MICROPROPAGAREA LA SPECIA PISTACIA LENTISCUS L.-OPTIMIZAREA PROTOCOLULUI DE STERILIZARE A SUPRAFEȚEI ȘI VENTILAȚIE FORȚATĂ ÎN CULTURA DE IMERSIE TEMPORARĂ MICROPROPAGATION OF PISTACIA LENTISCUS L.-OPTIMIZATION OF THE SURFACE STERILIZATION PROTOCOL AND FORCED VENTILATION IN TEMPORARY IMMERSION CULTURE

Pistacia lentiscus L., belonging to Anacardiaceae family, is a typical species of the Mediterranean maquis and it is widely grown in Greece and Italy mainly for its aromatic resin extraction or as ornamental plant and also as Pistacia vera L. rootstock. Its propagation is difficult either by seed or by cuttings. The current study was carried out to optimize the micro propagation of Pistacia lentiscus L. starting from seedlings and woody explants. For the surface sterilization two different protocols were evaluated for woody explant and 6 treatments with combinations of different sterilizing agent types and concentrations were used for mature seeds. For woody explants, no significant differences could be evidenced on contamination percentage and plant survival but the initial growth in vitro of the explant was better in case of the first treatment (1.5% NaOCl for 30 min and 70% Ethanol for 1 min) than opposite combination. The highest seed contamination percentage occurred in case of treatment with 1% NaOCl for 30 min. The treatment with Ethanol (70%) for 30 second followed by three times washing with distilled water then use of NaOCl (1%) for 30 min permitted to obtain 100% of sterility. The highest seed germination (100% after 3 days) was obtained in seeds treated with Ethanol (70%) for 30 second then NaOCl (1%) for 30 min. In order to study the proliferation two different procedures were compared in liquid and agar-based media. Our results proved that proliferation rate increased 6.5 % by forced ventilation system. Longer shoots (10.5 cm) were obtained in temporary immersion system using RITA boxes. This culture system induced also the highest shoot weight which is the increasing of the 29.56% respect common vessels and agar-based medium. Cuvinte cheie: arbore de mastic, micropropagare, agenți de sterilizare, cultură de imersie, ventilație forțată.

serious problems were evidenced for primary sterilization and in vitro multiplication of adult lentisk plants (Mascarello et al., 2007;Yildirim, 2012;Kilinc et al., 2014). It was well documented that, starting from adult plants of Pistachio species like lentisk, leading to a severe occurrence of phenolic compounds and contaminations in vitro at least in the first phase. (Dolcet-Sanjuan and Claveria, 1995; Barghchi and Alderson, 1989). Many attempts were tested for the reduction of the contamination problems for example Onay (2000) proposed meristem tips excision; Parfitt and Almehdi (1994) stated that the reduction of sugar content in the culture medium could prevent contamination. Tilkat et al (2006) showed that the use of 10% NaOCl for 30 min can be effective for surface sterilization of explants on adult Pistachio trees. Different procedures were used to eliminate seeds fungal and bacterial contamination in different plant species (Salehi and Khosh-Khui, 1997;Haldeman et al., 1987;Reed and Tanprasert, 1995;Seckinger, 1995; Sen et al., 2013 a and b).  reported that among different sterilizing agents, for seed sterilization of Achyranthes aspera plant, Sodium hypochlorite application for 30 min at 1% concentration was highly effective as a sterilizing agent with 83. 44 and 63.88% plant survival, accompanied by 100 and 60% germination percentage. Based on  reports, growth and development of explants were not negatively affected by NaOCl treatment, and other procedures of NaOCl treatment involve increased concentration and duration were less efficient also in respect to sterilization, because of negative consequences on growth of plantlets. They also reported that different concentration of sterilizing agents had different effects on contamination level and survival percentage. In a similar study, Tomaszewska-Sowa and Figas (2011) showed that for Cup plant (Silphium perfoliatum L.) seeds, among the tested methods, sodium hypochlorite solution proved to be the most effective for disinfestation. Different propagation aspects for lentisk were evaluated by Fascella et al (2004), Ruffoni et al (2004), Mascarello et al (2007) and Taskin and Inal (2005), and mainly have been focused on lentisk explants micro propagation by using agar-based systems. it is well documented that the commercial plant micro propagation in conventional agar-based media is labor intensive and costly (Ascough and Fennel, 2004;Berthouly and Etienne, 2005;Quiala et al., 2012). In vitro plant propagation using liquid based media may reduce production costs (Ziv, 2005). Gelling agents increase the production costs and restrict the possibility of automation (Quiala et al., 2012). Furthermore, the liquid culture improves multiplication successes in term of plant quality (Ascough and Fennel, 2004). Despite of all benefits of liquid culture media, the occurrence of hyperhydricity is the main disadvantage of these method (Ascough and Fennel, 2004;Berthouly and Etienne, 2005), So, some techniques were used to prevent this damage for example, membrane rafts, bubble and gassing bioreactors usage, shaking batch cultures applying and temporary immersion system (TIS). Based on previous reports, temporary immersion system has been used for different plant species successfully (Cabasson et  . Now TIS is used for the production of plant secondary metabolites in some rare herbs. RITA boxes can increase plant growth parameters and propagation yield by ventilation which is in direct relationship with Increasing growth parameters (Ibaraki,1992). In unventilated vessels the plant material shows several physiological disorder, such as inability to photosynthesize, open stomata and lack of a cuticle layer (Fuchigami et al., 1981). It has been demonstrated that in common vessels there is low transpiration rate, minimum water and nutrient uptake, low photosynthesizing enzymes activity and decrease of growth parameters (Jeong et al., 1995). Forced or natural ventilation can create better conditions for plantlets growth by increasing Leaf cuticule layer and also stomatal function (Zobayed et al., 2001). To our knowledge, there are no cited references on Lentisk micro propagation using TIS, furthermore, there is poor information on micro propagation of woody plants using . The present study aims to improve the efficiency of surface disinfestation and initial in-vitro establishment of lentisk by comparing sterilization treatments and in vitro culture initiation of seeds and woody explants from adult lentisk trees. Moreover, In current study in order to develop an efficient micro propagation method for the Lentisk plant, using RITA boxes, a growth comparison versus agarbased media were assessed.

Plant material and sterilization treatments
Woody cuttings were collected in December from lentisk plant, grown at the CREA OF Institute, in Sanremo, a province located in the North of Italy. Immature seeds were collected from plants grown in the wild in Strada Mulattiera San Lorenzo, Liguria region, 163 meter above sea level. Woody explants, 2 cm long with three buds were washed with tap water for 1 hour and were sterilized following the combinations reported in Table 1. Solutions of 1 and 1.5% of sodium hypochlorite (NaOCl) and 70% Ethanol were prepared with distilled water. After sterilization treatment, the basal part of the shoot tips was cut and explants were cultured in the culture initiation medium. The seeds were sterilized according to different treatments ( Table 2). The culture initiation medium was Murashige and Skoog (1962) base medium containing mineral nutrients, vitamins, and sucrose (30 g L ) for seed culture. The pH was adjusted to 5.7 before autoclaving. Woody explants were cultured aseptically in jars containing 40 ml of medium and shelled seeds were cultured in petri dishes containing 25 ml of medium. Data about percentages of contamination survival and growth were recorded after 30 days. After seed sowing Petri dishes were kept in dark condition for 4 days and vessels containing woody explants were kept at 25  ). Data about vitality and contamination of the woody micro-cuttings were recorded after 14 days; visual evaluation of eventual presence of phenolic compounds was also performed and data about germination percentages and days for germination were recorded every 3 days up to 12 days. Final germination percentage (FGP) and mean germination time (MGT) calculated based on Oroiard (1977) method and coefficient of velocity of germination (CVG) calculated according to Jones and Sanders (1987) based on the following equations: (FGP): final number of seeds germinated/total seed number × 100 (MGT)=∑fx/∑f, (f: seeds germinated on day x) (CVG)=∑n/∑nt, (n: number of seeds germinated each day, t: number of days corresponding to n)

Proliferation phase using Temporary immersion system (TIS) and agar-based media
Four weeks after the seed germination, shoot tips (seedlings epicotyl) were transferred to WPM media, supplemented with 0.5 mg L -1 BA (6-benzyladenine) and 100 mg L -1 Ascorbic acid solidified with Agar (7 g L ) at 25 ± 2, After 6 weeks, the proliferation rate (number of shoots per initial explant), plantlet height and weight were recorded and analyzed.

Statistical analysis
Data were subjected to analysis of variance (ANOVA) and means were compared using Duncan's test (P≤ 0.05). Before media comparison the percentages were transformed in angular values. For the two different sterilization methods and ventilation treatments, T-tests were used to show differences among treatments. The experimental design was a CRD with 8 replicates, each replicates 1 petri dish containing 3 seeds, and the analysis was performed using SAS 9.

The effects of sterilization method on phenolic compound exudation of woody explants
After 5-7 days from initial establishment, at the base of the Lentiscus woody explants, exudation of phenolic compounds was evidenced together with media in browning and this was the main inhibitor factor for explant establishment. in current experiment exudation of phenolic compounds were not observed as a consequence of different sterilization treatments.

The effect of sterilization treatments on asepsis of woody explants
The applied treatments improved sterilization percentage of woody explants. The first treatment (immersion in 1.5% NaOCl for 30 min and after treatment with 70% Ethanol for 1 min) showed more contaminations than the second one but this difference was not statistically significant. Furthermore, the treatments had no significant effect on plant survival but the percentage was higher (46.62%) in the first treatment. The explant initial growth expressed in elongation and initial development of the buds was affected by treatments at P≤ 0.05, in fact the explants following the treatment 1 grew in the 20.87% respect to the 6.68% after the other one. Contamination percentage remained for both treatments from 30 to 37.5% (Table 3).

The effects of treatment on FGP, MGT and CVG seed parameters
The treatments had no significant effect on mean germination time (MGT) and on the coefficient of velocity of germination (CVG) but lentisk seeds final germination percentage (FGP) was affected by treatments at P≤ 0.05 (Table 4). As already mentioned the highest seed germination percentage (85.59%) was seen in seeds treated with 70% Ethanol for 30 seconds washed three times by distilled water then 1% NaOCl used for 30 min with no significant difference with revers state (treatments 5 and 6 - Table 4). The minimum value for mean germination time (MGT) was 4.37 days after the treatment 6, followed by 4.56 days after treatment 1. The maximum value for CVG was again after treatment 1 (0.125), which means more speed germination.

The effect of treatment on development of normal seedlings
Our results demonstrated that different sterilization methods had significant effects on percentage of germination of regular seedlings (with normal root system, stem and leaves) ( Table 4). The highest percentage of seedling with a regular behavior was achieved after treatment 6 (45.93% -table 4). The minimum percentage of normal seedlings was achieved after the reverse treatment (treatment 5) indicating that the use of ethanol after NaOCl could inhibit the germination of normal seeds.

Proliferation treatment results
The different parameters evaluated in the experiment showed a significance at P≤ 0.05. P. lentiscus L. shoot tips on liquid WPM media containing 0.5 mg L -1 BA and 100 mg L -1 Ascorbic acid showed an average of 6.18 shoot explant and plant proliferation rate was increased by 6.5 % using forced ventilation system than agarised medium (Table 5). Agar-based WPM medium containing 0.5 mg L -1 BA and 100 mg L -1 Ascorbic acid showed an average of 5. 8 shoots explant. Longer shoots (10.5 cm) were obtained in RITA. Plant height in TIS was higher than in agar based medium calculating a significant increase of 16.5%. Temporary immersion system was also more efficient considering the total plant weight (1.49 g, Table 5), mainly due to the high number of shoots per each explant. Plant weight in RITA containers increase by 29.56% in comparison to the other culture types. An example of shoots grown in the two systems is presented in fig. 1.

Discussions
According to some published results there have been some problems for primary sterilization and in vitro multiplication of mature lentisk plant materials (Mascarello et al., 2007;Yildirim, 2012;Kilinc et al., 2014) The results we obtained in our work are in agreement with the papers of Dolcet-Sanjuan and Claveria (1995) and Barghchi and Alderson (1989), who reported the extreme difficulty to establish aseptic culture of some Pistacia species because of high phenolic compounds level and contamination problems. The browning of the woody explants and the medium surrounding the explant base occurred during initial explant establishment and it is known as the main inhibiting factor for woody explant in vitro establishment especially for the Pistacia species (Ozden-Tokatli et al., 2005;Tilkat, 2006). Previous researches proposed different antioxidant treatments for preventing exudation of phenolic compound like PVP, ascorbic acid and other antioxidants; almost no significant differences were observed from the point of browning control view (Wessel et al., 1976;Roy and Sarkar, 1991) but in this study, the preparation procedure of the explants was efficient and permitted to avoid phenolic compound exudation. The results presented in Table (3) showed that either the rate of shoot survival and their initial growth in vitro were high in treatment 5 (immersion in 1.5% NaOCl for 30 min and after treatment with 70% Ethanol alcohol for 1 min) than after treatment 6 which is the reverse treatment that showed also the higher contamination percentage. Our result proved that beneficial effects of NaOCl treatment also on lentisk seed asepsis, and this is in agreement with , which reported that between different sterilizing agents, for seed sterilization of another species as Achyranthes aspera plant, sodium hypochlorite solution application for 30 min was the most effective treatment and Tomaszewska-Sowa and Figas (2011) which stated that, sodium hypochlorite solution can be the most effective for disinfection of the species Silphium perfoliatum L. seeds. Our result showed that seeds treated with 70% Ethanol alcohol for 30 second followed by 1% NaOCl for 30 min were exhibited lower contamination. This emphasize the role of 70% Ethanol alcohol in the disinfection of lentisk seeds. It seems that the use of tap water before sterilizing agents has a negative effect on asepsis procedure which can refer to contamination agent penetration in the seed cuticle. Our results indicated that different sterilizing agents had different effects on the evaluated parameters; for example, final germination percentage (FGP), mean germination time (MGT) and coefficient of velocity of germination (CVG) showed different values. Plant micro propagation using bioreactor system, in the liquid medium is a strategy to prevent some limitations that occurs in plant micro propagation using glass vessels without forced ventilation in agarised substrate. (Paek et al., 2001(Paek et al., , 2005. Liquid media could decrease the production costs related to manual labor and enhance the possibility of automation (Quiala et al., 2012). In agreement with our finding, previous result showed that temporary immersion system performed with RITA containers could improve plant quality and production with high yield in comparison to medium containing agar (Preil 2005). Based on our results, P. lentiscus L. shoot tips in liquid WPM medium showed high multiplication rate. The liquid medium that temporary bubble around the shoot tips permitted a high cell hydration inducing longer and heavier shoots rather than agarised medium. In agreement with our finding previous studies showed that bioreactor system used to different plant species successfully, (Cabasson et

Conclusions
To our knowledge, this is the first study on the micro propagation of the lentisk plant by temporary immersion system. Growth parameters comparison between liquid medium and agar-based medium showed that, bioreactor system permitted to increase quality and yield of the plants in proliferation phase. The use of WPM salts confirmed the good performances of the shoots in combination with BA and Ascorbic acid for an effective method for Lentisk micro propagation. The data about the surface sterilization of lentisk seeds and woody explants permitted to establish an efficient procedure for lentisk micro propagation.  Significance n.s n.s * *Means followed by the same letters in column are not significantly different (P≥ 0.05).