Relationship between pollen emission and fruit production in olive (Olea europaea L.)

Aerobiological data of pollen emission concentrations is used to predict fruit production. The principal aim of this work was to study the relationship between pollen emission patterns, emission homogeneity and fruit production in olive (Olea europaea L.). Data of daily pollen concentrations in the atmosphere during the flowering period, collected over a 20‐year period in Perugia (Central Italy), and the corresponding fruit production data were analysed. Correlation and regression analyses on the partial pollen amounts (subdivisions of the whole flowering period), their statistical variability (expressed as coefficients of variation of the daily pollen concentration), and the production values in the different years demonstrate that pollen emission, during the seven to ten day period immediately preceding the maximum pollen emission day, appears to be most closely related to fruit production. Moreover, the pollen emission homogeneity (minimal variability in daily pollen concentrations) during the “critical” flowering period is very important for fertilization.     

Morphology and discrimination features of pollen from Italian olive cultivars (Olea europaea L.)

Pollen morphology of 14 cultivars of Olea europaea subsp. europaea var. europaea was analysed in order to discriminate main pollen types. The cultivars were selected from the most spread and early flowering crops grown in Italy. Morphometric parameters were observed on acetolysed pollen by means of light microscopy and scanning electron microscopy. Polar axis (P), equatorial diameter (E), P/E ratio, maximum distance between colpi in mesocolpium, distance between the apices of two colpi, exine thickness, maximum length of lumina in mesocolpium and in apocolpium, and exine reticulum thickness in mesocolpium have been measured. According to P and E, the 14 olive cultivars of this study can be divided into the three groups of small (P: 21.75 µm, E: 22.55 µm; ‘Manna’ and ‘Tonda di Cagliari’), large (P: 25.1 µm, E: 26.1 µm; ‘Pescarese’ and ‘Rotondella di Sanza’) and medium size (P: 23.49 µm, E: 24.54 µm, ‘Carolea’, ‘Grossa di Cassano’, ‘Giarraffa’, ‘Nocellara messinese’, ‘Nocellara del Belice’, ‘Santagatese’, ‘Intosso’, ‘Maiatica di Ferrandina’, ‘Nostrale di Fiano Romano’, ‘Santa Caterina’). Maximum length of lumina and exine thickness are useful parameters for further distinction of olive pollen groups, since these parameters are able to provide a specific pollen profile for each cultivar.     

Behavior of storage lipids during development and germination of olive ( Olea europaea L.) pollen

Summary.  The presence of abundant oil bodies in the mature olive pollen grain has led us to focus on the behavior of these lipid bodies during pollen development and in vitro pollen germination. The appearance, increase, and accumulation of lipid bodies have been determined by following the sequential development of the pollen grain. Semithin slices of anthers and pollen grains were stained with Sudan Black B in order to identify neutral lipids. Ultrastructural studies were also carried out. Our results show a notable increase in lipid bodies between the young-pollen-grain stage and the mature-pollen-grain stage. Substantial polarization of lipid bodies was observed after 1 or 2 h of pollen incubation in germination medium. During pollen tube growth, the lipid bodies are located near the germinative aperture after 3 h of incubation, as well as inside the pollen tube, thus suggesting that the lipid bodies move from the pollen grain to the pollen tube. After 7 h of germination the presence of lipid bodies inside the pollen tube is no longer substantial. Our results support the idea that lipid bodies are involved in pollen germination, stigma penetration, and pollen tube growth. These results are discussed in connection with their implications for the pollen germination process. Received June 4, 2002; accepted October 29, 2002; published online April 8, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Profesor Albareda 1, 18008 Granada, Spain.     

Cytoplasmic male sterility in the olive (Olea europaea L.)

The olive tree is usually hermaphrodite but self-incompatible. In the Western Mediterranean some cultivars are totally male-sterile. Three different male-sterile phenotypes have been recognised. To infer the genetic basis of male sterility we studied its inheritance and cytoplasmic diversity in wild (oleaster) and cultivated Mediterranean olive. In the cross Olivière×Arbequina, the male-sterile trait was maternally inherited and affected all progenies. We also checked that both chloroplast and mitochondrial DNAs are maternally inherited. RFLP studies on chloroplast and mitochondrial DNAs revealed several cytotypes: two chlorotypes and four mitotypes in cultivars and oleaster (wild or feral Mediterranean olive). Furthermore, a total linkage desequilibrium between the CCK chlorotype and the MCK mitotype in cultivars and oleaster from different regions supports the fact that paternal leakage of organelles was not observed. The male sterility (ms 2) displayed by Olivière, plus six other cultivars and three oleaster was strictly associated with the CCK chlorotype and the MCK mitotype. These facts suggest that Olivière carries cytoplasmic male sterility. Male-fertile and male-sterile oleasters carrying this cytotype showed the presence of restorer alleles. This CMS might be due to a distant cross between olive taxa. The two other male-sterile phenotypes displayed by Lucques (ms 1) and Tanche (ms 3) were associated with the ME1 mitotype but we have not demonstrated CMS. Received: 26 July 1999 / Accepted: 27 August 1999     

Somatic embryogenesis and plant regeneration in olive (Olea europaea L.)

Leaf discs from olive (Olea europaea L.) grown in vitro and immature zygotic embryos collected at 50, 75, 90 and 105 days after full bloom were tested for their somatic embryogenic capacity. The embryos were grown in half-strength MS medium and half-strength OM medium with BAP combinated with either 2,4-D or NAA. Incubation was either in an initial dark period followed by 16h daylight or in 16h daylight throughout. Somatic embryogenesis, approx. 40%, mostly directly from the embryos, was observed only in 75-day-old embryos in medium containing low cytokinin and auxin concentrations. Differentiation was inhibited by 2,4-D whereas NAA did not. In leaf discs and younger and older zygotic embryos, only callus and root formation was observed. Somatic embryos were germinated and then potted-up to soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA naphtaleneacetic acid     

Chloroplast-DNA variation in cultivated and wild olive (Olea europaea L.)

Polymorphism in the lengths of restriction fragments of the whole cpDNA molecule was studied in cultivated olive and in oleaster (wild olive) over the whole Mediterranean Basin. Seventy two olive cultivars, 89 very old trees cultivated locally, and 101 oleasters were scored for ten endonucleases. Moreover, maternal inheritance of cpDNA in olive was shown by analysing the progeny of a controlled cross between two parents which differed in their cpDNA haplotypes. In the whole species, three site- and three length-mutations were observed, corresponding to five distinct chlorotypes. The same chlorotype (I) was predominant in both oleasters and cultivated olive trees, confirming that these are closely related maternally. Three other chlorotypes (II, III and IV) were observed exclusively in oleaster material and were restricted either to isolated forest populations or to a few individuals growing in mixture with olive trees possessing the majority chlorotype. An additional chlorotype (V) was characterised by three mutations located in distinct parts the cpDNA molecule but which were never observed to occur separately. This chlorotype, more widely distributed than the other three, in both cultivated and wild olive, and occurring even in distant populations, was observed exclusively in male-sterile trees showing the same specific pollen anomaly. However, in the present study, no evidence was provided for a direct relationship between the occurrence of the cpDNA mutations and male sterility. It is suggested that the large geographic distribution of chlorotype V may be related to the high fruit production usually observed on male-sterile trees. These may be very attractive for birds which are fond of olive fruit and spread the stones efficiently. Probably for the same reason, people preserved male-sterile oleasters for long periods and, in several places, used male-sterile cultivars over large areas. Received: 25 November 1998 / Accepted: 19 December 1998     

Advances in the pollination biology of olive (Olea europaea L.)

The olive originated in the Mediterranean region, and is most extensively cultivated oleiferous tree species in the world due to its high economic value. The olive has been introduced to China for more than 50 years, and some cultivars have showed good ecological suitability. The plantation of olive in China is increasing rapidly. However, many olive trees during the fruiting age showed the low fruit-setting rates and unstable fruit yields. Studies on pollination biology of olive are important to improve olive fruit-setting rates, genetic resources, and clonal selection and cross-breeding. Overseas much have been done about studies on pollination biology of olive, which are still scarce in China. In this paper, we presented a review of the recent advances in the pollination biology of olive, with the emphasis on blossom phenological characteristics, floral morphology, pollen germination, mating system, as well as their impacts on the fruit set of olive. The future researches in China were subsequently suggested as follows: (1) Strengthen the basic researches of olive pollination biology, including the ratio between monoecious and andromonoecious flowers in main introduced cultivars, the mechanisms for the maintenance of the olive sexual system, and the functional differences between monoecious and andromonoecious flowers and their relationship with wind-pollination, (2) Both field artificial pollination and molecular technology should be combined to elucidate the olive breeding system of cultivars, to explore the genetic and physiological mechanisms of self-infertility and cross-pollination, and to find the determinative factors of affecting the pollination compatibility of various cultivars, and (3) Studies on both olive pollination biology and breeding system should be combined to analyze comprehensively the effects of pollination and fertilization processes on fruit setting and early fruit development.     

Isolation,culture and division of olive (Olea europaea L.) protoplasts

Protoplasts from Olea europaea L. have been compared in terms of their yield, viability, cell division and callus differentiation. Viable protoplasts were isolated from in vitro cultured leaves and cotyledons by an overnight incubation in an enzyme solution containing 1–1.5% driselase and 0.5M sucrose. This method allowed high yield of purified protoplasts, which floated and formed a dark green band at the meniscus, after centrifugation. Purified protoplasts were diluted to 3×10⁴ protoplasts·ml^–1 in culture medium. After cell wall regeneration, protoplasts gradually increased their volumes under appropriate conditions. The first divisions occurred during the second week in culture. Division efficiency ranged from 5.2 to 9.8% after 20 days in culture. Two weeks later visible microcolonies developed only from cotyledon protoplasts. After 6 weeks in culture, the microcalli were transferred to a solidified culture medium with 0.6% agarose, which induced active callus growth.Abbreviations OM olive proliferation medium, Rugini 1984 - Omg OM for the germination of olive embryos - OMr=OM for root induction - OMp=OM for protoplasts - OMc=OM for callus - BN Bourgin and Nitsch medium 1967 - IBA indol-3-butyric acid - NAA naphthalene acetic acid - 2,4-D dichlorophenoxyacetic acid.     

SNP-based markers for discriminating olive (Olea europaea L.) cultivars.

A set of 11 polymorphic markers (1 cleaved amplified polymorphic sequence (CAPS), 2 sequence-characterized amplified regions (SCARs), and 8 single-nucleotide polymorphism (SNP)-derived markers) was obtained for olive cultivar identification by comparing DNA sequences from different accessions. Marker development was more efficient, using sequences from the database rather than cloning arbitrary DNA fragments. Analyses of the sequences of 3 genes from 11 diverse cultivars revealed an SNP frequency of 1 per 190 base pairs in exons and 1 per 149 base pairs in introns. Most mutations were silent or had little perceptible effect on the polypeptide encoded. The higher incidence of transversions (55%) suggests that methylation is not the major driving force for DNA base changes. Evidence of linkage disequilibrium in 2 pairs of markers has been detected. The set of predominantly SNP-based markers was used to genotype 65 olive samples obtained from Europe and Australia, and was able clearly to discriminate 77% of the cultivars. Samples, putatively of the same cultivar but derived from different sources, were revealed as identical, demonstrating the utility of these markers as tools for resolving nomenclature issues. Genotyping data were used for constructing a dendrogram by UPGMA cluster analysis using the simple matching similarity coefficient. Relationships between cultivars are discussed in relation to the route of olive's spread.     

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.     

In vitro propagation of olive (Olea europaea L.) cv. Koroneiki

Single node explants of 'Koroneiki' olive trees werecultured for one month on a modified Driver-Kuniyuki for Walnut medium, lackinggrowth regulators. The explants were subcultured once a month on a mediumsupplemented with zeatin riboside, 6-(--dimethylallylamino)purine,6-benzyladenine or thidiazuron. Zeatin riboside proved to be superior to othercytokinins in inducing shoot proliferation. The combination of olive knotextract at 25 or 50 mg l^–1 with cytokininssuppressed shoot proliferation. After two months at the proliferation stage,theexplants were cultured for one week in the dark in 1 ml liquidWoody Plant Medium supplemented with IBA, -NAA or IBA+-NAA. Theexplants were then transferred to the same solid medium lacking growthregulators, with a small layer of perlite on the surface. The combination ofthetwo auxins at 1+1 mg l^–1 resulted in almost 76%rooting. The combination of olive knot extract at 50 mgl^–1 with auxins increased the rooting percentage up toalmost 87%. Artificial infection of explants with the bacteriumPseudomonas savastanoi pv. savastanoiinhibited rhizogenesis, even in the presence of auxins. Rooted explants weresuccessfully acclimatised under a mist system, with the survival rate reachingalmost 75%.     

Biochemical characterization of a lipase from olive fruit (Olea europaea L.)

Lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is the first enzyme of the degradation path of stored triacylglycerols (TAGs). In olive fruits, lipase may determine the increase of free fatty acids (FFAs) which level is an important index of virgin olive oil quality. However, despite the importance of virgin olive oil for nutrition and human health, few studies have been realized on lipase activity in Olea europaea fruits. In order to characterize olive lipase, fruits of the cv. Ogliarola, widely diffused in Salento area (Puglia, Italy), were harvested at four stages of ripening according to their skin colour (green, spotted I, spotted II, purple). Lipase activity was detected in the fatty layer obtained after centrifugation of the olive mesocarp homogenate. The enzyme exhibited a maximum activity at pH 5.0. The addition of calcium in the lipase assay medium leads to an increment of activity, whereas in the presence of copper the activity was reduced by 75%. Furthermore, mesocarp lipase activity increases during olive development but declined at maturity (purple stage). The data represent the first contribution to the biochemical characterization of an olive fruit lipase associated to oil bodies.     

Oxidative reactions in axenically seedling roots of olive (Olea europaea, L.)

The production of reactive oxygen species (ROS) plays important roles in the life cycle and in the stress response and defence mechanisms of plants. Various enzyme systems are involved in the formation of ROS in the apoplast, including plasmalemma NADPH oxidase and apoplastic peroxidases. The production of O ₂ ^·? and apoplastic peroxidase and exogenous NADH oxidation activities are all strongly dependent on the age of roots??the younger the root, the greater the activity. Apoplastic production of ROS is shown in the root by using specific histochemical probes, this ROS production is growing zone dependent. In the present study, using olive seedlings, differences were also observed between cultivars, especially in O ₂ ^·? production by the Verdial cultivar which was well above that of other cultivars studied. In all the cultivars, treatment of roots with methyl jasmonate (MeJA) or methyl salicylate (MeSA) increased O ₂ ^·? production. Similar results were observed for peroxidase activity, but not for the oxidation of exogenous NADH which was either unaffected (MeJA) or even partially inhibited (MeSA). A conclusion was that MeJA or MeSA induced apoplastic production of ROS does not use exogenous NADH. Treatment with diphenylene iodonium (DPI) reduced the formation of O ₂ ^·? , but affected neither peroxidase nor NADH oxidation activities. Cyanide inhibited O ₂ ^·? production and peroxidase and NADH oxidation activities. Treatment with MnCl₂ had a strong stimulatory effect on peroxidase and NADH oxidation activities, but much less on O ₂ ^·? production. Finally, azide greatly reduced all activities, but especially O ₂ ^·? production. Together, these results indicate a relationship between oxidative activities and the processes of root growth, and that those activities are also dependent on the cultivar, as well as an involvement of peroxidases and plasmalemma NADPH oxidase in apoplast ROS production which is sensitive to DPI, azide, and cyanide but relatively insensitive to MnCl₂, while exogenous NADH oxidation is linked to peroxidase activity.     

Identification of simple sequence repeats (SSRs) in olive ( Olea europaea L.)

A small insert genomic library of Olea europaea L., highly enriched in (GA/CT)n repeats, was obtained using the procedure of Kandpal et al. (1994). The sequencing of 103 clones randomly extracted from this library allowed the identification of 56 unique genomic inserts containing simple sequence repeat regions made by at least three single repeats. A sample of 20 primer pairs out of the 42 available were tested for functionality using the six olive varieties whose DNA served for library construction. All primer pairs succeeded in amplifying at least one product from the six DNA samples, and ten pairs detecting more than one allele were used for the genetic characterisation of a panel of 20 olive accessions belonging to 16 distinct varieties. A total of 57 alleles were detected among the 20 genotypes at the ten polymorphic SSR loci. The remaining primer pair allowed the amplification of a single SSR allele for all accessions plus a longer fragment for some genotypes. Considering the simple sequence repeat polymorphism, 5.7 alleles were scored on average for each of the ten SSR loci. A genetic dissimilarity matrix, based on the proportion of shared alleles among all the pair-wise combinations of genotypes, was constructed and used to disentangle the genetic relationships among varieties by means of the UPGMA clustering algorithm. Graphical representation of the results showed the presence of two distinct clusters of varieties. The first cluster grouped the varieties cultivated on the Ionian Sea coasts. The second cluster showed two subdivisions: the first sub-cluster agglomerated the varieties from some inland areas of Calabria; the second grouped the remaining varieties from Basilicata and Apulia cultivated in nearby areas. Results of cluster analysis showed a significant relationship between the multilocus genetic similarities and the geographic origin of the cultivars. Received: 2 February 2001 / Accepted: 1 June 2001     

Cell wall reformation by pollen tube protoplasts of olive (Olea europaea L.): structural comparison with the pollen tube wall

. Mature pollen grains of olive (Olea europaea L.) were germinated in vitro in Brewbaker and Kwack medium, and emerging pollen tubes were then enzymatically digested in the presence of high osmoticum. This treatment resulted in simultaneous degradation of pollen tube walls and fragmentation of their cytoplasm, giving rise to numerous protoplasts of different sizes and different numbers of nuclei. After the protoplasts had been purified, they were cultured in Murashige and Skoog medium supplemented with auxin and cytokinin. The initial steps of cell wall reformation were studied after 12 h and 24 h of culture with a series of cytochemical techniques including periodic acid-Schiff reagent and phosphotungstic acid, as well as with electron microscopy and immunocytochemical techniques using monoclonal antibodies directed against pectins and #-(1̅)-glucan (callose). Among the components of new wall in the protoplasts, callose proved to be the earliest and most abundantly secreted polysaccharide, whereas the deposition of pectins recognized by the antibody JIM7 started several hours later. Pectins that bind JIM5 antibody were not detected in this early stage of development. Cell wall components deposited by protoplasts were compared with those present in growing pollen tubes. Callose secreted by protoplasts formed a relatively thicker layer than that found in the tubes, and pectins recognized by JIM7 were highly abundant, mostly within the cytoplasm and in the apical zone of the tubes.     

Peroxynitrite mediates programmed cell death both in papillar cells and in self-incompatible pollen in the olive (Olea europaea L.)

Programmed cell death (PCD) has been found to be induced after pollination both in papillar cells and in self-incompatible pollen in the olive (Olea europaea L.). Reactive oxygen species (ROS) and nitric oxide (NO) are known to be produced in the pistil and pollen during pollination but their contribution to PCD has so far remained elusive. The possible role of ROS and NO was investigated in olive pollen-pistil interaction during free and controlled pollination and it was found that bidirectional interaction appears to exist between the pollen and the stigma, which seems to regulate ROS and NO production. Biochemical evidence strongly suggesting that both O(2)(-) and NO are essential for triggering PCD in self-incompatibility processes was also obtained. It was observed for the first time that peroxynitrite, a powerful oxidizing and nitrating agent generated during a rapid reaction between O(2)(-) and NO, is produced during pollination and that this is related to an increase in protein nitration which, in turn, is strongly associated with PCD. It may be concluded that peroxynitrite mediates PCD during pollen-pistil interaction in Olea europaea L. both in self-incompatible pollen and papillar cells.     

A new repetitive DNA sequence family in the olive (Olea europaea L.)

Two families of repeated DNA sequences were cloned from Olea europaea ssp sativa cv. "Picual". The first repetitive DNA is organized in a tandem repeat of monomers of 178 bp. Sequencing of several clones showed that it is relatively A-T rich (54.49%) and possesses short direct and inverted subrepeats as well as some palindromic sequences. Comparison between the monomers revealed heterogeneity of the sequence primary structure. This repetitive DNA is present in several cultivars of olive cultivates. Comparison of sequences with other repetitive DNAs described in Olea europaea has been carried out. No significant similarity was found. All the obtained results suggest that this repetitive DNA described here is a new family of repetitive DNA. The second repetitive DNA is organized in a tandem repeat of monomers of 78 bp. This second family of repetitive DNA showed significant similarity with other repetitive DNAs previously described in Olea europaea. Their existence in new cultivars of olive is shown.     

In vitro olive (Olea europaea L.) cvs Frantoio and Moraiolo microshoot tolerance to NaCl

Abstract

In vitro culture of rooted and unrooted olive microshoots, established from seed lines of free-pollinated “Frantoio” and “Moraiolo” cultivars, were evaluated for NaCl tolerance. The aim was to use growth and physiological parameters in order to identify salt-adapted genotypes. Leaf tissue elemental distribution of Na, Cl and K was also investigated in unrooted plantlets by cryo-scanning electron microscopy and energy-dispersive X-ray microanalysis. Both in unrooted and rooted plantlets, increased concentrations of NaCl reduced shoot growth, whereas plant survival was not affected. However, no significant interactions between line and NaCl concentration were found. Elemental distribution showed that Moraiolo J and Frantoio Z accumulated more Na and Cl inside leaves, and that these elements followed a tissue-dependent pattern. Rooting capacity was reduced at the higher levels of NaCl. Significant interactions between seed line and salt treatment were found. Seed lines showed different abilities to develop roots at different salt levels. In particular, Frantoio Z showed a significant and different behaviour relative to the other seed lines at 50 mM NaCl with regard to both length and dry weight of roots. The results obtained suggest that rooting parameters are the most useful tools in the evaluation and screening of salt-tolerant olive genotypes through in vitro shoot culture.     

Histological and immunohistochemical studies on flower induction in the olive tree (Olea europaea L.)

The aim of this research was to study flower bud differentiation processes in two oil olive cultivars from Tuscan germplasm (Leccino and Puntino). The effect of fruit-set was studied using 'ON' (with fruits) and 'OFF' (without fruits) shoots. Axillary buds were periodically collected at different phenological stages, from endocarp sclerification (July) until budbreak in the following spring. Thin sections were analysed using histology (apex size), histochemistry (RNA, starch and soluble carbohydrates) and cytokinin immunocytochemistry (zeatin localisation). The micromorphological observations and histochemical procedures did not allow us to distinguish axillary buds sampled from 'ON' and 'OFF' shoots. Cytokinin immunocytochemistry revealed early different localisation patterns between 'ON' and 'OFF' samples. Zeatin accumulated only in 'OFF' axillary bud meristems, particularly in July, when endocarp sclerification of fruits from the previous flowering is taking place. At this time, a strong RNA signal was also observed. Both these signals were correlated with floral evocation, and their coincidence with a phenological stage of development provided a useful tool to determine the time when axillary buds switch from the vegetative to the reproductive phase.