Phenotypic diversity and volatile composition of Iranian Artemisia dracunculus

Industrial Crops and Products 65 (2015) 315–323

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Phenotypic diversity and volatile composition of Iranian Artemisia dracunculus Ali Karimi a , Javad Hadian a,∗ , Mohsen Farzaneh a , Abdollah Khadivi-Khub b,∗ a b

Department of Agriculture, Medicinal Plants and Drug Research Institute, Shahid Beheshti University, G.C., Evin, Tehran 1483963113, Iran Department of Horticultural Sciences, Faculty of Agriculture and Natural Resources, Arak University, Arak 38156-8-8349, Iran

a r t i c l e

i n f o

Article history: Received 9 September 2014 Received in revised form 27 November 2014 Accepted 1 December 2014 Keywords: Tarragon Artemisia dracunculus Volatile oil Morphological variables Cluster analysis

a b s t r a c t Artemisia dracunculus (tarragon) has been used orally as an antiepileptic remedy in Iranian folkloric medicine. In the current study, the morphological variation and the essential oil composition from the aerial parts of the plant were evaluated. The results indicated significant differences among genotypes for morphological traits and clustering based on these traits classified the genotypes into two clusters. The essential oil content ranged from 1.42 to 2.53 v/w. Analysis of the essential oil revealed the presence of methyl chavicol (68.21–81.11%), limonene (7.18–16.73%), terpinolene (0.01–7.68%), (Z)-␤-ocimene (0.89–4.99%), (E)-␤-ocimene (0.81–4.52%), methyl eugenol (0.90–2.67%) and ␣-pinene (0.43–1.91%) as the main components. Variables related to leaf were found to be associated with all phytochemical compositions, indicating a main role of leaf on production of these compounds. These data collectively demonstrated large phenotypic and chemical diversity among the selected genotypes which can be considered as a valuable gene pool for breeding programs. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Plants may be a main source to provide drugs in traditional medicine. Aromatic plants are often applied in traditional medicine as antimicrobial agents and their essential oils, mixtures of natural volatile compounds extracted by steam distillation, have been known since antiquity to possess antifungal and antibacterial characteristics. Some essential oils have indicated important antimicrobial activity against dermatophyte, yeasts, bacteria, and Aspergillus strains (Lawrence, 2005; Lopes-Lutz et al., 2008), and have therapeutic potential, mainly in diseases involving cutaneous, mucosal, and respiratory tract infections. Phenolic compounds (terpenoids and phenylpropanoids) including thymol, carvacrol or eugenol, are the main components for many of these essential oils, of which have antioxidant and antimicrobial activities (Lawrence, 2005). Besides, the essential oils are widely used in different industries such as perfumery, cosmetics, and food. Asteraceae is the greatest family in the flowering plants with about 1535 genera comprising approximately 23,000 species, among which many are applied for their medicinal properties. One of the important genuses in this family is Artemisia. This genus has approximately 800 species which are widely distributed

∗ Corresponding authors. Tel.: +98 21 29903023; fax: +98 21 29903023. E-mail addresses: j [email protected] (J. Hadian), [email protected] (A. Khadivi-Khub). http://dx.doi.org/10.1016/j.indcrop.2014.12.003 0926-6690/© 2014 Elsevier B.V. All rights reserved.

throughout the world. Artemisia genus is industrially important due to its antifungal, insecticidal, allelopathic, antibacterial, and other characteristics. Furthermore, the plant is useful in Unani, Homeopathy, Ayurveda, and Siddha (Ved and Goraya, 2008). Biological activities and phytochemical composition for essential oils of different species of Artemisia have been recently reported (Kordali et al., 2005a,b; Lopes-Lutz et al., 2008). Artemisia dracunculus L. (tarragon) is an important species in Artemisia genus and has high phenotypic and genotypic diversity. Also, this species has high variability in reproductive behavior and component of essential oils. This plant is widely distributed in Japan, India, Iran, Europe, North America and China (Hooker, 1882). This species is considered for its aromatic values in medicinal properties, edibles, salads, and in tarragon vinegar preparation which is cultivated for long time in Iran. Tarragon is used in functional foods or as dietary supplements (Poulev et al., 2004). Biological properties and useful characteristics of A. dracunculus are reviewed in a report currently (Aglarova et al., 2008). The species is useful as radical-scavenging activities and insecticide (Saadali et al., 2001; Parejo et al., 2002). Essential oil compositions of A. dracunculus have antibacterial (Deans and Svobada, 1988), antitumor, antifungal, and DNA damaging effects (Zani et al., 1991 Meepagala et al., 2002). The dried aerial parts of A. dracunculus are applied to treat epilepsy in traditional medicine in Iran (Aqili Khorasani, 1992). Moreover, several studies indicated that methanolics of A. dracunculus had anticonvulsant benzodiazepines (Kavvadias et al., 2000). Furthermore, monoterpenes of essential oils in A. dracunculus have sedative

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A. Karimi et al. / Industrial Crops and Products 65 (2015) 315–323

Table 1 Descriptive statistics for the measured morphological variables among the studied genotypes of A. dracunculus. No.

Character

Abbreviation

Unit

Min.

Max.

Mean

SDa

CV (%)b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Plant height Plant diameter Total fresh weight Main branch no. Lateral branch no. (more than 5 cm) Lateral branch no. (less than 5 cm) Stem diameter Leaf length Leaf width Internode length Leaf area Total dry weight Fresh/dry weight Leaf dry weight Stem dry weight Leaf dry/stem dry weight

PlHe PlDi TFrWe MaBrNo LaBrNo LaBrNoS SrDi LLe LWi InLe LA TDrWe Fr/DrWe LDrWe StDrWe LDr/SDrWe

cm cm g Number Number Number mm mm mm mm cm2 g Ratio g g Ratio

37.22 34.00 46.67 3.44 8.28 6.78 2.50 33.81 3.10 7.80 0.39 11.12 3.07 7.93 3.19 1.48

59.62 64.61 170.89 9.00 22.50 12.58 4.26 47.83 5.14 10.54 1.78 43.47 4.23 27.19 16.60 2.87

46.25 45.93 106.53 5.87 13.09 9.44 3.24 39.25 3.97 9.16 0.80 28.55 3.73 19.12 9.43 2.15

6.19 7.18 32.40 1.69 3.35 1.54 0.47 2.96 0.47 0.75 0.27 8.25 0.23 5.22 3.39 0.38

13.38 15.64 30.41 28.85 25.61 16.33 14.60 7.55 11.92 8.21 34.03 28.89 6.22 27.33 35.88 17.48

a b

SD: Standard deviation. CV: Coefficient of variation = (Standard deviation/Mean) × 100.

and anticonvulsant effects (Sayyah et al., 2004). Also, ethenolic extract of the species supports the antidiabetic properties (Ribnicky et al., 2004). Several studies have been published on phytochemical components of A. dracunculus from different countries (Pino, 1996; Irena and Krystyna, 1996; Pappas and Sturtz, 2001). However, although tarragon is distributed across Iran (Mozafarian, 1996), very few chemical reports (Sayyah et al., 2004; Ayoughi et al., 2011) and no morphological analyses have been conducted with this plant species from this region. Thus, the goal of the current work was to detect variability of morphological and volatile oil properties from cultivated genotypes of Iranian A. dracunculus.

three replicates for each genotype. To compare these genotypes independent from environmental conditions, the selected plants materials were vegetatively cultivated into the experimental field collection at Shahid-Beheshti University (Tehran, Iran), where the plants were grown in uniform environmental conditions, so that the soil was soft, well drained, with a pH value close to 6.80 and fertilized with 60 kg N/ha, 30 kg P/ha and 30 kg K/ha. The cultural operations included manual weeds elimination and frequent irrigation to maintain the soil wet. During the growing season, no pest and disease were observed. The studied plants were harvested individually at full flowering time.

2.2. Morphological evaluation 2. Materials and methods 2.1. Plant material In the present work, morphological and phytochemical diversity of 26 cultivated genotypes of A. dracunculus was investigated, with

Morphological assessments were carried out on fully flowered fresh plants. Sixteen morphological characteristics were determined from fresh materials. Some of the phenotypic traits were evaluated at the experimental collection, and then the plant samples were transported to the laboratory for further analyses.

Table 2 Mean of morphological traits for the studied genotypes of A. dracunculus. Genotype

PlHe (cm) PlDi (cm) TFrWe (g) MaBrNo LaBrNo LaBrNoS SrDi (mm) LLe (mm) LWi (mm) InLe (mm) LA (cm2 ) TDrWe (g) Fr/DrWe LDrWe (g) StDrWe (g) LDr/SDrWe

Unknown1 45.94 49.08 Abadeh Neyshabour 45.44 52.89 Chenaran 47.89 Zarand 46.06 Esfahan1 Khoramabad 49.28 40.92 Hamedan Shahr-Rey 38.33 49.61 Yazd 58.50 Ferdos 43.32 Birjand Kermanshah 50.83 Kashmar 43.44 38.50 Kerman1 Unknown2 42.33 Estahbanat 46.11 37.22 Qom1 Qom2 40.67 59.62 Varamin 59.00 Ardestan 46.83 Khomein 46.50 Esfahan2 42.00 Tabriz 41.83 Kerman2 40.28 Dezfoul

47.39 49.58 43.67 48.69 47.22 42.89 49.53 40.50 39.44 49.83 56.44 50.28 52.11 45.47 37.29 44.89 44.33 35.25 36.28 64.61 57.31 49.22 45.25 34.00 44.75 37.94

98.11 116.17 75.44 91.44 128.67 134.22 137.67 62.50 82.11 145.56 162.00 84.22 136.33 78.89 85.00 100.44 133.78 73.11 46.67 145.44 170.89 85.44 111.78 79.00 118.33 86.44

4.33 6.58 5.33 5.56 7.67 8.56 6.44 3.50 4.61 9.00 8.00 5.22 6.67 4.44 4.67 6.56 8.56 4.33 3.67 7.11 7.33 4.89 6.56 3.50 6.00 3.44

11.44 17.00 16.53 8.28 15.89 14.56 13.72 8.83 9.17 16.28 14.56 11.28 12.78 13.72 10.08 16.72 22.50 13.28 10.28 14.56 16.33 11.94 10.61 10.25 9.50 10.22

7.67 12.58 9.22 9.06 9.17 10.72 9.06 8.17 6.78 9.39 10.22 7.56 10.89 9.78 8.00 10.67 11.50 11.44 9.72 11.44 11.17 9.44 8.56 7.00 8.00 8.22

3.58 3.04 2.90 3.72 2.50 2.63 3.34 3.27 3.55 2.62 3.24 2.97 3.90 4.07 3.06 2.78 2.65 3.42 2.88 3.24 3.90 3.67 3.10 3.07 3.03 4.26

47.83 39.61 40.44 39.99 33.81 41.16 38.86 38.74 41.26 36.60 37.95 36.57 44.33 38.34 41.18 36.64 34.78 38.58 35.89 38.63 38.39 40.01 39.17 37.62 41.21 42.85

4.70 3.53 3.84 4.13 3.13 4.01 4.09 4.01 4.14 3.27 3.85 3.92 5.14 4.20 4.03 3.17 3.10 4.14 4.42 3.95 3.88 4.58 4.11 3.72 4.01 4.14

9.52 8.11 8.83 10.09 9.14 9.51 9.08 9.32 10.37 9.27 9.89 10.54 8.99 9.36 8.75 7.80 8.16 8.48 9.01 10.26 9.73 9.01 8.35 7.97 8.79 9.89

0.99 0.62 0.80 0.67 0.39 0.63 0.81 0.76 1.18 0.46 0.63 0.69 1.78 0.87 0.87 0.59 0.52 0.92 0.76 1.04 0.76 0.90 0.80 0.53 0.87 0.94

25.91 32.39 21.18 26.27 34.19 35.33 36.59 16.75 22.02 37.52 40.82 24.71 34.87 20.90 22.38 28.28 35.94 18.11 11.12 40.71 43.47 23.19 28.50 22.18 30.79 28.20

3.78 3.57 3.56 3.51 3.73 3.78 3.74 3.73 3.74 3.87 4.00 3.41 3.90 3.78 3.78 3.51 3.64 4.07 4.23 3.59 3.96 3.70 3.93 3.56 3.84 3.07

16.96 22.09 15.16 16.04 23.67 24.71 23.57 11.35 15.02 27.19 24.37 16.83 22.42 14.40 14.45 20.81 25.26 12.37 7.93 25.04 26.88 15.59 19.80 14.68 19.61 20.79

8.95 10.30 6.02 10.23 10.53 10.62 13.02 5.40 6.99 10.34 16.45 7.87 12.45 6.49 7.93 7.47 10.68 5.74 3.19 15.67 16.60 7.60 8.69 7.50 11.18 7.41

1.93 2.14 2.56 1.62 2.40 2.36 1.95 2.10 2.16 2.69 1.48 2.15 1.83 2.22 1.86 2.87 2.34 2.27 2.54 1.60 1.67 2.20 2.43 1.96 1.75 2.81

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Table 3 Bivariate correlations among the measured morphological variables in the studied genotypes of A. dracunculus. Variable

PlHe

PlDi

TFrWe MaBrNo LaBrNo LaBrNoS

SrDi

LLe

LWi

InLe

LA

TDrWe Fr/DrWe LDrWe StDrWe LDr/SDrWe

PlHe PlDi TFrWe MaBrNo LaBrNo LaBrNoS SrDi LLe LWi InLe LA TDrWe Fr/DrWe LDrWe StDrWe LDr/SDrWe

1.00 0.90** 0.79** 0.63** 0.37 0.47* 0.09 −0.07 −0.06 0.31 −0.03 0.78** 0.09 0.68** 0.87** −0.524**

1.00 0.74** 0.60** 0.36 0.43* 0.10 −0.02 −0.02 0.40* 0.10 0.75** 0.01 0.66** 0.81** −0.48*

1.00 0.87** 0.54** 0.46* −0.11 −0.08 −0.26 0.12 −0.07 0.98** 0.14 0.94** 0.94** −0.36

1.00 0.54** 0.65** 0.38 0.61** −0.08 −0.15 −0.17 0.07 −0.30

1.00 0.71** 0.20 0.67** −0.07 −0.10 −0.11 0.01 −0.25

1.00 0.30 0.81** −0.30 0.24 −0.40* −0.11 −0.33

1.00 0.23 0.15 −0.14 0.07 0.26 −0.34

1.00 −0.08 0.12 −0.16 0.04 −0.27

1.00 −0.04 0.97** 0.93** −0.32

1.00 0.70** 0.54** −0.46* −0.31 −0.48* −0.03 −0.28 0.84** 0.17 0.87** 0.70** −0.06

1.00 0.71** −0.39* −0.42* −0.59** −0.33 −0.34 0.54** 0.01 0.62** 0.36 0.24

1.00 −0.09 −0.24 −0.19 −0.26 −0.03 0.44* 0.22 0.44* 0.39 −0.01

1.00 −0.11 0.08 −0.22

1.00 0.83** −0.10

1.00 −0.61**

1.00

For explanation of variable symbols, see Table 1. * Correlation is significant at the 0.05 level. ** Correlation is significant at the 0.01 level.

Quantitative measurements were carried out on 30 leaves per sample. Some variables were measured by using laboratory equipment. For instance, dimensions (length, width and diameter) were measured by digital caliper. Weight for different organs was measured by electronic balance with 0.01 g precision.). Also, leaf area was measured using Leaf Area Meter device (Delta T, England). 2.3. Phytochemical evaluation The aerial parts of the studied genotypes were air-dried in the shade at the room temperature and placed to oil extraction after grinding to a fine powder. The essential oil of drug fraction of each genotype (30 g in three replications) was extracted by a Clevenger-type apparatus using hydro-distillation for 3 h according to the method described in British pharmacopoeia (1993). The oils extracted were dried over anhydrous sodium sulfate and kept in tightly closed dark vials at 4 ◦ C until analysis. Analysis of GC was carried out with a flame ionization detector (FID) using a Thermoquest gas chromatograph. The analysis was performed on fused silica capillary DB-5 column (30m × 0.25 mm i.d.; film thickness 0.25 ␮m). The detector and injector temperatures were kept at 300 ◦ C and 250 ◦ C, respectively. Nitrogen was applied as the carrier gas at a

1.1 mL/min as flow rate; oven temperature program was 60–250 ◦ C at the rate of 4 ◦ C/min and finally held isothermally for 10 min; split ratio was 1:50. Analysis of GC–MS was performed using Thermoquest-Finnigan gas chromatograph equipped with fused silica capillary DB-5 column (60 m × 0.25 mm i.d.; film thickness 0.25 ␮m) coupled with a TRACE mass (Manchester, UK). Helium was used as carrier gas with ionization voltage of 70 eV. Ion source and interface temperatures were 200 ◦ C and 250 ◦ C, respectively. Mass ranged from 35 to 456 amu. Program of oven temperature was the same as mentioned above for the GC. The essential oil constituents were identified using calculation of their retention indices under temperature-programmed conditions for n-alkanes (C6–C24) and the oil on a DB-5 column under the same chromatographic conditions. Identification of genotype compounds was performed by use of comparison of their mass spectra with those of the internal reference mass spectra library (Adams and Wiley 7.0) or with authentic compounds and confirmed by comparing their retention indices with authentic compounds or with those of reported in the literature (Adams, 2007). Relative area percentages obtained by FID were applied for quantification purpose.

Chenaran Unknown2 Abadeh Esfahan2 Kerman2 Neyshabour Kashmar Birjand Khomein Shahr-Rey Kerman1 Tabriz Dezfoul Hamedan Qom1 Qom2 Zarand Estahbanat Esfahan1 Khoramabad Kermanshah Yazd Ferdos Ardestan Varamin 4.44

18.92

33.40

47.88

Coefficient

Fig. 1. UPGMA cluster analysis for the studied genotypes of A. dracunculus based on morphological variables.

62.35

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A. Karimi et al. / Industrial Crops and Products 65 (2015) 315–323

Table 4 Eigenvectors of the principal component axes from PCA analysis of morphological variables in the studied genotypes of A. dracunculus. Component Variable

1

Plant height Plant diameter Total fresh weight Main branch no. Lateral branch no. (more than 5 cm) Lateral branch no. (less than 5 cm) Stem diameter Leaf length Leaf width Internode length Leaf area Total dry weight Fresh/dry weight Leaf dry weight Stem dry weight Leaf dry/stem dry weight Total % of Variance Cumulative % **

**

0.90 0.88** 0.96** 0.80** 0.51 0.50 0.01 −0.04 −0.19 0.28 −0.01 0.96** 0.02 0.90** 0.96** −0.45 6.62 41.37 41.37

2

3

4

0.02 0.10 −0.08 −0.37 −0.36 0.00 0.80** 0.83** 0.87** 0.23 0.90** −0.08 −0.01 −0.15 0.04 −0.28 3.34 20.90 62.27

−0.13 −0.15 0.11 0.30 0.67** 0.67** −0.24 −0.10 −0.21 −0.72** 0.01 0.09 0.15 0.21 −0.12 0.52** 2.02 12.64 74.91

0.16 0.07 0.06 0.07 −0.10 0.21 −0.13 −0.13 0.29 −0.13 0.13 −0.11 0.93** −0.24 0.10 −0.45 1.39 8.65 83.56

Eigenvalues are significant ≥ 0.52.

2.4. Statistical analyses Analysis of variance (ANOVA) was carried out for phenotypic characters and phytochemical components by SAS software. Coefficients of variation (CV%) were estimated as variability indicators. The relationships between the studied phenotypic traits and between phytochemical components were determined using simple correlation coefficient by SPSS software individually. Relationships among the genotypes were detected using principal component analysis (PCA). PCA was performed for the studied morphological traits and for phytochemical components using SPSS statistics software individually. Mean values were used to create a correlation matrix from which standardized principal component Table 5 Descriptive statistics for phytochemical compositions among the studied genotypes of A. dracunculus. No.

Composition

Min.

Max.

Mean

SD

CV (%)

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

Essential oil ␣-Pinene Camphene Sabinene ␤-Pinene Myrcene Limonene (Z)-␤-Ocimene (E)-␤-Ocimene Terpinolene Linalool Allo-Ocimene Methyl chavicol Bornyl acetate ␦-Elemene Eugenol Methyl cinnamate-E Methyl eugenol Caryophyllene-E ␣-Humulene ␥-Decalactone Germacrene D ␣-Zingiberene Bicyclogermacrene (E,E)-␣-Farnesene ␤-Sesquiphellandrene Spathulenol Phytol

1.42 0.43 0.01 0.01 0.01 0.10 7.18 0.89 0.81 0.01 0.01 0.01 68.21 0.01 0.01 0.01 0.01 0.90 0.01 0.01 0.01 0.01 0.01 0.11 0.01 0.01 0.01 0.01

2.53 1.91 0.10 0.42 0.52 0.27 16.73 4.99 4.52 7.68 1.40 0.21 81.11 0.25 0.56 1.57 0.37 2.67 0.21 0.27 0.40 0.50 0.50 0.80 0.26 0.50 0.45 0.12

1.91 1.22 0.02 0.17 0.24 0.19 12.56 3.28 2.95 1.96 0.19 0.14 73.53 0.06 0.11 0.21 0.14 1.55 0.04 0.03 0.10 0.21 0.15 0.44 0.03 0.04 0.15 0.02

0.31 0.53 0.03 0.13 0.13 0.06 3.82 1.46 1.33 3.02 0.25 0.06 4.08 0.08 0.12 0.41 0.10 0.52 0.06 0.06 0.12 0.13 0.13 0.14 0.06 0.10 0.10 0.03

16.10 43.29 143.73 77.54 54.43 29.38 30.41 44.54 45.01 153.64 135.45 47.38 5.55 145.23 112.06 195.47 76.02 33.69 146.24 205.43 122.10 62.94 86.61 32.26 169.49 242.45 64.61 157.85

(PC) scores were extracted. The morphological and phytochemical distance coefficients were individually calculated according to Euclidean (Ed) method using the SIMINT program of the numerical taxonomy multivariate analysis system NTSYS-pc version 2.10 (Rohlf, 2000) and the dendrograms were constructed through SAHN clustering program using the unweighted pair group method with arithmetic means (UPGMA). Regression analysis between morphological date (as independent variables) and phytochemical compositions (as dependent variables) was performed using multiple regression analysis (MRA) to identify association between them. Multiple regression analysis (MRA) was conducted using “stepwise” method of “linear regression analysis” option of SPSS® version 16.

3. Results and discussion 3.1. Morphological analysis Morphological variability was observed among the studied plant genotypes. Coefficient of variation was the lowest for fresh/dry weight (CV = 6.22%), while it was the highest for stem dry weight (CV = 35.88%) (Table 1). Variability was recorded in plant height (37.22–59.62 cm), plant diameter (34.00–64.61 cm), main branch number (3.44–9.00), leaf length (33.81–47.83 mm), leaf width (3.10–5.14 mm), leaf area (0.39–1.78 cm2 ). Rzepka-Plevneˇs et al. (2009) reported 96 mm for leaf length of A. dracunculus in Poland. Also, total fresh weight ranged between 46.67 (for Qom2 genotype) and 170.89 g (in Ardestan genotype), while total dry weight varied from 11.12 (Qom2 genotype) to 43.47 g (in Ardestan genotype). Leaf dry weight ranged between 7.93 (for Qom2 genotype) and 27.19 g (in Yazd genotype), while stem dry weight varied from 3.19 (Qom2 genotype) to 16.60 g (Ardestan genotype) (Tables 1 and 2). Pearson correlation analysis indicated the existence of significant positive and negative correlations between traits measured (Table 3). It was found that plant height was in significant positive correlation with plant diameter (r = 0.90), total fresh weight (r = 0.79), main branch number (r = 0.63), total dry weight (r = 0.78), leaf dry weight (r = 0.68) and stem dry weight (r = 0.87). Total dry weight was positively correlated with total fresh weight (r = 0.98), leaf dry weight (r = 0.97) and stem dry weight (r = 0.93). Leaf width was positively correlated with leaf length (r = 0.71), stem diameter (r = 0.65) and leaf area (r = 0.81). Factor analysis was applied based on principal components to provide a reduced dimension model indicating differences measured among groups. PCA allows to evaluate multicollinear data and to determine the traits most suitable for classification (Iezzoni and Pritts, 1991). For each factor, loading value above 0.52 was considered as significant that indicated four components with explaining 83.56% of the total variance (Table 4). The first three components explained 74.91% of the total variability observed. PCA results indicated that the first component (PC1) related to plant height, plant diameter, total fresh weight, main branch number, total dry weight, leaf dry weight and stem dry weight and accounted for 41.37% of the total variation. The second component (PC2) which explained 20.90% of the total variation is dominated by four characteristics including stem diameter, leaf length, leaf width and leaf area. Furthermore, the characteristics lateral branch number (more than 5 cm), lateral branch number (less than 5 cm), internode length and leaf dry/stem dry weight performed as the third main factor (PC3) and explained 12.64% of the total variance. The most appropriate approach for classification purposes is the group average clustering method (Peeters and Martinelli, 1989). Morphological cluster analysis based on UPGMA showed two distinct groups with high variations (Fig. 1). The first major cluster contained 17 genotypes including Unknown1,

Table 6 Variation of the phytochemical compositions [% (w/w) based on dry weight] among the studied genotypes of A. dracunculus. Adams (RI)

Unknown1

Abadeh

Neyshabour

Chenaran

Zarand

Esfahan1

Khoramabad

Hamedan

Shahr-Rey

Yazd

Ferdos

Birjand

Kermanshah

Esential oil ␣-Pinene Camphene Sabinene ␤-Pinene Myrcene Limonene (Z)-␤-Ocimene (E)-␤-Ocimene Terpinolene Linalool Allo-Ocimene Methyl chavicol Bornyl acetate ␦-Elemene Eugenol Methyl cinnamate-E Methyl eugenol Caryophyllene-E ␣-Humulene ␥-Decalactone Germacrene D ␣-Zingiberene Bicyclogermacrene (E,E)-␣-Farnesene ␤-Sesquiphellandrene Spathulenol Phytol

– 935 950 975 979 990 1033 1041 1051 1089 1098 1127 1210 1285 1337 1356 1381 1402 1420 1454 1465 1482 1491 1497 1504 1522 1579 1933

– 932 946 969 974 988 1024 1032 1044 1086 1095 1128 1195 1284 1335 1356 1376 1403 1417 1452 1465 1484 1493 1500 1505 1521 1577 1942

2.26 1.46 T 0.10 0.16 0.22 15.58 4.99 4.52 0.19 0.10 0.17 69.76 T 0.10 T T 1.23 T T T 0.26 0.18 0.42 T T 0.10 T

1.72 0.43 T 0.31 0.34 0.11 8.04 1.00 0.90 5.80 0.11 0.12 77.05 T T 0.90 0.28 2.60 0.13 T 0.15 0.20 0.34 0.62 T T T T

1.89 0.53 T 0.35 0.43 0.11 7.74 1.00 0.90 6.54 0.10 0.07 79.08 T 0.21 T T 2.11 T 0.10 T T 0.21 0.28 T T 0.10 T

1.79 1.75 T 0.12 0.19 0.23 15.50 4.32 3.91 0.12 0.15 0.16 70.89 T 0.14 T 0.14 1.00 T T T 0.24 0.21 0.60 T T 0.15 T

1.49 0.51 T 0.41 0.48 0.12 7.18 1.05 0.95 7.16 T 0.10 78.47 T T T 0.27 2.09 T T 0.13 T 0.21 0.40 T T 0.12 T

1.57 0.53 T 0.40 0.52 0.14 7.71 1.12 1.01 7.61 0.11 0.10 76.78 0.22 T 0.49 0.27 1.80 T T 0.14 T 0.50 0.37 T T T T

2.37 1.57 T 0.11 0.18 0.22 12.50 4.01 3.63 0.15 0.21 0.15 74.32 0.16 T T 0.12 1.38 T T T 0.18 0.13 0.34 T 0.13 0.13 T

2.53 1.63 0.10 0.11 0.20 0.23 14.64 4.36 3.94 0.35 0.16 0.15 71.46 T T T 0.14 1.26 T T T 0.18 0.19 0.41 T T 0.13 T

1.91 1.49 T 0.12 0.20 0.21 15.03 3.65 3.31 0.12 0.20 0.16 72.86 T T T 0.10 0.97 0.14 T T T 0.39 0.50 T T 0.21 T

2.04 0.47 T 0.27 0.34 0.10 7.25 0.89 0.81 5.51 T T 81.11 T T T T 2.02 T T T T 0.25 0.35 T T T T

1.57 1.55 T 0.10 0.18 0.21 15.19 3.85 3.48 0.13 0.17 0.20 70.61 T 0.13 1.57 T 1.14 T T T 0.50 T 0.46 T T 0.17 T

2.09 1.65 T 0.10 0.18 0.22 15.15 3.92 3.55 0.24 0.16 0.15 72.19 T 0.11 T 0.15 0.90 T T 0.24 0.19 T 0.47 T T T T

2.21 1.56 T 0.10 0.17 0.23 15.23 4.33 3.92 0.18 0.26 0.19 71.31 T 0.13 T 0.11 1.28 T T T 0.16 0.12 0.32 0.10 T 0.10 T

Composition

RI

Adams (RI)

Kashmar

Kerman1

Unknown2

Estahbanat

Qom1

Qom2

Varamin

Ardestan

Khomein

Esfahan2

Tabriz

Kerman2

Dezfoul

Esential oil ␣-Pinene Camphene Sabinene ␤-Pinene Myrcene Limonene (Z)-␤-Ocimene (E)-␤-Ocimene Terpinolene Linalool allo-Ocimene Methyl chavicol Bornyl acetate ␦-Elemene Eugenol Methyl cinnamate-E Methyl eugenol Caryophyllene-E ␣-Humulene ␥-Decalactone Germacrene D ␣-Zingiberene Bicyclogermacrene (E,E)-␣-Farnesene ␤-Sesquiphellandrene Spathulenol Phytol

– 935.45 950.26 974.60 979.37 990.48 1032.93 1040.65 1050.81 1089.43 1098.37 1127.10 1209.89 1284.79 1336.68 1355.98 1381.08 1402.44 1419.92 1454.47 1464.63 1481.71 1490.65 1497.15 1503.81 1521.61 1578.81 1933.33

– 932 946 969 974 988 1024 1032 1044 1086 1095 1128 1195 1284 1335 1356 1376 1403 1417 1452 1465 1484 1493 1500 1505 1521 1577 1942

1.93 1.31 T 0.10 0.17 0.20 12.65 4.11 3.71 0.10 0.18 0.13 73.69 0.20 0.16 T 0.17 1.35 T T 0.27 0.21 T 0.51 0.10 T 0.21 T

1.70 1.58 0.10 0.10 0.18 0.22 16.44 4.77 4.32 0.13 0.12 0.21 68.21 0.25 0.56 0.17 T 1.16 T T T 0.34 0.14 0.33 T T 0.32 T

1.54 0.49 T 0.39 0.49 0.14 7.21 0.92 0.83 7.68 0.14 T 78.58 T T T 0.17 2.10 T 0.10 T 0.19 T 0.28 T T 0.12 T

1.52 0.55 T 0.42 0.46 0.13 8.50 1.20 1.10 7.20 1.40 T 72.50 T 0.14 1.26 0.28 2.67 0.21 0.27 T 0.32 0.11 0.80 T T 0.23 T

2.01 1.91 0.10 0.13 0.23 0.27 16.31 4.57 4.13 0.29 0.12 0.21 68.77 0.17 T 0.11 0.11 1.36 T T T 0.18 0.21 0.43 T 0.10 0.12 T

1.81 1.47 T 0.11 0.18 0.23 16.48 3.89 3.51 0.19 0.16 0.16 70.05 0.10 T T T 1.30 0.16 T 0.29 0.30 0.12 0.53 T T 0.28 T

2.04 1.74 T 0.12 0.20 0.24 16.73 4.24 3.83 T 0.20 0.18 69.40 T 0.13 0.10 T 1.11 T T T 0.34 0.19 0.59 T T 0.16 T

2.02 1.72 T 0.11 0.20 0.25 15.44 4.59 4.16 0.22 0.13 0.19 69.95 T 0.16 T 0.17 1.30 T T 0.25 0.18 T 0.41 T 0.12 0.14 T

2.47 0.89 T T 0.12 0.23 11.18 3.33 3.00 0.23 0.13 0.16 77.97 T 0.23 T 0.11 1.24 T T 0.12 0.21 T 0.28 T T 0.16 T

1.84 1.65 T 0.11 0.19 0.23 16.70 4.11 3.71 T 0.13 0.19 69.56 T 0.12 T 0.14 1.37 T T 0.40 0.22 0.14 0.46 T T 0.18 T

2.13 1.82 T 0.12 0.21 0.26 16.38 4.56 4.12 0.15 0.18 0.19 68.90 T 0.11 T 0.11 1.16 T T 0.31 0.24 0.11 0.52 0.10 T 0.16 T

1.68 0.80 T T 0.10 0.12 7.63 3.52 3.00 0.35 0.12 0.17 79.18 T 0.23 0.28 0.27 2.25 0.12 T 0.14 0.30 T 0.11 0.26 T 0.45 0.11

1.42 0.73 T T T 0.10 8.27 3.00 2.33 0.38 0.10 T 79.05 0.22 T 0.45 0.37 2.23 0.14 T T 0.50 T 0.60 0.10 0.50 0.18 0.12

RI: Retention indices relative to C6–C25 on the DB-5 column. T: Trace < 0.10%.

319

RI

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Composition

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Unknown2, Chenaran, Hamedan, Shahr-Rey, Birjand, Kashmar, Kerman1, Qom1, Qom2, Khomein, Esfahan2, Tabriz, Kerman2, Dezfoul, Abadeh and Neyshabour, characterized by lower values for plant height, plant diameter, main branch number, total fresh weight, total dry weight, leaf dry weight and stem dry weight and also higher values for stem diameter and leaf length. The second major cluster contained nine genotypes including Zarand, Esfahan1, Estahbanat, Khoramabad, Kermanshah, Yazd, Ferdos, Ardestan and Varamin, characterized by higher values for plant height, plant diameter, main branch number, total fresh weight, total dry weight, leaf dry weight and stem dry weight and also lower values for stem diameter and leaf length. This observed morphological diversity in the studied A. dracunculus germplasm may be accompanied by phenotypic plasticity and genetic variability (Nemeth, 2000). 3.2. Phytochemical analysis In the present study, high variation was observed in essential oil content and composition of the studied genotypes of A. dracunculus. The content of essential oil varied between 1.42 (in Dezfoul genotype) and 2.53% (in Hamedan genotype) with an average of 1.91% v/w. Twenty-seven volatile components were identified in the studied genotypes. The percentage of each component within the essential oils is presented in Table 5. The table also includes retention indices (RI). The volatile components identified accounted for 99.63% of the oils composition. The major component of essential oil was methyl chavicol in the studied genotypes of A. dracunculus, ranged from 68.21 (in Kerman1) to 81.11% (in Yazd) with an average of 73.53% (Tables 5 and 6). Also, limonene ranged from 7.18 (in Zarand) to 16.73% (in Varamin) with an average of 12.56%. Other important constituents were terpinolene (0.01–7.68%), (Z)-␤-ocimene (0.89–4.99%), (E)-␤ocimene (0.81–4.52%), methyl eugenol (0.90–2.67%) and ␣-pinene (0.43–1.91%) (Tables 5 and 6). ␣-pinene and ␤-pinene present in our genotypes has shown anticonvulsant activity (Sayyah et al., 2004). It is reported that some analogs of pinene prevent the audiogenic seizures in susceptible rats (Consroe et al., 1981). Moreever, methyl eugenol present in the essential oil possesses considerable anticonvulsant activity (Dallmeier and Carlini, 1981). In Iran, Sayyah et al. (2004) reported the presence of transanethole (21.10%), ␣-trans-ocimene (20.60%), limonene (12.40%), ␣-pinene (5.10%), allo ocimene (4.80%), methyl eugenol (2.20%), ␤pinene (0.80%), ␣-terpinolene (0.50%), bornyl acetate (0.50%) and bicyclogermacrene (0.50%) as the main components in oil of A. dracunculus. Also, Ayoughi et al. (2011) reported (Z)-anethole (51.72%), (Z)-␤-ocimene (8.32%), methyleugenol (8.06%), limonene (4.94%) and linalool (4.41%) as the main components in oil of A. dracunculus. In other countries, Kordali et al. (2005a,b) reported that Turkish A. dracunculus oil contained mainly (Z)-anethole (81.00%), (Z)␤-ocimene (6.50%), (E)-␤-ocimene (3.10%), limonene (3.00%) and methyl eugenol (1.80%). Besides, Chauhan et al. (2010) reported that capillene (58.38%) was the major component of essential oil in A. dracunculus from north–west Himalaya, India and followed by Z-␤-ocimene (8.63%), ␤-phellandrene (7.03%), terpinolene (5.87%), camphene (4.16%), spathulenol (2.02%), and ␤-pinene (1.02%). Furthermore, Eisenman et al. (2013) reported that (Z)-␤-ocimene, methyl chavicol, methyl eugenol and ␣-terpinolene were found to be the most common primary components in populations of A. dracunculus from North America. The Pearson correlations showed positive and negative significant correlations between phytochemical compounds (not shown). Methyl chavicol as the major component, was negatively correlated with ␣-pinene (r = −0.88), limonene (r = −0.93), (Z)-␤-ocimene (r = −0.78) and (E)-␤-ocimene (r = −0.80), and also positively

Table 7 Eigenvalues and cumulative variance for six factors obtained from principal component analysis (PCA) based on phytochemical compositions for the studied genotypes of A. dracunculus. Component Composition ␣-Pinene Camphene Sabinene ␤-Pinene Myrcene Limonene (Z)-␤-Ocimene (E)-␤-Ocimene Terpinolene Linalool Allo-Ocimene Methyl chavicol Bornyl acetate ␦-Elemene Eugenol Methyl cinnamate-E Methyl eugenol Caryophyllene-E ␣-Humulene ␥-Decalactone Germacrene D ␣-Zingiberene Bicyclogermacrene (E,E)-␣-Farnesene ␤-Sesquiphellandrene Spathulenol Phytol Eigenvalue % of Variance Cumulative % **

1 **

0.97 0.29 −0.69** −0.64 0.95** 0.99** 0.93** 0.94** −0.86** −0.08 0.83** −0.90** 0.02 0.23 −0.21 −0.50 −0.88** −0.26 −0.40 0.23 0.34 −0.20 0.19 −0.13 −0.11 0.18 −0.30 9.52 35.26 35.26

2

3

4

5

6

0.01 −0.13 −0.61 −0.69** −0.15 −0.08 0.25 0.19 −0.44 −0.14 −0.11 0.20 0.25 −0.04 0.11 0.35 0.10 0.29 −0.24 −0.08 0.63 −0.52 0.02 0.54 0.81** 0.33 0.85** 4.06 15.05 50.31

−0.07 −0.05 0.24 0.15 −0.09 0.00 −0.12 −0.12 0.16 0.89** −0.24 −0.27 −0.04 0.02 0.68** 0.25 0.31 0.66** 0.75** −0.10 0.47 −0.09 0.76** −0.13 0.07 0.22 −0.02 3.57 13.24 63.55

−0.01 0.23 −0.20 −0.14 0.00 −0.01 0.17 0.17 −0.14 0.14 0.24 −0.03 0.02 0.78** 0.01 −0.14 0.05 0.03 0.18 −0.01 0.21 −0.39 −0.49 0.52 −0.32 0.76** 0.23 2.38 8.81 72.36

0.05 0.69** 0.04 0.04 −0.01 0.02 0.06 0.05 0.02 −0.08 0.07 −0.12 0.80** 0.09 0.00 0.20 0.05 0.07 −0.15 −0.21 −0.04 0.45 0.04 −0.07 0.29 0.10 0.12 1.59 5.89 78.25

−0.01 −0.31 −0.04 −0.04 −0.02 −0.02 0.02 0.02 −0.05 −0.01 0.17 0.00 −0.02 −0.20 −0.13 0.47 0.11 0.36 −0.17 0.76** −0.18 0.14 0.11 0.44 −0.09 0.24 0.20 1.52 5.64 83.89

Eigenvalues are significant ≥ 0.66.

correlated with terpinolene (r = 0.61) and methyl eugenol (r = 0.69). Furthermore, limonene was positively correlated with ␣-pinene (r = 0.96), (Z)-␤-ocimene (r = 0.89) and (E)-␤-ocimene (r = 0.91), and also negatively correlated with terpinolene (r = −0.80) and methyl eugenol (r = −0.87). Beside, ␣-pinene was in significant positive correlation with (Z)-␤-ocimene (r = 0.93) and (E)-␤-ocimene (r = 0.94) and negative correlation with terpinolene (r = −0.84) and methyl eugenol (r = −0.87). To determine the degree of phytochemical variations, a principal component analysis (PCA) was performed using a correlation matrix of essential oil compositions. For each factor, a principal component loading of more than 0.66 was considered as being significant that 83.89% of the variability observed was explained by six components (PC1–PC6) (Table 7). The first three components explained 63.55% of the total observed variability. PC1 represented ␣-pinene, sabinene, myrcene, limonene, (Z)-␤-ocimene, (E)-␤-ocimene, terpinolene, allo-ocimene, methyl chavicol and methyl eugenol and accounted for 35.26% of the variance. PC2 represented mainly ␤-pinene, ␤-sesquiphellandrene and phytol and accounted for 15.05% of the variance. PC3 included linalool, eugenol, caryophyllene-E, ␣-humulene and bicyclogermacrene and accounted for 13.24% of the variance. The remaining components (PC4–PC6) explained less variability (20.34% of total variance) and included other variables. On the other hand, methyl cinnamate-E, germacrene-D, ␣-Zingiberene and (E,E)-␣-Farnesene showed low variation between studied genotypes and were not placed in PCs that seemed to be less important when applying this analysis. According to the aforementioned six factors, phytochemical cluster analysis using UPGMA showed two major clusters (Fig. 2). The cluster I contained 16 genotypes (Unknown1, Chenaran, Khoramabad, Hamedan, Shahr-Rey, Ferdos, Birjand, Kermanshah, Kashmar, Kerman1, Qom1, Qom2, Varamin, Ardestan,

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321

Table 8 Phytochemical compositions associated with morphological characters in A. dracunculus revealed using MRA and coefficients. Phytochemical composition

Morphological trait

r

R2

Standardized ␤ coefficients

t value

P value

␣-Pinene

Leaf dry/stem dry weight Main branch number

0.605a 0.758b

0.37 0.57

−0.63 −0.46

−4.64 −3.35

0.00 0.00

Limonene

Leaf dry/stem dry weight Leaf dry weight

0.557a 0.732b

0.31 0.54

−0.61 −0.48

−4.24 −3.34

0.00 0.00

(Z)-␤-Ocimene

Leaf width Leaf dry/stem dry weight Main branch number

0.674a 0.796b 0.883c

0.45 0.63 0.78

0.27 −0.56 −0.45

2.19 −5.08 −3.80

0.04 0.00 0.00

(E)-␤-Ocimene

Leaf width Leaf dry/stem dry weight Main branch number

0.666a 0.799b 0.879c

0.44 0.64 0.77

0.27 −0.57 −0.43

2.12 −5.14 −3.61

0.05 0.00 0.00

Terpinolene

Lateral branch number Stem dry weight Leaf dry/stem dry weight

0.726a 0.839b 0.892c

0.53 0.70 0.80

0.50 −0.38 0.32

4.75 −3.51 3.14

0.00 0.00 0.01

Methyl chavicol

Leaf dry/stem dry weight Leaf dry weight

0.561a 0.674b

0.31 0.46

0.60 0.38

3.87 2.43

0.00 0.02

Methyl eugenol

Lateral branch number Leaf dry/stem dry weight

0.613a 0.725b

0.38 0.53

0.52 0.40

3.52 2.69

0.00 0.01

Esfahan2 and Tabriz), characterized by higher values for ␣pinene (1.31–1.91%), limonene (12.50–16.73%), (Z)-␤-ocimene (3.65–4.99%) and (E)-␤-ocimene (3.31–4.52%) and lower values for terpinolene (0.01–0.35%), methyl chavicol (68.21–74.32%) and methyl eugenol (0.90–1.38%). Cluster II contained 10 genotypes including Dezfoul, Abadeh, Neyshabour, Zarand, Esfahan1, Yazd, Estahbanat, Khomein, Kerman2 and Unknown2, characterized by lower values for ␣-pinene (0.43–0.89%), limonene (7.18–11.18%), (Z)-␤-ocimene (0.89–3.52%) and (E)-␤-ocimene (0.81–3.00%) and higher values for terpinolene (0.23–7.68%), methyl chavicol (72.50–81.11%) and methyl eugenol (1.24–2.67%). Cluster analysis of essential oil composition based on chromatographic data can be used to determine specific differences between the individual chemotypes (Echeverrigaray et al., 2001).

Quantitative and qualitative composition of essential oil of A. dracunculus in the present work was much different from other studies in this species (Pino, 1996; Irena and Krystyna, 1996; Pappas and Sturtz, 2001; Sayyah et al., 2004; Kordali et al., 2005a,b; Chauhan et al., 2010; Ayoughi et al., 2011 Eisenman et al., 2013). Observed variations in essential oil content and components can be attributed to factors related to ecotype, chemotype, phenophases and the environmental condition including temperature, relative humidity, irradiance and photoperiod (Fahlen et al., 1997). The quantitative composition of the essential oils of many aromatic plants is greatly affected by the genotype and agronomic conditions, such as harvesting time, plant age and crop density (Marotti et al., 1994). In the present study, the composition of the essential oils of the studied genotypes was measured at full flowering stage as described by Chauhan et al. (2010) and Eisenman et al. (2013),

Unknown1 Ardestan Kerman1 Qom1 Tabriz Qom2 Varamin Esfahan2 Chenaran Hamedan Kermanshah Shahr-Rey Birjand Ferdos Khoramabad Kashmar Abadeh Esfahan1 Neyshabour Zarand Unknown2 Yazd Estahbanat Khomein Kerman2 Dezfoul 0.53

3.54

6.55

9.56

Coefficient

Fig. 2. UPGMA dendrogram for the studied genotypes of A. dracunculus based on phytochemical compositions.

12.57

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who reported higher the quantity for the essential oils composition in A. dracunculus at this stage. The Artemisia plants are different with essential oils of varying compositions, leading to the distinct flavors of each herb. For instance, the essential oil of the French tarragon (A. dracunculus) consists predominantly of estragole (60%), which is responsible for the carcinogenicity and genotoxicity of the oil (Ribnicky et al., 2004). The essential oil obtained in this study may have low toxicity in this regard. The A. dracunculus that we used in this work had no estragole, just as the Russian tarragon does not (Ribnicky et al., 2004). Methyl eugenol is another component of A. dracunculus, which possesses carcinogenic and genotoxic properties at high doses (Burkey et al., 2000; Ribnicky et al., 2004). The range of methyl eugenol in the essential oil of A. dracunculus in this study was 0.90–2.67%, which is lower than the quantity of methyl eugenol (9.00%) in the essential oil of A. dracunculus reported by Meepagala et al. (2002). This finding further supports the low toxicity of our essential oil. 3.3. Association between morphological and phytochemical characters Multiple regression analysis (MRA) revealed that major phytochemical compositions as dependent variable, showed statistically significant correlation and in association with some morphological traits as independent variable, especially variables related to leaf (Table 8). ␣-pinene was found to be associated with leaf dry/stem dry weight (R2 = 0.37) and main branch number (R2 = 0.57). Also, limonene showed association with leaf dry/stem dry weight (R2 = 0.31) and leaf dry weight (R2 = 0.54). Both (Z)-␤ocimene and (E)-␤-ocimene showed strong correlations with leaf width, leaf dry/stem dry weight and main branch number with high combinations. Terpinolene was found to be associated with lateral branch number (R2 = 0.53), stem dry weight (R2 = 0.70) and leaf dry/stem dry weight (R2 = 0.80). Besides, methyl chavicol was associated with leaf dry/stem dry weight (R2 = 0.31) and leaf dry weight (R2 = 0.46). Furthermore, methyl eugenol had association with lateral branch number weight (R2 = 0.38) and leaf dry/stem dry weight (R2 = 0.53) (Table 8). According to these results, variables related to leaf had association with all phytochemical compositions, indicating a main role of leaf in production of these compounds and agreed with finding of other researchers that reported that leaf is important in production of phytochemical compositions in Artemisia spp. (Pino, 1996; Sayyah et al., 2004; Kordali et al., 2005b). Some of morphological variables were found to be associated with more than one phytochemical composition in multiple regression analysis. For instance, leaf dry/stem dry weight was associated with all of the compositions, indicated that leaf weight had a vital role in production of these compounds. Thus, understanding association between phytochemical compositions and morphological variables can help breeders for selection and crosses (Khadivi-Khub et al., 2014). 4. Conclusion The present investigation is the primary study of phytochemical and phenotypic variability in Iranian A. dracunculus germplasm. The A. dracunculus genotypes characterized in this study exhibited a great deal of morphological and phytochemical variations and they seem to have a high potential for breeding programs. All of the genotypes studied were principally composed methyl chavicol. The essential oil of this A. dracunculus germplasm from Iran may have important medicinal values because of high methyl chavicol percentage. Also, this methyl chavicol-rich germplasm may be useful in the development of a new line of tarragon with both the desirable

flavor characteristics of tarragon, as well as desirable traits of wild tarragon, such as the ability to produce seeds. Moreover, it may provide promising genetic materials for selection and breeding efforts. According to result of multiple regression analysis (MRA), variables related to leaf were found to be associated with all of the phytochemical compositions, indicating a main role of leaf in production of these compounds; thus, the selection for a higher leaf values would result in the improvement of phytochemical compositions. The integration of the data regarding phenotypic variation determined in the experimental field and phytochemical data can be interesting in breeding programs for this species, since they can be applied to select individual plants during cultivation. 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