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http://journals.tubitak.gov.tr/biology/

Turkish Journal of Biology Turk J Biol(2014) 38: 653-663© TÜBİTAKdoi:10.3906/biy-1401-69

In vitro regeneration and conservation of the lentisk (Pistacia lentiscus L.)

İbrahim KOÇ1, Ahmet ONAY2, Yelda ÖZDEN ÇİFTÇİ1,* 1Department of Molecular Biology and Genetics, Faculty of Science, Gebze Institute of Technology, Gebze, Kocaeli, Turkey

2Department of Biology, Faculty of Science, Dicle University, Diyarbakır, Turkey

* Correspondence: ozden@gyte.edu.tr

1. IntroductionPistacia lentiscus L., which is also known as the lentisk or mastic tree, is widespread from Asia Minor to the Canary Islands and throughout the Mediterranean region (Ak and Parlakcı, 2009). It is an evergreen shrub with lovely red berries and dark green foliage and is often used as an ornamental plant (Mascarello et al., 2007). Lentisk can adapt to several climatic and pedological conditions (Zohary, 1952), such as severe drought (Correia and Catarino, 1994) and calcareous soil; it also exhibits regrowth after forest fires or deforestation (Ladd et al., 2005).

P. lentiscus is also the source of a unique resin called mastic gum or mastic. It is obtained by wounding the trunk and thick branches of the male trees, since the females produce low levels of mastic (Acar, 1988). The cultivated male trees and mastic production have been exclusively maintained on the Greek island of Chios for a long time. Mastic production is also carried out in the adjacent Çeşme Peninsula of İzmir, Turkey, where ecological conditions are similar to Chios, Greece; however, mastic production in Çeşme is very restricted and does not match the quantity of the production on Chios Island (Baytop,

1968; Isfendiyaroglu, 2000). The agricultural activities and tourist facilities established in Çeşme have resulted in the continuous loss of mastic trees (Dogan et al., 2003). Therefore, the Turkish Foundation for Combating Soil Erosion for Reforestation and the Protection of Natural Habitats has been leading a project to protect the native mastic trees and establish new plantations on the Çeşme Peninsula to revive viable commercial production (www.tema.org.tr). In addition, a forest fire recently occurred on Chios Island and destroyed a total of 12,740 ha, which resulted in massive damage not only to the island’s agricultural economy, but also to the world supply of Chios mastic (http://effis.jrc.ec.europa.eu). Mastic gum has a long tradition in folk medicine and has been used by healers for several treatments, such as for hypertension, coughs, sore throats, eczema, stomachaches, kidney stones, and jaundice (Ljubuncic et al., 2005b; Gardeli et al., 2008).

The essential oil of mastic contains several secondary metabolites such as myrcene, limonene, terpinen-4-ol, alpha-pinene, beta-pinene, alpha-phellandrene, sabinene, para-cymene, and gamma-terpinene (Castola et al., 2000). Antimicrobial and antifungal activity of the essential oil of the resin (Magiatis et al., 1999) and inhibition of in vitro

Abstract: Pistacia lentiscus L. (lentisk or mastic tree) is an economically important member of the genus Pistacia due to its valuable mastic resin. Shoot tips and nodal segments were used as explant sources from in vitro-germinated seeds of lentisk. Shoot tips were found to be suitable for multiple shoot formation. Thereafter, the influences of different growth regulators [N6-benzyladenine (BA), gibberellic acid (GA3), naphthalene acetic acid (NAA), or jasmonic acid (JA)] together with various elicitors [silver nitrate (AgNO3) or phloroglucinol (PG)] were assessed to develop efficient micropropagation protocol. Our results showed that the combination of all tested concentrations of GA3 with 1.0 mg/L BA resulted in enhancement of multiple shoot formation. The maximum number of shoots per explant (3.95) was recorded on MS medium containing 1.0 mg/L BA and 0.3 mg/L GA3. In contrast, JA had a negative influence, while AgNO3 had no significant effect on multiple shoot formation. In terms of synthetic seed production, it was possible to encapsulate shoot tips at 4 °C in darkness for up to 6 months with a frequency of 87.5% plant regrowth. In vitro-propagated microshoots, including plantlets derived from synthetic seeds, were transferred to MS medium containing different concentrations (1.0, 2.0, or 4.0 mg/L) of indole butyric acid for rooting and successfully acclimatized to ex vitro conditions. The presented data suggest an efficient in vitro regeneration system and conservation via synthetic seed production for lentisk.

Key words: Pistacia lentiscus L., lentisk, micropropagation, plant growth regulators, synthetic seed

Received: 22.01.2014 Accepted: 03.06.2014 Published Online: 05.09.2014 Printed: 30.09.2014

Research Article

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proliferation of colon cancer cells by compounds in mastic gum were also reported (Balan et al., 2007). Thus, nowadays, biologically active compounds in the resin (mastic) is of interest to the medicinal industry (Ljubuncic et al., 2005a; Paraschos et al., 2007; Kim and Neophytou, 2009; Dabos et al., 2010; Lemonakis et al., 2011; Triantafyllou et al., 2011). Apart from pharmaceutical exploitation, mastic resin has also been commonly used as a food and beverage additive (Calabro and Curro, 1974). This unique characteristic of mastic provides approximately 20% of the gross budget of Chios Island’s economy (Theodoropoulos and Apostolopoulos, 2004).

Lentisk is naturally propagated by seeds that might result in genetic variability due to the dioecious feature of the tree. Propagation by seed increases genetic variability, but this method also has problems, such as parthenocarpy, ovary abortion (Grundwag, 1976), and varying germination rates among genotypes (Mulas et al., 1998). Moreover, vegetative multiplication by cutting is very hard, since good induction of adventitious roots and large quantities of mastic tree production were not reported for any reforestation program (Yıldırım, 2012). In vitro multiplication and reintroduction into natural habitats of rare, endangered, or endemic plant species allows the preservation of these species and expands the natural population (Jarda et al., 2014). Thus, the development of in vitro propagation and conservation techniques, such as encapsulation technology, in lentisk could be a complementary strategy for industrial-level propagation and preservation of its germplasm.

Up to now, studies on micropropagation of the lentisk have been performed by using juvenile material obtained from in vitro-germinated seeds (Fascella et al., 2004; Ruffoni et al., 2004; Taşkın and İnal, 2005; Mascarello et al., 2007; Yıldırım, 2012). However, those studies still need further improvement due to a lack of information on proliferation rate in the remarks of the micropropagation protocol, proliferation potential of different explant types, combined effects of various plant growth regulators (PGRs; Sharmin et al., 2013), and elicitors on in vitro propagation of lentisk. Thus, the micropropagation protocols of lentisk could be improved by applying various PGRs and elicitors.

The aim of the present study concerns a contribution to the development of an efficient in vitro propagation procedure by assessing the influence of N6-benzyladenine (BA) incorporation with 1) jasmonic acid (JA), to reveal whether it stimulates or inhibits shoot multiplication; 2) naphthalene acetic acid (NAA), to evaluate the interaction of auxin and cytokinin for shoot induction; 3) silver nitrate (AgNO3), an ethylene action inhibitor, to assess whether it promotes shoot formation or not; 4) gibberellic acid (GA3), to ascertain multiple shoot formation and elongation, which did not induce multiple shoots in the study of

Yıldırım (2012); and 5) phloroglucinol (PG), a phenolic compound, to determine its influence on enhancement of shoot formation in P. lentiscus L. Moreover, the conservation of lentisk by using encapsulation technology, with the aim of preserving its germplasm, was also assessed for the first time.

2. Materials and methods2.1. Plant material, seed germination and initiation of cultureSeeds of the lentisk cultivar were harvested in the Çeşme district of İzmir Province in Turkey. Seeds were stored at 4 °C in a refrigerator until they were used for the trials. Seeds were sterilized according to the method developed by Ozden-Tokatli et al. (2005), with minor modifications. In short, seeds were surface-presterilized with 70% ethanol (v/v) for 5 min and disinfected in 10% hydrogen peroxide (w/v) for 5 min, followed by 20% (v/v) Domestos (commercial bleach containing approximately 2% available chlorine) for 20 min. After each step, seeds were rinsed with autoclaved distilled water (dH2O) for 5 min at least 3 times. Seed coats were removed and kernels were transferred to MS medium (Murashige and Skoog, 1962) containing different concentrations of either BA (1.0 and 2.0 mg/L) or GA3 (0.1, 0.3, and 0.5 mg/L) for germination. After 4 weeks of cultivation, the mature lentisk seeds germinated and produced actively growing shoot tips. Shoot tips and nodal segments were then excised from developing seedlings and were transferred to MS medium containing 1.0 mg/L BA. By subculturing explants (after 30 days of culture) for at least 6 months, in vitro plantlets were regenerated.

All media were supplemented with 3% sucrose (w/v) and solidified with 0.7% agar (w/v), and pH was adjusted to 5.8 before autoclaving (121 °C for 20 min at 1 atm pressure). GA3 was filter-sterilized and added to the autoclaved media. All cultures were maintained in a 16-h photoperiod at 25 ± 2 °C with light intensity of 40 µmol m–2 s–1 provided by cool white fluorescent lamps for 30 days.2.2. Influence of explant type and PGRs on proliferationShoot tips or nodal segments (approximately 0.5 mm in size) were transferred to MS medium containing 1.0 mg/L BA. The explants (shoot tips) that gave the highest shoot-forming capacity (SFC) index were transferred to various concentrations of NAA (0.1, 0.5, or 1.0 mg/L) supplemented with 1.0 mg/L BA. Moreover, the shoot tips were transferred to MS medium containing 1.0 mg/L BA and various concentrations (0.1, 0.3, or 0.6 mg/L) of GA3 in order to assess the influence of BA and GA3 on in vitro propagation. The combined effects of JA (0.1, 0.15, or 0.2 mg/L) plus 1.0 mg/L BA were also assessed in order to determine their influence on shoot development.

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2.3. Influence of different elicitors on proliferationIn the other experimental design, PGRs were replaced by various concentrations of PG (50, 100, or 150 mg/L) plus 1 mg/L BA to induce shoots in MS medium. Explants were also transferred to MS medium containing AgNO3 (an ethylene inhibitor) (2.0, 4.0, or 8.0 mg/L) and 1 mg/L BA in order to reveal if ethylene affected multiple shoot formation. 2.4. Influence of the combination of different PGRs and elicitors on in vitro proliferation of lentiskThe effect of the combination of various PGRs and elicitors on in vitro multiplication of lentisk was also assessed by incorporating GA3 (0.1 or 0.3 mg/L), AgNO3 (4.0 mg/L), and BA (1.0 mg/L) to MS medium.2.5. Influence of storage period on in vitro conservation of lentiskShoot tips were excised from in vitro-proliferated (in 1.0 mg/L BA containing MS medium) microshoots and transferred into 3% Na-Alg (w/v) MS liquid salt solution (Ca2+-free) containing 0.4 M sucrose. The explants were then sucked up with a pipette (whose tip had been trimmed to obtain a hole of 2–4 mm) together with the Na-alginate solution and released drop-by-drop (each drop containing a single explant) into a sterile solution of the complexing agent (100 mM CaCl2.2H2O). The beads containing shoot tips were held for 25 min in this complexing solution. After bead hardening, encapsulated shoot tips were washed 2 times with dH2O. The encapsulated shoot tips were transferred to MS medium and cultured at 4 °C in the dark in petri plates for 2, 4, 6, 9, or 12 months for in vitro conservation. Following each storage period, synthetic seeds were transferred to fresh MS medium containing 1.0 mg/L BA in petri plates and cultured at standard proliferation conditions for shoot growth.2.6. Influence of indole butyric acid on rootingAs the highest rooting percentage (92%) was obtained with full-strength MS medium in lentisk according to the previous report of Yıldırım (2012), the influence of 2-step rooting medium together with different concentrations of indole butyric acid (IBA) (0, 1.0, 2.0, or 4.0 mg/L) was tested in this study. Thus, shoots (approximately 2 cm in length) coming from in vitro propagation and synthetic seed-derived plantlets were transferred to Magenta vessels (GA-7, Sigma Ltd., approximately 50 mL) containing MS medium with or without various concentrations of IBA (1.0, 2.0, or 4.0 mg/L) for 7 days. Shoots were cultured in IBA containing MS medium for 7 days and were then transferred to auxin-free MS medium and cultured for an additional 23 days. 2.7. AcclimatizationThe rooted shoots were washed with tap water in order to remove traces of agar and then transferred to pots

containing a 1:1:1 mixture of autoclaved perlite, peat, and garden soil as mention by Yıldırım (2012) for acclimatization. Plantlets were transferred directly to greenhouse conditions [25 ± 2 °C with a photoperiod of 16 h provided by white fluorescent lamps (50 µmol m–2 s–1 photosynthetic photon flux density)] and acclimatized to ex vitro conditions by covering the pots with transparent polyethylene bags to sustain high humidity during hardening. Polyethylene bags were punctured gradually, at a rate of 2 holes per day, to progressively reduce humidity and to supply gas exchange for successful acclimatization. The bags were partially removed after 10 days and completely removed after 20 days to totally acclimatize the plants to natural conditions. The plants were irrigated with tap water every day.2.8. Data collection and statistical analysisAll experiments were carried out using a minimum of 15 replicates and repeated at least twice. Proliferation percentage, number of shoots proliferated per explant, and length of shoots were scored after 30 days of culture. SFC index (Lambardi et al., 1993) was also calculated by multiplying the proliferation percentage by the number of shoots proliferated per explant and then dividing the result by 100. In the case of rooting, a minimum of 10 shoots were used and the experiment was repeated at least 2 times. Percentage of rooting, number of roots formed per shoot, and root lengths were scored after a total of 30 days of culture. Root-forming capacity (RFC) index was also calculated by multiplying the rooting percentage by the number of roots formed per shoot and dividing the result by 100.

Statistical analysis of the percentages was carried out by the test for homogeneity of proportions and significant treatment differences were selected by a nonparametric statistical test, the post hoc multiple comparisons test (Marascuilo and McSweeney, 1977). Discrete data were subjected to analysis of variance (ANOVA), followed by the least significant difference (LSD) test at P ≤ 0.05 to compare mean values.

3. Results3.1. Seed germinationFollowing the surface sterilization, 16.5% of lentisk seeds were germinated in PGR-free MS medium (MS0, Table 1). BA was not effective on germination, as 8.0% or 11.0% germination was obtained (1.0 or 2.0 mg/L BA, respectively). Callus formation occurred in seeds cultured on cytokinin-containing media. However, the addition of GA3 to MS media resulted in an enhancement of germination, irrespective of the concentrations tested. Twenty-four percent of germination was achieved with the inclusion of 0.1 mg/L GA3; this value was improved by increasing the GA3 concentration in the medium. The

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highest germination percentage (35.3%) was obtained on MS medium containing 0.3 mg/L GA3. MS medium containing 0.5 mg/L GA3 resulted in a 34.2% germination rate. 3.2. Influence of BA on proliferation Shoot tips and nodal segments were tested in MS medium supplemented with 1.0 mg/L BA. Shoot tips resulted in relatively higher shoot proliferation (93.3%), whereas nodal segments showed 71.4% proliferation (Table 2). Moreover, shoot tips formed 1.93 shoots per explant, which was also slightly higher than nodal segments (1.87 shoots per explant). In terms of the average length of the shoots, the length of nodal segments and shoot tips was 4.0 mm or 3.3 mm long, respectively.

The SFC index enables assessment of both shoot proliferation frequencies and the average number of shoots formed per explant and provides information about the overall proliferation potential of the explants. The SFC index was 1.37 in nodal segments and 1.83 in shoot tips. The microshoots that proliferated from shoot tips were also healthier than the ones obtained from nodal segments. Moreover, some nodal segments were necrotic and some of them did not proliferate, as indicated by the proliferation percentage (71.4%). Thus, as we had enough shoot tips for our experiments, further trials were carried out using shoot tips as an explant source. Shoot tips were transferred to 1.0 mg/L BA-containing MS medium enriched with different

PGRs (NAA, JA, GA3) and various elicitors (AgNO3, PG) to see if the result obtained in previous trials could be improved further. 3.3. Influence of NAA on proliferationExperiments were designed to explore the effect of cytokinin (BA) augmented with various concentrations of auxin (NAA; 0.1, 0.5, and 1.0 mg/L) on shoot formation at shoot tips (Table 3). BA at 1.0 mg/L in combination with any NAA concentration led to high proliferation rates (93.3% and 100%). However, there were no significant differences in terms of mean number of shoots per explant for BA and NAA combinations. On the contrary, addition of NAA to BA resulted in relatively shorter shoots than 1.0 mg/L BA-enriched medium.3.4. Influence of JA on proliferationInclusion of JA reduced multiple shoot formation, as the number of shoots proliferated per explant was significantly lower than that obtained with BA-enriched medium, irrespective of the concentrations (Table 3). SFC indices were also low due to the relatively small number of shoots obtained per explant. Similar to NAA plus BA-containing medium, shoot length in JA plus BA medium was also relatively short (maximum of 2.42 mm in JA plus BA medium, while it was 3.28 mm in BA-containing medium). JA also did not improve the quality of the shoots, as proliferated shoots were unhealthy with poor leaf formation (data not shown).3.5. Influence of GA3 on proliferationThe addition of relatively higher GA3 concentrations (0.1, 0.3, or 0.6 mg/L) increased not only the proliferation rate but also multiple shoot formation. The maximum shoot proliferation (100%) together with the highest number of shoots proliferated per explant (3.95) was recorded on MS medium containing both 1.0 mg/L BA and 0.3 mg/L GA3 (Table 3). This growth regulator combination also resulted in the highest SFC index (3.95) (Figure 1A). In terms of shoot length, addition of GA3 (0.1, 0.3, or 0.6 mg/L) to BA did not enhance shoot length (1.99, 1.96, or 2.65 mm, respectively).3.6. Influence of AgNO3 on proliferationAll concentrations of AgNO3 with BA provided 100% shoot proliferation (Table 3) with greenish leaves and healthy

Table 1. Influence of different concentrations of BA and GA3 on germination of a minimum of 100 lentisk seeds after 30 days of culture.

MS medium Germination rate (%)

Hormone-free (MS0) 16.51.0 mg/L BA 8.02.0 mg/L BA 11.0 0.1 mg/L GA3 24.00.3 mg/L GA3 35.30.5 mg/L GA3 34.2

Table 2. Influence of explant type on in vitro proliferation of lentisk after 4 weeks of culture.

Explant type Proliferation(mm)

Shoots/explant*(mean ± SE)** SFC index Shoot length* (%)

(mean ± SE)**

Nodal segments 73.3 1.87 ± 0.24a 1.37 4.01±0.86aShoot tips 93.3 1.96 ± 0.08a 1.83 3.28±0.38a

*: Different lowercase letters following means in a column indicate that these values are significantly different at P ≤ 0.05 according to the LSD test. **: SE = Standard error.

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shoots. The combination of 2.0 and 8.0 mg/L AgNO3 with 1.0 mg/L BA induced fewer shoots (1.74 and 1.10 per explant, respectively) than 1.0 mg/L BA alone. However, 4.0 mg/L AgNO3 plus 1.0 mg/L BA formed a comparable number of shoots per explant (2.00) with BA alone. The SFC index was higher (2.00) in 4.0 mg/L AgNO3 and 1.0 mg/L BA-containing medium. No significant differences were obtained in terms of shoot length, except that relatively shorter shoots proliferated in 2.0 mg/L AgNO3-containing medium. 3.7. Influence of PG on proliferationThe maximum proliferation (100%) was obtained with the inclusion of 150 mg/L PG to BA-containing medium (Table 3). However, multiple shoot formation declined with the incorporation of PG. Thus, relatively low SFC indices were obtained, irrespective of the PG concentration. Length of the shoots also reduced as the maximum shoot length in PG-containing (100 mg/L) medium was 2.57 mm. 3.8. Influence of GA3, NAA, AgNO3, and BA together on in vitro proliferation of shoot tips of lentiskThe combination of GA3 (0.1 or 0.3 mg/L) or NAA (1.0 mg/L) with AgNO3 (4.0 mg/L) and BA (1.0 mg/L) lowered the proliferation (Table 4). GA3 (0.1 or 0.3 mg/L) plus AgNO3 (4.0 mg/L) and BA (1.0 mg/L) resulted in 80%

and 73.3% proliferation with 1.13 shoots and 1.00 shoot per shoot tips, respectively. Interestingly, GA3 at 0.1 or 0.3 mg/L concentrations in combination with 4.0 mg/L AgNO3 and 1.0 mg/L BA, produced the largest shoot size in the overall treatments (5.02 and 4.40 mm, respectively). In general, the shoots developed in AgNO3-, BA-, and GA3-containing media were not strong and had browning on the shoot tips. NAA (1.0 mg/L) plus AgNO3 (4.0 mg/L) and BA (1.0 mg/L) enhanced neither proliferation rate (83.3%) nor multiple shoot formation (1.00). Shoot length was also lower (1.82 mm) than in those obtained from BA alone (3.28 mm). According to SFC indices, the values that resulted from combination of GA3 or NAA with BA and AgNO3 were always below 1.00.

In all of the treatments, brown callus formation and phenolic accumulation was evident on the basal end of the explants due to wounding. However, phenolic compounds in the medium were lower with the inclusion of AgNO3 in the medium.3.9. Influence of storage period of encapsulated shoot tips on conservationWhen the synthetic seeds were stored at 4 °C in darkness, significantly lower rates of shoot regrowth were obtained with storage of encapsulated lentisk shoot tips for 2 and 4

Table 3. Influence of various PGRs on in vitro proliferation of shoot tips of lentisk after 4 weeks of culture.

PGR (mg/L) Proliferation(mm)

Shoots/explant*(mean ± SE)** SFC index Shoot length*

(mean ± SE)** (%)

1.0 BA 93.3 1.96 ± 0.08c 1.83 3.28 ± 0.38a1.0 BA + 0.1 NAA 100 1.85 ± 0.27cd 1.85 1.98 ± 0.15cd1.0 BA + 0.5 NAA 93.3 1.55 ± 0.27d 1.44 1.88 ± 0.11d1.0 BA + 1.0 NAA 100 2.35 ± 0.35bc 2.35 1.60 ± 0.11e1.0 BA + 0.10 JA 100 1.40 ± 0.16de 1.40 1.86 ± 0.10d1.0 BA + 0.15 JA 90 1.39 ± 0.16de 1.25 2.42 ± 0.21b1.0 BA + 0.20 JA 93.3 1.33 ± 0.13de 1.23 1.91 ± 0.14cd1.0 BA + 0.1 GA3 93.3 2.89 ± 0.34b 2.70 1.99 ± 0.18cd1.0 BA + 0.3 GA3 100 3.95 ± 0.46a 3.95 1.96 ± 0.13cd1.0 BA + 0.6 GA3 100 3.05 ± 0.35b 3.05 2.65 ± 0.20b1.0 BA + 2.0 AgNO3 100 1.74 ± 0.25d 1.74 2.25 ± 0.19c1.0 BA + 4.0 AgNO3 100 2.00 ± 0.21c 2.00 2.73 ± 0.28ab1.0 BA + 8.0 AgNO3 100 1.10 ± 0.07f 1.10 2.88 ± 0.24ab1.0 BA + 50 PG 93.3 1.42 ± 0.16de 1.32 1.99 ± 0.20cd1.0 BA + 100 PG 93.3 1.80 ± 0.25cd 1.68 1.99 ± 0.18cd1.0 BA + 150 PG 100 1.40 ± 0.13de 1.40 2.57 ± 0.19bc

*: Different lowercase letters following means in a column indicate that these values are significantly different at P ≤ 0.05 according to the LSD test. **: SE = Standard error.

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months (40.0% and 32.1%, respectively; Table 5). However, storage for up to 6 months increased the proliferation rate (87.5%) and the number of shoot proliferated per explant

(1.8); thus, the SFC index also increased (1.6). A significant decline, especially in shoot formation, was obtained with 9-month and 12-month storage. Although the proliferation

Figure 1. Micropropagation of P. lentiscus L. A) Plantlets of lentisk on 1 mg/L BA and 0.3 mg/L GA3 containing MS medium after 30 days of culture, bar = 3 mm; B) plantlet retrieval from 6-month conserved synthetic seed of lentisk on MS medium containing 1 mg/L BA after 30 days of culture at standard proliferation conditions, bar = 4 mm; C) root formation of lentisk microshoots that were treated with 2 mg/L IBA-containing MS medium for 7 days, followed by culture on auxin-free medium for an additional 21 days, bar = 4 mm; D) acclimatized seedlings of lentisk after 1 month, bar = 24 mm; E) acclimatized seedlings of lentisk after 3 months, bar = 30 mm; F) Acclimatized seedlings of lentisk after 6 months, bar = 35 mm.

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percentage was not sufficient, relatively higher numbers of shoots sprouted per explant in 12-month storage at low temperature. In the case of length of the shoots, the longest shoots were obtained in synthetic seeds stored for 6 months. Overall results showed that lentisk shoot tips can be efficiently stored for up to 6 months at 4 °C as synthetic seeds (Figure 1B).3.10. Influence of IBA on rooting and acclimatizationLentisk microshoots did not form adventitious roots in auxin-free media. A beneficial influence on rooting was observed when IBA was applied for 7 days (Table 6). IBA at 2.0 mg/L concentration had a positive influence on rooting, as the highest root formation rate (80.0%), the highest number of roots per shoot (4.3), the maximum RFC index (3.45), and the longest root length (8.3 mm) were seen after 4 weeks (Figure 1C). Elevation of IBA concentration to 4.0 mg/L decreased the rooting. After rooting, plantlets were transferred in pots containing a sterile peat, soil, and perlite mixture. In this compost, rooted plantlets tended to resume growth quickly (Figures 1D–1F) and showed approximately 90% plant survival (data not shown).

4. DiscussionPoor seed germination together with the release of phenolics at the base of axenic shoots were reported as most serious limitations for the initiation of in vitro cultures in lentisk (Mulas et al., 1998; Fascella et al., 2004; Ruffoni et al., 2004; Mascarello et al., 2007; Yıldırım, 2012). In this study, various PGRs (BA and GA3) in different concentrations were used to aid the germination of the seeds. The highest germination rate (35.3%) was obtained with the inclusion of 0.3 mg/L GA3. The effects of GA3 on lentisk germination also correlated with an earlier report (Abu-Qaoud, 2007)

Many studies indicated that the type and the concentration of PGRs, genotype, medium, and type of explant are important for the induction of shoot proliferation (Pati et al., 2006; Nhut et al., 2010). Thus, in the present study, we initially evaluated the explant types in 1.0 mg/L BA-containing MS medium as that concentration was found to be better than 2.0 and 4.0 mg/L in our previous study (unpublished data). In addition, the positive influence of BA on shoot proliferation of lentisk

Table 4. Influence of GA3, AgNO3, and BA on in vitro proliferation of shoot tips of lentisk after 4 weeks of culture.

PGR (mg/L) Proliferation(%)

Shoots/explant*(mean ± SE)** SFC index Shoot length*

(mean ± SE)** (mm)

1.0 BA 93.3 1.96 ± 0.08a 1.83 3.28 ± 0.38b1.0 BA + 0.1 GA3 + 4.0 AgNO3 80.0 1.13 ± 0.13b 0.90 5.02 ± 0.41a1.0 BA + 0.3 GA3 + 4.0 AgNO3 73.3 1.00 ± 0.00b 0.73 4.40 ± 0.47a1.0 BA + 1.0 NAA + 4.0 AgNO3 83.3 1.00 ± 0.00b 0.83 1.82 ± 0.15c

*: Different lowercase letters following means in a column indicate that these values are significantly different at P ≤ 0.05 according to the LSD test. **: SE = Standard error.

Table 5. In vitro conservation of encapsulated lentisk shoot tips at 4 °C in the dark. Data were collected after 4 weeks of culture in standard proliferation conditions following conservation.

Storage(months)

Proliferation*(%)

Shoot/explant**(mean ± SE)*** SFC index Shoot length**

(mean ± SE)*** (mm)

2 40.00b 1.50 ± 0.19b 0.60 2.13 ± 0.30b4 32.14c 1.56 ± 0.38b 0.50 2.14 ± 0.30b6 87.50a 1.81 ± 0.21ab 1.58 4.82 ± 0.61a9 30.00c 1.78 ± 0.28ab 0.53 1.91 ± 0.22b12 6.66d 2.50 ± 0.50a 0.16 1.92 ± 0.19b

*: Different lowercase letters following means in a column indicate that these values are significantly different at P ≤ 0.05 according to the post hoc multiple range test.**: Different lowercase letters following means in a column indicate that these values are significantly different at P ≤ 0.05 according to the LSD test. ***: SE = Standard error.

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was also reported previously (Mascarello et al., 2007; Yıldırım, 2012). However, in those studies, it was seen that lower BA (0.5 mg/L) concentration was more suitable in lentisk, but the shoot proliferation was better at 1.0 mg/L than at 0.5 mg/L (93.3% and 80%, respectively) in our unpublished results. Thus, trials were carried out in 1 mg/L BA-containing media. Furthermore, shoots sprouted from shoot tips were better than the ones obtained from nodal segments. Shoot tips were also used for micropropagation of other Pistacia species including P. vera L. (Dolcet-Sanjuan and Claveria, 1995) and P. khinjuk Stocks (Tilkat et al., 2005).

Auxin–cytokinin interactions are usually considered to be vital for regulating growth and organized development in plant tissue and organ cultures (Gaspar et al., 1996). However, the mode of interaction between cytokinin and auxin often depends upon the plant species and the organ being studied (Moubayidin et al., 2009). Adding 1.0 mg/L NAA to BA-containing medium improved the proliferation rate of lentisk in comparison to 1.0 mg/L BA-containing medium. The positive effect of NAA in combination with BA on shoot induction has been shown in several studies (e.g., Caboni et al., 2002; Koroch et al., 2003). Contrary to this, addition of IBA to BA did not improve shoot induction in P. lentiscus (Yıldırım, 2012).

JA and methyl jasmonate (MeJA), referred to as jasmonates, are regarded as PGRs; they also interact with other hormones (Wasternack and Hause, 2002). It was shown that the expression of wounding-related genes was also induced by the interaction of ethylene and jasmonates (Rojo et al., 2003). Therefore, the influence of JA was evaluated due to its potential to overcome stress conditions related to wounding during preparation of explants of lentisk in tissue culture. Although JA/MeJA had positive effects on shoot induction in several studies (Ravnikar et al., 1993; Dolcet-Sanjuan and Claveria, 1995), exposure to JA incorporated with 1 mg/L BA led to the reduction of

shoot formation in lentisk in this study. Similar to lentisk, the inhibitory effects of JA/MeJA on shoot formation were also reported on micropropagation of Pinus radiata (Tampe et al., 2001), Nicotiana tabacum (Capitani et al., 2005), and Pinus pinaster (Martin et al., 2009).

In terms of GA3, our results showed that the combination of GA3 (0.3 or 0.6 mg/L) with 1.0 mg/L BA significantly increased multiple shoot formation in P. lentiscus. Indeed, inclusion of 0.3 or 0.6 mg/L GA3 to 1.0 mg/L BA-containing medium resulted in the highest shoot proliferation and maximum multiple shoot formation in this study. Interestingly, the negative influence of GA3 (0.1 mg/L) in combination with BA (0.5 mg/L) on number of shoots proliferated per explant in lentisk was reported previously by Yıldırım (2012). However, different concentrations and combinations of PGRs should be tested to reveal their influence on the proliferation of the plant species. Thus, the different responses of shoot tips to BA- and GA3-containing MS medium obtained in this study and Yıldırım’s report (2012) may be due to the different concentrations of the PGRs tested and genotypic differences of the seeds utilized in the studies. Nevertheless, the positive influence of this growth regulator on multiple shoot formation was demonstrated previously in P. vera L. (Ozden-Tokatli et al., 2005), Terminalia chebula Retz. (Shyamkumar et al., 2003), and Quercus rubra L. (Vengadesan and Pijut, 2009).

Attempts were made to improve shoot regeneration capacity by incorporating different elicitors (AgNO3 and PG) to BA-containing medium in lentisk. However, our results showed that neither of the tested elicitors had a significant influence on multiple shoot development of explants, though lower levels of visible phenolic compounds (browning) were observed with the inclusion of AgNO3. The result obtained with AgNO3 could be attributed to nonexcessive ethylene production in the culture vessels (Chandra et al., 1997) of lentisk. Our results

Table 6. Influence of 7-day incubation of different IBA concentrations on rooting of lentisk

IBA (mg/L) Rooting*(%)

Root/shoot**(mean ± SE)*** RFC index Root length**

(mean ± SE)*** (mm)

0.0 0.0d 0.00 ± 0.00d 0.00 0.00 ± 0.001.0 30.0c 1.33 ± 0.33c 0.40 2.33 ± 0.33c2.0 80.0a 4.31 ± 0.74a 3.45 8.27 ± 1.41a4.0 70.0b 2.00 ± 0.26b 1.40 5.91 ± 0.45b

*: Different lowercase letters following means in a column indicate that these values are significantly different at P≤ 0.05 according to the post hoc multiple range test.**: Different lowercase letters following means in a column indicate that these values are significantly different at P ≤ 0.05 according to the LSD test. ***: SE = Standard error.

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also showed that the addition of PG was not essential for shoot proliferation or multiple shoot formation (Teixeira da Silva et al., 2013) in lentisk.

The effects of the highest shoot-promoting concentrations of GA3 (0.1, or 0.3 mg/L) or NAA (1.0 mg/L) individually combined with AgNO3 (4.0 mg/L) and 1.0 mg/L BA on shoot multiplication were also assessed. Their interactions had a negative influence on multiple shoot induction, probably due to the mutual inhibitory effect of the PGRs and elicitor. On the contrary, the highest shoot length response was recorded in the presence of GA3, AgNO3, and BA in the overall treatments. This result could be due to the beneficial influence of the combined usage of GA3 and AgNO3 on shoot length, as such improvement was not achieved in this parameter by the presence of GA3 or AgNO3 alone in BA-containing media. Thus, usage of GA3, AgNO3, and BA resulted in shoots longer in length but fewer in number. Similarly, usage of BA, AgNO3, and GA3 in combination resulted in longer shoots in P. vera L. (Ozden-Tokatli et al., 2005).

Regrowth of synthetic seeds after 2 and 4 months of storage at 4 °C was relatively lower than in the other tested storage periods. However, prolongation of storage for up to 6 months resulted in a high shoot-formation percentage. This result could be due to the improved tolerance of explants to low temperature conditions. Moreover, cold treatment of synthetic seeds could also result in faster and/or more complete elimination of bud dormancy of explants (Tsvetkov and Hausman, 2004). Our results demonstrated that encapsulated shoot tips could not resist 9- and 12-month storage, as lower shoot formation percentages were observed after conservation.

Regenerated shoots failed to produce roots on auxin-free medium. The incorporation of IBA to MS medium for 7 days

facilitated root formation, as IBA tends to be denaturated in media and rapidly metabolized in plant tissues (Gaspar et al., 1996). The use of IBA to attain optimum rooting response has been reported in P. lentiscus (Yıldırım, 2012) and other woody species, including P. vera L. (Ozden-Tokatli et al., 2005), Anacardium occidentale (Boggetti et al., 1999), Dendrocalamus asper (Arya et al., 1999), and Ziziphus jujuba (Hossain et al., 2003). Nevertheless, in our study, 2.0 mg/L IBA was found to be suitable for lentisk. Rooted shoots were transferred to sterilized compost containing peat, soil, and perlite and successfully acclimatized to greenhouse conditions.

Although the clonal propagation protocol for woody plants is generally constrained by several factors (e.g., optimization of PGRs and medium compositions; release of phenolic compounds to medium; choice of a suitable explant type), we can conclude that the results of this study, along with other studies, showed that in vitro culture of P. lentiscus can be easily achieved in MS medium containing BA. NAA, JA, AgNO3, and PG did not result in improved shoot proliferation. However, explants from different genotypes may respond to PGRs such as GA3 in a different manner. In addition, encapsulated shoot tips of lentisk provide an effective procedure for medium-term conservation of its germplasm. Thus, the method optimized in the present study may provide an efficient means of propagation and conservation of the lentisk as well as other woody species.

Acknowledgment This study was funded by a grant from the Scientific and Technological Research Council of Turkey (TÜBİTAK, No. KBAG-110T941).

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