Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 109, Issue 2, pp 201–211

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

  • Elena Palomo-Ríos
  • Araceli Barceló-Muñoz
  • José A. Mercado
  • Fernando Pliego-Alfaro
Original Paper

DOI: 10.1007/s11240-011-0086-5

Cite this article as:
Palomo-Ríos, E., Barceló-Muñoz, A., Mercado, J.A. et al. Plant Cell Tiss Organ Cult (2012) 109: 201. doi:10.1007/s11240-011-0086-5

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.

Keywords

Genetic transformationSomatic embryogenesisTransgenic fruit trees

Abbreviations

BA

6-Benzyladenine

GA3

Gibberellic acid

MS

Murashige and Skoog medium

MSP

Embryogenic avocado culture medium

SE

Somatic embryo

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Elena Palomo-Ríos
    • 1
  • Araceli Barceló-Muñoz
    • 2
  • José A. Mercado
    • 1
  • Fernando Pliego-Alfaro
    • 1
  1. 1.Departamento de Biología Vegetal, Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora” (IHSM-UMA-CSIC)Universidad de MálagaMálagaSpain
  2. 2.IFAPA Centro de ChurrianaChurriana, MálagaSpain