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Authors: ABSTRACT:
Ananth Kumar 1 And
Mohamed Abdul Kadher
Haniffa2. 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
Institution:
1. Department of Zoology, days. And the other groups were immersed with the hormone for 6h. The hormone
V.H.N.S.N College, incorporated feed at 500mg/l produced 68% males in koicarp and 80% in platy.
Viruthunagar 626001, According to immersion technique 400mg/l produced 85% in platy and 75% in koicarp.
Tamilnadu , India. Exposure to other concentrations of 17 - MTS at varying durations invariably
2. Centre for Aquaculture produced intersex individuals. From the present study it is concluded that Koicarp and
Research and Extension Platy are amenable to endocrine sex reversal by steroid immersion and dietary
(CARE), St.Xaviers treatments of fry.
College, Palayamkottai-
627002, Tamilnadu, India.
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cited.
Water was changed once in 2 days in order monosex populations and contained intersex
to keep maintain the dissolved oxygen content individuals were characterized by the intermingling
without much fluctuation. The experimental fish of spermatogonia and oocytes in the same gonad
were least disturbed except on certain occasions (Fig. 1 c & d). The effect of MTS treatment on the
like change of water. For each concentration of sex of C. carpio is shown in Table 3. The highest
hormone three tanks each with thirty fishes in each percentage conversion to males (80%) occurred in
were maintained as triplicate and triplicate was the treatment dose of 500g MTS / l of water. In
maintained for the control also. Owing the this batch 80% fish showed normal testes similar to
ambiguous nature of manual sexing, juvenile fish the control fish (Fig. 1 a & b). The 500g MTS /
were often sacrificed to determine the sex ratio. l and 600 g MTS / l treatment for 6h significantly
After 90 days the gonads were located after (P <0.001) altered the sex ratio 4M: 1F when
dissection under a dissection microscope. A portion compared to the control. None of the
of the gonadal tissue was removed with a pair of 100,200,300,400,500 and 600g MTS / l treatment
fine forceps and placed on a clean glass slide. A combinations produced cent percent monosex
few drops of acetic orcein or geimsa stain were populations in koicarp but produced 2 5%
added and the tissue was squashed after placing a intersex individuals characterized by the
cover slip on the glass slide (Guerrero and Shelton, intermingling of spermatogonia and oocytes in the
1974; Haniffa et al., 2004). The squashed tissue was same gonad. In the reversing gonads (i.e, intersex),
examined under a research microscope low spermatogenic cysts in early stages of development
magnification. Ovarian tissue was identified by the were predominant and were distributed throughout
presence of oocytes and the testicular tissue by the the entire gonad amist the stroma cells. The oocytes
spermatocytes on a uniform background of tissue. were reduced in number and only the small,
Sex ratio obtained in the experiments were developmentally younger ones were seen in areas
subjected to Chi-square test for goodness of fit (Zar between the spermatogenic cysts. Other cells
2000). included cells containing dense dark staining nuclei
and light staining cytoplasm. Sex ratio of platy
RESULTS administered with diet containing 17 methyl
The effect of MTS treatment on the sex of X. testosterone is reported in Table 2. After 30 days
maculatus is shown in Table 1. The highest gonad formation was not clear. Histological
percentage conversion to males (85%) occurred in observations of the gonad of fish given hormone
the treatment dose of 400g MTS / l of water. In diets were made after 90 days (Fig. 2 a & b).
this batch 85% fish showed normal testes similar to Experiment 2
the control fish. Sex ratio observed after 90 days of dietary
Experiment 1 hormone administration in platy revealed a male
The 400g MTS / l and 500 g MTS / l dominant population. In 400 g/l of 17 MTH
treatment for 6h significantly (P <0.001) altered the administered diet for 90 days the males were 75%
sex ratio from when compared to the control (4M: whereas the females were only 10%. Remaining
1F). None of the 100, 200, 300, 400, 500 and 600g 10% were died. At the same time 5% of fishes
MTS / l treatment combinations produced 2 5% were intersex. Platy fish fed with a diet containing
TABLE 1. Effect of different doses of discrete 17 MTS immersion treatment on sex ratio and survival on
Xiphophorous maculatus Platy.
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
TABLE 2. Effect of different doses of discrete 17 MTS through supplementary diet on sex ratio and survival
on Xiphophorous maculatus Platy.
TABLE 3. Effect of different doses of discrete 17 MTS immersion treatment sex ratio and survival on koicarp
Cyprinus carpio
TABLE 4. Effect of different doses of discrete 17 MTS through supplementary diet on sex ratio and
survival on Koicarp Cyprinus carpio
Demzka-Zakes K and Zakes Z. 1997. Effect of 17 Piferrer F and Donaldson EM. 1994. Uptake and
alpha methyltestosterone on gonadal differentiation in clearance of exogenous estradiol 17 and
pikeperch, Stizostedion lucioperca L. Aquaculture testosterone during the early development of Coho
Research 28:59-63. salmon (Oncorhynchus kisutch), including eggs,
alevins and fry. fish physiology and Biochemistry 13
Desprez D, Graz E, Hoareau MC, Mlard C, Bosc (3):219-232.
P, Baroiller JF. 2003b. Production of high
percentage of male offspring with natural androgen, Piferrer F. 2001. Endocrine sex control strategies for
11 hydroxyandrostenedione (11OHA4), in Florida the feminization of teleost fish. Aquculture 197:229-
red tilapia. Aquaculture 216:55-65. 281.
Fagerland UHM and Mc Bridge JRC. 1978. Rougeot C, Jacobs B, Kestemont P and Melard C.
Distribution and disappearance of radioactive in blood 2001 Sex control and sex determinism study in
tisues of coho salmon (Oncorhynchus kitsch) after oral Eurasian perch, Perca fluviatilis, by use of hormonally
admisnitration of H testosline J.Fish Res. Board Car sex reversed male breeders. Aquaculture 265-268.
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Salam A. Al-ablani and Ronald Phelps P. 1997. Sex
Feist G., Yeoh C.G., Fitzpatrick M.S., Schreck C.B. reversal in Black crappie Pomoxis nigromaculatus,
1995 The production of functional sex reversed effect of oral administration of 17a Methyltestosterone
male rainbow trout with 17 -methylotestosterone on two age classes. Aquaculture 158:155-65.
and 11-hydroxyandrostenedione Aquaculture 131:
145-152. Ohia H and Takano K. 1996. Testicular maturation
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Morphological and experimental study of gonadal sex
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Authors: ABSTRACT:
Ayandele AA and Adeniyi
SA.
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
Institution:
seven-nine different microorganisms were isolated from the naira notes, while the
Department of Pure and
Applied Biology, prevalent microorganisms include; Staphylococcus aureus (3.8 %), Bacillus subtilis
Ladoke Akintola (10.1%), Enterobacter aerogenes (8.9%), Staphylococcus epidermidis and Aspergillus
University of Technology, niger (11.4%), Bacillus megaterium (12.7%), Escherichia coli (3.8%), Pseudomonas
P.M.B. 4000, Ogbomoso. 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
Corresponding author: the fungal isolate that is susceptible to those antifungal agents is <15mm.
Ayandele AA
Keywords:
Email: Prevalence, Drug resistance, Susceptibility, Small Denominations,
lizdeley@yahoo.com Enteropathogenic Microorganisms.
Dates:
Received: 18 Oct 2011 /Accepted: 31 Oct 2011 /Published: 08 Dec 2011
Ficus Publishers.
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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.
(PDA) and placed on already prepared plates of Aspergillus niger (11.4%), Bacillus subtilis (10.1),
PDA. Different concentration of Mycoten and Enterobacter aerogenes (8.9%), Trichoderma
Nystatin were prepared (100, 250 and 500mg/ml) reesei and Colletotrichum gloesporoides (7.6%),
and sterile paper discs were dipped into each Aeromonas hydrophila (6.3%), Fusarium solani
concentration and placed aseptically on the plates and Colletotrichum truncatum (5.1%), and
that contained the test fungi and incubated at 25oC Escherichia coli and Staphylococcus aureus (3.8%).
for 2-3 days after which the zones of inhibition Similar works by Awe et al. (2010), Feglo and
were measured. Nkansah (2010), Matur et al. (2010), and Shakir et
al. (2010) had also reported the occurrence of
RESULTS AND DISCUSSION different microorganisms from the currency notes
Table 1 showed the Total bacterial counts of in their countries. The isolation of Staphylococcus
different denominations, #10 notes had the count of on the currency notes could have been
33.93 cfu/ml 104, followed by #5 notes (20.68 contamination from normal skin flora and soil
cfu/ml 104), while #50 notes showed the least (Igumbor et al., 2007; Larkin et al., 2009) and
bacterial load of 19.94 cfu/ml 104. Table 2 Staphylococci infection occur when Staphylococcus
revealed that #10 notes had the highest fungal count enter the body through breaks, cuts and abrasions in
of 22.60 cfu/ml 104, while #5 notes had the least the skin (Shakir et al., 2010) and it is also
fungal load of 5.16 cfu/ml 104. The Total coliform associated with impetigo, carbuncles and food
count was represented by Table 3, #10 also had the intoxication (Jensen et al., 1997). Escherichia coli
highest coliform count of 21.00 cfu/ml 104, and which is due to feacal contamination is also
followed by #20 notes with count of 18.12 cfu/ml responsible for many diseases when consumed in
104 and #5 notes had the least count of 9.58 cfu/ml large doses, other bacterial isolates like
104. Studies from other parts of the world have Enterobacter aerogenes, Bacillus subtilis,
shown that bank notes contained high load of Staphylococcus epidermidis, Pseudomonas putida
bacteria which could cause many diseases (Shukla, and Aeromonas hydrophila have been associated
1980; Oyler et al., 1996; Pachter et al., 1997; with different types of diseases especially in
Havas, 2000). Kawo et al. (2009) also reported high immunocompromised and immunosuppressed
load of bacterial and fungal count from abused naira patients (Kelly et al., 1993; Yang et al., 1996;
notes in Kano metropolis, Nigeria. Table 4 showed Aurlie et al., 2005), Fusarium solani may also
the occurrence of different microorganisms isolated cause a range of invasive mycoses and a range of
from the different naira notes analysed in this study. opportunistic infection in immunocompromised
Bacillus megaterium had the highest occurrence of patients (Zhang et al., 2006), the fungal isolates
12.7% followed by Staphylococcus epidermidis and could also produce mycotoxins in food, which is
dangerous to human and other animals (Grundy and contamination rate followed by #20 notes, these
Grundy, 1974). Table 5 shows the microbial rate of two naira notes are commonly found among the
naira notes in relations to the locations and the poor people and the children, the least
denominations of the different naira notes used in contamination rate observed with #5 notes may be
this study. The results revealed that Sabo area due to the fact that it is very rare to found most
which comprises of different artisans and beggars commodities sold at #5 nowadays. In a similar work
had the highest contamination rate followed by by Oyero and Emikpe (2007), they reported highest
LAUTech campus area which also comprised of level of microbial contamination for #10 notes
different food vendors, shop owners, different among Nigerian currencies notes in their work.
artisans and students, while Kuye area which is a Also, #10 notes are the most commonly used
residential area for Government workers and among the small denominations in Nigeria and it is
artisans had the least contamination. Similar work exchanged many times especially amidst the
by Shakir et al. (2010) also revealed that artisans and lower economic class people. Shakir et
Bangladesh paper currency notes collected from al. (2010) had also reported highest contamination
artisans groups had the highest contamination rate rate among the small denomination notes in
compared to money collected from educated Bangladesh currencies.
people. #10 notes used in this work had the highest Table 6 showed the Antibiotics sensitivity
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
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 isolated from Nigerian Naira notes were resistant to
antibiotics. All the bacteria isolates showed 100% first line antibiotics and this was also observed in
resistant to Augmentin, Nitrofurantoin and this work. The resistance observed in this work
amoxicillin, they had 87.5% resistant to might also be due to the indiscriminate use of
tetracycline, chloramphenicol and streptomycin, the antibiotics by people. The resistance to the
isolates showed 50% resistant to Ceftriazone, antifungal agents might be as a result of the carriers
Cotrimoxazole and Gentamycin, 37.3% and 25.0% of some diseases who might have been exposed to
resistant to Ofloxacin and Pefloxacin respectively. different antifungal agents especially mycoten
While all the bacterial isolates were susceptible to which is used by many women in the treatment of
Ciprofloxacin with zones of inhibition ranging from Candida albicans infection.
16 to 5mm. The fungal isolates also showed varied The findings from this work showed that
activities against the two antifungal (Mycoten and infections that may occur from the microorganisms
Fulcin) used for the sensitivity test at varying isolated from this work might be difficult to treat
concentrations (100, 250 and 500mg/ml). At 100, and also the treatment might be very expensive
250 and 500mg/ml concentrations for Fulcin, the because of the resistance of these isolates to the
resistant percentage was 80, 40 and 40% common and cheaper antibiotics drugs.
respectively, and for Mycoten, the resistance In conclusion, this study showed that naira
percentage were 80, 60 and 40% for 100, 250 and notes collected from different areas in Ogbomoso
500mg/ml respectively (Table 7). Similar work by North Local Government of Nigeria is
Emikpe and Oyero (2007) revealed that organisms contaminated with different pathogenic organisms
Table 6: Antimicrobial Sensitivity Pattern of Some Bacterial Isolates Against Selected Antibiotics
and the microbial load is also very high. The the Hungarian currency (notes and coins). Magya
isolated organisms showed high resistance to Allatoryosol Lapja 122(8):501-503.
available antibiotics and antifungal agents.
Therefore, there is need to educate the populace on Holt JG, Krieg NR, Sneath PHA, Stanley JT and
the effect of improper handling of naira notes and Williams ST. 1994. Bergeys Manual of
women most especially because of their children Determinative Bacteriology, Williams and Wilkins,
who always put money in their mouth. Baltimore.
Gadsby P. 1998. Filthy lucre: bugs, drugs and Matur BM, Malann YD and Edhomeriegue Y.
grime hitch a ride on the back of every buck. 2010. A survey of parasite cysts, eggs and bacteria
Discover 19:76-81. on Nigerian currency in FCT, Abuja. New York
Science Journal 3(1):10-13.
Goktas P and Oktav G. 1992. Bacteriological
examination of paper money. Mikrobiyol. Bull., Michaels B. 2002. Handling Money and Serving
26:344-348. Ready-to-eat food. Food Servo Technol., 2:1-3.
Grundy F and Grundy P. 1974. Community Oyero OG and Emikpe BO. 2007. Preliminary
health and social services. H.K. Lewis publishers, Investigation on the Microbial Contamination of
London. 108. Nigerian Currency. Int. J. Trop. Med., 2(2):29-32.
Havas F. 2000. About the bacteriological state of Oyler J, Darwin WD and Cone EJ. 1996. Cocaine
592 Journal of Research in Biology (2011) 8: 587-593
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contamination of United States paper currency. Talaro KP. 2005. In: Foundations in Microbiology.
Journal of Analytical Toxicology 20(4):213-216. 5th Ed. (McGraw-Hill Companies, Inc., New York,
USA). 407.
Pachter BR, Kozer L, Pachter SA and Weiner
M. 1997. Dirty money- A bacteriophage Xu J, Moore JE and Millar BC. 2005. Ribosomal
investigation of US currency. Infectious Diseases DNA (rDNA) identification of the culturable
14(7):574. bacterial flora on monetary coinage from 17
currencies. J. Env. Health 67(7):51-55.
Pope TW, Ender PT, Woelk WK, Koroseil MA
and Koroseil TM. 2002. Bacterial contamination Yang CH, Young T, Peng MY and Weng MC.
of Nigerian Currency. Int. J. Trop. Med., 2(2):29- 1996. Clinical spectrum of P. putida infection. J.
32. Formos. Med. Assoc., 95(10):754-761.
Shakir Md UA, Parveen S, Nasreen T and Zhang N, ODonnell K, Sutton DA, Nalim FA,
Feroza B. 2010. Evaluation of the Microbial Summerhall RC, Padhye AA and Geiser DM.
Contamination of Bangladesh Paper Currency 2006. Members of the Fusarium solani species
Notes (Taka) in Circulation. Advances in Biological complex that cause infections in both humans and
Research 4(5):266-271. plants are common in the environment. Journal of
Clinical Microbiology 44(6):2186-2190.
Shukla KA. 1980. Reservoir of organisms. Indian
Journal of Medical Sciences 32(7):168-170.
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.
Dates:
Received: 19 Sep 2011 /Accepted: 27 Oct 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.
nutrient broth-glucose medium were added into the organisms were able to reduce TTC to red fomazan
tubes, vortexed for 2 min and preincubated on a at variable rates and extent (Table 2). The gram
rotary incubator (150 rpm) at room temperature (28 negative Pseudomonas sp. RBD1 had higher rate of
2oC) for 30 min. dehydrogenase activity in all the exposure period
Viability assay time than gram positive Bacillus sp. RBD2. The
The assay is based on the inability of dead reason for these differences is not known. However,
cells or injured bacterial strains to reduce 2, 3, 5- variation may be due to differences in cell wall
TriphenylTetrazolium Chloride (TTC) into components or dehydrogenase systems, since
insoluble TriphenylFormazan (TPF) product. The different microorganisms have been reported to
ability of viable bacterial strains (by dehydrogenase have different dehydrogenase systems (Praveen-
enzyme activity) to reduce TTC to TPF provides an Kumar, 2003; Nweke et al., 2006). Moreso, Table 2
indication of membrane intact which in turn, may showed decrease in formazan formation with
be interpreted as a measure of cell viability. At the increase in exposure period to phenol stress. This
end of preincubation with nutrient broth-glucose decrease in formazan formation with increase in
medium, 0.2 ml of 0.1 % (w/v) TTC in deionized time is in line with observations of Ross (1971),
distilled water was added into the tubes. The who reported that a gradual decrease, in formazan
reaction mixture was incubated statically at room formation after an incubation time of 1 h. Griebe et
temperature (28 2oC) for 6 h in the dark. Since the al., (1997), also reported a decrease in the rate of
TPF produced by cellular dehydrogenases is formazan formation after 2 h incubation time when
insoluble in water, extraction of TPF was done in CTC was incubated with activated sludge and stated
3.0 ml amyl alcohol and determined that no significant increase was realized in
spectrophotometrically at 445 nm (max). The formazan formation after 4 h incubation time.
amount of formazan produced was determined in Estimation of viability of the organisms by
reference to a standard dose-response curve. inhibition/stimulation of dehydrogenase activity at
Viability [Dehydrogenase activity, (DHA)] was various exposure periods in different concentration
expressed as microgram of TPF formed per mg dry of phenol is shown in Figure 1. The results showed
weight of cell biomass per hour. decrease in stimulation of dehydrogenase activity
Calculation of viability/stimulation or inhibition upto 3 h of exposure at 250 mg/l of phenol in
of enzyme activity Pseudomonas sp. RBD1 while in Bacillus
The viability/stimulation or inhibition of sp.RBD2, stimulation was observed at 250 mg/l of
dehydrogenase enzyme activity was computed as phenol only for 1 h of exposure. Thereafter, there
percent deviation from the control. Where showed total inhibition in both organisms in all the
applicable, the IC50 of the test chemical (phenol) concentrations of phenol in the fourth hour.
was determined by fitting the TPF values produced Stimulation of dehydrogenase activity in both
to simple equations using Table 2D Curve (systat organisms at 1 - 3 h exposure periods indicates
Incorporation, USA) and calculating the viability of the organisms hence the utilization of
concentrations of the test chemical at 50% phenol as a growth substrate is possible. The
inhibition of dehydrogenase activity. The greater progressively decreasing stimulation of
the IC50 value, the lower the toxicity of phenol dehydrogenase activity with increasing exposure
sample at that exposure time period. period in Pseudomonas sp.RBD1 is in line with
Statistical analysis Table 2: Dehydrogenase activities in the absence of
Data obtained from the study were analyzed by the toxicant (phenol) at different exposure period
use of two-way analysis of variance (ANOVA) and Dehydrogenase activity
values for P < 0.05 were considered statistically (g Formazan/mg cell dry wt/h)
significant. Exposure Bacterial strain
period (h)
Pseudomonas sp. Bacillus sp.
RESULTS AND DISCUSSION RBD1 RBD2
Two bacterial strains comprising gram negative and 1 1.175 0.056 0.719 0.022
gram positive were isolated from the biological
treatment unit of wastewater sample and identified 2 1.097 0.029 0.666 0.009
as Pseudomonas sp. RBD1 and Bacillus sp. RBD2 3 1.014 0.021 0.606 0.012
organisms. Results obtained from the control
4 0.964 0.041 0.571 0.023
samples on viability assays showed that these
597 Journal of Research in Biology (2011) 8: 594-602
Nwanyanwu et al.,2011
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)
(2001) reported decrease in bioluminescence of thereby utilizing phenol as carbon and energy
Vibrio fischeri bacteria with exposure period to sources. At the fourth hour, total inhibition was
pentachlorophenol. Also, Okolo et al., (2007) and observed in both organisms. The inhibition may be
Stotzky and Norman (1961) observed that attributed to the disruption of membrane barrier
nitrophenols repressed soil respiration and function of the organisms that resulted in the
microbial activity with increasing exposure periods. inhibition of dehydrogenase activities in both
The effects of exposure to phenol on viability of organisms. The inhibition of dehydrogenase
the bacterial strains assessed via dehydrogenase activities is attributable to the toxicity of phenol.
activity are as shown in Figure 2. In both Dehydrogenase enzymes are membrane associated
organisms, dehydrogenase activity decreased with and phenols have been reported to affect
increasing exposure period and concentrations of membranes (Irya et al., 2003). The dehydrogenase
phenol. In Pseudomonas sp.RBD1, dehydrogenase activities correlated with exposure periods and
activity was stimulated at 250 - 500 mg/l of phenol phenol concentration are as shown in Figure 3. The
in the first hour while in 2 3 h of exposure, high R2 values ().6472 < R2 < 0.9809) for
stimulation was observed only in 250 mg/l of Pseudomonas sp. RBD1 and (0.7689 < R2 <
phenol. In Bacillus sp. RBD2, stimulation of 0.8483) for Bacillus sp.RBD2 indicated that phenol
dehydrogenase activity was observed only in the concentration as well as exposure period was a
first one hour at 250 mg/l of phenol. The strong determinant of metabolic activity in the
stimulatory effects observed at the first hour period organisms.
of exposure to phenol in both organisms may be The toxicity threshold of phenol
attributed to the metabolic activity at this period 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
(g TPF/mg dry cell weight/hour
A B
Log1
0
DH
A
Concentration (mg/l)
Figure 3: correlation of phenol concentrations with dehydrogenase activity in
(A) Pseudomonas sp.RBD1 and (B) Bacillus sp. RBD2 in response to different exposure
time to phenol toxicity
Afzal M, Iqbal S, Rauf S.and Khalid ZM. 2007. of phenol-degrading bacillus stearothermophilus
Characteristics of phenol biodegradation in saline and partial characterization of the phenol
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Authors: ABSTRACT:
Sidhu MC and Saini P.
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
Institution: plant has been studied in detail for its anatomical variations. Stem anatomy is almost
Department of Botany,
similar to other members of the family. The Transverse Section of the leaf shows the
Panjab University,
presence of accessory vascular bundles. Similarly Transverse Section of the root has
Chandigarh-160014-India.
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
Corresponding author: anatomical details has been worked out for this species in the past except one study
Sidhu MC 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.
Email: Keywords:
mcsidhu@hotmail.com Asteraceae, anatomy, leaf, root, stem, microscopy, Silybum marianum.
Dates:
Received: 21 Nov 2011 /Accepted: 23 Nov 2011 /Published: 12 Dec 2011
Ficus Publishers.
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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.
Fig. 1-a (T. S. Stem; Magnification - 4x) Fig. 1-b (T. S. Stem; Magnification - 10x)
Fig. 1-c (T. S. Stem; Magnification - 20x) Fig. 1-d (T. S. Stem; Magnification - 40x)
present a discontinuous peridermal layer which later present a small sized parenchymatous pith. It is
on differentiated into phellem and the phellogen surrounded by certain sclerenchymatous cells which
(Figure 2-a). Only two to three layers of cortical forms a portion of the xylem tissue (Figures 2-c &
cells are visible in reduced form. The formation of d). The occurrence of secondary growth in the root
distinct layers of secondary xylem and phloem seems to be an abnormal feature since Silybum
suggests the occurrence of secondary growth growing in the present investigation area occurs as
(Figure 2-b). Secondary phloem is red in colour an annual species and formation of growth rings in
while the primary phloem has been compressed and an annual plant is an exception. Fritz and Saukel
only some of its remnants can be seen. There are (2011) while studying the anatomy of subterranean
present distinct layers of cambial cells (2 layers) organs of some medicinally used species observed
which are responsible for the secondary growth. the presence of secondary phloem. They were of the
The major portion of root is occupied by the opinion that root anatomy of Silybum marianum is
secondary xylem which is distinguished by its similar to that of Onopordum acanthium and Cnicus
vessels or xylem tracheids. Xylem tracheids are benedictus but preceding secondary growth was the
usually two to three celled. There are present distinctive feature of Silybum marianum root. Their
distinct layers of thin walled ray parenchyma cells. findings substantiated the present observations
Both of these components (vessels and ray regarding the secondary growth in the root portion
parenchyma) form the secondary xylem while the only.
primary xylem is present towards the centre in a Leaf:
crushed miniature form. In the centre there is The leaves are alternate, large, white veined,
Fig. 2-a (T. S. Root; Magnification - 4x ) Fig. 2-b (T. S. Root; Magnification - 10x)
Fig. 2-c (T. S. Root; Magnification - 20x) Fig. 2-d (T. S. Root; Magnification - 40x)
glabrous and have strong spiny margins. The main are encircled by a parenchymatous bundle sheath
and an important feature is the presence of white cells (Figure 3-c). The upper and lower sides of the
streaks on the leaf surface. The leaf of Silybum is of vascular bundle are covered by bundle sheath
dorsiventral type. The Transverse Section shows the extensions and sclerenchymatous cells. In addition,
presence of single layered upper and lower there are present two to four layers of collenchyma
epidermis which is made up of compactly arranged cells near the upper and lower epidermis. Each
barrel shaped cells. Both the surfaces are covered vascular bundle consists of xylem present towards
up with a thick and wavy cuticle. It is more on the the upper epidermis and phloem close to the lower
upper epidermis and lesser on the lower epidermis epidermis. Xylem is differentiated into metaxylem
(Figure 3-a). The stomata are usually absent in the and protoxylem. Protoxylem vessels are smaller in
epidermal layers which is a distinct feature. Inner to size and facing towards the upper epidermis
the epidermis present mesophyll cells which is (Figure 3-d). Phloem has various components like
differentiated into two to three layered sieve tubes, companion cells and phloem
parenchymatous palisade cells. It consists of green parenchyma cells. Along with the largest vascular
coloured columnar cells which are arranged in bundle present in midrib, there are present certain
compact rows. Each palisade cell contains several accessory bundles which are smaller in size and
chloroplasts positioned around its walls. A they are present towards the upper epidermis near
prominent and large vascular bundle (collateral and the wing of the leaf (Figure 3-b). These accessory
closed type) is present in the region of midrib. A vascular bundles may be developed to meet the
layer of parenchymatous cells separates the vascular requirements in greater amount through
bundles from the epidermis. The vascular bundles translocation system. For example to provide
606 Journal of Research in Biology (2011) 8: 603-608
Sidhu et al.,2011
Fig. 3-a (T. S. Leaf; Magnification - 4x) Fig. 3-b (T. S. Leaf; Magnification - 10x)
Fig. 3-c (T. S. Leaf; Magnification 10x) Fig. 3-d (T. S. Leaf; Magnification - 20x)
similar to many other species. Leaf anatomy has Lilium polyphyllum D. Don ex Royle (Liliaceae).
shown the occurrence of some accessory vascular The Journal of American Science 5(5):85-90.
bundles. This character has already been reported
by many researchers and it is there to meet the Dilcher DL. 1991. The importance of anatomy and
translocation requirements during the unfavourable whole plant reconstruction in palaeobotany. Current
conditions. Science 61(9 & 10):627-29.
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.
Dates:
Received: 17 Nov 2011 /Accepted: 01 Dec 2011 /Published: 12 Dec 2011
Ficus Publishers.
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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.
taxonomic criteria based on the morphological d) Protein estimation: Protein was quantified as
characteristics were used for their identification g protein/ml algal suspension (Lowry, et al., 1951;
(Desikachary, 1959). Herbert, et al., 1971).
All chemicals used were of analytical e) Ammonium estimation: Ammonium as NH4Cl
reagent grade and prepared in distilled water. A (BDH, U.K.) was estimated calorimetrically
single beam visible spectrophotometer (Electronics (sensitivity range, 0.04-5.0 g NH4+/ml algal
India Ltd., India) was used for measuring suspension) using Nesslers reagent (Burris, et al.,
absorption data. 1957).
Parameters studied f) Nitrate estimation: NO3- concentration in the
a) Temperature measurement: It was measured water of two ponds was measured using
using a wet thermometer at 30 cm depth of the conventional colorimetric method (sensitivity
ponds. range, 10-100 g NO3-/ml algal suspension) using
b) pH measurement: The approximate pH was 4% Brucine (SISCO, India) in Chloroform (BDH,
estimated on the spot in the water of both the ponds India) and concentrated H2SO4 (Qualigens, India)
using pH paper and later verified through a digital (Nicholas, et al., 1957).
pH meter (pH range: 0-14; Electronics India Ltd., 3. Microphotography: Microphotography was
India). carried out using a microscope (Make: Suswox
c) Transparency estimation: The transparency of Optik, India), an electronic camera (Make: Nikon,
aquatic bodies was measured with the help of Model No. Coolpix S4000, Japan) and printed with
Secchi disc (20 cm in diameter) pointed alternating the help of a laser printer (Make: Canon, model No.
black and white. LBP-1210, Japan) connected to a computer.
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., were recorded towards lower sides in both years for
1999). both ponds, which favoured higher protein value of
At Pond-1, the high temperatures (30-320C), algal mass in pond-2 than pond-1. If one looks into
low pH (5.8-7.4), high transparency (50.2 to 52.2 the nitrogen balance of pond systems, the NH4+ &
cm), lower NH4+ (2.5-4.0 g NH4+/ml algal NO3- levels at Pond-1 were lower than that of Pond-
suspension) and NO3- levels (31.0-35.0 g NO3-/ml 2. On comparative grounds in pond-1 & 2, there
algal suspension) in the year 2009 favoured the was an increasing trend observed from the year
growth of unicellular cyanobacteria viz; Microcystis 2009 to 2010 with respect to NO3- levels. While
bl ooms, M eri smopedi a, Synechocyst i s, sampling in Aug-Sept 2009, the dominant
Chroococcus and Aphanothece along with desmids cyanobacterial flora were the filamentous forms like
like Pinnularia, Nitzschia, Nedium, and Navicula Oscillatoria, Anabaena, Lyngbya etc. and the algae
(Table-1; PLATE-III [A]). However, the pattern of other than the cyanobacteria were predominantly
cyanobacterial biodiversity was different at Pond-2 the Chara, Nitella and Coleochaete on Eichhornia
where higher aquatic macrophytes like Eichhornia, and other higher aquatic plants. The stairs get
Hydrilla, Azolla and Nelumbo etc. dominated the slippery due to the growth of organisms like
aquatic body. Prominent cyanobacterial blooms of Aphanothece. It was also strange to observe the
Microcystis were recorded during the summer balls of Anabaena iyengerii on the leaves of
months of both the calendar years (2009 & 2010). Nelumbo in Aug 2010. Six species of the
As evident (Table-2) on comparative cyanobacteria viz., Microcystis, Merismopedia,
grounds, during the summer months of 2009, except Aphanothece, Oscillatoria and Lyngbya were found
NO3- levels, the temperature, transparency and in the 1st pond (the religious one), whereas 2nd pond
NH4+ were towards lower side. This favoured (the non-religious one), was mainly dominated by
immense growth of filamentous cyanobacteria like the filamentous cyanobacterial species like
Oscillatoria, Lyngbya and Anabaena (Table-2; Oscillatoria, Anabaena and Lyngbya. However, in
PLATE-III [B]). During the same period in next comparison the unicellular forms like Chroococcus,
year 2010, the temperature was comparatively low Chlorella, Synechocystis and Aphanothece were
at Pond-1 with higher pH than that in year 2009, but also detected in smaller strength.
at Pond-2; it was just a reversed case (Table-1 & 2). As per the diversity and abundance of
It was interesting to note a marked decrease in the cyanobacteria, the members of the family
transparency from (51.0 - 53.5) to (16.2 - 19.4) in Oscillatoriaceae were dominating in the surveyed
Pond-2 as compared to Pond-1. This favoured to ponds. However, the Diatoms were in abundance in
record higher total protein content per ml algal both ponds together with some other algae like
suspension in pond-2 than that of Pond-1, yet, the Chara, Nitella, Coleochaete, Chlorella, and
NH4+ and NO3- levels of the algal suspensions were Batrachospermum (data not presented here).
towards higher side in Pond-2. A corollary of such Similarly, there was a clear cut variation in other
observations favoured much better growth of parameters studied (Table-1 and Table-2) for the
almost all phytoplanktons in the Pond-2. In the post Pond-1 and Pond-2 respectively. In May-June of
-monsoon season of the year (2009) which was both the calendar years (2009 & 2010), the nutrient
approximately the same for the same period in Pond concentrations get increased at both study sites due
-2 also. However the pH range and transparency to the availability of lesser quantity of water owing
to evaporation under hot climatic conditions several species in a course of time. Though, the
(temperature ranges, 40-450C). The transparency of observations and sample collections were made on
the two ponds was found high during this time a regular basis throughout the year, but the data has
period. Such aspects ultimately lead to the been depicted as summer and autumn seasons. It
eutrophication among the two aquatic bodies. has been observed that the cyanobacterial
In any ecosystem, not a single species grows dominance often occurs when water temperature
independently and indefinitely, because all the rises above 200C, when there is depletion of
species are interlinked and has cyclic dissolved inorganic N and free CO2 from the water
transformation of nutrients according to prevailing (Table-1 & 2). It is reported that low nitrogen to
environmental conditions. The physicochemical phosphorus ratios favour the dominance and
changes in the environment not only induce a production of high blue green algal biomass in
particular species but the entire biota. It similar aquatic bodies (Smith, 1983). In shallow,
considerably affects the growth and abundance of mixed water bodies with high total phosphate
other species, which leads to the succession of content organisms like Oscillatoria dominates,
Microcystis Aphanothece
Lowry OH, Rosebrough NJ, Farr AL, Randall Scheffer M, Rinaldi S, Gragnani A, Mur LR,
RJ. 1951. Protein measurements with the Folin- Van Nes EH. 1997. On the dominance of
phenol reagent. Journal of Biological Chemistry filamentous cyanobacteria in shallow turbid lakes.
193:265-275. Ecology 78:272-282.
Macedo MF, Miller Ana Zlia, Saiz-Jimenez Smith VH, Bennet. 1999. Nitrogen: phosphorus
Cesareo. 2009. Biodiversity of cyanobacteria and supply ratios and phytoplankton community
green algae on monuments in the Mediterranean structures in lakes. Archiv fr Hydrobiologie
Basin. An overview Microbiology 155:3476-3490. 146:37-53.
Murphy TP, Lean DRS, Nalewajko C. 1976. Blue Smith VH. 1983. Low nitrogen to phosphorus
green algae: their excretion of iron selective ratios favour dominance by blue-green algae in lake
chelators enables them to dominate other algae. phytoplankton. Science 221:669-671.
Science 192:900-902.
Soininen J, Eloranta P. 2004. Seasonal persistence
Nicholas DJ, Nason A. 1957. Determination of and stability of diatom communities in rivers: are
nitrate and nitrite. Methods in Enzymology. there habitat specific differences? European Journal
Academic Press, New York. III:981-984. of Phycology 39:153-160.
Pandey U, Dungarwal HK. 2004. Algal flora from Walsby AE. 1994. Gas Vesicles. Microbiological
tank of Ekling ji temple of Udaipur, Rajasthan. Reviews 58:94-144.
Plant Archives 4:483-485.
Zhao Hui W, Chun Lei F, Qiu qi L, Ren H, Bo-
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Authors: ABSTRACT:
Nwaogu LA1, Igwe CU1,
Ujowundu CO1, Arukwe
U1, Ihejirika CE2 and Biochemical changes in the tissues of thirty-six albino rats (Rattus rattus)
Iweke AV1. 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
Institution:
1 serum albumin, cholesterol and glucose, liver total protein, ascorbic acid and reduced
Department of
Biochemistry, Federal glutathione (GSH) as well as the activities of alanine amino transferase (ALT),
University of Technology, aspartate amino transferase (AST), alkaline phosphatase (ALP) and catalase (CAT) from
Owerri, Nigeria. the rats were determined using standard methods. The results indicate that there
2 were no significant (P>0.05) changes in these parameters from the rats fed with
Department of
Environmental Technology, 1.25% and 2.50% crude oil mixed feed when compared with the control. However,
Federal University of significant (P<0.05) reductions were observed in glucose, ascorbic acid and GSH
Technology, Owerri, concentrations as well as in catalase activity, with a concomitant significant (P>0.05)
Nigeria. 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.
Corresponding author:
Nwaogu LA
Email: Keywords:
nwogulinus@yahoo.com Albino rats, crude oil, liver enzymes, pollution.
Ficus Publishers.
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Fig. 1: Serum albumin concentration of albino rats Fig. 3: Liver protein concentration of rats fed
fed different concentrations of crude oil mixed feed. different concentration of crude oil mixed feed.
Fig. 4: Serum glucose concentration of rats fed Fig. 5: Serum ALT activities of rats fed different
effluent concentrations of crude oil mixed feed. concentration of crude oil mixed feed.
result of the exposure to crude oil. quenching their effects in the cellular compartments
Serum ALT, AST and ALP activities are and is in the process converted to dehydroascorbic
used as indicator of chemically induced liver acid (DHAA) ( Mckee and Mckee, 1999). Our
damage ( Drotman et al., 1978). Hepatotoxicity has results indicate that there was a significant (p<0.05)
been viewed as liver injury associated with reduction in the concentration of ascorbic acid as
impaired liver function caused by exposure to the concentration of crude oil in the feed increased.
xenobiotics ( drugs, petroleum hydrocarbons) and This significant reduction could be due to the
other non-infectious agent (Navarron, 2006). utilization of ascorbic acid in scavenging the
Results of this study revealed that there were reactive intermediates generated in the tissues of the
significant (p < 0.05) increases in the serum ALT, albino rats fed with the animal feed mixed with
AST and ALP activities in rats fed 5.0% crude oil- crude oil.
mixed feed. These enzymes usually leak out into Glutathione (GSH) is usually located in the
the blood stream in cases of liver damage or loss of cystol, nuclei and mitochondria. It is the major
integrity. The observed increase in serum activities soluble antioxidant in these cell organelles
of these enzymes indicate a sign of liver ( Masella et al., 2005). Reduced glutathione (GSH)
dysfunction as a result of ingestion of crude oil in and its oxidized form (GSSG) accumulate inside the
the feed (Delvin, 2006). cells. The ratio of GSH to GSSG is a good indicator
Ascorbic acid is a water-soluble antioxidant of oxidative stress in a living system (Mckee and
molecule found in the cytoplasm of the cells, which Mckee, 1999). The reduction in the concentration of
scavenges free radicals in the cytosol. It readily glutathione either by conjugation and removal from
donates electrons to free radicals thereby cell or oxidation to oxidized glutathione could
Fig. 8: Liver ascorbic acid concentration activities of rats Fig. 9: Liver glutathione (GSH) concentration of albino
fed different concentrations of crude oil mixed feed. rats fed different concentrations of crude oil mixed feed.
significantly affect the overall redox potential of observed in the activities of catalase from the liver
the cell (Hansen et al., 2001). Our result indicate tissues of rats fed with 1.25%, 2.25% and 5.0%
that glutathione concentration was significantly (p < crude oil polluted feed. However the reduction in
0.05) reduced in rats fed with 5% crude oil-mixed catalase activity was only statistically significant
feed when compared to the control (Figure 9). (p<0.05) obtained in rats fed with 5.0% crude oil
The reduction could be a compensatory mechanism mixed feed when compared to the control
by the animals fed with feed mixed with crude oil indicating a possible attenuation of oxidative stress.
to overcome the effect of the oxidant stress caused
by free radicals generated by crude oil. These CONCLUSION
observations are corroborated by earlier studies on This study showed that subchronic exposure
the effects of petroleum hydrocarbon on other to crude oil caused adverse changes in the tissues
animal species (Nwaogu et al., 2008; Ibrahim and of albino rats. These changes if unchecked could
Rizk, 2008). precipitate various disease conditions.
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622 Journal of Research in Biology (2011) 8: 617-623
Nwaogu et al.,2011
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
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
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
The lower concentrations of the extract also (Eds.). Academic Press Inc., New York. 149-150.
promoted the chlorophyll content of V. unguiculata
up to 20% when compared to the control while Bhosl NB, Untawale AG and Dhargalker VK.
higher concentrations (> 20%) decreased the 1975. Effects of seaweed extract on growth of
chlorophyll content. A similar observation was Phaseolus vulgaris. Indian Journal of Marine
made in Vigna mungo (Venkataraman and Mohan, Sciences 4:208-210.
1997) and when seaweed extract (15-20%) applied
as foliar spray enhanced the leaf chlorophyll level Blunden G. 1971. The effect of aqueous seaweed
in plants (Blunden et al., 1996). extract as fertilizer additives. Proceedings of Sixth
The highest total sugar content was recorded International Seaweed Symposium, Tokyo,
at 20% concentration of seaweed extract soaked (Blunden G (Ed.), 584-589.
treatment in V. unguiculata. The sugar content
increased up to 20% concentration of seaweed Blunden G, Jenkins T and Liu YW. 1996.
extract and decreased at higher concentrations. The Enhanced chlorophyll levels in plants treated with
increase in the total sugar content at lower seaweed extract. Journal of Applied Phycology
concentration of seaweed extract might be due to 8:535-543.
absorption of most of the necessary elements
(Kannan and Tamilselvan, 1990). The same trend Bokil KK, Mehta VC and Datat DS. 1974.
was observed in the H. musciformis with NPK Seaweed as manure: II pot culture manurial
application in black gram (Tamilselvan and experiments on wheat. Phykos 13:1-15.
Kannan, 1994), and D. biflorus (Anantharaj and
Venkatesalu, 2002). It has been observed that Booth E. 1969. The manufacture and properties of
amylase activity was higher than the amylase liquid seaweed extracts. Proceedings of Sixth
activity. Both amylase and -amylase activity International Seaweed Symposium, Tokyo,
increased at lower concentrations and decreased in (Blunden G (Ed.), 655-662.
higher concentrations. In conclusion, Ulva lactuca
extract was found to be a promising fertilizer. The Challen SB and Heminway JC. 1965. Growth of
extract act as a biological fertilizer for organic higher plants in response to feeding with seaweed
farming, which is non-toxic, non-flammable for extracts. Proceedings of Sixth International Seaweed
attaining better seed germination and growth. Symposium, Tokyo, (Blunden G (Ed.), 682-686.
Authors: ABSTRACT:
Sivasankaran K,
Babu Thangadurai T,
Ignacimuthu S.
Corresponding author:
Ignacimuthu S
Email: Keywords:
entolc@hotmail.com Catocalinae, Noctuidae, Genitalial morphology.
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.
Female genitalia: Ovipositor lobes slightly Anomis privata Walker, 1865 (Fig. 5a)
inferior, triangular setose. Both pairs of apophysis Material Examined:
well developed. Anterior apophysis hardly longer Tamilnadu (INDIA): Nilgiri District, Coonoor, 10.
than posterior apophysis with spatulate tip. Ductus iv. 2007, 2 ex. ; 8. xii. 2007, 3 ex.
bursae long. Bursae copulatrix elongate Male genitalia: Uncus short, stout curved broadly,
membranous with fine wrinkles. Signum represents sword-shaped, medially setose. Tegumen broad.
a narrow sclerotized band. (Fig. 2d). Tuba analis prominent. Vinculum broad Ushaped.
Valvae simple and sclerotized costal arms not well
Hypocala deflorata Fabricius, 1794 (Fig. 3a) developed; sacculus heavily sclerotized. Transtilla
Material Examined: membranous. Juxta U shaped. Aedeagus long,
Tamilnadu (INDIA): Nilgiri District, Coonoor, 29. broad with thorn-like cornuti. (Figs. 5b, 5c).
xi. 2006, 2 ex. ; 9. iv. 2007, 6 ex. ; 22. x. 2007, 4 Female genitalia: Ovipositor lobes well developed
ex. ; 8. xii. 2007, 4ex. clothed with setae. Both pairs of apophyses strongly
Male genitalia: Uncus bifurcate, fringed with hairs. developed. Posteriores longer than anteriores.
Tegumen broad. Vinculum V-shaped with narrow Ductus bursae long and stout. Bursae copulatrix
arms. Valvae short; cucullus triangular shaped; membranous, approximately rhomboid without
costa and sacculus well differentiated. Harpe spine- signum. (Fig. 5d).
like sclerotized. Juxta bowl-shaped with raised
margin. Aedeagus of moderate length, slender and Anomis mesogona Walker, 1858 (Fig. 6a)
slightly contricted into apical region; vesica bearing Material Examined:
a short spine like cornuti; ductus ejaculatorius Tamilnadu (INDIA): Nilgiri District, Coonoor, 18.
broad, long originated from vesica. (Figs. 3b, 3c) v. 2007. 7 ex.
Female genitalia: General aspect elongate Male genitalia: Uncus long curved, medially
ovipositor lobes inferior, clothed with hairs. Both broad. Tegumen short, broad. Scaphium well
pairs of apophyses strongly developed; anterior developed. Saccus broad. Valvae symmetrical,
apophysis longer than posterior apophysis with long; cucullus blunt, broad. Aedeagus long and
spatulate tip. Ductus bursae very long and broad. slender; vesica bearing rod-like cornuti. (Figs. 6b,
Bursae copulatrix membranous, broad oval and fine 6c).
wrinkles, signum ribbon -like. (Fig. 3d). Female genitalia: Ovipositor lobes prominent with
strong setose. Both pairs of apophyses well
Hypocala lativitta Moore, 1877 (Fig. 4a) developed. Ductus bursae long and broad. Corpus
Material Examined: bursae membranous, rectangular with broad
Tamilnadu (INDIA): Nilgiri District, Coonoor, 8. signum. (Fig. 6d).
xii. 2007, 4 ex.
Male genitalia: Uncus broad fringed with hairs, Polydesma lindsayi Hampson, 1893 (Fig. 7a)
composed of a curved terminal lobe and having a Material Examined:
subapical beak-like part. Genital capsule elliptical, Tamilndau (INDIA): Nilgiri District, Coonoor, 29.
tegumen broad, without penicular lobe. Vinculum xi. 2006, 5 ex.; 7. vii. 2010, 2 ex.
V shaped with narrow arms. Valvae symmetrical Male genitalia: Uncus short curved apically
short; cucullus broad, flat and clothed with hairs; hooked. Scaphium and tuba analis well developed.
costa and sacculus well differentiated; sacculus Tegumen broad. Vinculum long, broad U shaped.
narrow and heavily sclerotized. Juxta basket- Valvae asymmetrical apically narrow into a long
shaped, sclerotized, projected with two arms. lobe; a conical costal process; sacculus well
Aedeagus stout, rod-like cornuti; ductus sclerotized and tappering. Transtilla membranous.
ejaculatorius broad originate from vesica. (Figs. 4b, Juxta simple sclerotized. Aedeagus long and slender
4c). basally swollen, without cornuti. (Figs. 7b, 7c).
Female genitalia: Ovipositor lobes prominent. Both Female genitalia: Ovipositor prominent developed
pairs of apophyses strongly developed; anterior with long setose. Both pairs of apophyses well
apophysis longer than the posterior apophysis with developed; posterior apophysis longer than the
spatulate tip. Ductus bursae long and broad, with anterior apophysis. Ductus bursea short. Corpus
well sclerotized. Corpus bursae membranous, oval bursae triangular in shape without signum. (Fig.
shaped with broad signum. (Fig. 4d). 7d).
Lacera noctilio (Fabricius, 1774) (Fig. 8a). Y shaped strongly sclerotized. Aedeagus long and
Material Examined: stout in the anterior half and uniformly scobinate.
Tamilnadu (INDIA): Niligiri District, Coonoor, 12. (Figs. 10b, 10c).
xii. 2006. 5 ex. ; 27. xi. 2006. 3 ex. ; 5. i. 2007. 4 Female genitalia: Ovipositor lobes well developed
ex . ; 10. vii. 2007. 2 ex. ; 9. ix. 2007. 2 ex. ; 22. x. covered with hairs. Both apophyses strongly
2007 3 ex. ; 8. xii. 2007. 46 ex. ; Kodaikanal, 10. vi. developed. Posteriores longer than anteriores.
2008. 2 ex. Ostium bursae simple. Ductus bursae
Male genitalia: Uncus rather long, strong, apically dorsoventrally flattened, sclerotized and wider
dilated finally hooked, distally covered with hairs. posteriorly. Bursae copulatrix elongate broader
Tegumen rather long narrow with penicular lobe. anteriorly. (Fig. 10d).
Vinculum short, U shaped; cucullus broad outer
margin with setose. Valvae not evenly developed; Serrodes campana Guenee, 1852 (Fig. 11a)
harpe long narrow finally curved with setose. Juxta Material Examined:
club-shaped, long and broad. Aedeagus relatively Tamilnadu (INDIA): Nilgiri District, Coonoor, 10.
large curved and widening anteriorly; apical region iv. 2007, 1 ex.; 6. vii. 2007, 1 ex. ; 22. x. 2007, 2
of aedeagus with elliptical shape. (Figs. 8b, 8c) ex.
Female genitalia: General aspect elongate. Male genitalia: Uncus short and stout, with a
Ovipositor lobes prominent, triangular, clothed with curved hooked apex. Tegumen broad; penicularlobe
long setose. Both pairs of apophyses strongly absent. Juxta narrow inversely bifurcated.
developed; posterior apophysis longer than anterior Vinculum broad and heavily sclerotized, V- shaped
apophysis, thin. Ductus bursae stout. Bursae and extended to form a prominent saccus. Valvae
copulatrix elongate and broad. (Fig. 8d). long and slender well sclerotized with small harpe.
Aedeagus long and stout with strong double
Sphingomorpha chlorea Cramer, 1779 (Fig. 9a) cornutes. (Figs. 11b, 11c).
Material Examined: Female genitalia: Ovipositor lobes inferior. Both
Tamilnadu (INDIA): Nilgiri District, Coonoor. 22. pairs of apophyses strongly developed with
xi. 2007. 7ex spatulate tip. General aspect of bursae copulatrix
Male genitalia: Uncus strongly developed, broad massive; wall of corpus bursae membranous
apically hooked. Socii well developed. Genital without signum. Ductus seminalis arising from
capsule narrow; tegumen broad, long. Saccus very posterior side of corpus bursae. (Fig. 11d).
broad. Valvae symmetrical long and narrow;
sacculus broad sclerotized. Juxta bifurcated. Chrysopera combinans Walker, 1862 (Fig. 12a)
Aedeagus stout, acute posteriorly without cornuti. Material Examined:
(Figs. 9b, 9c). Tamilnadu (INDIA): Nilgiri District. Coonoor, 18.
Female genitalia: Ovipositor lobes well developed. v. 2007, 7 ex.
Both pairs of apophyses strongly developed. Male genitalia: Uncus long and slender, with
Posterior apophysis hardly longer than anterior acutely pointed. Tegumen short. Saccus broad.
apophysis. Ductus bursae short. Bursae copulatrix Juxta broad with heavily sclerotized between the
with irregular contour without signum. Ductus valvae. Valvae massive; harpe prominent.
seminalis arising from base of bursae copulatrix. Aedeagus slender, vesica bearing with rod-like
(Fig. 9d). cornuti. (Figs. 12b, 12c).
Female genitalia: Ovipositor lobes small covered
Oxyodes scrobiculata Fabricius, 1775 (Fig. 10a) with setae. Posterior apophysis short and thin;
Material Examined: anterior apophysis stout, longer than posterior
Tamilnadu (INDIA): Nilgiri District, Coonoor, 29. apophysis. Ductus bursae much longer. Bursae
xi. 2006, 15 ex. ; 18. xii. 2006, 13 ex. ; 1. i. 2007, copulatrix membaranous with delicate signum.
40 ex. ; 28. ii. 2007, 40 ex. ; 9. iv. 2007, 79 ex. ; 22. (Fig. 12d).
x. 2007, 31 ex. ; 8. xii. 2007, 98 ex. ; 23. i. 2008, 58
ex. Homoptera glaucinans Guenee, 1852 (Fig. 13a)
Male genitalia: Uncus long and sword-shaped. Material Examined:
Tegumen long, broad. Vinculum long and broad; Tamilnadu (INDIA): Nilgiri District. Coonoor, 8.
saccus U-shaped. Valva with a well differentiated xii. 2007, 5ex.
sacculus and costa; cucullus long and narrow. Juxta
634 Journal of Research in Biology (2011) 8: 631-642
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
640 Journal of Research in Biology (2011) 8: 631-642
Sivasankaran et al.,2011
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.
Email: Dates:
ericbkouam@yahoo.com
Received: 29 Nov 2011 /Accepted: 07 Dec 2011 /Published: 26 Dec 2011
Ficus Publishers.
Web Address: This Open Access article is governed by the Creative Commons Attribution License (http://
http://jresearchbiology.com/ creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
Documents/RA0156.pdf. commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.
dehydrogenase (FDH, one locus), fluorescent The FIT and FIS coefficients measure
esterase (FLE, two loci), isocitrate dehydrogenase deficiencies of homozygotes or heterozygotes
(IDH, two loci), malate dehydrogenase (MDH, four relative to the panmictic expectations within the
loci)), 6-phosphogluconate dehydrogenase (PGD, overall sample and within populations respectively.
two loci), phosphoglucose isomerase (PGI, three The FST coefficient estimates relative population
loci), Phosphoglucomutase (PGM, two loci), differentiation. Standard errors were estimated by
shikimate dehydrogenase (SDH, one locus). Stain jack-knifing using the Fstat program (Goudet
recipes were taken from Wendel and Weeden 1995). Neis (1972) genetic distances (D) were
(1989). calculated for each pair-wise combination of
Data analysis population and values were used to construct a
Putative loci were designated sequentially, dendrogram using the UPGMA (unweighted pair
with the most anodally migrating isozyme group method with arithmetic mean) method.
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 based on Wrights (1951) equation: Nm = 1/4(1/FST
the value 100, slower and faster bands on the - 1), where Nm is the average number of migrants
zymmogram relative to this common one, which exchanged per generation between populations. A
represent other alleles, were given lower and higher second estimate of Nm, using the distribution of
values corresponding to their relative migration private alleles (alleles found in only one
using the same nomenclature as in Pasquet (1999). population), was calculated using GenAlex
The genotype of each mother plant was estimated computer program (Peakall and Smouse, 2006)
from the progeny array following the method of following the procedure of Barton and Slatkin
Brown and Allard (1970) and using the MLTR (1986).
computer program, version 2.2 (Ritland 2002).
We calculated using Popgene software RESULTS
version 1.31 (Yeh et al. 1999) standard measures of Patterns of genetic diversity
genetic diversity for each population. This includes Of the twenty-one isozyme loci essayed,
the mean number of alleles per locus (A), the nine (42.9%) were polymorphic across the range of
percentage of polymorphic loci (P), and mean V. unguiculata. The remaining twelve loci (Fle-1,
observed (Ho) and expected heterozygosity (He) at Idh-2, Mdh-1, Mdh-2, Mdh-3, Mdh-4, Pgd-1, Pgd-
the species and within-population levels. Wrights 2, Pgi-1, Pgi-2, Pgm-1 and Sdh) were
fixation index (F) (Wright 1922) was used to monomorphic, displaying unique allele each, in all
calculate deviations from Hardy-Weinberg populations. An average of 11.4% of the loci was
equilibrium for each polymorphic locus within polymorphic within population, with individual
populations [F = (He Ho) / He]. Chi-square tests population values ranging from 0 to 19.1% (Table
were used to test for significant deviations in the 1). We found thirty-four distinct alleles overall
fixation indices from the expected value that is F = across the twenty-one loci. Six of the thirty-four
0 (Li and Horvitz 1953). Chi-square tests were also alleles were unique to a single population: Pgm2104
used to test for heterogeneity in allele frequencies and Idh1090 in SDN03; Pgi3096 in SDN09; Fle3098 in
among populations (Workman and Niswander, SDN17; Pgm2096 in SDN26 and Enp096 in SDN27.
1970). Total genetic heterozygosity (H T), The mean number of alleles per locus was 1.62 at
heterozygosity within populations (HS), genetic the species level. Across populations it ranges from
diversity among populations (DST), and the 1 to 1.19 with a mean at 1.128. (Table1). Gene
proportion of genetic diversity found among diversity or expected heterozygosity for the species
populations (GST) were calculated following the was somewhat low (0.084); in other words, only
equations of Nei (1973, 1977) and using GenAlex 8.4% of individuals are genetically expected to be
computer program (Peakall and Smouse, 2006). heterozygous at a given locus under random mating
The genetic structure within and among populations conditions. At the population level, expected
was also evaluated with F-statistics (FIT, FIS and heterozygosity ranges from 0 (SDN09) to 0.074
FST) following Weir and Cockerham (1984) and (SDN22) with the average at 0.049. Observed levels
using Fstat computer program (Goudet 1995). of heterozygosity in populations (Table 1),
These F-statistic values were tested for difference however, were almost always lower than Hardy-
from zero using permutation tests (Goudet 1995). Weinberg expectations, averaging 0.014.
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 of the nine polymorphic loci were significantly
Between populations, allele frequencies greater than zero (Table 2). The mean estimates of
were significantly different at eight of the nine these coefficients were significant (FIS = 0.684 P <
polymorphic loci as shown by Chi square test 0.001; FIT = 0.835 P < 0.001), reflecting that
(Table 2). The estimates of genetic structure using observed levels of heterozygosity within
Neis genetic diversity estimates are shown in populations and the entire study area were smaller
Table 2. The average of total heterozygosity (HT) than would have been expected in case of random
and within population genetic diversity (HS) were sexual reproduction. High FST values for most of
0.083 and 0.049, respectively. The inter-population the polymorphic loci (Table 2) indicate significant
genetic diversity (DST) and the coefficient of genetic differentiation at the population level (FST =
genetic differentiation among populations (GST) 0.477, P < 0.001). The majority of test for pair-wise
varied from 0.000 (Enp) to 0.172 (Fdh) and from - genetic differentiation among populations (60 out
0.028 (Enp) to 1.000 (Pgi-3), with a mean of 0.034 of 78) were statistically greater than zero; the
and 0.409, respectively. The result indicates that highest value (0.904) found between SDN 09 and
about 41% of the genetic variation in our sample SDN16 (Table 3). Genetic distance (D) values
can be attributed to variation among populations. between pair of populations are also shown in Table
The inbreeding coefficients (FIS and FIT) for eight 3. They ranged from 0.002 to 0.128 with a mean at
646 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 gene flow (NmS = 0.043). Both estimates of Nm
effect on geography as the correlation between being lower than unity suggest strong population
genetic and geographic distance was weak (r = differentiation.
0.10, P > 0.050). A UPGMA dendrogram
illustrating genetic relationships among populations DISCUSSION
were constructed (Figure 1). Indirect estimates of Genetic diversity is essential to avoid risk of
gene flow indicate low levels of pollen migration extinction and promote the long-term survival of
among V. unguiculata populations. The application plant species. The loss of genetic variation is
of Wrights (1951) model gave an estimate of NmW thought to decrease both the short-term and the long
= 0.274. The low value of Nm reflects the high -term adaptability of populations in variable and
value of FST. An independent estimate of gene flow changing environments (Hamrick 1994, Young et
(Barton and Slatkin 1986) based on the frequencies al. 1996). Populations of selfing species and animal
of private alleles gave an even lower estimate of -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
0.04 0.03 0.02 0.01 0.00
respectively) to the means for selfing species
Fig 1: An UPGMA clustering tree based on Neis (20.0%, 1.31, and 0.074) (Hamrick and Godt,
(1972) genetic distance calculated for 21 allozyme loci 1990). V. unguiculata, however, had at the species
in 13 populations of wild V. unguiculata
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
-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
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*
0.588*
0.278*
0.427*
0.022
0.016
0.003
0.025
0.493*
0.443*
0.234*
0.663**
0.309NS
0.323NS
SDN19
0.280*
0.411*
0.041
0.032
0.073
0.013
0.022
0.057
0.330NS
0.614NS
0.759*
0.049
0.073
0.075
0.095
0.066
0.077
0.057
0.407*
0.670**
SDN12
0.105*
0.038
0.047
0.048
0.053
0.038
0.059
0.037
0.039
0.035
0.092
0.050
0.128
0.106
0.124
0.054
0.068
0.084
0.064
0.071
0.027
0.062
0.049
0.010
0.087
0.050
0.073
0.010
0.024
0.031
0.016
0.033
high level of inbreeding can result from several among populations (Wright 1951). Between V.
causes: family structuring within a restricted unguiculata populations, gene flow (Nm) is very
neighbourhood causing mating between relatives low, less than unity like in other endemic and
(Levin and Kerster 1971, 1974), selection for endangered plant species (Hamrick and Godt 1996)
homozygotes, Wahlund effect and positive and appears insufficient to counter divergence due
assotative mating (preferential mating between to the effects of random genetic drift according to
individual of similar genotypes) (Crow and population genetics theory (Wright 1951, Real
Felsenstein 1968). Other authors have also observed 1994). Although both low, we noted that Nm W is
positive and significant inbreeding coefficient about 6 times higher than NmS. Such disparity
values and attributed it to populations substructure could be attributed to the general high frequencies
and inbreeding (Bi et al. 2003, Ueno et al. 2002). of private alleles (Idh1090 in SDN03 with 0.4
An important feature of the floral biology of V. frequency; Pgm2096 in SDN26 with 0.6 frequency
unguiculata is the position of stigma and anther that and Pgi3096 in SDN09 with 1 frequency). Similar
are in contact with the pollen that usually shed low level of gene flow were reported by Bi et al.
before the full opening of the flower (Lush 1979). (2003) and Hardy et al. (1997) after studying the
This may reduce cross-pollination and provide high population genetic structure of Phaseolus lunatus,
reproductive assurance even in the absence of an autogamous plant species. Two main
pollinators. Hence, the high levels of inbreeding in mechanisms of gene flow exist for cowpea: seed
V. unguiculata appear to be due to autonomous and pollen dispersal. Seed migration, is very
selfing due to the close proximity of the stigma and unlikely given the extensive spaces that generally
anthers in this species (Lush 1979). Geography did separate neighbouring wild populations. Pasquet et
not correlate significantly with the genetic al. (2008) reported that bees are able to disperse
relationship among populations of V. unguiculata. cowpeas pollen to few km with a low probability
The lack of significant association between of long pollen transport events. Pollen dispersal by
geographic and genetic distances found in V. bees clearly is the most likely mechanism of gene
unguiculata may be explained by the expectation of flow between populations of cowpea.
a model of differentiation by founder effects (Mayr
1963) where small number of individual from an ACKNOWLEDGEMENTS
existing population colonize a new habitat forming We thank the German Academic Exchange
a new population genetically close to the former. It Service (DAAD) and the ICIPE - ARPPIS training
might also be the result of genetic drift program. The study was also funded by the United
(Chakraborty et al. 1978) coupled with a highly States Agency for International Development
selfed-mating system and restricted pollen flow (USAID).
among local populations. These factors could all
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Authors: ABSTRACT:
Komathi S1, Rajalakshmi
G1, Savetha S2 and
Passiflora foetida is an angiosperm plant with high medicinal value and is
Ayyappadas MP2.
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
Institution: action in burn wounds and is recommended by Brazilian Drugs centre as an
1. Department of antirheumatic. The present study report an efficient in vitro regeneration protocol by
Biotechnology, Hindustan using leaf, node, internode and shoot tip explants for this species. Explants are surface
College of Arts & Science. 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
2. Department of medium containing BAP and NAA at 3.0 and 0.5 mg/l respectively. The nodal and
Biotechnology, R.V.S shoot tip segments derived shoots and rooted effectively (70% & 80%) in the basal
College of Arts and science medium containing the auxin, IBA and IAA alone at 1.0 mg/l respectively. The nodal
Sulur, Coimbatore -402. callus derived in vitro rooted shoots responded well (92%) in the hardening medium.
Ficus Publishers.
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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.
nutrient medium completely without causing onto the MS basal medium containing different
damage to roots. Then the plants were soaked in 1% concentrations of growth regulators.
(w/v) fungicide, methyl-3 benzimidizole carbomate The sub culturing experiments were
(Bavistin) solution for 10-15 minutes and conducted by using callus as the secondary explants
transferred to plastic pots (Bhojwani, S.S 1983). for shoot proliferation and root induction in the MS
Twenty five plantlets per potting mixture were basal medium with various concentrations of the
tested and growth rates were calculated after 30 growth regulators, BAP, NAA, IAA and IBA. The
days of hardening. During hardening process, results obtained are described below
initially the plantlets were kept under shade not In the basal medium, the responses of node
present inside the 75% shade house for ten days. derived callus for shoot initiation to various
After 10 days, they were gradually transferred to concentrations and combinations of BAP, NAA,
75% shade house and maintained for two weeks. IAA and IBA are given in (Table 2). The shoot
Then the plantlets were exposed to the natural formation during sub culturing was highly effective
environmental conditions. (80%) in the basal medium containing BAP and
NAA at 3.0 and 0.5 mg/l respectively followed by
RESULTS AND DISCUSSION 70% in BAP and NAA at 2.5 and 0.5 mg/l in the
In vitro regeneration basal medium. Similarly, the shoot initiation of
The number of days required for callus shoot tip derived callus was widely varied
induction of explants is 10-35 after inoculation of according to concentrations and combinations of
explants (Table 1). The percentage of explants growth regulators BAP, NAA, IAA and IBA in the
responding for callus formation with respect to basal medium (Table 3). The MS medium fortified
internode and leaves were lower, lies less than 15 with the growth regulators BAP and NAA at 3.0
and 18 only. However, it was noticed that the shoot and 0.5 mg/l respectively registered higher amount
tip and node explants responded well for callus (76%) of shoot initiation from the shoot tip derived
formation when cultured on MS medium callus. The results indicate that the cytokinin, BAP
supplemented with the growth regulators, BAP and in combination with the auxin, NAA was found to
NAA at 2.5 and 0.3 mg/l respectively. Similarly, be more suitable for the improved response of calli
higher amount of (60%) shoot tip explants to produce shoots during sub culturing. The number
responded well for callus formation in the MS of shoots produced was greater (13 shoots/callus)
medium containing the growth regulators BAP and and shoot length was higher (7.4 cm) in basal
NAA at 2.5 and 0.3 mg/l respectively. On the other medium containing BAP and NAA at 3.0 and 0.5
hand, all attempts with various combinations of mg/l respectively for node derived callus. Whereas
BAP and NAA in basal medium produced different the shoot tip derived callus produce more number
color of callus. Furthermore it was observed that the of shoots (17 shoots/callus) in the medium
explants such as node and internode produced light containing the growth regulators BAP alone at 3.0
brown and dark brown colored calli respectively, mg/l. The shoot length was higher (7.3 cm) in the
while the explants of shoot tip and leaf produced MS medium enriched with the growth regulators
green coloured calli when the explants inoculated 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.
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.
secondary metabolites, zygotic embryo culture was study cell growth, to produce secondary metabolites
attempted for 62 Passiflora species, starting from and a tool for rapid micro propagation of some
seeds mainly collected in the wild. Twenty nine of economically valuable crops. Using this technique,
these species produced calli, which had very we can understand cell growth parameters and
different growth rates. Plants were successfully establish an efficient protocol. In this study, we
regenerated from calli of 13 different species. For used callus derived from in vitro plantlets of yellow
25 of the responsive species this is the first report of passion fruit to initiate cell suspension culture.
in vitro culture (Flavia Guzzo et al., 2004). Cultures were maintained in MS medium
Passion fruit is a highly valuable species but supplemented with various NAA concentrations
it has some problems about pests and diseases. (0.0-2.0 mg l-1), 30 g l-1 sucrose. Cell growth was
There are some reports about shoot regeneration evaluated by counting cell numbers on 0, 5, 10, 15,
and plantlet formation of vine crops from various 20 and 25 days of culture. After 20 days of culture,
explants and using different techniques, but these 2 ml of suspension culture was transferred to
are not suitable. In this study, we used callus hormone-free MS medium containing 60 gl-1
derived from in vitro plantlets of yellow passion sucrose and 8 gl-1 agar to induce callus formation.
fruit to initiate cell suspension culture. Cultures These calli were used to regenerate shoots (Bui Le
were maintained in MS medium supplemented with Thanh Khiet et al., 2006).
various NAA concentrations (0.0-2.0 mg l-1), 30 g l
-1 sucrose. Cell growth was evaluated by counting CONCLUSION:
cell numbers on 0, 5, 10, 15, 20 and 25 days of Callus was induced efficiently from the leaf
culture. After 20 days of culture, 2 ml of suspension explants of Passiflora foetida L. (Passion fruit) on
culture was transferred to hormone-free MS Murashige and Skoog (MS) medium containing 2
medium containing 60 gl-1 sucrose and 8 gl-1 agar mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) and
to induce callus formation. These calli were used to 0.5 mg/l kinetin (KN) after 16 days. Maximum
regenerate shoots(Bui Le Thanh Khiet et al., 2006). callus (65% response, 610 mg fresh weight and 55
Passion fruit is a highly valuable species but mg dry weight) was formed on MS medium
it has some problems about pests and diseases. supplemented with low sucrose and salt
There are some reports about shoot regeneration concentration than that of high sucrose and salt
and plantlet formation of vine crops from various concentration (Rasool SN et al., 2011).
explants and using different techniques, but these
are not suitable. Cell suspension culture is a tool to
658 Journal of Research in Biology (2011) 8: 653-659
Komathi et al.,2011
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Nong Lam University Ho Chi Minh City. Research in Natural Products 4(1):1-10.
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Science 4(10):417-426. 67:33-38.
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.
used to drive the reduction of components of the Carotenoids = (1000 A470 1.90 Ca - 63.14 Cb)/214
electron transport chain. Statistical Analysis
Extraction of Photosynthetic pigments Data from the two selected sites for the plant
Extract photosynthetic pigments by grinding materials were subjected to the two way analysis of
1g of leaves, torn into small pieces, in a mortar with variance (ANOVA). Using ANOVA the
a pinch of clean sand and a total of 10 ml of 100% comparison made between control plant species and
acetone. Initially, add only a small amount of polluted plant species. Significance difference was
acetone to begin the grinding process. It is much calculated at 0.05%, 0.01% and 0.001% level as per
easier to grind the leaves if the extract is a pasty standard method of Gomez and Gomez (1984).
consistency. Add more solvent in small increments The present investigation has been
while continuing to grind the leaves. For some undertaken to study the effect of cement dust
species may need to add more than the suggested pollutant on total chlorophyll, carotenoids,
10ml of acetone. Pour the extract into a 15ml chlorophyll a and chlorophyll b of selected plant
centrifuge tube and centrifuge in the bench top species. In the present study, the cement pollution
centrifuge for 3 to 5 min. Remove the extract to a effects on the performance of selected plant species
10ml graduated cylinder using a Pasteur pipette. was observed and the total chlorophyll content
Transfer an aliquot of the clear leaf extract decreased significantly in response to cement dust
(supernatant) with a pipette to a 1-cm-pathlength pollutants in polluted plant leaves compared with
cuvette and take absorbance readings against a control of Azadirachta indica(L), Polyalthia
solvent blank in a UV-VIS spectrophotometer at longifolia(L), Ficus religiosa(L), Pongamia
four different wavelengths. pinnata(L) and Delonix regia(L).3.
750 nm (A750 = 0 for clear extract)
662 nm (chlorophyll a maximum using 100% acetone) RESULTS AND DISCUSSION
645 nm (chlorophyll b maximum using 100% acetone) The quantitative analysis of chloroplast
470 nm (carotenoids). pigments revealed difference between the selected
Apply measured absorbance values to plant species exposed to cement dust pollution and
equations given by Lichtenthaler (1987) for acetone those from control. The mean values of all
to determine pigment content (g/ml extract measured parameters are shown in Fig. 1 to 4.
solution).Once the baseline has been run from 700- Chlorophyll a content was higher in control site
400nm using acetone in the cuvette, run an compared with polluted site. The amount of
absorption spectrum for each pigment, rinsing the chlorophyll a varied from 1.18 to 1.78 mg g-1(FW)
sample cuvette with acetone between readings. The in control site and from 0.61 to 1.33 mg g-1(FW) in
peaks and valleys will be adjusted automatically by the polluted site (Table 1).
the Lamda-35 UV-VIS spectrophotometer, by The chlorophyll b content was lowered in
changing the range of percentage absorbance on the polluted site compared with control site in all the
y-axis. We can use the cursor to obtain the plant species. The values varied from 0.81 to 1.43
wavelengths of the spectral peaks, or estimate them mg g-1(FW) and 0.54 to 1.02 mg g-1(FW) in control
from the printed spectra. These peak wavelengths and polluted sites respectively (Table 2, fig. 2). The
will be useful for determining the identities of the amount of chlorophyll reduced in all the polluted
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 Figure 1. The mean value of chloroplast pigments in
Azhadirachta indica
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.
Figure 2. The mean value of chloroplast pigments in Figure 3. The mean value of chloroplast pigments in
Polyalthia longifolia Ficus religiosa
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.
Polyalthia Polyalthia
1.430.16 1.02**0.11 <0.01% 2.830.49 2.04**0.38 <0.01%
ongifolia(L) longifolia(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 experienced by the plant species. The average
chlorophyll was observed in Delonix regia (L) amount of chlorophyll a to chlorophyll b (a/b) is
whereas the lowest reduction (24.67%) was minimum in pongamia pinnata(L) (0.76 mg g-1fw)
recorded in Azadirachta indica(L). Similarly, in and maximum in Azadirachta indica(L) (1.80 mg g-
1
case of carotenoid contents, highest reduction fw) (Table 5). The total photosynthetic pigments
(65.55%) was observed in Pongamia pinnata(L) (Chl a+ Chl b+ carotenoid) were found to
and lowest in Ficus religiosa(L) (29.01%). minimum in polluted sites compared to the control.
Polyalthia longifolia(L) and Ficus religiosa(L) The average amount of total photosynthetic
showed significant reduction in chlorophyll pigments in polluted plants was less in Ficus
a (p<0.01) and other three species showed the religiosa (L) (1.37 mg g-1fw) and more in
significance level (p<0.05%). Azadirachta indica (L) (2.40 mg g-1fw) compared to
Mandre and Tuulmets (1997) reported a all the control plant species. The reduction in total
decrease of chlorophyll content in Norway spruce pigments is mostly caused by Chl a and Chl b
needles caused by cement dust. which have significant value and also the low
Total average amount of assimilating pigments: carotenoidic pigments. The low ratio value
The weight ratio of Chl a and Chl b indicates the plant species subjected to the dust
indicates the functional characters of photosynthetic pollution.
pigments. The ratio of Chl a and Chl b was The observed variation in photosynthetic
found to be higher for the polluted environment pigment was contributed due to the air pollutant and
exposed to the Azadirachta indica (L) compared to sensitivity of the plant. The ratio of Chl a+b to
the control. Similarly, the weight ratio of Chl a carotenodic pigments, (a+b)/c) has extremely low
and Chl b to total carotenoids indicates the stress 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
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
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Pollution injury to Vegetations A Pictorial Atlas.
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Pennsylvania. the use of electrothermal atomic absorption
spectrophotometry for the determination of
Lichtenthaler HK. 1987. Chlorophyll and Chromium and Copper in portland cement. Micro
carotenoids: Pigments of photosynthetic chemistry Journal 41(3):348-355.
biomembranes. Meth Enzyme 148:331-382.
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in Norway spruce induced by dust pollution. Water 213:142-167.
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and protective function of carotenoids in
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on photosynthesis of tomato plants at different CO 2
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Authors: ABSTRACT:
Sanjay Lal, Kinnari N.
Mistry, Smit D. Shah, The genetic relationship study was carried out among nine different cultivars
Riddhi Thaker, Parth B. of Catharanthus roseus using RAPD, ISSR and SSR markers. In RAPD analysis, out of
Vaidya. 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
Institution: separated the cultivars in two major groups. Blue pearl, cooler red, and pacifica
Ashok & Rita Patel Institute apricotwere in the same group, while rest was in other group. Amongst all Pacifica
of Integrated Studies in liac and Albus with red eye were found to be closely related. Our results showed
Biotechnology & Allied that all RAPD, ISSR and SSR markers are sensitive and effective tool for genomic
Sciences (ARIBAS), New analysis in Catharanthus roseus. This study provides key platform for further crop
Vallabh Vidhya Nagar improvement and cross breeding.
388121 (Gujarat) India.
Keywords:
Genetic diversity, RAPD, ISSR, SSR, Catharanthus roseus.
Abbreviations:
Corresponding author: Random amplified polymorphic DNA (RAPD), Inter simple sequence repeats
Sanjay Lal (ISSR), Simple sequence repeats (SSR), basepair (bp).
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.
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 annealing temperatures (Table-3) and an extension
removed by giving RNaseA treatment which was step of 2 min at 720C. The last cycle was followed
given after incubation at 650C in boiling water bath. by final extension step of 7 min at 72 0C. The
The isolated DNA was dissolved in 25l of T10E1 holding temperature was 40C.
buffer (Tris 10mM and EDTA 1mM, pH 8.0). The Simple Sequence Repeats (SSR)
DNA was checked through agarose gel In SSR analysis, the polymerase chain
electrophoresis and the quantity and purity was reactions were performed in 25l system containing
checked by Nanodrop-1000 (Thermo Fisher 2.5l of 10 X assay buffer (10mM Tris-Cl; pH 9.0,
Scientific, USA). 1.5mM MgCl2, 50mM KCl and 0.01% gelatin),
Random Amplified Polymorphic DNA (RAPD) 2.5mM of each dNTPs (dATP, dTTP, dCTP and
analysis dGTP) (Bangalore Genei Pvt. Ltd, Bangalore,
The RAPD analysis was India), 1.5M of primer (Sigma Aldrich,
carried out using the method of Williams et al., Bangalore), 0.2 unit of Taq DNA polymerase
(1990). Polymerase chain reactions were performed (Bangalore Genei Pvt. Ltd, Bangalore, India) and
in 25l system containing 2.5l of 10 X assay 25ng of template DNA. The reaction was carried
buffer (10mM Tris-Cl; pH 9.0, 1.5mM MgCl2, out using thermo cycler (Corbett research gradient
50mM KCl and 0.01% gelatin), 2.5mM of each automatic, UK). Reaction was carried out in three
dNTPs (dATP, dTTP, dCTP and dGTP) (Bangalore steps. The first step of initial denaturation was
Genei Pvt. Ltd, Bangalore, India), 1.5M of primer performed at 940C for 5 min. Following the initial
(Sigma Aldrich, Bangalore), 0.2 unit of Taq DNA denaturation step, PCR was carried out for 40
polymerase (Bangalore Genei Pvt. Ltd, Bangalore, cycles. Each cycles consisted of a denaturation step
India) and 25ng of template DNA. The reaction was of 1 min at 940C, annealing step of 1 min at various
carried out using thermo cycler (Corbett research annealing temperatures (Table-4) and an extension
gradient automatic, UK). Reaction was carried put step of 2 min at 720C. The last cycle was followed
in three steps. by final extension step of 7 min at 72 0C. The
The first step of initial holding temperature was 40C.
denaturation was performed at 94 0C for 5 min. Agarose gel electrophoresis
Following the initial denaturation step, PCR was The amplified products were checked on gel
carried out for 45 cycles. Each cycles consisted of a electrophoresis (1.5% agarose gel for RAPD and
denaturation step of 1 min at 920C, annealing step 2% for ISSR and SSR). The electrophoresis was
of 1 min at 370C and an extension step of 2 min at performed for four hours at a constant voltage of
720C. The last cycle was followed by final 100 volts. The bands were visualized under U.V.
extension step of 7 min at 72 0C. The holding light and the photographs were taken by gel
temperature was 40C. documentation system (Alpha Innotech, Alpha
Inter Simple Sequence Repeat (ISSR) analysis Imager EP, USA). 100bp ladder was used to
In ISSR analysis, the determine the size of the amplicons.
polymerase chain reactions were performed in 25l Data scoring and statistical analysis
system containing 2.5l of 10 X assay buffer The band was scored 1 for its presence and
(10mM Tris-Cl; pH 9.0, 1.5mM MgCl2, 50mM KCl 0 for its absence. This data were used to construct
and 0.01% gelatin), 2.5mM of each dNTPs (dATP, the binary matrix. The Jaccards coefficient
dTTP, dCTP and dGTP) (Bangalore Genei Pvt. Ltd, (Jaccard, 1908) was used to construct the similarity
Bangalore, India), 1.5M of primer (Sigma Aldrich, matrix among the nine cultivars of Catharanthus
Bangalore), 0.2 unit of Taq DNA polymerase roseus. The phylogram was constructed using the
(Bangalore Genei Pvt. Ltd, Bangalore, India) and unweighted pair group method using arithmetic
25ng of template DNA. The reaction was carried means (UPGMA) (Sneath and Sokal, 1973) and
out using thermo cycler (Corbett research gradient SAHN clustering. The entire analysis was
automatic, UK). Reaction was carried out in three performed using the NTSYS-pc (Numerical
steps. The first step of initial denaturation was taxonomy system, applied biostatistics, Inc., New
performed at 940C for 5 min. Following the initial York, USA, software version 2.02e) (Rohlf, 1997).
denaturation step, PCR was carried out for 40 The polymorphism information content (PIC) value
cycles. Each cycles consisted of a denaturation step was calculated as PIC=1-P2i ; Pi is the band
of 1 min at 940C, annealing step of 1 min at various frequency of the ith allele (Smith et al., 1997).
Groupings of cultivars were also evaluated by the matrix, the similarity index was observed
principle coordinate analysis (PCA) as reported by ranging from 0.1980.731 with the mean similarity
Thomas et al. (2006). PCA was performed by index of 0.56 indicating reasonable variability as
extracting Eigen value and Eigen vectors from a obtained by RAPD markers (data not shown). The
correlation matrix which was generated using a maximum PIC value (0.955) was observed in OPC
standardized data matrix 2-D and 3-D plots were 12 primer, while the minimum PIC value (0.900)
constructed to evaluate the groupings of C. roseus was observed in OPAF 05. The average PIC value
cultivars. observed from all six RAPD primers was 0.93.
In our study, RAPD markers were
RESULT AND DISCUSSION successfully used to differentiate all nine cultivars
In RAPD analysis, total twenty RAPD of Catharanthus roseus from each other. Thus, on
primers were used to check the genetic variation in the basis of RAPD, the findings of this study are
nine different cultivars of Catharanthus roseus. Out similar to the observations of Rajaseger et al.,
of these twenty, six primers gave satisfactory and (1990).
reproducible bands. The banding pattern of the In ISSR analysis, totally six ISSR primers
RAPD analysis for two primers has been shown in were used. Out of these six, three primers gave
Fig-1. The details of the banding pattern are shown satisfactory and reproducible bands. The banding
in Table-2. All these six primers gave pattern of one ISSR primer is shown in Fig-2 and
amplifications in all nine cultivars. Total 592 bands the details are given in Table-3. All these three
were observed. From these total bands, 466 were primers gave amplifications in all nine cultivars.
polymorphic while, 126 bands were monomorphic. These ISSR primers produced total 342 scorable
This resulted in total polymorphism of 78.71%. bands. From these total bands, 270 bands were
OPA-03 gave maximum number (146) of bands, polymorphic while, 72 bands were monomorphic.
while minimum number of bands (62) was observed This gave a total polymorphism of 78.94%. The
in OPAF-15 primer. The amplicons were observed maximum number of bands (142) was observed in
ranging from 190-2650bp. The largest amplicon (AGG)6 primer, while minimum number of bands
(2650bp) was amplified by OPA-03 primer, while (91) was observed in (GA)9T primer. The
the shortest amplicon (190bp) was amplified by amplicons were observed ranging from 190-
OPN-15 and OPC-12 primer. Similarity matrix was 1635bp. Both the largest and the smallest amplicons
plotted using the Jaccards coefficient. According to 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
matrix constructed using Jaccards coefficient Out of these five primers, only two primers gave
showed that the similarity index ranged from 0.257- satisfactory and reproducible results. The banding
0.648. The mean similarity index of 0.76 was pattern of one SSR primer is shown in Fig-2 (B)
observed indicating good variability (data not and the detailing of this banding pattern is given in
shown). All the ISSR primers showed same PIC Table-4. Both these primers gave amplifications in
value (0.94). all C. roseus cultivars. Total 231 scorable bands
In SSR study, five primers were analyzed. 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.
177 bands were polymorphic, while 54 bands were coordination analysis (PCA) supported the similar
monomorphic. This resulted in total polymorphism groupings (Fig-4).
of 76.62%. Primer (GAA)7 produced more number Good correlation (r value) was observed
of bands (142) than (AAGC)3 primer (89). The among all three markers. The average
amplicons were observed ranging from 145- polymorphism using all three markers was observed
1839bp. The largest amplicon (1839bp) was 78.09% which indicates high degree of
amplified by (AAGC)3 primer, while the smallest polymorphism among all nine cultivars.
amplicon (145bp) was amplified by (GAA)7 The dendogram constructed using all three
primer. Similarity matrix was plotted using the RAPD, ISSR and SSR markers successfully
Jaccards coefficient. According to the matrix, the differentiated all nine cultivars of Catharanthus
similarity index was observed ranging from 0.261 roseus. These results demonstrate the excellent
0.736 with the mean similarity index of 0.68 power of all these three markers in studying the
indicating reasonable variability as obtained by closely related taxa. This supports the findings of
SSR markers (data not shown). Primer (GAA)7 Raina et al., (2001). They studied that RAPD and
showed higher PIC value (0.96) than (AAGC)3 ISSR fingerprints as useful genetic markers for
primer (0.91). analysis of genetic diversity, varietal identification,
The combined RAPD, ISSR and SSR and phylogenetic relationships in peanut (Arachis
analysis detected high degree of genetic variations. hypogaea) cultivars and wild species.
The highest similarity index (0.632) was observed Lalhruaitluanga and Prasad (2009) studied
between Pacifica liac and Albus with red eye. comparative results of RAPD and ISSR markers for
The least similarity index (0.287) was observed genetic diversity assessment in Melocanna
between Cooler orchid and Blue peal. The mean baccifera (Roxb.) growing in Mizoram State of
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. Fig-3: Dendogram based on UPGMA method
First kiss podka dot and cooler orchid were in showing genetic relationship among nine C. roseus
the sub cluster A2. Cluster B was further divided cultivars as revealed by RAPD, ISSR and SSR
into two sub clusters B1 and B2. cooler red, and markers, PW=patricia white, GC= grape cooler,
CR= cooler red, FKPD= first kiss podka dot, PA=
pacifica apricot were in the sub cluster B1, while
pacifica apricot, CO= cooler orchid, PL= pacifica
Blue pearl was in sub cluster B2. The principal liac, PRW= albus with red eye, BP= blue pearl.
Table-4: Detailing of SSR 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
(GAA)7 54 145-1793 142 133 09 0.96
(AAGC)3 60 452-1839 89 44 45 0.91
Total 145-1839 231 177 54 Avg=0.93
672 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 Ajibade et al., (2000) and Galvan et al.,
(98.02%) was obtained in RAPD markers than (2003) concluded that ISSR would be a better tool
ISSR markers (84.1%). However, in our present than RAPD for phylogenetic studies. Nagaoka and
investigation it was found that slightly higher Ogihara (1997) have also reported that the ISSR
polymorphism (78.94%) was observed in ISSR primers produced several times more information
markers compared to RAPD markers (78.71%) and than RAPD markers in wheat. Our study also shows
SSR markers (76.62%). Similar results were that ISSR marker is better tool than RAPD markers
observed by Parsons et al., (1997) who studied the for phylogenetic study.
genetic diversity relationship in rice using different RK Shaw et al. (2008) studied the genetic
marker systems. They observed 56% polymorphism variation in cultivars of C. roseus using RAPD and
in ISSR markers while, 50% polymorphism was ISSR markers. They observed genetic variability
observed in RAPD markers. using 18 RAPD and ISSR markers. They concluded
Moreover, Lal et al., (2010) also reported that both markers are equally potential to
that higher polymorphism (95%) was obtained in differentiate the closely related cultivars of C.
ISSR markers than RAPD (87%) and SSR (93%) roseus. In our investigation also, the high
markers. They checked the efficiency of all these discriminating power of both RAPD and ISSR
three PCR based markers in Cicer arietinum L. and markers among C. roseus cultivars was observed.
Cajanus cajan L. Millspaugh and found that higher To best of our knowledge, no earlier reports
PIC value was observed in ISSR markers (0.70) are available regarding the genetic diversity
than in RAPD (0.49) and SSR (0.61) markers. In analysis in C. roseus using all these three markers.
same manner, in our investigation also, higher PIC The polymorphism data generated can be used for
value (0.94) was observed in ISSR markers than in plant breeding, crop improvement programs and
RAPD (0.93) and SSR (0.93) markers. also might be helpful in future strategies for
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