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Agrobacterium rhizogenes-mediated transformation of Prunus as an alternative for gene functional analysis in hairy-roots and composite plants

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Abstract

Resistant rootstocks offer an alternative to pesticides for the control of soil pests. In Prunus spp., resistance loci to root-knot nematodes (RKN) have been mapped and a transformation method is needed to validate candidate genes. Our efforts have focused on the generation of transformed hairy-roots and composite plants appropriate for nematode infection assays. An efficient and reliable method using the A4R strain of Agrobacterium rhizogenes for the transformation of Prunus roots with an Egfp reporter gene is given. The rooting efficiency, depending on the genotypes, was maximal for the interspecific hybrid 253 (Myrobalan plum × almond-peach), susceptible to RKN, that was retained for subsequent studies. From the agro-inoculated cuttings, 72% produced roots, mainly at the basal section of the stem. Transformed roots were screened by microscope detection of Egfp fluorescence and molecular analyses of the integration of the transgene. The absence of residual agrobacteria in the plants was checked by the non-amplification of the chromosomal gene chvH. Egfp was expressed visually in 76% of the rooted plants. Isolated hairy roots in Petri dishes and composite plants (transformed roots and non-transformed aerial part) in soil containers were inoculated with the RKN Meloidogyne incognita. In both cases, root transformation did not affect the ability of the nematodes to develop in the root tissues. Our results showed that isolated hairy-roots can be used to validate candidate genes and the conditions in which composite plants offer a complementary system for studying the function of root genes in physiological conditions of whole plants are discussed.

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Acknowledgments

All work with genetically transformed material was carried out in containment chambers under licence no. 4061 from the Ministry of Research. This work was partly funded by the European Union via the FAIR RTD Program (Research Project no. FAIR6-CT 984139; 1999–2004). The authors thank David Tepfer (INRA, Versailles, France) for kindly providing the A. rhizogenes A4R strain and Hervé Etienne (CIRAD, Montpellier, France) for his expert advices on transformation with agrobacteria.

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Authors and Affiliations

  1. INRA, UMR 1301, 06903, Sophia-Antipolis, France

    Nathalie Bosselut, Cyril Van Ghelder, Michel Claverie, Roger Voisin, Marie-Noëlle Rosso & Daniel Esmenjaud

  2. CNRS, UMR 6243, 06903, Sophia-Antipolis, France

    Nathalie Bosselut, Cyril Van Ghelder, Michel Claverie, Roger Voisin, Marie-Noëlle Rosso & Daniel Esmenjaud

  3. UNSA, UMR 1301, 06903, Sophia-Antipolis, France

    Nathalie Bosselut, Cyril Van Ghelder, Michel Claverie, Roger Voisin, Marie-Noëlle Rosso & Daniel Esmenjaud

  4. CIRAD, UMR DAP, 34398, Montpellier, France

    Michel Claverie

  5. INRA, Unité de Recherches Intégrées en Horticulture, 06903, Sophia-Antipolis, France

    Jean-Paul Onesto

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  1. Nathalie Bosselut
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  2. Cyril Van Ghelder
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  3. Michel Claverie
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  4. Roger Voisin
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  6. Marie-Noëlle Rosso
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  7. Daniel Esmenjaud
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Correspondence to Daniel Esmenjaud.

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Communicated by E. Guiderdoni.

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Bosselut, N., Van Ghelder, C., Claverie, M. et al. Agrobacterium rhizogenes-mediated transformation of Prunus as an alternative for gene functional analysis in hairy-roots and composite plants. Plant Cell Rep 30, 1313–1326 (2011). https://doi.org/10.1007/s00299-011-1043-9

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