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Genetic transformation of Gentiana macrophylla with Agrobacterium rhizogenes: growth and production of secoiridoid glucoside gentiopicroside in transformed hairy root cultures

  • Genetic Transformation and Hybridization
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Abstract

Hairy root cultures of Gentiana macrophylla were established by infecting the different explants four Agrobacterium rhizogenes strains namely A4GUS, R1000, LBA 9402 and ATCC11325, and hairy root lines were established with A. rhizogenes strain R1000 in 1/2 MS + B5 medium. Initially, 42 independent hairy root clones were maintained and seven clones belongs to different category were evaluated for growth, morphology, integration and expression of Ri T-DNA genes, and alkaloid contents in dry root samples. On the basis of total root elongation, lateral root density and biomass accumulation on solid media, hairy root clones were separated into three categories. PCR and Southern hybridization analysis revealed both left and right T-DNA integration in the root clones and RT-PCR analysis confirmed the expression of hairy root inducible gene. GUS assay was also performed to confirm the integration of left T-DNA. The accumulation of considerable amounts of the root-specific secoiridoid glucosides gentiopicroside was observed in GM1 (\({\rm T}_{\rm L}^ +\) and \({\rm T}_{\rm R}^ +\)) and the GM2 (\({\rm T}_{\rm L}^ +\) and \({\rm T}_{\rm R}^ -\) DNA) type clones in considerably higher amount whether as two \({\rm T}_{\rm L}^ -\) but \({\rm T}_{\rm R}^ +\) callus-type clones (GM3) accumulated much less or only very negligible amounts of gentiopicroside. Out of four media composition the 1/2 MS + B5 vitamin media was found most suitable. We found that initial establishment of root cultures largely depends on root:media ratio. Maximum growth rate was recorded in 1:50 root:media ratio. The maximum biomass in terms of fresh weight (33-fold) was achieved in 1/2 MS + B5 media composition after 35 days in comparison to sixfold increase in control. The biomass increase was most abundant maximum from 15 to 30 days. Influence of A. rhizogenes strains and Ri plasmid of hairy root induction, the possible role of the TL-DNA and TR-DNA genes on growth pattern of hairy root, initial root inoculum:media ratio and effect of media composition is discussed.

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Abbreviations

ags :

Agropine synthase

FW:

Fresh weight

HPLC:

High-performance liquid chromatography

MS:

Media Murashige and Skoog

NT:

Non-transformed

Ri:

Root-inducing

TL :

Left-terminus DNA

TR :

Right-terminus DNA

YEB:

Yeast extract broth

References

  • Aoki S, Syono K (1999) Synergistic function of rolB, rolC, ORF13 and ORF14 of TL-DNA of Agrobacterium rhizogenes in hairy root induction in Nicotiana tabacum. Plant Cell Physiol 40:252–256

    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 

  • Baumann K, De Paolis A, Costantino P, Gualberti G (1999) The DNA binding site of the Dof protein NtBBF1 is essential for tissue-specific and auxin-regulated expression of the rolB oncogene in plants. Plant Cell 11:323–333

    Article  PubMed  CAS  Google Scholar 

  • Bhattacharya SK, Ghosal S, Chaudhuri RK, Singh AK, Sharma PV (1974) Chemical constituents of Gentianaceae XI: antipsychotic activity of gentianine. J Pharm Sci 63(8):1341–1342

    Article  PubMed  CAS  Google Scholar 

  • Binns AN, Tomashow JV (1988) Cell biology of Agrobacterium infection and transformation of plants. Annu Rev Microbiol 42:575–606

    Article  CAS  Google Scholar 

  • Bourgaud F, Gravot A, Milesi S, Gontier E (2001) Production of plant secondary metabolites: a historical perspective. Plant Sci 161:839–851

    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

    Article  CAS  Google Scholar 

  • Capone I, Spanó L, Cardarelli M, Bellincampi D, Petit A, Costantino P (1989) Induction and growth properties of carrot roots with different complements of Agrobacterium rhizogenes T-DNA. Plant Mol Biol 13:43–52

    Article  PubMed  CAS  Google Scholar 

  • Chu CC (1978) The N6 medium and its applications to anther culture of cereal crops. In: Proceedings of the Symposium on Plant Tissue Culture, Beijing, China, pp 43–50

  • Duncan DB (1955) Multiple range and multiple F tests. Biometrics 11:1–42

    Article  Google Scholar 

  • Filippini F, Rossi V, Marin O, Trovato M, Downey PM, Costantino P, Lo Schiavo F, Terzi M (1996) A plant oncogene as a phophatase. Nature 379:499–500

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Giri A, Narasu ML (2000) Transgenic hairy roots: recent trends and applications. Biotechnol Adv 18:1–22

    Article  PubMed  CAS  Google Scholar 

  • Guivarc’h A, Boccara M, Prouteau M, Chriqui D (1999) Instability of phenotype and gene expression in long term culture of carrot hairy root clones. Plant Cell Rep 19:43–53

    Article  CAS  Google Scholar 

  • Hooykaas PJJ, Klapwijk PM, Nuti MP, Shilperoot RA, Horsch A (1977) Transfer of A. tumefaciens Ti plasmid to avirulent Agrobacterium and Rhizobium ex planta. J Gen Microbiol 98:477–484

    Google Scholar 

  • Hosokawa K, Matsuki R, 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 

  • Huffman GA, White FF, Gordon MP, Nester EW (1984) Hairy root-inducing plasmid: physical map and homology to tumor inducing plasmids. J Bacteriol 157:269–276

    PubMed  CAS  Google Scholar 

  • Ishiguro K, Yamaki M, Takagi S (1982) Studies on the iridoid related compounds. I. On the antimicrobial activity of aucubigenin and certain iridoid aglycones. Yakugaku Zasshi 102(8):755–759

    PubMed  CAS  Google Scholar 

  • Jefferson RA (1987) Assaying chimeric genes in plant: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405

    CAS  Google Scholar 

  • Jouanin L, Guerche P, 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 grown in vitro. Plant Sci 50:145–152

    Article  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 Biol Rep 17:1–7

    Article  Google Scholar 

  • Koltunow AM, Johnson SD, Lynch M, Yoshihara T, Costantino P (2001) Expression of rolB in apomictic Hieracium piloselloides Vill. causes ectopic meristems in planta and changes in ovule formation, where apomixis initiates at higher frequency. Planta 214:196–205

    Article  PubMed  CAS  Google Scholar 

  • Kondo Y, Takano F, Hojo H (1994) Suppression of chemically and immunologically induced hepatic injuries by gentiopicroside in mice. Planta Med 60:414–416

    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 

  • Momcilovic I, Grubisic D, Kojic M, Neskovic M (1997) Agrobacterium rhizogenes-mediated transformation and plant regeneration of four Gentiana species. Plant Cell Tissue Organ Cult 50:1–6

    Article  Google Scholar 

  • Moore L, Warren G, Strobel G (1979) Involvement of a plasmid in the hairy root disease caused by A. rhizogenes. Plasmid 2:617–626

    Article  PubMed  CAS  Google Scholar 

  • Mugnier J (1988) Establishment of new axenic hairy root lines by inoculation with Agrobacterium rhizogenes. Plant Cell Rep 7:9–12

    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 

  • Nilsson O, Olsson O (1997) Getting to the root: the role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots. Physiol Plant 100:463–473

    Article  CAS  Google Scholar 

  • Pengcheng L, Zhaohui Y, Yongchan L, Suqin Z, Fengzu H, Lanju J (2004) Content determination of Loganic acid and Gentiopicroside in Tibetan herbal medicines Gentiana macrophylla and G. straminea by HPLC. J Chin Med Mater 27(11):819–821

    Google Scholar 

  • Pomponi M, Spano L, Sabbadini MG, Costantino P (1983) Restriction endonuclease mapping of the root inducing plasmid of Agrobacterium rhizogenes 1855. Plasmid 10:119–129

    Article  PubMed  CAS  Google Scholar 

  • Rhodes MJC, Robins RJ, Hamill JD, Parr AJ, Hilton MG, Walton NJ (1990) Properties of transformed root cultures. In: Charlwood BV, Rhodes MJC (eds) Proceedings of the Phytochemical Society of Europe on Secondary Products from Plant Tissue Culture, Clarendon, Oxford, pp 201–225

    Google Scholar 

  • Riker AJ, Bandfield WM, Wright WH, Keitt GW, Sagen HE (1930) Studies on infectious hairy root of nursery apple tree. J Agric Res (Washington) 41:507–540

    Google Scholar 

  • Salzman RA, Fujita T, Zhu-Salzman K, Hasegawa PM, Bressan RA (1999) An improved RNA isolation method for plant tissues containing high levels of phenolic compounds or carbohydrates. Plant Mol Biol Rep 17:11–17

    Article  CAS  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 

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

    PubMed  CAS  Google Scholar 

  • Sevon N, Oksman-Caldentey KM (2002) Agrobacterium rhizogenes-mediated transformation: root cultures as a source of alkaloids. Planta Med 68:859–868

    Article  PubMed  CAS  Google Scholar 

  • Skrzypczak L, Wesolowska M, Skrzypczak E (1993) Gentiana species XII: In vitro culture, regeneration, and production of secoiridoid glucosides. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 21: Medicinal and aromatic plants IV. Springer-Verlag, Berlin, Heidelberg, pp 172–186

    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

    PubMed  CAS  Google Scholar 

  • Spanò L, Mariotti D, Cardarelli M, Branca C, Costantino P (1988) Morphogenesis and auxin sensitivity of transgenic tobacco with different complements of Ri T-DNA. Plant Physiol 87:479–483

    Article  PubMed  Google Scholar 

  • Tang W, Eisenbrand G (1992) Gentiana spp. In: Tang W, Eisenbrand G (eds) Chinese drugs of plant origin, chemistry, pharmacology, and use in traditional and modern medicine. Springer-Verlag, Berlin, Heidelberg, pp 549–553

    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

    Article  CAS  Google Scholar 

  • Tepfer D (1990) Genetic transformation using Agrobacterium rhizogenes. Physiol Plant 79:140–146

    Article  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  • Van Der Sluis WG, Van Der Nat JM, Labadie RP (1983) Thin-layer chromatographic bioassay of iridoid and secoiridoid glucosides with a fungitoxic aglucone moiety using b-glucosidase and the fungus Penicillium expansum as a test organism. J Chromatogr 259:522–526

    Article  PubMed  CAS  Google Scholar 

  • Vervliet G, Holsters M, Teuchy H, Van Montagu M, Schell J (1975) Characterization of different plaque forming and defective temperate phages in Agrobacterium. J Gen Virol 26:33–48

    Article  PubMed  CAS  Google Scholar 

  • White FF, Nester E W (1980). Hairy root: plasmid encodes virulence traits in Agrobacterium rhizogenes. J Bacteriol 141:1134–1141

    PubMed  CAS  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

  • White PR (1963) The cultivation of animal and plant cells. Ronald Press, New York

    Google Scholar 

  • Yoshikawa T, Furuya T (1987) Saponin production by cultures of Panax ginseng transformed with Agrobacterium rhizogenes. Plant Cell Rep 6:449–453

    CAS  Google Scholar 

  • Zehra M, Banerjee S, Sharma S, Kumar S (1999) Influence of Agrobacterium rhizogenes strains on biomass and alkaloid productivity in hairy root lines of Hyoscyamus muticus and H. albus. Planta Med 64:60–63

    Article  Google Scholar 

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Acknowledgments

We thank Prof. D. Tepfer, INRA, France for providing the Agrobacterium rhizogenes A4GUS strain. We also thank Dr. Simon Deroles, New Zealand Institute for Crop & Food Research, New Zealand, Dr. Mark Tepfer, International Center for Genetic Engineering and Biotechnology, Italy, for stimulating discussions. Financial assistance in the form of post-doc fellowship provided by the China Post-Doctoral Research Foundation (No. 224-415-203) and PDF fund of Lanzhou University (No. 505-415-104), Lanzhou, China is gratefully acknowledged.

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  1. Institute of Cell Biology, School of Life Sciences, Lanzhou University, Lanzhou, 73000, Gansu, P.R. China

    Rajesh Kumar Tiwari, Mala Trivedi, Zhang Chun Guang, Guang-Qin Guo & Guo-Chang Zheng

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  1. Rajesh Kumar Tiwari
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  2. Mala Trivedi
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  3. Zhang Chun Guang
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Correspondence to Rajesh Kumar Tiwari.

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Communicated by A. Atanassov

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Tiwari, R.K., Trivedi, M., Guang, Z.C. et al. 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 (2007). https://doi.org/10.1007/s00299-006-0236-0

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