Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 103, Issue 1, pp 61–69 | Cite as

Development of a high throughput system for genetic transformation of olive (Olea europaea L.) plants

  • Rocío Torreblanca
  • Sergio Cerezo
  • Elena Palomo-Ríos
  • José A. Mercado
  • Fernando Pliego-Alfaro
Original Paper

Abstract

Olive tree, Olea europaea L., is one of the most commercially important oil crops. A reliable protocol for the genetic transformation of this species has been developed. Embryogenic calli were infected with different Agrobacterium tumefaciens strains harboring pBINUbiGUSint or pGUSINT binary plasmids. These vectors contain the nos-nptII and the uidA gene driven by the maize polyubiquitin Ubi1 and CaMV35S promoter, respectively. Inoculated explants were cocultured for 2 days, and later selected in the presence of 200 mg l−1 paromomycin. The inclusion of a 3 weeks selection period in liquid medium supplemented with 50 mg l−1 paromomycin was critical for elimination of chimaeric calli. Agrobacterium strain AGL1 containing pBINUbiGUSint plasmid yielded higher transformation frequencies than EHA105 or LBA4404. Globular somatic embryos (SE), 1–2 mm diameter, cultured in the selection medium in groups of three, were the best explant for transformation. Using this protocol, transformation frequencies in the range of 20–45%, based on the number of infected explants proliferating in the selection medium, have been obtained. More than 100 independent transgenic lines were generated, and 16 of them converted to plants. Transgenic plants were acclimated and grown in the greenhouse, being phenotypically similar to wild type plants. The uidA gene was strongly expressed in transgenic material during the in vitro regeneration phase; however, β-glucuronidase (GUS) activity in pBINUbiGUSint transgenic plants was neither detected in shoots growing in vitro nor in acclimated plants. Transgenic leaves, however, contained high levels of NPTII protein. By contrast, plants transformed with the pGUSINT plasmid showed a strong GUS activity in leaves. The protocol here described will allow the genetic improvement of this traditional crop.

Keywords

Genetic transformation Somatic embryogenesis Transgenic fruit trees 

Abbreviations

2iP

6-(Dimethylallylamino)purine

BA

6-Benzyladenine

ECO

Olive cyclic embryogenesis medium

ELISA

Enzyme-linked inmunosorbant assay

GUS

β-Glucuronidase

IBA

Indole-3-butyric acid

MS

Murashige and Skoog medium

OM

Olive medium

PCR

Polymerase chain reaction

PMSF

Phenylmethanesulfonyl fluoride

SE

Somatic embryos

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 Dr. Ricardo J. Ordás, Universidad de Oviedo, Spain, for providing the AGL1 strain with the pBINUbiGUSint plasmid, and Isabel M.G. Padilla for technical assistance.

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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Rocío Torreblanca
    • 1
    • 2
  • Sergio Cerezo
    • 1
    • 2
  • Elena Palomo-Ríos
    • 1
    • 2
  • José A. Mercado
    • 1
    • 2
  • Fernando Pliego-Alfaro
    • 1
    • 2
  1. 1.Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC)MálagaSpain
  2. 2.Departamento de Biología VegetalUniversidad de MálagaMálagaSpain

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