|Austin-Phillips, Sandra - UNIVERSITY OF WISCONSIN|
Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: February 2, 2005
Publication Date: May 1, 2006
Citation: Samac, D.A., Austin-Phillips, S. 2006. Alfalfa (Medicago sativa L.). In: Wang, K., editor. Methods in Molecular Biology, Vol. 343, Agrobacterium Protocols. 2nd edition. Totowa, NJ: Humana Press. p. 301-311. Technical Abstract: 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 removed, and explants inoculated with Agrobacterium tumefaciens strain LBA4404 carrying the T-DNA vector of interest. The explants and bacteria are co-cultured for 7-8 days. 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 weeks with this protocol. Typically 60-80% of inoculated explants produce transgenic plants and escapes are rare.