Effects of cadmium and lead on seed germination, morphological traits, and essential oil composition of sweet basil (Ocimum basilicum L.)

Industrial Crops & Products 138 (2019) 111584

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Effects of cadmium and lead on seed germination, morphological traits, and essential oil composition of sweet basil (Ocimum basilicum L.)

T

Bahman Fattahia, Kazem Arzania, , Mohammad Kazem Souria, Mohsen Barzegarb ⁎

a b

Department of Horticultural Science, Tarbiat Modares University (TMU), P.O. Box 14115-336, Tehran, Iran Department of Food Science and Technology, Tarbiat Modares University (TMU), P.O. Box 14115-336, Tehran, Iran

ARTICLE INFO

ABSTRACT

Keywords: Environmental pollution Heavy metals Medicinal plant Stress Vegetable crop

The negative impact of contaminated soil with heavy metals on plant and human health is an important global concern. The aim of this study was to explore the influence of cadmium (Cd) and lead (Pb) on seed germination, morphological traits, and essential oil (EO) composition of sweet basil (Ocimum basilicum L.). Two months prior to the experiment, soils pre-treated using Cd (0, 5, 10, 20) and Pb (0, 100, 200, 400) in mg kg soil−1. Seeds were sown in the pots containing the contaminated soil under greenhouse conditions at 26 ± 6 °C and 60–70 %RH. The amount and composition of EO were determined using Gas chromatography–mass spectrometry (GC-MS). The contaminated soil had a negative impact on seed germination, leaf area, and flowering, stem growth and plant dry mass. The extracted EO yield was varied (P > 0.05) among the applied treatments from 0.28 to 0.39% (v/w). The GC-MS analysis of the EO identified the presence of 38 compounds. The major identified components of the EO were included estragole (18.80–50.32%), 2, 6- octadienal (3.2–11.95%), caryophyllene oxide (0.98–10.69%), caryophyllene (0.42–5.70%), phthalic acid (1.43–47.89%), and geranial (2.60–9.43%). The canonical correspondence analysis (CCA) showed a positive correlation between the plant height and phthalic acid contents. In conclusion, sweet basil cultivation in the Cd and Pb contaminated soils could cause undesirable effects on the seed germination and morphological traits, but might be have a positive influence on the EO yield, composition and phytoremediation of the soil.

1. Introduction Contamination of arable soils with heavy metals is a global challenge in many parts of the world. In addition, environmental and public health issues are the main concerns of human health regarding heavy metal pollution. This pollution can have negative effects on the vegetable crops and also consumers (Wuana and Okieimen, 2011). Chemical fertilizers and municipal waste can result in the heavy metal build-up in arable soil in big cities (Fattahi et al., 2017). Cadmium (Cd) and lead (Pb) are major toxic heavy metals with an unknown biological role in human life (Fattahi et al., 2017). Besides, it has been reported that Cd and Pb are toxic to any biological system even at the low concentrations (Shahid et al., 2017). The general toxic effects of Cd and Pb on plants include inhibition of shoot and root growth, and reduction in photosynthesis, and further crop yield (Lone et al., 2008; Majer et al., 2002). Moreover, accumulation of these heavy metals in different tissues of the affected plants, particularly in the fresh vegetables can simply transfer them into the human food chain, causing several health problems such as brain and kidney diseases (Lone et al., 2008; Majer et al., 2002).

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Sweet basil is considered as an essential fresh vegetable and a medicinal plant with various uses. According to some research results, basil species have antibacterial, antioxidant, antifungal, anti-inflammatory, and many other beneficial effects on consumer health (Tanrıkulu et al., 2018; Juliani et al., 2002). In addition, phenolic compounds such as rosemarinic and cynamic acids, flavonoids and anthocyanins, essential oils, and saponines are among the major biochemical’s of sweet basil (Filip et al., 2017; Jayasinghe et al., 2003). As reported by Chalchat and Özcan (2008), basil leaves and flowers have 1 and 0.5% oil, respectively. Estragole, 1,8-cineole, methyl eugenol, and linalole are recognized as the main oil components of sweet basil (Azzaz et al., 2018; Al Abbasy et al., 2015). Also, it has been reported that estragole is one of the predominant compounds in basil oil that has antiinflammatory and anti-edematogenic activities (Rodrigues et al., 2016). Different levels of Cd (0, 2.5, & 5 mg L−1) and Zn (0, 10, & 20 mg L−1) on sweet basil plants showed that large amounts of these metals were accumulated in the shoots and roots after increasing their concentrations in the soil. However, the content was considerably higher in the roots than shoots for both Cd and Zn (Chaiyarat et al.,

Corresponding author. E-mail address: [email protected] (K. Arzani).

https://doi.org/10.1016/j.indcrop.2019.111584 Received 1 February 2019; Received in revised form 16 June 2019; Accepted 17 July 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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2011). Furthermore, Zheljazkov et al. (2006) found that increasing copper (Cu) concentration (from 20 to 60 & 150 mg L−1) significantly reduced plant dry mass, plant height (PH), and oil yield in dill plants as compared to the control. They indicated that the concentration of 150 mg L−1 was phytotoxic and inhibited dill plant growth. Moreover, plant leaf area and dry weight of the aerial parts in peppermint were reduced under Cd and Pb treatments as compared to the control plant (Amirmoradi et al., 2012). As stated by Kranner and Colville (2011), heavy metals can induce toxicities at different growth stages of the plants starting from germination. According to them, heavy metals such as Cd, Cu, and arsenic (As) can inhibit seed germination even at the low concentrations. Likewise, Peralta et al. (2001) indicated that Lucerne seed germination was very susceptible to the presence of heavy metals as all the germination indices reduced under heavy metal treatments. The effect of contaminated soil with different Cd and Pb levels on the leaf colorophyl pigments and photochemical efficiency of photo system ІІ (Fv/Fm ratio) of sweet basil was evaluated and reported by Fattahi et al. (2017). This study was performed to evaluate the impact of different Cd and Pb levels on seed germination and some morphophysiological traits as well as EO composition of the sweet basil. In addition, the canonical correlation of the morphological traits and the oil compositions under Cd and Pb treatments were examined.

Besides, for uniform distribution of heavy metals, the soil was passed several times through the 2 mm sieve. The moisture content of the soil was adjusted at 60–65% field capacity (FC). Then, the soil was incubated for two months at 24 ± 4 °C. Thereafter, the soil of each treatment was placed into the black plastic pots containing 8–10 kg dry soil and the seeds were sown in 1 cm depth. A total amount of 15 plants per pot were allowed to grow while the remaining plants were removed. The pots were then kept under the greenhouse conditions at 26 ± 6 °C temperature and 60–70% RH. The pots were irrigated in a day intervals with purified tap water based on 80% FC. Therefore, the plants were left to grow under the aforementioned conditions for three months. 2.2. Germination experiment The germination of sweet basil seeds was prepared for evaluation using Cd and Pb treatments in the different experiment, under the laboratory conditions. The basil seeds were first disinfected with sodium hypochlorate for 20 min and then were placed on a tissue paper in the Petri dishes each containing 20 seeds. The tissue papers were wetted with different Cd (0, 1, 2, 4, 8, & 16 mg L−1) and Pb (0, 5, 10, 20, 40, & 80 mg L−1) solution concentrations (Table 1). Petri dishes were sealed with cellophane covers to prevent water loss and seed infestations, then placed in a germinator at 24 °C and 64% RH. The germinated seeds were counted every day, so the minimum radicle length emergance (RE) at 2 mm was considered as the germination count. The seeds germination counts were continued for 20 days. The quantity and quality traits for seed germination were evaluated as follows (Fattahi et al., 2011): Germination start (GS) = T2 – T1 (1), where T1 is the day of seed sowing, and T2 is the day of beginning of first seed germination; Germination percentage = (n/N)×100 (2), where n denotes to the total number of germinated seeds and N shows the number of total seeds sowed; Also, the Germination rate (GR) was calculated using the following equation as described by Burgert and Burnside (1972):Germination rate (GR) = ∑(number of germinated seeds till n-1)/n (3), where n is incubation days.

2. Materials and methods 2.1. Experimental setup This experiment was conducted under greenhouse conditions at the Department of Horticultural Science, Tarbiat Modares University (TMU), Iran. Sweet basil seeds of a local population were purchased from Pakan Seed Co., Isfahan, Iran. The suitable fertile soil with the following physical and chemical characteristics was used in the experiment. The soil texture was silty loam with 23, 29 and 48% clay, sand and silt, respectively. The pH of the soil was 7.6 with 3.28 dS m−1 EC and 0.07%, 0.68%, and 199 mg.kg-1 and 7.5 mg.kg-1 for nitrogen (N), carbon (C), potassium (K) and phosphorus (P), respectively. The concentration of cadmium and lead in the soil was 0.49 and 0.97 mg.kg1 respectively. Note that, two months prior to seed sowing, different concentrations of Cd (0, 5, 10 and 20 mg kg−1 dry soil) and Pb (0, 100, 200 and 400 mg kg−1 dry soil) were uniformly applied into the soil. The related codes for the applied treatments presented in Table 1. In order to prepare the contaminated soil for the experiment, nitrate salts of Cd and Pb were dissolved in distilled water (DW) and sprayed into the soil.

2.3. Determination of morphological traits Plants were grown for three months and thereafter various morphophysiological traits were measured. Morphological traits were recorded at the flowering stage, including plant height (PH, in cm), the secondary stems length (SSL, in cm), leaf length (LL, in cm), leaf width (LW, in cm), root and shoot fresh and dry weights (RFW, SFW, RDW, & SDW, in gr), internodes length (IL, in cm), flowering stem length (FSL, in cm), collar diameter (CD, in mm), stem diameter (SD, in mm), and leaf area index (LAI in cm2). The leaf area for each treatment was calculated using a digital leaf area meter (Nguy-Robertson et al., 2012).

Table 1 The related codes for the applied treatments on sweet basil (Ocimum basilicum L.). Treatments

Related Codes

Cadmium Cadmium Cadmium Cadmium Lead 0 Lead 100 Lead 200 Lead 400 Cadmium Cadmium Cadmium Cadmium Cadmium Lead 5 Lead 10 Lead 20 Lead 40 Lead 80

Control Cd5 Cd10 Cd20 Control Pb100 Pb200 Pb400 Cd1 Cd2 Cd4 Cd8 Cd16 Pb5 Pb10 Pb20 Pb40 Pb80

0 5 10 20

1 2 4 8 16

2.4. Soil and plant analysis for cadmium and lead The pre-contaminated soil for the experiment was analyzed for Cd and Pb before treatments applications. Also, at the end of the experiment and after destructive plants harvest, the pot soil was re-analyzed for the amount of Cd and Pb based on the DTPA (diethylene triamine penta-acetic acid) method that described previously by Beckett (1989). Note that, for Cd and Pb measurements in the leaves and roots, plants materials were first oven dried at 60 °C for 72 h and then ground powdered. Tissue powder (0.5 g) was digested in 25 ml nitric acid (65%), and Cd and Pb concentrations were determined according to the method described by Sekabira et al. (2011) using atomic absorption spectrometry (Flow Injection Analysis System 400AA 100; PerkinElmer, Waltham, MA, USA). 2

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2.5. Essential oil extraction (EO) The EO extraction was carried out in 50 g dried shoot samples after hydro-distillation using the Clevenger-type apparatus for 4 h. The EO extract was kept in the sealed vial under the dark at 4 °C until time of analysis. The amount of EO content (% v/w) was recorded based on the EO oil volume per air-dried sample (Fattahi et al., 2016). 2.6. Essential oil analysis Essential oil samples were analyzed using gas chromatograph (GC), according to the method described in detail for the type, column etc. by Fattahi et al. (2016). A 1 μL of the sample was dissolved in diethyl ether as a solvent then injected in the GC. Retention indices (RI) were calculated at the same conditions using the retention times of the injected n-alkenes (C6-C24). In order to identify the compounds, their RI was compared with those reported in the literature by Davies (1990) and Adams (2007). The mass spectra of the compounds were obtained using Agilent MSD Chemstation Libraries according to the installation of the national institute of standards and technology (NIST). The compound percentages were calculated using the area normalization method, excluding the response factors (Fattahi et al., 2016). 2.7. Statistical analyses The data were subjected to analysis of variance (ANOVA) using statistical analysis system software (SAS) version 9.02. The significant differences were calculated using the least significance difference (LSD) and the differences were considered statistically significant at P ≤ 0.05 (Varasteh et al., 2012). In addition, canonical correspondence analysis (CCA) was used to study phytochemical components as influenced by morphological traits (Fattahi et al., 2016). The CCA and related scatter plots were monitored by the Paleontological Statistics software (PAST).

Fig. 1. Effects of cadmium (Cd) and lead (Pb) on A; germination percentage B; germination rate and C; germination start of sweet basil (Ocimum basilicum L.). Mean values of four replications ± standard error of the mean.

3.2. Morphological traits 3. Results and discussion

The results of soil analysis showed that the soil used in this experiment had a relatively neutral pH with a low level of heavy metals such as Cd and Pb. In addition, there was a significant difference (P < 0.05) among the applied treatments in terms of morphological traits. The effects of different Cd and Pb concentrations on sweet basil growth are presented in Table 2. The variation coefficient was the lowest for PH (CV = 6.21%) while it was the highest for IL (CV = 32.46%). The IL ranged from 3.82 to 7.90 cm, with minimum and maximum values recorded for Cd5, Pb100, respectively. The LAI per plant varied from 190.25 to 211.25 cm2, with the maximum value found in the control. Note that, if basil is cultivated for fresh consumption, the high secondary stem length (SSL) is considered as an undesirable trait. However, cultivation of basil under the uncontaminated soil can be important in producing high quality vegetables. The SSL with minimum and maximum values were recorded in control (2.14 cm) and Pb400 (5.79 cm), respectively. Flowering stem in the control appeared later than the other treatments, which was due to the impact of contaminated soil for earlier flowering. Heavy metals stress is probably believed to produce the ethylene biosynthesis enzyme, ACC-Synthase, so ethylene stimulates flower buds in the plants (Apelbaum and Yang, 1981). In addition, early flowering from plants subjected to drought stress condition reported by Siddique et al. (2003). Note that, the crisp and freshness of the vegetables considered as qualitative trait. In the present research, stem diameter (SD) in Cd and Pb treatments was more than control treatment (Table 2). Although, the stem dry weight (SDW) was the highest and lowest in the control (15.2 g) and Cd10 (7.9 g) treatments, respectively (Table 2). It has been reported that the effect of

3.1. Effect of Cd and Pb on germination indices The different Cd and Pb concentrations were applied in-vivo in order to monitor the effect of these elements on the percentage, rate, and basil seeds germination (Fig. 1). Germination of basil seed in control treatment began after 4.66 days from the beginning of the experiment and an increase in Cd and Pb concentrations led to an increase in germination time to 7.66 and 10 days (Fig. 1A). Germination percentage of the control treatment was 81.33%. However, it reduced to 4% by increasing the Cd concentration up to 16 mg L-1. In addition, using Pb concentration up to 80 mg L−1 significantly decreased seed germination percentage to 9.33% (Fig. 1B). In other words, there was a general decrease in germination rate by increasing the concentrations of both elements since the highest germination rate was observed in control (1.69 seed per day). Besides, the lowest germination rate was detected in 16 mg L−1 Cd. Moreover, using Pb at 40 and 80 mg L−1 reduced the germination rate (0.19 seed per day) as compared to the control treatment (Fig. 1C). It has been reported a similar results in the reduction of seed germination rate along with the increase in the seed abscisic acid (ABA), following the increase in the Cd, Pb, and Hg application concentrations (Munzuroğlu et al., 2008). In addition, several researchers reported that using heavy metals can inhibit seed germination (Munzuroglu and Geckil, 2002; Li et al., 2005; Deng et al., 2016). Furthermore, when plants grow in low concentrations of heavy metals such as Cd, Cu, and As, their seeds dormancy were prolonged under the room temperature (Kranner and Colville, 2011). 3

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Table 2 Means comparisons of morphological traits (Mean values of four replications ± standard error of the mean) of sweet basil (Ocimum basilicum L.) under cadmium (Cd) and lead (Pb) concentrations (mg. kg−1 soil). R

Characteristics*

Unit

Control

Cd5

Cd10

Cd20

Pb100

Pb200

Pb400

CV (%)

1 2 3 4 5 6 7 8 9 10

PH SSL IL FSL LL LW CD SD SDW LAI

cm cm cm cm cm cm mm mm gr cm2

27.07 ± 0.49 ab 2.14 ± 0.52c 5.93 ± 0.44ab 4.98 ± 0.18b 2.35 ± 0.02ab 1.30 ± 0.02a 1.21 ± 0.05c 0.91 ± 0.02c 15.2 ± 0.95a 211.25 ± 1.31a

25.98 ± 0.14b 3.84 ± 0.79bc 3.82 ± 0.18b 9.60 ± 0.33a 1.73 ± 0.01c 0.88 ± 0.01b 1.37 ± 0.04bc 1.09 ± 0.01c 9.50 ± 1.31bc 198.00 ± 3.62b

27.20 ± 0.35ab 5.00 ± 0.55ab 4.21 ± 0.41ab 8.73 ± 0.22a 1.84 ± 0.06c 0.95 ± 0.04b 1.58 ± 0.19abc 1.38 ± 0.22ab 7.90 ± 0.29c 191.25 ± 1.93b

28.41 ± 1.38ab 4.18 ± 0.22ab 5.59 ± 0.47ab 8.93 ± 1.28a 2.50 ± 0.15ab 1.29 ± 0.09a 1.97 ± 0.07a 1.54 ± 0.07a 10.77 ± 0.52b 190.25 ± 3.52b

26.18 ± 0.99ab 4.48 ± 0.78ab 7.90 ± 3.38a 8.65 + 0.13 a 2.01 ± 0.13bc 1.00 ± 0.07a 1.77 ± 0.19ab 1.47 ± 0.22ab 8.92 ± 0.39bc 203.75 ± 4.15b

28.75 ± 1.05a 4.27 ± 0.65ab 5.24 ± 0.63ab 8.33 ± 0.81a 2.43 ± 0.22ab 1.26 ± 0.12a 1.79 ± 0.15a 1.42 ± 0.12ab 10.35 ± 0.90bc 202.50 ± 3.52b

26.27 ± 1.12ab 5.79 ± 1.83a 6.24 ± 0.48 ab 7.92 ± 1.54a 2.55 ± 0.31a 1.30 ± 0.12a 1.89 ± 0.16a 1.61 ± 0.14a 9.07 ± 1.31bc 201 ± 2.19b

6.21 30.82 32.46 22.00 15.66 14.59 16.18 19.11 18.34 10.37

*

PH = plant height; SSL = secondary stems length; IL = internode length; FSL = flowering stem length; LL = leaf length; LW = leaf width; CD = collar diameter; SD = stem diameter; SDW = shoot dry weight; LAI = leaf area index.

Cd and Pb in EO chemical compounds in peppermint reduced leaf area and dry weight of the aerial parts in Cd and Pb treatments in comparison with control (Amirmoradi et al., 2012).

3.4. The percentage of EO The EOs isolated among the samples in this experiment was yellow in color and showed over the range of 0.28-0.39% (v/w). In addition, the amount of EO was varied among the different treatments (P > 0.01). The highest and lowest amount of EO was belonged to the Cd20 (0.39%) and control samples (0.28%), respectively. An increase in Cd and Pb concentration enhanced the EO yield. The average EO percentage for different treatments shown in Fig. 3. Padalia et al. (2017) reported 0.53% EO yield that obtained from the basil at flowering stage. The report from the other experiment indicated that, the effect of 80% FC water stress in comparison with the 100% FC resulted to the increase in the basil EO content, while the 60% FC treatment reduced EO yield extract (Sirousmehr et al., 2014).

3.3. Soil and plant analysis for cadmium and lead Results showed that by increasing Cd and Pb concentrations in the soil, the higher content of these metals was observed in the leaf and root samples (Fig. 2a, 2b). In addition, Cd and Pb content in the leaf were higher than in the root samples. Also, lead concentration in plant tissues was higher than Cd concentration (Fig. 2). It has been reported that the world allowable standard levels of Cd and Pb in basil leaves are 0.1 and 0.3 mg.kg−1 plant dry weight, respectively (Sharma and Prasad, 2010). In the present research, Cd and Pb concentrations in the leaf and root tissues of basil plants were higher than the standard. So, it is not suggesting for fresh consumption, those basil plants that grown under heavy metals polluted soils. The higher concentrations of Cd and Pb in the leaf sample tissues that recorded from this experiment will suggest possible using basil plant for the purpose of phytoremediation of contaminated soils. It has been shown in the other experiment that some of medicinal plants can be used for phytoremediation of contaminated soils (Zheljazkov, et al. 2006). The high uptake rates of arsenic by different basil species (O. basilicum, O. tenuiflorum and O. gratissimum) showed high arsenic concentration in the leaves and roots, suggested that basil plantation might be a suitable plant for arsenic remediation (Siddiqui et al., 2013).

3.5. EO composition The identified chemical components of sweet basil are provided in Table 3. In the studied samples about 38 of these components represented about 90.14–97.74 % of the oil (Table 3). A sample GC-MS chromatogram of sweet basil and mass spectra of some components are shown in Fig. 4. Oxygenated monoterpene and sesquiterpene hydrocarbons formed the major part of the studied EO yield. Ladwani and Salman (2018) reported the similar results regarding oxygenated monoterpene (60.76%) and sesquiterpene hydrocarbons. In another study, monoterpene containing compounds were found as the pre dominant components of basil EO (more than 70%). In addition, Telci et al. (2006) evaluated the variation of basil oil chemical composition

Fig. 2. Cadmium (Cd) and lead (Pb) concentrations in soil, leaf and root tissues of basil plants in Cd treatments (A) and Pb treatments (B) after destructive plants harvest. Mean values of four replications ± standard error of the mean. 4

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and observed that CCA oxygenated monoterpene (63.7–85.6%) was the major compound of the EO. In the present research, the elevation of Pb contamination from 0 to 400 mg kg−1 of the soil increased the number of oxygenated monoterpene compounds from 40.6 to 61.8%. Moreover, as shown in Table 3, after Cd treatment, Cd5 (42.74) and Cd10 (55.24) showed higher monoterpenic compounds than Cd20 (26.87%). It has been reported that the application of Pb (0, 50, 100, 500, and 1000 ppm) enhanced oxygenated monoterpenic compounds in Cymbopogon citrates (Lermen et al., 2015). In addition, in the present research an increase in Cd and Pb concentration led to the reduction of the amounts of sesquiterpene hydrocarbons. Also, results obtained in the control plants indicated that estragole, phthalic acid, 2,6- octadienal, caryophyllene oxide, and caryophyllene are the main components of the sweet basil EO yield. In addition, in the present research the increase in soil Pb from 0 to 400 mg kg−1 has resulted in 110% increase in estragole production. Despite, the proven role of estragole as medicinal treatment, its higher concentration on rodent animals showed the carcinogen effects. However, the carcinogenic effect of estragole on human has not been yet reported (Clarke, 2008). Estragole in the presence of other EO components has less toxicity, and can be used without problem for its inflammatory effects as cream or ointment application

Fig. 3. The quantity of essential oil (EO) of sweet basil (Ocimum basilicum L.) grown under cadmium (Cd) and lead (Pb) treatments. Mean values of four replications ± standard error of the mean.

Table 3 Essential oil components percentage of sweet basil (Ocimum basilicum L.) grown under cadmium (Cd) and lead (Pb) concentrations (mg. kg−1 soil). R

Compounds

Empirical Formula

RI

Control

Cd5

Cd10

Cd20

Pb100

Pb200

Pb400

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

α- Thujene α-Pinene 1- Octan-3-ol 6-Methyl-5- hepten β-myrcene β-pinene L-Limonene 1,8- Cineole Octanal Octanol Linalool Fenchone Heptadienal Estragole 2,6- Octadienal Nerol Fenchole Cyclohexanedim ethanol Geraniol Geranial Neryl acetate Geranyl acetat α-Copaene Methyl eugenol Caryophyllene β- Farnesene β- Selinene Germacrene D β- Bisabolene γ-Cadinene Cis-α-Bisabolene Caryophyllene oxide Farnesole Neophytadiene Humulene epoxide II Oxabicyclo dodeca 2-Pentadecanone Phthalic acid Monoterpene hydrocarbons Oxygenate monoterpene Sesquiterpene hydrocarbons Oxygenate Sesquiterpene Diterpede hydrocarbons Others Total (%) Eo quantity % (v/w)

C10H16 C10H12O C8H16O C8H14O C10H16 C10H16 C10H16 C10H18O C8H16O C8H18O C10H18O C10H16O C7H10O C10H12O C8H12O C10H18O C10H18O C8H16O2 C10H18O C10H16O C12H20O2 C12H20O2 C15H24 C11H14O2 C15H24 C12H20O2 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24O C15H26O C20H38 C15H24O C15H24O C15H30O C8H6O4 – – – –

845 928 963 995 981 984 1028 1032 1005 1010 1099 1093 1124 1213 1232 1234 1242 1254 1252 1274 1366 1368 1369 1442 1451 1459 1491 1495 1498 1514 1544 1584 1592 1605 1610 1625 1642 1680 – – – –

– – –

– – –

0.84 2.46 0.84 0.49 t 0.54 t 0.22 0.19 0.10 0.18 t t 23.69 6.93 2.48 t 0.22 2.32 9.43 0.22 0.39 0.54 2.47 5.70 – 2.28 0.66 0.96 t 2.58 7.39 t 0.52 2.66 0.21 0.17 20.06 1.38 40.60 12.72 3.43 0.52 39.09 97.74 0.28

0.61 0.51 0.27 0.35 0.58 0.2 0.2 0.1 0.12 0.21 0.42 – – 24.87 11.95 2.27 0.10 0.25 4.52 8.95 1.89 0.97 0.91 2.37 3.98 0.68 0.94 t 0.48 0.16 2.15 10.69 0.21 0.65 1.26 2.41 0.22 8.32 1.59 42.74 12.51 10.90 0.65 26.38 94.77 0.34

0.52 0.41 0.14 0.21 t t 0.12 t 0.10 0.25 0.25 t 0.1 41.99 11.20 2.19 0.15 – 3.21 6.92 0.62 0.46 t 3.29 4.67 0.67 1.16 1.06 2.40 0.21 1.85 7.41 0.41 1.2 t 1.30 0.14 1.43 0.52 55.24 12.65 8.63 1.20 17.80 96.04 0.34

0.23 0.25 0.12 t t 0.10 t 0.12 0.10 0.33 0.38 t t 18.80 4.06 1.68 0.18 – 2.53 2.60 0.85 0.61 t 0.45 1.21 0.21 0.23 t t 0.25 1.33 0.99 1.67 3.13 0.32 1.12 0.12 47.89 0.45 26.87 1.81 4.22 3.13 55.38 91.86 0.39

0.93 0.61 0.14 0.22 1.21 1.32 1.14 1.13 1.70 0.40 0.20 2.12 2.18 27.76 4.26 2.59 0.15 t 2.13 6.24 0.45 0.61 t 0.94 1.79 0.24 1.42 1.45 – 1.31 0.32 1.21 1.21 1.75 t 0.56 0.12 20.33 4.6 44.72 4.50 3.10 1.75 31.47 90.14 0.30

0.71 0.53 0.22 0.12 t 0.25 0.13 t 0.11 0.58 0.32 0.38 0.21 28.78 14.76 2.42 – t 0.91 8.54 0.25 0.55 0.62 1.50 3.20 0.92 0.83 0.42 0.88 0.33 1.81 0.98 0.42 1.85 0.21 1.77 t 14.57 0.71 42.26 8.09 3.38 1.85 32.79 90.08 0.32

0.52 0.39 0.32 t t 0.20 t 0.15 t 0.36 0.38 t 0.15 50.32 3.2 2.20 – – 1.93 6.31 0.44 0.48 t 1.79 0.42 1.32 0.33 t 0.49 1.44 0.45 9.28 0.65 1.92 2.27 2.15 t 0.42 0.52 61.8 2.71 14.35 1.92 8.98 90.28 0.38

RI*: retention index; t: trace (0.1 >). 5

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Fig. 4. Gas chromatography–mass spectrometry (GC-MS) peaks and mass spectra spectrum of some essential oil (EO) major components of sweet basil (Ocimum basilicum L.).

6

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(Yadav et al., 2013). The safe range of 40–87% chavicol (estragole) in basil oil has been reported by Clarke (2008). Basil plants containing 22–88% estragole can be effectively used for aromatherapy (Clarke, 2008). In addition, estragole reported that has anti insect activity, and can be used as a natural insecticide (Ling Chang et al., 2009). In present research, all other EO compounds are listed in Table 3. In addition, the obtained data showed that the raise in the Cd concentration in the soil was led to the increase of the compounds such as linalool, octanol, nerol, neryl acetate, γ-cadinene, caryophyllene oxide, farnesol, and neophytadiene. Besides, it decreased compounds likeαthujene, α-pinene, 1, 8- cineole, octanal, geranial, methyl eugenol, βSelinene, cis-α-bisabolene, humulene epoxide II, and phthalic acid (Table 3). Furthermore, increasing Pb concentration enhanced the compounds such as octanol, linalool, nerol, neryl acetate, caryophyllene oxide, neophytadiene, and oxabicyclododeca while decreasing other compounds like α-pinene, 1- Octan-3-ol, 6-methyl-5hepten, β-pinene, 1,8- cineole, octanal, geranial, geranyl acetat, methyl eugenol, caryophyllene, β- selinene, germacrene D, β- bisabolene, cis-αbisabolene, and phthalic acid compared the control treatment (Table 3). Prasad et al. (2011) examined the influence of heavy elements on basil oil found that treatment of Cr (chromium), Cd, Pb, and Ni (nickel) increased methyl chavicol whereas decreasing the amount of linalole in sweet basil. The Cr treatment in Ocimum tenuiflorum L. increased the biosynthesis of the eugenol (Amirmoradi et al., 2012(. However, Siddiqui et al. (2013) found no specific process on arsenic treatment in Ocimum tenuiflorum L., Ocimum basilicm L., and Ocimum gratissimum L.

Table 4 Canonical coefficients, Eigen-values and variance for three canonical correspondence analysis (CCA) sets between phytochemicals with morphological traits of sweet basil (Ocimum basilicum L.). Traits

Essential oil compounds Estragole Nerol 2,6- Octadienal Geraniol Geranial Methyl eugenol Caryophyllene Cis-α-Bisabolene Caryophyllene oxide Phthalic acid Control Cd5 Cd10 Cd20 Pb100 Pb200 Pb400 Morphological factor Plant Height Sub Stems Length Internodes’ Length Flowering Stem Length Leaf Length Leaf Width Collar Diameter Stem Diameter Dry Weight Shoot Leaf Area Index Eigen-value Variance (%)

3.6. Principal component analysis (PCA) The PC1 and PC2 scatter plots were applied to monitor the phytochemical distance (Fig. 5). The scatter plots showed the phytochemical distances among the samples within the plot, influenced by their relationships, the studied populations were divided into three groups. The EO of the control, Pb100, and Cd20 formed an individual treatment separated from other samples and characterized by higher phthalic acid (38). In treatment, Cd5 and Pb200 formed other isolated groups with higher 2, 6- octadienal (15). The rest of the EO compounds formed in another group (Fig. 5).

CCA sets of Morphological factors 1

2

3

1.61 0.84 0.21 0.38 −0.34 0.41 −1.13 −1.40 3.66 −3.00 0.20 0.12 0.26 −0.46 −0.04 −0.01 0.32

−0.56 1.57 3.80 1.73 0.86 2.91 4.92 4.25 2.54 0.13 0.01 0.17 0.10 −0.12 −0.14 0.07 −0.11

−1.09 −1.48 −2.36 4.24 2.50 1.15 1.13 0.57 1.14 −1.75 0.04 0.08 0.01 −0.17 0.01 −0.03 0.03

−0.53 0.59 −0.23 0.68 −0.06 −0.19 0.51 0.71 −1.01 −0.07 0.031 76.3

−0.01 −0.27 −0.84 0.04 −0.58 −0.47 −0.84 −0.96 0.03 −0.31 0.009 23.7

−0.75 −0.10 −0.11 −1.46 −1.12 −0.97 −2.12 −2.23 1.43 0.25 4.750 0.001

presented a positive amount of nerol (0.84) while showing a negative amount of phthalic acid (-3.00) (Table 4). In other words, the basil plant with higher SSL and FSL had high nerol versus low phthalic acid contents. In the second canonical sets, treatments with low IL, SD, and CD had higher caryophyllene and cis-α-bisabolene contents. The third canonical is demonstrated in Table 4. The scatter plots of CC1 and CC2 were employed to determine more associations between the morphological and phytochemical traits. As it is illustrated in Fig. 6, a positive correlation was found between PH of the morphological trait and phthalic acid contents. Estragole positively correlated with plants

3.7. The CCA among morphological and EO traits The CCA was employed to explore the influence of morphological characters on the phytochemical composition. The first studied canonical sets of EO to morphological traits (more than 76%) indicated that the treatments with positive and notable values of SSL and FSL

Fig. 5. Classification of seven treatments applied on sweet basil (Ocimum basilicum L.) based on the essential oil (EO) chemical composition with biplot of first two components. 7

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Fig. 6. Canonical correspondence analysis biplot in the percentage of the major essential oil (EO) components with the morphological traits of sweet basil (Ocimum basilicum L.).

containing higher SSL. In addition, methyl eugenol, 2, 6- octadienal, geraniol, and nerol had a negative correlation with the IL (Fig. 6).

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