Articles
AGROBACTERIUM-MEDIATED TRANSFORMATION OF OLIVE (OLEA EUROPAEA L.) EMBRYOGENIC CULTURES
Article number
839_50
Pages
387 – 391
Language
English
Abstract
Olive (Olea europaea L.) breeding programs are hampered by the long juvenile period shown by this species.
Genetic transformation would allow incorporation of interesting agronomical traits in much shorter times.
Our group has developed an Agrobacterium-mediated transformation protocol for this species using embryogenic cultures as explants.
In this protocol, somatic embryos derived from radicle explants are co-cultured with A. tumefaciens strain AGL1 containing the binary vector pBINUbiGUSint with the nptII gene for kanamycin selection and uidA gene as reporter.
After 12 weeks of selection in the presence of 200 mg/L paromomycin, an average transformation rate of 17.5±5.2 was obtained, based on the number of infected explants proliferating in the selection medium.
Almost 32% of the paramomycin resistant calli showed GUS activity.
Somatic embryos from different transgenic lines were maturated and germinated under a constant selection pressure of 200 mg/L paramomycin, and the shoots obtained proliferated in DKW medium.
Putative transgenic shoots were challenged to proliferate in the presence of increasing concentrations of paramomycin.
Control shoots did not grow at low antibiotic concentration (25 mg/L). However, transgenic shoots showed good proliferation rates at 25 and 50 mg/L paramomycin, and shoot length was only reduced to 62% compared to the length of control shoots at 100 mg/L paramomycin.
Transgenic olive plants have been acclimated in the greenhouse and are currently being analyzed at the molecular level.
Genetic transformation would allow incorporation of interesting agronomical traits in much shorter times.
Our group has developed an Agrobacterium-mediated transformation protocol for this species using embryogenic cultures as explants.
In this protocol, somatic embryos derived from radicle explants are co-cultured with A. tumefaciens strain AGL1 containing the binary vector pBINUbiGUSint with the nptII gene for kanamycin selection and uidA gene as reporter.
After 12 weeks of selection in the presence of 200 mg/L paromomycin, an average transformation rate of 17.5±5.2 was obtained, based on the number of infected explants proliferating in the selection medium.
Almost 32% of the paramomycin resistant calli showed GUS activity.
Somatic embryos from different transgenic lines were maturated and germinated under a constant selection pressure of 200 mg/L paramomycin, and the shoots obtained proliferated in DKW medium.
Putative transgenic shoots were challenged to proliferate in the presence of increasing concentrations of paramomycin.
Control shoots did not grow at low antibiotic concentration (25 mg/L). However, transgenic shoots showed good proliferation rates at 25 and 50 mg/L paramomycin, and shoot length was only reduced to 62% compared to the length of control shoots at 100 mg/L paramomycin.
Transgenic olive plants have been acclimated in the greenhouse and are currently being analyzed at the molecular level.
Authors
R. Torreblanca, E. Palomo-Ríos, S. Cerezo, J.A. Mercado, F. Pliego-Alfaro
Keywords
genetic transformation, somatic embryogenesis, transgenic woody plants
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