquid chromatography. J Chromatogr 955:87–93

    Article  CAS  Google Scholar 

  • Toivonen L, Balsevich J, Kurz WGW (1989) Indole alkaloid production by hairy root cultures of Catharanthus roseus. Plant Cell Tissue Organ Cult 18:79–93

    CAS  Google Scholar 

  • Toivonen L, Laakso S, Rosenqvist H (1992) The effect of temperature on hairy root cultures of Catharanthus roseus: growth, indole alkaloid accumulation and membrane lipid composition. Plant Cell Rep 11:395–399

    CAS  Google Scholar 

  • Verpoorte R, van der Heijden R, van Gulik WM, ten Hoopen HJG (1991) Plant biotechnology for the production of alkaloids: present status and prospects. In: Brossi A (ed) The alkaloids, vol 40. Academic, San Diego, pp 1–187

  • White FF, Taylor BH, Huffman GA, Gordon MP, Nester EW (1985) Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes. J Bacteriol 164:33–44

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We sincerely thank the Department of Biotechnology, Government of India, for financial support. We gratefully acknowledge Dr. S. Dwivedi, Central Institute of Medicinal and Aromatic Plants, Lucknow, India for kindly providing the seeds of C. roseus var. Prabal.

Junior Research Fellowship to JB by CSSR, Govt. of India, is thankfully acknowledged.

Author information

Authors and Affiliations

  1. National Centre for Plant Genome Research, Jawaharlal Nehru University Campus, P.O. Box 10531, New Delhi, 110 067, India

    Jyoti Batra, Ajaswrata Dutta, Digvijay Singh, Sushil Kumar & Jayanti Sen

Authors
  1. Jyoti Batra
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ajaswrata Dutta
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Digvijay Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sushil Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jayanti Sen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jayanti Sen.

Additional information

Communicated by P. Lakshmanan

Rights and permissions

Reprints and permissions

About this article

Cite this article

Batra, J., Dutta, A., Singh, D. et al. 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 (2004). https://doi.org/10.1007/s00299-004-0815-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-004-0815-x

Keywords

Search

Navigation