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 The olive (Olea europaea L.) is considered as one of the oldest fruit tree in the Mediterranean basin and it has nutritional benefits in human health. It is an indigenous and a traditional tree crop cultivated for oil and canned fruit. Cultivated olive is the sixth most important oil crop in the world, presently spreading from the Mediterranean region of origin to new production areas such as China, Japan, and USA due to the beneficial nutritional properties of olive oil and to its high economic value.

 In Tunisia, the olive is an important crop mainly cultivated for olive oil production and widely extends from the north to the south regions of the country for its high economic value, where two-third of total olive area are localized in arid and semi-arid conditions (Abdelhamid et al., 2013). Tunisia’s olive resources are estimated at over 70 million olive trees, grown on 1,680,000 ha, almost 2 out of 3 farmers cultivate olives in the country. Olive is widely spreading over the country from the north to the south and mainly cultivated under rainfed conditions (less than 250 mm of rain-fall). Tunisia is the 4^th largest producer of olive oil in the world and oil exports represent 40% of the overall value of agronomic exports and 5.5 % of aggregate exports, making it the fifth largest source of foreign currency earnings for the country (Fig. 1).

Fig. 1. Production, consumption and export of Tunisian olive oil.

 Tunisian olive groves are essentially dominated by two olive oil varieties:”Chetoui” in the north and “Chemleli” in the center and south of the country. Furthermore, those two varieties have been chosen by olive growers.

 The recent boom in demand for olive oil around the world requires Tunisian producers to improve fruit and oil quality to maintain their competitiveness on the international oil market and to meet consumer demands. A strategy was adopted for the development of the olive sector aimed primarily at improvement of hectare yields, raising productivity and oil quality. New orchards are being planted with high yield cultivars and cultural techniques such as fertigation and foliar sprays of micronutrients and better growth habit adapted for mechanized harvest. Olive orchards have become a new and promising market for suppliers of agronomic inputs and are critically dependent on the quality of olive oil and the availability of genetically superior cultivars.

 In Tunisia, breeders and researchers are focused on improving the quality of olive oil (lipogenesis, acidic composition and minor components) of some commercial varieties and other local types by self- and controlled crossing which is a frequent mode of cross using foreign and local varieties as pollinators (Mehri et al., 2003).

 Since 1990, the National Olive Genetic Resources Conservation located in Boughrara-Sfax, Tunisia, collected worldwide diverse germplasm resources of olive, which constitute available sources of plant genetic material for breeding and improvement of olive cultivars.

 Cultivation of olive in Tunisia requires much effort to develop new strategies for olive improvement, to preserve and to promote the potential value of this resource. Tunisian cultivars should be improved for the competitiveness of olive oil in international market and protection of the commercial varieties quality label to develop typical olive oil products such as Protected Designation of Origin (PDO), Protected Geographical Indication (PGI) and Traditional Specialty Guaranteed (TSG) in varietals identification.

 In order to exploit genetic diversity for targeted olive cultivar improvements, several Tunisian research teams have used PCR-based markers for basic and applied research to assess the genetic diversity of Tunisian olive cultivars. These markers types include RAPD, AFLP (Grati-Kamoun et al., 2006), SSR (Hannachi et al., 2008) and SNP (Rekik et al., 2010).

 In recent years, the olive industry is getting attractive considerable interest due to the growing perception of olive oil which induced an intensive wave of olive planting.

 Most of olive oil production has been made by traditional techniques and no irrigation but it changed to extremely erratic production levels depending on the year.

 Research on genetic improvement and varietal selection takes into account the response on abiotic stress, the productivity and the quality of oil content was established since twenty years.

 To boost the olive oil industry, the government support and stimulate industry of the sector by creating a fund for the promotion of olive oil and table olive, by support producers and by the establishment of the National Development Strategy for the Olive to improve and to develop the sector. Breeding and selection of olive tree cultivars has been traditionally carried out. The necessity to target olive cultivar identification led researchers to undertake new studies of varietal identification based on genetic markers such as DNA

MOLECULAR MARKERS IN OLIVE STUDY

 Olive is a diploid species (2n = 2x = 46), predominantly allogamous, showing a high degree of outcrossing, with a genome size of about 1,800 Mb (Rugini et al., 1996).

 The distribution of Olea varieties in the Mediterranean basin gave rise to a very complex and highly articulated structure of olive culture which was marked by the existence of a considerable number of different olive cultivars (Bartolini et al., 2005). The cultivated olive is an evergreen, out-crossing, vegetatively propagated tree with a very wide genetic patrimony that is the result of both plant longevity and the scarcity of genotype turnover through centuries of cultivation (Bracci et al., 2011). The great number of existing varieties have the problem like the existence of homonyms and synonyms (Poljuha et al., 2008), which make cultivar identification very difficult and complex.

 Powerful method of genetic analysis for the development of conservation management strategies for the genetic resources and for the protection of the commercial varieties quality label are necessary.

 Investigation of olive germplasm resources in Tunisia has been motivated to increase knowledge of the genetic patrimony and to insure its identification, conservation, selection and genetic improvement of it. Tunisian olive groves are essentially dominated by two olive oil varieties: ”Chetoui” in the north and “Chemleli” in the centre and south of the country. Furthermore, those two varieties are mainly used by olive growers. Research on genetic improvement and varietal selection takes into account the response on biotic and abiotic stress, the productivity and the quality of oil content was established for last twenty years. A national breeding program was established to increase the tolerance of abiotic stress and acidic composition of cultivars within the national genetic resources (Trigui, 1996). The breeding program based on controlled crosses has been carried out and 1,600 hybrids have been planted at a density of 1,250 plants/ha in three orchards for conservation and assessment. These progenies are evaluated for early fruit bearing and for qualitative and quantitative traits or their products (olives and oils). 

 The identification of olive tree cultivars has been traditionally carried out by morphological markers that may be detected visually, include for example plant height, flower and fruit shape (Trigui & Msallem, 2002; Hannachi et al., 2008) and by biochemical markers that are allelic variants of proteins based on iso-enzymes and alloenzyme markers. These markers have shown a differential expression depending on the environment conditions and the type of tissue (Ouazzani et al., 1995).

 Many different approaches have been used molecular markers to assess the genetic diversity of Tunisian olive cultivars and wild forms such as RAPD, AFLP (Grati- Kamoun et al., 2006), SSR (Rekik et al., 2008; Abdelhamid et al., 2013). Recently, Rekik et al (2010) discovered nine new SNPs by direct sequencing of the lupeol synthase (OEW) and cycloartenol synthase (OEX) genes in 16 Tunisian olive cultivars.

 Despite its high economical importance, the available datasets and information on structural and functional genomics of olive tree development, tolerance to biotic and abiotic stress, and fruit development, are still unknown. Efforts to improve the identification and annotation of genes are prevalently based on EST identification, which are predominantly related to pollen allergens and characteristics of olive fruit.

 Many genes encoding key enzymes for fatty acid biosynthesis and modification including enoyl-ACP reductase, stearoyl-ACP desaturase, omega 6 plastidial desaturase, omega 3 plastidial desaturase, cytochrome b5, omega 6 cytoplasmatic desaturase, omega 3 cytoplasmic desaturase, acyl-CoA diacylglycerol acyltransferase and oleosin enzymes, triacylglycerol synthesis and storage have been isolated (Baldoni et al., 1996). Recently, the molecular bases of water transport in olive have been studied and three aquaporin (AQPs) genes have been isolated from cv Leccino tissues (Secchi et al., 2007). Many molecular studies were undertaken to show the molecular mechanisms of flower induction, fruit growth and development, ripening processes, and the identification of common transcription factors, including genes related to defense, abiotic and biotic response.

 Recently, genomic studies in cultivated olive were lead to the sequencing of the whole chloroplast genome and DNA sequencing of the entire plastome of the Italian cultivar ‘Frantoio’ was generated (Mariotti et al., 2010).

 Chloroplast sequence information will be used to understand the domestication of olive, the function of plastid genes on plant metabolism and will be applied in olive cultivar identification.

TUNISIAN OLIVE OIL FEATURES

 In Tunisia, olive oil export represents 40% of total agricultural exports and 5.5% of total exports and is the fifth largest source of foreign exchange.

 The olive provides 40 million working days per year and plays a role of leading regional development and social balances. Finally, from an environmental perspective, the olive tree is the only crop of fruit trees has been valued marginal soils abandoned saw its enormous plasticity and strong climate and soil adaptation.

 Nevertheless, it is essential to plant olive trees in conditions that are favourable to ensure profitability.

 With respect to its importance in more than one respect, the sector has a national strategy development to reduce annual fluctuations in production and improve the quality of olive oil in order to meet our commitments to the European Union quota which allows export of 56,000 tons of olive oil on favourable terms.

 Currently, according to data from the Tunisian ministry of agriculture and the national office of oil, there are 1,723 working olive oil mills with a crushing capacity of 44,077 tones per day (DGPA, 2012). The extraction system with continuous system is the most dominant, accounting for 65% of total crushing capacity. The processing sector also comprises 15 refineries, 14 units pomace oil extraction, more than 100 exporters and 35 units of packaging which offered great potential for boosting the expansion of the sector.

 Tunisia is ranked 4th in the world in terms of production and exports of olive oil after Spain, Italy and Greece. It exports an average of 113,000 tonnes per year or 72% of its production. More than 85% of exports are in bulk and focus on the European market (96%).

 Olive oil is the first agricultural product for export. The olive oil exports represent 30% of agri-food exports and 4% of the total value of Tunisian exports of goods.

 The Tunisian olive oil is well positioned at the intervention price and the cost of crushing and lagging behind in terms of quality, marketing and packaging.

 The strategic option for the development of the industry "Tunisian olive oil" is based on the production of oil quality and improved value to strengthen the position of Tunisian olive oil on the international market, since the 'Tunisian olive oil is well known throughout the world. Therefore different national projects focus on the development of this market in order to improve the rate of production of high quality extra virgin oil (25% in Tunisia against 80% in Italy, 78% in Spain and 70% in Greece).

 To preserve the good quality reputation of Tunisian olive oil and to prevent fraud, some commercial Tunisian company used advances technology in fats and oils analysis recommended by the International Olive Council for the effective control of the purity and quality of the olive oils. Tunisian accredited laboratories and producers applied physico-chemical and organoleptic analysis of extra virgin olive oil, i.e., free fatty acids, total phenols content and UV absorption characteristics at 232 and 270 nm, were carried out following analytical methods described in the EEC 2568/91 and EEC 1429/92 European Union Regulations. These analyses aimed to differentiate between each grade and to check product authenticity.

PRODUCTS OF OLIVE TREE AND USES

 The economic importance and the nutritional value of olive oil and table olives are well-known. In fact, consumption of olive oil and table olives was shown to be associated with a variety of health benefits, including a lower incidence of heart disease and certain types of cancer (Tuck & Hayball, 2002; Aruoma, 2003; Pérez- Jiménez, et al., 2005).

 However, the derived products from this species and the olive oil industry, in general, produce large amounts of wastes, which remain most of potentially interesting compounds that can valorised in different domains (Fig. 2). This has prompted considerable research into the valorisation of such products, and the study of their effects on the ecosystem.

Fig. 2. By-products of the olive tree and olive oil industry.

 The extraction of olive oil generates generally three products: olive oil (20%), solid waste (30%) and aqueous liquid (50%). The solid waste (so called pomace or crude olive cake) is a combination of olive pulp and stones. The aqueous liquid comes from the vegetation water and the soft tissues of the olive fruits, with water added during processing, so-called olive-mill wastewater (OMW).

 These percentages for the different phases varied according to the extraction process:

 - For the traditional press olive oil extraction system one ton of olive generates 450 litres of mill waste water and 330 kg of pomace where humidity varied from 25 to 30 %.

 -With the three-phase centrifugal olive oil extraction the same olive quantity produces about 870 litres of mill waste water

 - Finally, for the two-phase centrifugal olive oil extraction the liquid waste is reduced in two-phase centrifugation system which generated 650 to 700kg of semi-solid commonly referred to as two phase pomace with a humidity rate of 68%. This product is high humidity residue with thick sludge consistency that contains 80 % of the olive fruit, including skin, seed, pulp and pieces of stones, which is later separated and usually used as solid fuel (Vlyssides et al., 2004).

 Around 740,000 tons of olive mill wastewater and 490,500 tons of pomace are produced annually in Tunisia. For that, the recovery of high value compounds or the utilization of these wastes as raw matter for new products is particularly attractive way to reuse it. This, added to the alternative proposals to diminish the environmental impact, will allow the placement of olive market in high competitive position and these wastes should be considered as by-products (Niaounakis & Halvadakis, 2004).

 Moreover, some characteristics of OMW are profitable for agriculture, since this effluent is rich in organic matter, nitrogen, phosphorous, potassium and magnesium (Paredes et al., 1999). The organic fraction of the OMW contains a complex consortium of phenolic substances, some nitrogenous compounds (especially amino acids), organic acids, sugars, tannins, pectins, carotenoids, polyphenols and almost all of the water soluble constituents of the olives (Mulinacci et al., 2001; Lesage-Meessen et al., 2001).

 The inorganic fraction contains chloride, sulfate, and phosphoric salts of potassium as well as calcium, iron, magnesium, sodium, copper, and other trace elements in various chemical forms. The inorganic constituents at the concentration levels found in OMW are not toxic; quite the reverse, they may potentially serve as good sources of plant nutrients and thereby rendering this effluent potentially suitable for recycling as a soil amendment (Rinaldi et al., 2003). Additionally, in organic and sustainable farming, the nutritional value of OMW as well as its potential herbicidal activity (Ghosheh et al., 1999), and ability to induce suppression against soil-borne plant pathogens are of extra value (Kotsou et al., 2004).

 In addition to the industrial olive processing by-products the Tunisian olive groves procure others resulting from pruning and old orchards conversion. It has been estimated that pruning produces 25 kg of by-products (twigs and leaves) per tree per year in addition to an important biomass from big branches(Martin-Garcia et al., 2004). The total biomass produced depends on many parameters regarding the tree age, training system, crop load, product use, environmental conditions, soil fertility, and farm structure. In fact, in heavy cropping years, the growth of the tree is reduced, so pruning is limited to the elimination of water sprouts and weak shoots. Alternatively, trees are pruned more severely after years of low yields. Thus in our conditions annually the olive orchards yield approximately a total biomass value of 2,400,000 tons with 940,000 tons of twigs and leaves and 1,460,000 tons from middle and big size branches.

PERSPECTIVES

 New measures have recently been taken to reorganise and consolidate the sector, particularly by adopting new strategies and developing a package of process to stimulate the expansion of the sector. The Tunisian Government has set a series of developmental goals for the olive sector:

 - Raising orchard productivity per hectare and olive oil production by restructuring old olive groves and maintenance of existing plantations

 - Improving olive oil and table olive quality and lowering costs of production to stimulate exports

 - Creating a fund to promote Tunisian exporters in the international olive oil trade

 - Consolidating research and valorisation of by-products of olive tree

 - Creation of intensive plantations and development of nurseries for the production of softwood cuttings

ACKNOWLEDGMENT

 This work was supported by the Korean-Africa Food & Agriculture Cooperation Initiative (KAFACI) program and National Academy of Agricultural Science, RDA ( PJ008598).