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

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. pCGUD1 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.

Trocas gasosas influenciam na morfogênese in vitro de duas cultivares de oliveira (Olea europaea L.)

“…Vários programas de melhoramento genético estão sendo executados em todo o mundo, visando ao aumento de produção, à melhoria do teor e da qualidade do óleo, à modificação do comportamento do crescimento vegetativo e à tolerância a vários estresses bióticos e abióticos (PÉREZ-BARRANCO et al, 2009). No entanto, a escassez de pesquisas com espécies do gênero Olea sp.…”

Response, Tolerance and Adaptation to Abiotic Stress of Olive, Grapevine and Chestnut in the Mediterranean Region: Role of Abscisic Acid, Nitric Oxide and MicroRNAs

“…Although there is widespread belief that particle bombardment generates large, multi-copy loci prone to instability and silencing, refinements of the technology to produce clean transgene loci have demonstrated clearly that this is not the case, and that particle bombardment has many advantages for the production of commercial transgenic plants that perform well in the field and comply with all relevant regulatory processes (Altpeter et al, 2005). Protocol for olive somatic embryos genetic transformation by particle bombardment has been recently established (Perez-Barranco et al, 2009). The induction of stress tolerance through engineering for over-expression of transcriptional factor genes is emerging as an attractive proposition (Yang et al, 2009).…”

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

“…To develop a transformation system for olive SE, we previously evaluated the tolerance of these explants to different aminoglycoside antibiotics for nptII selection (Pérez- Barranco et al 2009). Olive SE showed a relatively high tolerance to antibiotics when growing in solid medium, consequently high levels of paromomycin or kanamycin, ca.…”

“…However, studies dealing with the effect of critical factors of the Agrobacterium transformation procedure, such as Agrobacterium strain or selection procedure, on the transformation efficiency are lacking. Using a different approach, Lambardi et al (1999) and Pérez-Barranco et al (2009) evaluated the particle bombardment system for the transformation of olive somatic embryos. In both cases, transgenic calli showing GUS expression were obtained but they were unable to regenerate plants.…”

“…Picual, has been developed by Pérez-Barranco et al (2007). Furthermore, the tolerance of somatic embryos (SE) to different aminoglycoside antibiotics for nptII selection has been assessed (Pérez-Barranco et al 2009). Recently, a preliminary work on the transformation of this material using Agrobacterium tumefaciens showed that embryogenic masses can be successfully transformed through this system ).…”