Abstract: Germination, coefficient of velocity of germination (CVG), lipid and sugar compositions were determined in whole ungerminated seed of three cowpea cultivars. The cultivars were chosen for biochemical characteristics based on seed germination and coefficient of velocity of germination tests. Texas cream 40 was able to germinate at very high and low temperatures. Black Crowder demonstrated acceptable germination at high temperatures but negatively affected at low temperature. Mississippi Purple obtained low germination percent and CVG at all temperatures studied. The main sugars present in cowpea seed are sucrose, raffinose and stachyose. Sugar contents were affected by cultivar. Sugar compositions were higher in the cultivars with high percent germination and reduced in the cultivar with lower percent germination suggesting the use of sugar for seed germination process. The most abundant fatty acids in cowpea seed were palmitic acid [CH3(CH2)14COOH; (16:0)], palmitoleic acid [CH3(CH2)5CH = CH(CH2)7COOH; (16:1)], stearic acid [CH3(CH2)16COOH; (18:0)], oleic acid [CH3(CH2)7CH = CH(CH2)7COOH; (18:1)], linoleic acid [CH3(CH2)4CH = CHCH2CH = CHCH2(CH2)6 COOH; (18:2)], linolenic acid [CH3CH2CH = CHCH2CH = CHCH2CH = CH(CH2)7COOH; (18:3)] and arachidic acid [CH3(CH2)18COOH; (20:0)]. The result shows that the long-chain fatty acid appears to be important in the cowpea seed germination process. Thus, the information provided by this research will facilitate future plant physiological and genetic studies of cowpea cultivars.
INTRODUCTION
Cowpea (Vigna unguiculata L. Walp), an indigenous African annual legume, is commonly known as southern pea, blackeye pea, crowder pea, lubia, niebe, coupe or frijole, (Craufurd et al., 1996; Hall et al., 1997). It is a high protein (25%), fiber (6.3%) and low fat (1.9%) vegetable crop having good nutritional qualities (Ricardo, 1985). It is a chilling sensitive crop (Ehlers and Hall, 1977; Hall et al., 1997) and is adapted to warm weather and humid conditions (Craufurd et al., 1996; Hall et al., 1997). Cowpea is cultivated for food (Hall et al., 1997), silage, green manure for soil improvement, livestock feed and pasture. Due to its nutritional content, versatility, adaptability and high yield, cowpea is chosen by the United States National Aeronautical and Space Administration as one of the few crops to be tested for cultivation in the space station (Ehlers and Hall, 1997). Furthermore, it is one of the mandated crops by the International Institute of Tropical Agriculture (Smartt and Hymowitz, 1985; Quin, 1997; Fery, 2002). Cowpea is a major agronomic crop in the United States during the early part of the 20th century, with production peaking at 2.4 million ha in 1937 (Fery, 1990). By the early 1980s, however, annual cowpea production in the United States was estimated at 197,680 acres (Fery, 1981). Cowpea has long been valued in the southern United States as a vegetable crop and an extensive industry currently distributes fresh, canned, frozen and dry-pea products nationwide. Additionally, cowpea has long been a popular item with home gardeners throughout the south. There is a broad range of characteristics among cowpea cultivars grown for horticultural use in the United States (Fery, 1990, 2002; Islam et al., 2006).
Lipid contents have been related to seed vigor in some seed (Perl et al., 1987). Seed of broad bean (Vicia faba L.), evening primrose (Oenothera lamarckiana De Varies) and carrot (Daucus carota L.) showed a drop of approximately 20 to 30% in the lipid fraction when subjected to accelerated aging; however, in cucumber (Cucumis sativus L.), squash [Praecitrullus fistulosus (Stocks) Pang] and pea (Pisum sativum L.) seed, an increase in lipid content was observed. In pepper (Capsicum annum L.) seed, the lipid content did not change with seed vigor level, but noticeable changes in fatty acid composition are observed. In the labeling of the lipids present in pea seed, phospholipids (70%), triglycerides (20%), waxes (8%), monoglycerides (2%) and diglyceride are detected (Harwood and Stumpf, 1970). Free fatty acids are seldom detected. The most common fatty acids in the phospholipids are palmitic acid (C16:0), palmitoleic acid (C16:1), oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3). The longer chain fatty acids are more common in the wax fraction. The endogenous fatty acids present in the seed were not always related to those synthesized during germination (Harwood and Stumpf, 1970). In germinating seed, de novo synthesis of long chain fatty acids occurs at early stages of germination. Fatty acids including palmitic (C16:0), stearic (C18:0), oleic acid (C18:1), linoleic (C18:2) and linolenic (C18:3), have been detected in cotyledons and axes of water oak (Quercus nigra L.), white oak (Quercus alba L.), americam muskwood [Guarea guidonia (L.) Sleumer and Carapa guianensis Aubl.], silver maple (Acer saccharinum L.) and red buckeye (Aesculus pavia L.) (Connor et al., 1996; Connor and Bonner, 1998; Connor et al., 1998; Connor et al., 2000; Connor and Bonner, 2001). Significant changes in the amounts of individual fatty acids in the seed and the subsequent changes in the total fatty acid content have been reported in these species.
Seed vigor has also been associated with carbohydrate content. In corn (Zea mays L.), the decline of sucrose and raffinose contents during aging was closely correlated with the loss of ability to germinate and rate of germination (Bernal-Lugo and Leopold, 1992). Furthermore, the quantity of oligosaccharides larger than sucrose (C12H22O11) decreased as the period of imbibition during the germination process increased (Koster and Leopold, 1988). As a chilling-sensitive crop, cowpea is affected during germination and early seedling development if planted late in the spring when temperatures are still low in subtropical areas. Good stand establishment is necessary to avoid loss of yield, thus it would be beneficial to plant a cultivar able to tolerate low temperatures. The objective for this research was to determine the germination, coefficient of velocity of germination, lipid and sugar compositions of ungerminated whole seed of different cowpea cultivars.
MATERIALS AND METHODS
Cultivars/Genotypes Used in this Study
Trials were carried out over two years using 25 cowpea cultivars/genotypes
grown to initially screen for germination and coefficient of variability
of germination tests (Islam et al., 2005, 2006). Three cultivars
were chosen from 25 for further study for biochemical characteristics
such as Texas cream 40 which showed ability to germinate at very high
(40°C) and low (10°C) temperature; Black Crowder which had acceptable
high germination at 40°C, but reduced germination at 10°C and
Mississippi Purple which exhibited lower germination at all temperatures
tested (Islam et al., 2005, 2006). The seeds were obtained from
Native Seed Search, Tucson, AZ, USA. The experiment was conducted during
2000 to 2002 at the Mississippi State University, USA. To get seeds from
the same lot, cultivars were grown in the same year, under a common set
of environmental conditions, handled using the same harvesting/seed processing
equipment and stored under the same conditions.
Seed Germination
Germination percentages of all cultivars were evaluated under the
methods described by Islam et al. (2006), except for the number
of seed used per replication and the inclusion of different temperatures.
Seeds were germinated using the between-paper method, in which two sheets
of germination paper (Anchor brown germination paper, Anchor Paper Co.,
St. Paul, Minnesota) were placed in the bottom of 48x50 cm trays and an
additional sheet of the same paper was used to cover the seed. The paper
sheets were previously saturated with 2.5 times their weight of water.
Four replications of twenty seeds of each cultivar were randomly assigned
to trays.
Coefficient of Velocity of Germination (CVG)
The same procedures and conditions as used in the germination study
were used in CVG evaluation. The numbers of germinated seed were evaluated
every day for a period of 8 day. The CVG was calculated using the formula
proposed by Islam et al. (2006).
Extraction of Carbohydrates
Seed carbohydrates were extracted and identified following the methodology
suggested by Connor and Sowa (2000). To determine the initial content
of carbohydrates in cowpea seed, whole dry seed were ground using a handy
mill machine and 0.5 g of flour were used for carbohydrate extraction.
For extraction from the germinating seed, after radical protrusion before
first leaf expansion, the seed were dissected into cotyledons and axes.
Tissues from each cultivar germinated at each temperature were separately
dried in a freeze-dry system (Laconic Freezone 4.5 LABCONCO Corporation,
Kansas City, MO). After removing all moisture, the tissue samples were
finely-ground using a mortar and pestle. A 0.2 to 0.5 g dry tissue sample
was used for each carbohydrate extraction. The tissue samples were placed
in test tubes with 10 mL of an 80% ethanol solution and then boiled in
a 75°C water bath for 1 h. The samples were then passed through a
piece of filter paper in filter funnel. Additional ethanol solution was
used to rinse the samples. The extracts were poured into evaporation flasks
and then rotor-evaporated to dryness in a flask-evaporator (Buchler Instruments.
Fort Lee, NJ). The evaporation flasks were rinsed with 10 mL of distilled
water and the samples were freeze-dried overnights in a stoppered vacuum
flask attached to the freeze-dry apparatus. When all the moisture was
removed from the flasks, the samples were dissolved in 1 mL of Trimethylsilylimidiazole
(TMSI), then heated in a 75°C water bath for 30 min and aspirated
to dryness. The samples were then resuspended in 2 mL of chloroform and
stored refrigerated until analysis.
Analysis of Carbohydrates
The analysis of carbohydrates was performed in a Hewlett Packard (HP)
gas chromatograph (GC) equipped with a flame ionization detector and using
a 125-5037 DB-5 column (30 m lengthx0.53 mm ID and 0.5 μm film thickness)
(J and W Scientific Folsom, CA). The program in the GC preset a detector
and injector temperature of 230°C. An initial oven temperature of
210°C was held for 7 min and then increased 15°C min-1
until a temperature of 270°C was reached and held for 25 min. The
flow was kept at 1.76 nn mL-1. Data were collected and printed
in a HP printer attached to the GC. A graph and numerical map with the
respective percentage peak area of each sugar was obtained. The carbohydrates
were identified by comparing with standards of pure sugars prepared in
a similar manner to the tissue samples and injected in the GC.
Extraction of Lipids
Fatty acids in the seed were extracted and identified through a modification
of the methodology suggested by Whitaker (1986). To determine the content
of fatty acids in cowpea seed, whole dry seed were ground using a glass
homogenizer and pestle. For extractions from germinating seed, after radicle
protrusion and before first leaf expansion, the seed were dissected into
cotyledons and embryos. Two to five grams of each type of tissue from
each cultivar germinated at each temperature were finely chopped and transferred
to Pyrex culture tubes and were then covered with isopropanol using a
pasteur pipette. Tubes were then boiled in a water bath for 3 to 5 min,
removed from the water bath and allowed to cool at ambient temperature.
The supernatant (isopropanol fraction), was transferred to another tube
and then dried under nitrogen to prevent airoxidation. The crude remains
of the extract were resuspended with approximately 2 mL of 2:1 chloroform:
methanol. To prevent autooxidation, the supernatants were then aspirated
under nitrogen atmosphere until dry. Three milliliter of chloroform and
1.5 mL of methanol were added to these fractions, followed by the addition
of 1.5 mL of 0.85% sodium chloride. This wash procedure described by Folch
et al. (1957) was used to remove non-lipid contaminants from the
samples. The tubes were then sealed under nitrogen and centrifuged at
1500x g (510.638 rpm) for 5 min to obtain better separation. The pellet
parts were removed with a pasteur pipette and dried under nitrogen. The
residues were taken up in 1.5 mL of 2:1 chloroform: methanol. The extracts
from sample were combined. A second addition of 1.5 mL of 0.85% sodium
chloride and a second centrifugation at the 1500 g for 5 min was performed
to improve separation. The pellets were then removed and dried under nitrogen.
The remaining portions were taken up in 2 mL of chloroform.
Transesterification of Lipids
The individual polar fractions were dried under nitrogen at 40°C.
The samples were then resuspended in 0.5 mL of chloroform followed by
the addition of 0.5 mL of 0.6 N KOH in dry methanol. The tubes were sealed
under nitrogen and placed on a rototorque rotator in the dark at room
temperature for 2 h. After the addition of 0.5 mL of distilled water and
50 μL 6 N HCl, the Fatty Acid Methyl Esters (FAME) were recovered
by extraction with 2 mL of hexane.
Analysis of Lipids
Fatty acids compositions were identified and analyzed as described
by Whitaker (1986). Lipids were analyzed in a Varian 3300 Gas chromatograph
(Gas Chromatography, Varian Associates, Sugarland, TX) equipped with a
flame ionization detector and using a Supelcowax 10 fused-silica wide-bore
capillary column (30 m lengthx0.53 mm I.D. and 1.0 μL film thickness)
(Supelco, Inc., Bellenfonte, PA). The program used established an injector
temperature of 250°C and a detector temperature of 300°C. The
initial column temperature was set at 190°C and a holding time of
3 min. The column temperature was increased 3°C min-1 until
the final temperature of 220°C was reached and a holding time of 20
min. Data were transferred to a Varian 4290 integrator (Varian Instrument
Division, Walnutcreek, CA), where they were expressed as percentage (%)
area under the peak. Identification of the FAME was achieved by comparing
them with the standards of FAME mixture reference No. 625006, 625007 and
625009 (Alltech Association Inc., Deerfield, IL).
Statistics
Each experiment was arranged in a completely randomized design (CRD)
with five replicates of 20 seed each for each cultivar at each temperature
(for germination and CVG). The data were analyzed as a combined series
of CRDs. Data were subjected to analysis of variance (ANOVA) using the
general linear models (GLM) procedure of SAS version 8.1 (SAS Instit.,
Inc. Cary, North Carolina, USA). Mean separations were done using Fisher`s
protected least significant difference (LSD) test.
RESULTS AND DISCUSSION
Seed Germination and CVG
The germination percent and coefficient of velocity of germination
(CVG) were demonstrated in Table 1. Usually one of the
suitable tests to describe seed vigor is the CVG and this test combines
the germination percentage and rate of germination into a single equation
(Islam et al., 2006). The three cowpea cultivars were chosen for
biochemical evaluation based on physiological performance of the seed
namely performance in the germination and CVG tests under temperature
stress conditions (Islam et al., 2005, 2006). Texas cream 40 which
performed well at 10, 30 and 40°C temperatures; Black Crowder which
was more sensitive to chilling temperature than high temperature (Islam
et al., 2005, 2006) and Mississippi Purple with low germination
and CVG at all temperatures such as 10, 30 and 40°C (Islam et al.,
2005, 2006).
Sugar Compositions
Sugar compositions namely sucrose, raffinose and stachyose content
were significantly differed among the cultivars (Table 2).
Black Crowder had the highest sucrose, raffinose and stachyose contents.
This cultivar also exhibited high germination percentage. The lowest content
of sucrose, raffinose and stachyose was in Mississippi Purple, which had
low germination percentage. The sucrose content in ungerminated seed was
highest in Black Crowder, followed by Texas cream 40 and lowest in Mississippi
Purple. Sucrose is a primary carbon source in cells of higher plants to
support as energy resources (Xu et al., 1989). During seed development
changes in carbohydrates occur in such way that monosaccharide content
decreases whereas oligosaccharide content increases, but during the germination
processes this pattern is reversed (Vertucci and Farrant, 1995). In this
study, Black Crowder seed had faster and higher percent germination than
Texas cream and Mississippi Purple; thus, the higher sucrose content in
the above cultivar suggest faster and more efficient use of sucrose in
germination process and seedling development in this cultivar. Xu et
al. (1989) indicated that during germination seed have a dual role:
first as a source of sucrose produced in cotyledons and secondly as sink
when the young seedling uses sucrose for development. The highest raffinose
content mean was in black crowder (2.46 mg g-1) and lowest
content in Texas cream 40 (1.53 mg g-1) and Mississippi Purple
(1.25 mg g-1). The highest stachyose content mean in the whole
ungerminated seed was in black crowder (10.54 mg g-1), followed
by Texas cream 40 (7.78 mg g-1) and Mississippi Purple (5.77
mg g-1). There were a significant (p<0.01) positive correlation
between sugar contents and
| Table 1: | Germination percentage and coefficient of velocity of germination (CVG) of three selected cowpea cultivars evaluated at different temperatures |
| Data are means of five replications±standard
error. z: Analysis performed on arcsine transformed data.
*: Means within a column not followed by a common letter are significantly
(p<0.05) different according to Fisher s protected LSD test |
|
| Table 2: | Sugar compositions of the three cowpea cultivars |
*: Means within a column not followed by a common letter(s) are significantly (p<0.05) different according to Fisher`s protected LSD test |
|
| Fig. 1a: | Relationship between germination (%) and sucrose content of cowpea seed |
| Fig. 1b: | Relationship between germination (%) and raffinose content of cowpea seed |
| Fig. 1c: | Relationship between germination (%) and stachyose content of cowpea seed |
germination percentage of cowpea seed. Sucrose has the most intimate
relationship (R = 0.917) with the seed germination percentage of cowpea
seeds followed by stachyose (R = 0.764) and raffinose (R = 0.708) (Fig.
1a-c). The results indicate that sucrose de novo synthesis was higher
in cowpea seeds and it played a vital role in cowpea seed germination
process. Koster and Leopold (1988) demonstrated that raffinose as well
as stachyose plays an important role in desiccation tolerance of pea,
corn and soybean seed using sucrose crystallization. The sugar accumulation
in the ungerminated seed and in cotyledon tissues of germinated seed at
low temperature indicated either de novo synthesis or convection
of other products to sucrose. Miguel and Browse (1995) pointed out that
seed storage components such as lipids, proteins and carbohydrates are
converted to sucrose in order to support embryo development.
| Table 3: | Lipid profiles of three cowpea cultivars |
| *: Means within a column not followed by a common letter(s)
are significantly (p<0.05) different according to Fisher?s protected
LSD test |
|
| Table 4: | Ratio of the different fatty acids in three cowpea cultivars |
| *: Means within a column not followed by a common letter(s) are significantly (p<0.05) different according to Fisher`s protected LSD test | |
Lipid Compositions
The main fatty acids present in cowpea seed and seedlings were palmitic
acid (C16:0), palmitoleic acid (16:1), stearic acid (18:0), oleic acid
(18:1), linoleic acid (18:2), linolenic acid (18:3) and arachidic acid
(20:0). The lipid compositions of the cowpea cultivars were presented
in Table 3. The palmitic acid (16:0) was highest in
Mississippi Purple. This cultivar had a lower germination percentage,
suggesting that palmitic acid was not essential to the cowpea seed germination
process. Palmitoleic acid (16:1) was extracted in small quantity in the
seed of Texas cream 40. Stearic acid (18:0) was highest in the seed of
Texas cream 40 which was highest germination percentage, while Mississippi
Purple was lowest and showed a low germination. Thus this long-chain fatty
acid would appear to be important in germination process. Oleic acid (18:1)
was highest in cream 40, the cultivar with high germination. Linoleic
acid (18:2) was highest in the cultivar with high germination. Linolenic
acid (18:3) was higher in the cowpea seed of higher germination (Texas
cream 40 and Black Crowder) and lower in Mississippi Purple. Arachidic
acid (20:0) was higher in both cultivars with high and low germination
[Mississippi Purple (a) and Black Crowder (ab) (Table 3)].
Harwood and Stumpf (1970) pointed out that the initial production of fatty
acid in germinating pea seed showed a lag which depended on temperature.
They also observed that production of fatty acids during the first 30
h of germination initiated with palmitate and stearate, followed later
by oleic acid. Polyunsaturated acids were undetected during this period
of pea seed germination. Stearic acid is found in fatty acid composition
of important oilseed plants such as soybean (Glycine max L. Merr.),
rapeseed (Brassica napus L.), sunflower (H. annuus L.),
palm oil (Elais guinenis L.), cotton (G. hirsutum L.), peanut
(A. hypogaea L.) and coconut (Cocus nucifera L.) in proportions
of approximately 4% (Miguel and Browse, 1995). Thus, this long-chain fatty
acid appears to be important in germination process.
Ratio of Fatty Acids
Table 4 shows the ratio of fatty acid composition
in the seed of three cowpea cultivars. The C18:2/C18:1 ratio was higher
in Black Crowder followed by Mississippi Purple and Texas cream 40. The
18-carbon unsaturated to 18-carbon saturated fatty acid ratio was lower
in Texas cream 40 than in Black Crowder and Mississippi Purple. The ratios
of unsaturated to saturated fatty acids were higher in Black Crowder and
in Texas cream 40 than in Mississippi Purple (Table 4).
Miguel and Browse (1995) reported that long-chain fatty acids are common
in seed from the Brassicaceae and other species such as meadowfoam
(Limnanthes alba Benthan) and jojoba (Simmondsia chinensis
L.). Membrane lipids contain mainly unsaturated fatty acids such as 18:2
(Linoleic acid) and 18:3 (Linolenic acid). When plants or plant parts
are exposed to low temperature, the desaturation of fatty acids occurs
mainly from 18:2 to 18:3 (Murata and Los, 1997). The importance of the
desaturation of fatty acids of membrane lipids in tolerance to low temperatures
has been demonstrated in transgenic systems. When relative levels of saturated
molecular species of phosphatidylglycerol are reduced by transformation
with glycerol-3-phosphate acyltransferase from a chilling resistant plant,
plants become more tolerant to low temperatures (Murata and Los, 1997).
The presence and composition of fatty acids in the seed affected its ability
to germination procedures. The differences in germination capacity may
be related to over expression or inhibition of genes encoding synthesis
of relevant molecules that reflect the composition differences demonstrated
in this study (Khan and Ungar, 1997; Castellon et al., 2003). These
changes could be explained by the possible translocation of fat-like substances
from reserves (cotyledons) to the sink where the respiratory process is
more active, or due to lipid peroxidation.
CONCLUSIONS
From the above results and discussion it is apparent that the main sugars present in cowpea seed were sucrose, raffinose and stachyose. The cowpea cultivars with high germination percentage showed higher sucrose, raffinose and stachyose contents in the seed. The differences in germination capacity may be related to over expression or inhibition of genes encoding synthesis of relevant molecules that reflect the composition differences. These changes could be explained by the possible translocation of fat-like substances from reserves to the sink where the respiratory process is more active. The results may help in future breeding for specific constituents to enhance or reduce the fatty acid and sugar contents and for improvements of the desired quality criteria of cowpea seeds.