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

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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.

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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

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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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Authors and Affiliations

  1. Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Málaga, Spain

    Rocío Torreblanca, Sergio Cerezo, Elena Palomo-Ríos, José A. Mercado & Fernando Pliego-Alfaro

  2. Departamento de Biología Vegetal, Universidad de Málaga, 29071, Málaga, Spain

    Rocío Torreblanca, Sergio Cerezo, Elena Palomo-Ríos, José A. Mercado & Fernando Pliego-Alfaro

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  1. Rocío Torreblanca
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  2. Sergio Cerezo
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  3. Elena Palomo-Ríos
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  4. José A. Mercado
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  5. Fernando Pliego-Alfaro
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Corresponding author

Correspondence to José A. Mercado.

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Rocío Torreblanca and Sergio Cerezo contributed equally to the work.

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Torreblanca, R., Cerezo, S., Palomo-Ríos, E. et al. Development of a high throughput system for genetic transformation of olive (Olea europaea L.) plants. Plant Cell Tiss Organ Cult 103, 61–69 (2010). https://doi.org/10.1007/s11240-010-9754-0

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  • DOI: https://doi.org/10.1007/s11240-010-9754-0

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