Efficient, Antibiotic Marker-Free Transformation of a Dicot and a Monocot Crop with Glutamate 1-Semialdehyde Aminotransferase Selectable Marker Genes

  • Nicoletta Ferradini
  • Angelica Giancaspro
  • Alessandro Nicolia
  • Agata Gadaleta
  • Fabio Veronesi
  • Daniele RoselliniEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1385)


Antibiotic-free, efficient in vitro selection in plant genetic engineering can improve risk perception and speed up pre-market scrutiny of genetically modified crops. We provide a protocol for genetic transformation of two important crops, durum wheat and alfalfa, using a bacterial and a plant-derived selectable marker gene encoding mutated, gabaculine-insensitive glutamate 1-semialdehyde aminotransferase (GSA) enzymes. These methods can potentially be applied, with minor adaptations, to many other monocot and dicot crop plants.


Alfalfa Durum wheat Gabaculine GMO HemMsGSAgr Non-antibiotic selection Plant genetic engineering 


  1. 1.
    Rosellini D (2012) Selectable markers and reporter genes: a well furnished toolbox for plant science and genetic engineering. Crit Rev Plant Sci 31:401–453CrossRefGoogle Scholar
  2. 2.
    Hill CM, Pearson SA, Smith AJ, Rogers LJ (1985) Inhibition of chlorophyll synthesis in Hordeum vulgare by 3-amino-2,3-dihydrobenzoic acid (gabaculine). Biosci Rep 5:775–781CrossRefPubMedGoogle Scholar
  3. 3.
    Gough KC, Haves WS, Kilpatrick J, Whitelam GC (2001) Cyanobacterial GR6 glutamate-1-semialdehyde aminotransferase: a novel enzyme-based selectable marker for plant transformation. Plant Cell Rep 20:296–300CrossRefGoogle Scholar
  4. 4.
    Rosellini D, Capomaccio S, Ferradini N, Savo Sardaro ML, Nicolia A, Veronesi F (2007) Non-antibiotic, efficient selection for alfalfa genetic engineering. Plant Cell Rep 26:1035–1044CrossRefPubMedGoogle Scholar
  5. 5.
    Ferradini N, Nicolia A, Capomaccio S, Veronesi F, Rosellini D (2011) A point mutation in the Medicago sativa GSA gene provides a novel, efficient, selectable marker for plant genetic engineering. J Biotechnol 156:147–152CrossRefPubMedGoogle Scholar
  6. 6.
    Giancaspro A, Rosellini D, Blanco A, Gadaleta A (2012) Gabaculine selection using bacterial and plant marker genes (GSA-AT) in durum wheat transformation. Plant Cell Tissue Organ Cult 109:447–455CrossRefGoogle Scholar
  7. 7.
    Gadaleta A, Giancaspro A, Blechl AE, Blanco A (2008) A transgenic durum wheat line that is free of marker genes and expresses 1DY10. J Cereal Sci 48:439–445CrossRefGoogle Scholar
  8. 8.
    Bingham ET (1991) Registration of alfalfa hybrid Regen-SY germplasm for tissue culture and transformation research. Crop Sci 31:1098CrossRefGoogle Scholar
  9. 9.
    Rosellini D, LaFayette P, Barone P, Veronesi F, Parrott WA (2004) Kanamycin resistant alfalfa has a point mutation in the plastid 16S rDNA. Plant Cell Rep 22:774–779CrossRefPubMedGoogle Scholar
  10. 10.
    Brown DCW, Atanassov A (1985) Role of genetic background in somatic embryogenesis in Medicago. Plant Cell Tissue Organ Cult 4:111–122CrossRefGoogle Scholar
  11. 11.
    Samac DA, Temple SJ (2004) Development and utilization of transformation in Medicago species. In: Liang GH, Skinner DZ (eds) Genetically modified crops: their development, uses, and risks. Haworth, Binghampton, NY, pp 165–202Google Scholar
  12. 12.
    Nitsch JP, Nitsch C (1969) Haploid plants from pollen grains. Science 163:85–87CrossRefPubMedGoogle Scholar
  13. 13.
    Rando RR (1977) Mechanism of the irreversible inhibition of γ-aminobutyric acid-α-ketoglutaric acid transaminase by the neurotoxin gabaculine. Biochemistry 16:4604–4610CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Nicoletta Ferradini
    • 1
  • Angelica Giancaspro
    • 2
  • Alessandro Nicolia
    • 1
    • 3
  • Agata Gadaleta
    • 2
  • Fabio Veronesi
    • 1
  • Daniele Rosellini
    • 1
    Email author
  1. 1.Department of Agricultural, Environmental and Food ScienceUniversity of PerugiaPerugiaItaly
  2. 2.Department of Soil, Plant and Food ScienceUniversity of Bari “Aldo Moro”BariItaly
  3. 3.ENEA Casaccia Research CenterRomaItaly

Personalised recommendations