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.