HORTSCIENCE 40(1):193–196. 2005. cultivars (‘ZDH4’ and ‘Lucques’), four cul- tivars with medium rooting ability (‘Haouzia and ‘Dahbia’, two traditional Moroccan cul- Micropropagation of Eight Moroccan tivars, and the French cultivars ‘Amellau’, and ‘Salonenque’), and finally ‘Picholine and French Olive Cultivars marocaine’and ‘Picholine du Languedoc’, as difficult-to-root cultivars. The local types Saïda Sghir ‘ZDH4’ and ‘Picholine marocaine’ were found Equipe Olivier, Département d’Arboriculture, Ecole Nationale d’Agriculture during searches conducted in north Morocco de Meknès BP S/40, 50 000 Meknès, Morocco (Ouazzani et al., 1996). Although no obvious virus symptoms were Philippe Chatelet observed on the mother plants, actual virus UMR BEPC. Equipe Architecture et Fonctionnement des Espèces Fruitières status was not determined which, especially INRA, Agro M., Montpellier, France in terms of latent viruses (OEPP/EPPO, 1997), might impact on the plant rooting ability as Noureddine Ouazzani recorded in other species (e.g., Kano and Equipe Olivier, Département d’Arboriculture, Ecole Nationale d’Agriculture Nagata, 1999). Nodes were taken from 1-year growth in de Meknès BP S/40, 50 000 Meknès, Morocco young plants originating from semi-hardwood Françoise Dosba cuttings and grown either in a glasshouse and regularly sprayed with fungicide (French UMR BEPC, Equipe Architecture et Fonctionnement des Espèces Fruitières cultivars) or under open shade (Moroccan INRA, Agro M., Montpellier, France cultivars). 1 Methods. Two disinfection methods were Ilham Belkoura used. In Moroccan cultivars, explants were pre- Equipe Olivier, Département d’Arboriculture, Ecole Nationale d’Agriculture pared as described by Rugini and Fontanazza de Meknès BP S/40, 50 000 Meknès, Morocco (1981) whereas an unpublished protocol (Brachet, personal communication) designed Additional index words. genotype effect, growth regulators, in vitro, microcutting, Olea and routinely performed in the laboratory was europaea, rooting used for French cultivars: 70% ethanol (1 min), Abstract. The responses of several Moroccan and French olive (Olea europaea L.) cultivars mercryl (1 min), and 2% calcium hypochlorite to various strategies for in vitro establishment and culture were compared. A cultivar effect (3 min). Both disinfection procedures were was clearly observed with ‘Haouzia’ cultivar being more readily multiplied. ZR produced followed by three 10-min washes with sterile the best response in all the cultivars studied, in particular when considering the time elapsed distilled water. between explant inoculation and budbreak for 50% of the explants (lag phase), growth Shoot growth was induced from single node of the primary shoot and the multiplication rate. Treatments with BA alone or combined explants using cultivation on Rugini basal with NAA increased the number of axillary buds and internodes without improving their medium (Rugini, 1984) with 3% sucrose, auto- growth. Root induction with IBA in the dark using a two-phase scheme resulted in the claved for 20 min at 121 °C. Growth regulators best rooting rate in shoots obtained in vitro, and this for all cultivars. Chemical names (BA, NAA, ZR) were added as necessary. Cultures were maintained under fluo- used: 6-benzyladenine (BA), indole-3-butyric acid (IBA), alpha-naphthalene acetic acid –2 –1 (NAA), zeatin riboside (ZR). rescent light (40 µmol·m ·s ) following a 16-h photoperiod with a matching 25/22 °C More than 70% of olive trees multiplied in in olive is related mainly to both genotype thermoperiod. the circum-mediterranean area are propagated (Mencuccini and Rugini, 1994; Rugini and In vitro shoots were rooted in OM medium through semi-hardwood cuttings (Cimato, Pannelli, 1993) and culture medium (Cozza (Rugini, 1984) containing 5.37 µM NAA or 24.6 1999). Although this technique ensures genetic et al., 1997; Grigoriadou, 2002; Rugini, 1984; µM IBA (single phase) or with a two-phase homogeneity, it cannot be successfully applied Santos et al., 2003), particularly growth regu- protocol developed in the laboratory (after to difficult-to-root cultivars and the demand lators (Chaari Rkhiss et al., 2003; Rugini and Druart, 1997) with a 5-d induction phase in for healthy planting material, particularly in Fedeli., 1990) and carbon source (Garcia et liquid 24.6 µM IBA solution in the dark with respect to viral contamination (Martelli et al., al., 2002; Leva et al., 1994). Studies on these further cultivation on regulator-free Rugini 2001), is currently not met. parameters have been limited to a small range medium. Micropropagation, as a possible solution, of olive cultivars (Rugini, 1984; Wallali, 1993; The following data were recorded in the has been relatively recently applied to olive Zuccherelli and Zuccherelli, 2002) including growth phase one month after culture initiation: multiplication. Previous studies have illus- only a few southern mediterranean clones. As the time elapsed between explant inoculation trated the difficulties inherent to applying a a result, little data on the micropropagation and budbreak for 50% of the explants, hereafter universal multiplication scheme and underlined potential of these clones is available. called the lag phase; the mean number of shoots the heterogeneity of the responses obtained The study reported here present original originating from a single node; the growth re- (Bartolini et al., 1990; Garcia-Fèrriz et al., data concerning the responses of eight Moroc- corded as the mean length of in vitro developed 2002; Leva et al., 1992; Rugini and Fontanazza, can and French olive cultivars to different strat- shoots; the mean number of internodes per in 1981). In fact, in vitro multiplication efficiency egies for in vitro establishment, and attempts vitro shoot; and the number of subcultured to highlight factors likely to improve in vitro shoots 1 month after culture initiation. Received for publication 17 Feb. 2004. Accepted multiplication rates. It also includes possible Rooting response was evaluated 1 month for publication 31 May 2004. Paper presented as multiplication schemes for these cultivars. after rooting initiation and expressed as the partial requirement for obtaining a PhD degree mean rooting and callusing rates, the mean by S. Sghir. This work was completed within the Materials and Methods root number and length, and the mean leaf framework of the project Projet de Recherche pair number. Agronomique pour le Développement 01-13. We Data were analyzed by ANOVA (Statistica thank Boutaïna Mokhless and Chantal Brachet for Plant material. Eight among interesting their expert technical assistance, and Mike Jones for Moroccan and French olive cultivars, differing (version 6) Statsoft) and means were separated English reviewing. in their response to semi-hardwood cutting using Newman and Keuls test (Miller, 1981) 1To whom reprint requests should be addressed; (Barranco et al., 2000; Sghir et al., 2003), at a 0.95 confidence level. Alternatively, a e-mail [email protected]. were used in these studies: two easy-to-root chi-square homogeneity test was performed HORTSCIENCE VOL. 40(1) FEBRUARY 2005 193 lower cytokinin concentration (Table 3). Genotype response to in vitro rooting. In vitro shoots were rooted using two different protocols as described previously. In the single phase rooting experiment, NAA stimulated root formation only in “Pi- choline marocaine” cultivar whereas IBA–con- taining treatments gave rise to variously sized calli at the base of the explants, without root formation. By contrast, the two-phase protocol pro- duced various rooting percentages in all geno- types (Table 4 and Fig. 3). The highest rate was observed in ‘Salonenque’ (70%) and ‘Picholine marocaine’ (65%). The ‘Haouzia’, ‘Dahbia’ and ‘ZDH4’ cultivars reached about 50% rooting, Picholine de Languedoc reached 40% rooting while ‘Lucques’ and ‘Amellau’ never exceeded Fig. 1. Budbreak rate over time after culture ini- tively, while falling to 1.5 and 1 in ‘Dahbia’ 30% root formation. Callusing was high for all tiation and ‘ZDH4’. cultivars, ranging between 70% (e.g., ‘Haouzia’) Mean shoot growth reached a maximum and 100% (e.g., ‘Salonenque’). Table 1. Growth parametersz in four Moroccan of 23 and 20 mm in length in ‘Haouzia’ and If all genotypes are considered, the average olive cultivars. ‘Picholine marocaine’, with a corresponding root number was 3.2 (reaching 5 in ‘Picholine marocaine’) for a 2 cm mean length (reach- Cultivar SN SL (mm) MIN internode number of 2.5 and 1.8. Intermediate ing 4 in ‘Salonenque’). The average number Dahbia 1.5 aby 15 a 2.3 ab results were obtained for the ‘Dahbia’ cultivar Haouzia 2.0 b 23 b 2.5 b (15 mm mean length and 2.3 internodes) and of newly formed leaves ranged from three Picholine marocaine 1.7 b 20 b 1.8 a for local type ‘ZDH4’ (14 mm length and 1.8 pairs in the ‘Haouzia’ variety to two pairs in ZDH4 1.0 a 14 a 1.8 a internodes). ‘Salonenque’ and ‘Picholine marocaine’ and zSN = average shoot number, SL = mean shoot Stimulation of multiplication by growth 1 for all remaining cultivars. length, MIN = mean internode number. regulators. The presence of growth regulators yMeans followed by different letters within the same clearly increased the multiplication response Discussion column are significantly different according to a in all tested cultivars (Table 2). No significant Newman and Keuls test at 5% error level interaction treatment × genotype was observed The multiplication rates observed in this when considering effects on mean shoot and study ranged