An Efficient Transformation Method to Regenerate a High Number of Transgenic Plants Using a New Embryogenic Line of Medicago truncatula cv. Jemalong

TLDR: Isolation of late torpedo/cotyledonary-stage embryos to lower carbenicillin/agar media reduced secondary embryogenesis and prevents hyperhydricity, improving embryo conversion and inheritance of the transgenes is shown to be stable in the T1 generation.

Abstract: A simple and efficient regeneration–transformation method was established to obtain transgenic plants of the model legume Medicago truncatula cv. Jemalong. This method takes advantage of a new highly embryogenic line (M9-10a) isolated in our laboratory. Leaflets of in vitro grown M9-10a plants were co-cultured with Agrobacterium tumefaciens EHA105. Plasmid constructs containing the oat arginine decarboxylase gene, Adc and the GUS reporter gene (p35SAdc–Gus) or ELIP-like drought stress protein 22 (DSP22) encoding gene from Craterostigma plantagineum (p35SDsp22) were used. Both constructs include the nptII gene as selection marker. Embryogenic calli (100–97%) were obtained on embryo induction medium containing 100 mg l −1 kanamycin and 500 mg l−1 carbenicillin. Using a two-fold increase in kanamycin concentration, instead of 50 mg l−1 usually used, we reduced the number of emerging false kanamycin-resistant (KanR) embryos, which is an important improvement to the method, making it less laborious and very efficient. Isolation of late torpedo/cotyledonary-stage embryos to lower carbenicillin/agar media reduced secondary embryogenesis and prevents hyperhydricity, improving embryo conversion. Primary transformants (T0) were regenerated within 3–4 months and those that were able to root in a 50 mg l−1 kanamycin medium were transferred to the greenhouse to produce seeds. Southern blot hybridisation analysis confirmed the integration of either the Adc or Dsp22 transgenes in the genome of the T0 transformants. Detection of β-glucuronidase (GUS) activity in Adc–Gus T0 plants demonstrated the expression of the inserted transgene. In average, 1–2 independent transgenic lines are obtained per KanR embryogenic callus, independently of the plasmid construct used for transformation. Inheritance of the transgenes is shown to be stable in the T1 generation.