Studies on genetic transformation of olive (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) somatic embryos: I. Evaluation of different aminoglycoside antibiotics for <Emphasis Type="Italic">nptII</Emphasis> selection; II. Transient transformation via particle bombardment |
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Authors: | Gema Pérez-Barranco Rocío Torreblanca Isabel M G Padilla Carolina Sánchez-Romero Fernando Pliego-Alfaro José A Mercado |
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Institution: | (1) Departamento de Biología Vegetal, Universidad de Málaga, 29071 Málaga, Spain |
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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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Keywords: | Biolistics Genetic transformation Olive Particle bombardment Selectable marker Somatic embryos |
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