Strategies for Olive (Olea europaea L.) Breeding: Cultivated Genetic Resources and Crossbreeding

Abstract

Olive cultivars represent an invaluable heritage of genetic variability selected over more than 5500 years of cultivation . This high diversity of local cultivars is a common feature in traditional olive -producing countries. Most cultivars are old and continue to be cultivated around areas where they have likely been selected. Crossbreeding in olives was only initiated in the second half of the twentieth century and currently represents the most promising strategy to provide farmers with new cultivars that are well adapted to the new high density olive plantations spreading in traditional and new olive -growing countries. This chapter focuses on cultivated genetic resources and crossbreeding strategies in olive . Exploration, cataloguing and authentication for the conservation and sustainability of true-to-type cultivars by morphological and DNA markers in the Network of Germplasm Banks promoted by the International Olive Council, is the most extensive and worldwide initiative to date. The strategies, methodologies and advances in crossbreeding programs worldwide are reviewed. Shortening the juvenile period , early selection and other strategies for the evaluation of valuable agronomical traits are integrated into the framework of alternative protocols that also provide information regarding the variability and heritability of these traits. In addition, the possibilities provided by new genomics tools to shorten the protracted crossbreeding process are also presented. Finally, new developments on in vitro culture and genetic transformation as well as the feasibility of using these tools in breeding programs are discussed.

1 Introduction

Olive ( Olea europaea L.) is a Mediterranean tree species that was probably domesticated in the Middle East and in Central Mediterranean approximately 5500 years B.P. Since that time, the crop has expanded along both shores of the Mediterranean Sea. Olive growing was well-established in Roman times, as witnessed by the agricultural treatises of Pliny and Columela. Beginning in the fifteenth century, the transoceanic voyages of Christopher Columbus, Ferdinand Magellan and Juan Sebastián Elcano helped olives reach and spread throughout the New World. They are currently also grown in South Africa, China, Japan and Australia.

Empiric local selection within wild olives and crosses between the previous selected or introduced cultivars , and other local cultivars or wild olives, in all growing areas have yielded a huge number of local cultivars . This high diversity of local cultivars is therefore a common trait in traditional olive -producing countries. Most cultivars are old and are cultivated around areas where they were likely selected. In most cases, cultivars are self-rooted. Grafted trees are found only in the case of difficult-to-root cultivars or due to top grafting onto wild olives or onto other obsolete cultivars (Díez et al. 2015). Most cultivars are exclusively used for oil production . However, certain cultivars are used for table olives, and others may be used for both oil and table olives.

According to data published by the International Olive Council (IOC); (www.international.oliveoil.org), the world’s olive -growing area reached approximately 11.4 million ha in 2015, which was mostly located (>96%) in the Mediterranean Basin. Seventy-eight percent of this area is rainfed, and 22% is irrigated. Nonbearing trees represent 13% of the total orchards. The average production and consumption per year for the periods 2012/2013 to 2016/2017 was approximately 2,760,000 and 2,951,700 mt of olive oil , respectively. For table olives, the average production and consumption for the same period were 2,632,100 and 2,542,300 mt, respectively. Production and consumption are currently balanced; the large price variations between years and seasons are usually due to the biennial bearing habit of olive crops.

Olives have traditionally been a long-lived rainfed crop that is well adapted to the Mediterranean climate, which is characterized by a low and irregular yield , a high demand for labor for harvesting , empirical technology and low investment. However, since World War II, olive growing has been changing in both Northern Mediterranean countries and in new olive -growing regions. Human migration from rural to urban areas has required more productive and mechanized orchards. The accumulated changes since that time have led to new olive plantations (Fig. 14.1). These new olive orchards bear earlier; however, their overall life span is shorter than in the past. Improved agricultural practices to avoid soil erosion and contamination of the environment are increasingly being employed. Annual cultural practices are being simplified, and pruning and harvesting have become more mechanized. Yield has increased through the implementation of irrigation and high-density plantations (Fig. 14.1). In many countries, traditional and local practices of selection and propagation of cultivars by farmers are being replaced by a few cultivars multiplied by nurseries. Finally, olive oil is now considered an important agricultural product for health, and its consumption has steadily increased. Extensive and comprehensive books on olive growing have been published in Italy (Fiorino 2003) and Spain (Barranco et al. 2017). An earlier edition of the latter book has been translated into English (Barranco et al. 2010).

Fig. 14.1

Traditional and new olive plantations. a Traditional rainfed orchard on high-slope soil, b Traditional rainfed orchard on low-slope soil, c Intensive high density irrigated orchard, d Narrow hedgerow (super intensive) orchard, e Mechanical harvest in intensive orchard, f Mechanical harvest in super intensive orchard. Photos a, c, e, f by P. Valverde; b by D. Barranco; d by D. Cabello

During this time of change, the conservation and sustainable use of genetic resources and breeding have become necessary. For the first time in history, olive growers face an increasing risk of genetic erosion, and coordinated efforts to establish an International Network on Olive Germplasm Banks have been developed by the IOC since 1994 (Rallo et al. 2011). Additionally, the need for new improved cultivars has prompted the first olive -breeding programs in the world (Bellini et al. 2002a; Lavee 1990). Furthermore, recent advances in genomics and biotechnology (Rugini et al. 2016) will allow early genetic selection of progenies and the development of transformation protocols. This review focuses on the state of the art of the conservation of cultivated germplasm and breeding with a particular focus on crossbreeding and the pervasive concern of how to reduce the time needed to create and release new cultivars .

2 The Genetic Resources: First Strategy for Breeding

Exploration, conservation , evaluation and sustainable use of genetic resources have become a priority in most plant species of interest for agriculture and food. The diversity of cultivars in olive -growing countries represents a legacy of genetic variability from more than 5500 years of cultivation for the breeding and future growth of olives.

A recent, extensive and updated review of olive genetic resources (Belaj et al. 2016) stated, as a first conclusion, that “A better knowledge, management, and exploitation of cultivated, wild, and ancient trees are still needed, establishing common protocols for the molecular identification and for cultivar naming,”. This section will focus on the works exploring the conservation of true-to-type cultivars , which have been historically used as the first strategy for breeding. However, this critical aspect is still missing in olive cropping. Furthermore, true-to-type cultivars represent an urgent and compulsory requirement for olive growing in the world due to the global exchange of plant material. Within the requirement of trueness to type, we also review the works on clonal selection in olive .

For other aspects of the management of genetic resources , we refer the reader to the above-cited review (Belaj et al. 2016).

2.1 Exploring and Cataloguing Cultivars

To date, efforts to explore and catalogue true-to-type olive cultivars in different countries are incomplete. Until the beginning of the twentieth century, only a few scattered studies on olive cultivars had been published. At the VII Congreso Internacional de Olivicultura (olive growing ) held in Seville in 1924, Coupin proposed a world catalogue of cultivars , their agronomic evaluation , and the need to breed new cultivars . However, catalogues of true-to-type cultivars are still pending in many countries; the common drawback of establishing synonyms and homonyms has not yet been solved in most cases. Furthermore, agronomic evaluation of cultivars is fragmentary and confusing because traits are assigned to varietal denominations (accessions ) rather than to specific cultivars (Bartolini et al. 1998). As such, olive breeding is still in its infancy.

2.2 Olive World Germplasm Bank (OWGB) of Córdoba, Spain

The case of exploring and cataloguing true-to-type olive cultivars in Spain represent a long-lasting work on the exploration, cataloguing and conservation of true-to-type cultivars (Rallo et al. 2005). Initial works before the 1970s (Priego 1935) demonstrated incomplete sampling and a lack of representativeness of the characterized plant material, insufficient description methodologies and confusion in cultivar naming.

The Olive World Germplasm Bank of Cordoba (OWGB) was established in 1970 on the Alameda del Obispo Farm of INIA (National Institute for Agrarian Research ), as a first attempt toward the conservation of olive cultivated genetic resources through an FAO-INIA project. Since the beginning, this collection has been cared for by INIA and the University of Córdoba (UCO). The collection was enlarged through prospecting surveys and cataloguing of cultivars in Andalucía (Barranco and Rallo 1984), Spain (Barranco et al. 2005a) and other international prospecting surveys and by exchanges with other germplasm banks (Caballero et al. 2006). The Alameda del Obispo collection was incorporated into the Andalusia Institute for Agricultural and Fishing Research (IFAPA in 2003 (Fig. 14.2a) and new exchanges with other banks have increased its accessions (Belaj et al. 2016). In 2011, a duplicate of true-to-type cultivars of this collection was established in the UCO experimental Rabanales Farm in a soil free from Verticillium dahliae to guarantee the conservation of the cultivated genetic resources in the OWGB (Fig. 14.2b). In 2009, UCO, in cooperation with the CAP (Andalusia Agricultural and Fishing Administration) and IFAPA, established an isolated repository to conserve true-to-type and pathogen-free authenticated commercial accessions of the Alameda del Obispo collection (Fig. 14.2c). An agreement between the CAP, UCO and IFAPA for the management of the OWGB was signed in 2013. The OWGB was acknowledged by the International Olive Council (IOC) as an International Bank of Reference of its Network of Germplasm Banks in 2015.

Fig. 14.2

The Olive World Germplasm Bank (OWGB) of Córdoba, Spain. a Collection in Alameda del Obispo Farm (IFAPA), b Collection in Rabanales Farm (UCO), c Isolated repository of true-to-type and pathogen-free cultivars (CAP-UCO-IFAPA). See text above. Photos: a by P. Valverde; b, c by P. Morello

2.3 Identification and Authentication

The identification of existing cultivars represents the first step in their cataloguing. Naming is the main difficulty in olive -cultivar identification because it has been historically based on common morphological traits (particularly of the fruit ), toponyms or practical uses of varieties (Barranco and Rallo 1984; Barranco et al. 2005a). Until the 1980s, only morphological descriptors were used for identification purposes. The effect of environment on the expression of these traits was the main shortcoming for the use of these characteristics. However, morphological descriptors have been a useful tool for naming true-to-type cultivars , synonyms, homonyms and inaccurate denominations for the most explored and sampled genotypes in Andalusia and Spain (Barranco and Rallo 1984; Barranco et al. 2005a), also establishing criteria for unambiguously naming genotypes carrying the same name (homonyms). Both catalogues represent the most complete morphological characterization of Spanish cultivars listing true-to-type cultivar names, homonyms, synonyms and inaccurate denominations. To distinguish among different genotypes carrying the same name, these authors proposed to add to the generic name of the cultivar the site of main diffusion (e.g. Manzanilla de Sevilla, Manzanilla de Jaén and Manzanilla Cacereña). True-to-type cultivars catalogued in this work were planted in the ex situ collection at Alameda del Obispo in Córdoba.

The use of molecular markers for genotyping olive cultivars started with isozymes in the 1980s (Pontikis et al. 1980; Trujillo et al. 1995). Molecular markers have provided a powerful tool to manage ex situ germplasm and distinguish between genotypes. The advent of DNA markers and their use for genotyping olives started in the mid-1990s with random amplified polymorphic DNA (RAPD) (Bogani et al. 1994; Fabbri et al. 1995).

Belaj et al. (2016) reviewed the use and DNA markers for molecular identification , the most used tool for studies of genetic variability worldwide. Their use has facilitated the management of the OWGB (CAP-UCO-IFAPA) (Table 14.1), evidencing duplications, synonyms and homonyms (Atienza et al. 2013; Belaj et al. 2003a, c; Fendri et al. 2010; Noormohammadi et al. 2007; Trujillo et al. 2014). The complementary use of SSRs and morphological characteristics of the endocarp have proved to be a powerful and discriminant method to authenticate accessions (true-to-type cultivars ), duplications, homonyms, synonyms and inaccurate denominations in the OWGB of Córdoba (Trujillo et al. 2014). Comparison of endocarps between genotypes and true-to-type control samples allows the discrimination of molecular variants of a cultivar (i.e. genotypes with very high SSR similarity and undistinguished by endocarp morphological traits) and different cultivars (genotypes with a very high SSR similarity and very different endocarp characteristics) as shown in Fig. 14.3.

Table 14.1 Works on the exploration, cataloguing, conservation and authentication of olive cultivars jointly developed by the University of Córdoba and the Andalusia Institute for Agricultural and Fishing Research (IFAPA)

Fig. 14.3

Identification (a) and authentication (b) in the Olive World Germplasm Bank (OWGB) of Córdoba. Photo b from: Trujillo et al. (2014) used by permission of Springer

2.4 The International Olive Council (IOC) Network

Prospecting surveys in many countries and the exchanges of cultivars between countries have contributed to the very high number of conserved accessions in ex situ collections. Bartolini et al. (1998, 2008) reviewed for FAO the accessions conserved in approximately 100 regional and national collections in 53 countries, which include more than 4000 accessions . This inventory is indicative of the cultivated accessions conserved worldwide. However, the criteria for the choice of accessions are diverse, and their identification and authentication are still lacking in most collections. We currently do not know whether such accessions correspond to true-to-type cultivars and both corresponding homonyms, synonyms and inaccurate denominations. Consequently, data on the phenotyping of many agronomic traits in these collections are not concordant for the same denominations, thus generating confusion. This finding demonstrated a need to authenticate the accessions as a first step in any collection to avoid inconsistent phenotyping data in many publications based on the names of the accessions in any collection .

Since 1994, the IOC has promoted a network that currently includes National Germplasm Banks in 22 countries focusing on the exploration and conservation and cataloguing of local cultivars . The network conserves more than 1100 accessions , which were collected and have been partially characterized http://www.internationaloliveoil.org/resgen/index.html). The IOC published a first partial World Catalog of Cultivars (Barranco et al. 2000), an incentive publication of some additional national catalogues (Barranco et al. 2005a; Hosseini-Mazinani et al. 2013; Mendil and Sebai 2006; Moutier et al. 2004; Muzzalupo et al. 2010; Trigui et al. 2002, 2006).

The IOC has recognized three International Olive World Germplasm Banks in Córdoba, Spain, Marrakech, Morocco and Izmir, Turkey. These banks are enriched by the interchange of material with other banks, particularly those of the IOC Network. Currently, the OWGB of Córdoba conserves more than 1000 accessions from 25 Countries (Belaj and Barranco, pers comm), the OWGB of Marrakech conserves 591 accessions that have been partially identified (El Bakkali et al. 2013; Haouane et al. 2011) and the OWGB of Izmir has established 183 identified cultivars from 13 different countries to date (Gurbuz, pers comm).

Currently, the IOC aims to authenticate the accessions of all collections and to conserve them true-to-type and pathogen-free in all the acknowledged National Germplasm Bank of its network. Thus, the IOC has recently proposed a project to the UCO to cooperatively authenticate the accessions of the National Germplasm Banks by the morphological and SSR protocol proposed by Trujillo et al. (2014). This initiative will also guarantee the absence of devastating effects by diseases such as verticillium wilt (Jiménez-Díaz et al. 2012) and Xylella fastidiosa (Saponari et al. 2013). Thus, this project will ensure, over the short term, the conservation of true-to-type and pathogen-free of: (a) main commercial cultivars of the world in a first step. Afterwards other projects will progressively authenticated accessions in the IOC Networks.

2.5 Clonal Selection

Most of the traditional olive varieties grown today are the result of the selection of singular seedlings from wild, feral or cultivated trees with outstanding characteristics such as a larger fruit size, greater production , higher oil content or adaptation to certain climatic zones, among others, and the clonal propagation of those trees (Barranco and Rallo 1984). All current cultivars are in fact selected clones.

There is evidence that cultivars often contain somatic mutations. This phenomenon has been described in several fruit crops, such as grape , apple , peach , pear and plum (Badenes and Byrne 2012). Somatic mutations might not have phenotypical consequences but they might also become the basis for new varieties, as it has been the case for citrus and grape , among others (Moore 2001; Pelsy et al. 2010; This et al. 2006).

Somatic mutations have also been reported in olives by using different molecular markers such as RAPD , AFLP and SSR (Charafi et al. 2008; Cipriani et al. 2002; Diez et al. 2011; Garcia-Diaz et al. 2003; Nikoloudakis et al. 2003; Trujillo et al. 2014). These mutations were responsible for subtle genetic differences among several genotypes and their closest cultivar (Diez et al. 2011; Trujillo et al. 2014). No morphological differences were observed among these genotypes and they were considered molecular variants of the standard genotype (Trujillo et al. 2014).

Variability within a cultivar due to somatic mutations should not be confused with a case of homonym; this is the existence of different cultivars sharing the same name. Clonal mutations happen at a very low rate and normally affect few loci (Heinze and Fussy 2008; Sarkar et al. 2017) leading to very close genetic similarity indexes (similarity index ~0.8 to 0.9) between the clones and the cultivar from which they derived. Because of this reason, the description of extensive genetic variation within a putative cultivar (Gemas et al. 2004; Martins-Lopes et al. 2009) should be treated with caution. In these cases, an accurate genetic , morphological and agronomical characterization of the accessions involved (Ben-Ari et al. 2014) should be required to corroborate the clonal origin of the observed variability.

Sampling surveys aimed to look for phenotypic diversity within olive cultivars showed that intra-cultivar phenotypic diversity seems to be an unusual phenomenon in olive . Since the 1970s, clonal selections within the main olive cultivars have been performed, such as cvs. Manzanilla de Sevilla, Picual, Arbequina and Arbosana in Spain (Garcia Berenguer 1988; Suarez et al. 1990; Tous et al. 1999); Chemlali de Sfax in Tunisia (Kamoun et al. 2002; Khlif and Trigui 1990); Picholine Marrocaine in Morocco (Boulouha 1986); Moraiolo, Canino, Biancolilla, Giarraffa Moresca, Cresuola, Tonda Iblea and Nera in Italy (Caruso et al. 2014; D’Hallewin et al. 1990); Souri in Israel (Ben-Ari et al. 2014; Lavee et al. 2008); and Cobrançosa, Santulhana, Verdeal Trasmontana, Azeitera, Blanqueta, Carraquenha, Redondil, Galega Vulgar and Macanilha Algarvia in Portugal (Fernandes Serrano 1990; Gomes et al. 2008; Leitao et al. 1999; Martins et al. 1998).

All these works were based on the selection of outstanding trees for some agronomic traits , usually the production or fruit size, within major cultivars in various locations. Comparison of these phenotypic selections in one comparative field trial with few replications did not provide a consistent genetic basis for selection .

To date these works have provided few new commercial selected clones. To our knowledge, only Arbequina IRTA i.18^R (Tous et al. 1999, 2005) selected from cv. Arbequina trees in Catalonia and Haouzia, Menara and Dhabia cvs. selected from cv. Picholine Marocaine trees in three distinct locations (Amal Hadiddou, pers comm) have been commercially propagated. However, Arbequina i.18^R did not show any significant difference, with the standard Arbequina in long-term experiments at 7 and 14 years after planting (De la Rosa et al. 2007; Diez et al. 2016). Additionally, Amal Hadiddou (pers comm) did not observe significant differences between Haouzia, Menara Dhabia and Picholine Marocaine cvs. in yield and oil content in 5-year crops in a comparative field trial in Marrakech. In both cases, only minor differences for DNA markers leading to very close similarity indexes were detected. These differences were not associated with any morphological difference (Belaj et al. 2004a; Trujillo et al. 2014) (Fig. 14.3).

3 Crossbreeding

The first olive crossbreeding programs in the world were initiated between 1960 and 1971 in Israel (Lavee 1990) and Italy (Bellini et al. 2002a), respectively. Since the 1980s, new crossbreeding programs have been developed in different countries (Arias-Calderon et al. 2014; Bellini 1992; Dabbou et al. 2012; Lavee 2012, 2013; Lavee and Avidan 2011; Ozdemir et al. 2016; Rallo 1995; Rallo et al. 2008b; Trapero et al. 2013a; Zeinanloo et al. 2009). Most of the programs aim to obtain cultivars for olive oil , and only a few for table olives or both, with a special focus on early bearing, high yielding and adaptability to the new planting systems designed for mechanical harvesting (Rallo 2014a; Rallo et al. 2013). Recently, breeding programs are underway for resistance to verticillium wilt (Arias-Calderón et al. 2015a, b; Trapero et al. 2013a, b, 2015). Pre-breeding evaluation of cultivars and new genotypes from breeding programs for resistance to the bacteria Xylella fastidiosa are also under development (Landa B pers comm).

A previous revision of the olive -breeding protocol has been published (Rallo et al. 2011). Recently, Rugini et al. (2016) and Rugini and De Pace (2016) published two protocols focusing on biotechnological approaches.

3.1 Strategies for Crosses

The design of crosses requires previous knowledge of the genetics of the desired traits and information on pollen pistil compatibility. In their absence, the initial crosses of the current programs include progenitors with the desired phenotypic traits to incorporate or complement the other selected progenitors. In these programs, the progenitors are cultivars derived from the same or different regions of origin . Recently, crosses of cultivated olive ( Olea europaea ssp. europaea var. europaea) with wild olive (O. europaea ssp. europaea var. sylvestris) and with other subspecies (O. europaea ssp. cuspidata and O. europaea ssp. laperrini) have been reported (Caceres et al. 2015; Klepo et al. 2014; Trapero et al. 2015), with the aim of enlarging the basis of genetic variability for olive breeding.

Olive is an allogamous species, in which self-incompatibility is the general rule with some cases of inter-incompatibility (Díaz et al. 2007; Koubouris et al. 2014; Rodriguez-Castillo et al. 2009; Seifi et al. 2011; Selak et al. 2014; Wu et al. 2002). The pollen tube of alien pollen grows faster than the pollen of the same genotype , triggering an earlier growth of both the seed and the fruit in cross-pollination than in self-pollination (Rallo et al. 1990). Therefore, emasculation it is usually not necessary for performing crosses. However, there may be some problems related to contamination by undesirable alien pollen due to the fast and long distance airborne transport of pollen that may have contaminated the limbs before they were bagged (De la Rosa et al. 2004; Díaz et al. 2007; Rallo 2000b). Currently, paternity testing by different SSRs is being used in breeding programs (De Rosa et al. 2013; Díaz et al. 2007), representing a compulsory step for any genetic study.

3.2 Strategies to Shorten the Juvenile Phase

The time elapsed from seed germination to first flowering of a seedling is known as the juvenile phase (JP). This phase is usually very long in woody species and represents one of the major drawbacks for olive tree breeding by crossings since the JP in this species can reach up to 15–20 years (Bellini 1992; Fontanazza and Baldoni 1990). For example, in an olive crossbreeding program initiated in the early 1970s in Italy, many seedlings were still juveniles 25 years after planting (Bellini et al. 2002a). The cost of maintaining in the field a large number of trees that will take many years to flower is extremely high. Consequently, many efforts have been undertaken in different olive -breeding programs to shorten the JP to initiate evaluations of fruit traits in the progenies as soon as possible (Lavee et al. 1996).

The first strategies to shorten the JP consisted of forcing the growth of the seedlings to achieve a certain size in a short time, since the onset of first flowering in many woody species is reported to be related to the size of the plant (Hackett 1985). Santos-Antunes et al. (1999, 2005) established a forced-growth protocol in which seedlings are grown under continuous light and fertigation in the greenhouse for approximately 7–8 months, after which the plants are transplanted to field conditions. This protocol has been shown to be very efficient, obtaining approximately 50% of flowering seedlings by the third year (Fig. 14.4). Other management techniques to reduce JP have been studied regarding plant canopy height (Moreno-Alías et al. 2010a) or soil solarization (El Riachy et al. 2011), among other traits.

Fig. 14.4

Steps to shorten the juvenile period . a Cross, b Seed germination , c Forcing seedlings growth in greenhouse under continuous light, d Forcing growth in field until first flowering and fruiting. Photos by P. Valverde

The relationship between seedling growth traits and the time of first flowering has been widely studied (De la Rosa et al. 2006; Pritsa et al. 2003; Rallo et al. 2008b; Santos-Antunes et al. 2005) to establish preselection criteria to cull seedlings with a long juvenile period in early stages, before transplantation to the field. Seedling vigor traits, mainly plant height and stem diameter, measured after the forced growth period in the greenhouse are strongly correlated to the earliness of first flowering and, thus, are currently used in breeding programs to preselect the seedlings to be planted. Threshold values based on a specific seedling height (approximately 100 cm; Moreno-Alías et al. 2010b) have been proposed, whereas Rallo et al. (2008b) suggest culling up to 35–40% of the shortest seedlings since growth performance may vary among progenies and years.

Differences in the earliness of first flowering among progenies have been reported, indicating a strong influence of the genitors on the length of the JP of the progenies (Bellini et al. 2002a; De la Rosa et al. 2006; Moral et al. 2013; Rallo et al. 2008b; Santos-Antunes et al. 2005; Suárez et al. 2011). Thus, parental selection of certain genitors is a good strategy to accelerate first flowering in progenies. Furthermore, the unproductive period of genotypes in the adult stage has been positively associated with the JP of the same genotypes (Leon et al. 2007). Therefore, early bearing is enhanced when selection for a short JP is performed, and similarly, the use of early-bearing parents will help to reduce the length of the JP of progenies.

The transition from the juvenile to the adult stage is not yet well understood in the olive tree. As described by Hackett (1985) and Hartmann et al. (2002), phase changes in woody species follow a cone-like pattern, known as the juvenility cone, during which the basal and inner parts of the adult seedling remain juvenile. In the case of the olive tree, the position of the first flower in seedlings has been studied as a marker of the end of the JP, and a juvenile cone has been confirmed in this species (Moreno-Alías et al. 2010a; Suárez et al. 2011). The attainment of a certain distance from the root to the meristem is required to flower, which was determined at approximately 200 cm for olive by Moreno-Alias et al. (2010a), although significantly different values among progenies were reported by Suarez et al. (2011). The latter authors also found correlations with the length of the juvenile period such that early flowering genotypes required a lower minimum distance to the ground to overcome juvenility .

In addition to the onset of flowering , other markers of the phase change from the juvenile to the adult stage have been studied, including proteins, morphological and anatomical traits and NIR (near-infrared) spectra of leaves, and rooting ability (Casanova et al. 2014; Garcia et al. 2000; Leon and Downey 2006; Moreno-Alías et al. 2009). Recently, much effort is being focused on the discovery of genes involved in the transition from juvenile to the adult stages and in flowering in olive species (Fernández-Ocaña et al. 2010; Garcia-Lopez et al. 2014; Haberman et al. 2017; Jiménez-Ruiz et al. 2015; Muñoz-Mérida et al. 2013; Sgamma et al. 2014). A better understanding of the complex genetic control of the juvenile-adult phase change will elucidate new avenues for the development of molecular tools for early transcriptomic selection of short JP seedlings.

3.3 Strategies for the Evaluation of Genotypes: Crossbreeding Steps

Crossbreeding in fruit trees is an extended process due to the length of the JP and the required multistep protocol to evaluate seedling progenies and their successive clonally-propagated selections (Hancock 2008). After crossbreeding pollination , seed germination and forced seedling growth to shorten the JP, selected genotypes are submitted to several selection steps for further evaluation . In each step, the number of genotypes is reduced and the number of replications per genotype and characters under evaluation increases up to the final release of new cultivars . The total number of evaluated genotypes and selection steps are usually determined by the available human and financial resources and facilities.

Figure 14.5 shows a general overview of the selection steps followed at the Spanish olive -breeding programs : UCO-IFAPA (University of Córdoba- Andalusia Institute for Agricultural and Fishing Research ) and US (University of Sevilla).

Fig. 14.5

Proposed breeding scheme in olive breeding programs in two (blue arrow) or three (red arrows) selection steps. JP: Early selection for the short juvenile phase

Two alternative protocols are used. Two steps of evaluation in the first crosses (Rallo et al. 2016a; Fig. 14.5 blue arrow): (1) for progenies, and (2) a final step for selected genotypes. As an example, 15 of 748 genotypes were selected in the first step of evaluation , and one new cultivar (Sikitita/Chiquitita) was registered after a comparative trial, 15 years after plantation of the progenies (Rallo et al. 2008a). After some additional comparative trials, two selections will soon be registered. In the USA’s table olive breeding program, 40 of 1800 genotypes were selected in the first step, 20 of which are currently in field trials for final evaluation (Rallo P, pers comm).

The second protocol proposes three steps (Leon et al. 2015; Figure 14.5 (red arrows): (1) initial progenies, (2) intermediate for preliminary selections and (3) final for advanced selection . Following this protocol, 108 of 1548 genotypes were selected in the first step, 14 of which remained after the intermediate step (15 years after plantation of the progenies). The final evaluation of the 14 advanced selections in comparative field trials ended approximately 20 years after planting of the seedlings.

These data indicate that the choice between both protocols will depend on available human, financial and logistic resources. Cooperation with stakeholders may facilitate a two-step protocol and accelerate the breeding process (Fig. 14.7c).

3.3.1 Step 1: Initial (Evaluation of Progenies)

Initially, the breeder has to manage a large number of non-replicated genotypes per cross. Rigorous criteria and techniques for early or indirect selection according to the main objectives of the program must drastically reduce the initial population of genotypes in this first step to save time, labor and money. The earliness of bearing, oil content and composition and adaptation to new plantation systems are systematically evaluated in all progenies in all crosses of the UCO-IFAPA Breeding Program (Rallo et al. 2016a).

Early criteria for selection in the first period of seedling growth must be a general concern in any olive breeding program. Early selection tests have also been an excellent and general strategy to shorten the JP and to eliminate late flowering genotypes in any cross (see Sect. 14.3.2). Additionally, early selection for Verticillium dahliae resistance has be conducted in 2-month-old seedlings, eliminating more than 90% of them for later evaluation in field plantations in highly infested soils (Trapero et al. 2013a) (Fig. 14.6). Currently, newly-developed genomic tools are providing some efficient tests for early genetic -based selection of relevant agronomic traits (see Sect. 14.4).

Fig. 14.6

Two-step selection for verticillium wilt resistance . a Young orchard devastated by this disease, b Evaluation of inoculated seedlings in greenhouse, c Evaluation of pre-selected seedlings in field. Photos: a by C. Trapero; b, c by P. Valverde

The evaluation of progenies usually persists for 5 years, i.e. the three first crops after overcoming the JP. Many traits have been evaluated in the initial step. Earliness of bearing (see Sect. 14.3.2), plant architecture , oil content and composition (see Sect. 14.3.6) of breeding oil cultivars are usually assessed in the initial step based on values correlated with those in subsequent steps and on moderate to high heritability values (Ben Sadok et al. 2013, 2015; Leon et al. 2015). Additionally, different fruit traits have also been accurately evaluated during this initial step for breeding table olives (see Sect. 14.3.6).

The simplification of recording and the use of suitable methods to evaluate small samples have been used in the initial step. Categorical data for the number of flower seedlings have demonstrated a fast, easy and reliable record for earliness of bearing that has been routinely adopted for seedling evaluation (Leon et al. 2015). Non-destructive and multicomponent trait evaluation by NIR spectrometry has allowed the evaluation of oil content , fruit traits, ripening index and oil composition in a fast and accurate manner (Bellincontro et al. 2013; Leon et al. 2003, 2004a; Morales-Sillero et al. 2011) in small samples, particularly in fruits, allowing an efficient way to evaluate the progeny variability in any segregating population for multiple traits.

The evaluation of progenies also represents a basic step for genetic and genomic studies that are undergoing active development (see below and Sect. 14.4).

3.3.2 Step 2. Intermediate (Evaluation of Preliminary Selections)

Vigor and yield are critical traits for final selection . Intermediate steps attempt to evaluate these traits with some replicates to first obtain and compare records for preliminary selections that cannot be accurately evaluated in the initial step. Additionally, the intermediate step may confirm the consistency of previously evaluated traits in the initial selection step.

Correlations between data recorded for seedling and intermediate steps indicate that selection for the earliness of bearing, fruit size and oil content can be efficiently performed at the seedling stage, whereas evaluation at the intermediate step would be necessary to select for yield and vigor . Similar conclusions were obtained from analyses of heritability estimated for these characteristics at the intermediate step of selection . Additionally, a high number of genotypes was retained in the intermediate step, allowing large variability for both previously evaluated and non-evaluated traits such as fatty acid composition, tocopherol and phytosterol contents and the phytosterol profile (Leon et al. 2015).

3.3.3 Step 3. Final (Evaluation of Advanced Selections)

Genotype × environment (G × E) evaluation is the main objective of the final evaluation of advanced selections before new cultivars are registered. In addition to vigor and yield , the most important traits in any new cultivar, other traits related to the breeding objectives must be re-evaluated at this step in several locations. Therefore, cooperation with growers in different sites provides information about the selection performance for different environments to other growers from those zones, thus promoting new potential registered cultivars . The UCO-IFAPA program has developed this strategy since the first crosses. Currently, more than a dozen stakeholders, including nurseries, participate in this step of evaluation .

Different field experiments corresponding to initial, intermediate and final evaluations are shown in Fig. 14.7.

Fig. 14.7

Field evaluation steps for crossbreeding . a Initial evaluation of unreplicated genotypes from progenies, b Intermediate evaluation of preliminary soft selection , many genotypes with some replications, c Final selections of genotypes in a comparative precommercial field trial. Photos: a, b by P. Valverde; c courtesy of TODOLIVO

3.3.4 The Components of Variability

Research on the genetics of agronomic traits in olive is in its infancy. Nevertheless, studies examining the components of genetic variability in cultivars and genotypes from crossbred progenies have been increasing in recent years. Several patterns have been observed in these studies.

A large variability among genotypes has been a general pattern for any evaluated trait in cultivars from germplasm banks and in genotypes from any crossbred progeny . This is the case for all reported traits in this review (Sects. 14.3.2, 14.3.4–14.3.6). As a rule, the values of the descendants transgress the genitor values in the segregating progeny . In all analyzed models, the genetic factor within crosses is the main component of the variability of the evaluated traits, as expected due the extensive heterozygosity of this species. This is a favorable situation from a clonally-propagated species such as the olive .

Few studies have examined heritability in olive . Hassani and Tombesi (2008) measured the growth characteristics in the greenhouse of a full diallel cross among nine cultivars . The heritability of characters such as dry weight and internode length in progenies indicates high values, (0.75 and 0.97), respectively. Zeinanloo et al. (2009) designed one-way diallel crosses with six genotypes for nine quantitative characteristics. Broad sense heritability ranged from 0.31 to 0.86 and narrow-sense heritability from 0.17 to 0.28. Zard and Roghani cvs. were good combiners for fruit weight, percentage of dry matter, fruit length, fruit width, and percentage of oils.

Architectural traits have attracted attention due to the search for compact and early bearing trees for new high-density systems. Hammami et al. (2011, 2012) proposed five traits (main vertical axis, preferential distribution of lateral shoots, dominant length of lateral shoots, branch orientation and branch bending) as the most relevant descriptors for the olive seedling architecture based on the high capacity to indicate diversity , a strong influence of the parent genotype , a lack of correlation with each other, and a demonstrated value for agronomic performance. They also showed that cvs. Picual, Arbosana and Sikitita were promising cultivars for use as genitors because of their tendency to produce offspring with desirable growth habit traits.

Ben Sadok et al. (2013, 2015) studied the heritability of tree architecture and reproductive traits in a progeny of 120 trees from the Oliviere × Arbequina cross in two contrasting growing sites. After selection of a model, broad sense heritabilities were estimated. Despite strong environmental effects on most traits, no G × E interaction was found. Moreover, the internal structure of the trait covariation was similar in both sites. Ontogenetic growth variation, related to (i) the overall tree form and (ii) the growth and branching habit at growth unit scale was not altered by the environment. Finally, moderate to strong genetic control was identified for traits at the whole tree scale and at the internode scale. Among all studied traits, the maximal internode length exhibited the highest heritability (H2 = 0.74).

A detailed variance analysis of oil content components and fruit morphology traits in an olive progeny issued from the cross Oliviere × Arbequina (Leon et al. 2016) showed the lowest values of heritability for fruit moisture (0.40) and the highest values for oil content in fruit fresh weight (0.94). An estimation of heritability and environmental variation over varying numbers of years and tree replications revealed that the inclusion of more annual replications could be more effective than the addition of tree replications. Finally, this study provides a global view of adequate spatial and temporal replications required to accurately develop criteria for both conventional and marker -assisted selection on fruit traits from olive crossing progenies.

An evaluation of resistance to the defoliating pathotype of Verticillium dahliae Kleb was performed in 12 cultivars and 52 genotypes previously selected from a wider initial population based on their agronomic performance (Arias-Calderon et al. 2014) and 6017 genotypes derived from 48 crosses obtained by open pollination and crosses between olive cultivars , wild olive genotypes and other Olea species and Olea europaea subspecies (Trapero et al. 2015). In both studies, high genetic variability and wide segregation of resistance were observed. The estimates of heritability suggest that it is possible to breed for verticillium wilt resistance in the olive .

3.4 Strategies for Tolerance to Abiotic Stress Selection

Environmental adaptation is a cornerstone to successfully grow a crop in a specific region. Olive cultivars that are able to tolerate many abiotic limiting factors such as cold, heat, water stress or nutrient-deficient soils can be found due to unintentional selection over thousands of years. However, these cultivars often lack other desired agronomic characteristics. Although the combination of both characteristics seems possible, the development of new cultivars with increased tolerance to abiotic stresses is not a major objective in current olive breeding programs worldwide, and research studying the genetic variability of tolerance to abiotic stresses is generally not extensive. Rootstock breeding also appears to be an opportunity to overcome some of these abiotic stresses.

Olive growth and productivity can be seriously limited by low temperatures, especially temperatures below −7 °C (Palliotti and Bongi 1996). Physiological responses related to cold damage in olive have been identified (Ruiz et al. 2006), and methods to quickly assess tolerance to damaging temperatures have also been established (Azzarello et al. 2009). This screening method, combined with the existence of genetic diversity regarding tolerance to cold conditions (Barranco et al. 2005b; Bartolozzi and Fontanazza 1999; Gómez-del-Campo and Barranco 2005), suggests that breeding for this trait should be feasible.

Chilling requirements in olive is an increasing limiting factor because of the expansion of olive plantations to new areas (Rapoport 2014), as well as the global temperature increase in established olive growing areas (Gabaldón-Leal et al. 2017). This trait is genetically dependent on field observations (Hartmann and Porlingis 1957; Zouari et al. 2017) and modeling (Garcia-Mozo et al. 2009). A method to determine the dormancy period has been recently developed (Ramos et al. 2018) and could accelerate the identification of previously initiated olive genotypes with low-chilling requirements (Cabello, unpublished data).

Olive is quite tolerant to drought stress in comparison to other tree species (Fernández and Moreno 1999). Some of the physiological factors that control drought tolerance are known (Sofo et al. 2004), and an important array of studies have reported genetic differences between olive cultivars in terms of tolerance to drought (Bacelar et al. 2009; Guerfel et al. 2007; Tugendhaft et al. 2016). Wild germplasm has also been identified as a possible source of tolerance to this abiotic stress (Besnard et al. 2012). Prospects for the breeding of this trait in the future are positive due to the development of a technique using chlorophyll fluorescence to screen for this factor (Faraloni et al. 2011) and the identification of a gene that enhances drought tolerance (Chiappetta et al. 2015). Tolerant genotypes may be used as rootstocks to alleviate this problem.

Chlorosis due to iron deficiency can result in important yield losses. This condition is common in plants growing in calcareous soils. Although olive species are considered quite tolerant in comparison to other plant species (De la Guardia and Alcántara 2002), they are grown in calcareous soils throughout the world and are therefore affected by iron chlorosis. Olive cultivars present notable genetic differences in their tolerance to this abiotic stress (Alcántara et al. 2003). As in other species, tolerance can be achieved by the use of rootstocks. A few other studies have reported genetic variability in the tolerance to other abiotic factors such as salinity (Marin et al. 1995) or heat stress (Mancuso and Azzarello 2002), although sources of tolerance have not been deeply investigated.

3.5 Strategies for Resistance to Biotic Agents

Diseases and pests have always been limiting factors for olive growing . Olive genotypes with a higher level of resistance to major pests and diseases have been selected intentionally and unintentionally for a long time, and therefore sources of resistance to a range of pests and diseases are available, as shown in Table 14.2. However, their use in breeding programs is limited due to other agronomic and adaptive traits that normally comprise the main breeding objectives (Leon et al. 2007). Breeding for disease or pest resistance aims to combine the best agronomic and adaptation traits with resistance to the desired disease or pest in a single genotype . It is a slow process due to the time necessary for plant generation evaluations, as well as the lack of knowledge about the mechanisms and genetics underlying the resistant expression (Johnson and Jellis 1992).

Table 14.2 Sources of resistance available against the major diseases and pests that affect olive and their current situation in breeding programs

Verticillium wilt of olive is a vascular wilt caused by the soil-borne fungus Verticillium dahliae. It has recently become the main disease in many olive -producing countries and is threatening olive production in many areas (Jiménez-Díaz et al. 2012; López-Escudero and Mercado-Blanco 2011). Factors such as the inefficacy of chemical compounds to control the disease make the use of plant material that is resistant to verticillium wilt especially important. However, most of the olive cultivars evaluated to date have been identified as susceptible or extremely susceptible to this disease in the field (Trapero et al. 2013b) and under controlled conditions (López-Escudero and Mercado-Blanco 2011). For many years, efforts to develop new olive material that is resistant to V. dahliae have been quite limited (Hartmann et al. 1971; Wilhelm and Taylor 1965). However, due to recent increases in the importance of the disease in the last 10 years, verticillium -wilt resistance has been incorporated into breeding programs , sometimes as a major objective. Mass screening methodologies (Trapero et al. 2013a) have allowed the evaluation of large numbers of genotypes generated by open pollination and targeted crosses in an attempt to combine high levels of resistance and positive agronomic traits (Arias-Calderón et al. 2015b; Colella et al. 2008; Trapero et al. 2015). According to these studies, complete resistance to verticillium wilt in olive is unlikely to exist, although high levels of resistance have been identified in olive cultivars , wild populations and related Olea species (Table 14.2). New varieties with higher level of resistance are likely to be generated in the upcoming years, once they have been assessed for a number of years in fields infested by the pathogen. The selection of undomesticated material that is resistant to disease for use as rootstock should first be evaluated for efficacy for controlling the disease. Verticillium wilt resistance is believed to be highly polygenic (Leyva-Pérez et al. 2017) and molecular tools to select for this trait are currently not available.

The main foliar diseases attacking olive are peacock spot (caused by Venturia oleginea), anthracnose (Colletotrichum spp.), and cercospora leaf spot (Pseudocercospora cladosporioides). These pathogens cause tree defoliation, premature fruit drop and fruit rot, which can be devastating under favorable weather conditions (Viruega et al. 2011). Although traditionally managed by cultural and chemical methods, genetic resistance has been demonstrated to be highly effective, especially when disease pressure is high. The identification of sources of resistance has been based on both field assessment (Moral and Trapero 2009) and artificial inoculation methods (López-Doncel et al. 1999). Genotypes generated from crossings between varieties with different levels of resistance have been selected in several breeding programs (Ciccarese et al. 2002; Moral et al. 2015; Rhouma et al. 2013) and one cultivar has been released with a reported high level of resistance to peacock spot generated by the self-pollination of a moderately resistant cultivar (Lavee et al. 1999). The inheritance of resistance to these diseases is likely to be mostly polygenically controlled according to the cited studies.

Olive knot is a disease characterized by the formation of cankers on the olive trunk, branches and shoots. It is caused by the bacteria Pseudomonas savastanoi pv. savastanoi. Although it is not a major objective in olive breeding programs , susceptible genotypes are usually identified in the field and culled. By using inoculation methods under controlled conditions, (Marcelo et al. 1999; Penyalver et al. 2006) identified a high level of resistance among olive cultivars (Young et al. 2004) that could be useful for breeding.

Xylella fastidiosa is a bacterium that infects and colonizes the xylem of a broad range of plant species and causes leaf scorch and quick decline syndrome in the olive . This disease was of little importance to olive until the recent outbreak in southern Italy (Saponari et al. 2013). Currently, the level of resistance of different cultivars is largely unknown, apart from recent field observations and studies using a small number of cultivars (Frisullo et al. 2014; Luvisi et al. 2017), which suggest a potential variability in resistance to the pathogen. Xylella fastidiosa is currently a major concern in Europe as it is likely to spread to other growing regions (Stokstad 2015). Therefore, breeding efforts are likely to arise in the near future.

The olive fly (Bactrocera oleae) is the major pest affecting olive worldwide, causing severe damage to fruits and reducing their quantity and quality (Daane and Johnson 2010). Differences in the resistance level of a number of olive cultivars have been reported in several studies using preference or oviposition experiments (Garantonakis et al. 2017; Gonçalves et al. 2012; Iannotta et al. 2007; Malheiro et al. 2015). Fruit traits such as size, maturity date and hardness usually explain a great proportion of the level of resistance , although Grasso et al. (2017) have shown that resistance to olive fly can also be induced. To date, resistance to this pest has not been targeted as a major objective by any olive breeding program.

3.6 Strategies for Quality

Quality is becoming a prime target for plant breeders since consumer demand is moving toward food products with improved sensorial, nutritional and nutraceutical value. Table olives and virgin olive oil , the two main products obtained from olive fruits, are staple foods of the Mediterranean diet, and the benefits of their consumption in human health have been extensively documented. Additionally, their extraordinary organoleptic properties are also responsible for their increasing demand worldwide (IOC 2017).

Olive quality is a wide and complex concept involving many traits that may be important for table olives, olive oil or both (Rallo et al. 2011, 2017). For instance, fruit appearance is essential for table olives, which encompass traits such as fruit size, shape, symmetry, color or absence of bruising, whereas oil content components and fatty acid profiles are particularly important for olive oil . Recently, a special emphasis has been placed on both products with respect to minor bioactive compounds such as tocopherols, phenolics , squalene, sterols or triterpenic acids, among others. Table 14.3 lists quality traits that have been evaluated in olive -breeding programs based on fresh unprocessed fruits, oil or processed table olives. High levels of variability have been observed for most of these traits, with cases of transgressed segregation and significant differences among crosses and/or genotypes within crosses (Arias-Calderon et al. 2014; El Riachy et al. 2012a; Lavee and Avidan 2011; Medina et al. 2012; Perez et al. 2014, 2016).

Table 14.3 Examples of quality traits evaluated in olive breeding programs

Breeding for quality in olive is more complex than in other fruit species since the olive drupe may not be consumed directly but may need to undergo industrial processing to obtain table olives or olive oil . Although some of the abovementioned quality traits may be evaluated in fresh unprocessed fruits, most should be assessed in the final elaborated products. This procedure hampers olive breeder work, especially in the early stages of evaluation , because processing a large number of genotypes is time-consuming and expensive. Furthermore, the low yield of genotypes in this stage limits the amount of fruit required for processing . In this regard, the strategy applied to correlate traits in fresh fruit with quality traits of the final product, for indirect selection based solely on fresh olive evaluation , has been studied. High correlations have been found between fruit flesh and extracted oils for the main fatty acids, tocopherols, sterols and squalene (De la Rosa et al. 2016; Velasco et al. 2014), and between oleuropein content in fresh fruit and different quality traits of Spanish-style green olives and black olives (Rallo et al. 2018 P, pers comm).

The complex methodologies needed to assess many of the abovementioned quality traits , along with the large number of genotypes to be screened, have encouraged olive breeders to explore the use of alternative fast and non-destructive methods, such as NIR spectrometry, to evaluate both olive oil and table olive quality traits (Giovenzana et al. 2015; Leon et al. 2003, 2012; Mailer 2004; Morales-Sillero et al. 2011). Good calibration models have been obtained for fruit weight, diameter and volume, flesh texture , oil content , moisture content, chlorophyll pigment, oleic and linoleic fatty acids.

The recent detection of loci associated with interesting quality traits (fruit weight, flesh/stone ratio, oil traits, fatty acid composition, among others) via classical QTL mapping approaches (Atienza et al. 2014; Ben Sadok et al. 2013; Hernandez et al. 2017) or through genome-wide association studies (GWAS) (Kaya et al. 2016) will soon enable marker -assisted breeding for olive quality , allowing very early selection of outstanding genotypes at the juvenile seedling stage.

4 Genomic Tools for Olive Breeding Programs

Despite the remarkable recent development of olive breeding programs , breeders lack the capacity to generate cultivars quickly in response to new growing systems, evolving consumer preferences and crises. Olive breeding is still based on classic methods using directed crosses between suitable cultivars followed by selection within the progenies, and finally the cloning of outstanding individuals. Non-conventional breeding approaches, such as protoplast technology or the selection of mutants from induced in vivo mutagenesis , have also been applied to olive with variable levels of success (Rugini et al. 2016). However, to our knowledge only cv. Briscola (Roselli and Donini 1982) obtained by gamma rays mutation of cv. Ascolana Tenera was released for its ornamental value. Thus, this section will be focused on genetic and genomic approaches that could accelerate classical breeding methods in the near future.

The olive tree is an extremely heterozygous species with high genetic variability. Thus, seedlings from breeding programs have been shown to undergo segregation for all the agronomical characters evaluated to date (Rallo 2014a, b), even the frequent presence of individuals showing transgressive segregation (seedlings with trait values exceeding the values of their genitors) (de la Rosa et al. 2016; Leon et al. 2004b; Trapero et al. 2015). Given this remarkable variability, breeding new olive cultivars with improved characteristics requires the germination and evaluation of thousands of seedlings. For example, more than 10,000 genotypes have been evaluated to obtain a new cultivar, two promising advanced selections and more than 30 preselections with high production , early bearing, and adaptation to mechanical harvesting and high-density at the UCO-IFAPA breeding program (Rallo et al. 2016a). In addition, olive -breeding cycles are extremely long (>12 years), mostly due to the length of the JP of long-lived perennials (van Nocker and Gardiner 2014). From an agronomic perspective, JP is the period between seed germination and first flowering , which are influenced by both environmental and genetic factors. As mentioned in Sect. 14.3.2, the length of the JP is inversely correlated with plant vigor . This association has led to the culling of approximately 40% of plants with a predictably long JP a few months after their germination (De la Rosa et al. 2006; Rallo et al. 2008b). Thus, the height of seedlings is currently the most effective early selection marker for the short juvenile phase in olive (Rallo 2014a, b). New biotechnological tools that are able to produce precocious flowering of juvenile plants via the action of the viral vector might also help to accelerate the breeding process of perennial crops in the near future (Haberman et al. 2017; Velázquez et al. 2016). Therefore, understanding the mechanisms controlling the JP transition in perennials and the detection of additional early selection markers related to other agronomic traits are important requirements to advance olive breeding.

The main use of genomics in breeding is marker -assisted selection (MAS ) for traits controlled by major genes or quantitative trait loci (QTLs). By MAS , genetic markers that either are known to cause a phenotype or are strongly linked to the causal genetic variant can be genotyped at the seedling stage, allowing a prediction of the phenotype of the adult plant. The construction of a high-density linkage map is a necessary prior step to localize QTLs and genes controlling agronomic traits . Several olive linkage maps have been developed using the following molecular markers and cultivar progenies: RAPD , AFLP , RFLP and SSR —Leccino × Dolce Agogia (De la Rosa et al. 2003); RAPD , SSR and SCAR —Frantoio × Kalamata (Wu et al. 2004); AFLP and SSR Picholine Marocaine × Picholine du Languedoc (Aabidine et al. 2010); AFLP , ISSR and SSR Oliviere × Arbequina (Aabidine et al. 2010); DArT and SSR —Picual × Arbequina (Domínguez-García et al. 2012); SNP —F2 from the selfing of Koroneiki (Marchese et al. 2016); and SNP , CAP and SSR —Gemlik × Edincik Su (İpek et al. 2017). These mapping approaches, combined with the phenotypical characterization of genitors and progenies, have allowed the location of QTLs related to flowering and fruiting traits (Ben Sadok et al. 2013), oil content , moisture, ratio pulp to stone, fruit weight and trunk diameter (Atienza et al. 2014) (see also Sect. 14.3.4).

The development of new high-throughput sequencing techniques (also known as next-generation sequencing ) has resulted in an increase in studies focused on olive transcriptomics. These studies have led to the identification of expressed sequence tags (ESTs) (Alagna et al. 2009), the assembly and annotation of the olive transcriptome (Muñoz-Mérida et al. 2013), the assessment of differential expression patterns among tissues and plant treatments (Bazakos et al. 2012, 2015; Carmona et al. 2015; Guerra et al. 2015; Jiménez-Ruiz et al. 2015) and the generation of saturated linkage maps (İpek et al. 2017; Marchese et al. 2016). Many of these studies have ultimately pursued the identification of candidate genes for agronomic traits such as the length of the juvenile period (Fernández-Ocaña et al. 2010; Jiménez-Ruiz et al. 2015).

Despite these diverse and numerous initiatives, the development of early markers to increase the efficiency of olive -breeding programs is still a pending task. The application of MAS to complex traits, such as yield or biennial bearing behavior, is not straightforward. Difficulties in manipulating these traits are derived from their genetic complexity, principally the number of genes involved, interactions between genes (epistasis) and environment-dependent expression of genes .

A recent publication concerning the reference genome of the cv. Farga (Cruz et al. 2016) and a wild olive (Unver et al. 2017) provides a landmark that can steer the generalization of genomic tools for the characterization of genetic resources and the selection of candidate genotypes. According to Cruz et al. (2016) and Unver et al. (2017), the olive tree, with 2n = 46 and a genome size ~1.3 to 1.4 Gb, has approximately 50,000 protein -coding genes and an evolutionary history marked by polyploidization events. The existence of these reference genomes along with the decreasing cost of sequencing will: (a) increase the number of sequenced cultivars , allowing the possibility of performing genome -wide association mapping and, ultimately, developing early markers to accelerate the breeding process; (b) allow an accurate evaluation of the germplasm variability for the classification of cultivars into gene pools to design crossbreeding strategies (Rugini et al. 2016) shed light on the controversial domestication history of olive (Besnard et al. 2013; Díez et al. 2015) by applying methodologies that have been previously used for annual plants with reference genomes (Gaut et al. 2015).

5 Biotechnology Applications

Olive is a species difficult to manipulate in vitro ; however, up to date, significant progress has been made in the understanding of its culture requirements under controlled conditions. In this section, the state of the art of micropropagation of important cultivars as well as key factors involved in adventitious regeneration, genetic transformation and in vitro storage of juvenile and adult tissues are reviewed, with indications regarding the possibilities of using these technologies as breeding tools.

5.1 Micropropagation

In olive micropropagation , explants of juvenile origin generally show high morphogenetic capacity (Cañas et al. 1992); however, working with this type of material is not of interest since agronomical traits are not known. Attempts to establish in vitro explants of adult origin has encountered several problems such as explant oxidation and necrosis, difficulties of disinfection, poor growth as well as a strong influence of the genotype (Lambardi et al. 2013; Rugini and Baldoni 2005). The obtainment of responsive material has required the use of explants from specific sources: (a) new growth obtained after severe pruning of field-grown trees (Peixe et al. 2007; Roussos and Pontikis 2002) shoots from rooted cuttings grown in the greenhouse (Sghir et al. 2005) suckers sprouting at the tree base (Rugini and Fontanazza 1981; Vidoy-Mercado et al. 2012), (d) new growth obtained after forcing ovules (Rama and Pontikis 1990) or hardwood cuttings (Vidoy-Mercado et al. 2012) to sprout in a controlled environment and (e) shoots from in vivo (Garcia-Férriz et al. 2002) or in vitro grafts (Revilla et al. 1996).

Explants used for culture establishment are generally nodal sections with lateral buds forced to elongate in vitro and subsequently propagated by segmentation of elongated shoots (Lambardi et al. 2013). This method is considered very reliable in terms of genetic stability of the obtained material (George 1993). Regarding nutritional requirements, Rugini (1984) developed the OM formulation, which, in comparison to the widely used MS (Murashige and Skoog 1962), contains higher levels of Ca, Mg, S, Cu and Zn, lower NH₄ as well as an additional supplement of reduced nitrogen in the form of glutamine. The OM formulation has been widely used in olive culture (Brhadda et al. 2003b; Chaari et al. 2002; Lambardi et al. 2013; Sghir et al. 2005; Vidoy-Mercado et al. 2012), although others mineral nutrient mixtures developed for woody plants have also shown to be adequate for olive ; e.g. WPM (Lloyd and McCown 1980) in cvs. Chondrodia and Chalkidikis (Grigoriadou et al. 2002) and cv. Kalamon (Dimassi-Theriou 1994) or a modified DKW (Driver and Kuniyuki 1984) in cvs. Koroneiki (Roussos and Pontikis 2002) and Arbequina (Vidoy-Mercado et al. 2012). In terms of carbon source, either sucrose (Grigoriadou et al. 2002; Rugini 1984; Sghir et al. 2005) or mannitol (Lambardi et al. 2013; Peixe et al. 2007; Roussos and Pontikis 2002) have been used; however, in a comparative study with cv. Maurino, Leva et al. (2013) showed that mannitol promoted shoot sprouting and growth more than sucrose.

Shoot elongation and proliferation require the presence of a cytokinin in the culture media, zeatin or zeatin riboside (4.6–13.6 µM) being the most widely used (Grigoriadou et al. 2002; Lambardi et al. 2013; Roussos and Pontikis 2002; Rugini 1984; Sghir et al. 2005); however, due to the high cost of these hormones, attempts have been made to replace them with other cytokinins ; along this line, Peixe et al. (2007) in cv. Galega Vulgar obtained good results with benzyladenine and a coconut milk supplement, while Peyvandi et al. (2009a) used 2-isopenteniladenine (2iP) in cv. Rowghani. In some cases, a GA₃ supplement has been used for shoot elongation either in combination with zeatin (Grigoriadou et al. 2002; Vidoy-Mercado et al. 2012) or as a separate treatment prior to rooting (Lambardi et al. 2013).

Rooting of olive microcuttings is generally carried out in two phases, e.g. Rugini (1984) recommended a 2-week exposure to 5.4 µM naphthaleneacetic acid (NAA) followed by transfer to another medium devoid of auxin but supplemented with zeatin and activated charcoal; however, in most cases, shorter exposures to auxin in liquid medium with subsequent transfer to either basal solid medium (Peixe et al. 2007; Sghir et al. 2005) or directly to the acclimatization substrate (peat moss/coco fiber/perlite, at different ratios, or jiffy pots) (García-Férriz et al. 2002; Peyvandi et al. 2009a) are preferred. In several cases, incubation in darkness and/or incorporation of putrescine to root induction medium have given positive results (Grigoriadou et al. 2002; Rugini et al. 1993). In addition, Peyvandi et al. (2009a) indicated that shoots which had proliferated in the presence of mannitol , rooted better than those previously multiplied in the presence of sucrose did. Acclimatization, a critical phase of the micropropagation process, is generally carried out on mist benches within a greenhouse with controlled light and temperature . Cozza et al. (1997) showed that survival of micropropagated plants of cv. Nocellara Etnea was linked to a higher level of vascular differentiation in comparison to those of Nocellara Belice. After hardening, Rugini (1984) recommends the use of GA₃ sprays to speed up regrowth of acclimatized plantlets.

Genetic stability of micropropagated olive plants has been evaluated through RAPDs analysis and conflicting results have been obtained, e.g. while García-Férriz et al. (2002), in cvs. Arbequina, Picual and Empeltre and Leva and Petruccelli (2012) in cv. Maurino, observed similar banding patterns in micropropagated material and the corresponding mother plants, Peyvandi et al. (2009b) and Farahani et al. (2011a, b) obtained opposite results in several Iranian cultivars ; in addition and, according to these authors, observed variations increased with the number of subcultures. Regarding field performance, while Leva (2009) found no differences between plants micropropagated through axillary buds and self-rooted controls of cv. Maurino in terms of vegetative and reproductive growth patterns, Briccoli-Bati et al. (2006) obtained different results depending on genotype , e.g. while plants of cv. Nocellara Etnea showed higher production and lower average fruit size than control-grafted plants, micropropagated material of cv. Carolea showed very low production throughout the 8-year evaluation period, leading these authors to indicate that further research is needed to elucidate whether epigenetic variations could have occurred during the long time the plants had spent in culture.

5.2 Organogenesis

Early studies on adventitious shoot formation in olive were carried out using juvenile explants; e.g. Cañas and Benbadis (1988) evaluated the in vitro organogenic capacity of basal and apical sections of cotyledons, observing a better response in the former. Shoot regeneration required a 3-week culture period on OMc medium ((OM formulation with BN macroelements (Bourgin and Nitsch 1967) and 1 g/l casein hydrolysate)) supplemented with 25 µM indole-3-butyric acid (IBA), followed by subsequent transfer to basal medium supplemented with the cytokinin 2iP. Petioles from apical and basal nodes of adult micropropagated shoots of cvs. Moraiolo and Dolce Agogia were used by Mencuccini and Rugini (1993) obtaining better response in apical nodes of Moraiolo. No response was observed when using petioles derived from adult plants grown either in the field or in the greenhouse. A hormonal supplement containing either 10 µM 2iP and 2.2-µM benzyladenine or 5–40 µM thidiazuron (TDZ) was recommended (Mencuccini and Rugini 1993). Interestingly, when regeneration took place in the presence of TDZ, MS formulation at full strength was required, although in cvs. Canino and Moraiolo adventitious shoots could also be obtained in ½ MS supplemented with 30 µM TDZ and 0.54 µM NAA (Rugini and Caricato 1995).

5.3 Somatic Embryogenesis

Somatic embryogenesis in olive was firstly observed when using immature zygotic embryos for culture establishment (Rugini 1988). Embryogenic callus could be induced in the absence of growth regulators although a cytokinin supplement was beneficial (Table 14.4). Orinos and Mitrakos (1991) in wild, as well as Mitrakos et al. (1992) and Cerezo et al. (2011) in domestic olives, recommended the use of isolated radicles from mature embryos to induce the process (Fig. 14.8a). In these cases, a medium with high auxin/cytokinin ratio was required to induce formation of embryogenic callus (Fig. 14.8b) followed by transfer to a medium without growth regulators or a very low auxin concentration, to enhance embryo differentiation (Fig. 14.8c). Similar requirements were found when using cotyledon (Brhadda et al. 2003a; Leva et al. 1995; Trabelsi et al. 2003) or root segments, from in vitro germinated seedlings (Rugini 1995; Rugini et al. 1995; Shibli et al. 2001), as explants (Table 14.4). Wounding the root surface and placing the explants in horizontal position enhanced callus formation (Rugini 1995).

Table 14.4 Somatic embryogenesis in olive

Fig. 14.8

Olive regeneration and transformation via somatic embryogenesis . a Radicle from mature embryo used for initiation of cultures, b Embryogenic callus , c Somatic embryos at torpedo and cotyledonary stages, d–f Germination of somatic embryo, g, h Transgenic plants, obtained after inoculation of globular somatic embryos with Agrobacterium tumefaciens , overexpressing the MtFT gene developed flowers in vitro (g) or after the acclimatization phase (h). Photos by S. Cerezo

Somatic embryogenesis has also been observed in adult material. Rugini and Caricato (1995) used petioles from shoots of adventitious origin of cvs. Canino and Moraiolo and cultured them in a medium with 2iP (0.5 µM), BA (0.44 µM), IBA (0.25 µM) and 0.42 mM cefotaxime. Resulting embryogenic structures could be maintained proliferating in the dark under the same conditions or in hormone-free medium supplemented with 0.1% activated charcoal; in the last case, proliferation of secondary embryos took place directly from epidermal or subepidermal cells of primary embryos (Benelli et al. 2001). In the Moroccan cv. Dahbia, Mazri et al. (2013) were able to induce embryogenesis following a 4-day exposure of leaf sections from in vitro grown shoots to liquid medium supplemented with 30 µM TDZ and 0.54 µM NAA followed by transfer to solid hormone-free medium for 8 weeks and final culture in the hormonal combination recommended by Rugini and Caricato (1995) (Table 14.4).

Mineral requirements for somatic embryogenesis seem to depend on explant used and genotype ; e.g. for radicles, the OMc formulation (Mitrakos et al. 1992; Orinos and Mitrakos 1991) has been used, while in the case of roots from seedlings, MS was preferred (Rugini 1995; Rugini et al. 1995; Shibli et al. 2001) (Table 14.4). For cotyledons, adult leaf fragments or petioles, either OMc (Rugini and Caricato 1995; Trabelsi et al. 2003), MS (Brhadda et al. 2003a; Capelo et al. 2010) or ½ MS (Mazri et al. 2013; Pritsa and Voyiatris 2004) have shown to be adequate (Table 14.4).

In a comparative study between the basal formulations OMc (Sect. 14.5.2) and ECO (¼ OM macroelements, ¼ MS microelements and 550 mg/l glutamine), Cerezo et al. (2011) observed similar callus proliferation rates, although embryogenic structures grown in ECO formulation, of lower mineral strength, showed a higher capacity to form mature embryos. The same authors also found that culture of embryogenic callus for 4 weeks in liquid medium followed by sieving though a 3 × 3 mm mesh, could help to synchronize cultures prior to undergo embryo maturation in the presence of semipermeable cellulose acetate membranes. Embryos under this treatment showed a much lower water potential and germinated at 37.8% rate in a modified MS basal medium with ½ macroelements and 10 g/l sucrose (Fig. 14.8d–f) (Cerezo et al. 2011).

5.4 Somaclonal Variation

It is widely known that phenotypic variations can appear in plants regenerated in vitro , with the chances to occur being higher when adventive regeneration protocols, including a callus phase, are used (Bairu et al. 2011; Larkin and Scowcroft 1981). In olive , Cañas and Benbadis (1988) found that plants regenerated from cotyledon fragments showed morphological alterations, such as dichotomy or presence of double leaves. In attempts to evaluate the effect of time in culture on regeneration capacity of olive embryogenic cells, Bradai et al. (2016a) studied the in vitro behavior of several embryogenic lines which had been kept in culture for 2 and 8 years. A strong genotypic effect was found, although embryos of aged lines showed a general decrease in maturation capacity and a reduced germination rate in comparison to younger ones; however, plants could be regenerated from material of all ages. Phenotypic evaluation of plants showed several alterations in vegetative traits (plagiotropic growth, fasciated stem and the appearance of 3 axillary shoots per whorl), phyllotaxy (4 leaves per verticil), leaf morphology (double leaves) and reproductive traits (flowers with 5–7 petals and 3–5 stamens). Again, genotype had a strong influence although, as expected, the frequency of variant phenotypes (19.78 vs. 4.0) and the percentage of plants showing these anomalies (37.36 vs. 10.0) were higher in aged than in younger lines (Bradai et al. 2016b). In plants obtained via somatic embryogenesis from immature cotyledons of cv. Frangivento, Leva (2009) characterized two types of somaclones: columnar and dwarf, differing in plant height, canopy projection, canopy volume and reproductive traits.

5.5 Genetic Transformation

Genetic transformation has been attempted in olive through biolistics and via Agrobacterium tumefaciens . Using the PDS-1000/He system to compare the efficiency of two promoters, Lambardi et al. (1999) found that the sunflower ubiquitin yielded higher Gus gene expression than the cauliflower 35S, in torpedo stage somatic embryos of cv. Canino, although opposite results were obtained with embryos at more advanced stages. Pérez-Barranco et al. (2009) using globular embryos derived from a mature embryo of cv. Picual reported similar results. These authors established a 6 cm target distance and a 900-psi bombardment pressure as optimum conditions for transformation . Gus expression could be observed 12 weeks after bombardment; however, as reported by Lambardi et al. (1999), no transgenic plants could be regenerated. Pérez-Barranco et al. (2009) also evaluated the response of olive cells to different antibiotics , pointing out the relatively high tolerance to kanamycin and paromomycin in solid medium while in liquid, antibiotics impaired cell growth at much lower concentrations.

Rugini et al. (2000) first attempted genetic transformation via Agrobacterium tumefaciens in somatic embryos of cv. Canino. In this protocol, after an initial 48 h exposure to the bacterial suspension , somatic embryos were transferred for 30 days to a medium deprived of antibiotics for recovery and subsequently exposed to 0.21 mM kanamycin in the dark; afterwards, embryogenic material was cultured in liquid medium in the light and only those embryos that turned green were selected. Finally, embryos were returned to the dark for secondary embryo production ; these embryos were then induced to germinate in liquid medium deprived of antibiotics and supplemented with 1.3 µM zeatin . A different approach was undertaken by Torreblanca et al. (2010), using the highly virulent strain AGL1 for inoculation of globular stage embryos, with continuous exposure to the antibiotic paromomycin 2 days after the co-culture phase. To avoid selection of chimeric tissues , a 3-week culture in liquid medium in the presence of antibiotic was used. For transgenic plant recovery the protocol of Cerezo et al. (2011), previously described (Sect. 14.5.3), was used.

Transformation via Agrobacterium tumefaciens has been used in attempts to improve agronomic traits as well as to undertake functional genomic studies. In an attempt to modify growth habit, somatic embryos of cv. Canino were transformed with rol ABC genes from A. rhizogenes; resulting transgenic plants showed hairy root phenotype , a long juvenile phase and maintained vegetative growth until late autumn (Rugini et al. 2008). To enhance stress tolerance, Rugini et al. (2000) were able to transform somatic embryos of the same genotype with the osmotin gene . Obtained transgenic plants have shown an outstanding resistance to water stress as well as enhanced tolerance to peacock spot (Spilocaea oleagina) although they are particularly attractive to the cribate weevil, Otiorhynchus cribricollis (Rugini et al. 2016). In order to elucidate the role of flowering locus T (FT) gene in olive , somatic embryos derived from a mature embryo of cv. Picual were transformed with FT-homologue from Medicago truncatula, following the protocol of Torreblanca et al. (2010). Some of the obtained transgenic lines flowered either during the in vitro phase (Fig. 14.8g) or following acclimatization (Fig. 14.8h); inflorescences were formed all year around although they were more abundant in spring. These plants also showed profuse axillary branching and reduced size (Haberman et al. 2017).

5.6 In Vitro Storage

5.6.1 Low-Temperature Conservation

Initial attempts to preserve olives under low temperature regimes were carried out by Micheli et al. (1998) through encapsulation of apical and lateral buds of cv. Moraiolo in sodium alginate beads; buds could be stored up to 45 days at 4 °C, although those of apical origin responded better in terms of sprouting. A few years later, Micheli et al. (2007) showed that 3–4 mm long nodal sections with lateral buds could be kept for 30 days at room temperature , in alginate nutrient solution kept in plastic cuvettes, suggesting that this could be an useful methodology to exchange material between countries. The encapsulation method has also been used by Cabello Moreno et al. (2013) using nodal sections of cv. Arbequina; these authors encapsulated buds at 4 °C over a 4-week period, indicating that pretreatments with growth regulators such as abscissic acid are beneficial for the conservation process.

Shoot cultures of cvs. Leccino and Frantoio, were maintained at 4 °C under darkness in OM basal medium for 8 months with a regrowth capacity above 80% (Lambardi et al. 2002), while in the case of cv. Arbequina maintained in RP medium (Roussos and Pontikis 2002) at 8 °C under light, shoots could be kept viable for 12 months (Imbroda et al. 2014).

5.6.2 Cryopreservation

Attempts at long-term conservation of buds have been carried out using the apical dome with 1–2 pairs of leaf primordia as explants. Martinez et al. (1999) obtained a 30% bud recovery in cv. Arbequina while Lambardi et al. (2002) reported a 15% in cv. Frantoio; however, in both cases, regrowth and further development or cryopreserved material occurred at a very low rate. Subsequent attempts to improve regrowth after cryopreservation through the inclusion of hormones in post-thaw medium, allowed Lynch et al. (2007) to maintain growth up to 10 weeks. Histological examinations showed the occurrence of damage in subapical cells, which could explain the failure in shoot recovery.

Somatic embryos seem to be a more suitable material for cryopreservation than buds. Shibli and AI-Juboory (2000) were able to successfully cryopreserve somatic embryos derived from seedling roots, via the encapsulation-dehydration or the encapsulation-vitrification methods; in both cases, inclusion of adequate dehydration steps (drying beads up to 21.1% moisture in the first case or keeping them for 3 h in plant vitrification solution) was critical for the process. In addition, exposure of embryogenic callus to 30 °C for 1 day allowed achievement of 58% survival rate when using encapsulation-dehydration or 68% in the case of encapsulation-vitrification protocol. Moreover, plants derived from cryopreserved cultures showed no morphological differences with controls. The importance of a correct dehydration step has also been emphasized by Sanchez-Romero et al. (2009) when using the droplet vitrification method after a 60 min dehydration in PVS2 (Sakai et al. 2008). In addition, Bradai and Sánchez-Romero (2017) indicated that preculturing embryogenic structures in a medium with high sucrose concentration was beneficial for culture recovery. Following regeneration, no morphological differences were observed between control plants and those derived from cryopreserved embryos. A different approach, including a 3-day sucrose pretreatment followed by incubation in a cry protectant mixture, prior to freezing at controlled rate (0.5 °C/min until −35 °C) and plunging into LN, allowed Lynch et al. (2011) to get noticeable callus regrowth and subsequent embryo recovery. Biochemical analysis indicated that applied pretreatments could enhance glutathione reductase, proline and sugar levels in the tissue, preparing them to withstand freezing.

6 Conclusions and Prospects

The choice of plant material is the first decision of the olive grower at the time of establishing a new plantation. A high diversity of local cultivars is a common trait in traditional olive -producing countries. Most olive cultivars are old, and they are grown around areas in which they were likely selected. In most cases, cultivars are self-rooted. Grafted trees are found only in the case of difficult-to-root cultivars or due to top grafting onto wild olives or onto other obsolete cultivars . In new olive -producing countries, imported cultivars represent most of the olive orchards. The intensification of olive plantations after World War II in producing countries is based on a few selected national or foreign cultivars . This change has heightened interest in the conservation of olive genetic resources and in breeding olives.

The exploration, cataloguing and conservation of true-to-type traditional olive cultivars are the first steps for the sustainable use of genetic resources in any olive -growing country. Currently, ex situ collections are common facilities for conserving cultivars in germplasm banks. In all banks, confusion between names of accessions and cultivars remain an unresolved issue. An International Network with 22 National Germplasm Banks coordinated by the International Olive Council (IOC) with more than 1300 accessions is attempting to identify and to authenticate them. Morphologic characterization and DNA markers, particularly SSRs, have been shown to be powerful and discriminant methods to authenticate accessions (true-to-type cultivars ), duplications, homonyms, synonyms and incorrect denominations. The identification and authentication of the IOC Network accessions is on course. The publication of a World Catalogue of True-to-Type Olive Cultivars will aid in solving the confusion in olive varietal denominations and developing International Plant Certification Protocols that will guarantee true-to-type and pathogen- and pest-free nursery plants in a time of global exchange of plant material. Other strategies, such as agronomic evaluation in banks and comparative trials, are in their infancy. The exploration and evaluation of wild and ancient olives is also under development to expand the genetic diversity to confront new breeding challenges such as climate change and devastating pests and diseases , as well as for studies related to the origin and domestication of the olive , among others.

New plantations systems have increased the need for new specific cultivars that have not been previously considered by empirical local breeders, i.e. the traditional farmer and breeder. The new systems require a high investment and early return, adaptation to mechanical harvesting , high yield , high oil content , resistance or tolerance to biotic and abiotic stresses, and quality of the olive oil and table olives, among others. In the last sixty years, crossbreeding has progressively developed in various olive growing countries. The breeding programs have demonstrated the following issues:

• (1) A consistent increase in trained qualified breeders and publications.

• (2) Evaluation and selection of parents for crosses will improve breeding efficiency.

• (3) Reduction of the juvenile period (JP) to 2–3 years after planting accelerates the breeding process.

• (4) Implementation of early selection methods of evaluation .

• (5) Genetic studies demonstrates transgressed segregation of any trait in any crossbred progeny , consistency among genotype values data during the different steps of evaluation for most evaluated traits, and gain in the evaluation of a smaller number of well-selected crossbred progenies rather than many crossbred progenies with a smaller size.

• (6) Moderate and high values of heritability for relevant agronomic, olive oil , and table olives traits is observed

• (7) Progressive and fast development of genomic tools, such as MAS and GWAS , in addition to a recent publication concerning the first sequenced genome will accelerate the process of crossbreeding .

• (8) Cooperation with farmers or other stakeholders during the final step of evaluation is critical for a sound G × E evaluation and for visibility among farmers of the new material advancements.

• (8) New materials from public breeding programs for olive oil and table olive cultivars and rootstock will be progressively released in the next decade, providing farmers with cultivars for new mechanized plantation systems and/or resistance or tolerance to verticillium wilt and other diseases .

• (10) New joint public-private consortia will develop new breeding programs .

Finally, registration represents the birth of a new cultivar. Marketing strategies appear as a major factor for its diffusion. This last step will determine whether any bred cultivar is well adapted to different environments in which it is dispersing, thus providing information about the real value of this innovation. A further requirement is the need for certification of nursery plants to ensure that newly-bred cultivars are true to type and free from pests and pathogens to demonstrate the potential performance of the new cultivar.

Regarding the possibilities of using in vitro regeneration and transformation technologies in breeding programs , micropropagation protocols are currently available for different olive cultivars ; hence, although the genotype has strong influence on in vitro behavior, the technology could be useful for rapid multiplication of new releases as well as for international exchange of material. Regeneration via somatic embryogenesis is well established for juvenile material and very promising results are being obtained for explants of adult origin . Optimizing this pathway would make it feasible the use of cryopreservation for long-term in vitro storage as well as to undertake transformation of selected genotypes with gene coding for important agronomical traits. In any case, it appears that the interest of private companies for genetic manipulation of commercial cultivars , will depend on changes of consumer’s acceptance of transgenic products.

Appendices

Appendix 1

Research institutes and online resources

Country	Institution	Specialization and research activities	Contact information and website
Albaniaa	Centre of Agricultural Technology Transfer. Centre of Agricultural Technology Transfer Shamogjin, Komuna Novosele, Vlorë. Phone.: 00355 33 404144/145 Fax: 00355 33 404144/145	Genetic resources	Ms. Aulona Veizi aulona10@gmail.com qttbvlore@yahoo.com
Algeriaa	ITAF. Tessala El Merdja - Birtouta -Alger. Phone: +213 023 58 38 60/61/66 Fax: +213 023 58 38 64/65	Genetic resources	M. Mahmoud Mendil mbmendil@gmail.com Itafv.dg@gmail.com webmaster@itafv.dz
Argentina	Laboratory of Genetic and Health Quality / Faculty of Agricultural Sciences/UNC. Faculty of Agricultural Sciences National University of Cuyo. Almirante Brown 500. Chacras de Coria - Luján de Cuyo. CPA M5528AHB - Mendoza - Argentina. Phone: (+54 261) 413-5010	Genetic resources Breeding Genomics	L.E. Torres itorres@agro.unc.edu.ar http://www.fca.uncu.edu.ar/
Argentina	IBAM/ CONICET/ INTA. Faculty of Agricultural Sciences National University of Cuyo. Almirante Brown 500. Chacras de Coria - Luján de Cuyo. CPA M5528AHB - Mendoza - Argentina. Phone: (+54 261) 413-5010	Genetic resources Breeding Abiotic stresses Biotic stresses. Oil quality Genomics	R. W. Masuelli masuelli@fca.uncu.edu.ar https://inta.gob.ar/mendoza
Argentinaa	EEA/CONICET/ INTA. Agricultural Experiment Station San Juan, Calle 11 y Vidart (5427) Villa Aberastain San Juan. Phone: (0264) 492 1079, (0264) 492 1191	Genetic resources Abiotic stresses	Dra. Mariela Torres mtorres@sanjuan.inta.gov.ar https://inta.gob.ar
Australia	WWAI/ NSW DPI. Wagga Wagga Agr Inst, EH Graham Ctr Agr Innovat, Wagga Wagga, NSW 2650, Australia. Phone: (02) 6938 1999 International: +61 2 6938 1999 Fax: (02) 6938 1809	Genetic resources	www.dpi.nsw.gov.au
Australia	UNE. Univ New England, Sch Environm & Rural Sci, Armidale, NSW, 2351 Phone: +61 2 6773 2323 Fax: +61 2 6773 2769	Genetic resources Breeding	msedgle2@une.edu.au ers@une.edu.au
Belgium	Ghent University. Univ Ghent, Dept Plant Biotechnol & Bioinformat, B-9052 Ghent, Belgium Phone. +32 9 331 38 00 Fax +32 9 331 38 09	Genomics	marc.vanmontagu@ugent.be
Croatia	Faculty of Agriculture/ University of Zagreb. University of Zagreb, Faculty of Agriculture, Svetošimunska cesta 25, 10000 Zagreb, Croatia, Phone: +385 (0)1 2393 777 Fax: +385 (0)1 2315 300	Genetic resources Breeding Genomics	Z. Satovic zsatovic@agr.hr http://www.agr.unizg.hr/en
Croatiaa	Institute for Adriatic Crops and Karst Reclamation. Inst Adriat Crops & Karst Reclamat, Put Duilova 11, Split 21000, Croatia. Phone: +385.21.43.44.44 Fax: +385.21.31.65.84	Genetic resources Biotic stresses	Mr. Slavko Perica Slavko.Perica@krs.hr info@krs.hr
Cyprusa	Agricultural Research Institute // Officer Olive Technology Laboratory. Agricultural Research Institute. P.O.Box 22016, 1516 Nicosia, Cyprus. Phone: ++357 22 403100 Fax: ++357 22 316770	Genetic resources	Ms. Dora Chimonidou dari@arinet.ari.gov.cy info@ari.gov.cy
France	AGAP/INRA/ Montpellier SupAgro. Montpellier SupAgro, 2 place Pierre Viala, 34060 MONTPELLIER Cedex 02. Phone: +33 (0)4 99 61 22 00 Fax: +33 (0)4 99 61 29 00	Genetic resources Breeding	L. Essalouh laila.essalouh@supagro.inra.fr https://www.supagro.fr
Francea	UMR-AGAP. Avenue Agropolis, 34398 Montpellier Cedex 5, France. Phone: +33 4 67 61 58 00	Genetic resources Breeding Oil Quality Genomics	B. Khadari khadari@supagro.fr https://umr-agap.cirad.fr
France	Université de Toulouse III/ EDB - UMR 5174. 118, route de Narbonne Bât. 4R131062 TOULOUSE cedex 9 Phone (+33) 05 61 55 73 84 Fax: (+33) 05 61 55 73 27	Evolution	G. Besnard guillaume.besnard@univ-tlse.fr http://www.edb.ups-tlse.fr/
Greece	Laboratory of Pomology/ Department of Crop Science/ Agricultural University of Athens. Agricultural University of Athens. Iera Odos 75, Athina 118 55, Greece. Phone.: +30 21 0529 4900 Fax. +30210-5294081.	Genetic resources Breeding	M. Hagidimitriou marianna@aua.gr http://www2.aua.gr
Greece	Institute of Viticulture, Floriculture and Vegetable Crops (I.V.F.V.H)/ NAGREF. Institute of Viticulture, Floriculture and Vegetable Corps of Herakleion // I.V.F.V.H // PO Box 2229 // 71003 Herakleion Phone: 2810 302 300// 245 851, 240 986. Fax 2810 245 873	Genetic resources Breeding Abiotic stresses Biotic Stresses Oil Quality	A.G. Doulis grandreas.doulis@nagref-her.gr http://www.nagref-her
Greecea	Olive Cultivation and Post Harvest Physiology Laboratory/ Institute for Olive Tree and Subtropical plants of Chania/NAGREF. Institute for Olive Tree and Subtropical plants of ChaniaLeof. Soudas 131, Chania 731 34, Greece. Phone:+30 2821 083472	Genetic resources Breeding Abiotic stresses Biotic Stresses Oil Quality	Dr. G.C. Koubouris koubouris@nagref-cha.gr Info@nagref-her.gr
Greece	Aristotle Univ. Thessaloniki. Aristotle Univ Thessaloniki, Dept Hort, Lab Biol Hort Plants, Thessaloniki 54124, Hellas, Greece	Biotic Stresses Breeding Oil Quality	Maria Tsimidou tsimidou@chem.auth.gr info@agro.auth.gr
Iran	National Institute of Genetic Engineering and Biotechnology . Shahrak-e Pajoohesh, km 15, Tehran - Karaj Highway, Tehran, Iran P.O. Box: 14965/161. Phone: +98 21 44787301-9. Fax: +98 21 44787399	Genetic Resources Breeding Genomics	M. Hosseini-Mazinani hosseini@nigeb.ac.ir nigeb_manager@nigeb.ac.ir
Iran	Horticulture Department/Gorgan University of Agricultural Sciences. Gorgan University of Agricultural Sciences and Natural Resources. Gorgan, 49138-15739, Iran. Phone: +98-171-2220320. Fax: + 98-171-2220640	Genetic Resources Breeding Genomics	M. Sharifani mmsharif2@gmail.com International@gau.ac.ir
Iran	SPII/HD. SPII, Hort Dept, Mahdasht Rd,POB 31359-33181, Karaj, Iran	Breeding	A. Zeinanloo info@abrii.ac.ir
Israel	The Robert H. Smith Faculty of Agriculture, Food and Environment The Hebrew University of Jerusalem. Hebrew Univ Jerusalem, Fac Agr, Inst Plant Sci, IL-76100 Rehovot, Israel. P.O Box 12, Rehovot 76100	Genetic resources Breeding Abiotic stresses Biotic stresses. Oil Quality Genomics	A. Samach alon.samach@mail.huji.ac.il http://departments.agri.huji.ac.il
Israela	ARO/ Volcani Centera. Agricultural Research Organization - the Volcani Center, 68 HaMaccabim Road, P.O.B 15159 Rishon LeZion 7505101, Israel Phone: +972-3-9683226 Fax: +972-3-9665327	Genetic Resources	Dr. Giora Ben Ari giora@agri.huji.ac.il http://www.agri.gov.il
Italy	IVALSA. National Research Council of Italy, Trees and Timber Institute Follonica (Grosseto) via Aurelia, 49 58022 - Follonica (GR) Phone. +39 056 652356	Genetic Resources Breeding Genomics	C. Cantini cantini@ivalsa.cnr.it http://www.ivalsa.cnr.it
Italy	CNR/Institute of plant genetics. Institute of plant genetics. Via Madonna Alta, 130-06128 Perugia (PG) - Umbria Phone: +39 0755014862 Fax: 0755014869	Genetic resources Breeding Genomics	L. Baldoni luciana.baldoni@ibbr.cnr.it http://www.ibbr.cnr.it
Italy	DEMETRA/ University of Palermo. University of Palermo. Piazza Marina, 61// 90133 - PALERMO. Phone: +39 091 238 93011	Genetic Resources Abiotic stresses Biotic stresses	T. Caruso f tiziano.caruso@unipa.it http://www.unipa.it
Italy	Management Department of Agricultural and Forestry Systems/ University of the Mediterranean Studies of Reggio Calabria. Mediterranea University of Reggio Calabria. Salita Melissari 89124 Reggio Calabria. Tel.: +39 0965 169 1207 Fax: +39 0965 332201	Genetic Resources Breeding	R. Mafrica rocco.mafrica@unirc.it http://www.unirc.it
Italy	UNIFI/DISPAA. Universita degli studi fii Firenze. Dipartimento di Scienze delle Produzioni Agroalimentari e dell’Ambiente Piazzale delle Cascine, 18 - 50144 Firenze Phone: +39 055275-5700	Genetic resources Breeding Abiotic stresses	http://dispaa.unifi.it
Italy	UNITU. Universita della Tuscia, DAFNE, Via San Camillo de Lellis Snc, I-01100 Phone +39 0761357581/554; Fax +39 0761357558/434	Biotechnology Genomics	Eddo Rugini rugini@unitus.it dafne@pec.unitus.it
Italy	CNR/ IVALSA. Sesto Fiorentino (Firenze) via Madonna del Piano, 10 50019 - Sesto Fiorentino (FI) Phone: +39 055 52251	Genetic Resources Genomics	M. Centrito centrito@ivalsa.cnr.it info@ivalsa.cnr.it
Italy	UNIBA/DISAAT. Universita di Bari, Dipartimento di Scienze Agro Ambiental e Territoriali (Di.S.A.A.T.) Amendola 165-A, I-70126 Bari, Italy	Genetic resources Biotic stresses	Franco Nigro franco.nigro@uniba.it info@agr.uniba.it
Italy	ENTECRA OLI. CRA OLI, I-06049 Spoleto, PG, Italy. Centro di ricerca per l’olivicoltura e l’industria olearia (Rende) Via Nursina 2 06049 - SPOLETO. Phonel: +39 0743-49743 Fax: +39 0743-43634	Genetic resources Biotic stresses	Adolfo Rosati rosati@entecra.it info@entecra.it
Italy	UNIPG/DSAAA. Univ Perugia, Dipartimento Sci Agr Alimentari & Ambientali, Via Borgo 20 Giugno 74, I-06121 Perugia, Italy	Oil quality	Maurizio Servili maurizio.servili@unipg.iti nfo@unipg.it
Italya	CRA-OLI Research center for olive growing and oil industry. Centro di ricerca per l’olivicoltura e l’industria olearia – Sede Scientifica di Città S.Angelo (OLI.PE) Viale Petruzzi 75 65013 - CITTA’ SANT’ANGELO	Genetic resources Breeding Abiotic stresses Biotic stresses Oil quality	Enzo Perri enzo.perri@crea.gov.it http://sito.entecra.it
Jordana	NCARE. National Center for Agricultural Research and Extension PO Box 639, Baq’a 19381, Jordania Phone: +962 (6) 4725071 Fax: +962 (6) 4726099	Genetic Resources Breeding Abiotic stresses Biotic stresses Oil quality	Dr. Salam Ayoub salamayoub@hotmail.com http://www.ncare.gov.jo/
Lebanon	Lebanese University/ Faculty of Agricultural Sciences. Faculty of Agricultural Sciences. PO Box 90775, Horst Tabet, Beirut - Lebanon Phone: 484130/01 484131/01 484132/01 Fax: 510870/01 510867/01	Genetic resources Breeding Genomics	L. Chalak lamis.chalak@gmail.com https://www.ul.edu.lb
Lebanona	LARI. Lebanese Agr Res Inst, Lab Olive Oil , Tal Amara, Bekaa, Lebanon	Genetic resources Oil quality	Milad El Riachy mraichy@lari.gov.lb info@lari.gov.lb
Montenegroa	Biotechnical Faculty/Centre For Subtropical Cultures. Biotechnical Faculty. Centre for Subtropical Cultures Bar- University of Montenegro. Ul. Bjelisi bb 85000 Bar// Montenegro Phone: (382) 69516165	Breeding Genomics	B. Lazovic biljanal@t-com.me http://www.ucg.ac.me
Moroccoa	INRA/ URAP. Centre Régional de la Recherche Agronomique de Marrakech Unité de Recherche sur l’Amélioration des Plantes et de la qualité B.P. 533 Menara MARRAKECH Maroc Phone: +212 524447882/ +212 524435175/ +212 524432627 Fax: +212 524446380	Genetic Resources Breeding	Sikaoui Lhassane sikaouilhassane@yahoo.fr http://www.inra.org.ma
Portugala	INIAV. UEI de Biotecnologia e Recursos Geneticos. Polo de Elvas Estrada de Gil Vaz, Apartado 67351-901 Elvas – Portugal Phone: (+ 351) 268 637 740	Genetic resources Breeding Oil quality Table olives quality Genomics	António M Cordeiro antonio.cordeiro@iniav.pt polo.elvas@iniav.pt
Sloveniaa	Experimental center for olive growing . Agriculture and Forestry Institute Nova Gorica.Ulica 15. maja 17, 6000 Koper Tel: ++386 (0)5 631 32 32/ ++386 (0)41 815 302	Genetic resources	Ms. Vesel Viljanka viljanka.vesel@siol.net www.kmetijskizavod-ng.si
Spaina	IFAPA Centro Alameda del Obispo. Centro Alameda del Obispo Avda. Menéndez Pidal s/n 14004- Córdoba Phone. +34 957016000	Genetic resources Breeding Abiotic stresses Biotic stresses. Oil quality Genomics	Raul De la Rosa raul.rosa@juntadeandalucia.es cordoba.ifapa@juntadeandalucia.es
Spain	IMIDRA. Finca El Encin, Autovía del Noreste A-2, Km. 38.200, 28805// Alcalá de Henares, Madrid Phone: +34 918 87 94 00	Genetic resources Breeding Oil quality Genomics	B. E. Sastre blanca.esther.sastre@madrid.org www.madrid.org/imidra/
Spain	Instituto de la Grasa/ CSIC. Instituto de la Grasa, CSIC. Ctra. de Utrera, km. 1. Campus Universitario Pablo de Olavide - Edificio 46. 41013 - SEVILLA (España) Phone:(+34) 95 461 1550 Fax:(+34) 95 461 6790	Genetic resources Breeding Oil quality Table olives quality Genomics	J. M. Martínez-Rivas mrivas@cica.es www.ig.csic.es
Spain	Plant Physiology/ Faculty of Science/ University of Extremadura. University of Extremadura. Avda. de Elvas, s/n. 06006 Badajoz Phone:+34 924 289 300 Fax.: +34 924 272 983	Genetic resources Abiotic stresses Biotic stresses. Oil quality Genomics	M. C. Gomez-Jimenez mcgomez@unex.es https://www.unex.es
Spain	IAS-CSIC. Instituto de Agricultura Sostenible Avenida Menéndez Pidal s/n Campus Alameda del Obispo 14004 Córdoba (España) Phone: +34 957 49 92 00 Fax:+34 957 49 92 52	Genetic resources Breeding Abiotic stresses Biotic stresses.	H. F. Rapoport hrapoport@ias.csic.es http://www.ias.csic.es/
Spaina	Department of Agronomy/ University of Córdobaa. Universidad de Córdoba Departamento Agronomía Campus Univ. de Rabanales Ctra. Madrid-Cádiz Km. 396 14071-Córdoba Phone +34 957218433/ 34/ 35 Fax +34 957218438	Genetic resources Breeding Abiotic stresses Biotic stresses. Oil quality Genomics	Diego Barranco ag1banad@uco.es infoetsiam@uco.es
Spain	IRTA Mas de Bover. Instituto de Investigación y Tecnología Agroalimentarias. Ctra. de Reus El Morell Km 4,5 Phone: 977 32 84 24 Fax: 977 34 40 55	Genetic resources Breeding Abiotic stresses Biotic stresses. Oil quality Genomics	Agusti Romero agusti.romero@irta.cat http://www.irta.cat
Spain	CSIC/EEZ. Plant Reproductive Biology Laboratory, Estacion Experimental del Zaidin (CSIC), Profesor Albareda 1, 18008 Granada, Spain. Phone +34 958572757 Fax +34958572753	Abiotic stresses Genomics	Juan D. Alche juandedios.alche@eez.csic.es info @csic.eez.es
Spain	US/ETSIA. Departamento Agroforestal. ETSIA. Universidad de Sevilla. Ctra. Utrera km 1. 41013 Sevilla. Phone +34 954486455 Fax +34 954486436	Breeding Quality table olives	Pilar Rallo prallo@us.es agroforestal@us.es
Spain	CNAG CRG. Barcelona Inst Sci & Technol, Ctr Genom Regulat, CNAG CRG, Baldiri i Reixac 4, Barcelona 08028, Spain Phone +34 93 316 01 00 Fax +34 93 316 00 99	Genomics	Toni Gabaldon toni.gabaldon@crg.eu. info@crg.esi
Spain	UJA/DBE. Univ Jaen, Dept Biol Expt, Campus Lagunillas S-N, Edif B-3, Jaen 23071, Spain. Phone:+34 953 212527	Genomics Oil Quality	Francisco Luque fjluque@ujaen.es info@ujaen.es
Spain	CSIC/IRNAS. CSIC, IRNAS, Irrigat & Crop Ecophysiol Grp, Ave Reina Mercedes 10, Seville 41012, Spain. Phone: +34 95 462 47 11	Abiotic Stresses	Enrique Fernandez Luque jeferse@irnase.csic.es www.irnas.csic.es
Spain	IHSM/UMA-CSIC, Inst Hortofruticultura Subtrop & Mediterranea, Univ Malaga-CSIC, Dept. Biol Vegetal, Fac Ciencias, E-29071 Malaga, Spain. Phone: +34 952131947	Biotechnology	F. Pliego, ferpliego@uma.es
Tunisia	IRESA. Univ Sousse, IRESA, High Agron Inst, B.P. n° 47 -4042 Chott Meriem Sousse, Tunisie Phone: (+216) 73 327 544/ (+216) 73 327.592. Fax: (+216) 73 327	Genetic resources	Ibtisissem Laribi ibtissem.laaribi@yahoo.fr isa.chott@iresa.agrinet.tn
Tunisiaa	Institut de l’Olivier Sfax, Route de l’Aéroport, B.P. 1087 3000 Sfax Tél: (+216) 74 241 240/ 74 241 589 Fax: (+216) 74 241 033	Genetic Resources Breeding Biotic Stresses	Monji Msallem msallemonji@yahoo.fr http://www.iosfax.agrinet.tn/
Turkey	Department of Bioengineering/Ege University. Ege University Faculty of Engineering Department of Bioengineering. Erzene Quarter, Ege Unv. No:180, 35040 Bornova/İzmir Phine: 0 232 311 58 11 Fax: 0 232 311 58 80	Genetic Resources and Breeding Genomics	B. Tanyolaç bahattin.tanyolac@ege.edu.tr http://biyomuhendislik.ege.edu.tr info@ege.edu.tr
Turkey	Bornova Olive Research Station. Olive Culture Research Station Üniversite Cd. No: 43 35100 Bornova/ İzmir Phone: +90 (232) 462-7073	Genetic Resources	Dr. Unal Kaya unal.kaya@gthb.gov.tr posta@zae.gov.tr
Turkeya	GFAR. Olive Research Institute - İzmir Universite Cd. No:43 35100 BORNOVA IZMIR Phone: +90 232 462 70 73 Fax: +90 232 435 70 42	Genetic Resources	Melek Gurbuz melekgurbuz11@gmail.com izmirzae@tarim.gov.tr
Uruguay	INIA/ National Agricultural Research Institute. National Agricultural Research Institute. Andes 1365 - piso 12 CP 11100 Montevideo, Uruguay Phone: + 598 2902 0550 Fax: + 598 2902 3666	Genetic Resources and Breeding Abiotic stresses Biotic stresses	P. Conde pconde@inia.org.uy inia@inia.org.uy
USA	National Clonal Germplasm Repository/USDA. Nat’l Clonal Germplasm Rep - Tree Fruit & Nut Crops & Grapes. Davis, CA One Shields Ave, UC Davis Davis, CA 95616 Phone: (530)752-7009 Fax: 530-752-5974	Genetic Resources Breeding Genomics	J. E. Preece John.Preece@ars.usda.gov https://www.ars.usda.gov

1. ^aIOC Network Germplasm Bank

Appendix 2

Genetic resources : Main native cultivars

Country	Cultivar	Use	Fruit weight (g)	Oil content
Albania	Kalinjot	Oil, Table	3.5	High
Algeria	Azeradj	Oil, Table	4	Medium
	Chemlal de Kabylie	Oil	2	Low
	Sigoise	Table, Oil	3	Medium
Argentina	Arauco	Table, Oil	8	Medium
Chile	Azapa	Table, Oil	8	Medium
Croatia	Lastovka	Oil	3	High
	Oblica	Table	5	Medium
Cyprus	Ladoelia	Oil	2.5	High
Egypt	AggeiziShami	Table	8	Very low
	Touffahi	Table	12	Very low
France	Aglandau	Oil, Table	2.5	Medium
	Bouteillan	Oil	5	High
	Grosanne	Table, Oil	2.5	Low
	Lucques	Table	3	Low
	Picholine du Languedoc	Table, Oil	3	Medium
	Salonenque	Table, Oil	3	Medium
	Tanche	Oil, Table	2.5	High
Greece	Adramitini	Oil	2.5	High
	Amigdaloia	Table, Oil	8	Medium
	Chalkidikis	Table	8	Medium
	Kalamata	Table	3.5	Medium
	Konservolia	Table	4	Medium
	Koroneiki	Oil	1	Very high
	Mastoidis	Oil, Table	2.6	Very high
	Megaritiki	Oil	2	High
	Valanolia	Oil	2.5	Medium
Iran	Fishomi	Table		Medium
	Mari	Table, Oil	3.5	High
	Roghani	Oil	4	High
	Zard	Oil, Table	4.5	Medium
Israel	Barnea	Oil, Table	3	Medium
	Nabali	Oil, Table	3	High
	Souri	Oil, Table	3	High
Italy	Ascolana	Table	7	Low
	Bosana	Oil	3	High
	Cellina di Nardo	Oil	2	Low
	Coratina	Oil	5	Medium
	Frantoio	Oil	3	High
	Leccino	Oil	2.5	Medium
	Moraiolo	Oil	2	High
	OgliarolaBarese	Oil	2	High
	Pendolino	Oil	2	Low
	Taggiasca	Oil	2	High
Jordan	Nabali Baladi	Table, Oil	3	High
	Rasei	Oil	3	Medium
Lebanon	Baladi	Oil, Table	3	High
	Souri	Oil, Table	3	High
Libya	Endory	Oil	1	High
	Hammudi	Oil	2.2	High
Malta	Bidni	Oil, Table	2	High
Montenegro	Zutica	Oil	3.2	High
Morocco	Picholine Marocaine	Oil, Table	3.5	Medium
Palestine	Nabali	Oil, Table	3.1	High
	Souri	Oil, Table	3	High
Perú	Criolla	Table	8	High
Portugal	Cobrançosa	Oil	3	Medium
	GalegaVulgar	Oil, Table	2.5	Medium
Slovenia	Buga	Oil
Spain	Arbequina	Oil	1.9	High
	Arbosana	Oil	2	Medium
	Cornicabra	Oil	3	High
	Empeltre	Oil	2.7	Medium
	Farga	Oil	2.4	Medium
	Gordal Sevillana	Table	12.5	Low
	Hojiblanca	Oil, Table	4.8	Medium
	Lechin de Sevilla	Oil	3	Medium
	Manzanilla de Sevilla	Table	4.6	Medium
	Picual	Oil	3.2	High
Syria	Doebli	Oil, Table	4.5	High
	Sorani	Oil, Table	3	High
	Zaity	Oil	2.5	Very high
	Kaissy	Table	5	Low
Tunisia	Chemlali Sfax	Oil	1	Very high
	Chetoui	Oil, Table	2.5	Medium
	Oueslati	Oil, Table	2	High
Turkey	Ayvalik	Oil	3.6	High
	Domat	Table	5.3	Medium
	Gemlik	Oil, Table	3.7	High
	Memecik	Oil, Table	4.8	Medium
USA	Mission	Oil, Table	3	Medium