Agrobacterium-Mediated Transformation of Olive (Olea Europaea L.) Embryogenic Cultures

Olive (Olea europaea L.) is one of the most important oil crops in the Mediterranean basin.Biotechnological improvement of this species is hampered by the recalcitrant nature of olive tissue to regenerate in vitro. In previous investigations, our group has developed a reliable Agrobacterium-mediated transformation protocol using olive somatic embryos as explants (Torreblanca et al., 2010). Embryogenic cultures derived from radicles of matured zygotic embryos are infected with Agrobacterium tumefaciens, AGL1 strain, containing a binary plasmid with the gene of interest and the nptII selection gene. After a meticulous selection procedure, carried out using solid and liquid media supplemented with paromomycin, the putative transformed lines are established. A preliminary confirmation of their transgenic nature is carried out through PCR amplification. Afterwards, plants can be obtained through an efficient regeneration protocol, whose main characteristics are the use of a low-ionic-strength mineral formulation, a phase in liquid medium for synchronization of cultures and the use of semi-permeable cellulose acetate membranes for embryo maturation (Cerezo et al., 2011). Final confirmation of transgene insertion is carried out through Southern or Northern analysis using leaf samples of regenerated plants.

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Generation and Selection of Transgenic Olive Plants

et al. 2017

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Genome Modification Approaches to Improve Performance, Quality, and Stress Tolerance of Important Mediterranean Fruit Species (Olea europaea L., Vitis vinifera L., and Quercus suber L.)

Cardoso

Figueiredo

Serrazina

et al. 2019

Advances in Plant Transgenics: Methods and Applications

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

Torreblanca

Cerezo

Palomo-Ríos

et al. 2010

Plant Cell Tiss Organ Cult

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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, b-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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Agrobacterium-Mediated Transformation of Olive (Olea Europaea L.) Embryogenic Cultures

Cited by 5 publications

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Generation and Selection of Transgenic Olive Plants

Generation and Selection of Transgenic Olive Plants

Genome Modification Approaches to Improve Performance, Quality, and Stress Tolerance of Important Mediterranean Fruit Species (Olea europaea L., Vitis vinifera L., and Quercus suber L.)

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

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