Plant Cell Reports

, Volume 29, Issue 9, pp 1013–1021 | Cite as

Backbone-free transformation of barrel medic (Medicago truncatula) with a Medicago-derived transfer DNA

  • Massimo Confalonieri
  • Roberto Borghetti
  • Anca Macovei
  • Claudia Testoni
  • Daniela Carbonera
  • Manuel Pedro Salema Fevereiro
  • Caius Rommens
  • Kathy Swords
  • Efisio Piano
  • Alma BalestrazziEmail author
Original Paper


In the present work, Agrobacterium tumefaciens-mediated genetic transformation of the model legume Medicago truncatula Gaertn. (barrel medic) was carried out using the pSIM843 vector that contains a Medicago-derived transfer DNA, delineated by a 25-bp sequence homologous to bacterial T-DNA borders. The transfer DNA contains an expression cassette for the nptII (neomycin phosphotransferase) gene and is flanked by an expression cassette for the backbone integration marker gene ipt (isopentenyl transferase). Our results demonstrate that the Medicago-derived RB-like elements efficiently support DNA mobilization from A. tumefaciens to M. truncatula. Kanamycin-resistant shoots with normal phenotype and ipt-shooty lines were recovered at a frequency of 11.7 and 7.8%, respectively. Polymerase chain reaction (PCR) analyses demonstrated that 44.4% of the independent transgenic lines were backbone-free and evidenced the occurrence of backbone-transfer events.


Backbone transfer ipt Medicago truncatula Morphological marker P-DNA 



This research was supported by Fondo di Ateneo per la Ricerca-University of Pavia. Authors are grateful to Dr. Cristina Ambroggi for excellent technical contribution and Dr. Lorenzo Concia for helpful suggestions concerning the interpretation of QRT-PCR data.


  1. Araujo SS, Duque SRL, Santos DMMF, Fevereiro MPS (2004) An efficient transformation method to regenerate a high number of transgenic plants using a new embryogenic line of Medicago truncatula cv Jemalong. Plant Cell Tissue Organ Cult 120:189–197Google Scholar
  2. Barker DG, Bianchi S, Blondon F, Dattlè Y, Duc G, Flament P, Gallusa P, Genier G, Guy P, Muel X, Tourner J, Denarue J, Huguet T (1990) Medicago truncatula a model plant for studying the molecular genetics of the Rhizobium-legume symbiosis. Plant Mol Biol Rep 8:40–49CrossRefGoogle Scholar
  3. Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193CrossRefPubMedGoogle Scholar
  4. Chabaud M, De Carvalho-Niebel F, Barker DG (2003) Efficient transformation of Medicago truncatula cv. Jemalong using the hypervirulent Agrobacterium tumefaciens strain AGL1. Plant Cell Rep 22:46–51CrossRefPubMedGoogle Scholar
  5. Confalonieri M, Cammareri M, Biazzi E, Pecchia P, Fevereiro MPS, Balestrazzi A, Tava A, Conicella C (2009) Enhanced triterpene saponin biosynthesis and root nodulation in transgenic barrel medic (Medicago truncatula Gaertn.) expressing a novel β-amyrin synthase gene. Plant Biotechnol J 7:172–182CrossRefPubMedGoogle Scholar
  6. Cook DR (1999) Medicago truncatula—a model in the making!. Curr Opin Plant Biol 2:301–304CrossRefPubMedGoogle Scholar
  7. Crane C, Dixon RA, Wang ZY (2006) Medicago truncatula transformation using root explants. Methods Mol Biol 343:137–142PubMedGoogle Scholar
  8. d’Erfurth I, Cosson V, Eschstruth A, Rippa S, Messinese E, Durand P, Trinh H, Kondorosi A, Ratet P (2003) Rapid inactivation of the maize transposable element En/Spm in Medicago truncatula. Mol Genet Genomics 269:732–745CrossRefPubMedGoogle Scholar
  9. Ebinuma H, Komamine A (2001) MAT (multi-auto-transformation) vector system The oncogenes of Agrobacterium as positive markers for regeneration and selection of marker-free transgenic plants. In vitro Cell Dev Biol Plant 37:103–113CrossRefGoogle Scholar
  10. Garbarino JE, Belknap WR (1994) Isolation of a ubiquitin-ribosomal protein gene (ubi3) from potato and the expression of its promoter in transgenic plants. Plant Mol Biol 24:119–127CrossRefPubMedGoogle Scholar
  11. Garbarino JE, Oosumi T, Belknap WR (1995) Isolation of a polyubiquitin promoter and its expression in transgenic potato plants. Plant Physiol 109:1371–1378CrossRefPubMedGoogle Scholar
  12. Hanson B, Engler D, Moy Y, Newman B, Ralston E, Gutterson N (1999) A simple method to enrich an Agrobacterium-transformed population for plants containing only T-DNA sequences. Plant J 19:727–734CrossRefPubMedGoogle Scholar
  13. Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacteria helper plasmids for gene transfer to plants. Transgenic Res 2:208–218CrossRefGoogle Scholar
  14. Huang S, Gilbertson LA, Adams TH, Malloy KP, Reisenbigler EK, Birr DH, Snyder MW, Zhang Q, Luethy MH (2004) Generation of marker-free transgenic maize by regular two-border Agrobacterium transformation vectors. Transgenic Res 13:451–461CrossRefPubMedGoogle Scholar
  15. Kamatè K, Rodriguez-Llorente ID, Scholte M, Durand P, Ratet P, Kondorosi E, Kondorosi A (2000) Transformation of floral organs with GFP in Medicago truncatula. Plant Cell Rep 19:647–653CrossRefGoogle Scholar
  16. Kim SR, Lee J, Jun SH, Park S, Kang HG, Kwon S, An G (2003) Transgenic structures in T-DNA-inserted rice plants. Plant Mol Biol 52:761–773CrossRefPubMedGoogle Scholar
  17. Kononov ME, Bassuner B, Gelvin SB (1997) Integration of T-DNA binary vector ‘backbone’ sequences into the tobacco genome: evidence for multiple complex patterns of integration. Plant J 11:945–957CrossRefPubMedGoogle Scholar
  18. Kuraya Y, Ohta S, Fukada M, Hiei Y, Murai N, Hamada K, Ueki T, Imaseki H, Komari T (2004) Suppression of transfer of non T-DNA ‘vector backbone’ sequences by multiple left border repeats in vector for transformation of higher plants mediated by Agrobacterium tumefaciens. Mol Breed 14:309–320CrossRefGoogle Scholar
  19. Lange M, Vincze E, Moeller MG, Holm PB (2006) Molecular analysis of the transgene and vector backbone integration into the barley genome following Agrobacterium-mediated transformation. Plant Cell Rep 25:815–820CrossRefPubMedGoogle Scholar
  20. Livak KJ, Flood SJ, Marmaro J, Giusti W, Deetz K (1995) Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 4:357–362PubMedGoogle Scholar
  21. Mason G, Provero P, Vaira AM, Accotto GP (2002) Estimating the number of integrations in transformed plants by quantitative real-time PCR. BMC Biotechnol 2:1–10CrossRefGoogle Scholar
  22. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  23. Neves LO (2000) Regeneration and transformation of barrel medic (Medicago truncatula Gaertn. cv. Jemalong). PhD Dissertation Thesis. Faculdade de Ciência da Universidade de Lisboa, LisbonGoogle Scholar
  24. Neves LO, Tomaz L, Fevereiro MPS (2001) Micropropagation of Medicago truncatula Gaertn cv. Jemalong and Medicago truncatula ssp. Narborensis. Plant Cell Tissue Organ Cult 67:81–84CrossRefGoogle Scholar
  25. Puchta H (2003) Marker-free transgenic plants. Plant Cell Tissue Organ Cult 74:123–134CrossRefGoogle Scholar
  26. Ramakers C, Ruijter JM, Lekanne Deprez RH, Moorman AFM (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339:62–66CrossRefPubMedGoogle Scholar
  27. Rommens CM (2007) Intragenic crop improvement: combining the benefits of traditional breeding and genetic engineering. J Agric Food Chem 55:4281–4288CrossRefPubMedGoogle Scholar
  28. Rommens CM, Humara JM, Ye J, Yan H, Richael C, Zhang L, Perry R, Swords K (2004) Crop improvement through modification of the plant’s own genome. Plant Physiol 135:421–431CrossRefPubMedGoogle Scholar
  29. Rommens CM, Bougri O, Yan H, Humara JM, Owen J, Swords K, Ye J (2005) Plant-derived transfer DNAs. Plant Physiol 139:1338–1349CrossRefPubMedGoogle Scholar
  30. Rommens CM, Haring MA, Swords K, Davies HV, Belknap WR (2007) The intragenic approach as a new extension to traditional plant breeding. Trends Plant Sci 12:397–403CrossRefPubMedGoogle Scholar
  31. Scaramelli L, Balestrazzi A, Bonadei M, Piano E, Carbonera D, Confalonieri M (2009) Production of transgenic barrel medic (Medicago truncatula Gaertn.) using the ipt-type MAT vector system and impairment of recombinase-mediated excision. Plant Cell Rep 28:197–211CrossRefPubMedGoogle Scholar
  32. Smigocki AC, Owens LD (1988) Cytokinin gene fused with a strong promoter enhances shoot organogenesis and zeatin levels in transformed plant cells. Proc Natl Acad Sci USA 85:5131–5135CrossRefPubMedGoogle Scholar
  33. Trinh TH, Ratet P, Kondorosi E, Durand P, Kamatè K, Bauer P, Kondorosi A (1998) Rapid and efficient transformation of diploid Medicago truncatula and Medicago sativa ssp falcata lines improved in somatic embryogenesis. Plant Cell Rep 17:345–355CrossRefGoogle Scholar
  34. van der Graaff E, den Dulk-Ras A, Hooykaas PJJ (1996) Deviating T-DNA transfer from Agrobacterium tumefaciens to plants. Plant Mol Biol 13:677–681CrossRefGoogle Scholar
  35. van Harten AM (1998) Mutation breeding: theory and practical applications. Cambridge University Press, CambridgeGoogle Scholar
  36. Wenck A, Czako M, Kanevski I, Marton L (1997) Frequent collinear long transfer of DNA inclusive of the whole binary vector during Agrobacterium-mediated transformation. Plant Mol Biol 34:913–922CrossRefPubMedGoogle Scholar
  37. Ye X, Williams EJ, Shen J, Esser JA, Nichols AM, Petersen MW, Gilbertson LA (2008) Plant development inhibitory genes in binary vector backbone improve quality event efficiency in soybean transformation. Transgenic Res 17:827–838CrossRefPubMedGoogle Scholar
  38. Zelasco S, Ressegotti V, Confalonieri M, Carbonera D, Calligari P, Bonadei M, Bisoffi S, Yamada K, Balestrazzi A (2007) Evaluation of MAT-vector system in white poplar (Populus alba L.) and production of ipt marker-free transgenic plants by ‘single-step transformation’. Plant Cell Tissue Organ Cult 91:61–72CrossRefGoogle Scholar
  39. Zhou Z, Chandrasekharan MB, Hall TC (2004) High rooting frequency and functional analysis of GUS and GFP expression in transgenic Medicago truncatula A17. New Phytol 162:813–822CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Massimo Confalonieri
    • 1
  • Roberto Borghetti
    • 2
  • Anca Macovei
    • 2
  • Claudia Testoni
    • 2
  • Daniela Carbonera
    • 2
  • Manuel Pedro Salema Fevereiro
    • 3
  • Caius Rommens
    • 4
  • Kathy Swords
    • 4
  • Efisio Piano
    • 1
  • Alma Balestrazzi
    • 2
    Email author
  1. 1.C.R.A.-Centro di Ricerca per le Produzioni Foraggere e Lattiero-CasearieLodiItaly
  2. 2.Dipartimento di Genetica e MicrobiologiaUniversità di PaviaPaviaItaly
  3. 3.Instituto de Tecnologia Quimica e BiológicaUniversidade Nova de LisboaOeirasPortugal
  4. 4.Simplot Plant SciencesJ.R. Simplot CompanyBoiseUSA

Personalised recommendations