In contrast to most other legumes, alfalfa is generally easy to transform and regenerate in tissue culture. Most alfalfa cultivars   have a low percentage of plants that will undergo somatic embryogenesis. To facilitate evaluation of transgenes and promoters, we optimized transformation of a single genotype of alfalfa, RegenSY27x (1). This plant was selected from Regen-SY, developed by Dr. Edwin Bingham (2). Using Agrobacterium tumefaciens-mediated transformation of leaf explants, we obtain transgenic plants rapidly, within 15 weeks. The method is highly efficient with practically every explant produces transgenic plants. We can provide RegenSY27x to any researcher interested in alfalfa transformation. Contact Dr. Deborah Samac (debby.samac@ars.usda.gov).

(1) Samac, D. A. and Austin-Phillips, S. 2006. Alfalfa (Medicago sativa L.). In: Methods in Molecular Biology, vol. 343 Agrobacterium Protocols, 2^nd Edition. K. Wang, ed. p. 301-311. Humana Press, Inc., Totowa, NJ.

(2) Bingham, E. T. (1991) Registration of alfalfa hybrid Regen-SY germplasm for tissue culture and transformation research. Crop Science 31, 1098.

The alfalfa transformation system has been used in our lab to identify promoters that have tissue specific expression, identify promoter elements, and to characterize promoters with high constitutive expression.

We have used the alfalfa transformation system to generate alfalfa plants that express proteins for novel traits for use in agriculture and bioprocessing.

Our current research focuses on developing genome-editing technologies in alfalfa.

Promoter characterization
Samac, D. A., Tesfaye, M., Dornbusch M., Purev, S., and Temple, S. J. 2004. A comparison of constitutive promoters for expression of transgenes in alfalfa (Medicago sativa). Transgenic Res. 13:349-361.

Trepp, G. B., van de Mortel, M., Yoshioka, H., Miller, S. S., Samac, D. A., Gantt, J. S., and Vance, C. P. 1999. NADH-glutamate synthase (GOGAT) in alfalfa root nodules: Genetic regulation and cellular expression. Plant Physiol. 119:817-828.

Pathirana, S., Samac, D. A., Roeven, R., Vance, C. P., and Gantt, S. J. 1997. Analyses of phosphoenolpyruvate carboxylase gene structure and expression in alfalfa. Plant J. 12:293-304.

Shi, L., Twary, S. N., Yoshioka, H., Gregerson, R. G., Miller, S., Samac, D. A., Gantt, J. S., Unkefer, P. T., and Vance, C. P. 1997. Nitrogen assimilation in alfalfa: Isolation and characterization of an asparagine synthetase gene showing enhanced expression in root nodules and dark adapted leaves. Plant Cell 9:1339-1356. 


Alfalfa biotechnology
Vail, A. W., Wang, P., Uefuji, H., Samac, D. A., Vance, C. P., Wackett, L. P., and Sadowsky, M. J. 2015. Biodegradation of atrazine by three transgenic grasses and alfalfa expressing a modified bacterial atrazine chlorohydrolase gene. Transgenic Res. 24:475-488.

Sullivan M. L., Hatfield R. D., Thoma S. L., and Samac, D. A. 2004. Cloning and characterization of red clover polyphenol oxidase cDNAs and expression of active protein in Escherichia coli and transgenic alfalfa. Plant Physiol. 136:3234-44.

Wang, L., Samac, D. A., Shapir, N., Wackett, L. P., Vance, C. P., Olszewski, N. E., Sadowsky, M. J. 2005. Biodegradation of atrazine in transgenic plants expressing a modified bacterial atrazine chlorohydrolase (atzA) gene. Plant Biotechnol. J. 3:475-486.

Tesfaye, M., Denton, M. D., Samac, D. A., and Vance, C. P. 2005. Transgenic alfalfa secretes a fungal endochitinase protein to the rhizosphere. Plant Soil 269: 233-243.
Samac, D. A., Litterer, L., Temple, G., Jung, H. G., and Somers, D. A. 2004. Expression of UDP-glucose dehydrogenase reduces cell-wall polysaccharide concentration and increases xylose content in alfalfa stems. Appl. Biochem. Biotechnol. 116:1167-1182.

Samac, D. A. and Smigocki, A. C. 2003. Expression of oryzacystatin I and II in alfalfa increases resistance to the root-lesion nematode (Pratylenchus penetrans). Phytopathology 93:799-804.

Saruul, P., Sreinc, F., Somers, D. A., and Samac, D. A. 2002. Production of a biodegradable plastic polymer, poly-b-hydroxybutyrate, in transgenic alfalfa (Medicago sativa L.). Crop Sci. 42:919-927.

Tesfaye, M., Temple, S. J., Allan, D. L., Vance, C. P., and Samac, D. A. 2001. Over-expression of malate dehydrogenase in transgenic alfalfa enhances organic acid synthesis and confers tolerance to aluminum. Plant Physiol. 127:1836-1844.