Brassica napus is also known as Canola. In strict sense canola
is that whose oil must contain less than 2% erucic acid and the solid
component of the seed must contain less than 30 μM of any one or
a mixture of 3-butenyl glucosinolate, 4-pentenyl glucosinolate, 2-hydroxy-3-butenyl
glucosinolate and 2-hydroxy-4-pentenyl glucosinolate per gram of air dry,
oil free solid (Cardoza and Stewart, 2002). Rapeseed/canola (Brassica
napus L. and B. campestris L.) recently moved up to the world`s
third most important edible oil source after soybean and palm (Downey,
The given triangle indicates the genetic relationships among the economically
most important Brassica species, B. napus (2n = 38, AACC), B.
juncea (2n = 36, AABB) and B. carinata (2n = 34, BBCC) species
resulting from combining chromosome sets of the low chromosome number
species B. nigra (2n = 16, BB), B. oleracea (2n = 18, CC)
and B. rapa (2n = 20, AA) (Morinaga, 1934; U.N., 1935).
B. napus contain 35% crude protein basis, having amino acids like
Alanine, Histidine, Arginine, Isoleucine, Aspartate, Leucine, Cystine,
Lysine, Glutamate, Methionine, Glycine, Methionine+cystine, Phenylalanine,
Tryptophan, Proline, Tyrosine, Serine, Valine and Threonine (Anderson
et al., 1993; Bell et al., 2000).
Brassica napus contains Calcium (0.63%), Phosphorus (1.08%), Sodium
(0.10%), Chlorine (0.10%), Potassium (1.22%), Sulphur (0.85%), Magnesium
(0.54%), Copper (5.8 mg kg-1), Iron (166 mg kg-1),
Manganese (52 mg kg-1), Molybdenum (1.4 mg kg-1),
Zinc (58 mg kg-1) and Selenium (1.1 mg kg-1) (Bell
and Keith, 1991; Bell et al., 1999).
Acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate
has become one of the most widely used techniques to separate and characterize
proteins (Laemmli, 1970). This technique offers two distinct advantages.
Polypeptides migrate according to molecular weight on SDS gels so that
molecular weight of polypeptides may be easily and rapidly estimated.
At the same time many insoluble proteins are solubilized by SDS so that
SDS gel electrophoresis has become the technique of choice for resolving
mixtures of insoluble proteins, especially membrane proteins. Electrophoresis
is the migration of charged molecules in solution in response to an electric
field. Their rate of migration depends on the strength of the field; on
the net charge, size and shape of the molecules and also on the ionic
strength, viscosity and temperature of the medium in which the molecules
are moving. The gel electrophoresis used support matrices such as paper,
cellulose acetate, starch gel, agarose or polyacrylamide gel separating
the molecules by size. Sodium Dodecyl Sulphate (SDS) is an anionic detergent
which denatures proteins by wrapping around the polypeptide backbone and
SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In
so doing, SDS confers a negative charge to the polypeptide in proportion
to its length i.e., the denatured polypeptides become rods of negative
charge cloud with equal charge or charge densities per unit length. It
is usually necessary to reduce disulphide bridges in proteins before they
adopt the random-coil configuration necessary for separation by size.
In denaturing SDS-PAGE separations therefore, migration is determined
not by intrinsic electrical charge of the polypeptide, but by molecular
weight. Aim of this research was to characterize B. napus seeds
and to compare seeds of different varieties for their compositions.
MATERIALS AND METHODS
The seeds of ten different varieties of Brassica napus namely
Cyclon, Star, Vangard, MLCP-048, Bullet, Altex, Con-III, Deffender, Hyola
and Dunkled were obtained from Agriculture Research Station North Swat
NWFP and National Agriculture Research Center (NARC) Islamabad and were
processed for analysis of different parameters like, pH, electrical conductivity,
thousand seed mass, sodium, potassium and vitamin C contents, proteins
were analyzed by SDS-PAGE. This study was conducted in Feb. 2007 in Department
of Biotechnology University of Malakand.
Na+ and K+ was determined by flame photometer using
acid digestion method (Jones et al., 1991) the instrument was standardized
by standard sodium and potassium solutions respectively (Sabir et al.,
Electrical conductivity of seeds was determined by imbibitions method.
Two gram of seeds were taken in beaker and with the help of calibrated
conductive meter, electrical conductivity was determined after 20, 40,
60, 80, 100 and 120 min. Thousand seeds were counted and their average
mass was measured by electronic balance.
Ascorbic acid also called vitamin C was determined by redox titration
method with iodine solution using starch as indicator. Two gram of seeds
was dissolved in 100 mL of distilled water, from this 10 mL was taken
and titrated against standard iodine solution. From this weight vitamin
C was determined by formula.
For pH values two grams of seeds were taken in powder form, dissolved
in 100 mL of distilled water and placed in shaker for 24 h, then filtered
and pH was determined by pH meter.
Brassica seed proteins were analyzed by SDS-PAGE (Sodium dodecyl
sulfate Polyacrylamide Gel Electrophoresis) using method describe by (Laemmli,
1970). For the preparation of 100 mL Protein extraction buffer, Tris (0.6057
g), Sodium Dodecyl Sulfate (SDS) 0.2 g and Urea (30 g) were dissolved
in distilled water. The pH was adjusted to 8 with concentrated HCl, 1
mL marcaptoethanol was added and then diluted to 100 mL with distilled
water. A little Bromophenol Blue (BPB) was added and stored in the refrigerator
at 5°C. A 500 mL electrode buffer was prepared by dissolving 15.15
g Tris, 0.5 g SDS and 7.2 g glycine in distilled water then diluted to
500 mL and stored at room temperature. Staining solution was prepared
by mixing methanol 440 mL, acetic acid (glacial) 60 mL and Coomassie Brilliant
Blue (CBB; R-250) 2.25 g in distilled water and diluted to 1 L. The solution
was stirred for 30 min, filtered and stored at room temperature. Destaining
solution was prepared by mixing methanol, acetic acid and distilled water
at a ratio of 20: 5: 75 mL, respectively. For extraction of proteins,
grains were ground to fine powder with pestle and mortar. A 400 μL
Protein Extraction Buffer (PEB) was added to 0. 01 g of seed flour of
Brassica napus and vertex thoroughly to homogenize, kept overnight
at 40°C. In order to purify the homogenate, samples were centrifuged
at 13000 rpm for 10 min at room temperature. The supernatant contained
extracted, that was transferred to new 1.5 mL eppendrof tubes and stored
at 4°C until they were run on the polyacrylamide gel. After electrophoresis
the gel was transferred to tray containing staining solution shake gently
for 40 min, followed by destaining until the background of gel disappeared.
The picture was taken by gel documentation system with white light illuminator.
Data analysis: The molecular weight of protein subunits was measured
by comparing sample bands to the standard protein molecular weight marker
bands in the electrophorogram.
Sodium and Potassium concentration (%) was found different for each variety,
Table 1 shows maximum Na+ concentration in
seeds of Cyclon and minimum in Vangard, while potassium was minimum in
Bullet variety and maximum (1.42%) in seeds of two varieties Star and
Con-III as compared to other varieties.
Analysis of Ascorbic acid (Vitamin C) showed maximum value in seeds of
Bullet variety while minimum value of ascorbic acid was found in seeds
of MLCP-048 variety (Table 1).
pH is an important parameter to detect the acidic or basic nature of
sample. The pH of juice from 2 g of seeds for variety was determined with
pH meter, the pH value for Altex was found minimum (6.63) while maximum
pH (6.93) was shown by MLCP-048 and HYOLA as compared to other varieties.
Table 1 shows maximum thousand seeds mass for Cyclon
(5.52 g) while minimum for BULLET variety as compared to other varieties.
The property of a sample to pass electric current due to the oozing
of ions from sample material in the solution is known as Electrical Conductivity
(EC). Electrical conductivity of Brassica napus seeds was determined
by conductometer after different time intervals i.e., 20, 40, 60, 80,
100 and 120 min. The data regarding electrical conductivity is show in
Table 2. Maximum EC value was shown by seeds of Vangard variety after
each interval of time.
||The presence of Na+, K+, Ascorbic Acid concentration
in seeds of B. napus varieties and also shows pH values and
thousand seeds mass
||Electrical conductivity (μ sec) mean values for seeds of different
varieties of Brassica napus
||Molecular weight, banding range, Number of protein bands and total
number of bands for different varieties of Brassica napus
Molecular characterization of proteins from seeds of ten different varieties
of Brassica napus
was determined using SDS-PAGE
. Molecular weight
and their corresponding band numbers of each variety of Brassica napus
have been shown in Table 3
and Fig. 1
. Banding pattern shows variation in
molecular weights for proteins among different varieties. Protein ladder
(molecular marker) is a mixture of highly purified proteins designed for
precise sizing of proteins by SDS-PAGE
. The proteins resolved into clearly
identifiable sharp bands from 10 to 200 kDa when analyzed by SDS-PAGE
The digits 01, 02, 03, 04, 05, 06, 07, 08, 09 and 10 represent the Brassica
napus varieties Cyclone, Star, Vangard, MLCP- 048, Bullet, Altex,
Con-III, Deffender, Hyola and Dunkled, respectively.
This study was carried out to evaluate the physicochemical, elemental
and molecular characterization of seeds of ten different varieties of
Brassica napus. The seeds were analyzed for various physiochemical
parameters such as, pH, Thousand Seeds Mass (TSM), electrical conductivity,
mineral composition (Sodium/Potassium) and vitamin C. Molecular characterization
of seeds was also carried out by studying the protein-banding pattern
The amount of sodium ranges from 0.020-0.039% in the present study while
using ten varieties of Brassica napus. The lowest value was 0.020%
for Vangard and highest value was 0.039% for Cyclone variety. Similar
results have also been found by Najib and Al-Khateeb (2004) by using Brassica
napus (canola) in their experiment and found that the amount was 0.02%.
The results of the present study also correlate with Leeson et al.
(1991) who worked on Brassica napus (canola) and found that the sodium
amount was 0.01%. However the present results have deviation from Bell
and Keith (1991) and Bell et al. (1999) who found sodium level
of 0.1%. This deviation may be due to difference in varieties used.
The minimum value 1.14% of Potassium was found in Bullet while the maximum
value 1.42% for Con-III. Same results were documented by Bell et al.
(1999) who found that potassium level in canola was 1.22% that correlates
with the present study. Leeson et al. (1991) also found that the
amount of potassium in canola was 0.81%, which shows narrows resemblance
with the study conducted. This slight variation may be due to differences
in species or varieties used. Najib and Al-Khateeb (2004) observed that
the amount of potassium found in canola was 0.71%, which does not satisfy
the present results variation may be due to difference in varieties used.
Vitamin C amount in seeds was found in the present study ranges from
30-96 mg/100 g. The maximum amount was found in Bullet variety while the
lowest amount was found in MLCP-048. Similar results have also been found
by Goldoni et al. (1983). They used Brassica olaracea as
an experimental plant and observed that the amount ranged from 12-112.5
mg/100 g that satisfy the present study.
MLCP-048 pH value was the highest as it was 6.933 while the lowest pH
value was found for Altex which was 6.632. The pH value of 6-6.8 was observed
by the Husted and Schjoerring (1995) in Brassica napus, which shows
complete correlation with the present study.
Thousand Seeds Mass (TSM) values showed variation due to different varieties
of Brassica napus used. In present study the high value of TSM
was 5.52 g for Cyclone variety and the low value range for Bullet variety
was 3.63 g. TSM values reported by Laoniste et al. (2004) in the
oil seed rape variety, ranging from 3.5-4.5 g, satisfying the present
The overall electrical conductivity of Brassica napus seeds in present
work ranged from 0.9-10.4 μ sec. Electrical conductivity reported
by Stephen et al. (2001) was 1.2 μ sec for nutrients seeds
of Brassica napus, which shows resemblance to the present study.
In contrast to nutrient seeds when saline water was used, the value recorded
was 11.2-24.9 μ sec. The variation was due to increased ion concentration.
The conductivity of Brassica napus seeds studied by Boem et
al. (1997) ranges from 5-8 μsec, which justifies the present
SDS-PAGE (Sodium Deodecyl Sulfate Polyacrylamide Gel Electrophoresis)
analysis of total seed protein of ten different varieties is shown in
Table 3. Maximum number of bands were found between the range of 10-50
kDa in majority of the varieties. Minimum number of bands were found in
the range of 120-200 kDa. In all varieties the number of bands were different.
Maximum number of bands were (15) for Bullet while minimum number of bands
(11) were found for Altex and Con-III. In the present study variation
was found among protein banding pattern of sees from different varieties
through SDS-PAGE. Similar results were documented by Benmoussa et al.
The lowest value of sodium was 0.020% for Vangard and the highest 0.039%
for Cyclone. While the potassium having lowest value for Bullet 1.14%
and the highest of 1.42% for Star. The highest amount of vitamin C (96
mg/100 g) was found in Bullet variety while the lowest concentration (30
mg/100 g) was found in MLCP-048.The pH values for all the varieties were
observed to be in the range of 6.632-6.933. The thousand seed mass determined
were 3.63-5.52 g. The overall electrical conductivity of Brassica napus
seeds were ranged from 0.9-10.4 μ sec. Brassica napus seed
proteins were analyzed by SDS-PAGE. The different numbers of bands of
the gel picture showed that variation exists among the different varieties.