Alfalfa (Medicago sativa L.)

Methods Mol Biol. 2006;343:301-11. doi: 10.1385/1-59745-130-4:301.


A protocol for rapid, highly efficient transformation of alfalfa is described. Leaf explants from growth chamber-grown plants of a highly regenerable genotype are surface-sterilized, the margins are removed, and explants are inoculated with Agrobacterium tumefaciens strain LBA4404 carrying the T-DNA vector of interest. The explants and bacteria are cocultured for 7 to 8 d. Bacteria are removed by rinsing explants in sterile distilled water and by culture on regeneration medium containing the antibiotics carbenicillin or ticarcillin. Transformed callus is selected using kanamycin. Somatic embryos are induced by culture of callus on medium lacking plant growth regulators. As mature cotyledonary stage embryos arise, they are transferred to a fresh medium for shoot development and finally to a medium lacking kanamycin for continued shoot and root development. Transgenic plants can be produced in 9 wk with this protocol. Typically 60 to 80% of inoculated explants produce transgenic plants, and escapes are rare.

Publication types

  • Review

MeSH terms

  • Agrobacterium tumefaciens / cytology
  • Agrobacterium tumefaciens / genetics*
  • Agrobacterium tumefaciens / growth & development
  • Anti-Bacterial Agents / pharmacology
  • DNA, Bacterial / genetics
  • Drug Resistance / drug effects
  • Drug Resistance / genetics
  • Gene Transfer Techniques*
  • Genetic Markers
  • Genetic Vectors
  • Genotype
  • Medicago sativa / cytology
  • Medicago sativa / genetics*
  • Medicago sativa / microbiology
  • Plant Leaves / cytology
  • Plant Leaves / genetics*
  • Plant Leaves / microbiology
  • Plants, Genetically Modified / embryology
  • Plants, Genetically Modified / genetics*
  • Plants, Genetically Modified / microbiology
  • Transformation, Genetic*


  • Anti-Bacterial Agents
  • DNA, Bacterial
  • Genetic Markers
  • T-DNA