Micropropagation of olive (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) and application of mycorrhiza to improve plantlet establishment

Micropropagation methods were developed for the three French varieties of olive (Olea europaea L.), Aglandau and Tanche, that have the Appelation d’Origine Contrôlée (AOC) status and one ecotype (05300, Laragne, France). Explants consisting of partially lignified nodal segments were collected from rejuvenated glasshouse-grown plant material. Nodal explants with axillary buds were cultured on different media. For AOC varieties, olive medium modified (OM mod) to contain half the concentration of macroelements was the most efficient in inducing bud break and growth when supplemented with 30 g l^?1sucrose and 4 mg l^?1 zeatin. The resulting shoot buds were further multiplied and maintained on OM mod medium. Rooting was best achieved on OM supplemented with 4 mg l^?1 indole-3-butyric acid (IBA). For the Laragne ecotype, maximal shoot proliferation occurred when explants were cultured on woody plant medium supplemented with 15 g l^?1 sucrose and 0.1 mg l^?1 zeatin. Efficient rooting was achieved with 1 mg l^?1 IBA combined with 0.75 mg l^?1 naphthaleneacetic acid. After acclimatization in the glasshouse, survival rates ranged from 57 to 92%, depending on the genotype. Inoculation of Laragne ecotype microplantlets with the arbuscular mycorrhizal fungus Glomus mosseae significantly improved plant survival and subsequent plant development and growth.     

In vitro effects of triterpenic acids from olive leaf extracts on the mitochondrial membrane potential of promastigote stage of Leishmania spp

Protozoan diseases, such as leishmaniasis, are a cause of considerable morbidity throughout the world, affecting millions every year. In this study, two triterpenic acids (maslinic and oleanolic acids) were isolated from Tunisian olive leaf extracts and their in vitro activity against the promastigotes stage of Leishmania (L.) infantum and Leishmania (L.) amazonensis was investigated. Maslinic acid showed the highest activity with an IC₅₀ of 9.32 ± 1.654 and 12.460 ± 1.25 μg/ml against L. infantum and L. amazonensis, respectively. The mechanism of action of these drugs was investigated by detecting changes in the phosphatidylserine (PS) exposure, the plasma membrane permeability, the mitochondrial membrane potential and the ATP level production in the treated parasites. By using the fluorescent probe SYTOX® Green, both triterpenic acids showed that they produce a time-dependent plasma membrane permeabilization in the treated Leishmania species. In addition, spectrofluorimeteric data revealed the surface exposure of PS in promastigotes. Both molecules reduced the mitochondrial membrane potential and decreased the ATP levels to 15% in parasites treated with IC₉₀ for 24 h. We conclude that the triterpenic acids tested in this study, show potential as future therapeutic alternative against leishmaniasis. Further studies are needed to confirm this.     

Hypoglycemic and antioxidant effect of oleuropein in alloxan-diabetic rabbits

Patients with diabetes mellitus are likely to develop certain complication such as retinopathy, nephropathy and neuropathy as a result of oxidative stress and overwhelming free radicals. Treatment of diabetic patients with antioxidant may be of advantage in attenuating these complications. Oleuropein, the active constituent of olive leaf (Olea europaea), has been endowed with many beneficial and health promoting properties mostly linked to its antioxidant activity. This study aimed to evaluate the significance of supplementation of oleuropein in reducing oxidative stress and hyperglycemia in alloxan-induced diabetic rabbits. After induction of diabetes, a significant rise in plasma and erythrocyte malondialdehyde (MDA) and blood glucose as well as alteration in enzymatic and non-enzymatic antioxidants was observed in all diabetic animals. During 16 weeks of treatment of diabetic rabbits with 20 mg/kg body weight of oleuropein the levels of MDA along with blood glucose and most of the enzymatic and non-enzymatic antioxidants were significantly restored to establish values that were not different from normal control rabbits. Untreated diabetic rabbits on the other hand demonstrated persistent alterations in the oxidative stress marker MDA, blood glucose and the antioxidant parameters. These results demonstrate that oleuropein may be of advantage in inhibiting hyperglycemia and oxidative stress induced by diabetes and suggest that administration of oleuropein may be helpful in the prevention of diabetic complications associated with oxidative stress.     

Supercritical carbon dioxide extraction of astaxanthin from Haematococcus pluvialis with vegetable oils as co-solvent

Soybean oil and olive oil were investigated as continuous co-solvents for supercritical carbon dioxide (SC-CO₂) extraction of astaxanthin from Haematococcus pluvialis. Without co-solvents, only 25.40 ± 0.79% efficiency was achieved with SC-CO₂ extraction at 70 °C and 40 MPa at a continuous flow rate of 3 mL min⁻¹ for 5 h. In the presence of soybean oil or olive oil as a co-solvent, the extraction efficiency was enhanced, with the most appropriate level of soybean oil in the solvent mixture being 10% by volume. At this concentration and the above extraction conditions, the highest extraction efficiency of 36.36 ± 0.79% was obtained for soybean oil, a 30% increase in extraction efficiency compared with SC-CO₂ extraction without soybean oil, whereas the 10% olive oil increased the extraction efficiency further to 51.03 ± 1.08%, which was comparable to that obtained using ethanol as co-solvent.     

Detoxification of olive mill wastewaters by Moroccan yeast isolates

A total of 105 yeast strains were isolated from Moroccan olive oil production plants and evaluated for their ability to grow in olive oil mill wastewaters (OMW). The 9 isolates that grew best on OMW were selected for further study to evaluate their effect on removal of organic pollutants and OMW phytotoxicity (barley seed germination test). The results showed that at least four yeast isolates effectively lowered the toxicity of this effluent in addition to providing very useful materials in terms of both yeast biomass (6 g/l DW) and an irrigation fluid. This group of yeast isolates significantly reduced the concentration of total phenols (44% removal) and Chemical Oxygen Demand, COD (63% removal). The best germination rate of 80% for undiluted OMW was obtained for strain Candida holstii that also increased the pH from 4.76 to 6.75. Principal component analysis of the results obtained for the best yeast strains confirmed the importance of COD and total phenol reduction along with increase of organic nitrogen and final pH for the improvement of germination rates and phytotoxic reduction. This study has highlighted the potential of indigenous yeasts in detoxification of olive mill wastewaters.     

Early markers of in vitro microspore reprogramming to embryogenesis in olive (Olea europaea L.)

Microspore embryogenesis to form haploid and double-haploid embryos and regenerated plants is an efficient method of producing homozygous lines for crop breeding. In trees, the process is of special interest since classical methods are impractical in many cases, as in Olea europaea L. Recently, a convenient method has been developed for microspore embryogenesis induction by stress in olive isolated microspores in vitro cultures. In the present work, the switch of the microspore developmental pathway and the formation of microspore-derived multicellular proembryos have been achieved and a cytochemical and immunocytochemical analysis was performed in the early stages. The young microspore proembryos displayed defined features different to both, the in vivo gametophytic, and the in vitro non-responsive microspores. Reprogrammed microspores showed an absence of starch, the occurrence of a first symmetrical division and cytokinesis, the presence of an abundant ribosomal population, and changes in cellulosic and pectic cell wall components which constituted early markers of the embryogenic microspore process. They provided new insights on the molecular and cellular events associated with the microspore reprogramming of woody plants, and specifically in olive, providing interesting knowledge which could guide future selection and regeneration strategies in this fruit tree of high economic interest.     

毕赤酵母组成型表达脂肪酶及其高通量筛选方法

【目的】获得组成型表达脂肪酶毕赤酵母,建立利用橄榄油罗丹明B平板高通量筛选组成型表达华根霉脂肪酶基因的有效方法。【方法】运用PCR技术从pGAPZαA表达载体上扩增得到GAP启动子片段,插入到表达载体pPIC9K-proRCL中,构建组成型表达载体pGAPK-proRCL。在保留含有同源双交换重组序列的诱导型启动子AOX1序列的基础上,电转化后华根霉Rhizopus chinensis CCTCC M201021脂肪酶基因proRCL表达盒在毕赤酵母基因组上发生双交换整合事件,从而组成型表达单拷贝的华根霉脂肪酶基因。【结果】重组菌发酵144 h后,脂肪酶最高酶活为130 U/mL。利用橄榄油罗丹明B平板高通量筛选组成型表达华根霉脂肪酶基因。【结论】该方法将初筛时间从12 d缩短为3 d,排除了多拷贝突变株的干扰,为后续脂肪酶的定向进化及筛选奠定了基础。     

Entrapment of lipase into K-carrageenan beads and its use in hydrolysis of olive oil in biphasic system

The porcine pancrease lipase was immobilized by entrapment in the beads of K-carrageenan and cured by treatment with polyethyleneimine (PEI) in the phosphate buffer. The retention of hydrolytic activity of lipase and compressive strength of the beads were examined. The activity of free and immobilized lipase was assessed by using olive oil as the substrate. The immobilized enzyme exhibited a little shift towards acidic pH for its optimal activity and retained 50% of its activity after 5 cycles. When the enzyme concentration was kept constant and substrate concentration was varied the K_m and V_max were observed to be 0.18 × 10⁻² and 0.10, and 0.10 × 10⁻² and 0.09 respectively, for free and for entrapped enzymes. When the substrate concentration was kept constant and enzyme concentration was varied, the values of K_m and V_max were observed to be 0.19 × 10⁻⁷ and 0.41, and 0.18 × 10⁻⁷ and 0.41 for free and entrapped enzymes. Though this indicates that there is no conformational change during immobilization, it also shows that the reaction velocity depends on the concentration. Immobilized enzyme showed improved thermal and storage stability. Hydrolysis of olive oil in organic–aqueous two-phase system using fixed bed reactor was carried out and conditions were optimized. The enzyme in reactor retained 30% of its initial activity after 480 min (12 cycles).     

Improvement of olive oil phenolics content by means of enzyme formulations: Effect of different enzyme activities and levels

The present work deals with the development of an enzymatic treatment aiming at producing high-quality olive oil with increased phenolics content and antioxidant activity. Three different enzyme formulations, specifically pectinase, hemicellulase and cellulase (A), pectinase and hemicellulase (B), and only pectinase (C), were used either at three different level of each or in ternary and binary mixtures at a constant level. All of them were added to the olive paste (Italian cultivar Coratina) at the beginning of the malaxation step. Results demonstrated that an increased enzyme level led to higher phenolics content in the oils, and such an effect was found to be enzyme dependent, being greater when using formulation A, followed by formulations B and C. Two significant correlations were obtained between total polyphenols and o-diphenols contents versus antiradical power (R² = 0.8743 and R² = 0.8635, respectively), which pointed out the effectiveness of the proposed enzymatic treatment to produce olive oils characterized by low susceptibility to oxidation. A synergistic effect between the different enzymatic activities contained in the single formulations was observed by combining enzymes A and B. The ternary mixture was selected as the most efficient enzymatic system ensuring the highest phenolics content increase (40 and 37% for total polyphenols and o-diphenols contents, respectively) compared to the other enzymes when used at the same level.     

Plant-parasitic nematodes associated with olive tree (Olea europaea L.) with a focus on the Mediterranean Basin: A review

The olive tree (Olea europaea ssp. europaea.) is one of the most ancient cultivated trees. It is an emblematic species owing to its ecological, economic and cultural importance, especially in the Mediterranean Basin. Plant-parasitic nematodes are major damaging pests on olive trees, mainly in nurseries. They significantly contribute to economic losses in the top-ten olive-producing countries in the world. However, the damages they induce in orchards and nurseries are specifically documented only in a few countries. This review aims to update knowledge about the olive-nematode pathosystem by: (1) updating the list of plant-parasitic nematodes associated with olive trees; (2) analysing their diversity (taxonomic level, trophic groups, dominance of taxa), which allowed us (i) to assess the richness observed in each country, and (ii) to exhibit and describe the most important taxa able to induce damages on olive trees such as: Meloidogyne, Pratylenchus, Helicotylenchus, Xiphinema, Tylenchulus, Rotylenchulus, Heterodera (distribution especially in the Mediterranean Basin, pathogenicity and reactions of olive trees); (3) describing some management strategies focusing on alternative control methods; (4) suggesting new approaches for controlling plant-parasitic nematodes based on the management of the diversity of their communities, which are structured by several environmental factors such as olive diversity (due to domestication of wild olive in the past, and to breeding now), cropping systems (from traditional to high-density orchards), irrigation, and terroirs.