Olive trees are one of the oldest and most economically relevant fruit trees and oilseed crops in the Mediterranean region, being most of the cultivars used for olive oil production [1
]. Two olive forms can be found in the Mediterranean basin, the wild olive (Olea europaea
L. ssp. europaea
) and the cultivated olive (Olea europaea
L. ssp europaea
), and, in many regions, the two forms coexist and are compatible [2
]. There are currently more than 2000 olive cultivars and this genetic diversity is the main factor contributing to the olive oil singularity of each country and region [4
]. Besides the organoleptic characteristics of fruits and oils produced, genetic diversity also represents a source of genetic information that could be further explored in breeding programs focused on specific agronomical traits [6
]. Development of new cultivars must be focused on solving issues such as modification of growth habit, development of self-fertile plants to increase yield, development of totally self-sterile plants for conventional breeding, increasing fruit oil content and quality, increasing abiotic stress tolerance, increasing disease and pest tolerance, and production of plants with a higher adventitious rooting ability [7
Olive transcriptomics, proteomics, and metabolomics [9
] have begun to provide indications of candidate genes that may be key to the definition of an interesting phenotype. However, data from these high throughput approaches only has physiological value if the functionality is confirmed, usually by knockout or overexpression of target genes [13
]. Functional validation requires the establishment of in vitro protocols for plant regeneration, where cells integrating the foreign DNA can regenerate an entirely new plant. Of the different strategies that could be followed for in vitro regeneration of a new plant, somatic embryogenesis (SE) is the most suitable tool for genetic transformation protocols. Somatic embryogenesis is recognized as an efficient morphogenic response upon external stress stimulus based on molecular and metabolic cell reprogramming that covers typically phases of dedifferentiation and de novo-differentiation [16
]. The selection of this regeneration system is mainly based on high proliferation rates [17
] and frequent single-cell origin of the differentiated somatic embryos, which avoids the problem of chimeras [8
Attempts to induce SE in olive started in the 1980s when Rugini and Tarini [23
] used seedling-derived roots to induce somatic embryos. Later, the first successful protocols were achieved by Rugini [24
] for cultivars ‘Frantoio’, ‘Moraiolo’, ‘Leccino’, and ‘Dolce Agogia’. Further research, mainly based on juvenile tissues of immature and mature zygotic embryos or petioles taken from in vitro seedlings as initial explants [25
] allowed the establishment of some effective protocols (see Cardoso et al. [32
] and [33
] for details). Recalcitrance is associated with the use of adult plant tissues with exceptions known for cvs. ‘Canino’, ‘Moraiolo’, ‘Chetoui’,‘Dahbia’, and ‘Picual’, for which were used petioles/leaf tissues taken from in vitro growing plants, to successfully induce SE and further embryos differentiation [34
]. Embryos conversion, known as a key step in an efficient SE protocol, was limited to cv. ‘Canino’ and ‘Moraiolo’ [34
]. Successful reports have also been described in some wild olive genotypes. The first report is known from Capelo and co-authors [38
], which demonstrated the possibility to use petioles/leaf tissues taken from plants established under greenhouse conditions (no juvenile or rejuvenated material), to induce SE response with efficient differentiation of somatic embryos. More recently, Narváez and co-authors [39
] described a protocol focused on different olive wild genotypes differing in their response to defoliating Verticillium dahlia
, in which was stated the use of shoot apex collected from in vitro plantlets as the most appropriated tissue to induce SE and further efficient embryos conversion.
The efficiency of SE induction depends on several factors with a complex interaction among them. The main factors affecting SE efficiency are the genotype of the donor plant, the type of explant taken for the establishment of in vitro cultures, the development and physiological stage, the growth conditions (mainly photoperiod and temperature), and the chemical composition of the culture media (including type and concentration of growth regulators) [40
]. The genotype strongly affects the ability of a tissue to dedifferentiate and de novo acquire meristematic competence and to differentiate embryogenic structures [39
]. Individual genotypes within the same species vary greatly in embryogenic capacity, reflecting substantial differences in the ability to activate key elements for the achievement of embryogenic competence [41
] which shows the need to develop improved methodologies for each genotype. Results reported by Narváez and co-authors [39
] emphasize this dependence, showing SE induction efficiency in two olive wild genotypes from the four initially considered, and embryos conversion from a single one.
Besides the genotype, the developmental stage and age of the explants used as start material, are key factors determining the success of SE induction. Several reports on woody plant species, Vitis vinifera
] Picea abies
], Prunus incisa
], Pinus radiata
], and Eucalyptus globulus
], emphasize the effect of explant developmental stage on embryogenesis efficiency.
Concerning the culture media formulations, the OM (Olive medium) [49
] medium with 1 g L−1
casein hydrolysate and devoid of glutamine or the MS (Murashige and Skoog) [50
] basal salts, are generally used in olive SE for both, juvenile and adult initial explants. High auxin/cytokinin ratios provided the best results for the culture induction phase [25
], while culture media lacking growth regulators, or with a low auxin concentration, were used for embryo differentiation and development [28
Somatic embryogenesis has not been routinely and widely used in the propagation of Olea
spp. where few cultivars have been used to collect plant material to induce SE response (see review in Cardoso et al. [32
] and Sánchez-Romero [33
]). The use of mature zygotic embryos as initial explants to attain this goal is far from optimal, but the information acquired from those trials is usually fundamental to obtain results when adult explants of a selected genotype are used. The cv. ‘Galega vulgar’ chosen for this study is recalcitrant for adventitious root formation when semi-hardwood cuttings are used, and previous work developed by our research group has been focused on understanding the mechanisms underlying that morphogenic process by following different omic approaches [53
]. Data achieved from the different omic platforms need functional validation as a step forward on this research topic, which required prior development of an effective protocol for SE. The present research reveals the establishment of an efficient protocol that will make available plant material to be used in genetic transformation and gene editing of olive varieties.
3. Materials and Methods
3.1. Plant Material
The fruits used for seed extraction were provided by Instituto Nacional de Investigação Agrária e Veterinária and were collected in Elvas, Portugal, in the Coleção Nacional de Referência de Cultivares de Oliveira, from 8-year-old trees of cv. ‘Galega vulgar’. The harvest was made at full ripeness, the pulp was removed manually, and after washing the seeds were kept in the cold (4 ± 1 °C) for 3 months to break the dormancy of the embryo. The endocarp was then broken with a manual press and the seeds isolated and placed in sterile water (autoclaved at 121 °C for 20 min) for 42 h in the dark at 24 ± 1 °C, before the surface disinfection process.
3.2. Seed Surface Disinfection and Explant Preparation
Seed disinfection consisted of the first wash with 70% (v/v) ethanol solution for 2 min followed by a wash with sterile bi-distilled water. Water was replaced by calcium hypochlorite solution (10% w/v) containing 0.1% Tween-20 (v/v), and the flasks were closed and kept under continuous agitation (~150 rpm) for 20 min. Finally, the solution was removed and seeds were rinsed three times with sterile bi-distilled water. In aseptic conditions, the radicles and cotyledons were excised from the interior of seeds and used as initial explants. The radicles were placed in culture as a whole while the cotyledons were separated and cut into distal and proximal portions before inoculation in culture media.
3.3. Embryogenesis Induction and Expression
For the embryogenesis induction phase, the explants were placed into Petri dishes (7 cm diameter) containing 25 ml of OMc culture medium [78
] supplemented with 2.5 µM 6-dimethylallylamino-purine (2iP), 25 µM indole-3-butyric acid (IBA) [55
] and jelled with 7 g L−1
Agar-Powder (VWR, Lisboa, Portugal). Cultures were maintained for 21 days at 25 ± 1 °C with a photoperiod of 16 h and a light intensity of 40–45 μmol m−2
, or alternatively in the dark.
After the induction phase, explants were transferred to hormone-free OMc culture medium to promote the differentiation of the embryogenic structures (expression phase). Thirty days after inoculation explants were subcultivated on OMc culture medium, and cultures were kept under the same growth conditions as described above. Two subcultures were considered and for the second one, only calli from radicles were used.
3.4. Cyclic Embryogenesis
After two sub-cultures on expression medium, all embryogenic calli were transferred to olive ECO medium to induce cyclic embryogenesis [30
]. The formulation of the ECO medium is based on the formulation of modified olive medium for SE (OMe) [78
] containing ¼ OM macroelements, ¼ MS microelements, ½ OM vitamins, 50 mg L−1
myo-inositol, 20 g L−1
sucrose, 550 mg L−1 l
-glutamine, and supplemented with 0.5 μM 2iP, 0.44 μM 6-benzyladenine (BA), 0.23 μM IBA, 1 g L−1
casein hydrolysate and 0.42 mM cefotaxime (sterilized by filtration as proposed by Rugini and Caricato [34
]). The culture medium was jelled with 2.5 g L−1
Cultures were maintained during eight months by calli sub-culture into fresh culture medium every month. The culture conditions were initially maintained as previously described for the expression phase, but after the second sub-culture, only the 16 h photoperiod was maintained.
3.5. Recover of Calli Embryogenic Capacity
To recover embryogenic capacity, all calli maintained in ECO medium for eight sub-cultures were transferred to the induction medium previously described (OMc). Cultures were kept at 25 ± 1 °C with a photoperiod of 16 h and a light intensity of 40–45 μmol m−2 s−1. After 21 days, calli were transferred back to ECO medium where they remained for 3 sub-cultures.
3.6. Embryos Conversion and Plants Acclimatation
To promote embryos conversion, embryos larger than 3 mm were removed from the embryogenic calli following the procedure previously described by Bradaï et al. [69
], and further cultivated in 180 mL glass bottles containing OMc medium devoid growth regulators. Cultures were kept at 25 ± 1 °C with a 16 h photoperiod and 40–45 μmol m−2
of light intensity.
Plantlets were maintained under in vitro conditions until the shoots reached about 10 cm in height. Young plants were then removed from the glass bottles and transferred to polypropylene honeycomb trays with 28 wells containing approximately 200 mL of substrate per well. The substrate consisted of a mixture of sand, perlite, and peat in the proportion of 1:1:3 (v/v).
To avoid dehydration, the trays were placed in stalls with a transparent plastic polyethylene cover to maintain high relative humidity. Plants were maintained under controlled conditions in a plant growth chamber with 24 °C /22 °C day/night temperature, 60% humidity, 16 h photoperiod and light intensity of 90 μmol m−2 s−1. After 15 days, the plastic cover was removed from the stalls and the plants remained for another 15 days under the same conditions.
Finally, the plants were transferred to 2 L pots containing a substrate with similar composition and transferred to a greenhouse.
3.7. Experimental Design and Statistical Analysis
For the experiments on the induction and expression of embryogenesis, three explant types (radicles, distal and proximal cotyledons) and two light conditions (16 h and 0 h photoperiod) were tested. The trial followed a complete factorial design, with each Petri dish having 10 explants acting as one replicate and with nine replications at least. Data on calli formation rates, development of adventitious structures, and the number of formed embryos were recorded.
For the experiments on cyclic embryogenesis, calli from radicles were transferred into ECO culture medium. Two photoperiod regimes were considered: 16 h and 0 h (dark). The trial also followed a complete factorial design, with 7 calli inoculated per Petri dish that acted as one replicate. The number of replicates varied for each culture condition tested, but at least 10 replications were used.
All data were tested for normality (by Shapiro-Wilk test) and submitted to analysis of variance (ANOVA) followed by Fisher’s (LSD) posthoc test. Significant differences were recorded for p ≤ 0.05. Values in percentage were transformed by arcsine of the square root before analysis.