Journal of Research in Biology
An International Online Open Access
Effect of 17 Methyltestosterone on sex reversal of Xiphophorous
maculatus platy and Cyprinus carpio Koicarp
Journal of Research in Biology
Authors:
Ananth Kumar 1 And
Mohamed Abdul Kadher
Haniffa2.
Institution:
1. Department of Zoology,
V.H.N.S.N College,
Viruthunagar 626001,
Tamilnadu , India.
2. Centre for Aquaculture
Research and Extension
(CARE), St.Xaviers
College, Palayamkottai-
627002, Tamilnadu, India.
Corresponding author:
Ananth kumar Y.
Email:
labeoshark@gmail.com.
Web Address:
http://jresearchbiology.com/
Documents/RA0072.pdf.
Journal of Research in biology
An International Open Access Online
Research Journal
Original Research Paper
Publication group
ABSTRACT:
Hormone sex manipulation role of the masculinizing hormone 17aMTS was
tested in platy Xiphophorous maculatus and koicarp Cyprinus carpio. The fry of platy
and koicarp of the first group were fed 17 - MTS hormone incorporated diet for 90
days. And the other groups were immersed with the hormone for 6h. The hormone
incorporated feed at 500mg/l produced 68% males in koicarp and 80% in platy.
According to immersion technique 400mg/l produced 85% in platy and 75% in koicarp.
Exposure to other concentrations of 17 - MTS at varying durations invariably
produced intersex individuals. From the present study it is concluded that Koicarp and
Platy are amenable to endocrine sex reversal by steroid immersion and dietary
treatments of fry.
Keywords:
Koicarp, platy, 17a methyltestosterone, sex reversal, gonads.
Article Citation:
Ananth Kumar And Mohamed Abdul Kadher Haniffa.
Effect of 17 Methyltestosterone on Sex Reversal of Xiphophorous maculatus Platy
and Cyprinus carpio Koicarp.
Journal of research in Biology (2011) 8: 580-586
Dates:
Received: 01 Aug 2011 /Accepted: 07 Nov 2011 /Published: 08 Dec2011
Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.
580-586 | JRB | 2011 | Vol 1 | No 8
Submit Your Manuscript
www.ficuspublishers.com www.jresearchbiology.com

INTRODUCTION
In aquaculture the production of single sex
populations offers several advantages, including
enhanced growth and prevention of unwanted
reproduction. Till date dietary administration is the
most accepted method for hormonal induction of
sex reversal in many ornamental fishes viz.,
poecilids, anabantids and others like cichlids but
this method is not widely accepted due to uneven
exposure to steroid by feeding hierarchies. Hence
immersion of fish fry in steroid solutions may be
another way to achieve masculinization or
feminization. Sex steroids, particularly androgens,
are known to stimulate growth in fish. Among the
androgens tested, 17 methyltestosterone (MTS)
was found to be the most potent. MTS is probably
the most widely studied natural androgen for
growth enhancement in fish. It was found to
accelerate growth in common carp (Matty and
Lone, 1979), coho salmon (McBride and Fagerlund,
1976; Fagerlund et al., 1978) and white prawn
(Vaitheeswaran and Ali, 1986). The major aim of
such hormonal manipulation has been the
production of mono sex populations displaying
enhanced growth and /or production of a desired
breeding line (George and Pandian, 1995; Blazquez
et al., 1999; Piferrer, 2001). Supply of feeds
supplemented with 17 MTS to sexually
undifferentiated fry is one of the methods of choice
for all male sex reversal (Abucay and Mair, 1997;
Ohia and Takano 1996; Demska- Zakes and Zakes,
1997). Moreover, sex of a fish, in which the sex
differentiation process has not yet commenced, can
be manipulated by administering steroid hormones
either through dietary supplementation or
immersion or through systematic transfer (injection,
silastic implantation) (Pandian and Sheela, 1995).
The role of different hormones in sex
reversal has been studied by number of earlier
investigators Piferrer and Donaldson (1994). Very
few attempts on endocrine sex reversal have been
made in ornamental fishes by hormonal immersion
and /or incorporation in diet during the fry stage.
The commercial production of Xiphophorous
maculatus is the latest in the series. In the present
work sex reversal in platy and koicarp were
attempted using different doses 17 -
methyltestosterone (MTS) of different doses by
immersion as well as by incorporation in diet with
the following two objectives. To study the
amenability of platy and koicarp to hormonal sex
reversal at the fry stage and to study the feasibility
of producing monosex population of platy and
581
Kumar et al.,2011
koicarp by immersion and dietary administration.
MATERIALS AND METHODS
Collection of hatchlings
Healthy hatchlings of koicarp and platy fish
were purchased from local ornamental fish market
and transported to CARE Aquafarm. The fishes
were stocked in rearing tanks and acclimatized to
laboratory conditions for one week before
commencing the experiment. They were provided
with well aerated water (dissolved oxygen: 5.4-
5.8mg/l; pH: 7.2-7.4; temperature: 24-26C) and
fed with plankton and boiled egg white Feeding
was stopped 24 hrs prior to the start of experiment
in view of making the gut empty.
Hormone preparation
For the present study androgenic steroid
hormone 17 alpha methyl testosterone (Sigma
Chemical co., USA) was selected. For hormone
preparation, initially stock solutions were prepared
by dissolving the appropriate amount of hormone in
dimethylsulphoxide [DMSO] (Ranbaxy, India).
The stock solutions of hormone were kept in cool
dark place and extreme care was taken to avoid the
hormone being exposed to light since these
hormones are photosensitive. (Haniffa et al. 2004).
The feed for the experimental fishes were prepared
using the ingredients viz. rice bran (25g), groundnut
oil cake (37g), fish meal (47.5g), and tapioca flour
(30.5g). The feed were prepared by incorporating
17 alpha-methyl testosterone at six different
dosages (0,100,200,300,400,500 & 600g/l) for
Platy and Koicarp.
Experiment 1
Each sample was mixed thoroughly in order
to distribute the hormone uniformly. One portion
of the feed was kept without hormone incorporation
which served as control feed. The sample of feed
was made into pellets and supplied to the test fish
as semi moist pellets. Two series of experiments
were carried out with 17 a MTS by immersion
technique and dietary administration. The fish
belonging to 7 groups including control each with
30 individuals in triplicate were treated for each
concentration (100,200,300,400,500 & 600g/l).
Experiment 2
The feeding trails were conducted in glass
tanks (181212) and each glass tank was
stocked with 30 fry (Length 0.2 0.4cm; Weight
0.05 0.08gm). The depth of the water in the glass
tank was maintained as 30 cm. Temperature,
dissolved oxygen, pH and ammonia contents were
recorded during the period of 90 days duration.
Journal of Research in Biology (2011) 8: 580-586
Kumar et al.,2011

Water was changed once in 2 days in order
to keep maintain the dissolved oxygen content
without much fluctuation. The experimental fish
were least disturbed except on certain occasions
like change of water. For each concentration of
hormone three tanks each with thirty fishes in each
were maintained as triplicate and triplicate was
maintained for the control also. Owing the
ambiguous nature of manual sexing, juvenile fish
were often sacrificed to determine the sex ratio.
After 90 days the gonads were located after
dissection under a dissection microscope. A portion
of the gonadal tissue was removed with a pair of
fine forceps and placed on a clean glass slide. A
few drops of acetic orcein or geimsa stain were
added and the tissue was squashed after placing a
cover slip on the glass slide (Guerrero and Shelton,
1974; Haniffa et al., 2004). The squashed tissue was
examined under a research microscope low
magnification. Ovarian tissue was identified by the
presence of oocytes and the testicular tissue by the
spermatocytes on a uniform background of tissue.
Sex ratio obtained in the experiments were
subjected to Chi-square test for goodness of fit (Zar
2000).
RESULTS
The effect of MTS treatment on the sex of X.
maculatus is shown in Table 1. The highest
percentage conversion to males (85%) occurred in
the treatment dose of 400g MTS / l of water. In
this batch 85% fish showed normal testes similar to
the control fish.
Experiment 1
The 400g MTS / l and 500 g MTS / l
treatment for 6h significantly (P <0.001) altered the
sex ratio from when compared to the control (4M:
1F). None of the 100, 200, 300, 400, 500 and 600g
MTS / l treatment combinations produced 2 5%
TABLE 1. Effect of different doses of discrete 17 MTS immersion treatment on sex ratio and survival on
Xiphophorous maculatus Platy.
monosex populations and contained intersex
individuals were characterized by the intermingling
of spermatogonia and oocytes in the same gonad
(Fig. 1 c & d). The effect of MTS treatment on the
sex of C. carpio is shown in Table 3. The highest
percentage conversion to males (80%) occurred in
the treatment dose of 500g MTS / l of water. In
this batch 80% fish showed normal testes similar to
the control fish (Fig. 1 a & b). The 500g MTS /
l and 600 g MTS / l treatment for 6h significantly
(P <0.001) altered the sex ratio 4M: 1F when
compared to the control. None of the
100,200,300,400,500 and 600g MTS / l treatment
combinations produced cent percent monosex
populations in koicarp but produced 2 5%
intersex individuals characterized by the
intermingling of spermatogonia and oocytes in the
same gonad. In the reversing gonads (i.e, intersex),
spermatogenic cysts in early stages of development
were predominant and were distributed throughout
the entire gonad amist the stroma cells. The oocytes
were reduced in number and only the small,
developmentally younger ones were seen in areas
between the spermatogenic cysts. Other cells
included cells containing dense dark staining nuclei
and light staining cytoplasm. Sex ratio of platy
administered with diet containing 17 methyl
testosterone is reported in Table 2. After 30 days
gonad formation was not clear. Histological
observations of the gonad of fish given hormone
diets were made after 90 days (Fig. 2 a & b).
Experiment 2
Sex ratio observed after 90 days of dietary
hormone administration in platy revealed a male
dominant population. In 400 g/l of 17 MTH
administered diet for 90 days the males were 75%
whereas the females were only 10%. Remaining
10% were died. At the same time 5% of fishes
were intersex. Platy fish fed with a diet containing

Hormone No. of Duration Sex ratio (%) Survival

S. No Duration
dosage(g/l) fishes (hours) Male Intersex Female (%)
(Days)
1 0 90 6 90 0.50 0.00 0.40 100
2 100 90 6 90 0.60 0.00 0.30 100
3 200 90 6 90 0.70 0.05 0.15 100
4 300 90 6 90 0.75 0.05 0.10 100
5 400 90 6 90 0.85** 0.02 0.03 100
6 500 90 6 90 0.80 0.02 0.08 100
7 600 90 6 90 0.75 0.02 0.13 100
The significance levels (**P<0.001) are for X2 values in comparison with a expected sex ratio of 4 M:1 F
Journal of Research in Biology (2011) 8: 580-586 582
 Kumar et al.,2011

500 g/l of 17 MTS showed males 70% and the females showed previtellogenic oocytes and
female population was also 18 %. Remaining 12% vitellogenic oocytes (Fig. 2 c & d) .Ovary with
were died. vitellogenic oocytes were characterized by the yolk
Histological sections of ovary of normal globules and peripheral yolk vesicles. The nucleus

583 Journal of Research in Biology (2011) 8: 580-586

Kumar et al.,2011

TABLE 2. Effect of different doses of discrete 17 MTS through supplementary diet on sex ratio and survival
on Xiphophorous maculatus Platy.

Hormone No. of Duration Sex ratio Survival

S. No Duration
dosage(g/l) fishes (hours) Male Intersex Female (%)
(Days)
1 0 90 6 90 0.60 -- 0.30 100
2 100 90 6 90 0.50 -- 0.40 100
3 200 90 6 90 0.65 0.02 0.13 100
4 300 90 6 90 0.70 0.05 0.15 100
5 400 90 6 90 0.75 ** 0.05 0.10 100
6 500 90 6 90 0.70 0.02 0.18 100
7 600 90 6 90 0.65 0.02 0.13 100
The significance levels (**P<0.001) are for X2 values in comparison with a expected sex ratio of 4 M:1 F
was in a central position. Yolk globules form a diet containing 600 g/l of 17 MTH the male
homogenous mass with some lipid droplets inside population was 62 % and the female population was
the cytoplasm. The nucleus and the germinal also 6%. The 500 g MT/ l and 600 g MT/ l
vesicle migrated from its central position to the treatment for 90 days significantly (P<0.001)
periphery. Mature oocytes were observed with large altered the sex ratio when compared to the control
accumulation of yolk denoting the growth of the (2 male and 1 female) whereas other treatment did
oocyte and completion of vitellogenesis. Ovary not.
sections showed characteristics where normal
differentiation of the ovary was suppressed. DISCUSSION AND CONCLUSIONS
Oocytes were in the previtellogenic stage and The present attempt to reverse sex in
failure of vitellogenesis was observed. Yolk Koicarp and Platy through immersion in steroid
globules fail to appear in the cytoplasm showed part solution and supplementary diet at doses ranging
of the ovarian tissue which was transformed into from 100 600 g/l of water to 6h around the time
testicular tissue. Testicular development was hatchlings induced a maximum production of 85%
elicited as an auto differentiation. Complete males and 40% females in the case of koicarp a
masculinization was also observed in the few maximum production of 75 % males and 60%
intersex gonads and the transformed testis showed females in platy. In the natural population of platy,
the germ cells which remained in the gonadal stage. the sex ratio is male biased (4males and 1 female)
In koicarp the sex ratio observed after 60 days of whereas in koicarp the natural population is female
dietary administration of hormone koicarp revealed biased (2 females and 1 male, Haniffa et al., 2007).
a male population. In 500 g/l of 17 MTS During the masculinization the fishes were destined
administered diet for 90 days; the males were 68 % to became females at the optimal treatment. In platy
whereas the females were only 30 % .At the same about 85% transformed into phenotypic males and
time 2 % of fishes were intersex. It was fed with a 20% could not be mascilinized in koicarp 75%

TABLE 3. Effect of different doses of discrete 17 MTS immersion treatment sex ratio and survival on koicarp
Cyprinus carpio

Hormone No. of Duration Sex ratio Survival

S. No Duration
dosage(g/l) fishes (hours) Male Intersex Female (%)
(Days)
1 0 90 6 180 0.30 -- 0.60 100
2 100 90 6 180 0.45 0.02 0.30 85
3 200 90 6 180 0.50 0.05 0.15 78
4 300 90 6 180 0.55 0.02 0.10 85
5 400 90 6 180 0.60 0.05 .0.10 83
6 500 90 6 180 0.80** 0.02 0.50 96
7 600 90 6 180 0.75 2 0. 50 91
2
The significance levels (**P<0.001) are for X values in comparison with a expected sex ratio of 2 M:1 F

Journal of Research in Biology (2011) 8: 580-586 584

 Kumar et al.,2011

TABLE 4. Effect of different doses of discrete 17 MTS through supplementary diet on sex ratio and
survival on Koicarp Cyprinus carpio

Hormone No. of Duration Sex ratio Survival

S.No Duration
dosage(g/l) fishes (hours) Male Intersex Female (%)
(Days)
1 0 90 6 180 0.30 -- 0.40 78
2 100 90 6 180 0.40 -- 0.35 83
3 200 90 6 180 0.45 0.02 0.10 85
4 300 90 6 180 0.50 0.02 0.15 74
5 400 90 6 180 0.60 0.04 0.5 76
6 500 90 6 180 0.68** 0.06 0.4 87
7 600 90 6 180 0.62 0.04 0.6 80
2
The significance levels (**P<0.001) are for X values in comparison with a expected sex ratio of 2 M: 1 F.
transformed into phenotypic males and 25% developing embryos with an extended supply of,
become females due to high dose of hormone and and exposure to, the steroid well after the treatment
time duration. In case of supplementary diet, in is completed. Thus full sex reversal with a single
platy about 75% males and 25 % females and in treatment requires further information on dose,
koicarp, 68% males and 32% females were timing and the retention of steroids in embryonic
produced due to supplementary diet. tissues. The presence of intersex individuals in
The results of the present study hormone induced sex reversal was not surprising,
demonstrated that significant masculinization can since it has been reported in previous studies
be achieved in platy and koicarp by immersion (Rougeot et al., 2002; Haniffa et al., 2004; Piferrer
treatment as well as through supplementary diet and Donaldson 1994). The present study also
using 17 a MTS concentration 400g/l in platy and demonstrated that the immersion treatment gave
in koicarp Methyltestosterone concentration 500g/ better results than the dietary method. Treatment of
l in supplementary diet the masculinization of platy Koicarp and platy hatchlings with 17a -
is 400g/l and in koicarp 500 g/l of hormone methyltestosterone at 400 500 g/l for 6 hours
concentration. The results obtained in the present duration and dietary supplementation resulted in an
study were similar to that of Haniffa et al., 2004 in obvious shift of the normal sex ratio in favor of
stinging catfish Heteropneustes fossilis and Salam males. From the present study it is concluded that
and Ronald (1997) in inducing sex reversal of Koicarp and Platy are amenable to endocrine sex
Black crappie (Pomoxis nigromaculatus).Usually reversal by steroid immersion and dietary
effectiveness of androgen treatment depends on treatments of fry.
hormonal doses. However, inhibitory effects of
hormone treatment at high dosage on gonadal ACKNOWLEDGMENT
development have also been demonstrated (Van We are thankful to our Principal and the
Den Hurk and Slof, 1981). Previous studies have authorities who provided facility, to carry out this
explored the use of this hormone through work i.
immersion as well as dietary supplement.
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 Journal of Research in Biology
Prevalence and antimicrobial resistance pattern of microorganisms isolated
from Naira notes in Ogbomoso North, Nigeria
Journal of Research in Biology
Authors:
Ayandele AA and Adeniyi
SA.
Institution:
Department of Pure and
Applied Biology,
Ladoke Akintola
University of Technology,
P.M.B. 4000, Ogbomoso.
Corresponding author:
Ayandele AA
Email:
lizdeley@yahoo.com
Web Address:
http://jresearchbiology.com/
Documents/RA0134.pdf.
Journal of Research in biology
An International Open Access Online
Research Journal
An International Online Open Access
Original Research Paper
Publication group
ABSTRACT:
Nigerian naira notes (#5, #10, #20 and #50) obtained from different locations
and occupational groups in Ogbomoso North Local Government Area in Oyo State,
Nigeria were analyzed microbiologically for evidence of microbial contamination using
MacConkey Agar, Nutrient Agar, Mannitol Salt Agar and Potato Dextrose Agar. About
seven-nine different microorganisms were isolated from the naira notes, while the
prevalent microorganisms include; Staphylococcus aureus (3.8 %), Bacillus subtilis
(10.1%), Enterobacter aerogenes (8.9%), Staphylococcus epidermidis and Aspergillus
niger (11.4%), Bacillus megaterium (12.7%), Escherichia coli (3.8%), Pseudomonas
putida and Aeromonas hydrophila (6.3%), Fusarium solani and Colletotrichum
truncatum (5.1%), and Trichoderma reesei and Colletotrichum gloesporoides (7.6%).
About 75% of the isolates were resistant to the broad spectrum antibiotics and the
Multi- Antibiotics resistance pattern among the bacterial isolates ranged from 7 to 11.
The fungi isolates also showed resistance to different concentration of Fulcin and
Mycoten used (500Mg/l, 250Mg/ml and 100Mg/ml). Zone of inhibition observed with
the fungal isolate that is susceptible to those antifungal agents is <15mm.
Keywords:
Prevalence, Drug resistance, Susceptibility, Small Denominations,
Enteropathogenic Microorganisms.
Article Citation:
Ayandele AA and Adeniyi SA.
Prevalence and antimicrobial resistance pattern of microorganisms isolated from Naira
notes in Ogbomoso North, Nigeria.
Journal of research in Biology (2011) 8: 587-593
Dates:
Received: 18 Oct 2011 /Accepted: 31 Oct 2011 /Published: 08 Dec 2011
Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.
587-593 | JRB | 2011 | Vol 1 | No 8
Submit Your Manuscript
www.ficuspublishers.com www.jresearchbiology.com

INTRODUCTION
Paper money was introduced into Nigeria in
1945 and since then several changes have been
made to different denominations we are using as
legal tender. Paper currency can act as an
environmental vehicle for the transmission of
potential pathogenic microorganisms (Abrams and
Waterman, 1972), since the currency is transferable
from person to person and from one country to
another.
Although paper money is impregnated with
disinfectant to inhibit microorganisms, however
pathogens have been isolated from currency notes
and coins (Talaro, 2005). Research has shown that
currency paper provides a large surface area as
breeding ground for pathogenic microorganisms.
Money on which pathogenic microorganisms can
survive represents an often overlooked reservoir for
enteric diseases (Michaels, 2002) but recent studies
from different parts of the world have revealed that
currency, either as coins or paper have high rate of
microbial contamination (Goktas and Oktav, 1992).
Environmental organisms such as Bacillus
sp., Staphylococcus aureus have been identified as
common contaminants isolated from paper money
(Xu et al., 2005). While other organisms like,
Micrococcus sp., Corynebacterium sp., Vibrio
cholerae, Mycobacterium tuberculosis and
members of the family Enterobacteriacea have been
isolated from money too. Different microorganisms
have been isolated from money worldwide
including developed countries, pathogenic microbes
like Staphylococcus aureus, E. coli, Klebseilla
enterobacter have been isolated from United State
coins and paper bills currencies (Abrams and
Waterman, 1972; Gadsby, 1998). Another survey
also isolated a total of 93 different types of bacteria
belonging to the genera; Staphylococcus,
Streptococcus, Acinetobacter, Pseudomonas,
Bacillus, Klebseilla pneumoniae and E. vuluneris
(Pope et al., 2002). Improper handling of money by
food vendors in which food vendors serve food
with the hands and at the same time handle
currency notes as they sell (Michaels, 2002;
Lamichhane et al., 2009) can transfer bacteria from
currency notes to humans through food
(Lamichhane et al., 2009). Emikpe and Oyero
(2007) had reported that most of the bacteria
isolated from these currencies notes were resistant
to the first line antibiotics which are cheap and
common.
This study then focused on the prevalent
bacteria and fungal contaminants present in small
588
Ayandele et al.,2011
denominations naira notes from Ogbomoso North
in Nigeria and the antimicrobial resistant pattern of
the isolates.
MATERIALS AND METHODS
Sources and Collection of Nigeria Currency
Notes: -
Samples were collected from different four
locations in Ogbomoso North Local Government
Area namely; LAUTECH campus area, Kuye, Sabo
and Takie areas. Four different denominations that
include #5, #10, #20, and #50 were collected at
random from different classes of people in those
four areas.
Microbial Isolation: -
Four different media, namely; Nutrient
Agar, Potato Dextrose Agar, MacConkey Agar and
Mannitol Salt Agar were used for the isolation of
bacteria, fungi, coliforms and Staphylococcus sp.
respectively.
Naira notes were washed in 50ml of sterile
distilled water, serial dilution was carried out and
0.2ml was plated out from appropriate diluents.
Pour plate method was used for the isolation of the
microorganisms. All plates were incubated at 37oC
for 24hrs with the exception of fungi plates that
were incubated at 270C for 2 to 5 days depending
on the visible growth on the plates. The isolated
microorganisms were identified by using
microscopic and biochemical tests as described by
Holt et al (1994). Lactophenol cotton Blue was
used for microscopic identification of fungi, using
both microscopic and macroscopic observations;
the probable identities of fungi were determined by
using Alexopoulous and Mims, (1970) and Domsch
et al., 1980.
Antimicrobial Sensitivity
Antimicrobial sensitivity test was carried out
to determine the sensitivity profiles of the isolates
to selected antibiotics discs. Disc diffusion method
using Nutrient Agar was used for the sensitivity
test. Antibiotics discs containing Tetracycline
(30g), Augmentin (30g), Nitrofurantoin (200g),
Cotrimozazole (25g), Amoxycillin (25g),
Streptomycin (30g), Pefloxacin (5g), Ceftriazone
(30g), Gentamycin (10g), Ofloxacin (5g),
Ciprofloxacin (10g), and Chloramphenicol (10g)
were used for the test. The plates were incubated at
37oC for 48 hours. After incubation, zones of
inhibition were examined and interpreted
accordingly.
While mycelia plug was cut off from the
previous grown plates of Potato Dextrose Agar
Journal of Research in Biology (2011) 8: 587-593
Ayandele et al.,2011

(PDA) and placed on already prepared plates of
PDA. Different concentration of Mycoten and
Nystatin were prepared (100, 250 and 500mg/ml)
and sterile paper discs were dipped into each
concentration and placed aseptically on the plates
that contained the test fungi and incubated at 25oC
for 2-3 days after which the zones of inhibition
were measured.
RESULTS AND DISCUSSION
Table 1 showed the Total bacterial counts of
different denominations, #10 notes had the count of
33.93 cfu/ml 104, followed by #5 notes (20.68
cfu/ml 104), while #50 notes showed the least
bacterial load of 19.94 cfu/ml 104. Table 2
revealed that #10 notes had the highest fungal count
of 22.60 cfu/ml 104, while #5 notes had the least
fungal load of 5.16 cfu/ml 104. The Total coliform
count was represented by Table 3, #10 also had the
highest coliform count of 21.00 cfu/ml 104, and
followed by #20 notes with count of 18.12 cfu/ml
104 and #5 notes had the least count of 9.58 cfu/ml
104. Studies from other parts of the world have
shown that bank notes contained high load of
bacteria which could cause many diseases (Shukla,
1980; Oyler et al., 1996; Pachter et al., 1997;
Havas, 2000). Kawo et al. (2009) also reported high
load of bacterial and fungal count from abused naira
notes in Kano metropolis, Nigeria. Table 4 showed
the occurrence of different microorganisms isolated
from the different naira notes analysed in this study.
Bacillus megaterium had the highest occurrence of
12.7% followed by Staphylococcus epidermidis and
Aspergillus niger (11.4%), Bacillus subtilis (10.1),
Enterobacter aerogenes (8.9%), Trichoderma
reesei and Colletotrichum gloesporoides (7.6%),
Aeromonas hydrophila (6.3%), Fusarium solani
and Colletotrichum truncatum (5.1%), and
Escherichia coli and Staphylococcus aureus (3.8%).
Similar works by Awe et al. (2010), Feglo and
Nkansah (2010), Matur et al. (2010), and Shakir et
al. (2010) had also reported the occurrence of
different microorganisms from the currency notes
in their countries. The isolation of Staphylococcus
on the currency notes could have been
contamination from normal skin flora and soil
(Igumbor et al., 2007; Larkin et al., 2009) and
Staphylococci infection occur when Staphylococcus
enter the body through breaks, cuts and abrasions in
the skin (Shakir et al., 2010) and it is also
associated with impetigo, carbuncles and food
intoxication (Jensen et al., 1997). Escherichia coli
which is due to feacal contamination is also
responsible for many diseases when consumed in
large doses, other bacterial isolates like
Enterobacter aerogenes, Bacillus subtilis,
Staphylococcus epidermidis, Pseudomonas putida
and Aeromonas hydrophila have been associated
with different types of diseases especially in
immunocompromised and immunosuppressed
patients (Kelly et al., 1993; Yang et al., 1996;
Aurlie et al., 2005), Fusarium solani may also
cause a range of invasive mycoses and a range of
opportunistic infection in immunocompromised
patients (Zhang et al., 2006), the fungal isolates
could also produce mycotoxins in food, which is

Table 1: Total Bacterial Counts on Different Denominations

#5 #10 #20 #50
Locations
Cfu/ml 104 Cfu/ml 104 Cfu/ml 104 Cfu/ml 104
Sabo 8.30 18.00 3.30 13.30
LAUTECH 2.32 3.00 4.00 1.20
Kuye 3.96 9.60 3.72 4.04
Takie 6.10 3.33 9.33 1.40
Total 20.68 33.93 20.35 19.94
Table 2: Total Fungal Count on Different Denominations
#5 #10 #20 #50
Locations
Cfu/ml 104 Cfu/ml 104 Cfu/ml 104 Cfu/ml 104
Sabo 2.00 10.00 8.30 6.60
LAUTECH NG 3.33 3.00 1.33
Kuye 1.00 NG NG 3.00
Takie 2.16 9.33 6.70 6.00

Total 5.16 22.66 18.00 16.93

Journal of Research in Biology (2011) 8: 587-593 589

 Ayandele et al.,2011

Table 3: Total Coliforms Count on Different Denominations

#5 #10 #20 #50
Locations
Cfu/ml 104 Cfu/ml 104 Cfu/ml 104 Cfu/ml 104

Sabo 7.23 11.0 8.45 7.80

LAUTECH NG 2.50 3.42 2.16
Kuye NG 1.30 2.50 1.76
Takie 2.35 6.20 3.75 5.20
Total 9.58 21.00 18.12 16.92
Key: NG- No Growth.

dangerous to human and other animals (Grundy and
Grundy, 1974). Table 5 shows the microbial rate of
naira notes in relations to the locations and the
denominations of the different naira notes used in
this study. The results revealed that Sabo area
which comprises of different artisans and beggars
had the highest contamination rate followed by
LAUTech campus area which also comprised of
different food vendors, shop owners, different
artisans and students, while Kuye area which is a
residential area for Government workers and
artisans had the least contamination. Similar work
by Shakir et al. (2010) also revealed that
Bangladesh paper currency notes collected from
artisans groups had the highest contamination rate
compared to money collected from educated
people. #10 notes used in this work had the highest
contamination rate followed by #20 notes, these
two naira notes are commonly found among the
poor people and the children, the least
contamination rate observed with #5 notes may be
due to the fact that it is very rare to found most
commodities sold at #5 nowadays. In a similar work
by Oyero and Emikpe (2007), they reported highest
level of microbial contamination for #10 notes
among Nigerian currencies notes in their work.
Also, #10 notes are the most commonly used
among the small denominations in Nigeria and it is
exchanged many times especially amidst the
artisans and lower economic class people. Shakir et
al. (2010) had also reported highest contamination
rate among the small denomination notes in
Bangladesh currencies.
Table 6 showed the Antibiotics sensitivity

Table 4: The Occurrence of Microorganisms Isolated from Naira Notes

KEYS:
Alphabets Locations
A - Sabo (Market and Beggars Area) B - LAUTech (Students and Civil Servants Area)
C - Kuye (Indigenes Area but no market) D - Takie Square (Motor Park and Commercial Area)

Numbers Denomination
1 - #5 2 - #10 3 - #15 4 - #20

590 Journal of Research in Biology (2011) 8: 587-593

Ayandele et al.,2011

Table 5: Microbial Contamination Rate of Naira Notes in Relation to Different Locations and Denominations
LOCATION #5 #10 #20 #50 TOT AL CONTAMINATION
(cfu/ml 104) (cfu/ml 104) (cfu/ml 104) (cfu/ml 104) (cfu/ml 104) RATE (%)
SABO 10.30 28.00 11.60 19.90 69.80 44.30
LAUTECH 2.33 12.33 16.03 2.53 33.22 21.08
KUYE 4.96 9.60 3.72 7.04 25.32 16.07
TAKIE 8.16 6.66 7.00 7.40 29.22 18.55
TOTAL 25.75 56.99 38.35 36.87 157.56
CONTAMINATION 15.30 35.08 24.40 23.34
RATE (%)

pattern of the isolated bacteria against selected
antibiotics. All the bacteria isolates showed 100%
resistant to Augmentin, Nitrofurantoin and
amoxicillin, they had 87.5% resistant to
tetracycline, chloramphenicol and streptomycin, the
isolates showed 50% resistant to Ceftriazone,
Cotrimoxazole and Gentamycin, 37.3% and 25.0%
resistant to Ofloxacin and Pefloxacin respectively.
While all the bacterial isolates were susceptible to
Ciprofloxacin with zones of inhibition ranging from
16 to 5mm. The fungal isolates also showed varied
activities against the two antifungal (Mycoten and
Fulcin) used for the sensitivity test at varying
concentrations (100, 250 and 500mg/ml). At 100,
250 and 500mg/ml concentrations for Fulcin, the
resistant percentage was 80, 40 and 40%
respectively, and for Mycoten, the resistance
percentage were 80, 60 and 40% for 100, 250 and
500mg/ml respectively (Table 7). Similar work by
Emikpe and Oyero (2007) revealed that organisms
Table 6: Antimicrobial Sensitivity Pattern of Some Bacterial Isolates Against Selected Antibiotics
isolated from Nigerian Naira notes were resistant to
first line antibiotics and this was also observed in
this work. The resistance observed in this work
might also be due to the indiscriminate use of
antibiotics by people. The resistance to the
antifungal agents might be as a result of the carriers
of some diseases who might have been exposed to
different antifungal agents especially mycoten
which is used by many women in the treatment of
Candida albicans infection.
The findings from this work showed that
infections that may occur from the microorganisms
isolated from this work might be difficult to treat
and also the treatment might be very expensive
because of the resistance of these isolates to the
common and cheaper antibiotics drugs.
In conclusion, this study showed that naira
notes collected from different areas in Ogbomoso
North Local Government of Nigeria is
contaminated with different pathogenic organisms

Table 7: Antimicrobial Sensitivity Test of Fungal Isolates Against Antifungal Agents

ORGANISM FULCIN MYCO TIN
100 mg/ml 250 mg/ml 500 mg/ml 100 mg/ml 250 mg/ml 500 mg/ml
Aspergillus niger R R R 8 R R
Trichoderma reesei 9 6 9 R 8 8
Colletotrichum gloesporoides R 8 14 R R 12
Fusarium solani R R R R R R
Colletotrichum truncatum R 10 8 R 9 6

Resistance Rate (%) 80 40 40 80 60 40

Journal of Research in Biology (2011) 8: 587-593 591
 Ayandele et al.,2011

and the microbial load is also very high. The
isolated organisms showed high resistance to
available antibiotics and antifungal agents.
Therefore, there is need to educate the populace on
the effect of improper handling of naira notes and
women most especially because of their children
who always put money in their mouth.
the Hungarian currency (notes and coins). Magya
Allatoryosol Lapja 122(8):501-503.
Holt JG, Krieg NR, Sneath PHA, Stanley JT and
Williams ST. 1994. Bergeys Manual of
Determinative Bacteriology, Williams and Wilkins,
Baltimore.

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 Journal of Research in Biology
An International Online Open Access
Original Research Paper
Publication group

Assessment of viability responses of refinery effluent bacteria after

exposure to phenol stress
Journal of Research in Biology

Authors: ABSTRACT:
Nwanyanwu CE1 and Abu
GO2. Toxicity of phenol on two bacterial strains isolated from biological treatment
unit of petroleum refinery effluent was assessed based on viability responses after
exposure to increasing doses of phenol. Viability was based on the reduction of TTC
Institution:
via dehydrogenase activity in viable cells but not by dead cells after 1, 2, 3 and 4 h
1. Department of
Microbiology, Federal exposure to phenol. The viability responses of the organisms to phenol are time
university of Technology, P. dependent. At 1, 2 and 3 h of exposure, there was a stimulation of dehydrogenase
M. B. 1526, Owerri, Nigeria. activity in Pseudomonas sp. RBD1 and Bacillus sp. RBD2. In Pseudomonas sp. RBD1,
dehydrogenase activity was progressively inhibited at phenol concentrations greater
. than 250 mg/l at 1, 2 and 3 h. In Bacillus sp. RBD2, dehydrogenase activity was also
2 Department of
Microbiology, University of progressively inhibited at concentrations greater than 250 mg/l at 1h. Total inhibition
Port Harcourt, P. M. B. occurred in 4 h exposure at all concentrations of phenol in both organisms. The IC 50
5323, Port Harcourt, Nigeria. ranges from 190.605 9.786 to 1070.546 23.127 mg/l. The results of the viability
responses assessment of the bacterial strains indicated that the toxicity of phenol
depends on the duration of exposure of the organisms to the test chemical.
Corresponding author:
Nwanyanwu CE

Email: Keywords:
cnwanyanwu2000@yahoo.com Phenol toxicity, Exposure period, Viability, Bacteria.

Web Address: Article Citation:

http://jresearchbiology.com/
Documents/RA0110.pdf.
Nwanyanwu CE and Abu GO.
Assessment of viability responses of refinery effluent bacteria after exposure to
phenol stress
Journal of research in Biology (2011) 8: 594-602

Dates:
Received: 19 Sep 2011 /Accepted: 27 Oct 2011 /Published: 12 Dec 2011

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594-602 | JRB | 2011 | Vol 1 | No 8

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INTRODUCTION
The number, diversity and complexity of
synthetic chemical compounds are overwhelming
and its use has permeated every aspects of modern
life. A particular widespread group of these
chemical is phenol and its derivatives. Phenol
enters the environment through wastewater
discharges from a variety of industries such as
leather, textile, petroleum refinery, coking-plants,
pharmaceuticals, coal conversion, etc and also
through the decomposition of attached algae and
phytoplanktons (Afzal et al., 2007, Hidalgo et al.,
2002). In refining of crude oil process in the
refinery, majority of phenols present in refinery
effluent originate from the catalytic cracking
process and chemicals added as additives during
physical and chemical treatment of the crude oil
(Jorgensen, 1979).
Phenol is very toxic to all forms of life even
to those aerobic mesophilic microorganisms that
utilize it as a sole source of carbon and energy
(Gurugeyalakshmi and Orial, 1989). This aromatic
compound is water soluble and highly mobile and
as such is likely eventually to reach down stream
drinking water sources where, even at very low
concentrations, it can cause severe odour and taste
problem. The toxic action of phenol is always
associated with loss of cytoplasmic membrane
integrity. This results in disruption energy
transduction, disturbance of membrane barrier
function, inhibition of membrane protein function
and subsequent cell death (Collins and Daugulis,
1997; Yap et al., 1999). Due to its toxicity to
microorganisms, phenol may often cause the
breakdown of wastewater treatment systems by
inhibition of microbial growth (Ren and Frymier,
2003). This sensitive bacterial assay for toxicity
assessments is important.
Many biological endpoints are used to
evaluate toxicity effect of phenol. This include: cell
proliferation (increase in cell number), membrane
damage (loss of ions or cofactors, NADPH),
incorporation of radioactive precursors (thymidyne
and DNA synthesis), cell viability (cell number, dye
reduction/uptake), etc. The results from any cellular
in vitro system are not only dependent on the
properties of the cell type used but also on
experimental conditions such as time, pH, medium
composition, etc (Skowron and Zapor, 2004). Long-
term assays are based on measuring cell survival
and cell proliferation over periods from 24 hour to
several days. Short-term assays involve exposure to
potential toxicants for periods from 1min to about 5
595
Nwanyanwu et al.,2011
hours and endpoint measurements focused on cell
viability and cell membrane damage. Short-term
assays determine toxic effects more effective than
long term assay when fast degradable organic or
inorganic substrates are considered which enhance
microbial growth.
Continuous biomass quantification using the
traditional methods such as gravimetric methods,
direct microscopic count, plate count, measuring
changes in the optical density and determination of
cell constituents are very difficult (Ghaly and
Mahomoud, 2006). Methods that are based on
measuring microbial enzymes activity are fast
gaining ground in the assessment of chemical
toxicity to microorganisms. Measurement of
microbial dehydrogenase activity is used in the
assessment of ecotoxicological impact of
environmental substrates as the enzymes are
intracellular (Matthew and Obbard, 2001; Rossel et
al., 1997). The use of dehydrogenase activity assay
for the detection, control and monitoring of the
presence of pollutants of both organic and inorganic
in soil, water as well as wastewater are recognized
as a useful indicator of the overall measure of
intensity of microbial metabolism (Friedel et al.,
1994; Quilchano and Maranon, 2002; Repetto et al.,
2001; Cordina et al., 1993). Dehydrogenases are the
group of endocellular enzymes that are present in
all living cells and are involved in catalyzing
biological oxidation of organic compounds. They
transfer hydrogen and electrons through a chain of
intermediate electron carriers to oxygen as a final
electron acceptor which then combines with them
and form water (Ghaly and Mahomoud, 2006).
Tetrazolium salts are reduced by viable cells and
not by dead cells. This property has been used in
agriculture to assess seed viability (Altman, 1976;
Brewer, 1949) and since then many other
applications were reported including the use of
triphenyltetrazolium chloride (TTC) in
microbiology (Hurwitz and McMcarthy, 1986;
Jones and Prasad, 1969).
This paper aims at assessing the viability
responses of refinery effluent bacteria based on the
reduction of 2, 3, 5-triphenyltetrazolium chloride by
dehydrogenase enzyme activity after exposure to
increasing doses of phenol.
MATERIALS AND METHODS
Isolation and identification of bacterial strains
Bacterial strains used in this study were
isolated from the outlet of biological treatment unit
(Rotary biodisk, RBD) of effluent from Nigeria oil
Journal of Research in Biology (2011) 8: 594-602
Nwanyanwu et al.,2011
refinery company, Port Harcourt, Nigeria. They
were isolated based on the morphological,
biochemical and sugar fermentation tests. All
microbial isolations were performed on mineral
salts medium (MSM) containing (g/l): K2HPO4,
0.750; KH2PO4, 0.840; (NH4)2SO4, 0.400;
MgSO4.7H2O, 0.060; NaCl, 0.060; CaCl2, 0.060;
FeCl3.6H2O, 0.060. Phenol (2.5mM) was used as a
sole source of carbon and energy. 15.0g of agar was
added and the pH adjusted to 6.9 using phosphate
buffers. This was followed by the addition of sterile
aliquot of ketoconazole (50g/ml) into the medium
after sterilization to exclude fungi. The medium
was then poured into Petri dishes. Bacterial strains
were isolated by spreading aliquots of decimally
diluted (10-4) effluent sample onto the surface of the
medium and incubated at 30oC for 72 h (Kahru et
al., 2002). Morphologically distinct strains were
isolated and were individually purified on nutrient
agar. Characterizations were done using standard
microbiological methods. Identification to the
genus level followed the schemes of Holt et al.
(1994).
Characteristics of the refinery wastewater
which is the source of the organisms are described
in Table 1. The pH, Total Dissolves Solids (TDS),
Chemical Oxygen Demand (COD), Total
Hydrocarbon (THC), Phenol concentration and
Phosphate (PO42-) and Nitrate (NO3-) in the
wastewater sample were determined according to
the standard methods (APHA, 1998). Heavy metals
in the samples were estimated using Atomic
Absorption Spectrophotometer (Perkins Elmer
3110, Walthan, Massachusetts, USA)
Table 1: Characteristics of Biological treated effluent
of the oil refinery samples
Parameter/unit Wastewater (value)
pH 7.64
TDS (mg/l) 250
THC (mg/l) 15.0
BOD (mg/l) 8.0
COD (mg/l) 76.0
Phenol (mg/l) 13.6
PO42-(mg/l) 0.14
-
NO3 (mg/l) 1.20
Cu2+(mg/l) <0.01
3+
Pb (mg/l) <0.01
Journal of Research in Biology (2011) 8: 594-602
Test chemicals
The following items were purchased from
the indicated sources: 2, 3, 5-TriphenylTetrazolium
Chloride (TTC); phenol (C6H5OH, 99% pure) from
sigma chemical Co (St. Louis, Mo, USA). Amyl
alcohol from Wako Pure, chemical industries
Osaka, Japan); nutrient broth and glucose from
Difco (Aldrich chemical company, Milwaukee,
Wisconsin, USA).
Preparation of inoculum
Seed cultures of the selected bacterial strains
were grown in 100ml nutrient broth medium (0.8
%) in 250 ml Erlenmeyer flasks, covered with
cotton wool wrapped in Aluminum foil and
incubated on a rotary shaker (150 rpm) for 24 h at
room temperature (28 2oC). Cells were recovered
by centrifugation at 4,000 rpm for 15min.
Harvested cells were washed twice in deionized
distilled water to avoid carry over and resuspended
in the same deionized distilled water. The
resuspended cells were standardized in a
spectrophotometer to an optical density of 0.90 at
540nm. The dry weight of the standardized cells
was determined by drying 15 ml of the cell
suspensions to a constant weight in an oven
(Gellankamp. England) at 110oC. The standardized
cell suspensions were used as inoculum in the
assay.
Preparation and treatment of inoculum with test
chemical
Exposure (contact) period assay technique as
described by Hidalgo et al., (2002) was employed
with little modification. Briefly, a range of
increasing doses of exposure solution (phenol) was
prepared in 1.65 ml of physiological saline (0.85 %)
medium in separate 15.0 ml screw capped test glass
tubes arranged in four groups that represent various
exposure time period. Aliquots (0.35 ml) of the
bacterial suspensions were seeded into the triplicate
glass tubes bringing the total volume of the mixture
in the tubes to 2.0 ml to obtain final phenol
concentrations of 250, 500, 750, 1000, 1500 and
2000 mg/l in different tubes. The tubes were
incubated on a rotary incubator (150 rpm) at room
temperature (28 2oC) for appropriate exposure
time period. The control consisted of the organism
and physiological saline medium without the
toxicant (phenol). Once the exposure period
(contact time) to phenol had elapsed (1, 2, 3 or 4 h),
the medium was removed by centrifugation for 15
min and the pelleted cells are washed twice with
deionized distilled water. Thereafter, 2.0 ml of
596

nutrient broth-glucose medium were added into the
tubes, vortexed for 2 min and preincubated on a
rotary incubator (150 rpm) at room temperature (28
2oC) for 30 min.
Viability assay
The assay is based on the inability of dead
cells or injured bacterial strains to reduce 2, 3, 5-
TriphenylTetrazolium Chloride (TTC) into
insoluble TriphenylFormazan (TPF) product. The
ability of viable bacterial strains (by dehydrogenase
enzyme activity) to reduce TTC to TPF provides an
indication of membrane intact which in turn, may
be interpreted as a measure of cell viability. At the
end of preincubation with nutrient broth-glucose
medium, 0.2 ml of 0.1 % (w/v) TTC in deionized
distilled water was added into the tubes. The
reaction mixture was incubated statically at room
temperature (28 2oC) for 6 h in the dark. Since the
TPF produced by cellular dehydrogenases is
insoluble in water, extraction of TPF was done in
3.0 ml amyl alcohol and determined
spectrophotometrically at 445 nm (max). The
amount of formazan produced was determined in
reference to a standard dose-response curve.
Viability [Dehydrogenase activity, (DHA)] was
expressed as microgram of TPF formed per mg dry
weight of cell biomass per hour.
Calculation of viability/stimulation or inhibition
of enzyme activity
The viability/stimulation or inhibition of
dehydrogenase enzyme activity was computed as
percent deviation from the control. Where
applicable, the IC50 of the test chemical (phenol)
was determined by fitting the TPF values produced
to simple equations using Table 2D Curve (systat
Incorporation, USA) and calculating the
concentrations of the test chemical at 50%
inhibition of dehydrogenase activity. The greater
the IC50 value, the lower the toxicity of phenol
sample at that exposure time period.
Statistical analysis
Data obtained from the study were analyzed by the
use of two-way analysis of variance (ANOVA) and
values for P < 0.05 were considered statistically
significant.
RESULTS AND DISCUSSION
Two bacterial strains comprising gram negative and
gram positive were isolated from the biological
treatment unit of wastewater sample and identified
as Pseudomonas sp. RBD1 and Bacillus sp. RBD2
organisms. Results obtained from the control
samples on viability assays showed that these
597
Nwanyanwu et al.,2011
organisms were able to reduce TTC to red fomazan
at variable rates and extent (Table 2). The gram
negative Pseudomonas sp. RBD1 had higher rate of
dehydrogenase activity in all the exposure period
time than gram positive Bacillus sp. RBD2. The
reason for these differences is not known. However,
variation may be due to differences in cell wall
components or dehydrogenase systems, since
different microorganisms have been reported to
have different dehydrogenase systems (Praveen-
Kumar, 2003; Nweke et al., 2006). Moreso, Table 2
showed decrease in formazan formation with
increase in exposure period to phenol stress. This
decrease in formazan formation with increase in
time is in line with observations of Ross (1971),
who reported that a gradual decrease, in formazan
formation after an incubation time of 1 h. Griebe et
al., (1997), also reported a decrease in the rate of
formazan formation after 2 h incubation time when
CTC was incubated with activated sludge and stated
that no significant increase was realized in
formazan formation after 4 h incubation time.
Estimation of viability of the organisms by
inhibition/stimulation of dehydrogenase activity at
various exposure periods in different concentration
of phenol is shown in Figure 1. The results showed
decrease in stimulation of dehydrogenase activity
upto 3 h of exposure at 250 mg/l of phenol in
Pseudomonas sp. RBD1 while in Bacillus
sp.RBD2, stimulation was observed at 250 mg/l of
phenol only for 1 h of exposure. Thereafter, there
showed total inhibition in both organisms in all the
concentrations of phenol in the fourth hour.
Stimulation of dehydrogenase activity in both
organisms at 1 - 3 h exposure periods indicates
viability of the organisms hence the utilization of
phenol as a growth substrate is possible. The
progressively decreasing stimulation of
dehydrogenase activity with increasing exposure
period in Pseudomonas sp.RBD1 is in line with
Table 2: Dehydrogenase activities in the absence of
toxicant (phenol) at different exposure period
Dehydrogenase activity
(g Formazan/mg cell dry wt/h)
Exposure Bacterial strain
period (h)
Pseudomonas sp. Bacillus sp.
RBD1 RBD2
1 1.175 0.056 0.719 0.022
2 1.097 0.029 0.666 0.009
3 1.014 0.021 0.606 0.012
4 0.964 0.041 0.571 0.023
Journal of Research in Biology (2011) 8: 594-602
Nwanyanwu et al.,2011

well documented inhibitory nature of phenol at microbial processes on exposure to phenolic

lower concentrations. Variations in response of compounds have been reported. Repetto et al.,
A B
Inhibition/stimulation

Concentration (mg/l)
Figure 1: Phenol inhibition of dehydrogenase activity in (A) Pseudomonas sp.RBD1 and (B) Bacillus
sp. RBD2. (>0% = Inhibition; <0 = Stimulation/viability)

Journal of Research in Biology (2011) 8: 594-602 598


(2001) reported decrease in bioluminescence of
Vibrio fischeri bacteria with exposure period to
pentachlorophenol. Also, Okolo et al., (2007) and
Stotzky and Norman (1961) observed that
nitrophenols repressed soil respiration and
microbial activity with increasing exposure periods.
The effects of exposure to phenol on viability of
the bacterial strains assessed via dehydrogenase
activity are as shown in Figure 2. In both
organisms, dehydrogenase activity decreased with
increasing exposure period and concentrations of
phenol. In Pseudomonas sp.RBD1, dehydrogenase
activity was stimulated at 250 - 500 mg/l of phenol
in the first hour while in 2 3 h of exposure,
stimulation was observed only in 250 mg/l of
phenol. In Bacillus sp. RBD2, stimulation of
dehydrogenase activity was observed only in the
first one hour at 250 mg/l of phenol. The
stimulatory effects observed at the first hour period
of exposure to phenol in both organisms may be
attributed to the metabolic activity at this period
(g TPF/mg dry cell weight/hour
Dehydrogenase activity
Concentration (mg/l)
Figure 2: Effect of exposure time on reduction of
TTC by the bacterial strains in response to
increasing doses of phenol
599
Nwanyanwu et al.,2011
thereby utilizing phenol as carbon and energy
sources. At the fourth hour, total inhibition was
observed in both organisms. The inhibition may be
attributed to the disruption of membrane barrier
function of the organisms that resulted in the
inhibition of dehydrogenase activities in both
organisms. The inhibition of dehydrogenase
activities is attributable to the toxicity of phenol.
Dehydrogenase enzymes are membrane associated
and phenols have been reported to affect
membranes (Irya et al., 2003). The dehydrogenase
activities correlated with exposure periods and
phenol concentration are as shown in Figure 3. The
high R2 values ().6472 < R2 < 0.9809) for
Pseudomonas sp. RBD1 and (0.7689 < R2 <
0.8483) for Bacillus sp.RBD2 indicated that phenol
concentration as well as exposure period was a
strong determinant of metabolic activity in the
organisms.
The toxicity threshold of phenol
concentrations against the bacterial strains are
shown in Table 3. The organisms showed variable
response pattern to prolong exposure period
incubation. In both Pseudomonas sp. RBD1 and
Bacillus sp. RBD2 the toxicity threshold increased
steadily upto the 2nd hour of exposure period and
thereafter decreased progressively as the exposure
period increased to the 4th hour. The median
inhibitory concentration of phenol (IC50) against the
organisms ranged from 590.870 14.211 mg/l in
Bacillus sp. RBD2 to 1070.546 46.111 mg/l in
Pseudomonas sp. RBD1 within 1h of exposure
period. Cenci et al., (1987) had reported an IC50 of
636 mg/l for phenol against dehydrogenase activity
of a gram negative Escherichia coli. However,
Pseudomonas sp. RBD1 with IC50 ranging from
1070.546 23.127 to 1654.903 51.234 mg/l
phenol appears to have high tolerance to phenol.
Findings from this study shows that much as
the dependence on the dehydrogenase activity assay
is a measure of the microbiological viability,
Table 3: Median inhibitory concentrations (IC50) of
phenol at different exposure period against bacterial
strains
Inhibitory concentrations (mg/l)
Exposure
period (h) Pseudomonas sp.
Bacillus sp. RBD
RBD
1 1240.100 46.111 590.870 14.211
2 1654.903 51.234 1609.829 48.781
3 1409.674 34.564 1593.605 34.451
4 1070.546 23.127 190.605 9.786
Journal of Research in Biology (2011) 8: 594-602
Nwanyanwu et al.,2011

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DH
A

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Figure 3: correlation of phenol concentrations with dehydrogenase activity in
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time to phenol toxicity

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 Journal of Research in Biology
Anatomical investigations in Silybum marianum (L.) Gaertn.
Journal of Research in Biology
Authors:
Sidhu MC and Saini P.
Institution:
Department of Botany,
Panjab University,
Chandigarh-160014-India.
Corresponding author:
Sidhu MC
Email:
mcsidhu@hotmail.com
Web Address:
http://jresearchbiology.com/
Documents/RA0149.pdf.
Journal of Research in biology
An International Open Access Online
Research Journal
An International Online Open Access
Original Research Paper
Publication group
ABSTRACT:
Anatomical studies have been carried out for various parts of species Silybum
marianum (L.) Gaertn. According to the available literature very little work has been
done on this aspect. Keeping this in view presently, the root, stem and leaf of this
plant has been studied in detail for its anatomical variations. Stem anatomy is almost
similar to other members of the family. The Transverse Section of the leaf shows the
presence of accessory vascular bundles. Similarly Transverse Section of the root has
revealed the occurrence of secondary growth. In normal conditions, secondary growth
if occur, has to be present in other plant parts also e.g., stem. But it has not been
observed in stem during present investigation. Further, stomata have not been found
on any of the two epidermal layers. In addition, to best of our knowledge, no
anatomical details has been worked out for this species in the past except one study
made on root anatomy as discussed in the results. So present findings related to the
anatomy of this particular species may be treated as pioneer.
Keywords:
Asteraceae, anatomy, leaf, root, stem, microscopy, Silybum marianum.
Article Citation:
Sidhu MC and Saini P.
Anatomical Investigations in Silybum marianum (L.) Gaertn.
journal of research in Biology (2011) 8: 603-608
Dates:
Received: 21 Nov 2011 /Accepted: 23 Nov 2011 /Published: 12 Dec 2011
Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
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603-608 | JRB | 2011 | Vol 1 | No 8
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INTRODUCTION:
Silybum marianum (L.) Gaertn. was
previously known as Carduus marianus. It is a
member of family Asteraceae (Compositae)
commonly known as Daisy family. Its synonyms
are Carduum marianum L., Carduus marianum L.
and Cnicus marianum L. Its botanical name is
derived from a Greek word Silybonor Silybos obtained manually. Different sections were stained
which means tassel or tuft. It is an annual or
biennial species occurring throughout the world.
But Eeastwood (1901) designated it as a stout
annual species having white patched leaves with
prickly margins. Silybum marianum is an annual
herb and many of its aerial parts bear thorns. Plant
height may vary from place to place. It also grows
as biennial species in certain areas. The stem is tall,
erect and branched towards the apex. The leaves are
alternate, large and glabrous. The seeds bear very
fine, long silky and creamy white pappus. The fruits
are referred to as achenes. Anatomical studies
illustrate the internal organisation of the species and
it has been placed between morphology and cell
biology. According to Dilcher (1991), anatomical
details are important for Palaeobotanists for the
reconstruction of complete plant. Anatomical
details can also be helpful in studying the
systematic position and evolutionary relationship
between the species (Dengler, 2002). Dhyani et al.
(2009) while analysing the anatomical features of
Lilium polyphyllum suggested that these types of
studies are important for botanists, medical
researchers and taxonomists. In case of a species
like Silybum marianum which is of high medicinal
importance, anatomical characterisation has great
significance to avoid adulteration (Eltahir and
AbuEReish, 2010). Silybum marianum has
anticancer, antidiabetic, antidote to mushroom
poisoning, anti-fibrotic, antihepatotoxic,
antiinflammatory, antioxidant, hepatoprotective,
hypocholesterolaemic, immunomodulatory, kidney
protective, neuroactive and neuroprotective etc.
properties (Corchete, 2008). Besides this it has also
been used in various veterinary medicines. In
addition to its benefits for human health, it also has
some negative aspects. In several parts of the world,
it is regarded as a toxic weed. To the best of our
knowledge, till today the internal organisation of
Silybum marianum has not been worked out in
detail. Hence this area needs a thorough and
extensive exploitation. Attempts have been made to
study the internal structure of root, stem and leaves.
604
Sidhu et al.,2011
MATERIAL AND METHODS:
The plant material was collected from its
natural habitats in Chandigarh, India. The root,
stem and leaf were fixed in the fixative for their
proper preservations. For longer duration these
samples were shifted to 70% alcohol. The
Transverse Sections of root, stem and leaf were
using safranin and fast green, then observed under
the microscope. Photographs of the well stained
sections showing cellular differentiation have been
taken.
RESULTS AND DISCUSSION:
Attempts have been made to study the
internal organization of root, stem and leaf of this
species. The results obtained from the present
investigations are as follows:
Stem:
The outline of Transverse Section of
Silybum marianum stem is nearly circular. The
outer most layer of the stem is epidermis which is
single layered and cuticularised (Figure 1-a). The
cortex is made up of collenchymatous and
parenchymatous tissues. There is present a
continuous layer of Chlorenchyma cells and
Collenchymatous cells above the cortical cell
layers. Endodermis is quite distinct. Its cells contain
large starch grains and hence referred as the starch
sheath. Multilayered pericycle is irregular and made
up of large distinct sclerenchymatous cells (Figure
1-c). The pericycle encloses the vascular bundles.
Vascular bundles are arranged in a ring (Figures 1-
a & b). They are conjoint (xylem and phloem are
lying on the same radius), collateral (xylem lies
inwards and phloem outwards), closed (absence of
cambial strip between the xylem and the phloem)
and exarch (metaxylem towards centre and
protoxylem faces the periphery) type. Xylem is
further differentiated into protoxylem and
metaxylem (Figure 1-d). The parenchymatous
tissues constitute the conjuctive tissue between the
two vascular bundles. The well developed
parenchymatous pith is located in the centre
(Figure1-a).
Root:
In Transverse Section of the root, the outer
most layer is epidermis which is single layered and
consists of thin walled parenchymatous cells. Some
of these cells get elongated to form the new roots
which are later designated as the lateral roots.
Cortex is being depressed and reduced due to the
occurrence of secondary growth in roots. There is
Journal of Research in Biology (2011) 8: 603-608
Sidhu et al.,2011
Fig. 1-a (T. S. Stem; Magnification - 4x)
Fig. 1-c (T. S. Stem; Magnification - 20x)
present a discontinuous peridermal layer which later
on differentiated into phellem and the phellogen
(Figure 2-a). Only two to three layers of cortical
cells are visible in reduced form. The formation of
distinct layers of secondary xylem and phloem
suggests the occurrence of secondary growth
(Figure 2-b). Secondary phloem is red in colour
while the primary phloem has been compressed and
only some of its remnants can be seen. There are
present distinct layers of cambial cells (2 layers)
which are responsible for the secondary growth.
The major portion of root is occupied by the
secondary xylem which is distinguished by its
vessels or xylem tracheids. Xylem tracheids are
usually two to three celled. There are present
distinct layers of thin walled ray parenchyma cells.
Both of these components (vessels and ray
parenchyma) form the secondary xylem while the
primary xylem is present towards the centre in a
crushed miniature form. In the centre there is
Journal of Research in Biology (2011) 8: 603-608
Fig. 1-b (T. S. Stem; Magnification - 10x)
Fig. 1-d (T. S. Stem; Magnification - 40x)
present a small sized parenchymatous pith. It is
surrounded by certain sclerenchymatous cells which
forms a portion of the xylem tissue (Figures 2-c &
d). The occurrence of secondary growth in the root
seems to be an abnormal feature since Silybum
growing in the present investigation area occurs as
an annual species and formation of growth rings in
an annual plant is an exception. Fritz and Saukel
(2011) while studying the anatomy of subterranean
organs of some medicinally used species observed
the presence of secondary phloem. They were of the
opinion that root anatomy of Silybum marianum is
similar to that of Onopordum acanthium and Cnicus
benedictus but preceding secondary growth was the
distinctive feature of Silybum marianum root. Their
findings substantiated the present observations
regarding the secondary growth in the root portion
only.
Leaf:
The leaves are alternate, large, white veined,
605

Fig. 2-a (T. S. Root; Magnification - 4x )
Fig. 2-c (T. S. Root; Magnification - 20x)
glabrous and have strong spiny margins. The main
and an important feature is the presence of white
streaks on the leaf surface. The leaf of Silybum is of
dorsiventral type. The Transverse Section shows the
presence of single layered upper and lower
epidermis which is made up of compactly arranged
barrel shaped cells. Both the surfaces are covered
up with a thick and wavy cuticle. It is more on the
upper epidermis and lesser on the lower epidermis
(Figure 3-a). The stomata are usually absent in the
epidermal layers which is a distinct feature. Inner to
the epidermis present mesophyll cells which is
differentiated into two to three layered
parenchymatous palisade cells. It consists of green
coloured columnar cells which are arranged in
compact rows. Each palisade cell contains several
chloroplasts positioned around its walls. A
prominent and large vascular bundle (collateral and
closed type) is present in the region of midrib. A
layer of parenchymatous cells separates the vascular
bundles from the epidermis. The vascular bundles
606
Sidhu et al.,2011
Fig. 2-b (T. S. Root; Magnification - 10x)
Fig. 2-d (T. S. Root; Magnification - 40x)
are encircled by a parenchymatous bundle sheath
cells (Figure 3-c). The upper and lower sides of the
vascular bundle are covered by bundle sheath
extensions and sclerenchymatous cells. In addition,
there are present two to four layers of collenchyma
cells near the upper and lower epidermis. Each
vascular bundle consists of xylem present towards
the upper epidermis and phloem close to the lower
epidermis. Xylem is differentiated into metaxylem
and protoxylem. Protoxylem vessels are smaller in
size and facing towards the upper epidermis
(Figure 3-d). Phloem has various components like
sieve tubes, companion cells and phloem
parenchyma cells. Along with the largest vascular
bundle present in midrib, there are present certain
accessory bundles which are smaller in size and
they are present towards the upper epidermis near
the wing of the leaf (Figure 3-b). These accessory
vascular bundles may be developed to meet the
requirements in greater amount through
translocation system. For example to provide
Journal of Research in Biology (2011) 8: 603-608
Sidhu et al.,2011
Fig. 3-a (T. S. Leaf; Magnification - 4x)
Fig. 3-c (T. S. Leaf; Magnification 10x)
nourishment to the densely crowded flowers
(Holroyd, 1928). The presently investigated species
show profuse growth during winter season and
flower nearly at the end of the winter season in this
study area. Accessory vascular bundles must have
been developed to overcome winters on reserve
food material.
In the available literature, there is hardly any
report on the complete anatomical studies of this
species. We have come across only one publication
(Fritz and Saukel, 2011) discussing the occurrence
of secondary phloem in the root portion of this
particular species. Since Silybum marianum is a
plant of high medicinal importance both in
traditional systems of medicines and in modern
medical treatments, its anatomical characterisation
is of great significance for its precise identification.
So, the main contribution of Silybum marianum is
its quite astonishing and vital medicinal properties
due to which it is gaining huge importance in
medical science. Due to large number of medicinal
applications, it proved to be highly beneficial for
Journal of Research in Biology (2011) 8: 603-608
Fig. 3-b (T. S. Leaf; Magnification - 10x)
Fig. 3-d (T. S. Leaf; Magnification - 20x)
human beings.
CONCLUSION:
Since in the present investigation area, plant
usually occurs as an annual weed, preferably in the
undisturbed regions, the exact reason behind the
preceding secondary growth in the root portion is
not clear. It can only be assumed that plant is
shifting from its annual habit to the perennial ones
as it has been reported (its biennial nature) in some
parts of the world. The initiation of secondary
growth in the root portion further confirm its
changing habit towards perennial species nature
since once the roots have retained viability, then it
can give rise to new plants in the next season. This
character (secondary growth) has not appeared in
the stem portion of the species. So this is a case
where only one plant part has shown the occurrence
of secondary growth. Further investigations are in
process to understand the reason behind this
characteristic feature of the root. However,
anatomical characterisation of this species is almost
607

similar to many other species. Leaf anatomy has
shown the occurrence of some accessory vascular
bundles. This character has already been reported
by many researchers and it is there to meet the
translocation requirements during the unfavourable
conditions.
ACKNOWLEDGEMENT:
Authors are thankful to the Chairperson,
Department of Botany, Panjab University,
Chandigarh for providing necessary facilities during
this work.
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Corchete P. 2008. Silybum marianum (L.) Gaertn.:
The Source of Silymarin. In: Bioactive Molecules
and Medicinal Plants, Ramawat K. G., Merillon J.
M. (eds.) 123-148.
Dengler NG. 2002. An Integral Part of Botany:
Book Review. American Journal of Botany 89(2):
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Journal of Research in Biology (2011) 8: 603-608
 Journal of Research in Biology
An International Online Open Access
Original Research Paper
Publication group

Dominant cyanobacterial flora of the Religious ponds at Holy Geetas

birthplace, Kurukshetra, India
Journal of Research in Biology

Authors: ABSTRACT:
Laxmi Biban and Chandra
Bhushan Singh The cyanobacterial biodiversity analysis from different fresh water of holy
aquatic bodies was conducted during summer (May-June, 2009) and autumn months
(Aug.-Sept., 2009) in the village Jyotisar of Kurukshetra, Haryana, India. Similar studies
were performed during the same periods of next year (2010). This place is popularly
Institution:
Cyanobacterial Research known as the birthplace of holy Bhagwat Geeta (=Geetas Janmsthal), which is
Laboratory, Department of geographically located at Lat. 2905753.54N; Long. 7604907.56E. There are mainly
Botany, Kurukshetra two ponds situated there viz. Pond-1 and Pond-2. Both of them were analyzed for
University, Kurukshetra - their physicochemical and biological properties. Our observations have revealed
136119, Haryana, India. various noticeable variations recorded in the cyanobacterial flora according to
seasonal and environmental variations among the two ancient ponds. Unicellular
cyanobacterial strains dominated the Pond-1, where people from all over India take
holy dip. Contrary to this, Pond-2 was dominated by mainly filamentous cyanobacteria
Corresponding author: along with massive growth of higher aquatic plants like Nelumbo. The entire microbial
Chandra Bhushan Singh community was dominated chiefly by cyanobacteria and diatoms.

Keywords:
Email: Cyanobacterial biodiversity, physicochemical properties, Kurukshetra, Geeta
cbsinghbhu@gmail.com Birthplace.

Web Address: Article Citation:

http://jresearchbiology.com/ Laxmi Biban and Chandra Bhushan Singh
Documents/RA0147.pdf. Dominant Cyanobacterial Flora of the Religious Ponds at Holy Geetas Birthplace,
Kurukshetra, India
Journal of research in Biology (2011) 8: 609-616

Dates:
Received: 17 Nov 2011 /Accepted: 01 Dec 2011 /Published: 12 Dec 2011

Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
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cited.

609-616 | JRB | 2011 | Vol 1 | No 8

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INTRODUCTION
Though, the persistence and stability of
various organisms in fresh water ecosystems has
been well documented (Scarsbrook, 2002; Soininen,
et al., 2004) yet, cyanobacteria need particular
attention owing to their habit of forming blooms up
to extremes throughout the summer in tropical
countries like India (Pearl, et al., 2001). The
taxonomic account of the freshwater algae (mainly
Cyanophyta, Bacillariophyta and Chlorophyta)
collected from the tank of Ekling Ji temple at
Udaipur; Rajasthan, India has also been well
documented (Pandey et al., 2004).
The present study site is mainly centered at
the northern part of India at the ancient and
historical place called Jyotisar at Dist.
Kurukshetra, Haryana, India (PLATE-I, actual
view & habitat of the study site). It is the birth place
of holy Geeta, where Lord Krishna preached the
lesson Geeta (the holy epic of Hindus) to Arjuna
during the war of Mahabharata and later became
famous as the Geeta Updesh Sthal. Its
geographical location is 2905753.54N Latitude
and 7604907.56E Longitude (PLATE-I, satellite
view). This place is presently a village called
Jyotisar, which is about 5 Km from Kurukshetra
University. It is believed that the same live holy
Akshya Vat Wriksha (the immortal Ficus
bengalensis tree) is still present here since the time
of Mahabharata. There are mainly two ponds
here. Pond-1 is used mainly for religious purpose,
while the 2nd one is from ecological point of view;
alternatively it is also used for either recreation
purposes, lying idle or occasionally for Nelumbo
cultivation by Govt. Agencies. Both the ponds are
interconnected via a small bridge. This non-
religious adjacent pond is the breeding grounds for
some higher genera like Eichhornia, Hydrilla and
Nelumbo etc. The preliminary studies on the
biological diversity of cyanobacterial flora in this
area have inspired to go for the present
investigation (Biban, et al., 2010). The
cyanobacterial biodiversity of this study site was
enumerated during two seasons: summer (May-June
2009) and autumn months (Aug.-Sept. 2009). It was
repeated during the same periods of next year, 2010
(PLATE-II & III). Similar work has been done on
various aquatic habitats of India and in other
countries (Pingale, et al., 2005; Samad, et al., 2008;
Macedo, et al., 2009), but an ancient place of such
historical importance has yet to be touched upon.
Therefore, the study site is an interesting religious
place to work on the studies of cyanobacterial and
610
Singh et al.,2011
other algal flora. The interest towards the study of
the cyanobacterial communities present in various
water reservoirs of the ancient places at
Kurukshetra, fuelled to enumerate the richness in
their biodiversity among the holy ponds. Such
freshwater habitats are the breeding grounds for
various cyanobacterial blooms and diatoms.
However their population fluctuates according to
the seasonal variations in summer, rainy, autumn
and winter seasons. The changes in biomass of N2-
fixing cyanobacteria and density of heterocysts are
strongly coupled with the depletion of dissolved
inorganic N or N-limitation with high irradiance
and high N2- fixation rates (Phlips, et al., 1997).
Hence the present work was aimed to understand
the biological diversity of cyanobacteria of the two
major fresh water ponds at Jyotisar and other
nearby ancient areas of Kurukshetra for exploring
their innate potentials; because such aquatic
systems also receive rain water, run off of
surrounding agricultural land containing the
agrochemicals (like pesticides), affect the algal
biodiversity of the pond by ameliorating their
ecophysiology (Kalia, et al., 2011).
MATERIALS AND METHODS
Study area and sample collection
The observations and sampling was done
twice in the calendar year of 2009 and 2010 during
summer season, as well as, after the Monsoon rains
in autumn season with a regular interval of
fortnight. There are several other natural and
artificial fresh water ponds distributed in and
around Kurukshetra. They are dominated with
various seasonal algal blooms. Planktonic
cyanobacterial samples were collected from the
epilimnion at the two different fresh water ponds of
Jyotisar in the month of May-June and Aug-Sept of
the year 2009 and 2010 from the two aquatic bodies
of Jyotisar using plastic plankton nets (0.1mm
pore size), forceps and knifes. The collected
cyanobacterial samples were isolated and
transferred to conical flasks with Allen and Arnons
nitrogen free medium (AA- medium) to grow them
in a culture room (2410C) with a16/8 h light/dark
cycle and 14.4 Watt/m2 cool fluorescent light on the
surface of the culture vessels for further
ecophysiological studies (Allen, et al., 1955). The
AA- medium was found to be most suitable to
isolate and maintain the strains like Anabaena,
Nostoc and Merismopedia etc. However, Chlorella,
Vaucheria and Cladophora were later maintained in
Chu-10 liquid medium (Chu, 1942). The traditional
Journal of Research in Biology (2011) 8: 609-616
Singh et al.,2011

taxonomic criteria based on the morphological
characteristics were used for their identification
(Desikachary, 1959).
All chemicals used were of analytical
reagent grade and prepared in distilled water. A
single beam visible spectrophotometer (Electronics
India Ltd., India) was used for measuring
absorption data.
Parameters studied
a) Temperature measurement: It was measured
using a wet thermometer at 30 cm depth of the
ponds.
b) pH measurement: The approximate pH was
estimated on the spot in the water of both the ponds
using pH paper and later verified through a digital
pH meter (pH range: 0-14; Electronics India Ltd.,
India).
c) Transparency estimation: The transparency of
aquatic bodies was measured with the help of
Secchi disc (20 cm in diameter) pointed alternating
black and white.
d) Protein estimation: Protein was quantified as
g protein/ml algal suspension (Lowry, et al., 1951;
Herbert, et al., 1971).
e) Ammonium estimation: Ammonium as NH4Cl
(BDH, U.K.) was estimated calorimetrically
(sensitivity range, 0.04-5.0 g NH4+/ml algal
suspension) using Nesslers reagent (Burris, et al.,
1957).
f) Nitrate estimation: NO3- concentration in the
water of two ponds was measured using
conventional colorimetric method (sensitivity
range, 10-100 g NO3-/ml algal suspension) using
4% Brucine (SISCO, India) in Chloroform (BDH,
India) and concentrated H2SO4 (Qualigens, India)
(Nicholas, et al., 1957).
3. Microphotography: Microphotography was
carried out using a microscope (Make: Suswox
Optik, India), an electronic camera (Make: Nikon,
Model No. Coolpix S4000, Japan) and printed with
the help of a laser printer (Make: Canon, model No.
LBP-1210, Japan) connected to a computer.

PLATE -I. (i) Satellite view of the study sites:

Geographical location: Lat. 2905753.54N; Long. 7604907.56E.

(ii) Actual view of the site and their Habitat:

Pond -1 (Religious Pond - Actual worship and Pond -2 (Non-religious Pond):

pilgrimage area):

Journal of Research in Biology (2011) 8: 609-616 611

 Singh et al.,2011

RESULT AND DISCUSSION being reduced, leading to the eutrophication of such

The cyanobacterial as well as algal flora
were observed as being attached not only to stairs
of Pond-I but also in the free floating zones of both
the ponds (PLATE-II). Unicellular cyanobacterial
strains along with few filamentous genera
dominated the Pond-1, where people from all over
India take holy dip (PLATE-I, Pond-1). During the
summer sampling time of May-June 2009, the
dominant cyanobacterial floras were the unicellular
forms like Synechocystis, Merismopedia,
Microcystis and Chroococcus and Desmids. In this
pond (Pond-1), low diversity of cyanobacteria (May
-June 2010) was attributed to a massive bloom of
Microcystis, which imparted significant
ameliorating impacts on other algal populations.
During summer season, condensed thick
populations of cyanobacterial blooms as observed,
were due to the higher concentrations of nutrients
like NH4+ and NO3- getting increased and pH level
aquatic bodies (Table-1 & 2). On the other hand,
during/after monsoon season when the samples
were collected in Aug.-Sept. (autumn season) in
both the calendar years, the cyanobacterial blooms
revealed less populations among the entire algal
community at the study sites. During the sampling
period (Aug-Sept 2009), the dominant
cyanobacterial flora were the filamentous forms like
Oscillatoria, Anabaena, Lyngbya etc. and the algae
other than the cyanobacteria were predominantly
the Chara, Nitella and Coleochaete on Eichhornia
growing either submerged or on other higher
aquatic plants. Other genera like Aphanothece,
Merismopedia, Oscillatoria and Lyngbya were
observed in lesser populations, from Pond-1 which
has been attributed to the high pH and lesser
bioavailability of the nitrogenous nutrients like
NH4+ and NO3-. Consequently, the protein level of
collected cyanobacterial bloom samples also varied

Table No.-1 (POND-1: Religious Pond):

Year 2009 Year 2010
PARAMETERS
May-June August-September May-June August-September

Temperature (oC) 30.00.8-32.00.9 28.01.0-30.00.9 27.00.8-31.00.8 27.00.9-29.00.8

pH range 5.8-7.4 7.5-8.4 5.9-7.4 7.6-8.4

Transparency (cm) 50.20.7-52.20.8 30.10.8-34.00.8 51.00.9-53.50.9 32.20.9-35.70.8

Protein
0.110.4-0.140.3 0.160.5-0.190.8 0.100.3-0.150.7 0.170.7-0.200.8
(g protein/ml algal suspension)
NH4+
2.50.4-4.00.3 1.50.5-3.00.4 3.50.3-4.50.4 1.40.2-2.80.2
(g NH4+/ml algal suspension)
NO3-
31.00.8-35.00.7 24.00.8-28.00.8 32.00.8-37.00.7 26.00.6-29.00.8
(g NO3-/ml algal suspension)
Values are mean 3SD.

Table No.-2 (POND-2: Non-religious Pond):

Year 2009 Year 2010
PARAMETERS
May-June August-September May-June August-September
Temperature (oC) 28.00.8-31.00.9 29.00.8-30.00.8 29.00.7-32.00.8 28.00.9-29.00.8

pH range 6.0-7.0 7.5-9.5 5.5-7.5 7.5-9.0

Transparency (cm) 45.40.6-49.20.5 25.30.7-29.10.8 16.20.6-19.40.2 23.10.8-27.30.9

Protein
(g protein/ml algal 0.130.1-0.170.1 0.180.1-0.210.1 0.120.1-0.160.1 0.190.1-0.220.1
suspension)
NH4+
2.00.3-3.50.5 1.40.3-2.50.4 2.50.4-4.00.5 1.20.2-2.30.5
(g NH4+/ml algal suspension)
NO3-
30.00.8-34.00.8 23.00.5-27.00.9 31.00.8-36.00.6 25.00.8-29.00.9
(g NO3- /ml algal suspension)
Values are mean 3SD.
612 Journal of Research in Biology (2011) 8: 609-616
Singh et al.,2011
PLATE -II. Cyanobacterial and algal flora attached on the stairs of Pond-1 and in the free floating zone of Pond-1 & 2
according to the seasonal variations (Smith, et al.,
1999).
At Pond-1, the high temperatures (30-320C),
low pH (5.8-7.4), high transparency (50.2 to 52.2
cm), lower NH4+ (2.5-4.0 g NH4+/ml algal
suspension) and NO3- levels (31.0-35.0 g NO3-/ml
algal suspension) in the year 2009 favoured the
growth of unicellular cyanobacteria viz; Microcystis
bl ooms, M eri smopedi a, Synechocyst i s,
Chroococcus and Aphanothece along with desmids
like Pinnularia, Nitzschia, Nedium, and Navicula
(Table-1; PLATE-III [A]). However, the pattern of
cyanobacterial biodiversity was different at Pond-2
where higher aquatic macrophytes like Eichhornia,
Hydrilla, Azolla and Nelumbo etc. dominated the
aquatic body. Prominent cyanobacterial blooms of
Microcystis were recorded during the summer
months of both the calendar years (2009 & 2010).
As evident (Table-2) on comparative
grounds, during the summer months of 2009, except
NO3- levels, the temperature, transparency and
NH4+ were towards lower side. This favoured
immense growth of filamentous cyanobacteria like
Oscillatoria, Lyngbya and Anabaena (Table-2;
PLATE-III [B]). During the same period in next
year 2010, the temperature was comparatively low
at Pond-1 with higher pH than that in year 2009, but
at Pond-2; it was just a reversed case (Table-1 & 2).
It was interesting to note a marked decrease in the
transparency from (51.0 - 53.5) to (16.2 - 19.4) in
Pond-2 as compared to Pond-1. This favoured to
record higher total protein content per ml algal
suspension in pond-2 than that of Pond-1, yet, the
NH4+ and NO3- levels of the algal suspensions were
towards higher side in Pond-2. A corollary of such
observations favoured much better growth of
almost all phytoplanktons in the Pond-2. In the post
-monsoon season of the year (2009) which was
approximately the same for the same period in Pond
-2 also. However the pH range and transparency
Journal of Research in Biology (2011) 8: 609-616
were recorded towards lower sides in both years for
both ponds, which favoured higher protein value of
algal mass in pond-2 than pond-1. If one looks into
the nitrogen balance of pond systems, the NH4+ &
NO3- levels at Pond-1 were lower than that of Pond-
2. On comparative grounds in pond-1 & 2, there
was an increasing trend observed from the year
2009 to 2010 with respect to NO3- levels. While
sampling in Aug-Sept 2009, the dominant
cyanobacterial flora were the filamentous forms like
Oscillatoria, Anabaena, Lyngbya etc. and the algae
other than the cyanobacteria were predominantly
the Chara, Nitella and Coleochaete on Eichhornia
and other higher aquatic plants. The stairs get
slippery due to the growth of organisms like
Aphanothece. It was also strange to observe the
balls of Anabaena iyengerii on the leaves of
Nelumbo in Aug 2010. Six species of the
cyanobacteria viz., Microcystis, Merismopedia,
Aphanothece, Oscillatoria and Lyngbya were found
in the 1st pond (the religious one), whereas 2nd pond
(the non-religious one), was mainly dominated by
the filamentous cyanobacterial species like
Oscillatoria, Anabaena and Lyngbya. However, in
comparison the unicellular forms like Chroococcus,
Chlorella, Synechocystis and Aphanothece were
also detected in smaller strength.
As per the diversity and abundance of
cyanobacteria, the members of the family
Oscillatoriaceae were dominating in the surveyed
ponds. However, the Diatoms were in abundance in
both ponds together with some other algae like
Chara, Nitella, Coleochaete, Chlorella, and
Batrachospermum (data not presented here).
Similarly, there was a clear cut variation in other
parameters studied (Table-1 and Table-2) for the
Pond-1 and Pond-2 respectively. In May-June of
both the calendar years (2009 & 2010), the nutrient
concentrations get increased at both study sites due
to the availability of lesser quantity of water owing
613
 Singh et al.,2011

to evaporation under hot climatic conditions
(temperature ranges, 40-450C). The transparency of
the two ponds was found high during this time
period. Such aspects ultimately lead to the
eutrophication among the two aquatic bodies.
In any ecosystem, not a single species grows
independently and indefinitely, because all the
species are interlinked and has cyclic
transformation of nutrients according to prevailing
environmental conditions. The physicochemical
changes in the environment not only induce a
particular species but the entire biota. It
considerably affects the growth and abundance of
other species, which leads to the succession of
several species in a course of time. Though, the
observations and sample collections were made on
a regular basis throughout the year, but the data has
been depicted as summer and autumn seasons. It
has been observed that the cyanobacterial
dominance often occurs when water temperature
rises above 200C, when there is depletion of
dissolved inorganic N and free CO2 from the water
(Table-1 & 2). It is reported that low nitrogen to
phosphorus ratios favour the dominance and
production of high blue green algal biomass in
similar aquatic bodies (Smith, 1983). In shallow,
mixed water bodies with high total phosphate
content organisms like Oscillatoria dominates,

PLATE -III. SAMPLES COLLECTED: [A] Unicellular Cyanobacterial Strains

Chroococcus Synechocystis Merismopedia

Microcystis Aphanothece

[B] Filamentous Cyanobacterial Strains:

Oscillatoria Lyngbya Anabaena

614 Journal of Research in Biology (2011) 8: 609-616
Singh et al.,2011
whereas in stable water columns showing depletion
of dissolved inorganic N and high temperature, N2-
fixing organisms like Anabaena dominates
(Reynolds, et al., 1987). Contrary to this, Pond-2
was dominated by mainly filamentous
cyanobacteria along with few unicellular ones.
However, the adjacent site, Pond-2 was dominated
by mainly filamentous cyanobacteria like
Oscillatoria and Lyngbya, along with
Synechocystis, Aphanothece, Chroococcus and
Anabaena. The accessory pigments which support
the net growth to occur even at low irradiance
(Scheffer, et al., 1997) and the buoyancy (Walsby,
1994) allowing certain taxa to bloom at the water
surface appeared to have played crucial role(s)
towards the dominance of cyanobacteria (Reynolds,
et al., 1987). Microcystis is one of the dominant and
persistent organisms, invariably associated with the
stability of almost permanent blooms in tropical
freshwaters that are exposed to constant sunshine,
warmth, and nutrients (Scarsbrook, 2002). The
development of cyanobacterial blooms in any
ecosystem, the siderophore mediated iron uptake is
believed to be a contributing factor in their ability
to dominate other microalgae (Murphy, et al., 1976;
Bailey, et al., 1980).
A phytoplankton and water quality survey
was conducted to evaluate the trophic state and blue
-green algal pollution during flood and dry seasons
in the year 2000 for the 19 typical reservoirs of
Guangdong Province in China (Zhao Hui et al.,
2004). During intense blooms, the photosynthetic
activity depletes free CO2 from the water body and
pH is driven up. The phytoplankton community of
Ranital, India was dominated by the tolerant
species of Cyanophyceae, Chlorophyceae and
Diatoms (Arjariya, 2003). Their distribution was
markedly influenced by the physicochemical as
well as biological nature of the aquatic system.
CONCLUSION
Based on our findings, it has been concluded
that in the aquatic ecosystems as studied, the
seasonal variations in the physicochemical and
biological profile of the fresh water aquatic bodies
governs the phytoplankton community in general
and entire aquatic microbial community in
particular. It was dominated mainly by the
unicellular cyanobacteria, followed by filamentous
ones and other algal organisms.
Journal of Research in Biology (2011) 8: 609-616
ACKNOWLEDGMENTS
The authors are thankful to the Department
of Botany, Kurukshetra University, Kurukshetra
(Haryana) for providing basic laboratory
infrastructure. One of the authors (Laxmi Biban) is
also thankful to CSIR, New Delhi for providing
financial support in the form of SRF.
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Biochemical changes in tissues of albino rats following subchronic
exposure to crude oil
Journal of Research in Biology
Authors:
Nwaogu LA1, Igwe CU1,
Ujowundu CO1, Arukwe
U1, Ihejirika CE2 and
Iweke AV1.
Institution:
1 serum albumin, cholesterol and glucose, liver total protein, ascorbic acid and reduced
Department of
Biochemistry, Federal
University of Technology,
Owerri, Nigeria.
2 were no significant (P>0.05) changes in these parameters from the rats fed with
Department of
Environmental Technology,
Federal University of
Technology, Owerri,
Nigeria.
Corresponding author:
Nwaogu LA
Email:
nwogulinus@yahoo.com
Phone No:
+234-8037510952
Web Address:
http://jresearchbiology.com/
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ABSTRACT:
Biochemical changes in the tissues of thirty-six albino rats (Rattus rattus)
following subchronic exposure to 1.25% , 2.50% and 5% of crude oil polluted feed
was investigated. There were age and sex matched with another twelve albino rats fed
without crude oil. The exposure period lasted for thirty days. The concentrations of
glutathione (GSH) as well as the activities of alanine amino transferase (ALT),
aspartate amino transferase (AST), alkaline phosphatase (ALP) and catalase (CAT) from
the rats were determined using standard methods. The results indicate that there
1.25% and 2.50% crude oil mixed feed when compared with the control. However,
significant (P<0.05) reductions were observed in glucose, ascorbic acid and GSH
concentrations as well as in catalase activity, with a concomitant significant (P>0.05)
increases in serum ALT, AST and ALP activities in the rats fed 5% crude oil polluted
feed, in comparison with the control. The results indicate that crude oil ingestion at
levels of up to 5% may have serious adverse effect on animal tissues.
Keywords:
Albino rats, crude oil, liver enzymes, pollution.
Article Citation:
Nwaogu LA, Igwe CU, Ujowundu CO, Arukwe U, Ihejirika CE and Iweke AV.
Biochemical changes in tissues of albino rats following subchronic exposure to crude
oil.
Journal of research in Biology (2011) 8: 617-623
Dates:
Received: 24 Oct 2011 /Accepted: 01 Nov 2011 /Published: 12 Dec 2011
Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.
617-623 | JRB | 2011 | Vol 1 | No 8
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 Nwaogu et al.,2011

INTRODUCTION (Pub. No. 85-23, Revised, 1985). After

The growth of the petroleum industry in acclimatization, the animals were divided into four
Nigeria and the marketing of petroleum products (4) groups (A,B,C,D). Groups A, B and C received
have made oil pollution a serious environmental 1.25%, 2.50% and 5.0% feed containing-crude oil
problem (Amund et al., 1987). Pollution of the respectively while group D received feed without
environment may result from the exploration, crude oil and served as the control. Feeding period
transportation or storage of petroleum products or lasted for 30 days.
from accidental events (spills). In some cases it Preparation of Blood and Liver Samples:
may be a consequence of careless disposal practices After 30 days, each rats was euthanized by
of residues like the oily sludge that accumulate in anesthesia using chloroform, and 5ml of Blood was
storage tanks (Morgan and Watkinsona, 1989). collected by cardiac puncture. The blood sample
Crude oil spill is the release of crude was allowed to stand for 10min for clotting to take
petroleum hydrocarbons into the environment due place. The serum was separated by centrifugation.
to human activities and are classified into two main The serum obtained was used for the determination
types; the land (on shore) and marine (off-shore) of serum albumin, chlorosterol and glucose
oil spills. Land oil spill occurs when crude oil is concentrations and assay of the serum (ALT, AST
released on the land which affects soil ecosystem. and ALP). Each rat was then surgically dissected,
The different ways by which crude oil enter the the liver extracted immediately, washed with ice-
environment are from natural seep (1%), cold 1.15% KCl and stored at -10C. Chilled liver
atmospheric input (1%), off-shore production (1%), tissues were later homogenized using a
coastal and estuarine effluents (3%), non-refinery homogenizer ( Janke and Kunkel, Germany). The
industrial wastes (5%), municipal waste (5%), homogenate was centrifuged and was used for the
urban run off (5%), rivers ( 26%) and oil water determination of liver protein, ascorbic acid and
discharge from oil industry (53%)(Carla, 2006; glutathione concentrations, and assay for the
Levorsen, 2008). These pollutants (crude oil and activity of catalase (CAT).
their products) are considered recalcitrant to natural
Determination of Biochemical Parameters:
biodegradation and persist in the ecosystem due to Serum and cholesterol concentrations were
their hydrophobicity and low volatility ( Ademoroti, determined using the methods of Doumas et al.
1996) (1971) and Allian et al. (1974) respectively.
Animals and plants growing in crude oil Glucose concentration was determined based on
polluted environment, take in large doses of glucose oxidase method as described by Trinder
harmful pollutants over the years. These pollutants (1969). The serum activities of ALT and AST were
and any product of their degradation products can assayed using the method of Reitman and Frankel
be carcinogenic, mutagenic and are potent (1957), while that of ALP was assayed as described
immunotoxicants which affect the biological in Tietz (1991). Hepatic Protein concentration was
systems of living things. (Boonchan et al., 2009; determined by Biuret method as described by
Rao and Panya, 1978). This study sought to Gornall et al. (1949). Ascorbic acid concentration
investigate the subchronic effect of different was determined using the method of Roe and
percentage concentrations of crude oil ingestion on Kuether (1961). Ascorbic acid is converted to
the tissues of albino rats. dehydroascorbic acid by shaking with Norit and
this was coupled to 2, 4-dinitrophenyl hydrazine in
MATERIALS AND METHODS the presence of thiourea (a mild reducing agent).
Experimental Animals: Sulphuric acid was then used to convert the
Forty-eight (48) adult male albino rats dinitrophenyl hydrazine to a coloured compound
(Rattus rattus) weighing between 210-380 grammes w h o s e a b s o r b a n c e w a s d e t e r m i n e d
were used for the experiments. The animals were spectrophotometrically at 540nm. Reduced
allowed to acclimatize for 7 days, and were glutathione (GSH) was determined by the method
maintained under laboratory condition of humidity, of Jollow et al. (1974). The method is based on the
temperature ( 25 2 C) and light (12 hr light/ dark formation of a relatively stable chromophoric
cycle) with free access to food and water ad product on reacting a sulphurhydryl compound
libitum. All experimental protocols were in (GSH) with Ellmans reagent. Catalase activity
compliance with the National Institute of Health (CAT, E.C. 1.11.1.1.) was assayed by measuring
Guide for Care and Use of Laboratory Animals spectrophotometrically at 570nm the rate of
618 Journal of Research in Biology (2011) 8: 617-623
Nwaogu et al.,2011
decomposition of hydrogen peroxide (H2O2) over a
period of 30 minutes at ( 1 minute interval ) as
Cholesterol Conc. Mg/100ml
described by Sinha (1972). The enzyme activity for
each tissue was expressed in terms of Katalase
feiahigkeit (kat. f) as ks-1 mg-1 protein where K is
the first order rate constant.
Statistical Analysis
Data generated were expressed as means
standard deviation and analyzed using one way
Analysis of Variance (ANOVA) with p 0.05 taken
to be significant.
RESULTS AND DISCUSSION
There has been an increased incidence of
petroleum hydrocarbon pollution from crude oils,
gas flaring, diesel oil and motor engine oil over the
years ( Odu, 1996; Nwaogu et al., 2008; Nwaogu
and Onyeze 2010). It has been considered by many
researchers that petroleum hydrocarbon-induced
oxidant stress is a critical pathologic mechanism
that initiates various cascades of metabolic
perturbations ( Rao and Pandya, 1978; Romieu et
al., 1999).
Induced changes in liver and blood
parameters of albino rats subchronically exposed to
different percentage concentrations of crude oil
revealed that there were no significant (p>0.05)
reductions in the concentrations of serum albumin,
cholesterol and liver protein of albino rats fed with
feed polluted with crude oil at 1.25% , 2.50% and
5.0% in comparison with the control (Figures 1-3).
There were also no significant ( p > 0.05) reduction
in the concentrations of glucose, ascorbic acid and
glutathione as well as the activities of hepatic
catalase obtained for rats fed with 1.25%, 2.50%
and 5% crude oil polluted feed. (Figures 4, 8-10).
On the other hand, the 1.25% and 2.50% crude oil
Fig. 1: Serum albumin concentration of albino rats
fed different concentrations of crude oil mixed feed.
Journal of Research in Biology (2011) 8: 617-623
Fig. 2: Serum cholesterol concentrations of Albino rats
fed different concentrations of crude oil mixed feed.
polluted feed caused non significant (p>0.05)
increases in serum activities of ALT, AST and ALP
(figures 5-7) . However there were significant
(p<0.05) changes in the mean concentrations
obtained for glucose, ascorbic acid, glutathione and
the activities of ALT, AST and ALP and catalase of
rats fed with feed polluted with 5.0% crude oil
concentrations in comparison with the control
(figures 4-10) indicating that crude oil at that
percentage concentration caused changes in the
blood and liver parameters in albino rats.
The results for glucose concentration (Figure
4) show that there was a dose-dependent reduction
in the blood glucose concentrations as the
percentage of crude oil in the feed increased.
During hypoglycemia, the brain suffers from
substrate deficiency since glucose is the major fuel
for the brain cells ( Nelson and Cox, 2000). This
leads to a decrease in anabolic processes (Patoekora
et al., 2003). The results indicate that the rats fed
with 5% crude oil mixed feed suffered stress as a
Fig. 3: Liver protein concentration of rats fed
different concentration of crude oil mixed feed.
619

Fig. 4: Serum glucose concentration of rats fed
effluent concentrations of crude oil mixed feed.
result of the exposure to crude oil.
Serum ALT, AST and ALP activities are
used as indicator of chemically induced liver
damage ( Drotman et al., 1978). Hepatotoxicity has
been viewed as liver injury associated with
impaired liver function caused by exposure to
xenobiotics ( drugs, petroleum hydrocarbons) and
other non-infectious agent (Navarron, 2006).
Results of this study revealed that there were
significant (p < 0.05) increases in the serum ALT,
AST and ALP activities in rats fed 5.0% crude oil-
mixed feed. These enzymes usually leak out into
the blood stream in cases of liver damage or loss of
integrity. The observed increase in serum activities
of these enzymes indicate a sign of liver
dysfunction as a result of ingestion of crude oil in
the feed (Delvin, 2006).
Ascorbic acid is a water-soluble antioxidant
molecule found in the cytoplasm of the cells, which
scavenges free radicals in the cytosol. It readily
donates electrons to free radicals thereby
Fig. 6: Serum AST activities of rats fed different-
620
Nwaogu et al.,2011
Fig. 5: Serum ALT activities of rats fed different
concentration of crude oil mixed feed.
quenching their effects in the cellular compartments
and is in the process converted to dehydroascorbic
acid (DHAA) ( Mckee and Mckee, 1999). Our
results indicate that there was a significant (p<0.05)
reduction in the concentration of ascorbic acid as
the concentration of crude oil in the feed increased.
This significant reduction could be due to the
utilization of ascorbic acid in scavenging the
reactive intermediates generated in the tissues of the
albino rats fed with the animal feed mixed with
crude oil.
Glutathione (GSH) is usually located in the
cystol, nuclei and mitochondria. It is the major
soluble antioxidant in these cell organelles
( Masella et al., 2005). Reduced glutathione (GSH)
and its oxidized form (GSSG) accumulate inside the
cells. The ratio of GSH to GSSG is a good indicator
of oxidative stress in a living system (Mckee and
Mckee, 1999). The reduction in the concentration of
glutathione either by conjugation and removal from
cell or oxidation to oxidized glutathione could
Fig. 7: Serum ALP activities of rats fed different
concentrations of crude oil mixed feed.
Journal of Research in Biology (2011) 8: 617-623
Nwaogu et al.,2011
Fig. 8: Liver ascorbic acid concentration activities of rats
fed different concentrations of crude oil mixed feed.
significantly affect the overall redox potential of
the cell (Hansen et al., 2001). Our result indicate
that glutathione concentration was significantly (p <
0.05) reduced in rats fed with 5% crude oil-mixed
feed when compared to the control (Figure 9).
The reduction could be a compensatory mechanism
by the animals fed with feed mixed with crude oil
to overcome the effect of the oxidant stress caused
by free radicals generated by crude oil. These
observations are corroborated by earlier studies on
the effects of petroleum hydrocarbon on other
animal species (Nwaogu et al., 2008; Ibrahim and
Rizk, 2008).
Catalase is an enzyme found in nearly all
living organisms (plants and animals) that are
exposed to oxygen where it functions to catalyse
the decomposition of hydrogen peroxide (H2O2) to
water and oxygen ( Diniz et al., 2004; Pigeolet et
al., 1990). A dose-dependent reduction was
Fig. 10: Liver catalase (CAT) activities of albino rats
fed different concentrations of crude oil mixed feed.
Journal of Research in Biology (2011) 8: 617-623
Fig. 9: Liver glutathione (GSH) concentration of albino
rats fed different concentrations of crude oil mixed feed.
observed in the activities of catalase from the liver
tissues of rats fed with 1.25%, 2.25% and 5.0%
crude oil polluted feed. However the reduction in
catalase activity was only statistically significant
(p<0.05) obtained in rats fed with 5.0% crude oil
mixed feed when compared to the control
indicating a possible attenuation of oxidative stress.
CONCLUSION
This study showed that subchronic exposure
to crude oil caused adverse changes in the tissues
of albino rats. These changes if unchecked could
precipitate various disease conditions.
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 Journal of Research in Biology
An International Online Open Access
Original Research Paper
Publication group

Effect of Ulva lactuca extract on growth and proximate composition of

Vigna unguiculata l. Walp.
Journal of Research in Biology

Authors: ABSTRACT:
Gireesh R1+, Haridevi CK2
and Salikutty Joseph3
As organic farming gains more attention, seaweed cultivation and its
Institution:
1, 3 utilization may be an economical approach in agricultural production. The effect of
Department of
seaweed Ulva lactuca extract on growth and proximate composition of Vigna
Horticulture, Kerala
Agricultural University, unguiculata L. Walp was studied. Seedlings of V. unguiculata soaked with seaweed
Vellanikkara P.O., Thrissur, extract performed better when compared to seedlings with water soaked control. The
Kerala, India 680656. low concentration (20%) of aqueous seaweed extract promoted seedling growth in
terms of shoot length, root length, fresh and dry weight and chlorophyll and
2 carotenoids content. The biochemical constitutents show similar patterns with protein
National Institute of
Oceanography, RC- Kochi, content of shoot and root, amino acid of shoot and root, -amylase and -amylase
Ernakulam North P.O., activities being higher with seaweed extract in V. unguiculata. The seaweed extract in
Ernakulam, Kerala, India these experiments showed as biological fertilizer, which is a nontoxic and ecofriendly
682018. supplement to chemical fertilizer for many crops intended to attain higher yields.
+
Present address: Central
Marine Fisheries Research
Institute, North P.O.,
Ernakulam, Kerala, India Keywords:
682018. Crop, seaweed extract, biochemical composition, fertilizer, organic farming

Corresponding author: Article Citation:

Gireesh R Gireesh R, Haridevi CK and Salikutty Joseph
Effect of Ulva lactuca extract on growth and proximate composition of Vigna
unguiculata l. Walp.
Journal of research in Biology (2011) 8: 624-630
Email:
girmsr@gmail.com
Dates:
Received: 17 Nov 2011 /Accepted: 26 Nov 2011 /Published: 13 Dec 2011

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 Gireesh et al.,2011

INTRODUCTION hour, and filtered through GF/C paper. The filtrate

Seaweeds have been used as green manure, was taken as 100% concentration of the seaweed
cattle feed, food for human consumption and as a extract and from this stock solution, different
source of phycocolloids such as sugar, alginic acid concentrations (5%, 10%, 20%, 30%, 40% and
and carrageenan (Chapman, 1970). Besides their 50%) were prepared using distilled water (Bhosle et
application as green manure, liquid extracts al, 1975). As the seaweed liquid fertilizers
obtained from seaweeds (SLF) have gained contained organic matter, they were refrigerated
importance as foliar sprays for several crops (Thivy, between 0 and 4 C. The colour, pH, calcium,
1961, Bokil et al, 1974) because the extract magnesium, sodium, potassium, iron, chloride,
contains growth promoting hormones auxins (IAA, sulphur, silicon, aluminium, zinc, copper and nitrate
IBA), cytokinins, trace elements, vitamins and content were analyzed by the method described by
amino acids (Challen and Hemingway 1965). Thus, American Public Health Association (APHA,
these extract when applied to seeds or when added 1995).
to the soil, can stimulate growth of plants (Blunden, The crop plant selected for the present study
1971). Green manure was found to be better than was Vigna unguiculata belonging to the family
chemical fertilizers because of the high level of Fabaceae. Seeds with uniform size, colour and
organic matter and colloidal compounds that aids in weight obtained from Seed Bank of Kerala
retaining soil moisture and minerals in the upper Agricultural University chosen for the experimental
soil level that are available to the roots (Wallen, purpose.
1955). Booth (1969) observed that the value of Experiment I (water soaked)
seaweeds as fertilizers was not only due to nitrogen, Eight hundred uniform sized seeds were
phosphorus and potash content, but also because of soaked in water for 24 hours. After soaking, they
the presence of trace elements and metabolites. were divided into batches of 100 seeds each and
Aqueous extract of Sargassum wightii when applied were placed in 8 Petri dishes with filter paper. One
as a foliar spray on Zizyphus mauritiana showed an batch of seeds was considered as the control and
increase yield and quality of fruits (Ramarao, they were watered with 10 ml of tap water every 24
1991). Seaweed extracts are now available hours. The reminder of batches were treated once
commercially as Maxicrop, Algifert Marinure, with 10 ml/Petri dish of 5%, 10%, 20%, 30%, 40%
Goemar GA14, Kelpak 66, Seaspray, Cytex, Seasol and 50% of aqueous seaweed extract every 24
and Seacrop 16. Recent, research demonstrated that hours.
seaweed fertilizers can compete with other Experiment II (seaweed extract soaked)
fertilizers and are very economical (Gandhiyappan One hundred seeds were soaked for each
and Perumal, 2001). This study was undertaken to concentration of aqueous seaweed extracts for 24
investigate the effect of seaweed liquid extract on hours and then were placed in various Petri dish
the growth and biochemical characteristics of Vigna plates with filter paper and sprayed water regularly
unguiculata. every 24 hours. Water soaked seeds were used as
controls. Samples were taken from each set 15 days
MATERIALS AND METHODS after sowing. The growth parameters including
Seaweed Ulva lactuca (Chlorophyceae) used germination percentage, fresh and dry weight and
in the present study was collected from the coastal shoot and root length was calculated. Seven
area of Rameswaram, India (925 N and 7915 E). biochemical constituents, chlorophyll content
The algal species were washed thoroughly with (Arnon, 1949), carotenoid (Mackinney, 1941),
seawater to remove all unwanted impurities, protein (Lowry et al, 1951), amino acids (Moore
adhering sand particles and epiphytes. and Stein 1948), total sugar content (Nelson, 1944)
Morphologically distinct thallus of algae were and -amylase and -amylase activities (Bernfeld,
placed separately in new polythene bags and were 1955) were estimated from V. unguiculata
kept in an ice box containing slush ice and seedlings. Data were statistically analyzed using
transported to the laboratory. Samples were washed correlation of coefficient method and all the
thoroughly using tap water to remove the salt on measurements were made triplicate.
thallus surface. The water was drained off and the
algae were spread on blotting paper to remove RESULTS
excess water. One kg of seaweed was cut into small The physico-chemical properties of seaweed
pieces, boiled with one litre of distilled water for an Ulva lactuca extract was analyzed (Table I). The
625 Journal of Research in Biology (2011) 8: 624-630
Gireesh et al.,2011

Table I. Physico-chemical properties of seaweedsoaked and seaweed extracts soaked seeds. The
Ulva lactuca extract germination percentage increased with
concentration up to 20% and thereafter it declined.
Parameters Seaweed extract
No germination was seen at concentrations above
Physical parameters 50%. The lowest germination percentage (19%)
Colour Yellow was found in water soaked seeds at 50%
pH 7.3 concentration. The highest shoot length (15.7 cm/
Chemical parameters (mg.l) seedling), root length (5.41 cm/seedling), fresh and
Sodium 185.00
Potassium 113.00
dry weight (3.970, 0.807 g/seedling) was observed
Magnesium 108.30 at 20% concentration of seaweed extract soaked
Calcium 195.26 plants. The lowest shoot length (8.20 cm/seedling),
Phosphorous 51.35 fresh and dry weight (1.980, 0.701 g/seedling) were
Iron 0.37 found at 50% concentration of seaweed extract
Chloride 415.55 soaked seeds. The biochemical constituents
Sulphate 16.84 increased with concentration levels up to 20% and
Silica 38.12 thereafter declined (Tables III a-c). The highest
Copper 0.38 values of chlorophyll content (2.268 mg/g fr. Wt.),
Zinc 1.01
carotenoid (0.852 mg/g fr. wt.), amino acid content
Nitrate 19.05
of shoot (1.343 mg/g fr. wt.), and amylase
pH of the yellow coloured extract was 7.3 and had (1.639, 1.613 g/min/mg protein respectively), total
high levels of calcium, sodium, potassium, sugar content of shoot and root (11.207, 8.602 mg/g
magnesium, phosphorous, iron and chloride. The fr. wt.) were recorded at 20% seaweed extract
effect of extract on germination percentage and soaked seedlings. The lowest values were observed
growth of V. unguiculata are presented in Tables at 50% U. lactuca extract soaked seedlings (Tables
IIa and IIb. Maximum seed germination (99%) III a-c). There was a significant difference in
was found at 20% concentration in both water growth and biochemical status at different

Table IIa. Effects of seaweed extract on germination and growth of V. unguiculata seedlings
Germination (%) Shoot length (cm/seedling) Root length (cm/seedling)
I II I II I II
Control 85 2.65 85 2.65 7.0 0.20 7.0 0.20 2.5 0.06 2.5 0.06
5% 93 0.58 97 0.58 10.7 0.20 11.7 0.06 2.6 0.12 2.6 0.06
10% 94 1.0 97 1.0 13.3 0.15 13.9 0.06 3.0 0.12 3.5 0.12
20% 99 0.58 99 0.58 14.2 0.06 15.7 0.21 4.2 0.06 5.4 0.06
30% 91 1.7 93 0.58 12.4 0.06 13.1 0.20 4.4 0.10 4.8 0.06
40% 65 0.58 78 1.00 8.60 0.10 9.3 0.20 3.0 0.12 4.6 0.07
50% 19 1.15 37 11.0 8.20 1.15 8.7 0.06 2.9 0.05 2.9 0.20
I Water soaked; II- Seaweed extract soaked

Table IIb. Effects of seaweed extract on germination and growth of V. unguiculata seedlings
Seedling (g/seedling)
Fresh weight Dry weight
I II I II
Control 1.82 0.06 1.82 0.06 0.679 0.00 0.679 0.00
5% 1.99 0.58 2.06 0.12 0.719 0.01 0.731 0.00
10% 2.07 0.6 3.93 0.05 0.782 0.00 0.873 0.01
20% 2.52 0.03 3.97 0.04 0.807 0.01 0.882 0.01
30% 2.12 0.01 3.73 0.36 0.800 0.00 0.875 0.01
40% 1.78 0.02 1.98 0.01 0.701 0.15 0. 790 0.00
50% 1.85 0.05 1.98 0.01 0.688 0.01 0.701 0.01
I Water soaked; II- Seaweed extract soaked
Journal of Research in Biology (2011) 8: 624-630 626
 Gireesh et al.,2011

Table IIIa. Effects of seaweed extract on proximate composition of V. unguiculata leaf

Leaf (mg/g fr. wt.)
Chlorophyll Carotenoids
I II I II
Control 1.253 0.11 1.253 0.11 0.667 0.01 0.667 0.01
5% 1.293 0.02 1.793 0.02 0.790 0.00 0.826 0.01
10% 1.370 1.01 1.960 0.00 0.826 0.00 0.840 0.01
20% 1.427 0.01 2.268 0.02 0.850 0.00 0.852 0.00
30% 1.110 0.00 1.226 0.00 0.604 0.00 0.647 0.02
40% 0.980 0.01 1.000 0.00 0.355 0.01 0. 422 0.01
50% 0.595 0.00 0.624 0.01 0.310 0.01 0.711 0.01
Leaf (mg/g fr. wt.)
- amylase - amylase
I II I II
Control 1.388 0.003 1.388 0.003 1.354 0.000 1.354 0.000
5% 1.402 0.001 1.522 0.020 0.378 0.001 1.404 0.010
10% 1.477 1.064 1.530 0.001 0.383 0.001 1.466 0.010
20% 1.636 0.005 1.639 0.010 1.566 0.005 1.613 0.031
30% 0.852 0.045 0.896 0.010 1.125 0.576 0.864 0.010
40% 0.396 0.003 0.509 0.031 0.668 0.005 0.765 0.011
50% 0.289 0.003 0.288 0.001 0.606 0.005 0.734 0.030
I Water soaked; II- Seaweed extract soaked

Table IIIb. Effects of seaweed extract on proximate composition of V. unguiculata shoot

Shoot (mg/g fr. wt.)
Total sugar Protein Amino acid
I II I II I II
Control 8.340 0.003 8.340 0.003 1.548 0.001 1.548 0.001 0.434 0.001 0.434 0.005
5% 10.004 0.01 10.280 0.02 2.513 0.002 2.632 0.004 0.627 0.003 0.646 0.003
10% 10.632 0.003 10.670 0.004 2.915 0.004 3.084 0.004 0.847 0.003 0.864 0.005
20% 11.270 0.003 11.290 0.016 3.156 0.005 3.338 0.005 1.279 0.002 1.343 0.002
30% 8.276 0.002 8.340 0.002 1.315 0.004 1.503 0.012 0.787 0.002 0.858 0.001
40% 6.369 0.010 6.391 0.006 0.983 0.001 1.114 0.040 0.691 0.001 0.752 0.001
50% 0.509 0.008 0.553 0.003 0.863 0.006 0.983 0.007 0.602 0.002 0.616 0.001
I Water soaked; II- Seaweed extract soaked

Table IIIc. Effects of seaweed extract on proximate composition of V. unguiculata root

Root (mg/g fr. wt.)
Total sugar Protein Amino acid
I II I II I II
Control 5.803 0.003 8.340 0.003 1.124 0.005 1.124 0.005 0.407 0.003 0.407 0.003
5% 7.481 0.002 7.533 0.010 2.124 0.004 2.120 0.056 0.430 0.002 0.480 0.010
10% 7.816 0.003 7.906 0.005 2.131 0.002 2.142 0.002 0.493 0.006 0.554 0.004
20% 8.496 0.001 8.602 0.001 2.542 0.008 2.721 0.008 0.554 0.003 0.684 0.010
30% 6.433 0.003 6.627 0.037 0.551 0.026 0.555 0.003 0.506 0.006 0.633 0.010
40% 4.394 0.007 4.620 0.011 0.429 0.001 0.452 0.016 0.451 0.002 0.595 0.008
50% 3.492 0.003 3.001 0.021 0.400 0.003 0.413 0.003 0.408 0.002 0.440 0.002
I Water soaked; II- Seaweed extract soaked
627 Journal of Research in Biology (2011) 8: 624-630
Gireesh et al.,2011

concentration levels. Correlation of coefficient was 1994), maize, ragi and kambu (Rajkumar and
carried out to find the significance level (*p<0.05 Subramanian, 1999) and Dolichos buflorus
and ** p<0.01). High significance was observed in (Anantharaj and Venkatesalu, 2002). Statistically
20% concentration (Table IV). significant differences were observed for leaf
pigments and total sugar contents in both shoot and
DISCUSSION root. A positive response was observed for shoot
Vigna unguiculata seeds soaked with lower length at 10-20% seaweed extract soaked seedlings.
concentration (5-40%) of the seaweed extract Trace elements, especially calcium that exists in
showed high germination rates (85-95%), while this seaweed extract are in a naturally chelated
higher concentrations ( 40%) have inhibited form, which can absorb more readily than from soil.
germination. The increased seedling growth may be The higher concentrations ( 40%) showed a
due to the presence of phenyl acetic acid and other decreasing for V. unguiculata. Similar results were
closely related compounds in the extract (Taylor recorded in C. cajan (Mohan et al., 1994) and
and Wilkinson, 1977) as well as the presence of Vigna radiata (Venkataraman et al., 1993) where
growth promoting hormones like auxins, maximum seedling growth occurred at lower
gibberellins, cytokinins, trace elements, vitamins concentrations of Padina extracts. Dhargalkar and
and amino acids (Challen and Hemingway, 1965). Untawale (1983) also reported comparable results
The present findings agree with field trials in other with red and brown algal extracts on the growth of
crops such as in Cajanus cajan (Mohan et al., chillies, turnips and pineapple.
Table IV. Coefficient values of seaweed extract with respect to different parameters
5% 10% 20% 30% 40% 50%
Seed Germination I 0.655 0.866* 1.000* 0.500 -1.000 -0.500
II 0.655 0.866* 0.500 0.500 -0.866 0.419
Seedling Fresh weight I -0.904 -0.999 -0.533 -0.999 -0.914 -0.983
II -0.604 -0.785 -0.997 -1.000 -0.821 -0.569
Seedling Dry weight I -0.329 0.245 -0.456 -0.381 0.381 -0.381
II -0.520 -0.381 0.536 0.610 -0.893 -0.810
Shoot Length I 0.500 -0.327 -0.866 -0.866 0.500 -
II -0.866 0.866* 0.961** 0.500 0.500 -0.866
Root Length I -0.500 0.500 -0.500 -0.500 -1.000 -0.500
II -0.866 -0.500 -0.500 -1.000 -0.500 -
Leaf Chlorophyll I -0.987 -0.172 -0.294 -0.939 -0.980 -0.985
II -0.987 -0.857 0.965** 0.284 -1.000 0.054
Carotenoids I 0.189 -0.500 0.933* -0.156 - -0.286
II 0.292 0.768* 0.933* 0.115 0.143 0.069
- amylase I -0.993 -0.915 0.999** -0.803 -0.300 -0.845
II 0.999** 0.981** 0.995** -0.826 -0.767 -0.596
- amylase I -0.189 -0.655 0.945* -0.945 -0.968 -0.866
II -0.945 -0.904 0.811* -0.918 -0.945 -0.951
Shoot Total sugar I -0.746 0.700 0.700 -0.803 0.684 0.545
II -0.797 0.717 0.765* -0.127 0.115 0.500
Protein I 0.655 0.500 0.101 0.500 0.500 0.545
II 0.427 0.277 0.819 0.126 0.062 0.352
Amino acid I -0.882 - -1.000 -0.891 -0.327 -0.756
II 0.945 0.945* -0.786 -0.945 -0.655 -0.189
Root Total sugar I -0.169 0.300 0.945* 0.655 0.655 -
II 0.933* 0.929* 0.982** -0.644 -0.810 -0.304
Protein I -0.349 -0.023 0.928 0.658 -0.600 -0.911
II -0.994 0.877* -0.890 -0.120 -0.970 0.392
Amino acid I -0.091 -0.583 0.529 -0.137 0.500 0.371
II -0.374 0.327 -0.693 -0.176 0.047 -0.619
I Water soaked; II- Seaweed extract soaked
Significant at * p<0.05 level; Significant **p< 0.01 level

Journal of Research in Biology (2011) 8: 624-630 628

 Gireesh et al.,2011

The lower concentrations of the extract also
promoted the chlorophyll content of V. unguiculata
up to 20% when compared to the control while
higher concentrations (> 20%) decreased the
chlorophyll content. A similar observation was
made in Vigna mungo (Venkataraman and Mohan,
1997) and when seaweed extract (15-20%) applied
as foliar spray enhanced the leaf chlorophyll level
in plants (Blunden et al., 1996).
The highest total sugar content was recorded
at 20% concentration of seaweed extract soaked
treatment in V. unguiculata. The sugar content
increased up to 20% concentration of seaweed
extract and decreased at higher concentrations. The
increase in the total sugar content at lower
concentration of seaweed extract might be due to
absorption of most of the necessary elements
(Kannan and Tamilselvan, 1990). The same trend
was observed in the H. musciformis with NPK
application in black gram (Tamilselvan and
Kannan, 1994), and D. biflorus (Anantharaj and
Venkatesalu, 2002). It has been observed that
amylase activity was higher than the amylase
activity. Both amylase and -amylase activity
increased at lower concentrations and decreased in
higher concentrations. In conclusion, Ulva lactuca
extract was found to be a promising fertilizer. The
extract act as a biological fertilizer for organic
farming, which is non-toxic, non-flammable for
attaining better seed germination and growth.
(Eds.). Academic Press Inc., New York. 149-150.
Bhosl NB, Untawale AG and Dhargalker VK.
1975. Effects of seaweed extract on growth of
Phaseolus vulgaris. Indian Journal of Marine
Sciences 4:208-210.
Blunden G. 1971. The effect of aqueous seaweed
extract as fertilizer additives. Proceedings of Sixth
International Seaweed Symposium, Tokyo,
(Blunden G (Ed.), 584-589.
Blunden G, Jenkins T and Liu YW. 1996.
Enhanced chlorophyll levels in plants treated with
seaweed extract. Journal of Applied Phycology
8:535-543.
Bokil KK, Mehta VC and Datat DS. 1974.
Seaweed as manure: II pot culture manurial
experiments on wheat. Phykos 13:1-15.
Booth E. 1969. The manufacture and properties of
liquid seaweed extracts. Proceedings of Sixth
International Seaweed Symposium, Tokyo,
(Blunden G (Ed.), 655-662.
Challen SB and Heminway JC. 1965. Growth of
higher plants in response to feeding with seaweed
extracts. Proceedings of Sixth International Seaweed
Symposium, Tokyo, (Blunden G (Ed.), 682-686.

ACKNOWLEDGMENT Chapman GJ. 1970. Seaweed and their uses.

The authors are grateful to the Head, Methuen and Company Limited, London.
Department of Olericulture, Kerala Agricultural
University, Thrissur, Kerala, India for the facilities Dhargalkar K and Untawale AG. 1983. Some
provided. observations of the effect of Seaweed Liquid
Fertilizer on higher plants. Indian Journal of Marine
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Plant physiology 12:1-15. Randall RJ. 1951. Protein measurement with the
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chlorophyll solutions. Journal of Biological the utilization of seaweeds as fertilizer for black
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Mohan VR, Venkataraman V, Murugeswari R
and Muthuswami S. 1994. Effect of crude and Taylor IEP and Wikinson AJ. 1977. The
commercial seaweed extracts on seed germination occurrence of gibberellins and gibberellins like
and seedling growth in Cajanus cajan L. Phykos substances in algae. Journal of Phycology 16:37-42.
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An International Online Open Access
Original Research Paper Publication group

Studies on external genitalial morphology of subfamily Catocalinae

(Lepidoptera: Noctuidae)
Journal of Research in Biology

Authors: ABSTRACT:
Sivasankaran K,
Babu Thangadurai T,
Ignacimuthu S.

Genitalial morphology of fifteen species of the subfamily Catocalinae of

Noctuidae was studied. The structure of uncus, saccus, valvae, Juxta and aedeagus of
Institution:
the male and corpus bursae, ductus bursae and signum of the female genitalia were
Entomology Research
Institute, Loyola College, analysed. The adult images, male and female genitalia were illustrated. A dichotomous
Chennai-600 034. key for the identification of species using genitalial structures is also presented.

Corresponding author:
Ignacimuthu S

Email: Keywords:
entolc@hotmail.com Catocalinae, Noctuidae, Genitalial morphology.

Phone No: Article Citation:

9964412704 Sivasankaran K, Babu Thangadurai T, Ignacimuthu S.
Studies on external genitalial morphology of subfamily Catocalinae
(Lepidoptera: Noctuidae).
Journal of research in Biology (2011) 8: 631-642
Web Address:
http://jresearchbiology.com/
Documents/RA0145.pdf.
Dates:
Received: 16 Nov 2011 /Accepted: 01 Dec 2011 /Published: 26 Dec 2011

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cited.

631-642 | JRB | 2011 | Vol 1 | No 8

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INTRODUCTION
Taxonomic differentiation of Lepidopteran
species has been on the basis of morphology
characters, such as labial palpi, antennae structure,
wing venation etc. On occasions these characters
were found to be less reliable for species
segregation particularly when sibling species of
geographical isolated were involved. Therefore
attempts were made to evaluate other characters
that are more reliable. The genital characters have
peculiar morphological pattern. They show great
divergence between species. The genitalial
characteristics play a major role in their
classification both in specific and higher taxonomic
level as well as in morphology based cladistic
phylogenies. Beuthine-Backer, (1914) explained the
significance of external genitalial morphology in
the Lepidopteran taxonomy and phylogeny. Several
investigations have been made on the general
external morphology of different groups. Busck and
Heinrich (1921) discussed the systematic
importance of the male genitalia in micro
Lepidoptera. Ogata et al. (1957) discussed the
morphological significance of uncus, socii in the
male genitalia of Lepidoptera. Pierce (1909),
Philpott (1927), Busk (1931), Diakonoff (1931),
Richards (1935), Forbes (1939) and Smith (1965).
Investigations had indicated that the structure of
genitalia in association with other morphological
and biological characters of the taxon would
provide a satisfactory basis for taxonomic
segregation. Among Lepidoptera, Noctuidae are
very important ecologically and economically. An
attempt was made to study the external genitalial
structures of 15 different species of Noctuid moths
belonging to Catocalinae subfamily. A dichotomous
key is also presented for the species level
identification of Catocalinae moths using genitalial
characters.
MATERIALS AND METHODS
The adult moths of the subfamily
Catocalinae were collected from Mercury light
sources during night from different localities
(Coonoor, Ooty, Doddabetta, Kothagiri and
Kodaikanal) in the Southern region of Western
Ghats of India. The collected moths were killed
with Ethyl acetate vapor in the killing bottle.
Genitalia were examined following the procedure
outlined in Clarke (1941), Hardwick (1950) and
Lafontaine (2004). The preparation of permanent
slide mounts of genitalia was as follows. The
abdomen was detached from the proximal segments
632
Sivasankaran et al.,2011
using microneedle and dropped into a boiling tube
containing a small quantity of 10% KOH solution.
The boiling tube containing KOH solution and
abdomen was gently boiled and the KOH treated
abdomen was washed in distilled water. The
genitalia were dissected out with the help of fine
needles. After proper dehydration the material was
stained in Acid Fuschin and cleared in Carbol-
Xylol solution prepared in 3:1 ratio. After the
material was mounted in Canada balsam, the
diagrams were made with the help of mirror type
Camera Lucida. For naming the various parts of the
genitalia the terminology proposed by Klots (1970)
and Tuxen 1970 has been followed.
Observations
The genitalial morphology of fifteen species
of the subfamily Catocalinae was studied. The
descriptions of male and female genitalia of these
fifteen species are given below.
Hypocala rostrata (Fabricius, 1794) (Fig. 1a)
Material Examined:
Tamilnadu (INDIA): Niligiri District, Coonoor, 7.
x. 2006, 4 ex. ; Kodaikanal, 8. x. 2006, 2 ex. ;
Coonoor, 15. v. 2007, 8 ex. ; Kodaikanal, 16. v.
2007, 3 ex. ; Coonoor, 8. xii. 2007, 2 ex.
Male genitalia: Uncus divided fringed with hairs.
Tegumen short, broad Vinculum long V shaped
with narrow arms. Valvae short and stumpy;
cucullus broad, clothed with hairs. Harpe spine-like
sclerotized, clasper rod- like. Juxta rectangular, cup
shaped. Aedeagus long narrow, cornuti rod like and
pointed. (Fig. 1b, 1c).
Female genitalia: Ovipositor lobes triangular. Both
pairs of apophyses well developed. Anterior
apophysis stronger than posterior apophysis and of
equal length. Ductus bursae long, slender. Corpus
bursae spherical signum present on the apical and
distal part. (1d).
Hypocala biarcuata Walker, 1858 (Fig. 2a)
Material Examined:
Tamilnadu (INDIA): Nilgiri District, Coonoor, 1. i.
2007, 4 ex.; 8. xii. 2007, 5 ex.
Male genitalia: Uncus fringed with hairs stout,
stalk having apical beak like part and sclerotized.
Tegumen short and broad. Vinculum long, V
shaped saccus conical. Valvae simple; cucullus
blunt; remaining part of valvae uniformarly
sclerotized and well differentiated. Transtilla, juxta
membranous. Aedeagus rather long and stout.
(Figs. 2b, 2c).
Journal of Research in Biology (2011) 8: 631-642
Sivasankaran et al.,2011
Female genitalia: Ovipositor lobes slightly
inferior, triangular setose. Both pairs of apophysis
well developed. Anterior apophysis hardly longer
than posterior apophysis with spatulate tip. Ductus
bursae long. Bursae copulatrix elongate
membranous with fine wrinkles. Signum represents
a narrow sclerotized band. (Fig. 2d).
Hypocala deflorata Fabricius, 1794 (Fig. 3a)
Material Examined:
Tamilnadu (INDIA): Nilgiri District, Coonoor, 29.
xi. 2006, 2 ex. ; 9. iv. 2007, 6 ex. ; 22. x. 2007, 4
ex. ; 8. xii. 2007, 4ex.
Male genitalia: Uncus bifurcate, fringed with hairs.
Tegumen broad. Vinculum V-shaped with narrow
arms. Valvae short; cucullus triangular shaped;
costa and sacculus well differentiated. Harpe spine-
like sclerotized. Juxta bowl-shaped with raised
margin. Aedeagus of moderate length, slender and
slightly contricted into apical region; vesica bearing
a short spine like cornuti; ductus ejaculatorius
broad, long originated from vesica. (Figs. 3b, 3c)
Female genitalia: General aspect elongate
ovipositor lobes inferior, clothed with hairs. Both
pairs of apophyses strongly developed; anterior
apophysis longer than posterior apophysis with
spatulate tip. Ductus bursae very long and broad.
Bursae copulatrix membranous, broad oval and fine
wrinkles, signum ribbon -like. (Fig. 3d).
Hypocala lativitta Moore, 1877 (Fig. 4a)
Material Examined:
Tamilnadu (INDIA): Nilgiri District, Coonoor, 8.
xii. 2007, 4 ex.
Male genitalia: Uncus broad fringed with hairs,
composed of a curved terminal lobe and having a
subapical beak-like part. Genital capsule elliptical,
tegumen broad, without penicular lobe. Vinculum
V shaped with narrow arms. Valvae symmetrical
short; cucullus broad, flat and clothed with hairs;
costa and sacculus well differentiated; sacculus
narrow and heavily sclerotized. Juxta basket-
shaped, sclerotized, projected with two arms.
Aedeagus stout, rod-like cornuti; ductus
ejaculatorius broad originate from vesica. (Figs. 4b,
4c).
Female genitalia: Ovipositor lobes prominent. Both
pairs of apophyses strongly developed; anterior
apophysis longer than the posterior apophysis with
spatulate tip. Ductus bursae long and broad, with
well sclerotized. Corpus bursae membranous, oval
shaped with broad signum. (Fig. 4d).
Journal of Research in Biology (2011) 8: 631-642
Anomis privata Walker, 1865 (Fig. 5a)
Material Examined:
Tamilnadu (INDIA): Nilgiri District, Coonoor, 10.
iv. 2007, 2 ex. ; 8. xii. 2007, 3 ex.
Male genitalia: Uncus short, stout curved broadly,
sword-shaped, medially setose. Tegumen broad.
Tuba analis prominent. Vinculum broad Ushaped.
Valvae simple and sclerotized costal arms not well
developed; sacculus heavily sclerotized. Transtilla
membranous. Juxta U shaped. Aedeagus long,
broad with thorn-like cornuti. (Figs. 5b, 5c).
Female genitalia: Ovipositor lobes well developed
clothed with setae. Both pairs of apophyses strongly
developed. Posteriores longer than anteriores.
Ductus bursae long and stout. Bursae copulatrix
membranous, approximately rhomboid without
signum. (Fig. 5d).
Anomis mesogona Walker, 1858 (Fig. 6a)
Material Examined:
Tamilnadu (INDIA): Nilgiri District, Coonoor, 18.
v. 2007. 7 ex.
Male genitalia: Uncus long curved, medially
broad. Tegumen short, broad. Scaphium well
developed. Saccus broad. Valvae symmetrical,
long; cucullus blunt, broad. Aedeagus long and
slender; vesica bearing rod-like cornuti. (Figs. 6b,
6c).
Female genitalia: Ovipositor lobes prominent with
strong setose. Both pairs of apophyses well
developed. Ductus bursae long and broad. Corpus
bursae membranous, rectangular with broad
signum. (Fig. 6d).
Polydesma lindsayi Hampson, 1893 (Fig. 7a)
Material Examined:
Tamilndau (INDIA): Nilgiri District, Coonoor, 29.
xi. 2006, 5 ex.; 7. vii. 2010, 2 ex.
Male genitalia: Uncus short curved apically
hooked. Scaphium and tuba analis well developed.
Tegumen broad. Vinculum long, broad U shaped.
Valvae asymmetrical apically narrow into a long
lobe; a conical costal process; sacculus well
sclerotized and tappering. Transtilla membranous.
Juxta simple sclerotized. Aedeagus long and slender
basally swollen, without cornuti. (Figs. 7b, 7c).
Female genitalia: Ovipositor prominent developed
with long setose. Both pairs of apophyses well
developed; posterior apophysis longer than the
anterior apophysis. Ductus bursea short. Corpus
bursae triangular in shape without signum. (Fig.
7d).
633

Lacera noctilio (Fabricius, 1774) (Fig. 8a).
Material Examined:
Tamilnadu (INDIA): Niligiri District, Coonoor, 12.
xii. 2006. 5 ex. ; 27. xi. 2006. 3 ex. ; 5. i. 2007. 4
ex . ; 10. vii. 2007. 2 ex. ; 9. ix. 2007. 2 ex. ; 22. x.
2007 3 ex. ; 8. xii. 2007. 46 ex. ; Kodaikanal, 10. vi.
2008. 2 ex.
Male genitalia: Uncus rather long, strong, apically
dilated finally hooked, distally covered with hairs.
Tegumen rather long narrow with penicular lobe.
Vinculum short, U shaped; cucullus broad outer
margin with setose. Valvae not evenly developed;
harpe long narrow finally curved with setose. Juxta
club-shaped, long and broad. Aedeagus relatively
large curved and widening anteriorly; apical region
of aedeagus with elliptical shape. (Figs. 8b, 8c)
Female genitalia: General aspect elongate.
Ovipositor lobes prominent, triangular, clothed with
long setose. Both pairs of apophyses strongly
developed; posterior apophysis longer than anterior
apophysis, thin. Ductus bursae stout. Bursae
copulatrix elongate and broad. (Fig. 8d).
Sphingomorpha chlorea Cramer, 1779 (Fig. 9a)
Material Examined:
Tamilnadu (INDIA): Nilgiri District, Coonoor. 22.
xi. 2007. 7ex
Male genitalia: Uncus strongly developed, broad
apically hooked. Socii well developed. Genital
capsule narrow; tegumen broad, long. Saccus very
broad. Valvae symmetrical long and narrow;
sacculus broad sclerotized. Juxta bifurcated.
Aedeagus stout, acute posteriorly without cornuti.
(Figs. 9b, 9c).
Female genitalia: Ovipositor lobes well developed.
Both pairs of apophyses strongly developed.
Posterior apophysis hardly longer than anterior
apophysis. Ductus bursae short. Bursae copulatrix
with irregular contour without signum. Ductus
seminalis arising from base of bursae copulatrix.
(Fig. 9d).
Oxyodes scrobiculata Fabricius, 1775 (Fig. 10a)
Material Examined:
Tamilnadu (INDIA): Nilgiri District, Coonoor, 29.
xi. 2006, 15 ex. ; 18. xii. 2006, 13 ex. ; 1. i. 2007,
40 ex. ; 28. ii. 2007, 40 ex. ; 9. iv. 2007, 79 ex. ; 22.
x. 2007, 31 ex. ; 8. xii. 2007, 98 ex. ; 23. i. 2008, 58
ex.
Male genitalia: Uncus long and sword-shaped.
Tegumen long, broad. Vinculum long and broad;
saccus U-shaped. Valva with a well differentiated
sacculus and costa; cucullus long and narrow. Juxta
634
Sivasankaran et al.,2011
Y shaped strongly sclerotized. Aedeagus long and
stout in the anterior half and uniformly scobinate.
(Figs. 10b, 10c).
Female genitalia: Ovipositor lobes well developed
covered with hairs. Both apophyses strongly
developed. Posteriores longer than anteriores.
Ostium bursae simple. Ductus bursae
dorsoventrally flattened, sclerotized and wider
posteriorly. Bursae copulatrix elongate broader
anteriorly. (Fig. 10d).
Serrodes campana Guenee, 1852 (Fig. 11a)
Material Examined:
Tamilnadu (INDIA): Nilgiri District, Coonoor, 10.
iv. 2007, 1 ex.; 6. vii. 2007, 1 ex. ; 22. x. 2007, 2
ex.
Male genitalia: Uncus short and stout, with a
curved hooked apex. Tegumen broad; penicularlobe
absent. Juxta narrow inversely bifurcated.
Vinculum broad and heavily sclerotized, V- shaped
and extended to form a prominent saccus. Valvae
long and slender well sclerotized with small harpe.
Aedeagus long and stout with strong double
cornutes. (Figs. 11b, 11c).
Female genitalia: Ovipositor lobes inferior. Both
pairs of apophyses strongly developed with
spatulate tip. General aspect of bursae copulatrix
massive; wall of corpus bursae membranous
without signum. Ductus seminalis arising from
posterior side of corpus bursae. (Fig. 11d).
Chrysopera combinans Walker, 1862 (Fig. 12a)
Material Examined:
Tamilnadu (INDIA): Nilgiri District. Coonoor, 18.
v. 2007, 7 ex.
Male genitalia: Uncus long and slender, with
acutely pointed. Tegumen short. Saccus broad.
Juxta broad with heavily sclerotized between the
valvae. Valvae massive; harpe prominent.
Aedeagus slender, vesica bearing with rod-like
cornuti. (Figs. 12b, 12c).
Female genitalia: Ovipositor lobes small covered
with setae. Posterior apophysis short and thin;
anterior apophysis stout, longer than posterior
apophysis. Ductus bursae much longer. Bursae
copulatrix membaranous with delicate signum.
(Fig. 12d).
Homoptera glaucinans Guenee, 1852 (Fig. 13a)
Material Examined:
Tamilnadu (INDIA): Nilgiri District. Coonoor, 8.
xii. 2007, 5ex.
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636 Journal of Research in Biology (2011) 8: 631-642

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Male genitalia: Uncus short, slender. Tegumen
broad narrow, arms without penicular lobe. Valvae
asymmetrical, elongate; costa and sacculus well
differentiated; costa with narrow arms; costal
process long and broad; sacculus broad sclerotized.
Vinculum V shaped long, much broad. Juxta
sclerotized between the valvae with irregular
contour. Aedeagus stout, vesica bearing a single
massive rod like cornuti. (Figs. 13b, 13c).
Female genitalia: Ovipositor lobes prominent
clothed with long setae. Both apophyses strongly
developed with equal length. Ductus bursae heavily
sclerotized. Bursae copulatrix massive. (Fig. 13d).
Hulodes caranea Cramer, 1779 (Fig. 14a)
Material Examined:
Tamilnadu (INDIA): Nilgiri District, Coonoor, 8.
xii. 2007, 5 ex.
Male genitalia: Uncus curved, acutely pointed and
setose. Tegumen broad without penicular lobes.
Vinculum markedly narrower and more strongly
sclerotized, V- to U shaped and extended to form a
prominent saccus. Juxta not prominent structure.
valae short and well sclerotized. Aedeagus long and
slender; vesica short with long cornuti. (Figs. 14b,
14c).
Female genitalia: Ovipositor lobes well developed
and sclerotized, covered with long hairs. Both pairs
of apophyses strongly developed. Anterior
apophysis shorter than posterior apophysis with
spatulate tip. Ductus bursae long and broad. Bursae
copulatrix broad without signum; ductus seminalis
originating from posterior part of corpus bursae.
(Fig. 14d).
Oraesia emarginata Fabricius, 1775 (Fig. 15a)
Material Examined:
Tamilndau (INDIA): Nilgiri District, Coonoor, 29.
xi. 2006, 2 ex.; 18. iv. 2010. 1 ex.; 7.vii. 2010, 3 ex.
Male genitalia: Uncus slender and curved. Genital
capsule narrow, tegumen shorter than vinculum,
bearing with penicular lobe. Vinculum much
longer, U- shaped and extended to form a
prominent saccus. Juxta small, bearing two pointed
process. Valvae long and slender with slender
harpe. Aedeagus much long and slender. (Figs. 15b,
15c).
Female genitalia: Ovipositor lobes and well
separated. Anterior apophysis shorter than the
posterior apophysis with spatulate tip. Ductus
bursae long and slender. Corpus bursae
membranous, elongated and emarginated at middle
without signum. (Fig. 15d).
638
Sivasankaran et al.,2011
RESULTS AND DISCUSSION
The subfamily Catocalinae was first
proposed by Boisduval, [1828]. Exhaustive treatise
on the Indian noctuids is that of Sir George
Hampsons Fauna of British India series, moths
volumes (II & III 1894, 1895). In this series, he
recorded the Noctuid moths under ten subfamilies.
He united the Catocalinae and Ophiderinae into one
group. Kitching (1984) united Catocalinae
(presence of spines on the mid-tibia) and
Ophiderinae (absence of spines on the mid-tibia) in
one group Catocalinae. Speidel et al. (1996)
suggested a division of the Catocalinae and
Ophiderinae complex into two groups. Male moth
genitalia are usually bilaterally symmetrical. Francy
and George Mathew (2005-2006) studied the
genitalial morphology of some species of the
subfamily Ophiderinae. They defined the uncus as
well developed in all the species of the subfamily
Ophiderinae. In this present study we noticed great
variation. Uncus was more prominent; the shape
and size varied in all species of subfamily
Catocalinae. It was long and slender in Anomis
mesogona, Chrysopera combinans, Oxyodes
scrobiculata and Oraesia emarginata, stout and
curved in A. privata, bifurcated in Hypocala
rostrata and Hypocala deflorata, beak-like in
Hypocala lativitta, short and hooked in Polydesma
lindsayi and Serrodes campana, long and dilated in
Lacera noctilio, swollen in Sphingomorpha
chlorea, very much short in Homoptera glaucinans
and moderately dilated in Hulodes caranea.
Francy and George Mathew (2005-2006)
described the tegumen as long and narrow in
Anticarsia irrorata Fabricius, Ischyja manila
Cramer and Ischyja infernae Walker, short and
broad in Speiredonia suffusoma Guenee. In our
study tegumen belonging to the subfamily of
Catocalinae was long and broad in Oraesia
emarginata, Hulodes caranea, Sphingomorpha
chlorea, L. noctilio and Oxyodes scrobiculata, short
and narrow in A. mesogana, short and broad in
Homoptera glaucinans, C. combinans, Anomis
privata, P. lindsayi, Serrodes campana and
shoulder shape in Hypocala rostrata, H. deflorata,
H. biarcuata and H. lativitta.
Francy and George Mathew (2005-2006)
demonstrated the structure of valvae with different
shapes and sizes from the subfamily Ophiderinae.
In the present study we observed that the valvae
was well developed in all the species of the
subfamily Catocalinae. It varied in shape and size.
Valvae was asymmetrical in P. lindsayi and
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Journal of Research in Biology (2011) 8: 631-642 639

 Sivasankaran et al.,2011

Homoptera glaucinans, and narrow in long and narrow in Oraesia emarginata, Anomis
Shingomorpha chlorea. mesogona and Oxyodes scrobiculata, somewhat
Juxta was defined in the species belonging broad in Hypocala rostrata, Hypocala deflorata,
to subfamily Ophiderinae by Francy and George Hypocala biarcuata, P. lindsayi and Serrodes
Mathew (2005-2006). It was highly sclerotised in campana, and short and stout in Hypocala lativitta,
Anticarsia irrorata Fabricius. In the present study Sphingomorpha chlorea and Homoptera
juxta was bowl shaped in A. privata and Hypocala glaucinans. Vesica was simple scobination in all
rostrata and basket shaped in Hypocala lativitta. It the species.
was highly sclerotized in Hypocala biarcuata, C. Francy and George Mathew (2005-2006)
combinans, Homoptera glaucinans. Juxta was defined the female genitalic structure of the
cylindrical in shape in Hypocala rostrata, small and subfamily Ophiderinae. It varied in shape and size.
bearing with two pointed process in Oraesia In the present study female genitalia ovipositor
emarginata. Vinculum was long and V shaped in lobes were well developed in all the species of the
Hypocala rostrata, Hypocala deflorata and subfamily Catocalinae. Corpus bursae was rounded
Hypocala rostrata. It was V- to U shaped in in H. rostrata, oval shaped in Hypocala lativitta
Homoptera glaucinans, Hulodes caranea and rectangular in Serrodes campana and L. noctilio,
Oraesia emarginata and U shaped in Polydesma and balloon-like in Homoptera glaucinans.
lindsayi, A. privata, Sphingomorpha chlorea and L. Key for identification of species
noctilio. Sexual segregation was the most important
Aedeagus was defined in the species of criteria between species; no two species contained
subfamily Ophiderinae by Francy and George the same type of genitalia. The genitalia of each
Mathew (2005-2006). It was long and narrow in species was distinct. Using characters unique to
Speiredonia suffusoma Guenee and short and stout each species, the following key had been devised to
in Episparis liturata Fabricius and Arcte modesta facilitate species segregation.
Van der Hoev. In the present study aedeagus was
Key to the species based on male genitalic characters
Catocalinae
1. Uncus long....Chrysophora combinans
- Uncus short.......................2
2. Uncus undivided.....3
- Uncus divided.....12
3. Uncus curved..4
- Uncus not curved.....9
4. Uncus stout.....5
- Uncus normal..Anomis mesogona
5. Uncus apically dilated..Sphingomorpha chlorea
- Uncus slender......6
6. Uncus apically hooked...7
- Uncus apically not hooked....Anomis privata
7. Valvae asymmetrical.Polydesma lindsayi
- Valvae symmetrical......8
8. Valvae long and broad; vinculum long.......Serrodes campana
- Valvae not long; vinculum short....Lacero noctilio
9. Tegumen short and broad.....10
- Tegumen long and narrow......11
10. Valvae asymmetrical; vinculum V shaped....Homoptera glaucinans
- Valvae symmetrical; vinculum U shaped.Oxyodes scrobiculata
11. Juxta prominent; vinculum long....Oraesia emarginata
- Juxta sclerotized; vinculum short..Hulodes caranea
12. Vinculum long......................................................................................................................13
- Vinculum short...14
13. Juxta cylindrical shape; valvae broad....Hypocala rostrata
- Juxta heavily sclerotized; valvae narrow...Hypocala biarcuata
14. Tegumen with penicular lobes; Aedeagus without cornuti.....Hypocala deflorata
-Tegumen without penicular lobes; Aedeagus with cornuti....Hypocala lativitta
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 Sivasankaran et al.,2011

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Baker GTB. 1914. Notes on the taxonomic value Noctuidae of the Lepidoptera of the British Islands.
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152. Smith JB. 1965. A revision of the West African
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Diakonoff A. 1937. Notes on Microlepidoptera on The phylogeny of Noctuidae (Lepidoptera).
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Forbes WTM. 1939. The muscles of the genitalia in Insects. Munksgaard. Copenhagan 359.
lepidopterous male genitalia. Ann. Entomol. Soc.
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India Moths vol. II: 160-581 III:1-107.

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of lepidopterous genitalia. The Canadian
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Br. Mus. Nat. Hist. (Ent.) 49(3):153-234.

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642 Journal of Research in Biology (2011) 8: 631-642
 Journal of Research in Biology
An International Online Open Access
Original Research Paper Publication group

Genetic diversity and population structure of Vigna unguiculata ssp.

unguiculata var. spontanea in Sudan
Journal of Research in Biology

Authors: ABSTRACT:
Kouam EBA,B,C, Pasquet
RSA, Elteraifi IA and Enzyme electrophoresis was used to estimate the genetic diversity and
Muluvi GMB. population structure of thirteen Vigna unguiculata ssp. unguiculata var. spontanea
populations in Sudan. Plant genotypes were homozygous at most loci and at several
Institution: populations. Nine of the twenty-one allozyme loci analysed (42.9%) showed
A. Molecular Biology and
detectable polymorphism, but only 11.4% of loci were polymorphic within local
Biotechnology
populations. Gene diversity at the species level and at the population level was low
Department, International
Centre of Insect (Hes = 0.084; Hep = 0.049, respectively). Analysis of fixation indices, calculated for all
Physiology and Ecology P.O polymorphic loci in each population showed a substantial deficit of heterozygotes
box 30772 Nairobi, Kenya. relative to Hardy Weinberg expectations. This deficit is partly associated with
inbreeding due to self and consanguineous mating. High inbreeding and strong genetic
B. Department of differentiation coefficients were found. Allele frequency data revealed a low degree
Biochemistry and of within population genetic diversity (Hs = 0.049) and a high degree of genetic
Biotechnology, Kenyatta heterogeneity among populations (Gst = 0.409). The indirect estimates of gene flow
University P.O Box 43844 were calculated based on the level of genetic differentiation between populations and
Nairobi, Kenya. frequencies of private alleles. These were 0.274 and 0.043 respectively. Genetic and
geographic distances were positively correlated although not significant, indicating
C. Department of that very little genetic variation is explained by difference in geographic pattern. This
Agriculture, Faculty of may be a result of inbreeding and genetic drift through a few founders coupled with
Agronomy and Agricultural limited pollen flow.
Sciences, University of
Dschang, P.O Box 222
Dschang, Western Region, Keywords:
Cameroon.
Enzyme, Genetic diversity, Population structure, Vigna unguiculata.

Corresponding author: Article Citation:

Kouam EB Kouam EB, Pasquet RS, Elteraifi I and Muluvi GM.
Genetic diversity and population structure of Vigna Unguiculata ssp. unguiculata var.
spontanea in Sudan
journal of research in Biology (2011) 8: 643-652

Email: Dates:
ericbkouam@yahoo.com
Received: 29 Nov 2011 /Accepted: 07 Dec 2011 /Published: 26 Dec 2011

Web Address:
http://jresearchbiology.com/
Documents/RA0156.pdf.
Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

643-652 | JRB | 2011 | Vol 1 | No 8

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INTRODUCTION
Taxonomic studies of the genus Vigna
divided cowpea (Vigna unguiculata L. Walp.) into
ten perennial subspecies and one annual subspecies
(ssp. unguiculata) ( Pasquet 1993, 1997, 1999).
These studies split the ssp. unguiculata into var.
unguiculata for the cultivated forms and var.
spontanea (Schweinf.) Pasquet for the wild and
weedy forms (Pasquet 1999). All member of
cowpea is a diploid plant species (2n = 2x = 22)
(Pasquet, 1999). In the Africa continent, var.
spontanea is quite wide spread and easily interbreed
with the cultivated forms (var. unguiculata) and
produce fertile offsprings through hybridisation
(Kouadjo 2007). Annual cowpea is primarily a self-
pollinated plant species (Fery 1985); however, cross
-pollination does exist and is mediated by bees of
the genus Xylocopa and Megachilidae family
(Tignegre et al. unpublished). These pollinators are
expected to do many more within flower patch
flights than between flowers patch flights (Levin
and Kerster 1974, Pasquet et al. 2008). The crop
plays a critical role in the live of millions of people
in the world, especially in Sub-saharan Africa
where it feed people, their livestock and the next
crop (Singh et al. 1997, Quin 1997).
Enzyme electrophoresis has been widely
used for the characterization of the genetic
variability, the evaluation of the genetic structure as
well as the determination of the genetic relationship
within and among plant populations (Hamrick and
Godt 1990). The genetic structure is one aspect of
the genetic organisation of a species, often cited to
play a key role in the speciation process (Wright
1965). The level of allozyme variation provides a
basis on which to build sound programs for the
conservation of the genetic diversity of rare and
endangered species (Hamrick et al. 1991). In
addition, allozyme diversity can be used as a
yardstick to measure the effectiveness of in situ and
ex situ conservation programs (Hamrick et al. 1991,
Hamrick and Godt 1996, Barrett and Kohn 1991).
Wild relatives of cultivated crops receive special
attention because of their poor representation in
gene banks and their high value as large stores of
genetic variation (Frankel 1974, Brown 1978,
Marshall 1990). Despite the importance of
knowledge on genetic variation for providing
information for conservation purposes, detailed
studies of genetic variation are not available for
cowpea populations. Pattern of geographical
variation in allele frequencies within a species may
provide valuable information on past and current
644
Kouam et al.,2011
evolutionary forces affecting the evolution of the
species such as gene flow, genetic drift, and natural
selection (Wright 1978, Loveless and Hamrick
1984, Slatkin and Barton 1989).
At the accession level, the genetic variation
of wild cowpea have been evaluated using allozyme
(Pasquet 1993, 1999, Panella and Gept 1992,
Vaillancourt et al. 1993), Random amplified
polymorphic DNA (Ba et al. 2004), Amplified
fragment length polymorphism (Coulibaly et al.
2002) and Restriction fragment length
polymorphism (Feleke et al. 2006). This genetic
diversity is yet to be studied at the population level.
Detailed analyses at the population level may
produce new insights and give a better
understanding of the distribution of the genetic
variation in cowpea populations. For the study of
thirteen wild cowpea (Vigna unguiculata ssp.
unguiculata var. spontanea) populations in
different localities in Sudan, the following
objectives were established: (1) To estimate the
level of genetic variation at the population and
species level. (2) To estimate the level of
inbreeding, differentiation and detect the structure
of cowpea populations. (3) To estimate the level of
gene flow among populations
MATERIAL AND METHODS
Sampling procedure and enzyme electrophoresis
Seeds of Vigna unguiculalata ssp.
unguiculata var. spontanea were collected from
thirteen natural populations in Sudan in October
2003. Eight to twelve plants were collected in each
population. One to three pods were sampled for
each plant and the seeds present in each pod were
kept in separate envelopes and store at 20oC until
analyses were carried out. For each plant, only one
pod was used and up to four seeds were analysed
for the study. Seeds were soaked in distilled - de-
ionized water and left overnight at room
temperature to initiate germination, prior to enzyme
expression. Germinating seeds were then crushed
with mortar and pestle and homogenized with
distilled de-ionized water. Enzyme extracts were
absorbed onto chromatography wicks and applied
into a 14% starch gels. Horizontal gel
electrophoresis was then done at a constant voltage
of 200 V at 4oC for approximately three hours using
a continuous histidine-citrate buffer system (Second
and Trouslot 1980). Gels were stained for ten
enzyme systems to resolve twenty one allozyme
loci: amino peptidase (AMP, three loci),
endopeptidase (ENP, one locus), formate
Journal of Research in Biology (2011) 8: 643-652
Kouam et al.,2011
dehydrogenase (FDH, one locus), fluorescent
esterase (FLE, two loci), isocitrate dehydrogenase
(IDH, two loci), malate dehydrogenase (MDH, four
loci)), 6-phosphogluconate dehydrogenase (PGD,
two loci), phosphoglucose isomerase (PGI, three
loci), Phosphoglucomutase (PGM, two loci),
shikimate dehydrogenase (SDH, one locus). Stain
recipes were taken from Wendel and Weeden
(1989).
Data analysis
Putative loci were designated sequentially,
with the most anodally migrating isozyme
designated 1, the next 2, and so on. The most Indirect estimates of gene flow were calculated
common allele at each locus was arbitrary assigned
the value 100, slower and faster bands on the
zymmogram relative to this common one, which
represent other alleles, were given lower and higher
values corresponding to their relative migration
using the same nomenclature as in Pasquet (1999).
The genotype of each mother plant was estimated
from the progeny array following the method of
Brown and Allard (1970) and using the MLTR
computer program, version 2.2 (Ritland 2002).
We calculated using Popgene software
version 1.31 (Yeh et al. 1999) standard measures of
genetic diversity for each population. This includes
the mean number of alleles per locus (A), the
percentage of polymorphic loci (P), and mean
observed (Ho) and expected heterozygosity (He) at
the species and within-population levels. Wrights
fixation index (F) (Wright 1922) was used to
calculate deviations from Hardy-Weinberg
equilibrium for each polymorphic locus within
populations [F = (He Ho) / He]. Chi-square tests
were used to test for significant deviations in the
fixation indices from the expected value that is F =
0 (Li and Horvitz 1953). Chi-square tests were also
used to test for heterogeneity in allele frequencies
among populations (Workman and Niswander,
1970). Total genetic heterozygosity (H T),
heterozygosity within populations (HS), genetic
diversity among populations (DST), and the
proportion of genetic diversity found among
populations (GST) were calculated following the
equations of Nei (1973, 1977) and using GenAlex
computer program (Peakall and Smouse, 2006).
The genetic structure within and among populations
was also evaluated with F-statistics (FIT, FIS and
FST) following Weir and Cockerham (1984) and
using Fstat computer program (Goudet 1995).
These F-statistic values were tested for difference
from zero using permutation tests (Goudet 1995).
Journal of Research in Biology (2011) 8: 643-652
The FIT and FIS coefficients measure
deficiencies of homozygotes or heterozygotes
relative to the panmictic expectations within the
overall sample and within populations respectively.
The FST coefficient estimates relative population
differentiation. Standard errors were estimated by
jack-knifing using the Fstat program (Goudet
1995). Neis (1972) genetic distances (D) were
calculated for each pair-wise combination of
population and values were used to construct a
dendrogram using the UPGMA (unweighted pair
group method with arithmetic mean) method.
based on Wrights (1951) equation: Nm = 1/4(1/FST
- 1), where Nm is the average number of migrants
exchanged per generation between populations. A
second estimate of Nm, using the distribution of
private alleles (alleles found in only one
population), was calculated using GenAlex
computer program (Peakall and Smouse, 2006)
following the procedure of Barton and Slatkin
(1986).
RESULTS
Patterns of genetic diversity
Of the twenty-one isozyme loci essayed,
nine (42.9%) were polymorphic across the range of
V. unguiculata. The remaining twelve loci (Fle-1,
Idh-2, Mdh-1, Mdh-2, Mdh-3, Mdh-4, Pgd-1, Pgd-
2, Pgi-1, Pgi-2, Pgm-1 and Sdh) were
monomorphic, displaying unique allele each, in all
populations. An average of 11.4% of the loci was
polymorphic within population, with individual
population values ranging from 0 to 19.1% (Table
1). We found thirty-four distinct alleles overall
across the twenty-one loci. Six of the thirty-four
alleles were unique to a single population: Pgm2104
and Idh1090 in SDN03; Pgi3096 in SDN09; Fle3098 in
SDN17; Pgm2096 in SDN26 and Enp096 in SDN27.
The mean number of alleles per locus was 1.62 at
the species level. Across populations it ranges from
1 to 1.19 with a mean at 1.128. (Table1). Gene
diversity or expected heterozygosity for the species
was somewhat low (0.084); in other words, only
8.4% of individuals are genetically expected to be
heterozygous at a given locus under random mating
conditions. At the population level, expected
heterozygosity ranges from 0 (SDN09) to 0.074
(SDN22) with the average at 0.049. Observed levels
of heterozygosity in populations (Table 1),
however, were almost always lower than Hardy-
Weinberg expectations, averaging 0.014.
645
 Kouam al.,2011

Table 1. Population coordinates, Proportion of polymorphic loci (P), number of allele per locus (A) Observed
heterozygosity (Ho), Expected heterozygosity (He) and Fixation index (F) for thirteen populations of wild V.
unguiculata in Sudan
Population Latitude Longitude P A (SE) Ho (SE) He (SE) F
1.143 0.000 0.053
SDN03 12 17 N 34 10 E 0.143 1.000 (0.000)***
(0.080) (0.000) (0.031)
1.095 0.010 0.039
SDN06 11 56 N 34 18 E 0.095 0.800 (0.062)***
(0.095) (0.013) (0.040)
1.000 0.000 0.000
SDN09 11 47 N 34 27 E 0.000 m
(0.000) (0.000) (0.000)
1.191 0.048 0.068
SDN12 11 45 N 34 22 E 0.191 0.303 (0.123)NS
(0.127) (0.044) (0.048)
1.048 0.010 0.009
SDN16 11 33 N 34 11 E 0.048 - 0.111 (0.010)NS
(0.069) (0.013) (0.012)
1.191 0.000 0.051
SDN17 12 48 N 30 06 E 0.143 1.000(0.000)***
(0.137) (0.000) (0.037)
1.191 0.024 0.073
SDN19 12 29 N 29 47 E 0.191 0.614 (0.124)***
(0.127) (0.025) (0.046)
1.191 0.027 0.074
SDN22 11 54 N 29 40 E 0.191 0.679 (0.082)***
(0.108) (0.022) (0.043)
1.048 0.010 0.020
SDN25 11 42 N 29 45 E 0.048 0.524 (0.022)*
(0.069) (0.013) (0.029)
1.048 0.000 0.023
SDN26 11 15 N 29 40 E 0.048 1.000 (0.000)**
(0.069) (0.000) (0.033)
1.191 0.020 0.053
SDN27 11 02 N 29 41 E 0.143 0.664 (0.117)**
(0.137) (0.018) (0.040)
1.143 0.005 0.043
SDN30 11 24 N 29 37 E 0.143 0.926 (0.028)***
(0.084) (0.006) (0.028)
1.191 0.024 0.045
SDN33 11 13 N 29 25 E 0.095 0.454 (0.025)*
(0.150) (0.018) (0.035)
1.128 0.014 0.049
Average Pop. level 0.114 0.662 (0.025)***
(0.096) (0.013) (0.033)
1.619 0.013 0.084
Specie level 0.429
(0.062) (0.002) (0.011)
*** :significance at the 0.1% nominal level; ** :significance at the 1% nominal level; * :significance at the 5%
nominal level
NS
: Not significant. m: Monomorphic.

Population structure and gene flow
Between populations, allele frequencies
were significantly different at eight of the nine
polymorphic loci as shown by Chi square test
(Table 2). The estimates of genetic structure using
Neis genetic diversity estimates are shown in
Table 2. The average of total heterozygosity (HT)
and within population genetic diversity (HS) were
0.083 and 0.049, respectively. The inter-population
genetic diversity (DST) and the coefficient of
genetic differentiation among populations (GST)
varied from 0.000 (Enp) to 0.172 (Fdh) and from -
0.028 (Enp) to 1.000 (Pgi-3), with a mean of 0.034
and 0.409, respectively. The result indicates that
about 41% of the genetic variation in our sample
can be attributed to variation among populations.
The inbreeding coefficients (FIS and FIT) for eight
646
of the nine polymorphic loci were significantly
greater than zero (Table 2). The mean estimates of
these coefficients were significant (FIS = 0.684 P <
0.001; FIT = 0.835 P < 0.001), reflecting that
observed levels of heterozygosity within
populations and the entire study area were smaller
than would have been expected in case of random
sexual reproduction. High FST values for most of
the polymorphic loci (Table 2) indicate significant
genetic differentiation at the population level (FST =
0.477, P < 0.001). The majority of test for pair-wise
genetic differentiation among populations (60 out
of 78) were statistically greater than zero; the
highest value (0.904) found between SDN 09 and
SDN16 (Table 3). Genetic distance (D) values
between pair of populations are also shown in Table
3. They ranged from 0.002 to 0.128 with a mean at
Journal of Research in Biology (2011) 8: 643-652
Kouam et al.,2011

Table 2. Chi square tests, Neis gene diversity, F-Statistics and gene flow estimates at nine polymorphic loci for
thirteen populations of wild V. unguiculata in Sudan
Gene 2 Hs Ht Dst Gst Fit Fis Fst NmW NmS
Amp2 56.840*** 0.120 0.122 0.002 0.014 0.502*** 0.430** 0.127NS 1.719
Amp3 62.720*** 0.285 0.401 0.116 0.290 0.846*** 0.748*** 0.389** 0.393
Amp4 54.691*** 0.079 0.170 0.091 0.535 0.728** 0.339* 0.587*** 0.176
NS NS NS NS
Enp 0.001 0.011 0.011 0.000 -0.028 -0.006 -0.070 0.066 3.521
Fdh 123.162*** 0.287 0.460 0.172 0.375 0.814*** 0.657*** 0.459** 0.295
Fle3 179.373*** 0.133 0.265 0.132 0.499 0.916*** 0.804*** 0.573*** 0.186
Idh1 95.452*** 0.044 0.060 0.016 0.266 1.000*** 1.000*** 0.381** 0.406
Pgi3 92.789*** 0.000 0.142 0.142 1.000 1.000*** 1.000*** 1.000*** 0.000
Pgm2 211.556*** 0.074 0.118 0.044 0.375 1.000*** 1.000*** 0.474*** 0.278
Overall 0.049 0.083 0.034 0.409 0.835*** 0.684*** 0.477*** 0.274 0.043
*** :significance at the 0.1% nominal level; ** :significance at the 1% nominal level; * :significance
at the 5% nominal level; NS: Not significant.

0.047. These distances did not show significant
effect on geography as the correlation between
genetic and geographic distance was weak (r =
0.10, P > 0.050). A UPGMA dendrogram
illustrating genetic relationships among populations
were constructed (Figure 1). Indirect estimates of
gene flow indicate low levels of pollen migration
among V. unguiculata populations. The application
of Wrights (1951) model gave an estimate of NmW
= 0.274. The low value of Nm reflects the high
value of FST. An independent estimate of gene flow
(Barton and Slatkin 1986) based on the frequencies
of private alleles gave an even lower estimate of
0.04 0.03 0.02 0.01
Fig 1: An UPGMA clustering tree based on Neis
(1972) genetic distance calculated for 21 allozyme loci
in 13 populations of wild V. unguiculata
gene flow (NmS = 0.043). Both estimates of Nm
being lower than unity suggest strong population
differentiation.
DISCUSSION
Genetic diversity is essential to avoid risk of
extinction and promote the long-term survival of
plant species. The loss of genetic variation is
thought to decrease both the short-term and the long
-term adaptability of populations in variable and
changing environments (Hamrick 1994, Young et
al. 1996). Populations of selfing species and animal
-pollinated species with mixed mating systems (i.e.,
partially selfed, partially outcrossed) have lower
SDN30 levels of genetic diversity than obligatory
SDN25
outcrossing species (Hamrick and Godt, 1990).
SDN27
Lush (1979) describe cowpea as a highly selfing
plant species. Although selfing would leads to
SDN19
individual homozygosity, this study showed that
SDN26
wild vigna unguiculata exhibit substantial allozyme
SDN03
variation and agrees with the report of Allard et al.
SDN16
(1968) indicating that selfing species are not
SDN12 necessarily nil in allelic variants. This study showed
SDN06 that V. unguiculata var. spontanea maintains lower
SDN33 diversity at the population than at species level as
SDN22 generally observed in plants (Hamrick and Godt,
SDN17 1990). The mean estimates of genetic diversity
SND09 parameters (P, A and He) within populations of V.
unguiculata were low (11.4%, 1.13 and 0.049
0.00
respectively) to the means for selfing species
(20.0%, 1.31, and 0.074) (Hamrick and Godt,
1990). V. unguiculata, however, had at the species

Journal of Research in Biology (2011) 8: 643-652 647

 Kouam et al.,2011

level close levels of overall diversity (42.9%, 1.62

0.314***

0.527***

Pairwise Fst significances: ***, significance at the 0.1% nominal level; **, significance at the 1% nominal level; *, significance at the 5% nominal level; NS
0.669**
0.297**

0.416**

0.318**
0.427**
0.319**
0.288**
0.113NS
SDN33

0.311*
0.466*
and 0.084) compared to other selfing species
(41.8%, 1.69 and 0.124) (Hamrick and Godt, 1990).
Table 3. Neis standard genetic distance (lower diagonal) and pairwise FST values (upper diagonal) between 13 wild V. unguiculata populations

At the species level, the total gene diversity

obtained (0.084) is low when compared to other

-0.069NS
0.255**
0.639**
0.331**

0.405**

0.463** 0.369**
reported data on wild V. unguiculata using
0.100NS

NS
SDN30
0.149*

0.549*
0.174*

0.014

0.026
allozymes. Panella and Gepts (1992) reported
0.110; Vaillancourt et al. (1993) 0.168 and Pasquet
(1999) 0.290. This difference is explained by the
0.270**

0.670**

0.380**
0.035NS

0.156NS
SDN27

fact that their studies included several subspecies,

0.279*

0.267*
0.324*
0.457*

0.007
0.036
representing much larger part of the crop gene pool
in contrast of the present study that involves only
one subspecies (ssp. unguiculata var. spontanea).
0.236**

0.813**

0.496**
0.428NS

0.389NS
SDN26

0.385*

0.464*

0.628*

0.543*

0.051 Limited allozyme variation may reflect restricted

0.032
0.038
habitat range or homogeneity of the environment
(Hedrick et al. 1976). The breeding system of a
species is an important determinant of variability at
-0.029NS
0.808**
0.058NS
0.250NS
SDN25

both the species and population levels. The

0.360*

0.588*
0.278*
0.427*

0.022
0.016
0.003
0.025

relatively low level of genetic variation found in V.

unguiculata is consistent with its breeding system.
marking depicts non-significant values.

V. unguiculata is a highly inbreed and insect-

0.455**
0.472**
0.678**
0.313**
SDN22

0.493*
0.443*
0.234*

pollinated species, a combination well-known to be

0.057
0.090
0.058
0.055
0.089

associated with low levels of allozyme variation

(Brown 1979, Gottlieb 1981, Hamrick and Godt,
1990).
0.356**

0.663**
0.309NS

0.323NS
SDN19

0.280*
0.411*

0.041
0.032
0.073
0.013
0.022
0.057

Genetic differentiation among populations is

principally a function of gene flow among
populations via pollen and seed dispersal (Loveless
0.359NS

0.330NS
0.614NS

and Hamrick 1984). Estimates of genetic

SDN17
0.547*

0.759*

0.049
0.073
0.075
0.095
0.066
0.077
0.057

differentiation indices (FST = 0.477; GST = 0.409)

indicated highly significant degree of differentiation
(P < 0.001) among populations of Vigna
0.909**
0.076NS
0.250NS
SDN16

0.407*

unguiculata in Sudan. Since the species of concern

0.074
0.045
0.064
0.002
0.018
0.026
0.009
0.020

is a tropical inbreed and bees pollinated (Lush

1979, Pasquet et al. 2008), this result may fit with
the general observation that inbreeding species
0.357**

0.670**
SDN12

0.105*

0.038
0.047
0.048
0.053
0.038
0.059
0.037
0.039
0.035

maintain high and significant level of population

differentiation (Hamrick and Godt 1990). Other
studies that have assessed genetic variation in
0.563**
SDN09

natural populations of tropical inbred plant species

0.706*

0.092
0.050
0.128
0.106
0.124
0.054
0.068
0.084
0.064
0.071

using allozyme exhibited similar high levels of

genetic differentiation: GST = 0.519 for wild lima
bean (Bi et al. 2003) and GST = 0.712 in bambara
0.233**
SDN06

groundnut (Pasquet et al. 1999). This high level of

0.065
0.018
0.015
0.042
0.044
0.079
0.019
0.032
0.032
0.025
0.013

genetic differentiation suggests that gene flow

among populations is low (NmW = 0.274). The
distribution of genetic diversity within and among
SDN03

0.027
0.062
0.049
0.010
0.087
0.050
0.073
0.010
0.024
0.031
0.016
0.033

populations of V. unguiculata, however, follows an

unexpected pattern. Most of the species genetic
variation (about 59%) occurs within populations.
We found highs, significant and positives F IS and
SDN03
SDN06
SDN09
SDN12
SDN16
SDN17
SDN19
SDN22
SDN25
SDN26
SDN27
SDN30
SDN33

FIT values (0.684 and 0.835 respectively) indicating

that homozygotes were significantly in excess. This
648 Journal of Research in Biology (2011) 8: 643-652
Kouam et al.,2011
high level of inbreeding can result from several
causes: family structuring within a restricted
neighbourhood causing mating between relatives
(Levin and Kerster 1971, 1974), selection for
homozygotes, Wahlund effect and positive
assotative mating (preferential mating between
individual of similar genotypes) (Crow and
Felsenstein 1968). Other authors have also observed
positive and significant inbreeding coefficient
values and attributed it to populations substructure
and inbreeding (Bi et al. 2003, Ueno et al. 2002).
An important feature of the floral biology of V.
unguiculata is the position of stigma and anther that
are in contact with the pollen that usually shed
before the full opening of the flower (Lush 1979).
This may reduce cross-pollination and provide high
reproductive assurance even in the absence of
pollinators. Hence, the high levels of inbreeding in
V. unguiculata appear to be due to autonomous
selfing due to the close proximity of the stigma and
anthers in this species (Lush 1979). Geography did
not correlate significantly with the genetic
relationship among populations of V. unguiculata.
The lack of significant association between
geographic and genetic distances found in V.
unguiculata may be explained by the expectation of
a model of differentiation by founder effects (Mayr
1963) where small number of individual from an
existing population colonize a new habitat forming
a new population genetically close to the former. It
might also be the result of genetic drift
(Chakraborty et al. 1978) coupled with a highly
selfed-mating system and restricted pollen flow
among local populations. These factors could all
result in the low levels of allozyme diversity within
populations and a high degree of genetic divergence
between populations of V. unguiculata. However, it
is difficult to determine which factor played a major
role in shaping population genetic structure in V.
unguiculata in the region.
Gene flow is the movement of gene within
and among populations. It has a significant
influence on the distribution of the genetic variation
(Hamrick 1989). Indirect population genetics
statistics estimates the number of individual
migrating from one population to the other (Nm).
The estimates of Nm based either on the level of
population differentiation or the private allele
approach were very low: NmW = 0.274 and NmS =
0.043. In general, if Nm is less than one, then local
differentiation of populations will result and if Nm
is grater than one, there will be little differentiation
Journal of Research in Biology (2011) 8: 643-652
among populations (Wright 1951). Between V.
unguiculata populations, gene flow (Nm) is very
low, less than unity like in other endemic and
endangered plant species (Hamrick and Godt 1996)
and appears insufficient to counter divergence due
to the effects of random genetic drift according to
population genetics theory (Wright 1951, Real
1994). Although both low, we noted that Nm W is
about 6 times higher than NmS. Such disparity
could be attributed to the general high frequencies
of private alleles (Idh1090 in SDN03 with 0.4
frequency; Pgm2096 in SDN26 with 0.6 frequency
and Pgi3096 in SDN09 with 1 frequency). Similar
low level of gene flow were reported by Bi et al.
(2003) and Hardy et al. (1997) after studying the
population genetic structure of Phaseolus lunatus,
an autogamous plant species. Two main
mechanisms of gene flow exist for cowpea: seed
and pollen dispersal. Seed migration, is very
unlikely given the extensive spaces that generally
separate neighbouring wild populations. Pasquet et
al. (2008) reported that bees are able to disperse
cowpeas pollen to few km with a low probability
of long pollen transport events. Pollen dispersal by
bees clearly is the most likely mechanism of gene
flow between populations of cowpea.
ACKNOWLEDGEMENTS
We thank the German Academic Exchange
Service (DAAD) and the ICIPE - ARPPIS training
program. The study was also funded by the United
States Agency for International Development
(USAID).
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 Journal of Research in Biology
In vitro regeneration of Passiflora foetida l.
Journal of Research in Biology
Authors:
Komathi S1, Rajalakshmi
G1, Savetha S2 and
Ayyappadas MP2.
Institution:
1. Department of
Biotechnology, Hindustan
College of Arts & Science.
2. Department of
Biotechnology, R.V.S
College of Arts and science
Sulur, Coimbatore -402.
Corresponding author:
Komathi S
Email:
komathipolur@gmail.com
Web Address:
http://jresearchbiology.com/
Documents/RA0158.pdf.
Journal of Research in biology
An International Open Access Online
Research Journal
An International Online Open Access
Original Research Paper Publication group
ABSTRACT:
Passiflora foetida is an angiosperm plant with high medicinal value and is
becoming less in natural communities. It is used as a bacteriocide, antidysentric and
antilithic. Aqueous extracts of leaves or whole plants have been used to treat colic,
colds, diarrhea, asthma, and sleeping problems. P. foetida has quick and effective
action in burn wounds and is recommended by Brazilian Drugs centre as an
antirheumatic. The present study report an efficient in vitro regeneration protocol by
using leaf, node, internode and shoot tip explants for this species. Explants are surface
sterilized and inoculated in to culture medium with different concentration of growth
regulators. The shoot tip derived callus effectively (76%) produced shoots in the MS
medium containing BAP and NAA at 3.0 and 0.5 mg/l respectively. The nodal and
shoot tip segments derived shoots and rooted effectively (70% & 80%) in the basal
medium containing the auxin, IBA and IAA alone at 1.0 mg/l respectively. The nodal
callus derived in vitro rooted shoots responded well (92%) in the hardening medium.
Keywords:
Passiflora foetida, MS medium, BAP, IAA.
Article Citation:
Komathi S, Rajalakshmi G, Savetha S and Ayyappadas MP.
In vitro regeneration of Passiflora foetida l.
Journal of research in Biology (2011) 8: 653-659
Dates:
Received: 01 Dec 2011 /Accepted: 12 Dec 2011 /Published: 26 Dec 2011
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653-659 | JRB | 2011 | Vol 1 | No 8
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INTRODUCTION
India for many years has enjoyed a moderate
harvest of purple passion fruit in the nilgiris in the
South and in various parts of northern India.
Passion fruit is an exotic and fast-growing
perennial. It is a creeping vine like other members
of the genus, and yields an edible fruit. The specific
name, foetida means stinking in Latin and refers
to the strong aroma emitted by damaged foliage
(Flavia Guzzo et al., 2004). It grows as much as 30
ft (10 m) tall, with a thick, woody stem. The stems
are thin and wiry, covered with minute sticky
yellow hairs (Figure. 1).The leaves are heart
shaped three- to five lobed, alternate, arranged
helically, with long-stalked glands and long fine
hairs on margins, producing a disagreeable smell
when crushed. The flowers are white to pale cream
coloured, about 5-6 cm in diameter and bisexual.
Passion fruit is mainly used in jams, jellies, and
fruit juices. It is used for medicinal purposes as a
sedative, as well as a food source. It is used for
mood disorders (depression, anxiety, and stress).
Young leaves and plant tips are edible. Dry leaves
are used in Vietnamese folk medicine to relieve
sleeping problems. The whole plant is used in the
treatment of insomnia and anxiety. In India, it is
traditionally used for diarrhea, throat and ear
infections, liver disorders, tumors, itches, fever,
skin diseases and for wound dressing (A. G. Ingale
et al., 2010).
MATERIALS AND METHODS
In vitro regeneration
The application of tissue culture technology
for the establishment of in vitro regeneration of
the study species has been done by following the
methods as described below.
Figure: 1 - Passiflora foetida L.
654
Komathi et al.,2011
Sources of plant materials
Healthy plant, Passiflora foetida were
collected from the habitat of occurrence of in and
around Coimbatore district.
Explant selection and sterilization
Healthy and immature leaves shoot tip,
nodal and intermodal segments of this species were
used as explants. These segments were washed
under running tap water followed by treatment with
a surfactant, tween 20 (5% w/v) for five minutes.
After repeated washes in double distilled water, to
eliminate the fungal contamination explants were
treated with the carbendazim (50% w/v), and with
fungicide (10%) also for 15 minutes and rinsed with
double distilled water 2 or 3 times. To eliminate
bacterial contamination explants were also treated
with 5% antibiotics (Ampicillin and Ribampicin)
for 30 minutes followed by three rinses in sterile
double distilled water (Shekhawat, M.S. et al.,
2007). Furthermore, surface sterilization was
carried out by dipping the explants in 0.1%
mercuric chloride for 3 minutes followed by 3-4
rinses in sterilized double distilled water.
Culture medium and its composition
MS (Murashige and Skoog, 1962) basal
medium contains 3% sucrose solidified with 0.8%
agar was used. The pH of the medium adjusted to
5.8-5.9 prior to the addition of the agar.
Preparation of medium
Double distilled water was used for
preparing the medium. The nutrient medium
basically consists of inorganic nutrients, carbon
source, vitamins, irons and amino acids. The
chemicals were weighed accurately in electronic
weighing balance. All the stock solutions were
prepared in and stored in well-stopped sterilized
bottles and preserved in refrigerator at 4C. Specific
quantity of the stock solutions of the chemicals and
growth regulators were pipette out onto a one liter
beaker and required sucrose was added. The final
volume was made up with distilled water and the
pH was adjusted to 5.8-5.9 with 0.1N NaOH or
0.1N HCL using a pH meter (M.E. Mohamed et al.,
1996). The bottles after covering with a
polyethylene cap were autoclaved at 1.06 kg
pressure/sq cm for about 20 minutes at 121C. The
autoclaved medium in the culture bottles were
cooled and allowed to solidify and it was stored in
dark for further use. The inoculations were done
after four days to ensure that the bottles were free
from contamination.
Journal of Research in Biology (2011) 8: 653-659
Komathi et al.,2011
Growth regulators and their preparation
Three important groups of growth regulators
such as auxins, cytokines and kinetins were used in
the experiments (Bui Le Thanh Khiet et al., 2006).
All the growth regulators were stored at 4C until
use.
Preparation of Growth Hormones
S. Growth Initial dissolving
No Regulators solution
1 IBA Absolute alcohol
2 GA3 Absolute alcohol
3 IAA 70% alcohol
4 NAA 1N Naoh
5 BAP 0.1N Hcl
6 2,4,D 70% alcohol
7 Kinetin 0.1N Hcl
Auxin and their preparation
Three auxin namely -naphthalene acetic
acid (NAA), indole 3-acetic acid (IAA) and indole
3-buteric acid (IBA) were used in the experiments
(Vikrant A. et al., 2001). The stock solution was
prepared by dissolving 10mg of auxin individually
in 1ml with sterile distilled water. The required
volumes of auxin were added to the nutrient media,
before autoclaving and were used in different
concentrations.
Cytokines and their preparation
The stock solution was prepared by
dissolving 10mg of 6-benzyl amino purine (BAP),
kinetin 6-furfurylaminopurine in 1ml of 0.1N
Hydrochloric acid (HCL) and the volume was made
up to 10 ml by adding sterile distilled water. The
different concentrations were used before
autoclaving.
Culture conditions
All the cultures were maintained in the
culture room at a temperature of 252C and
relative humidity of 65-70%. The cultures were
kept under white light intensity of 3000 lux
provided from white fluorescent lamps (Philips,
India) with 14 hours photoperiodic duration.
In vitro studies
The present investigation on in vitro
propagation of the study species were carried out in
the tissue culture laboratory of Department of
Biotechnology, Hindusthan college of Arts and
Science, Coimbatore. In vitro techniques like
callogenesis, organogenesis and micro propagation
were attempted.
Journal of Research in Biology (2011) 8: 653-659
Callogenesis
Leaf segments of 0.50.5cm, nodal
segments (below the two terminal nodes) and shoot
tip measuring about 0.4-0.6 cm length were used as
explants for the study species. They were cultured
on full strength MS medium supplemented with the
growth regulators BAP (6-benzylaminopurine),
NAA (-Napthalene acetic acid), Kn (Kinetin).
Twenty five explants were used for each culture and
the experiments were twiced. The percentage of
explants responding for callus multiple shoot
induction, time taken for this induction, total
number of shoots/ explants and length of the shoots
were recorded after 40 days of culture (Aitchison,
P.A. et al., 1977). In the subculturing experiments
for organogenesis, the callus and in vitro produced
shoots were harvested and used as secondary
explants of culture on the same medium. Sub
culturing was carried out at the regular interval of
22-30 days using the same medium. The callus
pieces (0.50.5cm) and the in vitro developed
micro-shoots were cut into segments (0.4-0.6cm
length with single internode). These secondary
explants were subcultured on MS medium
containing the same growth regulators as used in
callusing (Vikrant A. et al., 2001).
Rooting of in vitro multiplied shoots
The elongated shoots were transferred to MS
medium supplemented with different auxin like
IAA, IBA and NAA at different concentration for
root induction. The rooted plantlets were removed
from culture tubes and transferred for hardening.
Callus induction
MS medium enriched with auxin (NAA),
cytokinins (BAP), kinetin (Kn) in the concentration
range of 1.0-2.5mg/l were tested for callus
induction and morphogenesis. Percentage of callus
induction of five week old cultures was calculated
(Bhojwani,S.S 1983). Callus was sub cultured
regularly at an interval of three weeks.
Callus frequency
The percentage of callusing was recorded at
the end of the fifth week. Frequency of callus
induction was calculated by using the following
formula and expressed in percentage:
Number of explants responded
Callus frequency (%) = 100
Total no of explants cultured
Hardening
The well developed healthy explants were
removed from the culture flask and were thoroughly
washed in running tap water to remove the adhering
655
 Komathiet al.,2011

nutrient medium completely without causing
damage to roots. Then the plants were soaked in 1%
(w/v) fungicide, methyl-3 benzimidizole carbomate
(Bavistin) solution for 10-15 minutes and
transferred to plastic pots (Bhojwani, S.S 1983).
Twenty five plantlets per potting mixture were
tested and growth rates were calculated after 30
days of hardening. During hardening process,
initially the plantlets were kept under shade not
present inside the 75% shade house for ten days.
After 10 days, they were gradually transferred to
75% shade house and maintained for two weeks.
Then the plantlets were exposed to the natural
environmental conditions.
RESULTS AND DISCUSSION
In vitro regeneration
The number of days required for callus
induction of explants is 10-35 after inoculation of
explants (Table 1). The percentage of explants
responding for callus formation with respect to
internode and leaves were lower, lies less than 15
and 18 only. However, it was noticed that the shoot
tip and node explants responded well for callus
formation when cultured on MS medium
supplemented with the growth regulators, BAP and
NAA at 2.5 and 0.3 mg/l respectively. Similarly,
higher amount of (60%) shoot tip explants
responded well for callus formation in the MS
medium containing the growth regulators BAP and
NAA at 2.5 and 0.3 mg/l respectively. On the other
hand, all attempts with various combinations of
BAP and NAA in basal medium produced different
color of callus. Furthermore it was observed that the
explants such as node and internode produced light
brown and dark brown colored calli respectively,
while the explants of shoot tip and leaf produced
green coloured calli when the explants inoculated
onto the MS basal medium containing different
concentrations of growth regulators.
The sub culturing experiments were
conducted by using callus as the secondary explants
for shoot proliferation and root induction in the MS
basal medium with various concentrations of the
growth regulators, BAP, NAA, IAA and IBA. The
results obtained are described below
In the basal medium, the responses of node
derived callus for shoot initiation to various
concentrations and combinations of BAP, NAA,
IAA and IBA are given in (Table 2). The shoot
formation during sub culturing was highly effective
(80%) in the basal medium containing BAP and
NAA at 3.0 and 0.5 mg/l respectively followed by
70% in BAP and NAA at 2.5 and 0.5 mg/l in the
basal medium. Similarly, the shoot initiation of
shoot tip derived callus was widely varied
according to concentrations and combinations of
growth regulators BAP, NAA, IAA and IBA in the
basal medium (Table 3). The MS medium fortified
with the growth regulators BAP and NAA at 3.0
and 0.5 mg/l respectively registered higher amount
(76%) of shoot initiation from the shoot tip derived
callus. The results indicate that the cytokinin, BAP
in combination with the auxin, NAA was found to
be more suitable for the improved response of calli
to produce shoots during sub culturing. The number
of shoots produced was greater (13 shoots/callus)
and shoot length was higher (7.4 cm) in basal
medium containing BAP and NAA at 3.0 and 0.5
mg/l respectively for node derived callus. Whereas
the shoot tip derived callus produce more number
of shoots (17 shoots/callus) in the medium
containing the growth regulators BAP alone at 3.0
mg/l. The shoot length was higher (7.3 cm) in the
MS medium enriched with the growth regulators
BAP and IAA at 2.0 and 1.0 mg/l respectively

TABLE 1. Effect of different concentration of growth regulators on callus induction from node, shoot tip
internode and leaf explants of the species, Passiflora foetida.
Growth
Days required for callus
regulators Callus formation (%) Color of the callus
formation after inoculation
(mg/l)
Shoot Inter Shoot Inter Shoot Inter
BAP NAA Node Leaf Node Leaf Node Leaf
tip node tip node tip node
1.0 0.3 15 18 31 32 50 30 8 10 LB G DB G

1.5 0.3 15 18 29 29 60 50 13 13 LB G DB G

2.0 0.3 19 17 25 29 70 50 14 17 LB G DB G
2.5 0.3 10 10 20 27 75 60 15 18 LB G DB G
LB- Light Brown, DB- Dark Brown and G - Green.
656 Journal of Research in Biology (2011) 8: 653-659
Komathi et al.,2011

TABLE 2. Effect of different concentration of growth TABLE 3. Effect of different concentration of growth
regulators on shoot initiation, shoot number and regulators on shoot initiation, shoot number and the
shoot length after sub culturing the node derived node derived callus of the species, Passiflora foetida.
callus of the species, Passiflora foetida.

Growth regulators Growth regulators

Culture No.of Shoot Culture No.of Shoot
(mg/l) (mg/l)
Rsponse shoots length response shoots/ length
BA (%) /callus (cm) (%) callus (cm)
NAA IBA IAA BAP NAA IBA IAA
P
1.0 - - - 20 07 4.2 1.0 - - - 16 07 4.1
1.5 - - - 20 10 4.6 1.5 - - - 18 08 4.5
2.0 - - - 30 11 5.1 2.0 - - - 20 12 5.2
2.5 0.5 - - 70 13 5.8 2.5 0.5 - - 70 13 7.7
3.0 0.5 - - 80 13 7.4 3.0 0.5 - - 76 15 7.1
1.5 - 1.0 - 50 10 5.2 1.5 - 1.0 - 50 10 5.2
2.0 - 1.0 - 55 08 5.7 2.0 - 1.0 - 60 10 7.3
2.5 - - - 49 11 6.1 2.5 - - - 30 14 6.0
3.0 - - - 60 07 6.7 3.0 - - - 40 17 6.2
1.5 - - 1.0 57 13 6.2 1.5 - - 1.0 27 08 6.5
2.0 - - 1.0 36 12 4.1 2.0 - - 1.0 42 13 6.8
2.5 - - - 15 11 3.7
2.5 - - - 18 09 3.8
3.0 - - - 23 13 4.3
3.0 - - - 20 10 2.7
while sub culturing the callus. It indicates the fact node callus derived shoots rooted effectively (70%)
that the concentration of auxin (NAA) was needed in the basal medium either with IBA alone at 1.0
for the shooting attributes of node and shoot tip mg/l. whereas, the number of root/shoot was also
derived callus of this species. (Figure: 2). greater (20 roots/shoot) in the same concentration
The rooting characters like percentage of of auxin, IBA. However, the NAA concentration at
shoot producing roots, number of roots produced by 1.0 mg/l produced higher length of roots (4-7 cm).
shoot and root length as influenced and root length The rooting attributes like percentage of roots
as influenced by the individual addition of growth produced by shoots, number of root/shoot and root
regulators viz., IBA, IAA and NAA in basal length were varied widely for shoot tip callus
medium for the study species are exhibited in derived shoots of the study species. A higher
(Table 4 & 5). percent of shoots rooted well in the MS medium
The nodal callus derived green adventitious containing the growth regulator IAA alone at 1.0
shoots of the species are carefully removed and sub mg/l. The number of roots produced by shoots is
cultured on the MS medium supplemented with higher in the same concentration of growth
individual addition of auxins for the adventitious regulator auxin, IAA. However the root length was
root initiation (Table 4). It was observed that the greater (4.9 cm) in the medium containing the
growth regulator auxin, NAA at 1.0 mg/l. While the
other supplementations of basal medium with IAA
and NAA resuspended considerably to the rooting
attributes of both node and shoot tip derived shoots
of the study species.
The genus Passiflora comprises hundred
species, mainly native of the South American
tropics and rainforests, which are grouped into 21
subgenera. Some species are widely studied for
their economic importance and are chiefly
figure2 figure2a figure2b cultivated for production of fruit juice. To obtain a
Figure: 2 &2a -Micro propagation of Passiflora foetida continuous source of material for a screening of
Figure: 2b - Callus induction of Passiflora foetida

Journal of Research in Biology (2011) 8: 653-659 657

 Komathi et al.,2011

TABLE 4. Effect of different concentration of growth TABLE 5. Effect of different concentration of growth
regulators on root initiation, root number and root regulators on root initiation, root number and root
length after sub culturing the node derived callus of length after sub culturing the node derived callus of
the species, Passiflora foetida. the species, Passiflora foetida.

Growth regulators Culture Growth regulators Culture No. of Shoot

No.of Root response roots/ length
(mg/l) response (mg/l)
roots/ length (%) callus (cm)
(%)
IBA IAA NAA callus (cm) IBA IAA NAA
0.1 - - 30 12 3.2 0.1 - - 30 10 3.1
0.3 - - 40 13 3.3 0.3 - - 41 12 3.4
0.5 - - 60 17 3.2 0.5 - - 62 14 3.9
1.0 - - 70 20 4.2 1.0 - - 84 18 4.2
- 0.1 - 20 12 3.1 - 0.1 - 25 11 3.2
- 0.3 - 30 16 3.3 - 0.3 - 33 14 3.5
- 0.5 - 45 18 3.7 - 0.5 - 45 15 3.7
- 1.0 - 61 17 3.2 - 1.0 - 61 16 3.7
- - 0.1 20 09 4.1 - - 0.1 29 08 4.1
- - 0.3 30 11 4.2 - - 0.3 34 10 4.3
- - 0.5 40 13 4.5 - - 0.5 41 12 4.5
- - 1.0 65 14 4.7 - - 1.0 67 14 4.9

secondary metabolites, zygotic embryo culture was
attempted for 62 Passiflora species, starting from
seeds mainly collected in the wild. Twenty nine of
these species produced calli, which had very
different growth rates. Plants were successfully
regenerated from calli of 13 different species. For
25 of the responsive species this is the first report of
in vitro culture (Flavia Guzzo et al., 2004).
Passion fruit is a highly valuable species but
it has some problems about pests and diseases.
There are some reports about shoot regeneration
and plantlet formation of vine crops from various
explants and using different techniques, but these
are not suitable. In this study, we used callus
derived from in vitro plantlets of yellow passion
fruit to initiate cell suspension culture. Cultures
were maintained in MS medium supplemented with
various NAA concentrations (0.0-2.0 mg l-1), 30 g l
-1 sucrose. Cell growth was evaluated by counting
cell numbers on 0, 5, 10, 15, 20 and 25 days of
culture. After 20 days of culture, 2 ml of suspension
culture was transferred to hormone-free MS
medium containing 60 gl-1 sucrose and 8 gl-1 agar
to induce callus formation. These calli were used to
regenerate shoots(Bui Le Thanh Khiet et al., 2006).
Passion fruit is a highly valuable species but
it has some problems about pests and diseases.
There are some reports about shoot regeneration
and plantlet formation of vine crops from various
explants and using different techniques, but these
are not suitable. Cell suspension culture is a tool to
658
study cell growth, to produce secondary metabolites
and a tool for rapid micro propagation of some
economically valuable crops. Using this technique,
we can understand cell growth parameters and
establish an efficient protocol. In this study, we
used callus derived from in vitro plantlets of yellow
passion fruit to initiate cell suspension culture.
Cultures were maintained in MS medium
supplemented with various NAA concentrations
(0.0-2.0 mg l-1), 30 g l-1 sucrose. Cell growth was
evaluated by counting cell numbers on 0, 5, 10, 15,
20 and 25 days of culture. After 20 days of culture,
2 ml of suspension culture was transferred to
hormone-free MS medium containing 60 gl-1
sucrose and 8 gl-1 agar to induce callus formation.
These calli were used to regenerate shoots (Bui Le
Thanh Khiet et al., 2006).
CONCLUSION:
Callus was induced efficiently from the leaf
explants of Passiflora foetida L. (Passion fruit) on
Murashige and Skoog (MS) medium containing 2
mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) and
0.5 mg/l kinetin (KN) after 16 days. Maximum
callus (65% response, 610 mg fresh weight and 55
mg dry weight) was formed on MS medium
supplemented with low sucrose and salt
concentration than that of high sucrose and salt
concentration (Rasool SN et al., 2011).
Journal of Research in Biology (2011) 8: 653-659
Komathi et al.,2011
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An International Online Open Access
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The monthly changes of chloroplast pigments content in selected plant

species exposed to cement dust pollution
Journal of Research in Biology

Authors: ABSTRACT:
Sarala Thambavani D1,
Saravana Kumar R2
Chloroplast pigments were shown to be very sensitive to various
environmental influences. Changes in chlorophyll and carotenoid content were
investigated in selected plant species exposed to alkaline dust emitted by the cement
Institution:
1. Department of Chemistry, industry. Pigments were extracted consequently for 30, 60, 90, 120, 150 and 180 days
Sri Meenakshi Govt. Arts interval and quantified spectrophotometrically. In comparison with control site all
College for women, Madurai measured pigments were reduced in dust-exposed plant species. This is due to
- 625 002. Tamil Nadu, deceleration of the biosynthetic processes rather than degradation of pigments.
India. Chlorophyll b content appeared to be more sensitive than chlorophyll a in polluted
2. Department of Chemistry, plants. Total carotenoids needed a longer period of time to reach nearly the same
N.P.R. College of level as in controls. The progression of pigment decline in polluted sites appeared not
Engineering and to be dramatically accelerated. It might thus be concluded that polluted sites had
Technology, Natham, sufficient biosynthetic capacity to prevent irreversible damage by cement dust.
Dindigul 624401.
TamilNadu, India.

Keywords:
Corresponding author: Cement dust, Bio-indicators, Photosynthetic pigments, Chlorophyll,
Saravana Kumar R Carotenoid, assimilating pigments and acetone.

Email: Article Citation:

sarala_dr@yahoo.in Sarala Thambavani D, Saravana Kumar R.
gsivasaravanan@gmail.com The monthly changes of chloroplast pigments content in selected plant species
exposed to cement dust pollution
Journal of research in Biology (2011) 8: 660-666
Phone No:
99423-68797 Dates:
Received: 10 Dec 2011 /Accepted: 16 Dec 2011 /Published: 26 Dec 2011
Web Address:
http://jresearchbiology.com/ Ficus Publishers.
Documents/RA0165.pdf.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

660-666 | JRB | 2011 | Vol 1 | No 8

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INTRODUCTION
The impact of the cement industry on the
surrounding vegetation has been widely
investigated (Farmer, 1993) although research on
the effects of dust pollution on plants has never
received the same level of attention as that given to
phytotoxic pollutants such as SO2, NO2 and O3.
Results from research that has been undertaken,
together with repeated observations of dust deposits
on vegetation, suggest that the effects of dust may
be important and are worthy of greater investigative
attention. Rapid industrialization and addition of the
toxic substances to the environment are responsible
for altering the ecosystem (Mudd & Kozlowski,
1975: Nriagu & Davidson, 1986; Clayton &
Clayton, 1982).
The Cement industry plays a vital role in the
imbalances of the environment and produces air
pollution hazards (Stern, 1976). In comparison with
gaseous air pollutants, many of which are readily
recognized as being the cause of injury to various
types of vegetation, relatively little is known and
limited studies have been carried out on the effects
of cement dust pollution on the growth of plants.
Carotenoids are a class of natural fat-soluble
pigments found principally in plants, algae and
photosynthetic bacteria, where they play a critical
role in the photosynthetic process (Ong and Tee,
1992; Britton, 1995) and also protect chlorophyll
from photo oxidative destruction (Siefermann-
Harms, 1987). When plants are exposed to the
environmental pollution above the normal
physiologically acceptable range, photosynthesis
gets inactivated (Miszalski and Mydiarz, 1990).
The amount of solar radiation absorbed by a leaf is
a function of the photosynthetic pigment content;
thus, chlorophyll content can directly determine
photosynthetic potential and primary production
(Curran et al., 1990, Filella et al., 1995).
Furthermore, leaf chlorophyll content is closely
related to plant stress and senescence (Hendry 1987,
Merzlyak and Gitelson 1995, Penuelas and Filella
1998, Merzlyak et al., 1999). A periodical study
was carried out to study the effect of cement dust
pollution on the growth of selected plant species
such as Azadirachta indica(L), Polyalthia longifolia
(L), Ficus religiosa(L), Pongamia pinnata(L) and
Delonix regia(L).
MATERIALS AND METHODS
Study Area Description
Madurai city is grown on both sides of river
Vaigai and its terrain is mostly flat. The ground
661
Thambavani et al.,2011
rises from the city, towards outward, on all sides
except the south, which is a gradually sloping
terrain. It is surrounded on the outskirts by small
and prominent hills. The city is about 100 meters
above mean sea level and it is situated on 9 055 NL
and 7807 EL and the city is covering 51.96 Sq.kms.
The climate of Madurai town is hot and dry and the
temperature range between a maximum and
minimum of 420C and 210C respectively.
Experimental procedure
The experiment was conducted in pots under
natural conditions. Mature ripe seeds of five plant
species like Azadirachta indica (L), Polyalthia
longifolia(L), Ficus religiosa (L), Pongamia
pinnata (L) and Delonix regia (L) were collected.
The seeds of all the three species were sown in
medium sized clay pots with three parts fine sand
and one part of natural manure. When the seedlings
reached a suitable height, they were transferred to
pots, 23.0 cm in diameter and 21.0 cm in depth and
the five plants were planted in three replicates. One
gram of cement dust was sprinkled regularly on the
aerial parts of each plant twice a week, except the
control and all the plants were watered daily with
tap water.
Photosynthetic pigments Analysis
Chlorophyll a and b impart the green
color that one associate with plant leaves.
Carotenoids, which are yellow pigments, are also
present in leaves but are usually masked by the
chlorophylls. It is only in the fall when the
chlorophylls are degraded faster than the carotenoid
that the yellow color becomes visible to us. The
chlorophyll and carotenoid contents of plants can
vary markedly with its age or depend on
environmental factors such as light intensity or
quality during growth. Carotenoids and
chlorophylls are found in the chloroplasts and are
associated with the thylakoids, the internal
membrane network of these organelles. It is now
established that all chlorophylls are organized as
discrete chlorophyll-protein complexes within the
lipid matrix of the photosynthetic membrane. The
majority of chlorophyll a molecules (and all
chlorophyll b and carotenoid molecules) functions
as antenna pigments. In combination with proteins,
they form the light-harvesting complexes, which
absorb and funnel light energy to the reaction center
chlorophylls, thereby allowing the plant to utilize a
broad spectrum of wavelengths for photosynthesis.
Some of the chlorophyll a molecules serve
specialized functions in the reaction centers of
photo systems I and II, where the light energy is
Journal of Research in Biology (2011) 8: 660-666
Thambavani et al.,2011
used to drive the reduction of components of the
electron transport chain.
Extraction of Photosynthetic pigments
Extract photosynthetic pigments by grinding
1g of leaves, torn into small pieces, in a mortar with
a pinch of clean sand and a total of 10 ml of 100%
acetone. Initially, add only a small amount of
acetone to begin the grinding process. It is much
easier to grind the leaves if the extract is a pasty
consistency. Add more solvent in small increments
while continuing to grind the leaves. For some
species may need to add more than the suggested
10ml of acetone. Pour the extract into a 15ml
centrifuge tube and centrifuge in the bench top
centrifuge for 3 to 5 min. Remove the extract to a
10ml graduated cylinder using a Pasteur pipette.
Transfer an aliquot of the clear leaf extract
(supernatant) with a pipette to a 1-cm-pathlength
cuvette and take absorbance readings against a
solvent blank in a UV-VIS spectrophotometer at
four different wavelengths.
750 nm (A750 = 0 for clear extract)
662 nm (chlorophyll a maximum using 100% acetone)
645 nm (chlorophyll b maximum using 100% acetone)
470 nm (carotenoids).
Apply measured absorbance values to
equations given by Lichtenthaler (1987) for acetone
to determine pigment content (g/ml extract
solution).Once the baseline has been run from 700-
400nm using acetone in the cuvette, run an
absorption spectrum for each pigment, rinsing the
sample cuvette with acetone between readings. The
peaks and valleys will be adjusted automatically by
the Lamda-35 UV-VIS spectrophotometer, by
changing the range of percentage absorbance on the
y-axis. We can use the cursor to obtain the
wavelengths of the spectral peaks, or estimate them
from the printed spectra. These peak wavelengths
will be useful for determining the identities of the
pigments associated with the spectra. The studies
were conducted on Azadirachta indica(L),
Polyalthia longifolia(L), Ficus religiosa(L),
Pongamia pinnata(L) and Delonix regia(L) plants
growing under natural conditions. The plant
samples were analyzed at every 30-day of intervals.
The concentrations of photosynthetic pigments like
chlorophyll-a, chlorophyll-b and carotenoids (mg/g
fresh weight) we obtained using the following
formula given by Lichtenthaler 1987.
Quantification of pigments (For 100% Acetone)
Chl-a (g/ml) = 11.24 A661.6 2.04 A644.8
Chl-b (g/ml) = 20.13 A644.8 4.19 A661.6
Journal of Research in Biology (2011) 8: 660-666
Carotenoids = (1000 A470 1.90 Ca - 63.14 Cb)/214
Statistical Analysis
Data from the two selected sites for the plant
materials were subjected to the two way analysis of
variance (ANOVA). Using ANOVA the
comparison made between control plant species and
polluted plant species. Significance difference was
calculated at 0.05%, 0.01% and 0.001% level as per
standard method of Gomez and Gomez (1984).
The present investigation has been
undertaken to study the effect of cement dust
pollutant on total chlorophyll, carotenoids,
chlorophyll a and chlorophyll b of selected plant
species. In the present study, the cement pollution
effects on the performance of selected plant species
was observed and the total chlorophyll content
decreased significantly in response to cement dust
pollutants in polluted plant leaves compared with
control of Azadirachta indica(L), Polyalthia
longifolia(L), Ficus religiosa(L), Pongamia
pinnata(L) and Delonix regia(L).3.
RESULTS AND DISCUSSION
The quantitative analysis of chloroplast
pigments revealed difference between the selected
plant species exposed to cement dust pollution and
those from control. The mean values of all
measured parameters are shown in Fig. 1 to 4.
Chlorophyll a content was higher in control site
compared with polluted site. The amount of
chlorophyll a varied from 1.18 to 1.78 mg g-1(FW)
in control site and from 0.61 to 1.33 mg g-1(FW) in
the polluted site (Table 1).
The chlorophyll b content was lowered in
polluted site compared with control site in all the
plant species. The values varied from 0.81 to 1.43
mg g-1(FW) and 0.54 to 1.02 mg g-1(FW) in control
and polluted sites respectively (Table 2, fig. 2). The
amount of chlorophyll reduced in all the polluted
Figure 1. The mean value of chloroplast pigments in
Azhadirachta indica
662

Table 1. The monthly mean values of chlorophyll a (mg g -1 FW; SD) in control and polluted leaves of
selecteplant species. P(t)- percent of similarity; NS- not significant difference.
Plants control
Azhadiracha indica(L) 1.78 0.42
Polyalthia longifolia(L) 1.40 0.34
Ficus religiosa (L) 1.180.22
Pongamia pinnata(L) 1.54 0.42
Delonex regia(L) 1.490.47
plants than control plants due to alkaline dust
pollution. The highest value of total chlorophyll is
2.08 mg g-1(fw) in A.indica and lowest value 1.19
mg g-1(fw) in F.religiosa at polluted site compared
with control (Table 3, fig. 3).
The amount of total carotenoid in control
and polluted sites varied from 0.25 to0.51 mg g-1
and 0.15 to 0.33 mg g-1 respectively (Table 4, fig.
4). All the selected plant species showed the
significant (p<0.01 and p<0.05) reduction in
pigment content during the study period. In general,
plants showed a decrease in photosynthetic
pigments due to cement dust treatment. The
relatively reduction in Chl a, Chl b, total such as 38.78%, 37.65%, 65.55% and 41.2%
chlorophyll and total carotenoids for the selected
species such as Azadirachta indica(L), Polyalthia
longifolia(L) Ficus religiosa(L), Pongamia pinnata
(L) and Delonix regia(L) were related to cement
dust pollution. The following observations were
made.
Azadirachta indica (L) showed 22.92%
reduction in the Chlorophyll a when exposed to
the cement dust pollution. There were 25.39%
reduction in Chlorophyll b when compared with
control and cement dusted plant species.
Azadirachta indica (L) showed 42.06% reduction in
the total carotenoids. There were 24.6% reductions
Figure 2. The mean value of chloroplast pigments in
Polyalthia longifolia
663
Thambavani et al.,2011
Polluted P(t)
1.33* 0.31 <0.05%
1.02** 0.29 <0.01%
0.64** 0.14 <0.01%
0.94* 0.35 <0.05%
0.61*0.21 <0.05%
in the total chlorophyll. Cement dust reduced the
photosynthetic pigments such as chlorophyll a,
chlorophyll b, total chlorophyll and total
carotenoids. Polyalthia longifolia (L) exhibited
27.34%, 26.61%, 61.15% and 28.2% reduction in
chlorophyll a, chlorophyll b, total carotenoids
and total chlorophyll respectively. There were
43.32% reduction in chlorophyll a, 32.39%
reduction in chlorophyll b, 29.01% reduction in
total carotenoids and 40.1% reduction in total
chlorophyll in the selected species of Ficus
religiosa (L). Pongamia pinnata (L) exhibited the
decreasing trend in the photosynthetic pigments
reduction in chlorophyll a, chlorophyll b, total
carotenoids and total chlorophyll respectively.
Delonix regia (L) exhibited the maximum reduction
in the photosynthetic pigments such as
37.65%,41.85%, 38.37% and 51.8% in chlorophyll
a, chlorophyll b, total carotenoid and total
chlorophyll respectively.
There was maximum reduction (43.32%) of
Chlorophyll a in the leaves of Ficus religiosa (L)
and minimum (22.92%) reduction in Azadirachta
indica (L) while maximum reduction (41.85%) of
Chlorophyll b was depleted in Delonix regia (L)
and minimum reduction (25.39%) in Azadirachta
Figure 3. The mean value of chloroplast pigments in
Ficus religiosa
Journal of Research in Biology (2011) 8: 660-666
Thambavani et al.,2011

Table 2. The monthly mean values of chlorophyll b Table 3. The monthly mean values of total
(mg g-1 FW; SD) in control and polluted leaves of chlorophyll (mg g-1 FW; SD) in control and
selected plant species. P(t)- percent of similarity; NS- polluted leaves of selected plant species. P(t)- percent
not significant difference. of similarity; NS- not significant difference.

Plants control Polluted P(t) Plants control Polluted P(t)

Azhadiracha Azhadiracha
0.97 0.20 0.75**0.17 <0.01% 2.76 0.62 2.08**0.47 <0.01%
indica(L) indica(L)

Polyalthia Polyalthia
1.430.16 1.02**0.11 <0.01% 2.830.49 2.04**0.38 <0.01%
ongifolia(L) longifolia(L)

Ficus religiosa Ficus

0.810.12 0.54**0.08 <0.01% 1.990.32 1.19**0.22 <0.01%
(L) religiosa (L)

Pongamia Pongamia
1.130.24 0.70*0.14 <0.05% 2.670.65 1.64*0.46 <0.05%
pinnata(L) pinnata(L)

Delonex Delonex
1.250.29 0.74*0.21 <0.05% 2.740.64 1.35**0.41 <0.01%
regia(L) regia(L)

indica (L). The highest reduction (51.81%) in total
chlorophyll was observed in Delonix regia (L)
whereas the lowest reduction (24.67%) was
recorded in Azadirachta indica(L). Similarly, in
case of carotenoid contents, highest reduction
(65.55%) was observed in Pongamia pinnata(L)
and lowest in Ficus religiosa(L) (29.01%).
Polyalthia longifolia(L) and Ficus religiosa(L)
showed significant reduction in chlorophyll
a (p<0.01) and other three species showed the
significance level (p<0.05%).
Mandre and Tuulmets (1997) reported a
decrease of chlorophyll content in Norway spruce
needles caused by cement dust.
Total average amount of assimilating pigments:
The weight ratio of Chl a and Chl b
indicates the functional characters of photosynthetic
pigments. The ratio of Chl a and Chl b was
found to be higher for the polluted environment
exposed to the Azadirachta indica (L) compared to
the control. Similarly, the weight ratio of Chl a
and Chl b to total carotenoids indicates the stress
experienced by the plant species. The average
amount of chlorophyll a to chlorophyll b (a/b) is
minimum in pongamia pinnata(L) (0.76 mg g-1fw)
and maximum in Azadirachta indica(L) (1.80 mg g-
1
fw) (Table 5). The total photosynthetic pigments
(Chl a+ Chl b+ carotenoid) were found to
minimum in polluted sites compared to the control.
The average amount of total photosynthetic
pigments in polluted plants was less in Ficus
religiosa (L) (1.37 mg g-1fw) and more in
Azadirachta indica (L) (2.40 mg g-1fw) compared to
all the control plant species. The reduction in total
pigments is mostly caused by Chl a and Chl b
which have significant value and also the low
carotenoidic pigments. The low ratio value
indicates the plant species subjected to the dust
pollution.
The observed variation in photosynthetic
pigment was contributed due to the air pollutant and
sensitivity of the plant. The ratio of Chl a+b to
carotenodic pigments, (a+b)/c) has extremely low
values compared to the control plant species. It

Figure 4. The mean value of chloroplast pigments in Figure 5. The mean value of chloroplast pigments in
Pongamia pinnata Delonex regia

Journal of Research in Biology (2011) 8: 660-666 664

 Thambavani et al.,2011

Table 4. The monthly mean values of total carotenoid in cement dust might be responsible for the
(mg g-1 FW; SD) in control and polluted leaves of reduction in plant species pigments. Traces of toxic
selected plant species. P(t)- percent of similarity; metals such as Chromium and Copper are common
NS- not significant difference. in some varieties of Portland cement and are
Plants control Polluted P(t) harmful to human beings and other living systems
(Omar & Jasim, 1990). Cement dust pollution
Azhadiracha indica (L) 0.510.14 0.33*0.11 <0.05%
imparts more stress on the plant species. Bio-
Polyalthia longifolia (L) 0.330.12 0.210.10 NS monitoring of the plants is an important tool to
Ficus religiosa (L) 0.320.09 0.190.05 NS evaluate the impact of cement dust pollutants on
pollution.
Pongamia pinnata (L) 0.280.11 0.180.08 NS
Delonex regia (L) 0.250.17 0.150.11 NS

Table 5. The mean values of assimilating pigments (mg g-1fw) of selected Plant Species
(Chl-a + Chl-b + Ca-
Chlorophyll a/b ratio Chl (a + b)/Carotenoid
Plant Species rotenoid)
Control Polluted Control polluted Control polluted
A.indica(L) 3.26 2.40 1.75 1.80 5.98 9.16
Polyalthia longifolia(L) 3.15 2.24 0.92 0.95 11.70 8.88
Ficus religiosa (L) 2.30 1.37 1.43 1.12 8.31 6.78
Pongamia pinnata(L) 2.98 1.50 1.14 0.76 10.68 -27.70
Delonex regia(L) 2.94 1.83 1.28 1.25 17.73 5.31

indicates that the plant species are under stress and

also had damage due to cement dust pollution.
CONCLUSION
The Cement dust had a significant effect on
the photosynthetic pigments such as Chla, Chlb
and total carotenoid. Plant response varies between
plant species of a given genus and between varieties
within a given species. Plants do not necessarily
showed similar susceptibility to different pollutants.
Major variations in response to different species to
air pollutants have been documented by Jacobson
and Hill (1970). Studies of biochemical changes
and pollution effects on the plant metabolism, that
is, reduction in chlorophyll and completely clogged
stomates (Ahmed & Qadir, 1975) reveals that these
parameters are important in regulating the
productivity and also the number of flowers and
seeds produced. Although all the species showed
significant reduction in the photosynthetic
pigments, the extent up to which the plant species
were affected varied from species to species and
days to days. Almost all the species showed
maximum variation in photosynthetic pigments
(table1 and fig1). The results presented in the paper
shows that cement dust pollution significantly
reduced the photosynthetic pigments of Delonix
regia (L) compared to other four species. It is also
clear that Delonix regia (L) is very sensitive species
compared to the other four species.
It is concluded that the presence of toxic pollutants
665
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666
 Journal of Research in Biology
Genetic diversity assessment in nine cultivars of Catharanthus roseus from
Central Gujarat (India) through RAPD, ISSR and SSR markers
Journal of Research in Biology
Authors:
Sanjay Lal, Kinnari N.
Mistry, Smit D. Shah,
Riddhi Thaker, Parth B.
Vaidya.
Institution:
Ashok & Rita Patel Institute
of Integrated Studies in
Biotechnology & Allied
Sciences (ARIBAS), New
Vallabh Vidhya Nagar
388121 (Gujarat) India.
Corresponding author:
Sanjay Lal
Web Address:
http://jresearchbiology.com/
Documents/RA0163.pdf.
Journal of Research in biology
An International Open Access Online
Research Journal
An International Online Open Access
Original Research Paper Publication group
ABSTRACT:
The genetic relationship study was carried out among nine different cultivars
of Catharanthus roseus using RAPD, ISSR and SSR markers. In RAPD analysis, out of
twenty primers, six primers amplified 592 bands out of which 466 were polymorphic
while rest was monomorphic. This gave high (78.71%) polymorphism among nine
cultivars. In ISSR analysis, 78.94% polymorphism was observed, while in SSR analysis,
76.62% polymorphism was observed. The dendogram based on all three markers
separated the cultivars in two major groups. Blue pearl, cooler red, and pacifica
apricotwere in the same group, while rest was in other group. Amongst all Pacifica
liac and Albus with red eye were found to be closely related. Our results showed
that all RAPD, ISSR and SSR markers are sensitive and effective tool for genomic
analysis in Catharanthus roseus. This study provides key platform for further crop
improvement and cross breeding.
Keywords:
Genetic diversity, RAPD, ISSR, SSR, Catharanthus roseus.
Abbreviations:
Random amplified polymorphic DNA (RAPD), Inter simple sequence repeats
(ISSR), Simple sequence repeats (SSR), basepair (bp).
Article Citation:
Sanjay Lal, Kinnari N. Mistry, Smit D. Shah, Riddhi Thaker, Parth B. Vaidya.
Genetic diversity assessment in nine cultivars of Catharanthus roseus from Central
Gujarat (India) through RAPD, ISSR and SSR markers.
Journal of research in Biology (2011) 8: 667-675
Dates:
Received: 09 Dec 2011 /Accepted: 15 Dec 2011 /Published: 30 Dec 2011
Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.
667-675 | JRB | 2011 | Vol 1 | No 8
Submit Your Manuscript
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 Lal et al.,2011

INTRODUCTION al., 1998). SSR and ISSR markers are also being
Catharanthus roseus (L.) G. Don (family widely used to study genetic diversity in number of
Apocynaceae) is a beautiful ornamental plant with plant species.
enormous medicinal values. In India, its commonly The current investigation was carried out
known as periwinkle, sada bahar, sadaphul, with the aim to study the genetic diversity among
or barmasi. Catharanthus roseus (Vinca rosea) is C. roseus cultivars using RAPD, ISSR and SSR
native to Madagascar and spread throughout the markers.
tropics and subtropics (RK Shaw et al., 2010).
The plant is known to have more than 70 MATERIAL AND METHODS
different alkaloids like ajmalicine, vincrystine, Plant material
vinblastine etc. These alkaloids contribute to Nine different cultivars of Catharanthus
anticancer and anti diabetic activity of plant. roseus were collected for this study. All samples
Catharanthus roseus has been used traditionally for were collected from nearby area of Anand, Gujarat,
its calming effect and its ability to reduce the blood India (Table-1). Fresh and young leaves were
pressure (Flora of China: Catharanthus roseus). collected to isolate the DNA.
Assessment of genetic diversity in crop Genomic DNA isolation
species is an important component of crop The genomic DNA was isolated from young
improvement programs. Accurate analysis of the leaves by standard CTAB (Cetyl trimethyl
genetic diversity can be useful in plant breeding. ammonium bromide) method (Doyle and Doyle,
The detailed understanding of relationship between 1990) with slight modifications. Slight higher
inbred lines and pure lines can be useful in planning Table-1: Name, Petal color and Images of nine
crosses (Hallauer and Miranda, 1988). Analysis of cultivars of Catharanthus roseus (L.) G. Don
genetic diversity in germplasm collection can
facilitate reliable classification of accessions and Cultivar Colour of
No. Image
name petal
identification of subspecies which can be further
useful for plant breeding.
1 Patricia white Milky white
Various marker systems have been used for
genetic studies and characterization analysis. These
include morphological, cytological, biochemical
2 Grape cooler Light pink
and DNA marker systems. DNA markers are
considered the best tools for determining genetic
relationships/diversity, as they are unlimited in
3 Cooler red Cherry red
number, show high polymorphism and are
independent of environmental interaction i.e.,
highly heritable (Singh et al., 2004). Peppermint White with
4
There are various types of DNA markers cooler red center
like Restriction Fragment Length Polymorphism
(RFLP, Sambrook et al., 1989), Variable Number Pacifica
of Tandem Repeats (VNTRs, Nakamura et al., 5 Whitish pink
apricot
1987), Simple Sequence Repeats (SSRs, Jacob et
al., 1991), Inter Simple Sequence Repeats (ISSR, Dark pink
Zietkiewicz et al., 1994) and Random Amplied 6 Cooler orchid with white
Polymorphic DNA (RAPD, Williams et al., 1990). eye
Amongst all these techniques, RAPD
technique has gained importance due to its 7 Pacifica liac Dark pink
simplicity, efciency, relative ease to perform and
non-requirement of DNA sequence information
(Karp et al., 1997; Khanuja et al., 1998). The Milky white
Albus with red
8 with radiating
technique has been very useful in studies of genetic eye
red eye
diversity (Orozco-Castillo et al., 1994; Chalmers et
al., 1994), phylogeny and systematic (Millan et al.,
1996; Sun et al., 1998), genetic linkage mapping 9 Blue pearl Purple
(Cheung et al., 1997) and gene tagging (Tiwari et
668 Journal of Research in Biology (2011) 8: 667-675
Lal et al.,2011
concentration of detergent was used. RNA was
removed by giving RNaseA treatment which was
given after incubation at 650C in boiling water bath.
The isolated DNA was dissolved in 25l of T10E1
buffer (Tris 10mM and EDTA 1mM, pH 8.0). The
DNA was checked through agarose gel
electrophoresis and the quantity and purity was
checked by Nanodrop-1000 (Thermo Fisher
Scientific, USA).
Random Amplified Polymorphic DNA (RAPD)
analysis
The RAPD analysis was
carried out using the method of Williams et al.,
(1990). Polymerase chain reactions were performed
in 25l system containing 2.5l of 10 X assay
buffer (10mM Tris-Cl; pH 9.0, 1.5mM MgCl2,
50mM KCl and 0.01% gelatin), 2.5mM of each
dNTPs (dATP, dTTP, dCTP and dGTP) (Bangalore
Genei Pvt. Ltd, Bangalore, India), 1.5M of primer
(Sigma Aldrich, Bangalore), 0.2 unit of Taq DNA
polymerase (Bangalore Genei Pvt. Ltd, Bangalore,
India) and 25ng of template DNA. The reaction was
carried out using thermo cycler (Corbett research
gradient automatic, UK). Reaction was carried put
in three steps.
The first step of initial
denaturation was performed at 94 0C for 5 min.
Following the initial denaturation step, PCR was
carried out for 45 cycles. Each cycles consisted of a
denaturation step of 1 min at 920C, annealing step
of 1 min at 370C and an extension step of 2 min at
720C. The last cycle was followed by final
extension step of 7 min at 72 0C. The holding
temperature was 40C.
Inter Simple Sequence Repeat (ISSR) analysis
In ISSR analysis, the
polymerase chain reactions were performed in 25l
system containing 2.5l of 10 X assay buffer
(10mM Tris-Cl; pH 9.0, 1.5mM MgCl2, 50mM KCl
and 0.01% gelatin), 2.5mM of each dNTPs (dATP,
dTTP, dCTP and dGTP) (Bangalore Genei Pvt. Ltd,
Bangalore, India), 1.5M of primer (Sigma Aldrich,
Bangalore), 0.2 unit of Taq DNA polymerase
(Bangalore Genei Pvt. Ltd, Bangalore, India) and
25ng of template DNA. The reaction was carried
out using thermo cycler (Corbett research gradient
automatic, UK). Reaction was carried out in three
steps. The first step of initial denaturation was
performed at 940C for 5 min. Following the initial
denaturation step, PCR was carried out for 40
cycles. Each cycles consisted of a denaturation step
of 1 min at 940C, annealing step of 1 min at various
Journal of Research in Biology (2011) 8: 667-675
annealing temperatures (Table-3) and an extension
step of 2 min at 720C. The last cycle was followed
by final extension step of 7 min at 72 0C. The
holding temperature was 40C.
Simple Sequence Repeats (SSR)
In SSR analysis, the polymerase chain
reactions were performed in 25l system containing
2.5l of 10 X assay buffer (10mM Tris-Cl; pH 9.0,
1.5mM MgCl2, 50mM KCl and 0.01% gelatin),
2.5mM of each dNTPs (dATP, dTTP, dCTP and
dGTP) (Bangalore Genei Pvt. Ltd, Bangalore,
India), 1.5M of primer (Sigma Aldrich,
Bangalore), 0.2 unit of Taq DNA polymerase
(Bangalore Genei Pvt. Ltd, Bangalore, India) and
25ng of template DNA. The reaction was carried
out using thermo cycler (Corbett research gradient
automatic, UK). Reaction was carried out in three
steps. The first step of initial denaturation was
performed at 940C for 5 min. Following the initial
denaturation step, PCR was carried out for 40
cycles. Each cycles consisted of a denaturation step
of 1 min at 940C, annealing step of 1 min at various
annealing temperatures (Table-4) and an extension
step of 2 min at 720C. The last cycle was followed
by final extension step of 7 min at 72 0C. The
holding temperature was 40C.
Agarose gel electrophoresis
The amplified products were checked on gel
electrophoresis (1.5% agarose gel for RAPD and
2% for ISSR and SSR). The electrophoresis was
performed for four hours at a constant voltage of
100 volts. The bands were visualized under U.V.
light and the photographs were taken by gel
documentation system (Alpha Innotech, Alpha
Imager EP, USA). 100bp ladder was used to
determine the size of the amplicons.
Data scoring and statistical analysis
The band was scored 1 for its presence and
0 for its absence. This data were used to construct
the binary matrix. The Jaccards coefficient
(Jaccard, 1908) was used to construct the similarity
matrix among the nine cultivars of Catharanthus
roseus. The phylogram was constructed using the
unweighted pair group method using arithmetic
means (UPGMA) (Sneath and Sokal, 1973) and
SAHN clustering. The entire analysis was
performed using the NTSYS-pc (Numerical
taxonomy system, applied biostatistics, Inc., New
York, USA, software version 2.02e) (Rohlf, 1997).
The polymorphism information content (PIC) value
was calculated as PIC=1-P2i ; Pi is the band
frequency of the ith allele (Smith et al., 1997).
669
 Lal et al.,2011

Groupings of cultivars were also evaluated by
principle coordinate analysis (PCA) as reported by
Thomas et al. (2006). PCA was performed by
extracting Eigen value and Eigen vectors from a
correlation matrix which was generated using a
standardized data matrix 2-D and 3-D plots were
constructed to evaluate the groupings of C. roseus
cultivars.
RESULT AND DISCUSSION
In RAPD analysis, total twenty RAPD
primers were used to check the genetic variation in
nine different cultivars of Catharanthus roseus. Out
of these twenty, six primers gave satisfactory and
reproducible bands. The banding pattern of the
RAPD analysis for two primers has been shown in
Fig-1. The details of the banding pattern are shown
in Table-2. All these six primers gave
amplifications in all nine cultivars. Total 592 bands
were observed. From these total bands, 466 were
polymorphic while, 126 bands were monomorphic.
This resulted in total polymorphism of 78.71%.
OPA-03 gave maximum number (146) of bands,
while minimum number of bands (62) was observed
in OPAF-15 primer. The amplicons were observed
ranging from 190-2650bp. The largest amplicon
(2650bp) was amplified by OPA-03 primer, while
the shortest amplicon (190bp) was amplified by
OPN-15 and OPC-12 primer. Similarity matrix was
plotted using the Jaccards coefficient. According to
the matrix, the similarity index was observed
ranging from 0.1980.731 with the mean similarity
index of 0.56 indicating reasonable variability as
obtained by RAPD markers (data not shown). The
maximum PIC value (0.955) was observed in OPC
12 primer, while the minimum PIC value (0.900)
was observed in OPAF 05. The average PIC value
observed from all six RAPD primers was 0.93.
In our study, RAPD markers were
successfully used to differentiate all nine cultivars
of Catharanthus roseus from each other. Thus, on
the basis of RAPD, the findings of this study are
similar to the observations of Rajaseger et al.,
(1990).
In ISSR analysis, totally six ISSR primers
were used. Out of these six, three primers gave
satisfactory and reproducible bands. The banding
pattern of one ISSR primer is shown in Fig-2 and
the details are given in Table-3. All these three
primers gave amplifications in all nine cultivars.
These ISSR primers produced total 342 scorable
bands. From these total bands, 270 bands were
polymorphic while, 72 bands were monomorphic.
This gave a total polymorphism of 78.94%. The
maximum number of bands (142) was observed in
(AGG)6 primer, while minimum number of bands
(91) was observed in (GA)9T primer. The
amplicons were observed ranging from 190-
1635bp. Both the largest and the smallest amplicons
were amplified by (GACA)4 primer. The similarity

Table-2: Detailing of RAPD primers and analysis of amplified bands in nine C. roseus cultivars.
Range of Total
Nucleotide Total Total
Primer amplicons polymorphic PIC
sequence bands monomorphic bands
(inbp) bands
OPA 03 AGTCAGCCAC 230-2650 146 128 18 0.954
OPC 12 TGTCATCCCC 190-2040 136 109 27 0.955
OPD 20 AACCCGGTCA 400-1730 63 45 18 0.922
OPN 15 CAGCGACTGT 190-1740 117 108 09 0.954
OPAF 05 CCCGATCAGA 320-1550 68 23 45 0.900
OPAF 15 CACGAACCTC 270-1940 62 53 09 0.906
Total 190-2650 592 466 126 Avg=0.93

Table-3: Detailing of ISSR primers and analysis of amplified bands in nine C. roseus cultivars.
Annealing Range of Total Total
Total
Primer temperature amplicons polymorphic monomorphic PIC
bands
(0C) (in bp) bands bands
(GACA)4 49 190-1635 109 82 27 0.94
(GA)9T 53.1 162-1375 91 91 00 0.94
(AGG)6 45 263-1425 142 97 45 0.94
Total 190-1635 342 270 72 Avg=0.94

670 Journal of Research in Biology (2011) 8: 667-675

Lal et al.,2011

matrix constructed using Jaccards coefficient
showed that the similarity index ranged from 0.257-
0.648. The mean similarity index of 0.76 was
observed indicating good variability (data not
shown). All the ISSR primers showed same PIC
value (0.94).
In SSR study, five primers were analyzed.
Out of these five primers, only two primers gave
satisfactory and reproducible results. The banding
pattern of one SSR primer is shown in Fig-2 (B)
and the detailing of this banding pattern is given in
Table-4. Both these primers gave amplifications in
all C. roseus cultivars. Total 231 scorable bands
were produced by both SSR primers. Out of these,

Fig-1: RAPD banding pattern in nine C. roseus cultivars. (M1= 100bp ladder, M2=1000bp ladder, Lane A-I are
different cultivars as shown in table 1). Fig 1A amplification with OPA 03 primer, Fig 1B amplification with OPD
20 primer, Fig 1C amplification with OPAF 15 primer and Fig 1D amplification with OPN 15 RAPD primer.

Fig-2: Banding pattern in nine C. roseus cultivars using ISSR and SSR primers (M1= 100bp ladder,
M2=1000bp ladder, Lane A = patricia white, B= cooler orchid, C= Pacifica liac, D= Albus with red eye, E= blue
pearl, F= Grape cooler, G= cooler red, H= first kiss podka dot, I= pacifica apricot). Fig 1A amplification with
(GA)9T ISSR primer and Fig 1B amplification with (GAA)7 SSR primer.

Journal of Research in Biology (2011) 8: 667-675 671


177 bands were polymorphic, while 54 bands were
monomorphic. This resulted in total polymorphism
of 76.62%. Primer (GAA)7 produced more number
of bands (142) than (AAGC)3 primer (89). The
amplicons were observed ranging from 145-
1839bp. The largest amplicon (1839bp) was
amplified by (AAGC)3 primer, while the smallest
amplicon (145bp) was amplified by (GAA)7
primer. Similarity matrix was plotted using the
Jaccards coefficient. According to the matrix, the
similarity index was observed ranging from 0.261
0.736 with the mean similarity index of 0.68
indicating reasonable variability as obtained by
SSR markers (data not shown). Primer (GAA)7
showed higher PIC value (0.96) than (AAGC)3
primer (0.91).
The combined RAPD, ISSR and SSR
analysis detected high degree of genetic variations.
The highest similarity index (0.632) was observed
between Pacifica liac and Albus with red eye.
The least similarity index (0.287) was observed
between Cooler orchid and Blue peal. The mean
similarity was observed reasonably high (0.68).
This indicates high genetic variation among nine C.
roseus cultivars. Similar variations were observed
in dendogram constructed using UPGMA method
(Fig-3) as well as in 2-D and 3-D plots (Fig-4(A)
and 4(B)). In dendogram, two clear clusters were
observed (A & B). Cultivars patricia white, grape
cooler, pacifica liac, albus with red eye, First
kiss podka dot and cooler orchid and were in
cluster A , while blue pearl, cooler red, and
pacifica apricot were in the cluster B. Cluster A
was divided into two sub clusters A1 and A2. Sub
cluster A1 was further divided into A1a and A1b.
Cultivars patricia white and grape cooler were in
A1a, while pacifica liac and albus with red eye
were in A1b. pacifica liac and albus with red eye
were found to be most similar with 100% similarity.
First kiss podka dot and cooler orchid were in
the sub cluster A2. Cluster B was further divided
into two sub clusters B1 and B2. cooler red, and
pacifica apricot were in the sub cluster B1, while
Blue pearl was in sub cluster B2. The principal
Table-4: Detailing of SSR primers and analysis of amplified bands in nine C. roseus cultivars.
Annealing Range of
Total
Primer temperature amplicons
bands
(0C) (in bp)
(GAA)7 54 145-1793 142
(AAGC)3 60 452-1839 89
Total 145-1839 231
672
Lal et al.,2011
coordination analysis (PCA) supported the similar
groupings (Fig-4).
Good correlation (r value) was observed
among all three markers. The average
polymorphism using all three markers was observed
78.09% which indicates high degree of
polymorphism among all nine cultivars.
The dendogram constructed using all three
RAPD, ISSR and SSR markers successfully
differentiated all nine cultivars of Catharanthus
roseus. These results demonstrate the excellent
power of all these three markers in studying the
closely related taxa. This supports the findings of
Raina et al., (2001). They studied that RAPD and
ISSR fingerprints as useful genetic markers for
analysis of genetic diversity, varietal identification,
and phylogenetic relationships in peanut (Arachis
hypogaea) cultivars and wild species.
Lalhruaitluanga and Prasad (2009) studied
comparative results of RAPD and ISSR markers for
genetic diversity assessment in Melocanna
baccifera (Roxb.) growing in Mizoram State of
Fig-3: Dendogram based on UPGMA method
showing genetic relationship among nine C. roseus
cultivars as revealed by RAPD, ISSR and SSR
markers, PW=patricia white, GC= grape cooler,
CR= cooler red, FKPD= first kiss podka dot, PA=
pacifica apricot, CO= cooler orchid, PL= pacifica
liac, PRW= albus with red eye, BP= blue pearl.
Total Total
polymorphic monomorphic PIC
bands bands
133 09 0.96
44 45 0.91
177 54 Avg=0.93
Journal of Research in Biology (2011) 8: 667-675
Lal et al.,2011

Fig-4: Phylogenetic relationship among nine Catharanthus roseus cultivars revealed by RAPD, ISSR and SSR
primers. (A) 2-D plot (B): 3-D plot. PW=patricia white, GC= grape cooler, CR= cooler red, FKPD= first kiss
podka dot, PA= pacifica apricot, CO= cooler orchid, PL= pacifica liac, PRW= albus with red eye, BP= blue pearl.

India. They found that higher polymorphism
(98.02%) was obtained in RAPD markers than
ISSR markers (84.1%). However, in our present
investigation it was found that slightly higher
polymorphism (78.94%) was observed in ISSR
markers compared to RAPD markers (78.71%) and
SSR markers (76.62%). Similar results were
observed by Parsons et al., (1997) who studied the
genetic diversity relationship in rice using different
marker systems. They observed 56% polymorphism
in ISSR markers while, 50% polymorphism was
observed in RAPD markers.
Moreover, Lal et al., (2010) also reported
that higher polymorphism (95%) was obtained in
ISSR markers than RAPD (87%) and SSR (93%)
markers. They checked the efficiency of all these
three PCR based markers in Cicer arietinum L. and
Cajanus cajan L. Millspaugh and found that higher
PIC value was observed in ISSR markers (0.70)
than in RAPD (0.49) and SSR (0.61) markers. In
same manner, in our investigation also, higher PIC
value (0.94) was observed in ISSR markers than in
RAPD (0.93) and SSR (0.93) markers.
Ajibade et al., (2000) and Galvan et al.,
(2003) concluded that ISSR would be a better tool
than RAPD for phylogenetic studies. Nagaoka and
Ogihara (1997) have also reported that the ISSR
primers produced several times more information
than RAPD markers in wheat. Our study also shows
that ISSR marker is better tool than RAPD markers
for phylogenetic study.
RK Shaw et al. (2008) studied the genetic
variation in cultivars of C. roseus using RAPD and
ISSR markers. They observed genetic variability
using 18 RAPD and ISSR markers. They concluded
that both markers are equally potential to
differentiate the closely related cultivars of C.
roseus. In our investigation also, the high
discriminating power of both RAPD and ISSR
markers among C. roseus cultivars was observed.
To best of our knowledge, no earlier reports
are available regarding the genetic diversity
analysis in C. roseus using all these three markers.
The polymorphism data generated can be used for
plant breeding, crop improvement programs and
also might be helpful in future strategies for

Table-5: Similarity matrix based on Jaccards coefficient.

PW GC CR FKPD PA CO PL PRW BP
PW 1
GC 0.628 1
CR 0.443 0.391 1
FKPD 0.312 0.447 0.306 1
PA 0.440 0.408 0.462 0.358 1
CO 0.471 0.479 0.329 0.562 0.385 1
PL 0.575 0.568 0.407 0.426 0.453 0.434 1
PRW 0.631 0.600 0.449 0.407 0.509 0.461 0.632 1
BP 0.461 0.388 0.370 0.370 0.440 0.287 0.447 0.493 1

Journal of Research in Biology (2011) 8: 667-675 673


evolution of desired genotypes and further
development of new C. roseus cultivars.
CONCLUSION
All three markers (RAPD, ISSR and SSR)
proved potential tools for analyzing genetic
variation among nine closely related cultivars. So,
all the three markers can be used to design a
strategy to maintain or enhance the genetic diversity
of future varieties. The polymorphism data
generated can be used for plant breeding, crop
improvement programs and also might be helpful in
future strategies for evaluation of desired genotypes
and further development of new C. roseus cultivars.
ACKNOWLEDGMENT
Authors are grateful to Charutar Vidya
Mandal (CVM) Vallabh Vidyanagar, Gujarat for
providing platform for this research work. We are
also thankful to Dr. Pradip S. Patel director of
Ashok and Rita Patel Institute of Integrated Study
& Research in Biotechnology and Allied Sciences
(ARIBAS) New Vallabh Vidya Nagar, for
providing the facilities and their valuable
suggestions during our research work.
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