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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

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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.

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Abbreviations

DKW:

Driver and Kuniyuki medium

ECO:

Olive cyclic embryogenesis medium

MS:

Murashige and Skoog medium

OEC:

Olive embryogenic calli

OMe:

Olive medium

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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 to Dr. Luc-Henri Tessier for providing the pCGU∆1 plasmid.

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Authors and Affiliations

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

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

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  1. Gema Pérez-Barranco
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  2. Rocío Torreblanca
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  3. Isabel M. G. Padilla
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  4. Carolina Sánchez-Romero
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  5. Fernando Pliego-Alfaro
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  6. José A. Mercado
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Correspondence to José A. Mercado.

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Pérez-Barranco, G., Torreblanca, R., Padilla, I.M.G. et al. 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. Plant Cell Tiss Organ Cult 97, 243–251 (2009). https://doi.org/10.1007/s11240-009-9520-3

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