Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 120, Issue 2, pp 475–487 | Cite as

An efficient transformation method for estrogen-inducible transgene expression in Catharanthus roseus hairy roots

  • Noreen F. Rizvi
  • Miglia Cornejo
  • Kassi Stein
  • Jessica Weaver
  • Erin J. Cram
  • Carolyn W. T. Lee-ParsonsEmail author
Original Paper


Efficient genetic engineering of the medicinal plant, Catharanthus roseus, is essential in improving the production of the pharmaceutically important anticancer compounds, vinblastine and vincristine. Here we optimize several steps in the Agrobacterium-mediated transformation of C. roseus, including antibiotic selection and Agrobacterium elimination parameters. With the optimized protocol, stable transgenic hairy root cultures expressing green fluorescent protein (GFP) under control of the estrogen-inducible XVE system were established with a transformation efficiency of 33 %. The estrogen-inducible system has not been previously tested in C. roseus hairy roots but offers several advantages as an inducible system. Prior to induction, low basal levels of Gfp are observed. Upon induction with 5 mM 17β-estradiol, Gfp transgene expression is highly and rapidly induced within 1 h, increasing after 24 h. Strongest GFP expression is observed in the meristematic root tips and is sustained over 5 days in the presence of estradiol. Neither the 17β-estradiol induction nor the estrogen-inducible system elicits a defense response in C. roseus hairy roots.


Catharanthus roseus Agrobacterium-mediated transformation Genetic engineering Estrogen-inducible Hairy roots 



This work was supported by the National Science Foundation (NSF CBET Award #1033889). The authors thank Dr. Nam-Hai Chua (The Rockefeller University, New York, NY) for providing the pER8-GFP vector and Dr. Jacqueline V. Shanks (Iowa State University, Ames, IA), Dr. Ka-Yiu San (Rice University, Houston, TX) for providing the wild-type C. roseus hairy root cultures.


  1. Aird ELH, Hamill JD, Rhodes MJC (1988) Cytogenetic analysis of hairy root cultures from a number of plant species transformed by Agrobacterium rhizogenes. Plant Cell, Tissue Organ Cult 15:47–57CrossRefGoogle Scholar
  2. Aoyama T, Chua NH (1997) A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J 11:605–612PubMedCrossRefGoogle Scholar
  3. Bhadra R, Vani S, Shanks JV (1993) Production of indole alkaloids by selected hairy root lines of Catharanthus roseus. Biotechnol Bioeng 41:581–592PubMedCrossRefGoogle Scholar
  4. Cheng ZM, Schnurr JA, Kapaun JA (1998) Timentin as an alternative antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation. Plant Cell Rep 17:646–649CrossRefGoogle Scholar
  5. Choi PSK, 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 22:828–831PubMedCrossRefGoogle Scholar
  6. Ciau-Uitz R, Miranda-Ham ML, Coello-Coello J, Chi B, Pacheco LM, Loyola-Vargas VM (1994) Indole alkaloid production by transformed and non-transformed root cultures of Catharanthus roseus. In Vitro Cell Dev Biol Plant 30:84–88CrossRefGoogle Scholar
  7. de Ruijter NCA, Verhees J, Leeuwen WV, Krol AD (2003) Evaluation and comparison of the GUS, LUC and GFP reporter system for gene expression studies in plants. Plant Biol 5:103–115CrossRefGoogle Scholar
  8. Felenbok B (1991) The ethanol utilization regulon of Aspergillus nidulans: the alcA-alcR system as a tool for the expression of recombinant proteins. J Biotechnol 17:11–17PubMedCrossRefGoogle Scholar
  9. Goklany S, Rizvi NF, Loring RH, Cram EJ, Lee-Parsons CWT (2013) Jasmonate-dependent alkaloid biosynthesis in Catharanthus roseus hairy root cultures is correlated with the relative expression of Orca and Zct transcription factors. Biotechnol Prog 29:1367–1376PubMedCrossRefGoogle Scholar
  10. Haas JH, Moore LW, Ream W, Manulis S (1995) Universal PCR primers for detection of phytopathogenic Agrobacterium strains. Appl Environ Microbiol 61:2879–2884PubMedCentralPubMedGoogle Scholar
  11. Hong SB, Peebles CA, Shanks JV, San KY, Gibson SI (2006) Expression of the Arabidopsis feedback-insensitive anthranilate synthase holoenzyme and tryptophan decarboxylase genes in Catharanthus roseus hairy roots. J Biotechnol 122:28–38PubMedCrossRefGoogle Scholar
  12. Hughes EH, Hong S-B, Shanks JV, San K-Y, Gibson SI (2002) Characterization of an Inducible Promoter System in Catharanthus roseus Hairy Roots. Biotechnol Prog 18:1183–1186PubMedCrossRefGoogle Scholar
  13. Hughes EH, Hong SB, Gibson SI, Shanks JV, San KY (2004a) Expression of a feedback-resistant anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation of terpenoid indole alkaloid levels. Biotechnol Bioeng 86:718–727PubMedCrossRefGoogle Scholar
  14. Hughes EH, Hong SB, Gibson SI, Shanks JV, San KY (2004b) Metabolic engineering of the indole pathway in Catharanthus roseus hairy roots and increased accumulation of tryptamine and serpentine. Metab Eng 6:268–276PubMedCrossRefGoogle Scholar
  15. Kang HG, Fang Y, Singh KB (1999) A glucocorticoid-inducible transcription system causes severe growth defects in Arabidopsis and induceses defense-related genes. Plant J 20:127–133PubMedCrossRefGoogle Scholar
  16. Kittipongpatana N, Hock RS, Porter JR (1998) Production of solasodine by hairy root, callus, and cell suspension cultures of Solanum aviculare Forst. Plant Cell, Tissue Organ Cult 52:133–143CrossRefGoogle Scholar
  17. Kost B, Spielhofer P, Chua NH (1998) A GFP-mouse talin fusion protein labels plant actin filamentsin vivoand visualizes the actin cytoskeleton in growing pollen tubes. Plant J 16:393–401PubMedCrossRefGoogle Scholar
  18. Lee SY, Kim SG, Song WS, Kim YK, Park NI, Park SU (2010) Influence of different strains of Agrobacterium rhizogenes on hairy root induction and production of alizarin and purpurin in Rubia akane Nakai. Romanian Biotechnol Lett 15:5406Google Scholar
  19. Li CY, Leopold AL, Sander GW, Shanks JV, Zhao L, Gibson SI (2013) The ORCA2 transcription factor plays a key role in regulation of the terpenoid indole alkaloid pathway. BMC Plant Biol 13:155PubMedCentralPubMedCrossRefGoogle Scholar
  20. Magnotta M, Murata J, Chen J, De Luca V (2007) Expression of deacetylvindoline-4-O-acetyltransferase in Catharanthus roseus hairy roots. Phytochemistry 68:1922–1931PubMedCrossRefGoogle Scholar
  21. Memelink J, Verpoorte R, Kijne JW (2001) ORCAnization of jasmonate-responsive gene expression in alkaloid metabolism. Trends Plant Sci 6:212–219PubMedCrossRefGoogle Scholar
  22. Meng ZH, Liang AH, Yang WC (2007) Effects of hygromycin on cotton cultures and its application in Agrobacterium-mediated cotton transformation. In Vitro Cell Dev Biol Plant 43:111–118CrossRefGoogle Scholar
  23. Menke FLH, Champion A, Kijne JW, Memelink J (1999) A novel jasmonate- and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate- and elicitor-inducible AP2-domain transcription factor, ORCA2. EMBO J 18:4455–4463PubMedCentralPubMedCrossRefGoogle Scholar
  24. Messina MJ (1999) Legumes and soybeans: overview of their nutritional profiles and health effects. Am J Clin Nutrit 70:439s–450sPubMedGoogle Scholar
  25. Moreno-Valenzuela OA, Galaz-Avalos RM, Minero-Garcia Y, Loyola-Vargas VM (1998) Effect of differentiation on the regulation of indole alkaloid production in Catharanthus roseus hairy roots. Plant Cell Rep 18:99–104CrossRefGoogle Scholar
  26. Nauerby B, Billing K, Wyndaele R (1997) Influence of the antibiotic timentin on plant regeneration compared to carbenicilling and cefotazime in concentrations suitable for elimination of Agrobacterium tumefaciens. Plant Sci 123:169–177CrossRefGoogle Scholar
  27. Noble RL (1990) The discovery of the vinca alkaloids-chemotherapeutic agents against cancer. Biochem Cell Biol 68:1344–1351PubMedCrossRefGoogle Scholar
  28. Okuzaki A, Konagaya KI, Nanasato Y, Tsuda M, Tabei Y (2011) Estrogen-inducible GFP expression patterns in rice (Oryza sativa L.). Plant Cell Rep 30:529–538PubMedCentralPubMedCrossRefGoogle Scholar
  29. Ouwerkerk PB, de Kam RJ, Hoge HJ, Meijer AH (2001) Glucocorticoid-inducible gene expression in rice. Planta 213:370–378PubMedCrossRefGoogle Scholar
  30. Pauw B et al (2004) Zinc finger proteins act as transcriptional repressors of alkaloid biosynthesis genes in Catharanthus roseus. J Biol Chem 279:52940–52948PubMedCrossRefGoogle Scholar
  31. Peebles CA, Gibson SI, Shanks JV, San KY (2007a) Characterization of an ethanol-inducible promoter system in Catharanthus roseus hairy roots. Biotechnol Prog 23:1258–1260PubMedGoogle Scholar
  32. Peebles CA, Gibson SI, Shanks JV, San KY (2007b) Long-term maintenance of a transgenic Catharanthus roseus hairy root line. Biotechnol Prog 23:1517–1518PubMedCrossRefGoogle Scholar
  33. Peebles CA, Hughes EH, Shanks JV, San KY (2009) Transcriptional response of the terpenoid indole alkaloid pathway to the overexpression of ORCA3 along with jasmonic acid elicitation of Catharanthus roseus hairy roots over time. Metab Eng 11:76–86PubMedCrossRefGoogle Scholar
  34. Peebles CA, Sander GW, Hughes EH, Peacock R, Shanks JV, San KY (2011) The expression of 1-deoxy-d-xylulose synthase and geraniol-10-hydroxylase or anthranilate synthase increases terpenoid indole alkaloid accumulation in Catharanthus roseus hairy roots. Metab Eng 13:234–240PubMedCrossRefGoogle Scholar
  35. Rizvi N, Goklany S, Cram EJ, Lee-Parsons CWT (2013) Rapid increases of key regulators precede the increased production of pharmaceutically valuable compounds in Catharanthus roseus. Pharm Eng 33:1–9Google Scholar
  36. Roslan HA et al (2001) Characterization of the ethanol-inducible alc gene-expression system in Arabidopsis thaliana. Plant J 28:225–235PubMedCrossRefGoogle Scholar
  37. Runguphan W, Maresh JJ, O’Connor SE (2009) Silencing of tryptamine biosynthesis for production of nonnatural alkaloids in plant culture. Proc Natl Acad Sci USA 106:13673–13678PubMedCentralPubMedCrossRefGoogle Scholar
  38. Salter MG, Paine JA, Riddell KV, Jepson I, Greenland AJ, Caddick MX, Tomsett AB (1998) Characterisation of the ethanol-inducible alc gene expression system for transgenic plants. Plant J 16:127–132CrossRefGoogle Scholar
  39. Suttipanta N, Pattanaik S, Kulshrestha M, Patra B, Singh SK, Yuan L (2011) The transcription factor CrWRKY1 positively regulates the terpenoid indole alkaloid biosynthesis in Catharanthus roseus. Plant Physiol 157:2081–2093PubMedCentralPubMedCrossRefGoogle Scholar
  40. van der Fits L, Memelink J (2000) ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science 289:295–297PubMedCrossRefGoogle Scholar
  41. van der Heijden R, Jacobs DI, Snoeijer W, Hallard D, Verpoorte R (2004) The Catharanthus alkaloids: pharmacognosy and biotechnology. Curr Med Chem 11:607–628CrossRefGoogle Scholar
  42. Vanhala LH, Hilunen R, Oksman-Caldentey K-M (1995) Virulence of different Agrobacterium strains on hairy root formation of Hyoscyamus muticus. Plant Cell Rep 14:236–340Google Scholar
  43. Veena V, Taylor CG (2007) Agrobacterium rhizogenes: recent developments and promising applications. In Vitro Cell Dev Biol Plant 43:383–403CrossRefGoogle Scholar
  44. Wang C-T, Liu H, Gao X-S, Zhang H-X (2010) Overexpression of G10H and ORCA3 in the hairy roots of Catharanthus roseus improves catharanthine production. Plant Cell Rep 29:887–894Google Scholar
  45. Weaver J, Goklany S, Rizvi N, Cram EJ, Lee-Parsons CW (2014) Optimizing the transient Fast Agro-mediated Seedling Transformation (FAST) method in Catharanthus roseus seedlings. Plant Cell Rep 33:89–97PubMedCrossRefGoogle Scholar
  46. White FF, Nester EW (1980) Hairy root: plasmid encodes virulence traits in Agrobacterium rhizogenes. J Bacteriol 141:1134–1141PubMedCentralPubMedGoogle Scholar
  47. 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 164:33–44PubMedCentralPubMedGoogle Scholar
  48. Wilmink A, Dons JJM (1993) Selective agents and marker genes for use in transformation of monocotyledonous plants. Plant Mol Biol Rep 11:165–185CrossRefGoogle Scholar
  49. Xu M, Dong J (2007) Enhancing terpenoid indole alkaloid production by inducible expression of mammalian Bax in Catharanthus roseus cells. Sci China, Ser C Life Sci 50:234–241CrossRefGoogle Scholar
  50. Yukimune Y, Hara Y, Yamada Y (1994) Tropane alkaloid production in root cultures of Duboisia myoporoides obtained by repeated selection. Biosci Biotech Biochem 58:1443–1446CrossRefGoogle Scholar
  51. Zarate R, Verpoorte R (2007) Strategies for the genetic modification of the medicinal plant Catharanthus roseus (L.) G. Don. Phytochem Rev 6:475–491CrossRefGoogle Scholar
  52. Zhao L, Sander GW, Shanks JV (2013) Perspectives of the metabolic engineering of terpenoid indole alkaloids in Catharanthus roseus hairy roots. Adv Biochem Eng Biotechnol 134:23–54PubMedGoogle Scholar
  53. Zhou M-L, Zhu X-M, Shao J-R, Wu Y-M, Tang Y-X (2010) Transcriptional response of the catharanthine biosynthesis pathway to methyl jasmonate/nitric oxide elicitation in Catharanthus roseus hairy root culture. Appl Microbiol Biotechnol 88:737–750Google Scholar
  54. Zhou ML, Zhu XM, Shao JR, Wu YM, Tang YX (2012) A protocol for genetic transformation of Catharanthus roseus by Agrobacterium rhizogenes A4. Appl Biochem Biotechnol 166:1674–1684PubMedCrossRefGoogle Scholar
  55. Zuo J, Niu Q-W, Chua N-H (2000) An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J 24:265–273PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Noreen F. Rizvi
    • 1
  • Miglia Cornejo
    • 1
  • Kassi Stein
    • 1
  • Jessica Weaver
    • 2
  • Erin J. Cram
    • 2
  • Carolyn W. T. Lee-Parsons
    • 1
    • 3
    Email author
  1. 1.Department of Chemical EngineeringNortheastern UniversityBostonUSA
  2. 2.Department of BiologyNortheastern UniversityBostonUSA
  3. 3.Department of Chemistry and Chemical BiologyNortheastern UniversityBostonUSA

Personalised recommendations