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Agrobacterium-mediated transformation of European chestnut embryogenic cultures

  • Genetic Transformation and Hybridization
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Abstract

An innovative and efficient genetic transformation protocol for European chestnut is described in which embryogenic cultures are used as the target material. When somatic embryos at the globular or early-torpedo stages were cocultured for 4 days with Agrobacterium tumefaciens strain EHA105 harbouring the pUbiGUSINT plasmid containing marker genes, a transformation efficiency of 25% was recorded. Murashige and Skoog culture medium containing 150 mg/l of kanamycin was used as the selection medium. The addition of acetosyringone was detrimental to the transformation efficiency. Transformation was confirmed by a histochemical β-glucuronidase (GUS ) assay, PCR and Southern blot analyses for the uidA (GUS) and nptII (neomycin phosphotransferase II) genes. At present, 93 GUS-positive chestnut embryogenic lines are being maintained in culture. Low germination rates (6.3%) were recorded for the transformed somatic embryos. The presence of the transferred genes in leaves and shoots derived from the germinated embryos was also verified by the GUS assay and PCR analysis.

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Abbreviations

BA:

Benzyladenine

kan:

Kanamycin

NAA:

α-Naphthaleneacetic acid

X-Gluc:

5-Bromo-4-chloro-3-indolyl-β-d-glucuronide

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Acknowledgements

The authors would like to thank Dr. L. Gómez (ETSIM, UPM, Madrid, Spain) for the gift of strain C58C1 and Dr. J. Segura (F. Farmacia, U. Valencia, Spain) for the strain EHA105. We also thank Dr. C. Sánchez (CSIC, Spain) and Dr. E. Sales (EPS, U. Zaragoza, Spain) for their expert advice on molecular techniques, and A. Rial and M. J. Cernadas for their technical assistance. This research was partially supported by MCYT and Xunta de Galicia (Spain), through the projects AGL 2000-1073 and PGIDT01PXI4000IPN.

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Authors and Affiliations

  1. Instituto de Investigaciones Agrobiológicas de Galicia, CSIC, Apartado 122, 15080, Santiago de Compostela, Spain

    E. Corredoira, D. Montenegro, M. C. San-José, A. M. Vieitez & A. Ballester

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  1. E. Corredoira
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  2. D. Montenegro
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  3. M. C. San-José
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  4. A. M. Vieitez
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  5. A. Ballester
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Correspondence to A. M. Vieitez.

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Communicated by S.A. Merkle

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Corredoira, E., Montenegro, D., San-José, M.C. et al. Agrobacterium-mediated transformation of European chestnut embryogenic cultures. Plant Cell Rep 23, 311–318 (2004). https://doi.org/10.1007/s00299-004-0804-0

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  • DOI: https://doi.org/10.1007/s00299-004-0804-0

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