Agrobacterium-mediated transformation of alfalfa (Medicago sativa) using a synthetic cry3a gene to enhance resistance against alfalfa weevil

  • Masoud TohidfarEmail author
  • Naser Zare
  • Gholamreza Salehi Jouzani
  • Seide Maryam Eftekhari
Original Paper


To introduce genetic resistance against alfalfa weevil (Hypera postica), leaves and petiole explants of three commercial alfalfa genotypes, including Km-27, Kk-14 and Syn-18 were transformed with Agrobacterium tumefaciens strains GV101, LBA4404 and AGL01. All the Agrobacterium strains used harbored the recombinant binary vector pBI121 containing a synthetic cry3a gene under the control of CaMV35S promoter as well as the nptII gene as selectable marker. Transformed explants were cultured on callus-induction medium, and the germinated somatic embryos were then transferred to the regeneration medium. The primary transformants were evaluated by PCR and Southern blot analysis. The results indicated successful integration of the target gene into the genomes of primary transgenic lines. Moreover, the expression of Cry3a protein in the transgenic plants was confirmed by ELISA method. Three transgenic lines, including TL6, TL8 and TL11 showed significantly higher levels of insect resistance against H. postica larvae (mortality rate of 73–90 % after infestation), in comparison with the control plants during the two-year bioassays. All transgenic plants were fertile and no irregular behavior in terms of growth and the morphological traits were observed. Transgenic plants developed during the course of this study are currently being grown in greenhouse and will be crossed with each other for seed production.


Agrobacterium tumefaciens Alfalfa weevil cry3a Hypera postica Transgenic alfalfa 



Blaydes 1966 medium


Cauliflower mosaic virus




2,4-dichlorophenoxyacetic acid


Enzyme-linked immunosorbent assay


Murashige and Shoog medium (1962)


Neomycin phosphotransferase gene


Nopaline synthase


Optical density



This work was supported by a grant received from the Agricultural Research and Education Organization of Iran (AREO). We wish to thank all our colleagues in the Department of Plant Tissue Culture and Genetic engineering of ABRII for their support and technical assistance. The authors would also like to extend their appreciation to Assist. Prof. Meisam Tabatabaei for his critical review of the manuscript in terms of its academic English writing.


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Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Masoud Tohidfar
    • 1
    Email author
  • Naser Zare
    • 1
  • Gholamreza Salehi Jouzani
    • 2
  • Seide Maryam Eftekhari
    • 1
  1. 1.Department of Plant Tissue Culture and Gene TransformationAgricultural Biotechnology Research Institute of Iran (ABRII)KarajIran
  2. 2.Microbial Biotechnology and Biosafety DepartmentAgricultural Biotechnology Research Institute of Iran (ABRII)KarajIran

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