Flow cytometric analysis and molecular characterization of Agrobacterium tumefaciens-mediated transformants of Medicago truncatula |
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Authors: | S. Ochatt L. Jacas E. M. Patat-Ochatt S. Djenanne |
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Affiliation: | 1. UMR1347 Agroécologie, P?le GEAPSI, INRA C.R. de Dijon, B.P. 86510, 21065, Dijon Cedex, France 2. INRA, UMR1131 Santé de la Vigne et Qualité du Vin, 68000, Colmar, France
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Abstract: | 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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