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Variability in shoot cultures regenerated from hairy roots of Gentiana punctata

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Biologia Plantarum

Abstract

Differences among three clones of Gentiana punctata L. hairy root shoot regenerants were investigated in relation to their growth patterns, production of secondary metabolites and 2D protein profiles. Prominent differences in growth parameters were stable thus qualifying regenerant clones as true somaclones. Marked differences in protein spots were registered among the regenerant clones but not in comparison with the non-transformed control. Southern blot hybridization of regenerants showed the absence of rolA, B and C genes, initially present in the main hairy root lines. Orf13 and rolD were present and orf8 was missing in all three regenerant clones whereas orf3 was missing only in clone 2. Although lacking the three major rol genes, plants of regenerant clones retained characteristics of the hairy root phenotype.

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Abbreviations

BA:

6-benzyladenine

GA3 :

gibberellic acid

HR:

hairy root phenotype

IAA:

indole-3-acetic acid

NAA:

α-naphthaleneacetic acid

SRC:

shoot regenerant clone

TDZ:

thidiazuron

WPM:

woody plant medium

References

  • Batra, J., Dutta, A., Singh, D., Kumar, S., Sen, J.: 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.

    Article  PubMed  CAS  Google Scholar 

  • Bauer, N., Kiseljak, D., Jelaska, S.: The effect of yeast extract and methyl jasmonate on rosmarinic acid accumulation in Colleus blumei hairy roots. — Biol. Plant. 53: 650–656, 2009.

    Article  CAS  Google Scholar 

  • Birot, A.-M., Bouchez, D., Casse-Delbart, F., Durand-Tardif, M., Jouanin, L., Pautot, V., Robaglia, C., Tepfer, D., Tepfer, M., Tourneur, J., Vilaine, F.: Studies and uses of the rRi plasmids of Agrobacterium rhizogenes. — Plant Physiol. Biochem. 25: 323–335, 1987.

    CAS  Google Scholar 

  • Chaudhury, K.N., Ghosh, B., Tepfer, D., Jha, S.: 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, 2006.

    Article  Google Scholar 

  • Christey, M.C.: Transgenic crop plants using Agrobacterium rhizogenes mediated transformation. — In: Doran, P.M. (ed.): Hairy Roots: Culture and Applications. Pp. 99–111. Harwood Academic Publishers, Amsterdam 1997.

    Google Scholar 

  • Christey, M.C.: Use of Ri mediated transformation for production of transgenic plants. — In Vitro cell. dev. Biol. Plant. 36: 687–700, 2001.

    Google Scholar 

  • Christey, M.C., Braun, R.H., Reader, J.K.: Field performance of transgenic vegetable Brassicas (Brassica oleracea and B. rapa) transformed with Agrobacterium rhizogenes. — Sabrao J. Breed. Genet. 31: 93–108, 1999.

    Google Scholar 

  • David, C., Tempe, J.: Genetic transformation of cauliflower (Brassica oleracea L. var. botrytis) by Agrobacterium rhizogenes. — Plant Cell Rep. 7: 88–91, 1988.

    Article  CAS  Google Scholar 

  • Dmitrović, S., Mitić, N., Zdravković-Korać, S., Vinterhalter, B., Ninković, S., Ćulafić Lj.: Hairy roots formation in recalcitrant-to-transform plant Chenopodium rubrum. — Biol. Plant. 54: 566–570, 2010.

    Article  Google Scholar 

  • Filipecki, M., Malepszy, S.: Unintended consequences of plant transformation: a molecular insight. — J. appl. Genet. 47: 277–286, 2006.

    Article  PubMed  Google Scholar 

  • Gangopadhyay, M., Sircar, D., Mitra, A., Bhattacharya, S.: Hairy root culture of Plumbago indica as a potential source for plumbagin. — Biol. Plant. 52: 533–537, 2008.

    Article  CAS  Google Scholar 

  • Gheysen, G., Dhaese, P., Van Montagu, M., Zambrzsky, P.: Integration of Agrobacterium tumefaciens transfer DNA (T-DNA) involves rearangements of target plant DNA sequences. — Proc. nat. Acad. Sci. USA 84: 6169–6173, 1987.

    Article  PubMed  CAS  Google Scholar 

  • Guivarc’h, A., Boccara, M., Prouteau, M., Chriqui, D.: Instability of phenotype and gene expression in long term culture of carrot hairy root clones. — Plant Cell Rep. 19: 43–50, 1999.

    Article  Google Scholar 

  • Hosokawa, K., Matsuki, R., Oikawa, Y., Yamamura, S.: Genetic transformation using wild-type Agrobacterium rhizogenes. — Plant Cell Tissue Organ Cult. 51: 137–140, 1997.

    Article  Google Scholar 

  • Jouanin, L.: Restriction map of an agropine-type Ri plasmid and its homologies with Ti plasmid. — Plasmid 12: 92–102, 1984.

    Article  Google Scholar 

  • Jouanin, L., Bouchez, D., Drong, R.F., Tepfer, D., Slightom, J.L.: Analysis of TR-DNA / plant junctions in the genome of Concolvulus arvensis clone transformed with Agrobacterium rhizogenes strain A4. — Plant mol. Biol. 12: 75–85, 1989.

    Article  CAS  Google Scholar 

  • Lemke, K., Schmülling, T.: Gain of function assays identify non-rol genes from Agrobacterium rhizogenes TL-DNA that alter plant morphogenesis or hormone sensitivity. — Plant J. 15: 423–433, 1998.

    Article  Google Scholar 

  • Levesque, H., Delepelaire, P., Rouzé, P., Slightom, J., Tepfer, D.: Common evolution origin of the central portions of the Ri TL-DNA of Agrobacterium rhizogenes and Ti T-DNAs Agrobacterium tumefaciens. — Plant mol. Biol. 11: 731–744, 1988.

    Article  CAS  Google Scholar 

  • Lloyd, G.B., McCown, B.H.: Commercially feasible micropropagation of mountain laurel — Kalmia latifolia by use of shoot-tip culture. — Proc. int. Plant Propag. Soc. 30: 421–427, 1980.

    Google Scholar 

  • Mauro, M.L., Trovato, M., De Paolis, A., Gallelli, A., Costantino, P., Altamura, M.M.: The plant oncogene rolD stimulates flowering in transgenic tobacco plants. — Dev. Biol. 180: 693–700, 1996.

    Article  PubMed  CAS  Google Scholar 

  • Menković, N., Šavikin-Fodulović, K., Vinterhalter, B., Vinterhalter, D., Grubišić, D.: Secoiridoid content of naturally grown and in vitro cultured Gentiana punctata. — Pharm. Pharmacol. Lett. 8: 110–111, 1998.

    Google Scholar 

  • Menković, N., Šavikin-Fodulović, K., Vinterhalter, B., Vinterhalter, D., Janković, T., Krstić, D.: Secoiridoid content in hairy roots of Gentiana punctata. — Pharm. Pharmacol. Lett. 10: 73–75, 2000.

    Google Scholar 

  • Mishiba, K.-I., Nishihara, M., Abe, Y., Nakatsuka. T., Kawamura, H., Kodama, K., Takesawa, T., Abe, J., Yamamura, S.: Production of dwarf potted gentian using wild-type Agrobacterium rhizogenes. — Plant Biotechnol. 23: 33–38, 2006.

    Article  CAS  Google Scholar 

  • Mishiba, K.-I., Nishihara, M., Nakatsuka, T., Abe, Y., Hirano, H., Yokoi, T., Kikuchi, A., Yamamura, S.: Consistent transcriptional silencing of 35S’ driven transgenes in gentian. — Plant J. 44: 541–556, 2005.

    Article  PubMed  CAS  Google Scholar 

  • Momčilović, I., Grubišić, D., Kojić, M., Nešković, M.: Agrobacterium rhizogenes-mediated transformation and plant regeneration of four Gentiana species. — Plant Cell Tissue Organ Cult. 50: 1–6, 1997.

    Article  Google Scholar 

  • Momčilović, I., Grubišić, D., Nešković, M.: Transgenic Gentiana species (Gentian). — In: Bajaj, Y.P.S. (ed.): Biotechnology in Agriculture and Forestry. Vol. 48. Transgenic crops III. Pp. 123–138. Springer-Verlag, Berlin — Heidelberg — New York 2001.

    Google Scholar 

  • Murray, M.G., Thompson, W.F.: Rapid isolation of high molecular weight plant DNA. — Nucl. Acids Res. 8: 4321–4325, 1980.

    Article  PubMed  CAS  Google Scholar 

  • Nacry, P., Camilleri, C., Courtial, B., Caboche, M., Bouchey, D.: Major chromosomal rearangements induced by T-DNA transformation in Arabidopsis. — Genetics 149: 641–650, 1998.

    PubMed  CAS  Google Scholar 

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

    Article  Google Scholar 

  • Nikolić, R., Mitić, N., Ninković, S., Miljuš-Đukić, J., Nešković, M.: Efficient genetic transformation of Lotus corniculatus L. and growth of transformed plants in field. — Biol. Plant. 47: 137–140, 2003/4.

    Google Scholar 

  • Piispanen, R., Aronen, T., Chen, X., Saranpää, P., Häaggman, H.: Silver birch (Betula pendula) plants with aux and rol genes show consistent changes in morphology, xylem structure and chemistry. — Tree Physiol. 23: 721–733, 2003.

    PubMed  CAS  Google Scholar 

  • Rosić, N., Momčilović, I., Kovačević, N., Grubišić D.: Genetic transformation of Rhamnus fallax and hairy roots as a source of anthraquinones. — Biol. Plant. 50: 514–518, 2006.

    Article  Google Scholar 

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

    Article  Google Scholar 

  • Sevón, N., Oksman-Caldentey, K.M.: Agrobacterium rhizogenes- mediated transformation: root cultures as a source of alkaloids. — Planta med. 68: 859–868, 2002.

    Article  PubMed  Google Scholar 

  • Slightom, J.L., Durand-Tardif, M., Jouanin, L., Tepfer, D.: Nucleotide sequence analysis of TL-DNA of Agrobacterium rhizogenes agropine type plasmid. — J. biol. Chem. 261: 108–121, 1986.

    PubMed  CAS  Google Scholar 

  • Spano, L., Mariotti, D., Cardarelli, M., Branca, C., Constantino, P.: Morphologenesis and auxin sensitivity of transgenic tobacco with different complements of Ri T-DNA. — Plant Physiol. 87: 479–483, 1988.

    Article  PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  • Sretenović-Rajičić, T., Ninković, S., Miljuš-Đukić, J., Vinterhalter, B., Vinterhalter, D.: Agrobacterium rhizogenes-mediated transformation of Brassica oleracea var. sabauda and B. oleracea var. capitata. — Biol. Plant. 50: 525–530, 2006.

    Article  Google Scholar 

  • Subotić, A., Budimir, S., Grubišić, D., Momčilović, I.: Direct regeneration of shoots from hairy root cultures of Centaurium erythraea inoculated with Agrobacterium rhizogenes. — Biol. Plant. 47: 617–619, 2003.

    Article  Google Scholar 

  • Suginuma, C., Akihama, T.: Transformation of Gentian with Agrobacterium rhizogenes. — Acta Hort. 392: 153–160, 1995.

    CAS  Google Scholar 

  • Tax, F.E., Vernon, D.M.: T-DNA-associated duplication/ translocation in Arabidopsis. Implications for mutant analysis and functional genomics. — Plant Physiol. 126: 1527–1538, 2001.

    Article  PubMed  CAS  Google Scholar 

  • Teixeira da Silva, J.A.: Chrysanthemum: advances in tissue culture, cryopreservation, postharvest technology, genetics and transgenic biotechnology. — Biotechnol. Adv. 21:715–766, 2003.

    Article  PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  • Tiwari, R.K., Trivedi, M., Guang, Z.-C., Guo, G.-Q., Yang, G.-C.: 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.

    Article  PubMed  CAS  Google Scholar 

  • Tiwari, R.K., Trivedi, M., Guang, Z.-C., Guo, G.-Q., Zheng, G-C.: Agrobacterium rhizogenes mediated transformation of Scutellaria baicalensis and production of flavonoids in hairy roots. — Biol. Plant. 52: 26–35, 2008.

    Article  CAS  Google Scholar 

  • Veena, V., Taylor, G.C.: Agrobacterium rhizogenes: recent developments and promissing applications. — In Vitro cell. Dev. Biol. Plant 43: 383–403, 2007.

    Article  CAS  Google Scholar 

  • Vinterhalter, B., Momčilović, I., Vinterhalter, D.: High biomass producing root cultures of Gentiana punctata L. transformed with Agrobacterium tumefaciens C58C1 (pArA4b). — Arch. Biol. Sci. Belgrade 52: 85–90, 2000.

    Google Scholar 

  • Vinterhalter, B., Ninković, S., Cingel, A., Vinterhalter D.: Shoot and root culture of Hypericum perforatum L. transformed with Agrobacterium rhizogenes A4M70GUS. — Biol. Plant. 50: 767–770, 2006.

    Article  Google Scholar 

  • Vinterhalter, B., Orbović, V., Vinterhalter, D.: Transgenic root cultures of Gentiana punctata L. — Acta Soc. Bot. Polon. 68: 275–280, 1999.

    Google Scholar 

  • Vinterhalter, B., Savić, J., Platiša, J., Raspor, M., Ninković, S., Mitić, N., Vinterhalter, D.: Nickel tolerance and hyperaccumulation in shoot cultures regenerated from hairy root cultures of Alyssum murale Waldst et Kit. — Plant Cell Tissue Organ Cult. 94: 299–303, 2008.

    Article  CAS  Google Scholar 

  • Vinterhalter, B., Vinterhalter, D.: In vitro propagation of spotted gentian Gentiana punctata L. — Arch. Biol. Sci. Belgrade 50: 177–182, 1998.

    Google Scholar 

  • White, F.F., Taylor, B.H., Huffman, A., Gordon, M.P., Nester, E.W.: Molecular and genetic analysis of the transferred DNA regions of the root inducing plasmid of Agrobacterium rhizogenes. — J. Bacteriol. 164: 33–44, 1985.

    PubMed  CAS  Google Scholar 

  • Zdravković-Korać, S., Muhovski, Y., Druart, P., Ćalić, D., Radojević, L.: Agrobacterium rhizogenes-mediated DNA transfer to Aesculus hippocastamum L. and the regeneration of transformed plants. — Plant Cell Rep. 22: 698–704, 2004.

    Article  PubMed  Google Scholar 

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Acknowledgements

Research was funded by the Serbian Ministry of Education and Science, through the project No. 173015.

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Authors and Affiliations

  1. Plant Physiology Department, Institute for Biological Research “S. Stanković”, Belgrade University, Bulevard despota Stefana 142, 11000, Belgrade, Serbia

    B. Vinterhalter, S. Zdravković-Korać, S. Ninković, N. Mitić & D. Vinterhalter

  2. Institute for Medicinal Plants Research “Josif Pančić”, Tadeuša Košćuška 1, 11000, Belgrade, Serbia

    T. Janković

  3. Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444 a, 11000, Belgrade, Serbia

    J. Miljuš-Djukić

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  1. B. Vinterhalter
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  2. S. Zdravković-Korać
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  3. S. Ninković
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Correspondence to D. Vinterhalter.

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Vinterhalter, B., Zdravković-Korać, S., Ninković, S. et al. Variability in shoot cultures regenerated from hairy roots of Gentiana punctata . Biol Plant 55, 414–422 (2011). https://doi.org/10.1007/s10535-011-0105-8

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  • DOI: https://doi.org/10.1007/s10535-011-0105-8

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