The Accumulation of Essential Oils In Organ Culture of Acinos Graveolens Via Methyl Jasmonate And Salicylic Acid Exertion

The impact of Salicylic acid (SA) and Methyl jasmonate (MJ) on growth, and essential oil quantity and quality in organ culture of Acinos were investigated. In the present study, nodal segments were used as explants for shoot production using MS media with BA and NAA. Afterward, they were transferred to liquid MS media culture containing SA and MJ with 50, 100, and 150 µM and their combinations (SA 25 +MJ 25 , SA 50 +MJ 25 , SA 25 +MJ 50, and SA 50 +MJ 50 ). After three weeks, samples were collected to assess the morphological and some growth parameters, quantitative changes in pigment amounts as well as volatile contents. Accordingly, almost all treatments led to a notable decrease in growth parameters in comparison with control. The highest amount of Chl a, b and carotenoids were achieved by MJ 100 , SA 50, and SA 25 +MJ 50 , respectively. In addition, GC-MS results indicated 38 components of volatile products mainly alkane hydrocarbons and sesquiterpenes. The major identied compounds were Decane, Eicosane, and Germacrene D. Altogether, results asserted that SA 25 +MJ 25 and SA 100 were more ecient in the enhancement of essential oil content among all treatments.


Introduction
Acinos is one of the smaller but important genera in the Lamiaceae family from the economic point of view (Bonnier et al. 1927; Bown et al. 1995). This genus is represented by two species in the ora of Iran, Acinos arvensis and Acinos graveolens ). Acinos graveolens is an annual, dwarf, evergreen, woody, small (10-45 cm), and clumpy herb growing in Iran and neighboring countries (Hedge 1982;Stojanović et al. 2009).
Based on previous reports, the main component of essential oils of this plant is Germacrene D. It is also reported to be the major compound in the oil of A. arvensis, A. hungaricus, A. alpinus, and two subspecies of A. troodi . Some species of Acinos are reported to be used in medicine as an antiseptic, tonic, and antispasmodic since the e cacy effects on the melancholy, toothache, coughs, and gastrointestinal epizootic (Kaya et al. 1999;Soules et al. 1988;Velasco-Negueruela et al. 1993), however, more researches are needed to be accomplished about chemical composition, antibacterial and antioxidant activity as well as traditional applications of Acinos species in folk medicine to nd out their value as medicinal plants (Stojanović et al. 2009).
The medicinal plants are used on a global scale owing to their valuable components known as secondary metabolites by which they gain pharmaceutical importance; hence most of the medicinal plants are threatened due to exploitation at a higher rate. For decades, scientists have been searching for new alternatives to conventional methods in order to overcome this problem, protect plants and simultaneously improve their bioactive compounds accumulation (Singh and Dwivedi 2018). Recently, the applications of signal molecules such as elicitors have provided an effective and appropriate strategy to promote changes in secondary metabolism in plant cell cultures (Zhao et al. 2005); therefore, the necessary conditions to stimulate the production of main biologically active ingredients can be clari ed (Pasternak et al. 2005).
Salicylic acid (SA), phenolic in nature, has been found to increase plant growth, plant resistance against pathogens, ower induction, stomatal stroke, and the level of chlorophyll and carotenoid pigments. It also regulates the activity of some of the important enzymes as well as acts as a potent enhancer of some secondary metabolites (Arberg et al. 1981;Hayat et al. 2007;Pitta-Alvarez et al. 2000;Yin et al. 1997).
Jasmonic acid and its methyl ester (MJ), are fragrant volatile compounds (Demole et al. 1962) that play a role as a signal in plant responses and interactions (Cheong et al. 2003). These compounds function as important cellular regulators involved in various developmental processes, such as seed germination, root growth, and senescence (Creelman et al. 2002;Wasternack et al. 2002). Jasmonates activate plant defense mechanisms in response to biotic and abiotic stresses salinity (Wasternack et al. 1996). It has also been reported that Methyl jasmonate can induce the production of secondary metabolites under in vitro tissue cultures of various plants ( Dathe et al. 1981;Ueda et al. 1980). Based on the literature review, there is no extensive study on tissue culture and the production of secondary metabolites in Acinos graveolens. For this reason, the production of in vitro plants and the evaluation of essential oils in this medicinal species construct a new perspective on plant biotechnology. Therefore, the effect of different concentrations of methyl jasmonate and salicylic acid alone or their interaction on the chemical composition of the volatile compounds of Acinos graveolens was examined under in vitro conditions. Moreover, morphogenetic responses such as shoot, root and leave proliferation as well as photosynthetic pigments content were evaluated.

Plant Material and Shoot Production
The seeds of A. graveolens were accumulated from the botanical garden of East-Azerbaijan province in Iran and were surface sterilized by diving in 70 % ethanol and 20 % sodium hypochlorite for 3 and 15 min, respectively. After 3 times rinses with sterile distilled water, the seeds cultivated in the Murashige and Skoog (MS) basal media (Murashige and Skoog 1962). The pH of all media was adjusted at 5.6-5.8 with NaOH before autoclaving at 121 C for 20 min. The media were solidified with 8 gl -1 agar. Then all cultures were maintained under cool-white light illumination at 70 µmol m -2 s -1 with the daily photoperiod of 16 h at 25 ± 1 C. 35 days old seedlings grown on MS medium were used as the source explants.
In order to shoot proliferation, the nodal segments were cultured on 30 ml of hormone-free control medium and in MS medium supplemented with 1 mgl -1 6-benzyladenine (BA) and 0.2 mgl -1 α-naphthalene acetic acid (NAA). All cultures were incubated under the situation explained above. After 25 days of culture, explants were transferred to liquid medium culture.

MS liquid medium preparation with the elicitors
Three different concentrations (50, 100, and 150 µM) of MJ and SA were added to MS liquid medium (pH 5.8) supplemented with 1 mgl -1 BA and 0.2 mgl -1 NAA. Samples were allowed to grow at constant shaking (100 rpm) in the above-mentioned medium for about four weeks. The synergistic effect of the two elicitors was also studied by applying them as 25 µM SA + 25 µM MJ (SA 25 + MJ 25

Extraction of photosynthetic pigments
For the determination of total chlorophylls and carotenoids, 0.05 g of in vitro produced shoots were pressed in 2 ml of Dimethylsulphoxide using a pestle and mortar. The pigment extracts with a total volume of 2 ml were then centrifuged at 16300 x g for 15 minutes (Spectrafuge 24DF) in glass tubes to make the extract fully transparent. The resulting extracts (supernatant) were immediately assayed spectrophotometrically. Speci c absorption coe cients of chl a , chl b , and carotenoids were used. The amount of these pigments was calculated by following formulas (Wellburn 1994 3. C (x+c) = (1000 A 480 -2.14 C a -70.15 C b ) / 220 Where C a , C b, and C (X+C) are Chlorophyll a, Chlorophyll b, and total carotenoids, respectively. All of the pigments' content was expressed as mg g -1 of fresh weight. Here A is the absorbance. Gas chromatography-mass spectrometry (GC-MS) GC-MS analysis was carried out to identify the composition of the essential oils. A volume of 1 ml of the clear extract was syringed into GC-MS (an Agilent 6890 gas chromatography) using HP5-MS capillary column (30 m, 0.25 mm, 0.25 µm). Injection initiation temperature was 150 ° C, the column temperature was set at 70 C, for 3 min, and then heated to 120 C and kept for 5 min at a rate of 10 C˚ min -1 . Then reached 150° C at a rate of 10 C˚ min -1 , after two minutes heated to 240 ° C at a rate of 7 C˚ min -1 for 5 min. The carrier gas was helium with a ow rate of 1 ml/min. Recognition of compounds was accomplished by comparing their retention indices and their mass spectra and with those reported in the literature (Adams, 1995).

Statistical analysis
The experiments were executed under a randomized complete block design (RCBD) for three replicates. The data were statistically analyzed using analysis of variance (ANOVA) and the means were compared by Duncan's Multiple Range Tests (DMRT) at the confidence level of P ≤0.05.

Results And Discussion
The effect of elicitors on shoot production and in vitro plant growth Almost all growth parameters in terms of root and shoot number, average length, and fresh and dry weight were negatively affected by different concentrations of SA and MJ during experimentation. Detailed results have shown in Table 1

Composition of volatiles
The 38 identi ed components of volatile products induced from the in vitro shoots of Acinos graveolense are listed in Table 3. In control and major treatments except MJ 50 , MJ 100, and SA 50 + MJ 25 , alkane hydrocarbons were major volatile compounds, while most treatments were poor in the production of alcohol, fatty acid, oxygenated monoterpenes, monoterpenes, and sesquiterpenes. One diterpene, three oxygenated monoterpenes, two alcohol, and four fatty acids have been identi ed in all treatments. Among 11 studied treatments, SA 150 and MJ 100 had the highest number of compounds (12 compounds) and MJ 50 , SA 50 , and SA 50 + MJ 25 had the lowest number (5, 5, and 4, respectively). In the control treatment, Eicosane had the highest amount among all alkane hydrocarbons. Germacrene D was the main sesquiterpene found in all treatments except MJ 150 . 1, 2-Diethylcyclohexadecane was the only identi ed diterpene in control and was not detected in other treatments. Among studied treatments, MJ 100 had favorable performance due to more sesquiterpene hydrocarbon compounds compared to other treatments. SA 50 , SA 100 , MJ 50 , SA 50 + MJ 25 and SA 50 + MJ 50 did not produce any monoterpenes. Among all 11 examined treatments, SA 25 + MJ 25 and SA 100 were more effective in volatile compounds production, while control and SA 50 were poor in this case.
Based on the obtained results, alkane hydrocarbons and sesquiterpenes were the most plentiful of components and diterpenes had the least amount in all treatments. Decane and eicosane (two alkane hydrocarbon) and germacrene D (a sesquiterpene) were the major constituents of the oil obtained in treatments. However, survey literature showed some differences in the essential oil composition of A. graveolens from different origins. According to the studies in A. graveolens in the ora of Serbia, germacrene D and bicyclogermacrene were the compounds found in a signi cant amount (Golubovic et al. 2010). In another study on the A. graveolens in Iran, germacrene D and dillapiole were identi ed as the main compounds ). In the present work, beta-elemene, delta-cadinene, and germacrene D were identi ed which was similar to Javidnia et al. The results obtained in the present study suggest that SA and MJ improved the increase of the essential oils. The present results are in agreement with the results of Rowshan et al. (2010) and Lee and Yang (2005) who demonstrated that SA spray increased terpenes in Chrysanthemum and essential oil in Carum copticum, respectively. Exogenous application of SA has earlier been indicated to promote growth, yield, and essential oil content in important species of Lamiaceae such as basil, marjoram, and summer savory (Gharib 2007;Marandi et al. 2011). Moreover, Yao and Tian (2005)  . It has appeared that plant secondary metabolite's pathways are activated during some particular growth and development stages and or in periods of stress caused by excess or de ciency of some factors of the environment (Mann 1987). Despite other factors, it has been demonstrated that signal molecules such as elicitors are very potential for induction of plant secondary metabolites (Zhao et al. 2005). When plant cells are exposed to the exogenous application of elicitors, a signal transduction initiates from the surface of the plasma membrane that triggers ROS production, which in turn induces plant defense response, subsequently, cause the rise of key enzymes activity, catalyzing the biosynthesis of target secondary metabolites (Zhao et al. 2005). It can be concluded that the increment in essential oil contents has occurred as a defense mechanism.