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Stable transformation and actin visualization in callus cultures of dodder (Cuscuta europaea)

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Abstract

Agrobacterium tumefaciens-mediated transformation of callus culture, combined with a visual selection of GFP-tagged fimbrin actin binding domain (FABD2) expression is described for parasitic species (Cuscuta europaea). The conditions for callus induction from 1 cm-long explants from the basal part of 7-day-old dodder seedlings were defined. We obtained light-green calli, which were transformed with A. tumefaciens bacterial strain GV3101 carrying plasmid pCB302 (35S::ABD2:gfp) with neomycin phosphotransferase (nptII) gene. The limitations of selection procedures based on antibiotics were avoided using green fluorescent protein (GFP) detection, as a visual selection marker subcellularly targeted to the actin cytoskeleton. Fluorescence microscopy analyses demonstrated a network of nucleus-associated actin arrays and dense cortical actin arrangements in stably transformed Cuscuta callus cells. RT-PCR analyses confirmed gfp expression in transformed calli 7, 14 and 21 days after transformation. Although the GFP fluorescence associated with the actin cytoskeleton has retained for at least six months without silencing, no shoot regeneration was observed. It can be concluded that, C. europaea callus cells are competent for transformation, but under given conditions, these cells failed to realize their morphogenic and regeneration potentials.

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References

  • Alakonya A., Kumar R., Koenig D., Kimura S., Townsley B., Runo S., Garces H.M., Kang J., Yanez A., Schwartz R.D., Machuka J. & Sinhab N. 2012. Interspecific RNA Interference of SHOOT MERISTEMLESS-like disrupts Cuscuta pentagona plant parasitism. Plant Cell 24: 3153–3166.

    Article  CAS  PubMed  Google Scholar 

  • Arias R.S., Filichkin S.A. & Strauss S.H. 2006. Divide and conquer: development and cell cycle genes in plant transformation. Trends Biotechnol. 24: 267–273.

    Article  CAS  PubMed  Google Scholar 

  • Bakos A., Fari M., Toldi O. & Lados M. 1995. Plant regeneration from seedling-derived callus of dodder (Cuscuta trifolii Bab. Et Giggs). Plant. Sci. 109: 95–101.

    Article  CAS  Google Scholar 

  • Balážová R., Breznenová K. & Blehová A. 2011. Localization of thylakoid formation protein 1 (THF1) in Cuscuta europaea, pp. 17. In: Book of abstracts: Plant Biotechnology. Green for Good, Olomouc — Czech Republic.

    Google Scholar 

  • Bleho J., Obert B., Takáč T., Petrovská B., Heym C., Menzel D & Šamaj J. 2012. ER disruption and GFP degradation during non-regenerable transformation of flax with Agrobacterium tumefaciens. Protoplasma 249: 53–63.

    Article  CAS  PubMed  Google Scholar 

  • Borsics T., Mihalka V., Oreifig A.S., Barany I., Lados M., Nagy I., Jenes B. & Toldi O. 2002. Methods for genetic transformation of the parasitic weed dodder (Cuscuta trifolii Bab. et Gibs) and for PCR-based detection of early transformation events. Plant Sci. 162: 193–199.

    Article  CAS  Google Scholar 

  • Dawson J.H., Musselman L.J., Wolswinkel P. & Dörr, I. 1994. Biology and control of Cuscuta. Rev. Weed Sci. 6: 265–317.

    Google Scholar 

  • Deeks S.J., Shamoun S.F. & Punja Z.K. 1999. Tissue culture of parasitic flowering plants: Methods and applications in agriculture and forestry. In Vitro Cell Dev. Biol. 35: 369–381.

    Article  Google Scholar 

  • Fernández-Aparicio M., Rubiales D., Bandaranayake P.C.G., Yoder J.I. & Westwood J.H. 2011. Transformation and regeneration of the holoparasitic plant Phelipanche aegyptiaca. http://www.plantmethods.com/content/7/1/36 (accesed 8 November 2011).

    Google Scholar 

  • Furuhashi K. 1991. Establishment of a successive culture of an obligatory parasitic flowering plant, Cuscuta japonica, in vitro. Plant Sci. 79: 241–246.

    Article  Google Scholar 

  • Hussey P.J., Ketelaar T. & Deeks M.J. 2006. Control of the Actin Cytoskeleton in Plant Cell Growth. Annu. Rev. Plant Biol. 57: 109–25, 2006.

    Article  CAS  PubMed  Google Scholar 

  • Chalfie M., Tu Y., Euskirchen G., Ward W.W. & Prasher, D.C. 1994. Green fluorescent protein as a marker for gene expression. Science 263: 802–805.

    Article  CAS  PubMed  Google Scholar 

  • Ishida J.K., Yoshida S., Ito M., Namba S. & Shirasu K. 2011. Agrobacterium rhizogenes-mediated transformation of the parasitic plant Phtheirospermum japonicum. http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0025802 (accesed 3 October 2011).

    Google Scholar 

  • Lichtenthaler H.K. 1987. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods enzymol. 148: 350–382.

    Article  CAS  Google Scholar 

  • Machado M.A. & Zetsche K. 1990. A structural, functional and moleculal analysis of plastids of the holoparasites Cuscuta reflexa and Cuscuta europaea. Planta 181: 91–96.

    Article  CAS  Google Scholar 

  • Mellor K.E., Hoffman A.M. & Timko M.P. 2012. Use of ex vitro composite plants to study the interaction of cowpea (Vigna unguiculata L.) with the root parasitic angiosperm Striga gesnerioides. http://www.plantmethods.com/content/8/1/22 (28 June 2012).

    Google Scholar 

  • Murashige T. & Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Plant Physiol. 15: 473–497.

    Article  CAS  Google Scholar 

  • Prather L. & Tyrl J. 1993. The biology of Cuscuta attenuata waterfall (Cuscutaceae). Proc. Okla. Sci. 73: 7–13.

    Google Scholar 

  • Rousset A., Simier, P. & Fer, A. 2003. Characterisation of simple in vitro cultures of Striga hermonthica suitable for metabolic studies. Plant Biol. 5: 265–273.

    Article  CAS  Google Scholar 

  • Runo S., Macharia S., Alakonya A., Machuka J., Sinha N. & Scholes J. 2012. Striga parasitizes transgenic hairy roots of Zea mays and provides a tool for studying plant-plant interactions. http://www.plantmethods.com/content/8/1/20 (21 June 2012).

    Google Scholar 

  • Sheahan M.B., Staiger C.J., Rose R.J. & McCurdy D.W. 2004. A green fluorescent protein fusion to actin-binding domain 2 of Arabidopsis fimbrin highlights new features of a dynamic actin cytoskeleton in live plant cells. Plant Physiol. 136: 3968–3978.

    Article  CAS  PubMed  Google Scholar 

  • Srivastava S. & Dwivedi U.N. 2001. Plant regeneration from callus of Cuscuta reflexa — an angiospermic parasite — and modulation of catalase and peroxidise activity by salicylic acid and naphthalene acetic acid. Plant Physiol. Biochem. 39: 529–538.

    Article  CAS  Google Scholar 

  • Šamaj J., Bobák M., Blehová A. & Preťová A. 2005. Importance of cytoskeleton and cell wall in somatic embryogenesis, pp. 35–50. In: Mujib A. & Šamaj J. (eds), Somatic Embryogenesis. Plant Cell Monogr. 2, Springer, Verlang, Berlin, Heidelberg.

    Google Scholar 

  • Švubová R., Kulich I., Ovečka M. & Blehová A. 2012. Tissue expression and subcellular localization of THF1 in dodder and its host plant, p. 142. In: Book of abstracts: 41st Annual meeting of ESNA “Advances in agrobiology research and their benefits to the future”, Stará Lesná, High Tatras, Slovak Republic.

    Google Scholar 

  • Švubová R., Ovečka M., Pavlovič A., Slováková Ľ. & Blehová A. 2013. Cuscuta europaea plastid apparatus in various developmental stages: Localization of THF1 protein. Plant Signal. Behav. 8: eLocation ID: e24037.

  • Tomilov A., Tomilova N. & Yoder J.I. 2007. Agrobacterium tumefaciens and Agrobacterium rhizogenes transformed roots of the parasitic plant Triphysaria versicolor retain parasitic competence. Planta 225: 1059–1071.

    Article  CAS  PubMed  Google Scholar 

  • Voigt B., Timmers A.C.J., Šamaj J., Müller J., Baluška F. & Menzel D. 2005. GFP-ABD2 fusion construct allows in vivo visualization of the dynamic actin cytoskeleton in all cells of Arabidopsis seedlings. Eur. J. Cell Biol. 84: 595–608.

    Article  CAS  PubMed  Google Scholar 

  • Wang Y.S., Yoo CH.M. & Blancaflor E.B. 2008. Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C- and N-termini of the fimbrin actin-binding domain 2. New Phytol. 177: 525–536.

    CAS  PubMed  Google Scholar 

  • Wasteneys G.O. 2000. The cytoskeleton and growth polarity. Curr. Opin. Plant Biol. 3: 503–511.

    Article  CAS  PubMed  Google Scholar 

  • Wenzel C.L., Williamson R.E. & Wasteneys G.O. 2000. Gibberellin-induced changes in growth anisotropy precede gibberellin-dependent changes in cortical microtubule orientation in developing epidermal cells of barley leaves: Kinematic and cytological studies on a gibberellin-responsive dwarf mutant, M489. Plant Physiol. 124: 813–822.

    Article  CAS  PubMed  Google Scholar 

  • Wigchert S.C., Kuiper E., Boelhouwer G.J., Nefkens G.H., Verkleij J.A. & Zwanenburg, B. 1999. Dose-response of seeds of the parasitic weeds Striga and Orobanche towards the synthetic germination stimulants GR 24 and Nijmegen 1. J. Agric. Food Chem. 47(4): 705–710.

    Google Scholar 

  • Yu M., Yuan M. & Ren H. 2006. Visualization of actin cytoskeletal dynamics during the cell cycle in tobacco (Nicotian tabacum L. cv. Bright Yellow) cells. Biol. Cell 98: 295–306.

    Article  CAS  PubMed  Google Scholar 

  • Zambre M., Geerts P., Maquet A., Montagu M.V., Dillen W. & Angenon G. 2001. Regeneration of fertile plants from callus in Phaseolus polyanthus. Ann. Bot. 88: 371–377.

    Article  CAS  Google Scholar 

  • Zhang S. & Lemaux P.G. 2004. Molecular analysis of in vitro shoot organogenesis. Plant Sci. 23(4): 325–335.

    Google Scholar 

  • Zhou W.J., Yoneyama K., Takeuchi Y., Iso S., Rungmekarat S., Chae S.H., Sato D. & Joel D.M. 2004. In vitro infection of host roots by differentiated calli of the parasitic plant Orobanche. J. Exp. Bot. 55: 899–907.

    Article  CAS  PubMed  Google Scholar 

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  1. Department of Plant Physiology, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynská dolina, SK-84215, Bratislava, Slovakia

    Renáta Švubová & Alžbeta Blehová

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  1. Renáta Švubová
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  2. Alžbeta Blehová
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Correspondence to Alžbeta Blehová.

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Švubová, R., Blehová, A. Stable transformation and actin visualization in callus cultures of dodder (Cuscuta europaea). Biologia 68, 633–640 (2013). https://doi.org/10.2478/s11756-013-0188-0

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