A Unified Agrobacterium-Mediated Transformation Protocol for Alfalfa (Medicago sativa L.) and Medicago truncatula

  • Qingzhen JiangEmail author
  • Chunxiang Fu
  • Zeng-Yu Wang
Part of the Methods in Molecular Biology book series (MIMB, volume 1864)


Simplification of transformation procedures greatly improves work efficiency. In this chapter, we introduce a unified Agrobacterium-mediated transformation protocol that is used for both alfalfa (Medicago sativa L., Regen SY4D) and Medicago truncatula (ecotype R108). Whole trifoliates or leaflets are used as explants. Sonication is applied to enhance Agrobacterium infection and cytokinins are added to the medium to facilitate shoot regeneration. More than 90% transformation efficiency is achieved for alfalfa, while the relatively lower efficiency of up to 60% is obtained for M. truncatula, which depends on different selectable markers in the gene constructs. Transgenic plants are produced within 4–8 months with average timeline of 6 months. Using this unified protocol, the same types of media are used for both species which results in significant saving in time and resources.

Key words

Alfalfa Medicago sativa Medicago truncatula Agrobacterium tumefaciens Genetic transformation Regeneration Transgenic plant Somatic embryogenesis 



We would like to thank our previous and current group members Tim Hernandez, Yewei Wang, Steven Tudor, Mohammad Fereidouni, Guangming Li, Jianfei Yun, and Lishan Yang for their knowledge and suggestions in improving the procedure. We are also grateful to all the researchers and their principal investigators for their trust and support using the service.


  1. 1.
    Riday H, Brummer EC (2002) Forage yield heterosis in alfalfa. Crop Sci 42:716–723CrossRefGoogle Scholar
  2. 2.
    Wang ZY, Brummer EC (2012) Is genetic engineering ever going to take off in forage, turf and bioenergy crop breeding? Ann Bot 110:1317–1325CrossRefGoogle Scholar
  3. 3.
    Deak M, Kiss GB, Koncz C, Dudits D (1986) Transformation of Medicago by Agrobacterium mediated gene transfer. Plant Cell Rep 5:97–100CrossRefGoogle Scholar
  4. 4.
    Shahin EA, Spielmann A, Sukhapinda K, Simpson RB, Yashar M (1986) Transformation of cultivated alfalfa using disarmed Agrobacterium tumefaciens. Crop Sci 26:1235–1239CrossRefGoogle Scholar
  5. 5.
    Austin S, Bingham ET, Mathews DE, Shahan MN, Will J, Burgess RR (1995) Production and field performance of transgenic alfalfa (Medicago sativa L.) expressing alpha-amylase and manganese- dependent lignin peroxidase. Euphytica 85:381–393CrossRefGoogle Scholar
  6. 6.
    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 Press, New York, pp 165–202Google Scholar
  7. 7.
    Samac DA, Austin-Phillips S (2006) Alfalfa (Medicago sativa L.). In: Wang K (ed) Methods in molecular biology, Agrobacterium protocols, 2/e, volume 1, vol 343. Humana Press Inc., Totowa, NJ, pp 301–311Google Scholar
  8. 8.
    Bingham ET (1991) Registration of alfalfa hybrid Regen-SY germplasm for tissue culture and transformation research. Crop Sci 31:1098–1118CrossRefGoogle Scholar
  9. 9.
    Chen F, Srinivasa Reddy MS, Temple S, Jackson L, Shadle G, Dixon RA (2006) Multi-site genetic modulation of monolignol biosynthesis suggests new routes for formation of syringyl lignin and wall-bound ferulic acid in alfalfa (Medicago sativa L.). Plant J 48:113–124CrossRefGoogle Scholar
  10. 10.
    Chabaud M, Larsonneau C, Marmouget C, Huguet T (1996) Transformation of barrel medic (Medicago truncatula Gaertn) by Agrobacterium tumefaciens and regeneration via somatic embryogenesis of transgenic plants with the MtENOD12 nodulin promoter fused to the gus reporter gene. Plant Cell Rep 15:305–310CrossRefGoogle Scholar
  11. 11.
    Trinh TH, Ratet P, Kondorosi E, Durand P, Kamaté K, Bauer P, Kondorosi A (1998) Rapid and efficient transformation of diploid Medicago truncatula and Medicago sativa ssp. falcata in vitro lines improved in somatic embryogenesis. Plant Cell Rep 17:345–355CrossRefGoogle Scholar
  12. 12.
    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 22:46–51CrossRefGoogle Scholar
  13. 13.
    Cosson V, Durand P, d’Erfurth I, Kondorosi A, Ratet P (2006) Medicago truncatula transformation using leaf explants. In: Wang K (ed) Methods in molecular biology, Agrobacterium protocols, 2/e, volume 1, vol 343. Humana Press Inc., Totowa, NJ, pp 115–127Google Scholar
  14. 14.
    Trieu AT, Harrison MJ (1996) Rapid transformation of Medicago truncatula: regeneration via shoot organogenesis. Plant Cell Rep 16:6–11CrossRefGoogle Scholar
  15. 15.
    Wright E, Dixon RA, Wang Z-Y (2006) Medicago truncatula transformation using cotyledon explants. In: Wang K (ed) Methods in molecular biology, Agrobacterium Protocols, 2/e, volume 1, vol 343. Humana Press Inc., Totowa, NJ, pp 129–136Google Scholar
  16. 16.
    Crane C, Dixon RA, Wang Z-Y (2006) Medicago truncatula transformation using root explants. In: Wang K (ed) Methods in molecular biology, Agrobacterium Protocols, 2/e, volume 1, vol 343. Humana Press Inc., Totowa, NJ, pp 137–154Google Scholar
  17. 17.
    Kamaté K, Rodriguez-Llorente ID, Scholte M, Durand P, Ratet P, Kondorosi E, Kondorosi A, Trinh TH (2000) Transformation of floral organs with GFP in Medicago truncatula. Plant Cell Rep 19:647–653CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Noble Research InstituteArdmoreUSA
  2. 2.Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of SciencesQingdaoChina

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