The effect of temperature on the duration of the effective pollination period in ‘Oblica’ olive (Olea europaea) cultivar

The effective pollination period (EPP) was determined for ‘Oblica’ olive trees in response to sequential pollinations at anthesis and at increasing days after. The role played by each of three components of the EPP, namely the stigmatic receptivity, pollen tube growth and ovule longevity was examined and the duration of the EPP under different environmental conditions was calculated. An increase in temperature in the field was caused by enclosing bearing trees within polyethylene cages. The duration of the EPP varied between years and environmental conditions. Initial fruit set (IFS) declined gradually in response to sequential cross‐pollination. The significant decrease with respect to the maximum levels of IFS occurred 5 days after anthesis (DAA) in 2007 and between 4 and 5 DAA in 2010 at both environmental conditions. However, the fruit set decrease with sequential pollinations was sharper for the trees inside the cages than for trees growing at the open air, suggesting that high temperatures induce earlier and more pronounced decrease in flower fertility. The stigma receptivity was quite extended decreasing slowly with time. In 2010, the stigma receptivity was prolonged beyond sampling date (7 DAA) in both environmental conditions. Stigmas first lost the capacity to support pollen germination and later their capacity to adhere pollen grains. Pollen tube growth occurred rapidly and fertilization took place 1 or 2 days after pollination. Cross‐pollination resulted with higher fertilization levels, whereas fertilization was reduced and delayed under self‐pollination. Enclosing the trees within the cages had small effect on cross‐pollination but impaired the self‐pollination which resulted with reduced levels of fertilization. The ovule longevity was prolonged beyond the end of sampling in both years and conditions (beyond 9 DAA in 2009 or 7 DAA in 2010). Discrepancy between the estimates of the duration of the EPP based on analyses of its components and those indicated by the declines in fruit set from delayed pollination suggests that a different component of the pistil, the style, could have the critical role in determining the duration of the EPP.     

In vitro plant regeneration from cotyledon fragments of the olive tree (Olea europaea L)

Olive tree (Olea europaea L) plantlets were regenerated from cotyledon segment calli on a modified olive medium (OMc) supplemented with 2iP alone or in combination with indol-3-butyric acid (IBA). Cell division in the explants was initially induced on OMc medium with high auxin (5 mg·l⁻¹ of IBA) and low cytokinin (0.2–0.5 mg·l⁻¹ of 2-isopentenyladenine (2iP) or zeatin riboside) content. Calli were then transferred to the same medium with different levels of IBA and/or 2iP in order to promote further development and obtain calli bearing either roots or shoots. On OMc medium, 1 mg·l⁻¹ of IBA induced the maximum of rooting, while shoot induction was greater when the medium was supplemented with 4 mg·l⁻¹ of 2iP. Shoot induction mainly occurred from calli of cotyledon fragments proximal to the embryo axes. Whole plantlets were obtained when the regenerated shoots were stimulated to produce adventitious roots on OMr medium with 1 mg·l⁻¹ of IBA or naphthaleneacetic acid (NAA). After root elongation on OMe medium without auxin, plantlets were transfered to peat and soil conditions where about 75–80% were able to survive. A certain variability was detected between regenerated olive plants.     

First evidence of a retrotransposon-like element in olive (Olea europaea): implications in plant variety identification by SCAR-marker development

When developing SCARs by sequencing RAPD markers useful for olive variety identification, one RAPD sequence of 407 bp has been identified that shows significant DNA homology with more than 160 retrotransposon-like sequences. A generally coherent phylogenetic tree has been constructed based on the homologous retrotransposon-like sequences, reflecting genetic distances in 35 species belonging to eight kingdoms. The implications of this finding in the development of SCAR markers are discussed. In addition, specific oligonucleotide primers have been developed to amplify the DNA region and have a practical application as an internal amplification control in SCAR-based multiplexed PCRs. To our knowledge, this is the first report of a retrotransposon-like element in olive. Received: 31 July 2000 / Accepted: 22 September 2000     

Vegetative growth response of young olive trees (Olea europaea L., cv. Arbequina) to soil salinity and waterlogging

High-density olive orchards are increasing around the world, many of which may be potentially affected by salinity and waterlogging (hypoxia), two important stresses common in irrigated fields in arid and semi-arid climates. However, the response of olive to these stresses under field conditions is not well established. Therefore, our objective was to evaluate the vegetative growth response of young olive trees (Olea europaea L., cv. Arbequina) grown in a spatially variable waterlogged, saline-sodic field. We monitored the growth in trunk diameter of 341, 3-year-old olives between September 1999 and September 2000. Field contour maps were developed delineating soil salinity (ECa), relative ground elevation (RGE) and water table depth (WTD). Soil samples were also collected and analyzed for ECe and SARe in order to characterize the salinity and sodicity profiles and develop the ECa-ECe calibration equation. The infiltration rate (IR) of the crusted and uncrusted soil and the penetration resistance (PR) were also measured. The field was characterized by spatially variable ECe (2–15 dS m^–1), SARe (3–40), RGE (–4 to +4 cm) and WTD (0.5–1.9 m, with corresponding ground water EC values between 12 and 6 dS m^–1). Steady-state IR of crusted soil was only 7% of the uncrusted soil. Since the field was heavily irrigated by flooding, waterlogging conditions were related to low RGE values. Soil salinity was negatively correlated (R ² = 0.83, P<0.001) with RGE (ponded water) and WTD (upward flux), due to the evapo-concentration of water and salts at the soil surface. Thus, inverted salinity profiles developed in high salinity areas. Fifty-five percent of the olives were dead 3.5 years after planted, and most of them were located in areas of high ECe (> 10 dS m^–1), low RGE (< – 1.5 cm) and low WTD (< 1.2 m). The surviving trees had vegetative salinity tolerance values of ECe threshold = 4 dS m^–1 and slope = –12% (i.e., percent decline per unit increase in ECe above the treshold), indicating that the Arbequina olive is moderately tolerant to salinity. The RGE and WTD thresholds for olive's survival were > 0.1 cm and > 1.6 m, respectively. Thus, very small changes in ground elevation had a significant effect on olive's survival or death. The coupled effects of salinity and waterlogging (hypoxia) stresses were most detrimental for olive's growth.     

Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination

Background and Aims

A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized.

Methods

The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods.

Key Results

Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles.

Conclusions

In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma.     

Stomatal and photosynthetic responses of olive (Olea europaea L.) leaves to water deficits

The leaf gas exchange of mature olive trees (Olea europaea L.) was characterized over a wide range of water deficits in the field during 1998, in Cordoba, Spain. Leaf photosynthesis (A) and stomatal conductance (g_l) responded diurnally and seasonally to variations in tree water status and evaporative demand. In the absence of water stress, A and g_l were generally high during autumn and low in days of high vapour pressure deficits (VPD). Leaf A varied between 0 and 2 µmol m^?2 s^?1 under severe water deficits that lowered the stem water potential (Ψ_x) to ?8·0 MPa, but recovered rapidly following rehydration. Transpiration efficiency (TE) was curvilinearly related to VPD and not influenced by water deficits except in cases of severe water stress, where low TE values were observed at Ψ_x below ?4 MPa. Three models of leaf conductance were calibrated and validated with the experimental data; two were based on the model proposed by Leuning (L) and the other was derived from the widely used Jarvis (J) model. The L models performed better than the J model in two validation tests. The scatter of the predictions and the limited accuracy of all three models suggest that, in addition to the physiological and environmental variables considered, there are additional endogenous factors influencing the g_l of olive leaves.     

Development of SCAR markers in olive (Olea europaea) by direct sequencing of RAPD products: applications in olive germplasm evaluation and mapping

RAPD markers generated by mixtures of two different 10-mer primers were developed for eight different olive cultivars used as parental lines in olive-breeding programs. Two RAPD bands were converted into dominant SCAR markers by direct sequencing of the RAPD products, avoiding the costly and time-consuming cloning step. The SCARs generated have maintained the original RAPD polymorphism among the cultivars and segregated according to Mendelian inheritance. Preliminary results suggest the use of the SCAR SCOeMS-2 for the marker-assisted selection of the high flesh/stone ratio. This strategy provides a rapid method for the characterization of RAPD markers and for the development of PCR-based markers with applications in olive mapping, paternal testing and germplasm characterization. The use of these markers in multiplexed PCRs, and the direct ethidium bromide detection of the PCR products in the test tube, facilitate their efficient and reliable breeding applications. Received: 1 November 2000 / Accepted: 24 November 2000     

Polyploidy in the olive complex (Olea europaea): evidence from flow cytometry and nuclear microsatellite analyses

BACKGROUND: Phylogenetic and phylogeographic investigations have been previously performed to study the evolution of the olive tree complex (Olea europaea). A particularly high genomic diversity has been found in north-west Africa. However, to date no exhaustive study has been addressed to infer putative polyploidization events and their evolutionary significance in the diversification of the olive tree and its relatives. METHODS: Representatives of the six olive subspecies were investigated using (a) flow cytometry to estimate genome content, and (b) six highly variable nuclear microsatellites to assess the presence of multiple alleles at co-dominant loci. In addition, nine individuals from a controlled cross between two individuals of O. europaea subsp. maroccana were characterized with microsatellites to check for chromosome inheritance. KEY RESULTS: Based on flow cytometry and genetic analyses, strong evidence for polyploidy was obtained in subspp. cerasiformis (tetraploid) and maroccana (hexaploid), whereas the other subspecies appeared to be diploids. Agreement between flow cytometry and genetic analyses gives an alternative approach to chromosome counting to determine ploidy level of trees. Lastly, abnormalities in chromosomes inheritance leading to aneuploid formation were revealed using microsatellite analyses in the offspring from the controlled cross in subsp. maroccana. CONCLUSIONS: This study constitutes the first report for multiple polyploidy in olive tree relatives. Formation of tetraploids and hexaploids may have played a major role in the diversification of the olive complex in north-west Africa. The fact that polyploidy is found in narrow endemic subspecies from Madeira (subsp. cerasiformis) and the Agadir Mountains (subsp. maroccana) suggests that polyploidization has been favoured to overcome inbreeding depression. Lastly, based on previous phylogenetic analyses, we hypothesize that subsp. cerasiformis resulted from hybridization between ancestors of subspp. guanchica and europaea.     

Three-year field response of young olive trees (Olea europaea L., cv. Arbequina) to soil salinity: Trunk growth and leaf ion accumulation

Irrigated olive is rapidly increasing in arid and semiarid areas, many of which may be negatively affected by soil salinity. We evaluated changes in trunk growth and leaf Cl^–, Na⁺ and K⁺ concentrations in young Arbequina olives (Olea europaea L.) grown in a saline-sodic field over a three-year period. The trunk diameter was measured at the beginning and the end of the 1999 (70 trees), 2000 (59 trees) and 2001 (42 trees) growing periods. Leaves, sampled in August of each year, were analyzed for Cl^–, Na⁺ and K⁺ concentrations. Soil salinity (apparent electrical conductivity, ECa) of each monitored tree was measured 14 times during the 1999–2001 experimental period with an electromagnetic sensor and converted to root zone electrical conductivity of the soil saturation extract (ECe) based on ECa–ECe calibration curves. Salinity tolerance was determined using the Maas and Hoffman threshold–slope response model. Based on salinity thresholds (ECe_thr), the tolerance of olive in terms of trunk growth was high in 1999 (ECe_thr = 6.7 dS m^–1), but declined with age and time of exposure to salts by 30% in 2000 (ECe_thr = 4.7 dS m^–1) and by 55% in 2001 (ECe_thr = 3.0 dS m^–1). Based on the high absolute slopes obtained in all years (values between 16% and 23% dS^–1 m), olive was classified as very sensitive to ECe values above the threshold. Trunk growth thresholds based on leaf ion concentrations varied, depending on years, between 2.6 and 4.0 mg g^–1 (Cl_thr) and between 1.0 and 1.2 mg g^–1 (Na_thr), indicating that Arbequina olive was less sensitive to leaf Cl^– and much more sensitive to leaf Na⁺ than values reported as toxic in greenhouse studies. Leaf K⁺ slightly decreased with increasing salinity, whereas the K⁺/Na⁺ ratio sharply decreased with increasing salinity. We concluded that the initial salinity tolerance of olive was high, but declined sharply with time of exposure to salts and became quite sensitive due primarily to increasing toxic concentrations of Na⁺ in the leaves.     

Ultrastructural aspects of the cytoplasmic origin and accumulation of oil in olive fruit (Olea europaea)

The development of oil bodies and oil droplets in fruits of olive was examined at the ultrastructural level. Both oil bodies that form in young fruits and oil droplets that develop with fruit maturation are cytoplasmic bodies. The formation of the small oil bodies occurs in localized regions of the cytoplasm. These bodies are closely associated and fuse together, forming a small oil droplet that protruded against and indented the vacuolar membrane. As the fruit matures, new oil bodies appear to form and fuse with the oil droplet, resulting in the formation of a single large oil droplet of about 30 μm in diameter in most mature mesocarp cells. The cytoplasmic region where the oil bodies formed had a granulate, ultrastructural appearance, and cytoplasmic components such as membranes and ribosomes were noticeably absent in these regions. The granulate material coated the oil bodies and oil droplets, and appeared as a thin, compressed band between the round inner surface of the droplets and the indented tonoplast. We suggest that this granulate material is involved in the synthesis of the oil and, with enlargement of the oil bodies, this coat becomes thinner in regions where they are closely associated, resulting in zones where confluence of the oil occurs.     

An in vitro nutritive evaluation of olive tree (Olea europaea) pruning residues as affected by cutting regimen

Nutritive values of the branches from Olea europaea trees cut at 25, 50, 75 or 100 cm distance from the tip were evaluated by determination of the in vitro digestible organic matter (IVDOM), metabolizable energy (ME), net energy lactation (NEL), and presence nutritional and anti-nutritional components. The values of nutritive components, nitrogen forms, IVDOM, ME and NEL declined and concentrations of crude fiber and cell wall constituents increased with the increase in cutting length. Total phenols, hydrolysable tannins and condensed tannins amounted to 70, 17 and 0.6 g/kg DM, respectively. The addition of polyethylene glycol (PEG, 6000) to the plant samples incubated with rumen fluid at a ratio of (2:1 PEG:substrate) increased the values of IVDOM, ME and NEL by 40 g/kg DM, 0.59 MJ/kg DM and 0.42 MJ/kg DM, respectively. IVDOM, ME and NEL were negatively correlated with crude fiber and cell wall constituents but positively correlated with nitrogen forms and non-fiber carbohydrates. Olive pruning branches in diameter <3 mm could be used as sources of feeds for small ruminants.     

Identification of olive (Olea europaea) seed and pulp proteins by nLC-MS/MS via combinatorial peptide ligand libraries

Different types of extraction protocols are described for identifying proteins in seed and pulp of olive (Olea europea), by employing both conventional extraction methods and capture with ProteoMiner as well as with in house-made combinatorial peptide ligand libraries (HM-CPLLs) at pH 7.4 and at pH 2.2. Thanks to the use of CPLLs, able to dramatically amplify the signal of low-abundance species, a quite large number of compounds has been indeed identified: 61 in the seed (vs. only four reported in current literature) and 231 in the pulp (vs. 56 described so far), the deepest investigation up to the present of the olive proteome. In the seed, it highlights the presence of seed storage proteins, oleosins and histones. In the pulp, the allergenic thaumatin-like protein (Ole e 13) was confirmed, among the other 231, as the most abundant protein in the olive pulp. The present research has also been undertaken with the aim of identifying proteins in olive oil and ascertaining the relative contribution of seed and pulp proteins in their presence, if any, in oils.     

Effect of polyethylene glycol,urea and sunflower meal on olive (Olea europaea var. europaea) leaf fermentation in continuous fermentors

A continuous culture system, inoculated with rumen liquor from goats or sheep, was used to study fermentation characteristics of olive leaves (OL). The effects of adding polyethylene glycol (PEG 4000 MW; 0, 2 or 20 g/100 g OL) and/or supplementing with urea (U) or sunflower meal (SM) (1.0 g N/100 g OM) were also studied. Olive leaf fermentation promoted low VFA production (35.2 mmol/d), predominantly of acetic acid, and low efficiency of VFA production (4.91 mol/kg digestible carbohydrates, DCHO). Both values increased with N supplementation, but effects of PEG were variable. No differences ascribed to the rumen inoculum origin were observed. The ammonia N concentration was increased only by supplementation with U. Total and amino acid N output was low and increased with N addition, but it was not affected by PEG treatment. No differences ascribed to the inoculum origin were observed concerning microbial N production rate or efficiency (g N/kg DCHO). There was no clear difference between sources of supplementary N regarding bacterial protein synthesis. On the basis of PEG results, the effect of tannins on OL fermentation was not important.     

Techno-economic and environmental approaches of Cd2+ adsorption by olive leaves (Olea europaea L.) waste

Abstract

In this study, the techno-economic approach of olive leaves (Olea europaea L.) wastes for the removal of Cd²⁺ from aqueous solutions was demonstrated. The adsorption process was illustrated regarding batch experiments and scanning electron microscopy, energy dispersive X-ray, and Fourier-transform infrared characterization. The optimum pH and contact time were 6.6 and 123?min, respectively, giving Cd²⁺ removal efficiencies of 94.9% at C_o = 50?mg/L and 81.5% at C_o = 100?mg/L. The monolayer adsorption capacity of the Langmuir isotherm model was 32.6?mg/g (R² = 0.97). The adsorption mechanisms might be related to (a) ion exchange with cations (e.g., K⁺, Na⁺, and Ca²⁺), (b) formation of cadmium chloride complexes, (c) interaction with oxygen-containing functional groups, (d) physical agglomeration in the pore surface, and (e) precipitation interaction using inorganic minerals (i.e., carbonates, phosphates, and silicates). The total cost of the adsorption process for the treatment of ions-containing wastewater was 0.038 $USD/m³. Assuming a benefit-cost of tertiary treated water as 0.044 $USD/m³, the adsorption system could attain a payback period of 5.7?years. This period was shorter than the lifetime of the capital investment (i.e., 10?years), and hence, the project would be economically feasible for an application.     

Exploiting the anti-inflammatory properties of olive (Olea europaea) in the sustainable production of functional food and neutraceuticals

Olive oil is an important lipid source of the Mediterranean diet which has been associated with lower incidence of cardiovascular diseases whereas olive pomace (OP), a natural by-product of olive oil production, has been found to contain micro constituents with antioxidant, antithrombotic and antiatherogenic activities. The evaluation of OP in order to produce sustainable functional food and neutraceuticals has been the subject of research over the last years. All recent data, focusing on the anti-inflammatory properties of olive oil derived from olive (Olea europaea) and OP along with the potential production of sustainable functional food and neutraceuticals, are presented in this review.     

Historical biogeography of olive domestication (Olea europaea L.) as revealed by geometrical morphometry applied to biological and archaeological material

Aim This study intends to improve our understanding of historical biogeography of olive domestication in the Mediterranean Basin, particularly in the north-western area. Location Investigations were performed simultaneously on olive stones from extant wild populations, extant cultivated varieties from various Mediterranean countries, and archaeological assemblages of Spanish, French and Italian settlements. Methods A combination of morphometrics (traditional and geometrical) allowed us to study both the size and shape of endocarp structure. Concerning shape, a size-standardized method coupled with fitted polynomial regression analysis was performed. Results We found morphological criteria for discriminating between wild and cultivated olive cultivars, and established patterns of morphological variation of olive material according to the geographical origin (for extant material) and to the age of the olive forms (for archaeological material). Levels of morphological convergences and divergences between wild olive populations and cultivated varieties are presented as evidence. Main conclusions Morphological changes of endocarps of olive under domestication at both geographical and chronological scales provide new criteria for the identification of olive cultivars. They allow to determine the origins of cultivated forms created and/or introduced in the north-western Mediterranean regions and to understand how human migrations affected the rest of the Western Mediterranean regions. A model of diffusion of olive cultivation is proposed. It shows evidence of an indigenous origin of the domestication process, which is currently recognized in the north-western area since the Bronze Age.