Backbone-free transformation of barrel medic (Medicago truncatula) with a Medicago-derived transfer DNA
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In the present work, Agrobacterium tumefaciens-mediated genetic transformation of the model legume Medicago truncatula Gaertn. (barrel medic) was carried out using the pSIM843 vector that contains a Medicago-derived transfer DNA, delineated by a 25-bp sequence homologous to bacterial T-DNA borders. The transfer DNA contains an expression cassette for the nptII (neomycin phosphotransferase) gene and is flanked by an expression cassette for the backbone integration marker gene ipt (isopentenyl transferase). Our results demonstrate that the Medicago-derived RB-like elements efficiently support DNA mobilization from A. tumefaciens to M. truncatula. Kanamycin-resistant shoots with normal phenotype and ipt-shooty lines were recovered at a frequency of 11.7 and 7.8%, respectively. Polymerase chain reaction (PCR) analyses demonstrated that 44.4% of the independent transgenic lines were backbone-free and evidenced the occurrence of backbone-transfer events.
KeywordsBackbone transfer ipt Medicago truncatula Morphological marker P-DNA
This research was supported by Fondo di Ateneo per la Ricerca-University of Pavia. Authors are grateful to Dr. Cristina Ambroggi for excellent technical contribution and Dr. Lorenzo Concia for helpful suggestions concerning the interpretation of QRT-PCR data.
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