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Evaluation of key factors influencing Agrobacterium-mediated transformation of somatic embryos of avocado (Persea americana Mill.)

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Abstract

Key factors influencing the efficiency of transformation of embryogenic cultures, induced from immature zygotic embryos, of avocado cv. ‘Duke 7’ were evaluated. Initially, the sensitivity of somatic embryos to the antibiotics kanamycin, used for selection, carbenicillin, cefotaxime and timentin, all used for elimination of Agrobacterium cells, were evaluated. Isolated globular somatic embryos were more sensitive to kanamycin than embryogenic masses, and 25 mg l−1 kanamycin completely restricted callus proliferation. Cefotaxime at 500 mg l−1 partially inhibited proliferation of embryogenic cultures, while both carbenicillin and timentin did not affect callus growth. For genetic transformation, somatic embryos were infected with A. tumefaciens containing the pBINUbiGUSint plasmid. After 2 days, the embryos were transferred to selection medium supplemented with 50 mg l−1 kanamycin and 250 mg l−1 timentin for 2 months. Then, kanamycin level was increased to 100 mg l−1 for two additional months. The A. tumefaciens strain AGL1 yielded higher transformation rates, 6%, than EHA105 or LBA4404, 1.2%. The percentage of kanamycin resistant calli obtained was significantly influenced by the embryogenic line used as source of explants. Genetic transformation was confirmed by PCR and Southern blot analysis. A significant improvement in the germination rate was obtained when transgenic embryos were cultured in liquid MS medium with 4.44 μM BA and 2.89 μM GA3 for 3 days in a roller drum and later transferred to the same medium gelled with 7 g l−1 agar. Plants from five independent transgenic lines were acclimated and grown in the greenhouse, being phenotipically similar to control plants.

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Abbreviations

BA:

6-Benzyladenine

GA3 :

Gibberellic acid

MS:

Murashige and Skoog medium

MSP:

Embryogenic avocado culture medium

SE:

Somatic embryo

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Acknowledgments

This research was funded by Ministerio de Ciencia e Innovación of Spain and Feder European Union Funds (Grant No. AGL2008-05453-C02-01/AGR). The authors thank Dr. Ricardo J. Ordás, Universidad de Oviedo, Spain, for providing the AGL1 strain with the pBINUbiGUSint plasmid, and Dr. Clara Pliego for her valuable support in the molecular analysis.

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Authors and Affiliations

  1. Departamento de Biología Vegetal, Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora” (IHSM-UMA-CSIC), Universidad de Málaga, 29071, Málaga, Spain

    Elena Palomo-Ríos, José A. Mercado & Fernando Pliego-Alfaro

  2. IFAPA Centro de Churriana, 29140, Churriana, Málaga, Spain

    Araceli Barceló-Muñoz

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  1. Elena Palomo-Ríos
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  2. Araceli Barceló-Muñoz
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  3. José A. Mercado
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  4. Fernando Pliego-Alfaro
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Correspondence to José A. Mercado.

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Palomo-Ríos, E., Barceló-Muñoz, A., Mercado, J.A. et al. Evaluation of key factors influencing Agrobacterium-mediated transformation of somatic embryos of avocado (Persea americana Mill.). Plant Cell Tiss Organ Cult 109, 201–211 (2012). https://doi.org/10.1007/s11240-011-0086-5

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  • DOI: https://doi.org/10.1007/s11240-011-0086-5

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