Material and methods
Seeds of 50 fennel (Foeniculum vulgare var. vulgare ) landraces provided by the seed bank of College of Aburaihan, University of Tehran (Bahmani et al., 2016, 2015, 2013, and 2012), were planted in a field under a randomized complete block design with three replications in an experimental field of College of Aburaihan. Each landrace was sowed in a 1 m2 plot in sandy–clay soil. Manual elimination of weeds and regular irrigation in 50% field capacity were performed. During the growing season, no diseases or pests were observed. After seedling emergence, seedlings were thinned to a final plant density of 10 plants per m2 for each landrace in each plot (Khorshidi et al., 2010; Falzari et al., 2006; El-Gengaihi and Abdallah, 1978). Wheat was grown in this field the two years before the current study; the wheat residues were incorporated in the soil, and no supplemental fertilizer was applied.
After the second year of growth in the field, seeds were harvested for the 50 landraces and fatty acids content was determined. Fatty acids were extracted from the seeds by hexane in an accelerated solvent extraction system (ASE) (Richter et al., 1996) at the University of Tehran (Figure 1). For this, seeds were milled and kept overnight in the oven at 105°C to reduce moisture content below 10%, and then about 1.3 g of the dried milled seeds from each landrace was placed in an extraction cell. During the extraction process, the conditions were set to 105°C oven temper­ature, 10 min static time, 70% flush volume, 60 s purge time, two static cycles, and 6.89 MPa pressure. Afterward, the extracted oils were air-dried overnight, and then dry mass and oil percentage were calculated.
To identify oil compositions among Iranian fennels, twelve of the landraces were selected to represent different maturity habits and diverse regions of origin. The selection criteria within each maturity type were for landraces of diverse geographic origin to include a wide range of climate diversity. For each of the twelve landraces, three equal amounts of seeds, harvested from the three replications in the second year of a field study described above, were mixed and a single sample of seeds for each landrace was formed. These twelve seed samples were brought to Michigan State University in 2015, and their total fixed oil contents were extracted using the same method described above (ASE) and, after calculating the percentage of oil, the dry oil samples using 1 ml hexane were collected.
For methyl esterification of fatty acids, the collected oil samples were dried again by evaporating the hexane, and then 1 ml methanol: H2SO4 (5:1 by volume) was added to each sample and mixed for a few minutes. The samples were kept overnight at room temperature. The following day, 1 ml chloroform and 5 ml deionized water were added to each sample, and the supernatant phase containing fatty acid methyl esters (FAMEs) was separated. These fatty acids methyl esters were identified and quantified using Thermo TRACE gas chromatog­raphy Ultra coupled with DSQII mass spectrometry (GC-MS). C19:0 methyl ester as internal standard, and four concentrations (0.4 to 400 ng/ml) of a 37-components FAME standard mixture as external standard were used. For each sample, three technical replications were performed. The 37-component FAME mix standard had a C18:1-9cis isomer of C18:1, which is oleic acid.
In GC, a DB-23 column (30 m × 0.25 mm i.d. × 0.25 mm film thickness) was used, syringe washes done by ethyl acetate and hexane, injection volume was 1 ml, inlet temperature 250 °C, and helium flow rate of 1.3 ml/min. The MS system had an electron ionization source operated at 70 eV and a single quadrupole mass analyzer and was operated with a 3 min solvent delay and an ion source temperature of 250 °C. The raw GC-MS result was processed in the MassLynx program to obtain the final data. The protocol used for oil extraction and GC-MS analysis is described in detail by Alameldin et al. (2017).
Fixed oil contents and fatty acids concentrations were reported as a percentage (%), which for fixed oil contents means ml fixed oil per 100 g dry ripened seeds (seed mass), and for fatty acids concentrations means ml fatty acid per 100 ml total fixed oil. Analyses of variance were performed using SAS 9.0, based on a randomized complete block design for oil contents.