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

Abstract

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.

A possible role for flowering locus T‐encoding genes in interpreting environmental and internal cues affecting olive (Olea europaea L.) flower induction

“…Based on a relatively new olive transformation protocol (Torreblanca et al . ), we produced transgenic olive plants overexpressing the MtFTa1 gene. Sixteen independent transgenic lines were recovered, yielding a 2.56% transformation rate.…”

High-efficiency Agrobacterium-mediated transformation in Quercus robur: selection by use of a temporary immersion system and assessment by quantitative PCR

“…Thus, paramomycin decreased the percentage of transgenic olive embryos that underwent maturation (Torreblanca et al 2010), and kanamycin inhibited embryo development in transgenic cells of papaya (Kung et al 2010) and also decreased the germination rate of transgenic white spruce embryos (Le et al 2001) and hampered growth, differentiation, and rooting in transgenic black pepper (Varghese and Bhat 2011). Plant susceptibility to antibiotics varies widely among species, genotypes and plant tissues (Padilla and Burgos 2010), and there is no general rule about including selective agents in media when regenerating transgenic woody plants.…”

“…In citrus (Dutt and Grosser 2010), sandalwood (Shekhawat et al 2008), and yew (Zhang et al 2011), cell suspension cultures have been used to generate more responsive target explants, although putatively transformed explants were selected on semi-solid medium. Cell suspension cultures have been used during the selection phase in mango (Mathews et al 1992), avocado (Cruz-Hernandez et al 1998;Raharjo et al 2008) and olive (Torreblanca et al 2010), although no clear comparison was made with selection on semi-solid medium. An efficient transformation protocol involving the use of embryogenic suspension cultures throughout the whole procedure has been established for American chestnut (Fagaceae) (Andrade et al 2009).…”

“…However, quantification of the transgene copy number is necessary because the spectrum of transgenic insertions is occasionally quite large, with reports of up to three copies in olive (Torreblanca et al 2010), four copies in cork oak (Álvarez et al 2009), avocado (Palomo-Ríos et al 2012, and plum (Wang et al 2013), and as many as 10 copies in hybrid poplar (Yevtushenko and Misra 2010). Insertion of a large number of transgene copies can lead to unpredictable performance, ranging from excessive to undetectable expression levels (Gelvin 2003).…”

“…Plant susceptibility to antibiotics varies widely among species, genotypes and plant tissues (Padilla and Burgos 2010), and there is no general rule about including selective agents in media when regenerating transgenic woody plants. Torreblanca et al (2010) maintained the selection pressure during the entire regeneration process (including embryo maturation and germination), to prevent the formation of chimaeric olive transgenic embryos, and kanamycin has been used both in proliferation and rooting medium of transgenic green ash (Du and Pijut 2009) and poplar (Yevtushenko and Misra 2010). However, transgenic embryos of other woody plants have also been matured and germinated in the absence of selective agents, as reported for avocado (Palomo-Ríos et al 2012), rubber tree (Leclercq et al 2010), and other species of Fagaceae such as European and American chestnut , Andrade et al 2009) and cork oak Ordás 2007, Álvarez et al 2009).…”