Agrobacterium-mediated transformation of Medicago truncatula cell suspension culture provides a system for functional analysis

  • Anelia Iantcheva
  • Miglena Revalska
  • Grigor Zehirov
  • Valya Vassileva
Embryo Culture

Abstract

Over the past decade, Medicago truncatula has been adopted as a model legume species for a range of “omic” studies. The availability of different transformation techniques has greatly advanced functional genomic studies in this species. In the present work, an efficient procedure for Agrobacterium-mediated transformation of M. truncatula cv. “Jemalong 2HA” through a cell suspension culture was developed. This procedure resulted in transformed single cells or cell clusters, giving rise to stable transgenic plants within 4 mo. Transformation experiments were performed with a vector carrying two marker genes: β-glucuronidase (GUS) and green fluorescent protein (GFP) under the control of endogenous gene promoters from LIKE AUX1 3 (LAX3) and GRAS transcription factor (named after GD3BERELLIC ACID INSENSITIVE [GAI], REPRESSOR OF GA1 [RGA], and SCARECROW [SCR]), as well as with a binary destination vector for overexpressing the cyclin-like F-box gene fused to GFP. Maximum transformation efficiency was achieved under the following experimental conditions: acetosyringone at a concentration of 25 μM, bacterial suspension with an optical density of 0.3 at 600 nm, inoculation under agitation at 100 rpm for 24 h, co-cultivation periods of 48 h, and an uninterrupted selection with 50 mg/L kanamycin. Selection of positive transformation events was imposed early in the regeneration stage (after 48 h co-cultivation), following a large-scale screening for GFP activity. Histochemical GUS and GFP reporter activity was detected in single cells, embryogenic zones, emerging embryos, in vitro plantlets, and T1 progeny seedlings. The transgenic nature of transformed plants was further confirmed by nptII-specific PCR amplification of T0 and T1 plant lines. The transgenic plants grown under standard greenhouse conditions displayed a wild-type phenotype and the obtained progeny segregated in a classical Mendelian manner. The fundamental steps in the transformation procedure are outlined and discussed.

Keywords

Cell suspension culture Agrobacterium transformation Transcriptional reporters β-Glucuronidase Green fluorescent protein Transformation efficiency 

References

  1. Atif R. M.; Patat-Ochatt E. M.; Svabova L.; Ondrej V.; Klenoticova H.; Jacas L.; Griga M.; Ochatt S. J. Gene transfer in legumes. In: Lüttge U.; Beyschlag W.; Francis D.; Cushman J. (eds) Progress in botany, vol. 74. Springer, Berlin, pp 37–100; 2013.CrossRefGoogle Scholar
  2. Boisson-Dernier A.; Chabaud M.; Garcia F.; Bécard G.; Rosenberg C.; Barker D. G. Hairy roots of Medicago truncatula as tools for studying nitrogen-fixing and endomycorrhizal symbioses. Mol Plant Microbe Interact 14: 693–700; 2001.CrossRefGoogle Scholar
  3. Chabaud M.; Larsonneau C.; Marmouget C.; Huguet T. Transformation of barrel medics (Medicago truncatula Gaetrn.) by Agrobacterium tumefaciens and regeneration via somatic embryogenesis of transgenic plants with the MtENOD 12 nodulin promoter fused to gus reporter gene. Plant Cell Rep 15: 305–310; 1996.PubMedCrossRefGoogle Scholar
  4. Chabaud M.; Ratet P.; de Sousa Araújo S.; Duque A. S. R. L. A.; Harrison M.; Barker D. G. Agrobacterium tumefaciens-mediated transformation and in vitro plant regeneration of M. truncatula. In: Mathesius U.; Journet E. P.; Sumner L. W. (eds) The Medicago truncatula handbook. The Samuel Roberts Noble Foundation, Ardmore, pp 16–34; 2007.Google Scholar
  5. Crane C.; Wright E.; Dixon R. A.; Wang Z. Y. Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens-transformed roots and Agrobacterium rhizogenes-transformed hairy roots. Planta 223: 1344–1354; 2006.PubMedCrossRefGoogle Scholar
  6. De Rybel B.; Vassileva V.; Parizot B.; Demeulenaere M.; Grunewald W.; Audenaert D.; Van Campenhout J.; Overvoorde P.; Jansen L.; Vanneste S.; Möller B.; Wilson M.; Holman T.; Van Isterdaele G.; Brunoud G.; Vuylsteke M.; Vernoux T.; De Veylder L.; Inzé D.; Weijers D.; Bennett M.; Beeckman T. A novel Aux/IAA28 signaling cascade activates GATA23-dependent specification of lateral root founder cell identity. Curr Biol 20: 1697–1706; 2010.PubMedCrossRefGoogle Scholar
  7. de Susa Araújo S.; Duque A. S. R. L. A.; Santos D. M. M. F.; Fevereiro M. P. S. An efficient transformation method to regenerate a high number of transgenic plants using a new embryogenic line of Medicago truncatula cv. Jemalong. Plant Cell Tiss Organ Cult 78: 123–131; 2004.CrossRefGoogle Scholar
  8. Galibert F.; Finan T. M.; Long S. R.; Pühler A.; Abola P.; Ampe F.; Barloy-Hubler F.; Barnett M. J.; Becker A.; Boistard P.; Bothe G.; Boutry M.; Bowser L.; Buhrmester J.; Cadieu E.; Capela D.; Chain P.; Cowie A.; Davis R. W.; Dréano S.; Federspiel N. A.; Fisher R. F.; Gloux S.; Godrie T.; Goffeau A.; Golding B.; Gouzy J.; Gurjal M.; Hernandez-Lucas I.; Hong A.; Huizar L.; Hyman R. W.; Jones T.; Kahn D.; Kahn M. L.; Kalman S.; Keating D. H.; Kiss E.; Komp C.; Lelaure V.; Masuy D.; Palm C.; Peck M. C.; Pohl T. M.; Portetelle D.; Purnelle B.; Ramsperger U.; Surzycki R.; Thébault P.; Vandenbol M.; Vorhölter F.-J.; Weidner S.; Wells D. H.; Wong K.; Yeh K.-C.; Batut J. The composite genome of the legume symbiont Sinorhizobium meliloti. Science 293: 668–672; 2001.PubMedCrossRefGoogle Scholar
  9. Gamborg O. L.; Miller R. A.; Ojima K. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50: 151–158; 1968.PubMedCrossRefGoogle Scholar
  10. Hoffmann B.; Trinh T. H.; Leung J.; Kondorosi A.; Kondorosi E. A new Medicago truncatula line with superior in vitro regeneration, transformation and symbiotic properties isolated through cell culture selection. Mol Plant Microbe Interact 10: 307–315; 1997.CrossRefGoogle Scholar
  11. Iantcheva A.; Vlahova M.; Atanassov A. Genetic transformation of Medicago truncatula using system for direct somatic embryogenesis promoted by TDZ. Biotechnol Biotechnol Eq 7: 50–56; 2005.Google Scholar
  12. Iantcheva A.; Vlahova M.; Atanassov A. Cell suspension culture of M. truncatula cv. R 108 1 initiated from leaf and root explants. In: Mathesius U.; Journet E. P.; Sumner L. W. (eds) The Medicago truncatula handbook. The Samuel Roberts Noble Foundation, Ardmore, pp 2–5; 2006.Google Scholar
  13. Iantcheva A.; Chabaud M.; Cosson V.; Barascud M.; Schutz B.; Primard-Brisset C.; Durand P.; Barker D. G.; Vlahova M.; Ratet P. Osmotic shock improves Tnt1 transposition frequency in Medicago truncatula cv Jemalong during in vitro regeneration. Plant Cell Rep 28: 1563–1572; 2009.PubMedCrossRefGoogle Scholar
  14. Jefferson R.; Kannagh T.; Bevan M. GUS fusion: β-glucoronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907; 1987.PubMedCentralPubMedGoogle Scholar
  15. Kamate K.; Rodriguez-Llorente I. D.; Scholte M.; Durand P.; Ratet P.; Kondorosi E.; Kondorosi A.; Trinh T. H. Transformation of floral organs with GFP in Medicago truncatula. Plant Cell Rep 19: 647–653; 2000.CrossRefGoogle Scholar
  16. Karimi M.; Depicker A.; Hilson P. Recombinational cloning with plant gateway vectors. Plant Physiol 145: 1144–1154; 2007.PubMedCentralPubMedCrossRefGoogle Scholar
  17. Lai E. M.; Shih H. W.; Wen S. R.; Cheng M. W.; Hwang H. H.; Chiu S. H. Proteomic analysis of Agrobacterium tumefaciens response to the vir gene inducer acetosyringone. Proteomics 6: 4130–4136; 2006.PubMedCrossRefGoogle Scholar
  18. Murashige T.; Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497; 1962.CrossRefGoogle Scholar
  19. Nolan K. E.; Rose R. J.; Gorst J. G. Regeneration of Medicago truncatula from tissue culture increased somatic embryogenesis using explant from regenerated plants. Plant Cell Rep 8: 278–281; 1989.PubMedCrossRefGoogle Scholar
  20. Olhoft P. M.; Flagel L. E.; Donovan C. M.; Somers D. A. Efficient soybean transformation using hygromicin B selection in the cotyledonary-node method. Planta 216: 723–735; 2003.PubMedGoogle Scholar
  21. Paz M. M.; Shou H.; Guo Z.; Zhang Z.; Banerjee A. K.; Wang K. Assessment of conditions affecting Agrobacterium-mediated soybean transformation using cotyledonary node explant. Euphytica 136: 167–179; 2004.CrossRefGoogle Scholar
  22. Raja N. I.; Bano A.; Rashid H.; Chandhry Z.; Ilyas N. Improving Agrobacterium-mediated transformation protocol for integration of XA21 gene in wheat (Triticum aestivum L.). Pak J Bot 42: 3613–3631; 2010.Google Scholar
  23. Raveendar S.; Ignacimuthu S. Improved Agrobacterium mediated transformation in cowpea Vigna unguiculata L. Walp. Asian J Plant Sci 9: 256–263; 2010.CrossRefGoogle Scholar
  24. Revalska M.; Vassileva V.; Goormachtig S.; Van Hautegem T.; Ratet P.; Iantcheva A. Recent progress in development of Tnt1 functional genomics platform for Medicago truncatula and Lotus japonicus in Bulgaria. Curr Genomics 12: 147–152; 2011.PubMedCentralPubMedCrossRefGoogle Scholar
  25. Sheikholesman S. N.; Weeks D. P. Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Mol Biol 8: 291–298; 1987.CrossRefGoogle Scholar
  26. Trieu A. T.; Burleigh S. H.; Kardailsky I. V.; Maldonado-Mendoza I. E.; Versaw W. K.; Blaylock L. A.; Shin H.; Chiou T. J.; Katagi H.; Dewbre G. R.; Weigel D.; Harrison M. J. Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium. Plant J 22: 531–541; 2000.PubMedCrossRefGoogle Scholar
  27. Trieu A. T.; Harrison M. J. Rapid transformation of Medicago truncatula: regeneration via shoot organogenesis. Plant Cell Rep 16: 6–11; 1996.PubMedCrossRefGoogle Scholar
  28. Trinh T. H.; Ratet P.; Kondorosi E.; Durand P.; Kamaté K.; Bauer P.; Kondorosi A. Rapid and efficient transformation of diploid Medicago truncatula and Medicago sativa ssp. falcata lines improved in somatic embryogenesis. Plant Cell Rep 17: 345–355; 1998.CrossRefGoogle Scholar
  29. Vassileva V.; Zehirov G.; Ugrinova M.; Iantcheva A. Variable leaf epidermal morphology in Tnt1 insertional mutants of the model legume Medicago truncatula. Biotechnol Biotechnol Eq 24: 2060–2065; 2010.CrossRefGoogle Scholar
  30. Yamada T.; Teraishi M.; Hattori K.; Ishimoto M. Transformation of azuki bean by Agrobacterium tumefaciens. Plant Cell Tiss Organ Cult 64: 47–54; 2001.CrossRefGoogle Scholar
  31. Young N. D.; Debellé F.; Oldroyd G. E.; Geurts R.; Cannon S. B.; Udvardi M. K.; Benedito V. A.; Mayer K. F.; Gouzy J.; Schoof H.; Van de Peer Y.; Proost S.; Cook D. R.; Meyers B. C.; Spannagl M.; Cheung F.; De Mita S.; Krishnakumar V.; Gundlach H.; Zhou S.; Mudge J.; Bharti A. K.; Murray J. D.; Naoumkina M. A.; Rosen B.; Silverstein K. A.; Tang H.; Rombauts S.; Zhao P. X. et al. The Medicago genome provides insight into the evolution of rhizobial symbioses. Nature 480: 520–524; 2011.PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2013

Authors and Affiliations

  • Anelia Iantcheva
    • 1
  • Miglena Revalska
    • 1
  • Grigor Zehirov
    • 2
  • Valya Vassileva
    • 2
  1. 1.AgroBioInstituteSofiaBulgaria
  2. 2.Institute of Plant Physiology and GeneticsBulgarian Academy of SciencesSofiaBulgaria

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