Phenological growth stages of olive trees (Olea europaea)

This paper describes the phenological growth stages of olive trees using the BBCH (Biologische Bundesanstalt, Bundessortenamt, Chemische Industrie) scale. Eight principal growth stages for bud, leaf and shoot development, inflorescence emergence, flowering, fruit development, fruit maturity and senescence and 32 secondary growth stages are described. Advantages of the BBCH scale over other methods are discussed.     

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.     

Isolation and characterization of polymorphic microsatellites in olive (Olea europaea L.) and their transferability to other genera in the Oleaceae

Seven polymorphic microsatellites were developed in olive. Six of them came from a genomic library enriched for GA and CA repeat sequences. They showed single locus polymorphism in a set of 23 olive cultivars (from six to nine alleles per locus). Three different pairs of loci were sufficient to discriminate all cultivars. The other polymorphic primer pair was designed from a published sequence for olive lupeol sgutase and revealed just two alleles. The seven primer pairs were tested on two accessions of five other species of the Oleaceae and three, EMO2, EMO13 and EMO90, revealed polymorphism in two, four and three species, respectively.     

Changes in non-structural carbohydrates in olive (Olea europaea) leaves during root zone salinity stress

Self-rooted olive ( Olea europaea L.) plants were grown in hydroponics at various NaCl concentrations (from 0 to 200m M ) for 28 to 32 days followed by 28 to 30 days of relief from salinity over two growing seasons. Olive leaves accumulated both glucose and mannitol during the period of salinity stress. The concentrations of fructose, myo -inositol, galactose, galactinol, sucrose, raffinose, and stachyose were not significantly affected by salinity. Starch content was decreased by salinity. The mannitol/glucose and mannitol/soluble carbohydrates ratios increased as the external NaCl concentration was increased, but returned to the control levels during the relief period. The increase in mannitol or glucose molar concentrations, expressed on a leaf tissue water basis, was partially due to a reduction in leaf tissue water content under salinity stress. However, an increase in mannitol concentration was also observed when expressed on a dry weight basis. The accumulation of mannitol in leaf tissue preceded any reduction in leaf area rate or net assimilation rate. The increase in leaf mannitol or glucose concentration was positively correlated with the increasing level of salinity at the root zone, but not with the accumulation of Na⁺ in the shoot. The role of mannitol. a potential osmoregulator in leaf mesophyll during salinity stress, is discussed in relation to the complex carbohydrate composition of olive leaves.     

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     

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

Olive (Olea europaea L.) inflorescences, formed in lateral buds, flower in spring. However, there is some debate regarding time of flower induction and inflorescence initiation. Olive juvenility and seasonality of flowering were altered by overexpressing genes encoding flowering locus T (FT). OeFT1 and OeFT2 caused early flowering under short days when expressed in Arabidopsis. Expression of OeFT1/2 in olive leaves and OeFT2 in buds increased in winter, while initiation of inflorescences occurred i n late winter. Trees exposed to an artificial warm winter expressed low levels of OeFT1/2 in leaves and did not flower. Olive flower induction thus seems to be mediated by an increase in FT levels in response to cold winters. Olive flowering is dependent on additional internal factors. It was severely reduced in trees that carried a heavy fruit load the previous season (harvested in November) and in trees without fruit to which cold temperatures were artificially applied in summer. Expression analysis suggested that these internal factors work either by reducing the increase in OeFT1/2 expression or through putative flowering repressors such as TFL1. With expected warmer winters, future consumption of olive oil, as part of a healthy Mediterranean diet, should benefit from better understanding these factors.     

中国北亚热带油橄榄(城固32)开花生物学特性研究

为研究北亚热带内陆地区油橄榄（Olea europaea L.）开花物候、开花样式及花器官特征,探讨其有性繁殖系统的特点。试验对西秦岭南坡地区油橄榄（城固32）的开花生物学进行研究,统计开花进程,雄花和两性花在花序上的着生位置和数量,测定雄花和两性花形态指标并切片观察,电镜扫描分析雄花与两性花花粉粒。结果表明：（1）在北亚热带北缘内陆气候条件下油橄榄花期集中在5月,单株花期15~20 d,盛花期持续4~6 d;（2）油橄榄具有雄全同株的性系统且花器官较小,雄花及两性花都具有正常发育的雄蕊。通过制作石蜡切片观察,发现两性花的雌蕊正常发育,而雄花只有较小的子房和败育的柱头;（3）雌蕊是两种花型花器官生物量差异的主要部位,两性花雌蕊的生物量明显比雄花大（P<0.01）,树体分化出雄花投入的资源更少;（4）雄花与两性花在花粉量、花粉粒大小上差异不显著,在着生位置上,花序轴顶花全部为两性花,雄花出现在花序轴的中部和基部的几率分别是21.13%和30.77%。油橄榄品种城固32具有典型的雄全同株现象,雄花更倾向于在花序上出现的位置在行使它雄性功能方面并无优势可言,而且雄花花粉粒在数量、活力方面也没有优势,但是雄花的出现增加了花粉数量和P/O值,提高了植株的雄性适合度,雄花的出现也降低了两性花落花导致的树体资源浪费,保障其在资源有限的环境中能够繁殖最大化。     

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.     

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     

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.     

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     

Paternity tests support a diallelic self‐incompatibility system in a wild olive (Olea europaea subsp. laperrinei,Oleaceae)

Self‐incompatibility (SI) is the main mechanism that favors outcrossing in plants. By limiting compatible matings, SI interferes in fruit production and breeding of new cultivars. In the Oleeae tribe (Oleaceae), an unusual diallelic SI system (DSI) has been proposed for three distantly related species including the olive (Olea europaea), but empirical evidence has remained controversial for this latter. The olive domestication is a complex process with multiple origins. As a consequence, the mixing of S‐alleles from two distinct taxa, the possible artificial selection of self‐compatible mutants and the large phenological variation of blooming may constitute obstacles for deciphering SI in olive. Here, we investigate cross‐genotype compatibilities in the Saharan wild olive (O. e. subsp. laperrinei). As this taxon was geographically isolated for thousands of years, SI should not be affected by human selection. A population of 37 mature individuals maintained in a collection was investigated. Several embryos per mother were genotyped with microsatellites in order to identify compatible fathers that contributed to fertilization. While the pollination was limited by distance inside the collection, our results strongly support the DSI hypothesis, and all individuals were assigned to two incompatibility groups (G1 and G2). No self‐fertilization was observed in our conditions. In contrast, crosses between full or half siblings were frequent (ca. 45%), which is likely due to a nonrandom assortment of related trees in the collection. Finally, implications of our results for orchard management and the conservation of olive genetic resources are discussed.     

Abnormalities induced by agricultural pesticides in the microsporogenesis of olive tree (Olea europaea L.) cultivars

The study evaluated the microsporogenesis of olive trees subjected to different agricultural pesticide applications during flowering. Inflorescences of cultivars Arbequina and MGS GRAP541 were subjected to agricultural pesticides: mineral oil, neem oil, dimethoate and deltamethrin. The floral buds were fixed in Carnoy for the microsporogenesis analysis and in Karnovsky for scanning electron microscopy. The slides were prepared by squash technique and staining with propionic carmine. The pollen viability was determined by Alexander’s stain and in vitro germination. Results show that the quantification of abnormalities in meiosis in the two cultivars caused significant effect among the treatments, being that all differed statistically from the control group. Both methods showed a higher percentage of viable pollens in the control treatment and lower percentage of viability with the agricultural pesticides. The method of pollen viability by staining presented the highest averages of viable pollens, but when compared together, both methods presented a strongly related positive linear correlation. It was concluded that the used chemical products increased the percentage of chromosomal abnormalities during microsporogenesis, which interfered in the pollen viability of the two analyzed cultivars. The product deltamethrin caused the strongest effect on meiosis and on pollen viability.     

Genetic relationships in the olive (Olea europaea L.) reflect multilocal selection of cultivars

One hundred and two olive RAPD profiles were sampled from all around the Mediterranean Basin. Twenty four clusters of RAPD profiles were shown in the dendrogram based on the Ward’s minimum variance algorithm using chi-square distances. Factorial discriminant analyses showed that RAPD profiles were correlated with the use of the fruits and the country or region of origin of the cultivars. This suggests that cultivar selection has occurred in different genetic pools and in different areas. Mitochondrial DNA RFLP analyses were also performed. These mitotypes supported the conclusion also that multilocal olive selection has occurred. This prediction for the use of cultivars will help olive growers to choose new foreign cultivars for testing them before an eventual introduction if they are well adapted to local conditions. Received: 10 April 2000 / Accepted: 15 May 2000     

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.     

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