Dipartimento di Produzione Vegetale, sez Ortofloroarboricoltura, Università degli Studi della Tuscia,01100 Viterbo , Italy
Massimo Mencuccini,
Istituto per i Sistemi Agricoli e Forestali del Mediterraneo (ISAFoM-CNR)Sezione di Perugia Via Madonna Alta 128, 06128 Perugia, Italy
Rita Biasi,
Dipartimento di Produzione Vegetale, sez Ortofloroarboricoltura, Università degli Studi della Tuscia,01100 Viterbo , Italy
Maria Maddalena Altamura,
Dipartimento di Biologia Vegetale, Università di Roma “La Sapienza”P.le Aldo Moro, 5, 00185 Roma, Italy
1. INTRODUCTION
Olive (Olea europaea L.) is one of the oldest, most widespread and important crops of the Mediterranean basin. Many different olive genotypes are cultivated and a high degree of morphological and biological variation exists (Rugini and Lavee 1992). Olive cultivation from Mediterranean basin is presently expanding into areas of Australia, South and North America (Argentina, Chile, United States) and South Africa (Rugini and Fedeli 1990). The Mediterranean basin is the traditional area of olive cultivation and has 95% of the olive orchards of the world.
The olive species is originated most likely in Asia and then spread westwardsalong the Mediterranean Sea coasts (Blazquez 1996). The olive belongs to theOleaceae family, which comprises of 29 genera and the genera Olea is one of themwith 35 species (Heywood 1978). The domesticated olives belong to the genus Olea, species europaea, subspecies sativa and the number of the cultivated speciesis estimated more than 2500 cultivars. However a new classification is understudyas reported by Rugini and Baldoni [in press in Litz (ed), Hardwick T. Book Publisher]. All species of the genus Olea have the basic chromosome number 2n =46 (x = 23). In the same area, subspecies sylvestris.is widespread in the wild.
Olive oil is the first product and olives are also consumed as table olives in the Mediterranean countries. The consumption of olive oil has recently increased dueto its nutritional value. The wood has a negligible importance even though itsappreciation especially for artistic and building construction works.
The olive tree and its products can be damaged from many diseases and pests. Themost dangerous are the bacterium Pseudomonas savastanoi, that producetubercules forms on the branches and stems, the fungus Cycloconium oleaginum
that damage the leaves and fruits and Verticillum dahlie that is harmful for the root apparatus and the growth of the plants. Among phytophagous, most harmful are the olive fruit fly (Bactrocera olea(( Gmelin), the olive moth (Prays oleae(( Bernard)and black scale (Saissetia oleae Olivier). Olive fruit fly is the major pest and can cause severe economic damage to olive production, which effect oil extraction and table use. Olive is susceptible to several viruses, more than 70% of cultivated oliveplants seem to be affected by latent viruses. At present, about 20 viruses have beenisolated from olive (Martelli, 1998). More common symptoms, as described in olive plants, are twisted and narrow leaf lamina and poor growth and deformed fruits that has been demonstrated due to the presence of strawberry latent ringspot virus (SLRV) (Marte et al., 1985; Pasquini et al., 2002).
Olive trees were multiplied by using different explants including ovule(spheroblast) or sucker and subsequently leafy stem cutting and grafting onseedlings or clonal stocks. Vegetative reproduction potential varies, which is dependent on genotype, e.g. easy to rooting and recalcitrant to root initiation(Hartmann and Kester 1968). Micropropagation of the olive cultivar wassuccessful on OM medium (Rugini, 1984) and subsequently several other researchers sligthly modified the culture medium by adding different growthsubstances (Fiorino and Leva, 1986; Cozza et al., 1997; Mencuccini et al., 1997) or rooting conditions (Mencuccini, 2003). The micropropagated materials can be used to screen for resistance to biotic and abiotic stress (Sasanelli et al., 2000; Bartolozzi et al., 2001) and for genetic improvement activity (Rugini et. al, 1999). Olive callus has been established from different olive tissues such as shoot (Lavee and Messer, 1969), fruit mesocarp (Lavee, 1977), hypocotyl seeds (Bao et al.,1980). Shoots organogenesis was obtained from seedling explants (Gilad and Lavee, 1974; Cañas and Benbadis, 1988; Rugini, 1988) and adventitious buds frompetioles cultivars with plantlets development (Mencuccini and Rugini, 1992). In olive, somatic embryogenesis was induced from immature zygotic embryos (Rugini, 1988; Leva et al., 1995), cotyledons and radicles from mature embryos(Orinos and Mitrakos, 1991; Mitrakos et al., 1992; Shibli et al., 2001), petioles,excised from adventive buds derived from in vitro growing shoots of cultivars Canino and Moraiolo (Rugini and Caricato, 1995) and explants of young lateralshoots of in vitro growing cultures (Mencuccini and Pollacci, 2002).
Olive plants regeneration via somatic embryogenesis and DNA recombinant technique, both have enabled us to obtain transgenic olive cultivars. Currently, thegenes inserted in olive plants are rolA,B,C and ll osmotin (Rugini et al., 2000) and transgenic plants are in field under observation.
In this chapter, we have described protocols for somatic embryogenesis from mature and immature zygotic embryos, and mature tissue of olive cultivars, histological studies and plantlet regeneration.
2. MATERIALS
2.1. Embryogenesis from mature and immature zygotic embryos,
seedlings and mature tissue explants:
1. Sterile water, Commercial bleach (NaClO commercial product with 5.5% of chlorine)
2. Flow hood, Petri dishes, pipettes, forceps, scalpel, parafilm 3. Dissecting microscope 4. Mature and immature fruits (75 days after full bloom and use at once or store
them at 14-150 C for 2-3 months before use) 5. Zygotic embryos (germinated and non germinated)6. In vitro shoots of mature cultivar proliferated on OM medium
7. Aventitious buds from lef petioles of cultivar 8. Media (see Table 1)
Table 1: Basic culture media
Constituents OM MS OMeMacro elements mg/l mg/l mg/l
KNO3 1100 1900 950
NH4NO3 412 1650 720Ca(NO3)2
. 4H20 600 - -
KCl 500 - -CaCl2
. 2H20 440 440 166
MgSO4. 7H20 1500 370 92,5
KH2PO4 340 170 68
Micro elements
FeSO4.7H20 27.8 27.8 27.8
Na2EDTA 37.5 37.5 37.5
MnSO4. 4H20 22.3 22.3 22.3
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H3BO3 12.4 6.2 12.4
ZnSO4. 7H20 14.3 8.6 14.3
NaMoO4. 2H20 0.25 0.25 0.25
CuSO4. 5H20 0.25 0.025 0.25
CoCl2. 6H20 0.025 0.025 0.025
KI 0.83 0.83 0.83
Vitamins
myo-Inositol 100 100 100
Glycine 2 2 2 Thiamine.HCl 0.5 0.1 0.5
Pyridoxin.HCl 0.5 0.5 0.5
Nicotinic acid 5 0.5 5Biotin 0.05 - 0.05
Folic acid 0.5 - 0.5
Amino acid
L-Glutamine 2190 -
Sugars
Mannitol 30000 30000 30000
Solidifying agent
Agar 6000 6000 6000
Pectin of must wine 10000
Supplements
NAA - -
IBA 0.05
Zeatin 2-4 -
2iP 0.1
BA 0.1
Thidiazuron 0.05-2 -
GA3 5-10
Cefotaxime 200
Caseine hydrolisate 1.000
*The pH of all media are adjusted to 5.8
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3. METHOD
The success of inducing somatic embryogenesis depends on origin of material and age of tissues. Somatic embryos have been obtained from both immature and mature zygotic embryo explants, with or without callus interposition and frommature tissues of olive cultivars.3.1 Somatic Embryogenesis from immature zygotic embryo
3.1.1 Initiation of Embryogenic Cultures
1. Break the stones and remove the seeds from stony endocarp. 2. Surface-sterilize seeds with 10% sodium hypochlorite (NaClO)
commercial product (5.5% chlorine) for 10 minutes. 3. Wash seeds 2-3 times in sterile water.4. Soak for at least 24 hr in sterile water at room temperature 5. Remove with the scalpel the zygotic embryos by longitudinal or
transversal cut of the endosperm.6. Place embryos horizontally on half strengthen MS medium (Table 1)
supplemented with 0.5-2.5 M BA, 2% sucrose and agar in a Petri dishes and wrap the plates with a double layer of parafilm.
7. Place the cultures in the dark at 23°C for one month.
3.1.2. Maintenance of callus culture and maturation of Somatic
Embryos
1. Subculture the embryogenic callus to the same medium reducing BAconcentration (normally the callus looses the capability to produce newembryos after one subculture).
2. The embryos are visible after 5-6 weeks of initial culture, directly from theinitial explant (60-70%), and also from callus (20-30%). The remainder normally is not possible to identify the origin with the stereo-microscopic examination.
3. Secondary embryogenesis is normally observed from the epidermal tissue of neoformed embryos or teratoma.
3.1.3. Embryo germination and conversion to plant
1. Somatic embryos separated from callus or from original tissues cangerminate quickly on OM solid medium plus 0.5-1 mg/l zeatin or in OMe liquid medium plus 0.3 mg/l zeatin, and to convert into plantlets.
2. When the second pair of leaves are formed, transfer them to jiffy-pots and place in the greenhouse under high relative humidity
Note: Embryogenic cultures have also been induced from cotyledons of 126-day-
old zygotic embryos of ‘Chalkidikis’ (Pritsa and Voyatzis, 1999), from cotyledon
segments from mature zygotic embryos of wild olive (O. europaea var. sylvestris),
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from the radicle of mature embryos of (Rugini and Tarini, 1986; Mitrakos et al.,
1992; Rugini et al., 1995; Shibli et al., 2001) and from mature zygotic embryos
(Orinos and Mitrakos, 1991; Mitrakos et al., 1992). However no standard medium
can be used since the success is highly genotypic dependent and often not
repeatable, contrary to immature zygotic embryos.
3.2 Somatic embryogenesis from mature tissue explants
Induction of somatic embryogenesis from tissues of elite olive has been rather difficult to obtain. Up-to-now it has been reported for three cultivars, 'Canino', 'Moraiolo' and “Dolce Agogia” (Rugini and Caricato, 1995; Mencuccini and Pollacci, 2002). The type of initial of explant and genotype are essential for the success. Up to now two types of explants have been used: a) leaflets fromadventitious buds, 2-3 mm long (double regeneration) (Fig 1 left), and b) 3-10 mmlong young shoots, after bud sprout from uninodal explants on OM medium.
3.2.1. Initiation of Embryogenic Cultures
1. Collect leaf petioles from in vitro olive cultivar from micropropagated shoots with frequent subcultures (not more than 20-25 days intervals) on OM medium.
2. Place the petioles in Petri dish (25x90 mm) containing 20 ml half-strength MS medium (Table 1) and seal with parafilm with the aim to regenerate adventitios buds.
3. Dissect the leaflets with petiole from 1-3 mm long neoformed adventitious buds, regenerated from petioles, and place them individually in 25 multiwell plate, each well containing 3 ml OMe medium and place the cultures in the dark at 23°C.
3.2.2. Formation of Embryonal Callus Mass
After about 4 weeks, morphogenic callus mass is produced from petioles. Transfer it to Petri dishes containing 5 ml OMe liquid medium (Table 1) sufficient to soaking a filter paper (Whatman No 3). Every 3-week interval, add fresh medium and remove the equivalent part of the resulted spent medium by pipetting it. After about 20 days proembryo masses recognizable because it looks like a callus with yellowish smooth surface (Fig 1 right), continue to enlarge differentiate somatic embryos. If the callus results too aboundant reduce or remove completely the hormones from the medium.
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3.2.3. Production of cyclic secondary embryogenesis and embryo
Maturation
Place the neoformed embryos on agar OMe medium (table 1) for producing cyclic secondary embryogenesis both from normal and abnormal (teratoma) embryos.
3.2.4. Embryo Germination
Culture single embryos in multiwells (12 wells containing 2 ml OMe liquid medium supplemented with 0.3 mg/l zeatin per 5-6 embryos) and place on agyratory shaker, 80 rpm, in a growth chamber at 23°C in the light photoperiod (16 h light). Conversion rate declines with time. Embryo germination is difficult, although the hypocotyl elongation generally occurs. On contrary,epicotyl development rarely occurs. However, 1-week cold treatment (4°C)increases often the subsequent phase of embryos germination.
3.2.5. Synthetic seeds
Micheli et al. (2002) reported first time production of olive synthetic seeds by using somatic embryos of cv. Canino.
1. Use 2-4 mm long somatic embryos2. Immerse them in a 2.5% sodium alginate solution (encapsulation matrix)
and then drop them in a complexing solution of 100 mM CaCl2, for 30min.
3. After hardening, rinse the capsules twice for 10 min in distilled water towash away calcium chloride residues.
4. In order to insert an artificial endosperm into the beads, use half strength OM medium solution containing sucrose (87.6 M) without any growth regulator and agar.
5. For germination, sow them in Petri dishes containing half strength OMmedium containing 9.2 µM zeatin.
6. Transfer the germinated seedlings on Jiffy-7 pots with high humidity and under continuous light flux
3.2.6. In vitro preservation
Embryogenic ‘Canino’ cultures, consisting of Pro-Embryo-Mass and somatic embryos at various developmental stages are highly suitable for cryopreservationby vitrification (Lambardi et al., 2000). After incubation in vitrification solution, high percent (38%) of cryopreserved embryogenic cultures survive. Moreover, therecovered embryogenic tissue show enhanced proliferative and morphogenicactivity.
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3.3 Histology of somatic embryogenesis.
In order to identify conditions for improving the development of secondary embryos into normal plantlets, different culture conditions are studied, as well ashistological analysis is done. Secondary embryogenesis is induced under conditions differ in medium composition and temperature.
The tested media are standard hormone-free OMe (medium A), medium OMecontaining low plant growth regulators, i.e. 0.05 mg/l 2i-P plus 0.05 mg/l BA and 0.005 mg/l IBA and double concentration of K and P (medium B), and medium OMe supplemented with 0.3mg/l zeatin only plus double concentration of Calcium(medium C) (see Table 1). The cultures are either combined with a short-term low temperature (4°C for 7 days) treatment or maintained at 23 1°C for the entire period. After one month of culture, under continuous darkeness, transfer secondary embryos into liquid OMe medium for germination, and after 20 days samples aretaken for histological examination (Altamura et al, 1992).
Under the tested conditions, the highest number of normal and well-developed somatic embryos is obtained on hormone-free medium (figure 2A). Independent of the culture medium, low temperature treatment is relevant to induce regular embryo polarity, however, irrelevant to reduce morpho-structual anomalies when compared with cultures maintained at 23°C. In fact, even with exposure to lowtemperature exposure, somatic embryos with more than two cotyledons are obtained on all media, and the development of globular adventive embryoids oncotyledons (figure 2B-C). Another anomaly is the callusing of the hypocotyl (figure 2D-E). These anomalies occur in hormone-free medium (figure2B-D), but more frequently on the media supplemented with hormones (figure 2E-H). Moreover, some somatic embryos growing on medium B show drastic reduction in cotyledonary expansion and adventive embryoid formation on rudimentarycotyledons. The adventive embryoids develop up to the cotyledonary stage (figure2F). Another major problem is enhanced callusing of hypocotyls (figure 2G), and of the cotyledonary rudiments on medium C (figure 2H). The point to be noted isthat independent of medium composition, the primary root always develops, and regular differentiation of root apex and lack of adventive embryogenesis (figures2A-H and 3A).
The histological analysis show that the shoot apex of the abnormal embryos obtained under hormone-free conditions may exhibit strong alteration in the formation of leaf primordia, with localised events of cell lysis (figure 3B). On the cotyledons, the adventive embryoids are present at various developmental stages, from globular (figures 3B-D) to heart stage (figure 3E). Also, caulogenesis (shoot formation) occur infrequently from the cotyledons (figure 3F).
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Somatic embryos cultured on medium B, adventive embryogenesis occur on thecotyledons was more extended and irregular than under hormone-free condition (figures 3C and 3G, in comparison). Furthermore, also the histological structure of the cotyledons change due to increased xylem formation (figure 3H). The embryosalso show anomalous proliferation of cells along the hypocotyl (figure 3I), withformation of new globular embryoids on it, and occurrence of cell lysis (figure 3J). In the embryos cultured on medium C, the cotyledon structure is badly disturbed by adventive embryoid formation, and they are located deep in the callused mesophyll (figure 3K). Forthermore, fusion events also occur and are sopronounced to strongly affect adventive embyoid morphology (figure 3L).
3.3.1. Derived Plants
1. Transfer germinated somatic embryos (Figure 4) to Jiffy-7 pots and placethem under high humidity (95 % RH) and 50 mol.m-2.s-1 photon flux in a growth room at 23 1 °C for two months, before transplanting them to pots (8 cm diam.) containing a peat moss substrate held under standard glasshouse conditions.
2. Break dormancy by spraying with 400 mg/l GA3 solution.
3.3.2. Field trials
Plants derived from somatic embryogenesis (Figure 5) show a tap root and a relative long juvenile period, however not exceeding 3-4 years, 2-3 years superior than corresponding micropropagated plants by axillary bud stimulation, and phenotype not different than mother plants, although molecular analysis is needed to ascertain somaclonal variation.
4. CONCLUSION AND FUTURE PROSPECTIVE
Regardless of available efficient protocols for the induction of somatic embryos in olive tree, the possibility of somatic embryos develop into healthy plants is stilllow. Therefore, the germination rate of somatic embryos needs to be increased for commercial application for large-scale plant multiplication, cryopreservation and encapsulation. Since somatic embryos start from adult cells through an indirect process (i.e., formed from callus produced by the adult cells), they may express new traits due to somaclonal variation. Thus, the stabilization of the embryogenic process, as well as the canalization of embryos into a regular plant development program could represent a valid tool for using the possibly induced somaclonalvariation for biotechnological applications (Lambardi et al., 1999; 2002).
The instability of somatic embryogenesis under the standard conditions, as described earlier, has frequently led to abnormal embryo structures responsible for the altered plant development, the same as secondary embryo production (Benelli
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et al., 2001). The latter event would, however, be utilized. In fact, the capability toform secondary embryos is the way to obtain somatic embryonic clones useful for early selection to various conditions. Furthermore, secondary embryos are suitabletarget for Agrobacterium-mediated transformation, especially by particlebombardment method. Nowadays, the latter seems to be an important strategy of forest biotechnology (Altamura, 2002). In olive tree, secondary embryogenic process is not yet completely controlled for obtaining healthy plants. Further attention is needed to overcome frequent structural anomalies in secondary embryos, such as the lack of embryo polarity, the proliferation of adventiveembryoids at the primary root pole, and embryo fusion (Benelli et al., 2001).
5. ACKNOWLEDGEMENT
Financial support from Regione Lazio – Italy- “PRAL olivo vivaismo”
6. REFERENCES
Altamura M.M. (2002). Somatic and androgenic embryos in Angiosperm. Abstract of Lectures and Free Communications of the XLVIII Convegno del Gruppo Embriologico Italiano,Grottammare (AP) 4-7 June 2002: 23-25
Altamura M.M., A. Cersomino, C. Majoli, and M. Crespan (1992). Histological study of embryogenesis and organogenesis from anthers of Vitis rupestris du Lot cultured in vitro.Protoplasma 171: 134-141.
Bao Z.H., Y.F. Ma, J.F. Liu, K.J. Wang, P.F. Zhang, D.X. Ni and W.Q. Yang (1980). Induction of plantlets from the hypocotyl of Olea europaea L. in vitro. Acta Bot. Sin. 2:96-97. (Chinese).
Bartolozzi F., M. Mencuccini and. G. Fontanazza (2001). Induction of frost tolerance in olive plantsin vitro by cold acclimation and sucrose increase. Plant Cell, Tissue and Organ Culture 67 (3):299-302.
Benelli, C., Fabbri, A., Grassi, S., Lambardi, M. & Rugini, E. (2001) Histology of somaticembryogenesis in mature tissue of olive (Olea europaea L.). Journal of
Horticultural Science Biotech 76, 112-119. Blazquez J. M. (1996). The origin and expansion of olive cultivation. In: International Olive Oil
Council (ed) World Olive Encyclopedia. IOOC, Madrid, pp.19-20.Cañas, L.A. and A. Benbadis (1988). Plant regeneration from cotyledon fragments of the olive tree
(Olea europaea L.). Plant Sci. 54: 65-74.Gilad F., S. Lavee (1974). Callus formation and organogenesis from varius parts of developing olive
embryos. Abstract Book ‘III International Congress on Plant Tissue and Cell Culture’.Leicester, p. 87.
Hartmann H.T. and D. Kester (1968). Plant propagation. Prentice Hall, pp. 222-230.Heywood, V. H. (1978). Flowering Plants of the World. Oxford, London, Melbourne: Oxford
University Press. Lambardi M., S. Amorosi, G. Caricato, C. Benelli, C. Branca and E. Rugini (1999). Microprojectile-
DNA delivery in somatic embryos of olive (Olea europaea L.). Acta Horticulturae 474: 505-509.
354
Lambardi M., C. Benelli, A. De Carlo, A. Fabbri, S. Grassi and P.T. Lynch (2002). Medium and long-term in vitro conservation of olive germplasm (Olea europaea L.). Acta Horticulturae 586:109-112.
Lavee S. (1977) . The growth potential of olive fruit mesocarp in vitro (Olea europaea L.). ActaHorticulturare, 78: 115-12.
Lavee S. and G. Messer (1969) - The effect of growth-regulating substances and light on olive callusgrowth in vitro. J. Exp. Bot. 20: 604-614.
Martelli, G.P. (1998) Enfermedades infecciosas y certificacion del olivo: panorama general. PhytomaEspana 102, 180-186.
Mencuccini M. (2003). Effect of medium darkening on in vitro rooting capability and rootingseasonality of olive (Olea europaea L.) cultivars. Scientia Horticulturae vol 97/2: 129-139.
Mencuccini M., M. Micheli, A. Standardi (1997). Micropropagazione dell’olivo: effetto di alcunecitochinine sulla proliferazione. Italus Hortus vol. 4 n. 7: 32-37.
Micheli M., P. Dell'Orco, M. Mencuccini, and A. Standardi (2002). Preliminary studies on thesynthetic seed and encapsulation technologies of olive vitro-derived olive explants. Acta Hort.586: 911-914.
Mencuccini M., P. Pollacci (2002). Rigenerazione di embrioni somatici da cultivar di olivo e loroimpiego nella costituzione del seme sintetico e nella trasformazione genetica. Convegno Internazionale di Olivicoltura, Spoleto 22-23 Aprile, pp. 417-422.
Mencuccini, M. and E. Rugini (1993). In vitro shoot regeneration from olive cultivars tissues. PlantCell, Tissue and Organ Culture 32: 283-288.
Mitrakos K., A. Alexaki, P. Papadimitriou (1992). Dependence of olive morphogenesis on callusorigin and age. J. Plant Physiol. 139: 269-273.
Orinos, T. and K. Mitrakos (1991). Rhizogenesis and somatic embryogenesis in calli from wild olive(Olea europaea var. sylvestris (Miller) Lehr) mature zygotic embryos. Plant Cell Tissue and Organ Culture 27: 183-187.
Pasquini, G., L. Baldoni, L. Ferretti, G. Pannelli, F. Faggioli, M. Barba (2002). Evaluation of thestrawberry latent ringspot virus (SLRSV) in some olive cultivars. Atti Convegno InternazionaleOlivicoltura, Spoleto, Italy, pp. 462-465.
Pritsa T.S. and D.G. Voyiatzis (1999). The in vitro morphogenetic capacity of olive embryos, asaffected by their developmental stage and the L-arginine and L-glutamine concentration in thenutrient substrate. Acta Horticulturae 474: 87-90.
Rugini E. (1984). In vitro propagation of some olive (Olea europaea sativa L.) cultivars withdifferent root-ability, and medium development using analytical data from developing shootsand embryos. Sci. Hortic. 24: 123-134.
Rugini E. and E. Fedeli (1990). Olive (Olea europaea L. ) as an oilseed crop. In: Y.P.S. Bajaj (ed.)Legumes and oilseed crops I. Biotecnology in Agriculture and Forestry, vol. 10 Springer ,Berlin Heidelberg New York, pp. 593-641.
Rugini E. and S. Lavee (1992). Olive. In: F.A. Hammerschlag, R.E. Litz (eds.) Biotechnology of Periennal Fruit crops. Biotecnologi In Agriculture, vol. 8 C.A.B International, Wallingford, pp.371-382.
Rugini E., P. Gutierrez-Pesce, P.L. Spampinato, A. Ciaramiello and C. D’Ambrosio (1999). Newperspective for biotecnologies in olive breeding: morphogensis, in vitro selection and genetransformation. Acta Horticulturae. 474: 107-110.
Rugini, E. and P. Tarini (1986). Somatic embryogenesis in olive (Olea europaea L.). In: MoetHennessy (ed.), Proceedings Conference Fruit Tree Biotechnology. Paris (France), p.62.
Rugini E., A. Pezza, M. Muganu, and G. Caricato (1995). Somatic embryogenesis in olive (Olea
europaea L.). In: Y.P.S. Bajaj (ed), Somatic Embryogenesis and Synthetic Seed I.Biotechnology in Agriculture and Forestry, Vol. 30. Springer, Berlin Heidelberg New York, pp. 404-414.
355
Rugini E., R. Biasi, R. Muleo (2000). Olive (Olea europaea var. sativa) Transformation. In:Molecular Biology of Woody Plants Vol. 2. S.M. Jain and S.C. Minocha (eds), Kluwer Academic Publishers, pp 245-279.
Sasanelli N., N. D’Addabbo, P. Dell’Orco and M. Mencuccini (2000). The in vitro use of oliveexplants in screening trials for resistance to the root-knot nematode, Meloidogyne incognita.Nematropica, 30: 101-106.
Shibli R.A., M. Shatnawi, M. Abu-Ein and K.H. El-Juboory (2001). Somatic embryogenesis and plant recovery from callus of ‘Nabali’ Olive (Olea europaea L.). Scientia Horticulturae 88: 243-256.
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Figure 1 - Adventive buds from petioles of in vitro grown cv Canino (left); Proembryonic massoriginated from petioles of adventive buds, obtained by a “double regeneration system” (right).
Figure 2 – Stereomicroscope images of olive tree secondary embryos cultured on different media andexposed to low temperature. A-D, medium A; E-F, medium B, G-H, medium C. Description of embryo morphology in the text.
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Figure 3 – Histological analysis of olive tree secondary embryos cultured on different media andexposed to low temperature. A-F, medium A (bars: A= 200 m; B= 500 m; C=100 m; D= 250 m;E=100 m; F= 200 m); G-J, medium B (bars: G= 200 m; H= 100 m; I=500 m; J=500 m); K-L,medium C (bars: K= 200 m; L= 500 m). Description of embryo histology in the text.
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Figure 4 - Cyclic somatic embryogenesis showing different developmental stages of somaticembryos.
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Figure 5 - Field grown 4-year-old olive tree cv Canino derived from somatic embryogenesis (left);Root system of a potted plant obtained from somatic embryo of cv Canino (note the presence of taproot) (right).
