Flow cytometric analysis and molecular characterization of Agrobacterium tumefaciens-mediated transformants of Medicago truncatula
2013
Ochatt, Sergio | Jacas, Louis | Patat-Ochatt, Estela | Djennane, Samia | Agroécologie [Dijon] ; Institut National de la Recherche Agronomique (INRA)-Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement | Santé de la vigne et qualité du vin (SVQV) ; Institut National de la Recherche Agronomique (INRA)-Université Louis Pasteur - Strasbourg I | Eurostar (E.U.) [E4770: PEASTAR]
Sur la publication, l'auteur S. Djennane est mal orthographié (Djenanne).
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Show more [+] Less [-]English. Leaf explants from leaflets collected from either in vivo grown or in vitro grown seedlings of Medicago truncatula genotype R108-1 were co-cultivated with bacterial cells of Agrobacterium tumefaciens strains EHA105 or C58pMP90. Each of these strains was carrying the pCambia 1390 plasmid harbouring a hygromycin resistance gene cassette. Explants were then incubated on a medium containing 10 mg/l hygromycin and 800 mg/l augmentin to suppress Agrobacterium growth, and subcultured 4-5 times every 2 weeks for the proliferation of calli. After 8-10 weeks, callusing explants were transferred to hormone-free medium with 10 mg/l hygromycin and 400 mg/l augmentin for shoot regeneration. After rooting, a total of about 300 putative transformants were grown into plantlets, transferred to soil, acclimatized, and then moved to the greenhouse. Of these, a total of 43 independent PCR positive primary transformants and their T1 and T2 progeny were subjected to flow cytometric analysis, to assessing their trueness-to-type, as well as to southern blot analysis.
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