Growth, volatile oil production and genetic study of some fennel cultivars under
different compost levels in sandy soil
Omneya F Abu El-Leel*, Rabie.M.M.Yousef.
Medicinal and Aromatic Plant Research Department, Horticulture Research Institute (HRI), Agricultural Research Centre (ARC),
The present work was conducted during the two successive seasons of 2011/2012 and 2012/2013 at the experimental farm of El-
Quassassin Hort. Res. Station, Ismailia Governorate, and Biotechnology Laboratory, Horticulture Research Institute, Agricultural
Research Centre, Egypt. The aim of this study was to investigate the effect of three compost fertilizer levels 4, 6 and 8 ton per Fadden
using five cultivars of bitter fennel on growth, fruits yield and volatile oil production of fennel (Foeniculum vulgare Mill). These cultivars
were Netherland, Indian, Azoricum, German and Local fennel. Several trails were studied including growth and yield production,
biochemical (the volatile oil) and molecular genetic (RAPD- and ISSR-PCR) characteristics under Egyptian sandy soil. The results
showed that increasing compost level progressively (form 4 to 8 ton/ Fed) and significantly increased the values of such parameters.
Azoricum cultivar was superior to other cultivars under study, as it showed the best growth in terms, fruits yield, fruit volatile oil (%)
and volatile oil production per plant and Fadden. The main compounds in all fennel volatile oils were: Anethole, Estragole, Fenchone
and Limonene. The highest percentage of Anethole found in German cultivar, while the lowest percentage found in Local cultivar, where
the highest percentage of Estragole (= Methyl chavicol ) compound undesirable found in Local cultivar, while the lowest percentage
found in Netherland and German cultivars. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR)
molecular fingerprinting markers were employed as genetic markers for the assay of the genetic relationship of five fennel cultivars. In
RAPD analysis, 10 selected primers displayed a total of 98 amplified fragments, in which 60 (61.22%) were polymorphic fragments.
Thirty-one out of 98 RAPD-PCR fragments were found to be useful as cultivar-specific markers.
The largest number of RAPD-PCR
markers was scored for Indian variety (68 markers), while the lowest (49 markers) was scored for Netherland variety. In the meantime,
the highest number of RAPD-PCR cultivar-specific markers was generated by primer OP-C04 (7 markers), while the lowest number of
RAPD-PCR specific markers (1 markers) was generated by primers OP-A13 and OP-B04. In ISSR analysis, 4 of the tested ISSR primers
generated variable banding patterns. A total of 26 out of 34 ISSR fragments were polymorphic. Eleven DNA amplified fragments were
considered as cultivar-specific markers. The cultivars distribution on the consensus tree according to the banding patterns of RAPD
differed from that based on ISSR. This may be due to the possibility that each technique of amplified different parts of the genome.
Therefore, it would be useful to use a combination of the banding patterns of the two technique in order to use more segments sites of
the genome that verify the validity of the consensus tree.
Fennel cultivars, essential oil, DNA fingerprinting and genetic relationship.
Fennel is a plant belonging to the Umbelliferae (Apiaceae) Family,
known and used by humans since antiquity. Because of its flavor,
it was cultivated in the countries surrounding the Mediterranean
Sea. Fennel is one of the oldest field crop used by the Egyptian
for medicinal purposes. Most of the area cultivated with fennel
is located in Mid-southern Egypt mainly, El-Fayom, Menia and
Assiut Governorates. Only one strain of common or bitter
fennel (Foeniculum vulgare, Mill.) was cultivated in Egypt for
the national and international purposes. Exports of local fennel
(Foeniculum vulgare Mill.) from Egypt over the past few years
have been affected due to the high Estragole but low Anethole
content of the oil. Therefore, fennel seeds were imported from different countries to investigate the adaptability of such strains
in different locations in Egypt, in comparison with the local one
(Shalaby et al., 2011). The volatile oil is used as flavoring agent,
carminative antispasmodic, stomachic, diuretic, expectorant,
aromatic and lactagogue. Fruits are used as spice, in pickles,
candies, in liquors and in the preparation of alcoholic beverages.
They are also used as remedy for jaundice and menstrual troubles
(Kotb, F. T.1985). The cultivar yields big fruits and reasonable
percentage of essential oil with particularly high Estragole content,
but it is poor in Fenchone which is an important constituent of
the fennel essential oil. These, along with some other components,
provide the unique aroma and taste. Trans-anethole accounts
for the anise taste, Estragole(=Methyl chavicol) compound
undesirable, Fenchone the bitterness, and Limonene provides the
citrus taste. Commonly, Trans-anethole was used for flavor in the
food and liquors industry, which considered non-toxic (Barazani
et al., 2002). Moreover, essential oil of fennel has been shown to
have antioxidant, antibacterial and antiviral activities (Farag et al,
1989). The fresh leaves and dried fruits of this plant are used as a
spice for meat, baked and confectionery products (Davis, 1972).
Compost enhances the environmental sustainability of agriculture
by decreasing chemical inputs and increasing soil organic matter
(Mathur et al., 1993).
Many research workers gained best
growth, yield, oil percantage and yield and chemical constituents
when used compost for several medicinal and aromatic plants, as
(Ibrahim, 1999) on Ocimum sanctum; (Khalil, 2002) on rosmary
(Rosemarinus officinalis); (Khalil et al., 2002) on Tagetes erecta;
(Khalil and El-Sherbeny, 2002) on three Mentha species; (Naguib
and Aziz, 2004) on Hyosyamus muticus and (El-Sherbeny et al.,
2005) on Sidritis montana. Germplasm is a vital source in
generating new plant types having desirable traits that help in
increasing crop production with quality and thus improve the level
of human nutrition. The genetic diversity is analyzed by using
morphological as well as genetic based tools, DNA techniques
(Bennici et al., 2003) and advanced molecular methods etc.
(Barazani et al., 2002; Shiran et al., 2007). The organic fertilizers
is utilized for the change of soil texture, supplying nutrients to the
growing plants and organic acids, enhancing nutrients uptake as
reported by Lampkin (1990), Mohamed and Matter (2001), Badran
(2002) and Yousef (2002) and the organic fertilizers consider save
for human health. Organic fertilization is also one of the methods
used to reclaim sandy desert land and to improve the chemical and
physical characteristics of the soil (Gomaa1995, Yousef 2002 and
Yousef et al. 2008).
Random amplified polymorphic DNA (RAPD) markers are easier
and quicker to use and are preferred in application where the
relationships between closely related breeding lines are of interest
(Hallden et al., 1994). ISSR-PCR is a genotyping technique based
on variation found in the regions between microsatellites. It has
been used in genetic fingerprinting (Blair et al., 1999), gene
tagging (Ammiraju et al., 2001), detection of clonal variation
(Leroy and Leon, 2000), cultivar identification (Wang et al., 2009),
phylogenetic analysis (Gupta et al., 2008), detection of genomic
instability (Anderson et al., 2001), and assessment of hybridization
(Wolfe et al., 1998) in many plant and animal species.
The aims of this study were therefore to study the effect of
organic compost on growth and volatile oil quantity of some
cultivars of bitter fennel; determine the genetic variability of some
fennel cultivars by RAPD and ISSR analysis; determine whether
secondary metabolites such as volatile compounds would be used
as taxonomic markers in these cultivars and elucidate relationships
between genetic and chemical diversity by comparing their
Materials And Methods :
This study was conducted to investigate the effect of different
levels of compost fertilizer using five fennel cultivars on growth,
fruits production, volatile oil percentage and volatile oil yield.
These cultivars were Netherland, Indian, Azoricum, German,
and Local fennel. This investigation was carried out during
the two successive seasons of 2011 / 2012 and 2012/2013 at
the experimental farm of El-Quassassin Horticultural Research
Station, Ismailia Governorate, Agricultural Research Center,
Egypt. Table 1 shows the mechanical and chemical analyses of
farm soil.Table (1) : The mechanical and chemical analysis of the
Seeds of bitter fennel obtained from Sekem Company, were sown
on 1 November of both years. Seedlings were thinned to single
plants and irrigated 15 days after sowing. The experimental unit
was 5.4 m2; every unit contained three dripper lines with 3m
length. Every experimental unit contained 30 plants (about 22222
plants per Fadden). The experiment was carried out using three
replicates in a randomized split plot design where the levels of
compost were the main plot and varieties of fennel were the subplot.
The compost fertilizer was obtained from Arab Organization
for Industrialization (A.O.I.); the chemical composition of the
compost fertilizer is shown in Table 2
The organic fertilization treatments as compost fertilizer were
applied at the rate of 4, 6 and 8 ton per Fadden. The data of plant
height (cm), herb fresh and dry weights (g/plant), number of
umbels /plant and number of flowers/umbel were recorded at full
flowering stage. The fruits yield (g/plant) and fruits yield (kg/fed)
were recorded when harvested at fruit maturity stage.
Determination of volatile oil content and composition :
Volatile oil percentage was determined in fruits according to the
method described in the General Medical Council (1963). Volatile
oil yield per plant was calculated by multiplying volatile oil
percentage by fennel fruit yield/plant and expressed as ml/plant.
Samples taken for the volatile oil obtained in the second season
were analyzed using DsChrom 6200 Gas Liquid Chromatography
equipped with a flame ionization detector for separation of volatile
oil constituents. The analysis conditions were as follows:-
The chromatograph apparatus was fitted with capillary column
BPX-5, 5% phenyle(equiv.) polysillphenylene-siloxane 30m X
0.25 mm ID X 0.25μm film. Temperature program ramp increase
with a rate of 10°C/ min from 70º to 200º C. Flow rates of gases
were nitrogen at 1 ml / min, hydrogen at 30 ml/ min and 330 ml
/ min for air. Detector and injector temperatures were 300ºC and
250ºC, respectively. The obtained chromatogram and report of GC
analysis for each sample were analyzed to calculate the percentage
of main components of volatile oil. Volatile oil yield/feddan
was calculated by multiplying oil (%) by fennel fruit yield.
RAPD -PCR Analysis :
Polymerase Chain Reaction (PCR)
In order to obtain clear reproducible amplification products,
different preliminary experiments were carried out in which a
number of factors were optimized. These factors included PCR
temperature cycle profile and concentration of each of the template
DNA, primer, MgCl2 and Taq polymerase. A total of twenty
random DNA oligonucleotide primers were independently used
according to Williams et al. (1990) in the PCR reaction. Only ten
primers succeeded to generate reproducible polymorphic DNA
products. The PCR amplification was performed in a 25 μl reaction
volume containing the following: 2.5 μl of dNTPs (2.5 mM), 1.5μl
of Mg Cl2 (25 mM), 2.5 μl of 10x buffer, 2.0 μl of primer (2.5
μM), 2.0 μl of template DNA (50 ng/μl), 0.3 μl of Taq polymerase
(5 U/μl) and 14.7 μl of sterile ddH2O. The reaction mixtures were
overlaid with a drop of light mineral oil per sample. Amplification
was carried out in Techni TC-512 PCR System. The reaction
was subjected to one cycle at 95 ºC for 5 minutes, followed by 35
cycles at 96 ºC for 30 seconds, 37 ºC for 30 seconds, and 72 ºC for
30 seconds, then a final cycle of 72 ºC for 5 minutes. PCR products
were run at 100 V for one hour on 1.5 % agarose gels to detect
polymorphism between the fennel cultivars under study. Only ten
primers succeeded to generate reproducible polymorphic DNA
products. Table 3 lists the base sequences of these DNA primers
that produced informative polymorphic bands. The PCR products
were separated on a 1.5 % agarose gels and fragments sizes were
estimated with two 100bp ladder markers (1000, 900, 800, 700,
600, 500, 400,300,200 and 100bp) and (3000,2500,2000,1500,10
ISSR-PCR Analysis :
Polymerase Chain Reaction (PCR).
ISSR-PCR reactions were conducted using four primers.
Amplification was conducted in 25 μl reaction volume containing
the following reagents: 2.5 μl of dNTPs (2.5 mM), 2.5 μl MgCl2
(2.5 mM), and 2.5 μl of 10 x buffer, 3.0 μl of Primer (10 pmol),
3.0 μl of template DNA (25 ng/ μl), 1 μl of Taq polymerase (1U/
μl) and 12.5 μl of sterile dd H2O. the PCRs were programmed for
one cycle at 94º C for 4 min. followed by 45 cycles of 1 min. at
94 ºC, 1 min. at 57 ºC, and 2 min at 72 ºC the reaction was finally
stored at 72 ºC for 10 min. The PCR products were separated
on a 1.5 % agarose gels and fragments sizes were estimated with
the 100bp ladder marker. Only four primers succeeded to generate
reproducible polymorphic DNA products. Table 3 lists the base
sequences of these DNA primers that produced informative
The experimental design was factorial experiment between compost
fertilizer and the fennel varieties in split plots with three replicates.
The compost fertilizers were arranged in the main plots, while the
fennel cultivars were assigned at random in the sub plots. The data
were statistically analyzed according to Steel and Torrie (1960)
and L.S.D. at (5% level) for comparison the means of different
treatments. The DNA bands generated by each primer were
counted and their molecular sizes were compared with those of the
DNA markers. The bands scored from DNA profiles generated by
each primer were pooled together. Then the presence or absence of
each DNA band was treated as a binary character in a data matrix
(coded 1 and 0, respectively) to calculate genetic similarity and to
construct dendrogram tree among the fennel cultivars under study.
Calculation was achieved using Dice similarity coefficients (Dice,
1945) as implemented in the computer program SPSS-10.
Results and Discussion :
1- Effect of different compost levels on growth , fruits and Volatile
oil production of fennel plant:
Data in Table (4) and Table (5) show that organic compost
significantly increased fennel growth parameter, fruit production,
volatile oil % and volatile oil yield per plant (ml) and per feddan
(L) of fennel
Increasing compost level progressively and significantly increased the values of such parameters. Wherever the highest values of
increase resulted by the highest level of compost (8 ton/Fed): plant height (105.46, 100.06 cm), herb fresh (180.73, 169.33g) and dry
weights (38.74, 31.44g) of plant , number of umbels /plant (20.66,19.40) and number of flowers/umbel(23.87,23.13) in the first and
second season respectively. On the other side the lower percentage of increase resulted by the lower level of compost (4 ton/Fed.):
(88.80, 82.40 cm) for plant height, (124.51, 113.04g) for fresh weight of herb, (24.34, 21.16g) for dry weights, (11.53, 10.00) for number
of umbels /plant and (19.13, 16.13) for number of flowers/umbel in the first and second season respectively
Such results on fennel are in the same line with many researchers on different plants, (Kandil, 2002) on fennel (Foeniculum vulgare
Mill.); (Khalil et al., 2002) on Tagetes erecta; (Khalil and El-Sherbeny, 2003) on three Mentha species, who reported that compost at
different levels significantly increased the vegetative growth characters including plant height, number of branches, fresh and dry weight
of herb during vegetative growth and flowering stage
Data in Table 5 revealed that the differences between the various
levels of compost were significant in most cases. The highest
level of compost (8ton /Fed.) increased yield of fruits per plant,
yield of fruits per feddan, volatile oil (%),volatile oil yield per plant
and volatile oil yield per Fed, by (61.00g, 1342.00 Kg, 2.39%,
1.52ml and 33.43L.) respectively in the first season and (54.97g,
1209.41Kg, 2.62%, 1.46ml and 32.06L.) successively in the
second one, where as the values of increase due to the lower level
of compost (4 ton/fed.) were [(50.00,40.78g), (1100.00,897.16Kg),
( 1.62,1.85%), ( 0.82,0.76ml) and (18.11,16.69L)]for the same
parameter in the two seasons, consecutively. It is clear that
volatile oil yield per plant (ml) and per feddan (L.) attained a parallel
trend to volatile oil (%). The three compost levels significantly
raised volatile oil yield. Raising compost levels progressively
increased fennel volatile oil yield. These results agree with those
obtained by (Ibrahim, 1999) on Ocimum sanctum; (Ibrahim and
Ezz El-Din, 1999) on catnip (Nepta cataria L.); (Khalil, 2002) on
Rosemarinus officinalis; (Khalil et al., 2002) on Tagetes erecta;
(Khalil and El-Sherbeny, 2003) on three Mentha species and (El-
Sherbeny et al., 2005) on Sidritis montana L., who mentioned that
compost addition markedly improved volatile oil %, productivity
and volatile oil yield.
2- Performances cultivars for growth , fruits and Volatile oil
production of fennel plant over all levels of fertilizers at the two
Data in Table 6 and Table 7 show significant differences in plant
height (cm/plant) among different cultivars. Netherland cultivar
was generally the shortest (75.11 and 64.78cm) followed by Local
cultivar (87.56 and 80.33cm) then Indian cultivar (91.22 and
82.78cm) after that German cultivar (108.33 and 108.66cm) and
the tallest cultivar was Azoricum (118.22 and 116.11cm) in both
Concerning the fresh and dry weight, data in Table (3) represent
that, in general, the Local cultivar recorded the lowest value
(95.88 and 94.59 g) in fresh weight and (20.71 and 17.12g) in
dry weight in both seasons. While German cultivar recorded the
highest value (235.15 and 212.97 g) in fresh weight and (45.80 and
39.91g) in dry weight in the first and second season respectively.
Taking number of umbels (umbels/plant) into consideration, data
in Table (3) reveal significant differences among all cultivar.
Generally, it was noticed that Local cultivars was superior in number
of umbels/plant (21.44 and 17.56 umbels/plant). Moreover, it was
found that Netherland and German cultivars were the lowest in
number of umbels/plant (14.00 and 10.89 umbels/plant), (13.67
and 12.00umbels/plant) in the first and second season respectively.
As for the number of flower (flowers/plant), it was found also that
Local cultivar was superior in number of flowers/plant (25.89 and
24.33 flowers/plant) and Netherland cultivar recorded the lowest
value (13.78 and 13.33 flowers /plant ) in both seasons.
The data on fruits yield/plant and fruits yield/feddan as shown
in Table 4, show significant differences in these parameters. It
was noticed that the German cultivar showed the lowest value
of both parameters (47.55 and 41.24g) for fruits yield/plant and
(1046.20 and 907.38 Kg) for fruits yield/feddan in both seasons.
Furthermore, Azoricum cultivar showed the highest value in the
same parameters (73.00 and 57.83g) for fruits yield/plant and
(1606.00 and 1272.33 Kg) for fruits yield/feddan in both seasons.
Table4, demonstrate the volatile oil of the cultivar. It is generally
noticed that, the Azoricum exhibited the highest values in volatile
oil percentage (2.82 and 3.03%) in the first and second seasons
Moreover, Netherland cultivar exhibited the lowest values (1.61
and 1.88%) in both seasons.
Concerning volatile oil yield/plant and volatile oil yield/feddan,
data in table 4, show that generally speaking, the Azoricum
cultivar was the highest values (2.14 and 1.77 ml) for volatile oil
yield/plant and (46.98 and 38.92L) for volatile oil yield/feddan in
the first and second seasons respectively. Also, Netherland cultivar
was the lowest values (0.81and 0.81ml) for volatile oil yield/plant
and (17.75and 17.82 L) for volatile oil yield/feddan in the first and
second seasons respectively. The results revealed that Azoricum
cultivar was the highest values in volatile oil percentage, fruits
yield/plant and fruits yield/feddan. It is in the same line with
Lal (2007), Lopes et al. (2010) and Safaei et al. (2011) which
reported that there is a positive and significant correlation between
volatile oil content and grain yield of fennel. It is clear that,
the growth of different cultivars of fennel could be arranged in a descending order as follows; Azoricum; German; Local; Indian
then Netherland from production point of view. Generally, it can be
concluded that Azoricum and German cultivars were the suitable
for sandy soil conditions.
The mean effect interactions at the different compost levels and
the cultivars of fennel on growth, fruits and volatile oil production
at the two years:
Tables 8 and Table 9 conclude that, plant height (cm) of the studied
cultivars. Significant differences were noticed among compost
levels and the studied varieties. Also, the Azoricum cultivar
combined with the highest level of compost fertilizer (8 ton/fed)
were superior, it recorded (125.33, 122.00cm) in first and second
season respectively. While Netherland cultivar was the shortest
(66.00, 55.67cm) when applied the compost fertilization level at
(4 ton/fed) in first and second season respectively. Generally,
increasing compost level led to increase plant height in both
seasons. There was a significant interaction between compost
levels and cultivars of fennel for fresh and dry weight, (Table 8).
German cultivar when applied the highest level of compost (8 ton/
fed) appeared the highest value (266.56, 239.10g) for fresh weight
and (50.60, 44.93g) for dry weight in first and second seasons
respectively. Fresh and dry weight was gradually reduced when
compost level was reduced. The results found that Local cultivar
combined with compost fertilizer level (4 ton/fed) exhibited the
lowest value (74.13, 66.07g) for fresh weight and (15.40, 11.90g)
for dry weight in first and second seasons respectively. As for
number of umbels/plant and number of flowers/plant, it was
noticed that Local cultivar was superior in number of umbels/
plant (27.00, 23.66) and in number of flowers/plant (29.00, 29.00)
in both seasons. At the same time, German cultivar recorded
the lowest value (9.67, 7.00) for number of umbels/plant and
Netherland cultivar recorded the lowest value (11.67, 10.33) for
number of flowers/plant in both seasons.
Data in Table 9 revealed that Azoricum cultivar produced the
highest value in yield of fruits per plant (79.67, 65.90gm), yield of
fruits per feddan (1752.60, 1449.80kg.), volatile oil (3.50, 3.71%),
volatile oil yield per plant (2.81, 2.36ml.) and volatile oil yield
per feddan (61.75, 51.99 L) in both seasons at rate (8 ton/fed) of
compost , While German cultivar recorded the lowest value in
yield of fruits per plant (44.33,34.86gm), yield of fruits per feddan
(975.33, 767.07kg) and Netherland cultivar represented the lowest
value in volatile oil (1.43 , 1.66%), volatile oil yield per plant (0.65
, 0.60ml.) and volatile oil yield per feddan (14.23 , 13.27 L) in both
seasons at rate (4 ton/fed) of compost.
It was found that increasing fertilization levels gave the highest
values of the studied parameters. These results are in agreement
with those obtained by Haridy et al. (2001) on lemongrass and
El-Ghadban et al. (2002) on Origanum majora. In this respect,
it is possible that the favorable effect of compost on growth
characteristics may be due to their ability to enhance the physical,
chemical and biological properties of the soil. A similar suggestion
was made by Hanafy et al. (2002) on rocket plants. The ratios
of volatile oil from all cultivars under this study were between
(1.43 and 3.71%); our results are in line with (Miraldi, 1999) who
mentioned that volatile oil ratio in sweet and bitter fennel samples
were found on an average to be 3.26% and 1.47%, respectively.
As known, amounts of volatile oil in fennel, like other aromatic
plants, can be influenced by a lot of factors such as climatic and
environmental conditions, season of collection and the stage of
ripening of the fruits (Arslan et al., 1989; Miraldi, 1999).
Table 10 showed the volatile oil composition (%) for five cultivars
of fennel at rate of compost (6 ton /fed) in the second season. The
results found presence of 11 compounds, five are monoterpenic
hydrocarbons comprising between (6.95%) in German cultivar
volatile oil and (17.1%) in Local cultivar volatile oil and six
oxygenated compounds compromising between (82.89%) in Local
cultivar volatile oil and (93.04%) in German cultivar volatile oil.
The major components in fennel fruits volatile oil were Trans-
anethole, it represented (52.29%, 56.67%, 47.18% and 62.24%) in
Netherland, Indian, Azoricum and German cultivars respectively,
the highest values of Anethole found in German cultivar, while the
lowest values found in Local cultivar.
Estragole (= methyl chavicol) Compound undesirable (24.4%,
27.42%, 39.89% and 26.92%) in the same cultivars respectively,
the highest values of Estragole found in Local cultivar, while
the lowest values found in Netherland and German cultivars.
Limonene represented (9.81%, 9.05%, 6.19% and 5.12%) in the
same cultivars respectively, after that Fenchone at percentages
(4.77, 4.48, 5.5 and 3.79) in the same cultivars respectively.
Fenchone has a pungent and camphorate odour; it is present
especially in bitter fennel. One of the main components of
the fennel is 3-Carene which represents (2.53%, 1.41%, 0.67%,
and 0.80%) in the same cultivars respectively. While, in Local
cultivar the major components of its volatile oil were Estragole,
Trans-anethole, Limonene, Fenchone and 3-Carene at percentages
(57.96, 18.23, 13.9, 5.99 and 2.15) respectively. Furthermore,
the minor compounds were α-Pinene, β-Myrecene, P-Cymene and
Camphor at percentages (0.89, 0.21, 0.98, and 0.33) respectively,
in Netherland cultivar. While their existed in Indian cultivar oil
at percentages (0.33, 0.9, 0.26 and 0.16) respectively, in Azoricum
cultivar oil at percentages (0.12, 0.4, 0.11 and 0.14) respectively,
in German cultivar oil at percentages (0.03, 0.08, 0.92 and 0.07)
respectively, and their percentages in Local cultivar were (0.57,
0.08, 0.4 and 0.25) respectively. Noteworthy differences were
recorded in the precentage of Anisaldehyde an autoxidation
product of Trans-anethole, ranged from 0.01% in Indian, Azoricum
and Local cultivars to the intermediate 0.02% in German cultivar,
up to the highest 3.41% in Netherland cultivar. Percentage of
α- Fenchyl acetate ranged from 0.12% in Indian cultivar,0.15% in
Azoricum cultivar, 0.37% in Netherland cultivar up to the highest
0.45% in Local cultivar. However, it contents were not available
in German cultivar volatile oil.
Limonene and Fenchone were found to be main constituents in
the studied cultivars (Table 10). Similar results were recorded by
several researchers (Arslan et al., 1989; Charles et al., 1993). It
can be seen in table 10 that Local cultivar volatile oil formed the
highest percentage of Estragole, while the other cultivars volatile
oil formed the highest percentage of T-anethole. These results are
in the line with (Shalaby et al., 2011). It was reported that the
chemical composition of bitter fennel volatile oil is very variable.
The chemo cultivars and the environmental conditions cause
this variability. The major components from these were found
to be Methyl chavicol, Trans-anethole, Limonene, Fenchone,
γ-terpinene, and piperitonene oxide (Marotti. et al., 1994)
Foeniculum vulgare var. presents great composition differences
with varying populations with the aim of clarifying the status of
var. vulgare, the proposed to subdivide it into three chemotypes
according to their relative compositions (McDonald, 1999). They
called them chemotype Estragole, chemotype Estragole/Anethole
and chemotype Anethole. According to that our results divided
into two chemotypes.
1- chemotype. Estragole (Estragole is the major compound) such
as Local cultivar oil.
2- chemotype. Anethole (T-anethole is the major compound) such
as Netherland, Indian, Azoricum and German cultivars volatile oil.
Molecular genetic identification
Table (11) and Figures (1 and 2) show the results of total amplified
fragments (TAF), amplified fragments (AF) and specific markers
(SM) for each cultivars of Fennel using RAPD-PCR analysis with
ten random primers. A total number of 98 DNA fragments were
detected, in which 60 (61.22%) were polymorphic fragments.
However, 38 bands were common (monomorphic) for all cultivars.
Polymorphism levels differed from one primer to another, i.e.
The results found that (OP-C02, OP-M15, OP-B04, OP-C04
andOP-A10) primers exhibited high levels of polymorphism
(90.91%, 78.57%, 75.00%, 72.73% and 70.00%) respectively.
While, (OP-A02, OP-A13, OP-K10 and OP-C05) primers
exhibited moderate level of polymorphism (62.50%, 55.56%,
62.50% and 54.55%), and primer OP-G14 represented the lowest
level 37.50% as exhibited in Table (11). These results agree with the previously reported for other medicinal and aromatic species
like Lavndaula angustifolia ( Echeverrigaray and Agostini, 2000)
and Ocimom gratissimum (Vieria et al., 2001). The lowest
number of polymorphic fragments was detected for primer OP-
M15 (3 out of 14 amplified bands); while the highest number of
polymorphic fragments was detected for primer OP-C02 (10 out
of 11 amplified bands). Cultivar-specific markers generated from
RAPD-PCR analysis are shown in Table (11).
Thirty-one out of 98
RAPD-PCR fragments were found to be useful as cultivar-specific
markers. The largest number of RAPD-PCR markers was scored
for Indian cultivar (68 markers), while the lowest (49 markers)
was scored for Netherland cultivar. In the meantime, the highest
number of RAPD-PCR cultivar-specific markers was generated by
primer OP-C04 (7 markers), while the lowest number (1 markers)
was generated by primers OP-A13 and OP-B04. Moreover, OP-
G14 was generated no RAPD-PCR cultivar-specific markers.
In conclusion, all of the ten primers used allowed enough
distinction among the cultivars under study. These cultivar-specific
markers can be used in subsequent experiments to detect molecular
markers for polymorphic genes with economic importance among
these and other cultivars. Similar finding were obtained in mints
by Hassan (2005) and Momeni et al., (2006) and in other genera
(Choi et al., 1999 and Benedetti et al., 2000).
Genetic similarity and cluster analysis based on RAPD markers:
Genetic similarities among the five fennel cultivars were estimated
according to the RAPD data by using UPGMA computer analysis
(Table 12 and Fig. 1 and Fig. 2). Table 12 showed that the most two
closely related cultivars were Indian and Local with the highest
similarity index (1.000). On the other hand, the results indicated
that the two most distantly related cultivars were Netherland and
Local with low similarity index (0.411). The results showed that
there was no similarity between Azoricum cultivar and Local
cultivar. A dendrogram for the genetic relationship among the
five genotypes of fennel cultivars genotypes is exhibited in Fig.
4, which separated them into two major groups. The first group
included Indian cultivar only, while the second group included
two subgroups, the first subgroup involved German cultivar only
and the other subgroup included Local, Azoricum and Netherland
Inter Simple Sequence Repeats (ISSR) markers:
The four ISSR primers succeeded in amplifying DNA fragments
for the five fennel cultivars genotypes (Fig.3). Polymorphism
levels differed from one primer to another, i.e. HB-11and 44A
primers exhibited high levels of polymorphism (93.33% and
77.78%) respectively, while, HB-09 primer exhibited low level
of polymorphism (62.5%) as exhibited in Table 11. The number
of total amplified fragments (TAF), polymorphic fragments (PF),
monomorphic fragments (MF) and specific markers (SM) for each
primer of the four primers are shown in Table 11. 14A Primer
showed two DNA fragments with molecular size ranging from
279 to 332bp (Fig.3 and Table 11), those two fragments were
monomorphic, and there was not any polymorphic fragments or
44B primer showed nine DNA fragments with molecular sizes
ranging from 238 to 515bp, seven fragments were polymorphic
(77.78 %), and two of them were positive species- specific markers
at 429bp for Azoricum genotype and 238bp for Local genotype.
HB-09 primer showed eight DNA fragments with molecular size
ranging from 207 to 518bp, five fragments were polymorphic
(62.50 %), and two of them were positive species- specific markers
at (399bp) for Indian genotype and at (207bp) for Azoricum
genotype. HB-11 primer showed fifteen DNA fragments with
molecular size ranging from 102 to 631bp, fourteen fragments of
them were polymorphic (93.33 %), and seven of them were positive
species- specific markers at (631 and 357bp) for Netherland
genotype, (107bp) for Azoricum genotype,( 596, 294 and ,114bp)
for German genotype and at 102bp for Local genotype.
Genetic similarity and cluster analysis based on ISSR markers:
According to ISSR results, the most two closely related cultivars
were Indian and German (Table 13) with the highest similarity
index (1.000). On the other hand, the most two distantly related
cultivars were German and Local with low similarity index (0.550)
and the two cultivars located very far were Azoricum and Local
variety with similarity index (0.000).
Figure 5: indicated that the dendrogram revealed one main group
of three cultivars including two subgroups. Subgroup 1 included
both Azoricum and Local and subgroup 2 included German
cultivar only. The remaining cultivars (Indian and Netherland)
represented distant sequences.
Combined identification based on RAPD and ISSR
Varieties distribution on the consensus tree according to the
banding patterns of RAPD differed from that based on ISSR
banding patterns, which may be due to that each technique,
amplified different parts of the genome. So, it is better to use
the combination of the banding patterns of the two techniques to
use more segments of the genome that will increase the validity
of the consensus tree. Results of the combined data as shown
in Fig. 6 and Table14 exhibited that the most closely related
cultivars were Indian and both of Local and Azoricum with the
highest similarity index (1.000). On the other hand, the two
most distantly related cultivars were Netherland and Local with
low similarity index (0.604) and also the two cultivars located
very far were Azoricum and Local variety with similarity index
The results of the consensus tree indicated that the tree divided
the cultivars into two main clusters, the first included cultivars
Indian and German. The second one divided into two subgroups,
the first one included cultivars Local and Azoricum and the
other included Netherland. This study provides evidence that
RAPD and ISSR polymorphisms could be used as efficient tools
for the detection of similarities and phylogenetic relationships
of the studied genotypes. The same conclusion was obtained
by several authors (Alexander, 2002: Abdel-Tawab, et al.,
2001 and Heikal, et al 2007). RAPD technique also is an effective technique in studying inter and intra specific variation
in fennel. These results are in accordance with (Fu et al.,
2003) who reported that out of 92 RAPD primers, 64 gave
polymorphism which indicated that 51.2% of total diversity was
among populations and 48.8 % within populations of Changium
smyrnioides Wolff (Apiaceae)
The conclusion :
Increasing compost fertilizer level progressively and
significantly increased the values of such studied vegetative and
yield trials. Wherever the highest values of increase resulted by
the highest level of compost (8 ton/ Fed.) in sandy soils gave the
highest values of growth, fruits and volatile oil yield in fennel
- Azoricum cultivar gave the highest values of vegetative growth,
fruit yield and volatile oil content, while German cultivar gave
the highest values of Anethole were main constituents in volatile
oil, Estragole (= methyl chavicol) Compound undesirable found
in Local variety, while the lowest percentage found in Netherland
and German cultivars.
Can expand the cultivation of cultivars fennel of German and
Azoricum and breeding operations with a Local cultivar to
produce a distinctive cultivar between the previous cultivars.
- According to the dendrogram of RAPD, ISSR and the
combination between them the results revealed that Netherland
existed alone in group or sub group of each dendrogram. This
would explain the growth results of this study where Netherland
showed the lowest value in most growth parameter (plant height,
No of umbels/plant, No of flowers/umbel, volatile oil (%),
volatile oil yield per plant, volatile oil yield per). Also, the results
indicated that Netherland recorded the highest percentage of
Anisaldehyde oil (3.41%). Such results are found for the German
cultivar. From the dendrogram, it would be observed that German
cultivar existed alone in a group or in a subgroup. This would
explain the results indicated that German cultivar recorded the
highest value of fresh and dry weight. Also α- Fenchyl acetate
oil existed in all studied cultivars except Geman cultivar. It
can be observed from similarity tables that Local cultivar and
Azoricum cultivar represented distant sequences. It may explain
that Local cultivar scored the highest value of estragole oil
(57.96%) while, Azoricum recorded (39.89%) and Azoricum
cultivar scored higher trans-anethole percentage than Local
cultivar (47.18% and 18.23%) respectively. so that, they could
be induced into a breeding program in the future for commercial
- Abdel-Tawab, F.M., A. Abo-Doma, A.I. Allam and H.A. El-
Rashedy,(2001). Assessment of genetic diversity for eight sweet
sorghum cultivars (Sorghum bicolar L.) using RAPD analysis.
Egypt. J. Genet. Cytol., 30: 41-50.
- Ahmed S. Shalaby, Saber F. Hendawy &Mona Y.Khalil,
(2011). Evaluation of Some Types of Fennel (Foeniculum vulgare
Mill.) Newly Introduced and Adapted in Egypt. J. of Essential Oil
Research, 32: 35-42
- Alexander, A.J., (2002). Genetic diversity of populations of
Astragalus oniciformis using Intersimple sequence repeat (ISSR)
markers. M.Sc. Thesis in Botany and Plant Pathology, Oregon
State Univ., USA.
- Ammiraju J.S.S., Dholakia B.B., Santra D.K. (2001).
Identification of inter simple sequence repeat (ISSR) markers
associated with seed size in wheat. Theor Appl Genet 102:726–
- Anderson G.R., Brenner B.M., Swede H. (2001).
Intrachromosomal genomic instability
- in human sporadic colorectal cancer measured by genome-
wide allelotyping and inter-
- (simple sequence repeat) PCR. Cancer Res 61:8274–8283.
- Arslan, N., Bayrak, A., & Akgu ̈ l, A. (1989). The yield and
components of essential oil in fennels
- of different origin (Foeniculum vulgare Mill.) grown in Ankara
conditions. Herba Hungarica, 3, 27–30.
- Badran, M. S. S. (2002): Organic US. mineral fertilization
on yield and components of some carley varieties under sandy
soil conditions. Proc. Minia 1st Conf. for Agric. & Environ. Sci.,
Minia, Egypt, March 25 – 28 : 917 – 934.
- Barazani, O, Cohen, Y., Fait,A., Diminshtein, S., Dudai, N.,
Ravid, U., Putievsky, E., and
- Friedman, J.(2002). Chemotypic differentiation in indigenous
populations of Foeniculum vulgare var. vulgare in Israel.
Biochemical Systematics and Ecology, 30, 721-731.
- Benedetti, L., G. Burchi, A. Mercuri and T. Fchida, (2000).
Use of RAPD analysis for genotype
- identification in alstroemeria. Acta Horticulture, 508: 277-
- Bennici, A., A. Maria and G.V. Giovanni. (2003). Genetic
stability and uniformity of Foeniculum vulgare Mill., regenerated
plants through organogenesis and somatic embryogenesis. Plant
Sci., 161(1): 221-227.
- Blair M.W., Panaud O, McCouch S.R. (1999). Inter-
simple sequence repeat (ISSR) amplification for analysis of
microsatellite motif frequency and fingerprinting in rice (Oryza
sativa L.). Theor Appl Genet 98:780–792.
- Charles, D.J., Morales, M.R., & Simon, J.E. (1993). New
crops. In J. Janick, & J.E. Simon (Eds.), Essential oil content and
chemical composition of finocchio fennel (pp. 570–573). New
- Choi, H.S., K.S. Kim, J.K. Choi, K.K. Lee, D.K. Hong, W.H.
Kang and Y.S. Lee, 1999.
- Classification of Lilium using random amplified polymorphic
DNA (RAPD). Korean J. of Hort. Sci. & Technol., 17: 144-147.
- Davis, P. H. (1972). Flora of Turkey and the East Aegean
Islands, vol.4 University Press,
- Dice, L. R. (1945). Measures of the amount of ecologic
association between species.
- Ecology, 26: 297-302.
- Echeverrigaray, S. and G. Agostini.( 2000). Avaliacao da
variabilidade genetica em lavandas atraves de marcadores de
RAPD. Horticult. Bras., 185-186.
- El-Sherbeny, S.E., M.Y. Khalil and N.Y. Naguib, (2005).
Influence of compost levels and suitable spacing on the
productivity of Sideritis montana L. plants recently cultivated
under Egyptian conditions. Bull. Fac. Agric., Cairo Univ., 56(2):
- El-Ghadban, E. A. E., A. M. Ghallab, A. F. Abdelwahab,
(2002). Effect of organic fertilizer (Biogreen) and biofertilization
on growth, yield and chemical composition of Marjoram plants
growth under newly reclaimed soil conditions, 2nd Congress of
Recent Technologies in Agriculture, 2, 334-361.
- Farag RS, Daw ZY, Hewedi FM, El-Baroty GSA (1989):
Antimicrobial activity of some Egyptian spice essential oils. J
Food Protect 1989;52:665–667.
- Fu, C., Q.I.U Yingxiong and H. Kong. (2003). RAPD analysis
for genetic diversity in Changium smyrnioides (Apiaceae), an
endangered plant. Bot. Bull. Acad. Sci., 44: 13-18.
- General Medical council British (1963). Pharmacopoeia 1963;
1st Edn., The pharmaceutical press,London, Uk.,Pages;1210.
- Gomaa, A.M. (1995): Response of certain vegetable crops to
biofertilization. Ph.D. Thesis, Fac. Agric., Cairo Univ.
- Gupta S.K., Souframanien J., Gopalakrishna T. (2008).
Construction of a genetic linkage map of black gram, Vigna
mungo (L.) Hepper, based on molecular markers and comparative
- studies. Genome 51:628–637.
- Hallden C., Nilsson N. O., Ebding L. M. and Sdl T., (1994).
Evalution of RFLP and RAPD markers in a comparision of
Brassica napus breeding lines, Theor. Appl. Genet., 123-128.
- Hanafy Ahmed, A. H., M. K. Kahlil, A. M. Farrag, (2002).
Nitrate accumulation, growth, yield and chemical composition of
Rocket (Eruca vesicaria Sub sp. sativa) plant as affected by NPK
fertilization, kinetin and salicylic acid, Annal. Agric. Sci. Ain
Shams Univ., Egypt, 47, 1-26.
- Harridy, I. M. A., S. G. I. Soliman, T. A. Mervat,( 2001).
Physiological, chemical and biological studies on Lemongrass
Cymbopogon citratus (DC) stapf in response to diazotrophic
bacteria, Agric. J. Sci. Mansoura Univ., 26, 6131-6152.
- Hassan, A.H.M., 2005. Identification of molecular markers
for some morphological and
- biochemical characters in some medicinal plants. M.Sc.
Thesis. Ain Shams Univ., Fac. Agric.
38. Heikal, A. Hadia, Y. Mabrouk, O.M. Badawy, A. El-Shehawy
and Effa t A. Badr, (2007).
- Fingerprinting Egyptian Gramineae Species Using Random
Amplified Polymorphic DNA
- (RAPD) and Inter-simple Sequence Repeat (ISSR) Markers.
J. Cell and Molecular Biology (RJCMB). 1(1): 15-22.
- Ibrahim, M.E., (1999). Physiological and chemical studies on
Tusli plant (Ocimum sanctum). Egypt. J. Hort., 26(2): 147-165.
- Ibrahim, M.E. and A.A. Ezz El-Din, (1999). Cultivation
of Nepta cataria L. in Egypt: its growth yield and essential oil
content as influenced by some agronomic practices. Egypt. J.
Hort., 26(3): 281-302.
- Kandil, M.A.M., (2002). The effect of fertilizers for
conventioal and organic farming on yield and oil
- quality of fennel (Foeniculum vulgare Mill.) in Egypt.
Ph.D.Thesis. Fakultat der Techischen Univ.,
- Carlowilhelmina Univ.
- Khalil, M.Y., (2002). Influence of compost and foliar
fertilization on growth, chemical composition of Rosemarinus
officinalis L. Egypt. J. Appl. Sci., 17(10): 684-699.
- Khalil, M.Y., N.Y. Naguib, and S.E. El-Sherbeny, (2002).
Response of Tagtes erecta L. to compost and foliar application of
some microelements. Arab Univ. J. Agric. Sci., Ain shams Univ.,
Cairo, 10(3): 939-964.
- Khalil, M.Y. and S.E. El-Sherbeny, (2003). Improving the
productivity of three Mentha species recently cultivated under
Egyptian conditions. Egypt. J. Appl. Sci., 18(1): 285-300.
- Lal RK., ( 2007). Association among agronomic traits and
path analysis in fennel. Journal of Sustainable Agriculture 30,
- Lampkin, N. (1990): Organic Farming. Farming Press Book.
United Kingdom. P. 63.
- Leroy X.J., Leon K. (2000) A rapid method for detection of
plant genomic instability using
- unanchored-microsatellite primers. Plant Mol Biol Rep 18 (20
- Lopes VR, Barata AM, Farias R, Mendes MD, Lima AS,
Pedro LG, Barroso JG, Figueiredo AC.,( 2010). Morphological
and essential oil variability from nine Portuguese fennel
(Foeniculum vulgare Mille.) accessions. Acta Horticulturae, 860,
- Marotti M, Piccaglia R, Giovanelli E, Deans SG, Eaglesham
E; (19945): Effects of variety and ontogenic stage on the essential
oil composition and biological activity of fennel (Foeniculum vulgare Mill.). J Essent Oil Res 1994;6:57–62.
- Mathur, G., Owen, G., Dinel, H. & Schnitzer, M. (1993).
Determination of compost biomaturity.
- Biological Agriculture & Horticulture, 10, 65-85.
- McDonald, T. A., (1999). Evidence on the carcinogencity of
estragole, Ph.D. staff
- Toxicologist Reproductive and cancer Hazard Assessment
Section office of Environmental Health Hazard Assessment,
California Environmental Protection Agency.
- Miraldi, E. (1999). Comparison of the essential oils from ten
Foeniculum vulgare Miller samples of fruits of different origin.
Flavour and Fragrance Journal, 14, 379–382.
- Mohamed, S. A. and F. M. A. Matter (2001): Effect of
ammonium nitrate and organic fertilizers on growth, volatile oil
yield and chemical constituents of marigold (Tagetesminuta L.)
plant. Fayoum J. Agric. Res., & Dec. 15 (1): 95 – 107.
- Momeni, S., B. Shiran and K. Razmjoo, 2006. Genetic
variation in Iranian mints on the bases of RAPD analysis.
Pakistan J. of Biological Sciences, 1898-1904. URL http://www.
- Naguib, N.Y. and A.A. Aziz, (2004). Yield and Quality of
Hyoscyamus muticus L. in relation to some fertilizer treatments.
Egypt. J. Hort., 29(4): 115-126.
- Safaei L, Zeinali H, Afiuni D., (2011). Study of genetic
variation of agronomic characteristics in Foeniculum vulgare
Mill. Genotypes Iranian Journal of Rangelands and Forests Plant
Breeding and Genetic Research 19, 167-180. [with English
- Shiran, B., N. Amirbakhtiar, S. Kiani, Sh. Mohammadi, B.E.
Sayed-Tabatabaei and H. Moradi.
- (2007). Molecular characterization and genetic relationship
among almond cultivars assessed by RAPD and SSR markers.
Scientia Horticulturae, 111: 280-290.
- Steel, R. R. D. and J. H. Torrie (1960). Principles and
Procedures of Statistics. MC. Graw-Hill International Book
Company, 3rd, Ed. London, pp. 633.
- Vieria, R.F., R.J. Grayer, A. Paton and J.E. Simon. 2001.
Genetic diversity of Ocimum gratissimum L. based on volatile
oil constituents, flavonoids and RAPD markers. Cab. Int., 7(4):
- Wang H.Z., Wu Z.X., Lu J.J. (2009). Molecular diversity and
- Cymbidium goeringii cultivars based on inter-simple
sequence repeat (ISSR) markers.
- Genetica 136(3):391–399.
- Williams, J.G.K., A.R.K. Kubelik., T. Livak., J.A. Rafalski,
S.V. Tingey. (1990). Nucleic Acids Research. 18: 6531-6539.
- Wolfe A.D., Xiang Q-Y, Kephart S.R. (1998). Assessing
hybridization in natural populations of Penstemon
(Scrophulariaceae) using hypervariable inter simple sequence
- Mol Ecol 71:1107–1125.
- Yousef, R. M. M. (2002): Effect of irrigation and fertilization
on Matricariachamomilla L. growth and productivity in sandy
soil. Ph. D. Thesis, Fac. Agric., Zagazig Univ.
- Yousef, R. M. M. , A.M. A. Hamouda and G. G Nawal.
(2008): Effect of irrigation and organic fertilization on growth
and productivity of Majoranahortensis in sandy soil. Mansoura
University Journal of Agricultural Sciences 33(11):8039-8056.