Studies on genetic transformation of olive (Olea europaea L.) somatic embryos: I. Evaluation of different aminoglycoside antibiotics for nptII selection; II. Transient transformation via particle bombardment

  • Gema Pérez-Barranco
  • Rocío Torreblanca
  • Isabel M. G. Padilla
  • Carolina Sánchez-Romero
  • Fernando Pliego-Alfaro
  • José A. Mercado
Original Paper

Abstract

As a first step to the establishment of a genetic transformation protocol for olive somatic embryos obtained from the seeds of cv. ‘Picual’, the efficiencies of different aminoglycoside antibiotics as selective agents to be used with the nptII marker gene, and the particle bombardment technique for transient transformation have been evaluated. Among the three antibiotics tested, paromomycin and kanamycin showed a similar inhibitory effect and, at 200 mg l−1, both of them impaired callus growth after 8 weeks of culture. However, when isolated embryos were cultured in the presence of these antibiotics, a 20% of the embryos still remained viable at 400 mg l−1. Neomycin was discarded as a selective agent since it showed only a moderate toxic effect. Contrary to solid medium, when olive callus was cultured in liquid medium supplemented with different paromomycin concentrations for 3 weeks, the callus growth was impaired at the lowest antibiotic concentration, 3 mg l−1. Best conditions for transient transformation of olive callus using PDS-1000/He system were a 6 cm target distance and a 900 psi bombardment pressure. pCGU∆1 plasmid, containing the gus gene under the control of sunflower ubiquitin promoter yielded a significantly higher number of gus expression areas per bombarded explant than pGUSINT or pJGUS5 plasmids, where the gus gene is driven by CaMV35S promoter or CaMV35S with enhancer, respectively. Almost 45% of bombarded explants showed gus expression 12 weeks after bombardment.

Keywords

Biolistics Genetic transformation Olive Particle bombardment Selectable marker Somatic embryos 

Abbreviations

DKW

Driver and Kuniyuki medium

ECO

Olive cyclic embryogenesis medium

MS

Murashige and Skoog medium

OEC

Olive embryogenic calli

OMe

Olive medium

Notes

Acknowledgments

This research was funded by Dirección General de Investigación y Formación Agraria y Pesquera, Consejería de Agricultura y Pesca, Junta de Andalucía (project CAO00-018-C7-5) and Fundación Genoma España (Oleagen project). The authors thank to Dr. Luc-Henri Tessier for providing the pCGU∆1 plasmid.

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Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Gema Pérez-Barranco
    • 1
  • Rocío Torreblanca
    • 1
  • Isabel M. G. Padilla
    • 1
  • Carolina Sánchez-Romero
    • 1
  • Fernando Pliego-Alfaro
    • 1
  • José A. Mercado
    • 1
  1. 1.Departamento de Biología VegetalUniversidad de MálagaMálagaSpain

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