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Production of transgenic barrel medic (Medicago truncatula Gaernt.) using the ipt-type MAT vector system and impairment of Recombinase-mediated excision events

  • Genetic Transformation and Hybridization
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Abstract

Expression of the uidA reporter gene was tested in transformation experiments of barrel medic (Medicago truncatula Gaertn.) with the ipt-type control vectors pIPT5, pIPT10 and pIPT20 and distinct in vitro culture conditions. The highest GUS expression levels were obtained with the pIPT10 construct carrying the ipt gene under the control of the native ipt promoter and using kanamycin as selective agent. The ipt-shooty transformants, characterized by the absence of both rooting ability and apical dominance associated with vitrification, were easily identified by visual selection. Using only the ipt gene as selectable marker, we obtained a stable transformation frequency of 9.8% with pIPT10 construct. The ipt-type MAT vector pEXM2 was then used to monitor the excision events mediated by the yeast Recombinase and the consequent production of ipt marker-free transgenic plants. Transgenic ipt-shooty lines were recovered at a frequency of 7.9% in the absence of kanamycin-based selection. The ipt-shooty phenotype was maintained in all the transgenic lines and no reversion to the normal phenotype occurred. PCR analysis revealed the presence of the ‘hit and run’ cassette in the genome of all the regenerated ipt-shooty lines while RT-PCR experiments confirmed the expression of the R gene, encoding the yeast Recombinase. A detailed molecular investigation, carried out to verify the integrity of the RS sites, revealed that these regions were intact in most cases. Our results with barrel medic suggest that the MAT system must be carefully evaluated and discussed on a case by case basis.

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Acknowledgments

We are grateful to Antonio Ruscica and Massimo Sari for excellent technical assistance and for their participation during various stages of this work. We would like to thank Drs. Sergio Arcioni and Andrea Porceddu (IGV-CNR, Perugia, Italy) for providing R108-1 Medicago truncatula genotype and Dr. Pascal Ratet (ISV-CNRS, Gif sur Yvette, France) for helpful support during M. truncatula transformation stage. Authors are grateful to Dr. Keiko Yamada (Nippon Paper Industries, Tokyo, Japan) for providing MAT constructs.

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Authors and Affiliations

  1. Centro di Ricerca per le Produzioni Foraggere e Lattiero Casearie, C.R.A., Viale Piacenza 29, 26900, Lodi, Italy

    L. Scaramelli, E. Piano & M. Confalonieri

  2. Dipartimento di Genetica e di Microbiologia, Università degli Studi di Pavia, via Ferrata 1, 27100, Pavia, Italy

    A. Balestrazzi, M. Bonadei & D. Carbonera

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  1. L. Scaramelli
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  2. A. Balestrazzi
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  3. M. Bonadei
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  4. E. Piano
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  5. D. Carbonera
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  6. M. Confalonieri
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Corresponding author

Correspondence to M. Confalonieri.

Additional information

Communicated by H. Ebinuma.

L. Scaramelli, A. Balestrazzi and M. Confalonieri have contributed equally to this work.

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Scaramelli, L., Balestrazzi, A., Bonadei, M. et al. Production of transgenic barrel medic (Medicago truncatula Gaernt.) using the ipt-type MAT vector system and impairment of Recombinase-mediated excision events. Plant Cell Rep 28, 197–211 (2009). https://doi.org/10.1007/s00299-008-0634-6

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  • DOI: https://doi.org/10.1007/s00299-008-0634-6

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