Articles
GENETIC TRANSFORMATION OF OLIVE SOMATIC EMBRYOS THROUGH BIOLISTICS
Article number
738_59
Pages
473 – 477
Language
English
Abstract
Considerable effort is being directed at the recovery of superior olive cultivars through conventional breeding.
However, the development of biotechnological tools could speed up the development of new cultivars with superior traits, e.g., enhanced disease resistance.
In this investigation, attempts have been made to develop a protocol that would allow genetic transformation of juvenile material from the cultivar Picual. Initially, embryogenic cultures were established from mature seeds using radicle segments as explants.
The effects of two different mineral formulations OMc vs.
OMe on growth and maintenance of embryogenic cultures were evaluated.
Results showed that although higher growth rate could be obtained with the OMc mineral formulation, a more organized growth occurred with the formulation with lower macroelements content (OMe). Moreover, culturing the embryogenic cultures for 3 weeks in liquid OMe medium followed by subculture of the fine fraction derived from the resulting suspension on a medium without growth regulators but supplemented with activated charcoal, allowed the development of white-opaque embryos, some of which could be converted into plants.
For transformation of embryogenic cultures via particle bombardment, 3 different plasmids containing the GUS marker gene under the control of different promoters were used: pGUSINT (including the 35S promoter), pJGUS5 (35S promoter with an enhancer) and pCGUΔ1 (sunflower ubiquitin promoter). The highest number of blue spots was obtained with the ubiquitin promoter.
Hence, it has been selected for transformation of this cultivar.
Experiments are now in progress to establish the most adequate conditions for selection of fully transformed somatic embryos.
However, the development of biotechnological tools could speed up the development of new cultivars with superior traits, e.g., enhanced disease resistance.
In this investigation, attempts have been made to develop a protocol that would allow genetic transformation of juvenile material from the cultivar Picual. Initially, embryogenic cultures were established from mature seeds using radicle segments as explants.
The effects of two different mineral formulations OMc vs.
OMe on growth and maintenance of embryogenic cultures were evaluated.
Results showed that although higher growth rate could be obtained with the OMc mineral formulation, a more organized growth occurred with the formulation with lower macroelements content (OMe). Moreover, culturing the embryogenic cultures for 3 weeks in liquid OMe medium followed by subculture of the fine fraction derived from the resulting suspension on a medium without growth regulators but supplemented with activated charcoal, allowed the development of white-opaque embryos, some of which could be converted into plants.
For transformation of embryogenic cultures via particle bombardment, 3 different plasmids containing the GUS marker gene under the control of different promoters were used: pGUSINT (including the 35S promoter), pJGUS5 (35S promoter with an enhancer) and pCGUΔ1 (sunflower ubiquitin promoter). The highest number of blue spots was obtained with the ubiquitin promoter.
Hence, it has been selected for transformation of this cultivar.
Experiments are now in progress to establish the most adequate conditions for selection of fully transformed somatic embryos.
Authors
G. Pérez-Barranco, J.A. Mercado, F. Pliego-Alfaro, C. Sánchez-Romero
Keywords
Olea europaea, genetic engineering, somatic embryo, regeneration, particle gun
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