Beruflich Dokumente
Kultur Dokumente
Authors: ABSTRACT:
Gopinath SM,
Suneetha TB,
Ashwini patil GM. Rice chaff - a polished substance extracted from Oryza sativa L. cv.
Devamallige was used as a novel substrate for the production of the fibrinolytic
enzyme. This vital enzyme is used in thrombolytic therapy, as a clot buster. The
Institution: production was done by solid state fermentation of rice chaff by Penicillium
Department of chrysogenum SGAD12, locally isolated from vegetable markets. Of the 28 strains
Biotechnology, Acharya isolated and screened, Penicillium chrysogenum SGAD12 was found to give an
Institute of Technology, inhibition zone greater than 2 mm. Hence it was identified as the potential organism
Soldevanhalli, Bengaluru- showing maximum fibrinolytic activity under specified culture conditions. Activity
560090, Karnataka, India. optimization was done under the parameters: Time, Inoculum ratio, Moisture, particle
size. The fibrinolytic activity was favourably maximized at 104 hrs, 7% (v/v) inoculum
ratio, 35-45 % (v/w) moisture content and 500m particle size.
Corresponding author:
Gopinath SM
Email:
Keywords:
gopinath@acharya.ac.in Rice chaff, Fibrinolytic enzyme, Solid state fermentation, Penicillium
chrysogenum SGAD12.
Dates:
Received: 09 May 2011 /Accepted: 13 May 2011 /Published: 03 Aug 2011
Ficus Press.
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.
50
found to produce fibrinolytic enzyme on fibrin
plate. This potential trait was used for further 40
production on solid state fermentation. Culture was 30
maintained on Czepek Dox Agar at 4C and sub 20
cultured fortnightly. The basic medium contained 10
rice chaff (variety: Devamallige) (20g), KH2PO4
0
(0.5g), MgSO4 .7H2 O (0.5g), MnSO 4 .7H2O
(0.001g), ZnSO4.7H2O (0.002g), FeSO4.7H2O -10
(0.0005g) and 9 ml water. After autoclaving at
0
16
32
48
64
80
96
2
11
Fibrinolytic Activity(IU/ml)
uninnoculated control plate showed no detectable 70
change. During initial 24 h no fibrinolytic activity 60
was seen, thereafter the enzyme activity increased 50
reaching a maximum at 104 h. With further 40
30
incubation the enzyme activity decreased.
20
Effect of Inoculum Ratio: 10
While optimization for inoculum ratio it was 0
found that 7% to 14% (v/v) (based on the volume of -10
mineral solution) elicited the best enzyme activity, 20 22 24 28 30 34 38 42 45 50 54 58
with 7 %(v/v),as adopted for this experiment,
giving the maximum result (fig 2). Any inoculum Miosture Content(%v/w)
size beyond the optimal range showed lower
activity. An inference can be drawn that larger Fig. 3. Effect of moisture level on the productivity by
inoculum sizes containing more amount of water Penicillium chrysogenum SGAD12on Rice chaff
led to decrease in the enzyme activity. (variety: Devamallige). Fermentation time: 104 hours.
Temperature: 28 C. Particle size: Mixture of all sizes.
Fibrinolytic activity(IU/ml)
12
16
20
24
28
Inoculum ratio(%v/v) 80
activity(IU/ml)
Fibrinolytic
60
Fig. 2. Effect of inoculum size on production by
Penicillium chrysogenum SGAD12 on rice chaff. 40
(variety: Devamallige) Fermentation time: 104 hours.
20
Temperature: 28 C. Particle size: Mixture of different
sizes. 0
Effect of moisture level: 75 100 150 500 1000 >1000
Water has a profound effect on productivity Particle size( m )
and hence used in limited amount in solid state
fermentation (Lonsane et al, 1992). Moisture level
between 35% - 45% (v/w) (fig3) results in the Fig. 4. Effect of substrate particle size on production
maximum enzyme production. Any value beyond by Penicillium chrysogenum SGAD12. Fermentation
this was unable to give increase in production. A time: 104 hours, temperature: inoculum size-7%
conclusion can be drawn that lower moisture level (v/v), moisture content 45 %( v/w).
leads to dry culture, sparse growth and hence
reduced production. Higher moisture concentration CONCLUSION
also creates an unsuitable environment for solid The above work indicates that Rice chaff
mycelium growth and hyphal diffusion, leading to (variety: Devamallige) can be used as a potential
less production of enzyme. substrate for production of economically important
Effect of substrate particle size: fibrinolytic enzyme with Penicillium chrysogenum
The effect of specific surface area is of high SGAD12. This substrate is easily available and
importance in solid state fermentation. Maximum economically feasible. Penicillium chrysogenum
production was obtained at a particle size of 500m SGAD12 was identified as the potential organism
showing maximum fibrinolytic activity under El-Aassar SA. 1990. Appl Microbiol Biotechnol
specified culture conditions. Time, Inoculum ratio, 33:26-30.
Moisture content, Particle size; were found to be
ideal parameters for the activity optimization. The Haber E, Quettermous T, Matsueda GR, Runge
fibrinolytic activity was favourably maximized at MS. 1989. Science 243:51-56.
104 hrs, 7% (v/v) inoculum ratio, 35-45 % (v/w)
moisture content and 500m particle size. Lonsane BK, Castaneda GS, Raimbault M,
Roussos S, Gonzalez GV, Ghildyal NP,
ACKNOWLEDGEMENT Ramakrishna M and Krishnaiah MM. 1992.
The authors are thankful to Visveswaraya Proc. Biochem. 27:259-273.
Technological University, Belgaum for their
financial assistance; and Acharya Institute of Pandey A. (1992) Prac. Biochem.
Technology for all the facilities graciously extended
to carry out the project. Sumi H, Hamada H, Tsushima H, Mihara H and
Muraki H. 1987. Birkhuser Verlag, CH-4010.
REFERENCES Experentia 43.
Astrup T and Mllertz S. 1952. Archs Biochem.
Biophys 40:346. Sun Tao Li Peng, Liu Beihui, Liu Deming and Li
Zouhu. 1997. Biotechnology Letters , Li Peng, Liu
Collen D and Lijnen HR. 1991. Blood- 78:3114- Beihui, Liu Deming and Li Zouhu . Vol 19(5):465-
3124. 467.
Effects of protein and lipid content of three artificial foods on survival and growth of
common dentex during the on-growing phase (Dentex dentex Linneaus, 1758).
Journal of Research in Biology
Authors: ABSTRACT:
Ali Ait Ali,
Azeddine Abrehouch and The purpose of this work is to highlight the effects of three different foods
Kamal Chebbaki. containing protein and lipids on the survival and growth of common dentex during the
on-growing phase.
Common dentex fingerlings weighting 5-6g were grown for one year in
polyester tanks. Three diets were used (A 1, A2 and A3), respectively, had a protein
Institution:
BP.31. Centre spcialis en content of 55%, 45%, 33% and lipid level of 10%, 15% 23%. At the end of the
aquaculture de Mdiq. experiment, fishes have reached a weight of 310.20 7.76, 406.24 11.01, 230.20
INRH. Morocco. 6.41g, respectively, for A1, A2 and A3. The respective survivals were of 60%, 80% and
88%. The specific growth rates were of 1.04, 1.13 and 1.04 while the respective food
conversion rates were of 1.45, 1.30 and 2.0. Diet containing 45% protein and 15% lipid
gave the highest specific growth rate and the lowest food conversion rate with an
intermediate survival of 80%.
Corresponding author: Results obtained during this phase are encouraged to undertake large-scale
Ali Ait Ali farming in sea cages, knowing that to transfer the fries into cages must have a weight
greater than 6g. This study showed that during one year common dentex reached
commercial size despite a thermal profile whose values have not reached the
optimum temperatures for the majority of sparidae.
Email:
a.aitali@yahoo.fr,
abraze1@yahoo.fr, Keywords:
chebbakikamal@yahoo.fr Common dentex, nutrition, protein, lipid, specific growth rate.
Web Address:
http://jresearchbiology.com/
Documents/RA0053.pdf.
Article Citation:
Ali Ait Ali, Azeddine Abrehouch and Kamal Chebbaki.
Effects of protein and lipid content of three artificial foods on survival and growth of
common dentex during the on-growing phase (Dentex dentex Linneaus, 1758).
Journal of research in Biology (2011) 4: 246-252.
Dates:
Received: 29 Jun 2011 /Accepted: 02 Jul 2011 /Published: 03 Aug 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.
Table1. Composition of the three experimental diets (A1 and A2 compositions are given following
manufacturers technical sheet). DM = dry matter; WM = wet matter.
Ingredients
Dry matter (%) 92 90 66
Fish meal, (%WM) --- --- 34
Fresh fish (%WM) --- --- 48
Sardine oil (%WM) --- --- 14
Premix vitc (%WM) --- --- 4
Crud protein (%DM) 55 45 ---
Crud lipid (%DM) 12 14 ---
Crud fiber (%DM) 1.5 1.2 ---
Ash (%DM) 11 9.8 ---
Carbohydrates(%DM) 14.5 --- ---
Vit A 5000 IU Kg-1 20000 IU Kg-1 ---
Vit D 1000 IU Kg-1 3000 IU Kg-1 ---
Vit E 180 mg Kg-1 250 mg Kg-1 ---
Vit C --- 350 mg Kg-1 ---
a
Ingredients: fish products, seed oil products and by-product set, cereal seed products, vitamins and minerals,
antioxidants : ethoxyquin.
b
Ingredients: fish meal, soybean, horse bean, pea, fish oil, rapeseed, maize gluten, rapeseed oil, weat, vitamins
and minerals, antioxidants : ethoxyquin.
c
Premix (dose kg-1): Minerals, 75% ; Phosphor, 3 4% ; Calcium 25 30%, Vitamin A, 2.000000 IU ; Vitamin
D3, 400000 IU ; Thiamine B1, 500 mg ; Riboflavin B2, 1.000 mg; Calcium pantothenate B3, 7500 mg;
Pyridoxine B6, 500 mg; Vitamin B12, 1.5 mg; Tocopherol Acetate E, 2500 mg; Nicotinic Acid PP, 10000 mg;
Cholin (Chloride), 50000 mg.
Survival
Common dentexs survival for the three
diets were of 60% for A1 diet, 80% for A2 diet and
88% for A3 diet. Mortality starts at d35 because of
flexibacteriosis disease (Fig. 2).
Growth
The initial weight of dentexs fingerlings
was of 6g. A2 diet, containing 45% CP and 15% CL
gave a best growth results (final live weight and
SGR) followed by A1 and finally A3 which gave the
lowest growth (Table 2). In early, A1 gave a high
growth compared to A2 but from d200 A2 showed
superior growth (Fig.3). Probably it should use in
first stage a protein level of 55% and use after six
months a protein level of 45% during the on-
growing phase of common dentex.
Fig. 1. Temperature and salinity during the on-
growing period
70
60 (fig. 6). The optimum protein level was 45%; lipid
50 A1 level had only a linear effect on specific growth rate
40 A2 and food conversion ratio.
30 A3
20 1.15
1
450
400 A1
30 40 50 60
350 Protein level (CP)
300 A2
Weight (g)
50
0 2
0 100 200 300 400
Duration (Days) 1.8 y = 0.003x 2 - 0.318x + 8.875
R = 1
Fig. 3. Weight growth of common dentex. Bars 1.6
indicates meansSD. There is significant difference
(P<0.01) according to Neuwman Keuls test (n=25). 1.4
1.2
Concerning weight-length relation, correlation of
Pearson was very significant (fig. 4) (n=336) 1
30 40 50 60
Diet A1 A2 A3
same species by Company et al.(1999) but for differences between fish dentex (1.5 to 98g) fed
fishes weighing between 10-20 g. Koumoundouros with extruded feed containing 45% or 50% CP.
et al. (2004) worked on fish larger but survivals Differences in the results quoted by various authors
were lowest (On 9300 fishes transferred to sea regarding the optimum protein and lipid can be
cages, only 1346 have survived after a month of explained by the energy level and protein / energy
rearing). In comparison with the results obtained ratio. During this work, the optimum energy that
during this work. gave best results was obtained with food containing
The specific growth rate (SGR) are between 21.10 and 21.80 MJ kg-1 and these results
relatively low, the maximum SGR obtained during are similar to those cited by Espinos et al, 2003 and
this work (1.13) is lesser than the SGR obtained for Tibaldi et al, 1996. During this work, the CP/GE
the same species by Ait Ali et al, 2008 (1.7% 2.4% successful is 20.63 g MJ-1; lower values have
and 2.7% d-1) but to fish smaller, they are also yielded poor results. Espinos et al. (2003) reported
lower than those obtained by Company et al, 1999 that the values of CP/GE ratio lower than 21 g MJ -1
and Cardenete et al, 1997a. These results are gave lowest growth, the same remarks were cited
identical to those reported by Tibaldi et al, 1996. by Tibaldi et al, 1996 for values of CP/GE ratio
However, they are higher than those cited by lower than 23 g MJ-1. The protein and lipid content
Cardenete et al, 1997d. Some lower SGR were which gave best results during this work (45 CP/15
obtained in other species such as sea bass: 07-08% CL) were identical to the results obtained by
d-1, 0.4-0.5% d-1 (Dias et al, 1998; Mtailler et al, Company et al, 1999 (46 CP/17 CL). Recent work
1981) and sea bream. More optimum temperature has confirmed that the best protein level for
for this species is not achieved and more experience optimum growth of dentex juveniles seems to be
in the fishes was large (in our case up to 400g) around 50% and 12% (Toms et al, 2009).
more SGR is down accordingly.
The food conversion ratio (FCR), whose REFERENCES
values are included in this work between 1.30-2.0 Abellan E, Garcia-Alcazar A, Arizcun M,
are similar to those contained by Tibaldi et al, 1996 Delgado J and Martin P. 1997. Experiencias
and in the same game values (1.3-1.8) reported by preliminares sobre reproduction y cultivo del
Ait Ali et al, 2008 (1.06-1.14) by Company et al, denton (Dente dentex L.) In : Actas VI Congresso
1999 and (1.5-2.2) by Espinos et al, 2003. Similar Nac. Acuicult. De Costa J, Abellan E, Garcia B,
results were obtained for other fish species such as Ortega A, Zamora S. (eds), Cartagena. Ministerio
sea bass: 1.4-2.2 (Perez et al, 1997), 1.3-1.7 (Diaz de Agricultura, Pesca y alimentation, Madrid. 477-
et al, 1998), 1.5-1.6 (Hidalgo and Alliot 1988) and 482.
seabream: 1.4-1.6 (Vergara et al, 1996), 1.1
(Calduch-Giner et al, 1998). The optimum growth Ait Ali A, Rharbi N, Abrehouche A, Nhhala H.
was obtained with a protein content of 45%, in spite 2008. Effects of lipids and (n-3) highly unsaturated
of lipid levels (Fig.5). The protein content of 55% fatty acids compositions of three artificial foods on
and 33% lipid gave lower results in terms of SGR survival, growth and body composition of common
and final weight. Company et al. (1999) obtained dentexs fingerlings (Dentex dentex, L). African
similar growth using two diets protein content of Journal of Biochemistry Research 2:001-007.
45% and 55%, Jover et al, 1998 did not obtain
Calduch-Giner JA, Company R, Kaushik S and Dias J, Alvarez MJ, Diez A, Arjel J, Corraze G,
Perez-Sanchez J. 1981. Risk and benefits of lipid Bautista GM and Kaushik SJ. 1998. Regulation
enriched diets in Gilthead seabream (Sparus aurata), of hepatic lipogenesis by dietary protein/energy in
in: VIII Int. Symp. Nutrition and Feeding of Fish, juvenile European sea bass (Dicentrarchus labrax).
Las Palmas, Spain 32. Aquaculture 161:169-186.
Cardenete G, Abellan E, Hidalgo MC, Skalli A Efthimiou S, Divanach P and Rosenthal H. 1994.
and Arizcum M. 1997. Relation proteina en dietas Growth, food conversion and agonistic behaviour in
para juveniles de denton (Dentex dentex), in: De common dentex (Dentex dentex) juveniles fed on
Costa J., Abellan E. Garcia G.B., Ortega R.A., pellet moist and dry diets. Aquat. Living Ressour.
Zamora N.S. (Eds), Actas del VI Congresso 7:267-275.
Nacional de Acuicultura, Cartagena, Spain. 581-
586. Espinos FJ, Tomas A, Prez LM, Balasch S and
Jover M. 2003. Growth of dentex fingerlings
Cardenete G, Abellan E, Hidalgo M.C, Skalli A (Dentex dentex) fed diets containing different levels
and Garcia-Alcazar A. 1997b. Utilisation of protein and lipid. Aquaculture 218:479-490.
digestiva y nutritiva de niveles de crecientes de
carbohidratos en dieta por el Dentn (Dentex Hidalgo F, Alliot E. 1988. Influence of water
dentex). Resultados preliminares. Proceedings of temperature on protein requirement and protein
the Six National Congress on Aquaculture, utilization in juvenile sea bass, Dicentrarchus
Cartagena, Murcia, Spain. 587-592. labrax. Aquaculture 72:115-129.
Cardenete G, Abelln E, Skalli A and Massuti S. Jover M, Riera F, Grau A, Pastor E, Espinos F.J
1997a. Feeding Dentex dentex with dry diets: and Perez L. 1998. Resultados preliminares de
Growth response and diet utilisation. Cahiers Opt. crecimiento del denton (Dentex dentex) alimentado
Mediterranennes 22:141-151. con cuatro piensos extrusionados de differente
relation proteine/lipidos. Aquat Electron. J.
Cardenete G, Skalli A, Hidalgo MC, Palma MC University of Zaragoza, Spain.
and Massuti S. 1997d. Variation de los niveles
proteico y lipidico de la dieta. Effecto sobre el Katavic I, Grubisic L and Skakelia, N. 2000.
crecimiento y utilization nutritiva de la misma por Growth performance of pink dentex as compared to
el denton (Dentex dentex), in: De Costa J., Abellan other four sparid reared in marine cages in Croatia.
E., Garcia G.B., Ortega R.A., Zamora N.S. (Eds), Aquacult. Int. 8:455-461.
Actas del VI Congresso Nacional de Acuicultura,
Cartagena, Spain. 559-564. Koumoundourous G, Carrilllo J, Divanach P
and Kentouri M. 2004. The rearing of common
Company R, Calduch-Ginner JA, Prez-Sanchez dentex Dentex dentex (L.) During the hatchery and
J and Kaushik SJ. 1999. Protein sparing effect of on-growing phases. Aquaculture 240:165-173.
dietary lipids in common dentex (Dentex dentex): a
comparative study with sea bream (Sparus aurata) Metailler R, Aldrin JF, Messager JL, Mevel G
and sea bass (Dicentrarchus labrax). Aquat. Living and Stephan G. 1981. Feeding of European sea
251 Journal of Research in Biology (2011) 4: 246-252
Ait Ali et al.,2011
bass Dicentrarchus labrax: role of protein level and Sokal RR, Rohlf FG. 1981. Biometry. The
energy source. J. World. Mari. Soc. 12:117-118. principles and practice of statistics in biological
research. 2nd edn. WH Freeman, New york.
Perez L, Gonzalez H, Jover M, Fernandez-
Carmona G. 1997. Growth of European sea bass Tibaldi E, Beraldo P, Volpelli L.A and Pinosa M.
fingerlings (Dicentrarchus labrax) fed extruded 1996. Growth response of juvenile dentex (Dentex
diets containing varying levels of protein, lipid and dentex L.) to varying protein level and protein to
carbohydrate. Aquaculture 156:183-193. lipid ratio in practical diets. Aquaculture. 139:91-
99.
Riera F, Pastor E, Grau AM, Pou S, Grau A and
Massuti E. 1993. Experiencias en el cultivo del Toms A, Martnez-Llorens S and Jover M.
denton. In: Actas IV Congresso Nac. Acuicult., 2009. The effect of dietary soybean meal on
Vilanova de Aroussa, 1993, Cervino A., Landin A., growth, nutrient utilization efficiency, and
de Coo A., Guerra A. and Torre, M. (eds), Centro digestibility of juvenile common dentex, Dentex
de Invesigaciones Marinas , Pontoverda, Spain. 143 dentex (Actinopterygii: Perciformes: Sparidae).
-148. Acta Ichthyol. Piscat. 39:19-25.
Riera F, Pastor E, Grau AM, Pou S, Grau A, Tulli F, Tibaldi E. 1997. Changes in amino-acids
Massuti E, Valencia JM, Palmer G and Pou S. and essential fatty acids during early larval rearing
1995. Resultados preliminares del engorde del of dentex. Aquac. Int. 5:229-236.
denton (Dentex dentex), en jaulas flotantes con
differenttes tipos de dietas. Proceeding of the Fifth Vergara JM, Robaina L, Izquierdo MS and De
National Congress on Aquaculture, San Carles de la la Higuera M. 1996. Protein sparing effect of lipids
Rapita, Tarragona, Spain. 111-606. in diets for fingerlings of gilthead seabream. Fish.
Sci. 62:624-628.
Rueda FM, Martinez FJ. 2001. A review on the
biology and potential aquaculture of Dentex dentex.
Rev. Fish Biol. Fish. 11:57-70.
Authors: ABSTRACT:
Rajmohan D and
Logankumar K.
Leaf extract of the species, Chromolaena odorata was evaluated for the egg
Institution:
hatchability, larvicidal and pupicidal activity of mosquito, Aedes aegypti under the
PG and Research
Department of Zoology, room temperature in the laboratory. Dosage value as expressed in ppm was 10 to 140
Kongunadu Arts and for Aedes aegypti. A relationship was observed between the plant extract doses and
Science College, percentage mortality. The percentage of egg hatchability, larval and pupal mortality
Coimbatore-641 029, were found to be increased with increase in the dosage. Based on the probit analysis,
Tamilnadu, India. the LC50 value of egg (99.15), I instar (42.24), IV instar (101.49) and pupae (121.57)
were hence assumed.
Corresponding author:
Rajmohan D
Email: Keywords:
rajmohandevadass@gmail.com Aedes aegypti, Chromolaena odorata, LC50.
Dates:
Received: 20.Jul 2011 /Accepted: 25 Jul 2011 /Published:
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.
Table 1. Effect of crude sample of Chromolaena odorata against the egg hatchability of Aedes aegypti.
Concentration (ppm)
No of eggs
70 80 90 100 110
exposed
h uh h uh h uh h uh h uh
20 17 3 14 6 11 9 6 14 3 17
20 16 4 12 8 10 10 7 13 2 18
20 17 3 11 9 10 10 5 15 3 17
20 18 2 13 7 11 9 4 16 1 19
20 15 5 11 9 10 10 5 15 4 16
2.6
Mean 16.6 3.4 12.2 7.8 10.4 9.6 5.4 14.6 17.4
1.1
SD 1.14 1.14 1.30 1.30 0.55 0.55 1.14 1.14 1.14
4
Mean % 83 17 61 39 52 48 27 73 87
13
h - hatched, un - unhatched.
Table 2. Effect of crude sample of Chromolaena odorata against the I Instar larvae of Aedes aegypti.
No of larvae Concentration (ppm)
exposed 10 20 30 40 50
Alive dead Alive dead Alive dead Alive dead Alive dead
20 19 1 17 3 12 8 11 9 2 18
20 18 2 15 5 11 9 10 10 4 16
20 19 1 16 4 12 8 10 10 3 17
20 16 4 17 3 13 7 12 8 4 16
20 17 3 15 5 11 9 10 10 5 15
Mean 17.8 2.2 16 4 11.8 8.2 10.6 9.4 6.4 13.6
SD 1.30 1.30 1.00 1.00 0.84 0.84 0.89 0.89 1.14 1.14
Mean % 89 11 80 20 59 41 53 47 32 68
Table 3. Effect of crude sample of Chromolaena odorata against the IV instar larvae of Aedes aegypti.
No of Concentration (ppm)
larvae
exposed 80 90 100 110 120
Alive dead Alive dead Alive dead Alive dead Alive dead
20 19 1 15 5 11 9 8 12 3 17
20 18 2 12 8 10 10 9 11 2 18
20 16 4 11 9 10 10 8 12 1 19
20 19 1 13 7 11 9 7 13 2 18
20 17 3 12 8 12 8 6 14 4 16
Mean 17.8 2.2 12.6 7.4 10.8 9.2 7.6 2.4 2.4 17.6
SD 1.30 1.30 1.52 1.52 0.84 0.84 1.14 1.14 1.14 1.14
Mean % 89 11 63 37 54 46 38 62 12 88
Aedes aegypti was found to be 99.15, 42.24, 101.49 Kalyana Sundaram, 2004; Sun et al., 2006) they are
and 121.57 respectively (Fig.1). These results are in more eco-friendly. The results of the present study,
agreeing with the earlier findings made by many indicate that the leaf extract of the weed species,
workers with botanicals for various properties (for Chromolaena odorata caused low percentage of
oviposition avoidance, larvicidal, Halawa, 2001; egg hatchability and high percentage of larval and
Saleh, 1995, adulticidal, Choochote et al., 2004 and pupal mortality. Hence the large biomass of C.
repellent activities, Choochote et al., 2004; Prakash odarata available in Southern India can be used by
et al., 2000). As the botanical insecticides for the pharmacological industries to obtain effective
including the extract of C. odarata are repellent to control mosquito population is an
biodegradable and harmless to the environment, ecofriendly manner.
pest specific and relatively harmless to non-target
organisms (Su and Mulla 1998; Sivagnaname and
Table 4. Effect of crude sample of Chromolaena odorata against the pupae of Aedes aegypti.
No of Concentration (ppm)
larvae
100 110 120 130 140
exposed
Alive dead Alive dead Alive dead Alive dead Alive dead
20 18 2 15 5 12 8 9 11 5 15
20 19 1 14 3 10 10 8 12 3 17
20 18 2 11 9 10 10 9 11 2 18
20 17 3 12 8 10 10 6 14 3 17
20 16 4 11 9 11 9 7 13 4 16
Mean 17.6 2.4 12.6 6.8 10.6 9.4 7.8 12.2 3.4 16.6
SD 1.14 1.14 1.82 2.68 0.89 0.89 1.30 1.30 1.14 1.14
Mean % 88 12 63 34 53 47 39 61 17 83
Table 5. 24 hours LC50 values (ppm) and their 95 % Fiducial (upper and lower) regression equation and Chi-square (c2)
values of the crude extract of Chromolaena odorata for the different developmental stages of Aedes aegypi.
95% Fiducial limit
LC50 (ppm)
Stages c2 X SD SE
(ppm)
Upper Lower
Egg 99.15 103.74 95.65 2.57 52.8 24.79 3.88
I instar 42.24 45.58 39.75 6.66 30.2 13.22 11.52
IV instar 101.49 105.38 107.61 4.58 48.8 25.63 5.25
Pupa 121.57 125.43 117.14 4.25 47.4 24.02 3.11
100
90
y = 1.74x - 103.8
Percentage mortality
80
70
60
50
40
`
30
20
10
Concentration
Egg I Instar
100 90
90 80 y = 1.69x - 155.4
80 y = 1.79x - 130.2
Percentage mortality
70
Percentage mortality
70
60
60
50
50
40
40 ` `
30
30
20
20
10
10
0
0
40 60 80 100 120 140 160
0 20 40 60 80 100 120 140
Concentration
Concentration
IV Instar Pupae
Fig 1. Regression line of log concentration of Chromolaena odorata crude extract vs. percent egg hatchability,
mortality of larvae and puape of Aedes aegypti.
Saleh EH. 1995. Effect of some botanical extracts different strains of Culex pipiens pallens. J. Med.
as potential insecticides for the control of some Entomol., 43:258-261.
mosquitoes in Egypt, 1995. (Ph.D., Dept. Entomol.
Fac. Sci. Cairo. Univ., Egypt.
Authors: ABSTRACT:
Raut KS*, Shinde SE**,
Pathan TS*** and
Sonawane DL**.
The present study deals with the assessment of water quality of the Ravivar
Institution:
Peth Lake, Dist. Beed Marathwada Region, India. The physico-chemical characteristics
*Department of Zoology,
L.L.Deshmukh Mahila were studied and analyzed during January December of the year 2004. Seasonal
College Parli -V. variations at three different sampling sites of the Ravivar Peth Lake were observed
**Department of Zoology, Conductivity, pH, Total Dissolved Solids (TDS), Total Suspended Solids (TSS), Nitrate,
Dr. Babasaheb Ambedkar Sulphate and Phosphate were studied at these studies. The results revealed that the
Marathwada University, condition of this lake in different seasons showed fluctuations in physico-chemical
Aurangabad - 431004, India. parameters and showed pollution status of this lake.
*** Department of Zoology
Kalikadevi Arts, Commerce
and Science College, Shirur
(KA) Dist. Beed (M.S.)
India.
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cited.
maximum pH was recorded in summer 8.540.61 In the present investigation the higher values
and minimum was in winter 7.510.45 with slight of TDS in monsoon may be due to surface runoff,
increase in monsoon 8.050.38. The overall mean precipitation and decaying matter from catchments
was 8.030.64 (Table 1). area. Salve and Hiware, (2006) reported seasonal
In the present investigation, the maximum analysis and stated that low total dissolved solids
pH was recorded in summer may be due to recorded in winter season while maximum value in
decreased volume of water by evaporation and monsoon due to addition of solids from surface run
monsoon and minimum in winter season may be off in Wanparakalpa reservoir, Nagapur near Parali
due to short day length and decrease in Vaijanath Dist. Beed, Maharashtra.
photosynthetic activity. Salve and Hiware, (2006) Total Suspended Solids (TSS)
reported the maximum pH in summer and minimum Total suspended solids are the cause of
in winter with slight increase in monsoon. suspended particles into the water body influences
Yeole and Patil, (2005) reported the pH turbidity and transparency.
values in the range of 7.0 to 9.5 in Yedashi Lake. Total suspended solids were ranged from
Bai, (1989) recorded the pH of polluted water in the 35.1 to 182.3 mg/l. Total suspended solids were
range of 8.0 to 9.0. Pawar and Pulle, (2005) maximum during monsoon 146.320.87 mg/l and
observed the pH in range of 7.0 to 8.7 and stated were minimum during winter 70.0122.23 mg/l.
that the pH of water is important for the biotic The overall mean Total suspended solids were
communities because most of the plant and animal 99.7940.08 mg/l (Table 1).
species can survive in narrow range of pH from In the present investigation, the high values
slightly acidic to slightly alkaline condition. of total suspended solids during monsoon may be
Total Dissolved Solids (TDS) due to siltation, deterioration and heavy
These are composed of inorganic salts like precipitation. Khabade et al, (2002) recorded
calcium, magnesium, potassium, sodium, maximum TDS during monsoon and minimum
bicarbonates, chlorides, sulfates and some heavy during winter and summer at Lodhe water reservoir,
metal compounds; besides organic matter in small Tasgaon. Khanna and Bhutiani, (2003) reported
quantity also contributes the amount of total maximum TDS in monsoon, moderate in summer
dissolved solids in water. and minimum in winter, which supports the
Total dissolve solids were ranged for 493.4 findings of present observations under study.
to 942.3 mg/l. Total Dissolve Solids were Nitrate
maximum during monsoon 840.377.55 mg/l and Nitrogen is less soluble in water than
were minimum during winter 564.9359.10 mg/l. oxygen. But as it constitutes 78% of atmosphere, it
The overall mean Total Dissolve Solids were still accounts for 65% of the dissolved gases at
700.86128.31 mg/l (Table 1). equilibrium. Nitrogen is important as it is a
necessary element in the structure of protein,
Table-1. Seasonal variations in physico-chemical parameters of Ravivar Peth Lake, Dist. Beed Marathwada
Region, India. (During January - December 2004).
chlorophyll, RNA and DNA etc. It is essentially there is an entry of detergents in the water body and
required by all living organisms, being a necessary less water quantity and during summer season the
element of biochemical substances. relatively low level of Phosphate have been
The nitrate ranged from 8.3 to 21.9 mg/l. reported which may be attributed to abundance of
Nitrate values were maximum during winter Phytoplanktons.
18.733.31 mg/l and minimum during monsoon Saikh and Yeragi, (2004) have reported that,
12.174.50mg/l. The overall mean was 15.394.88 increase in phosphate is a result sewage
mg/l (Table 1). contamination record. Urban water bodies subjected
Johnson and Kauser, (2004) stated that to pollution from domestic sewage exhibit high
nitrate in piped municipal water remained constant levels of phosphate and show all the signs of
in summer and monsoon 0.2 mg/l. but increased in eutrophication Adarsh et al, (2006).
winter to 0.9 mg/l. High values of nitrates in winter
may be due to high rain fall observed during winter CONCLUSION
season 665 mm in October 2004 and 902 mm in The present study show detailed physico-
October 2004 and 2005. The increased Nitrate value chemical characteristics and quality of water in
due to Lake Runoff, land drainage and input of Ravivar Peth Lake, Dist. Beed Marathwada Region,
fertilizers from adjacent and agricultural fields and India.
oxidation of ammonia similar results have been The summer, monsoon and winter seasons
reported by Anbazhagan, (1988). shows different seasonal fluctuations in various
Sulphate physico-chemical parameters.
The most abundant form of sulphur is anion Data indicated that among the physico-
sulphate (So42). Sulphate is ecologically important chemical parameters of the lake water, the level of
for the growth of plants and its short supply may Conductivity, pH, TDS, TSS, nitrate and sulphate
inhibit the development of planktons. Sulphur is were above the permissible limits prescribed by ISI
also important in protein metabolism. and WHO for drinking water. So the lake water is
Sulphate ranged from 20 to 41.71 mg/l. The not suitable for drinking purpose.
higher values of sulphate were recorded in monsoon It is being polluted by different sources like,
36.563.22 mg/l and lower in summer 29.177.72 municipal wastes, agricultural wastes, domestic
mg/l. The overall mean was 31.956.28 mg/l (Table wastes etc.
1). The continuous biomonitering of Ravivar
In the present investigation, the Sulphate Peth Lake is needed as it affects the flora and fauna
values were maximum during monsoon and of the lake.
minimum during winter. Maximum Sulphate It can be concluded that physico-chemical
concentration during monsoon may be due to the parameters are important to determine the quality of
dilution and utilization of Sulphate by aquatic aquatic environment.
plants. However, the low Sulphate concentration
was noted during winter may be due to ACKNOWLEDGMENT
biodegradation and low water level. Similar results The authors are thankful to Head, Dept of
have been reported by Reddy et al, (2009); Zoology, Dr. Babasaheb Ambedkar Marathwada
Telkhade et al, (2008). University, Aurangabad India for providing
Phosphate laboratory and library Facilities and also thankful to
In water bodies phosphate occurs both in its principal, Dr. R. J. Parlikar madam for kind
inorganic and organic forms as organic assistance and all society members of Late L. D.
phosphorous and orthophosphate, Phosphate plays a Shikshan Prasarak Mandal; Parli Vaijnath Dist.
dynamic role by acting as the limiting nutrient Beed.
presence of phosphate in water and waste water
analysis has a great significance. REFERENCES
Phosphate concentration ranged from 0.23 to Adarsh Kumar, Qureshi TA, Alka Parashar and
0.88 mg/l. The phosphate values were maximum Patiyal RS. 2006. Seasonal variations in Physico-
during monsoon 0.770.08 mg/l and minimum chemical characteristics of Ranjit Sagar reservoir,
during winter 0.380.13 mg/l. The overall mean Jammu and Kashmir. J. Ecophysiol . Occup. Hlth.,
was 0.530.21 mg/l (Table 1). Maximum during 6.
monsoon might be due to the washing activities,
261 Journal of Research in Biology (2011) 4: 258-262
Raut et al.,2011
Anbazhagan P. 1988. Hydrobiology and benthic Pejaver Madhuri and Minakshi Gurav. 2008.
ecology of kodiokkarai castal an actuary southeast Seasonal variations of zooplanktons in Nirmalya
coast of India. Ph.D.thesis,Annamali University, (religious refuges) enclosure of Kalawa Lake,
India. 208. Thane, Maharashtra. J. Aqua. Biol., 23(1):22-25.
Anitha G. 2002. Hydrography in relation to benthic Reddy Vasumathi K, Laxmi Prasad K, Swamy
macro invertebrates in Mir Alam Lake Hyderabad, M and Ravinder Reddy T. 2009. Physico-
A.P. Indian. Ph.D. Thesis submitted to Osmania chemical parameters of Pakhal Lake of Warangal
University, Hyderabad. district andhra Pradesh, India. J. Aqua. Biol., 24
(1):77-80.
APHA. 2005. Standard methods for the
examination of water and waste waters, 21st Edn., Saikh N and Yeragi SG. 2004. Some
Washington. DC. USA. physicochemical aspects of Tansa River of Thane
district Maharashtra J. Aqua. Biol., 19(1):99-102.
Bai, MM. 1989. Pollution in polar River; A
preliminary assessment of various physico- Salve BS and Hiware CJ. 2006. Studies on water
chemical parameters. In: Environmental impact on quality of Wanparakalpa Reservoir, Nagapur, near
Biosystems. Eds Proceedings (Vol. 2): of the 10th Parli Vaijnath, dist. Beed, Marathwada region. J.
annual seminar on "Environmental impact on Aqua. Biol., 21(2):113-117.
Biosystems" at Loyala College, Madras 13-17.
Sharma KK, Nitasha Sawhney and Sarbjeet
Chandrasekhar SVA and Kodarkar MS. 1996. Kour. 2007. Some limnological investigations in
Biodiversity of zooplankton in saroor nagar lake, Ban Ganga stream, Katra, Jammu and Kashmir
Hyderabad. J. Aqua, Biol., 9(1 and 2):30-33. State. J. Aqua. Biol., 22(1):105-109.
Dhere RM and Gaikwad JM. 2006. Physico- Shinde SE, Pathan TS, Raut KS, More PR and
chemical characteristics of Karpara Reservoir dist. Sonawane DL. 2010. Seasonal variationss in
Parbhani, Maharashtra. J. Aqua. Biol., 21(2):86-88. physico-chemical characteristics of Harsool-
Savangi Dam, district Aurangabad, India. The
Ingole SB, Pawale RG and Wavde PN. 2009. Ecoscan., 4(1):37-44.
Water quality studies on Majalgaon dam, Beed
district, Maharashtra, J. Aqua. Biol., 24(1):71-76. Telkhade PM, Dahegaonkar NR, Zade SB and
Lonkar AN. 2008. Quantitative analysis of
Johnson Mary Easter and Rana Kauser. 2004. Phytoplanktons and zooplanktons of Masala Lake,
Chemical and microbial quality of different types of Masala; Dist. Chandrapur, Maharashtra. Envirn.
drinking water of Hyderabad, HiTech city. A.P. Cosr. Jou., 9(1 & 2):37-40.
India. J. Aqua. Biol., 19(1):93-97.
Trivedy RK and Goel PK. 1984. Chemical and
Khabade SK, Mule MB and Sathe SS. 2002. biological methods for water pollution studies,
Studies on physico-chemical parameters of Iodhe Environmental Publications, Karad, India. 1-122.
water reservoir from Tasgaon Teshil (Maharastra):
Indian. J. Environ and Ecoplan., 6(2):301-304. Yeole SM and Patil GP. 2005. Physico-chemical
status of Yedshi Lake in relation to water pollution,
Khanna DR and Bhutiani R. 2003. Ecological J. Aqua. Biol., 20(1):41-44.
status of Sitapur pond at Hardwar (Uttaranchai),
India. Indian J. Environ and Ecoplan., 72(1):175-
178.
Authors: ABSTRACT:
Sandeep R. Rathod and
Gulab D. Khedkar
India is very rich in biodiversity, India supports about 10% of the worlds
biological diversity, with just 2% of world land area. Therefore India is the seventh
richest biodiversity in the world. The India has Himalaya Mountain, Eastern Ghat,
Institution:
Aquaculture Research Western Ghats Indo-gangetic plain, Deccan plateau, desert, coasts are all present in it.
laboratory, In India many type of river also present, one of the river Godavari River is the second
Deparment of Zoology, largest river in India. Fish biodiversity studies were undertaken during January-2008 to
Dr. Babasaheb Ambedkar Decemeber-2008 to census and commercially important fishes in the Godavari River.
Marthwada The present paper deals with the variety and abundance of fresh water fishes in
University, Aurangabad. Godavari River with reference to the elevation, latitude and longitude. Two sampling
(Maharashtra) India. points were selected with different elevation latitude and longitude. The Results of
the present investigation reveal the occurrence of 53 fish species belonging to 9
orders, 21 families and 37 genera.
Among the collected species two sampling site species composition were so
Corresponding author: different. Species composition in Gangapur dam is lower than the Rajahmundry dam.
Sandeep R. Rathod In species composition order Cypriniformes was most dominant to both sampling site.
Email: Keywords:
rathod.sr@gmail.com Elevation, latitude and longitude Fish biodiversity, Percent wise compositions,
Godavari River.
Dates:
Received: 07 Aug 2011 /Accepted: 11 Aug 2011 /Published: 16 Aug 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.
Table 1: - The fish biodiversity of Godavari River in Gangapur dam during January 2008 December 2008.
Family Species Native/Introduce Species
abundance
1 Notopteridae Notopterus notopterus Native ++
2 Cyprinidae Puntius ticto Native +++
3 Cyprinidae Puntius fraseri Native +
4 Cyprinidae Puntius filamentosus Native -
5 Cyprinidae Puntius dorsalis Native -
6 Cyprinidae Salmostoma navacula Native +++
7 Cyprinidae Chela laubuca Native -
8 Cyprinidae Rasbora Daniconias Native +
9 Cyprinidae Thynicthys sandkhol Native +
10 Cyprinidae Osteobrama vigorsii Native +
11 Cyprinidae Catla catla Native ++
12 Cyprinidae Cyprinus carpio carpio Introduced ++
13 Cyprinidae Garra mullya Native ++
14 Balitoridae Nemachilus Moreh Native +
15 Balitoridae Nemachilus botia Native +
16 Chanidiae Chanda nama Native +++
17 Chanidiae Parambassis ranga Native +
18 Gobiidae Glossogobius giuris giuris Native ++
19 Channidae Channa marulius Native +
20 Channidae channa punctatus Native +
21 Channidae Channa oriantalis Native +
22 Bagiridae Aoirichthys aor Native ++
23 Bagiridae Aoirichthys cavasius Native +
24 Siluridae Wallago attu Native -
25 Heteropneustidae Heteropneustes fossilis Native -
26 Mastacembelidae Macrognathus pancalus Native ++
Journal of Research in Biology (2011) 4: 269-275 271
Rathod et al.,2011
Table 2: - The fish biodiversity of Godavari River in Rajahmundri dam during January 2008 December 2008.
Sr. no Family Species Native/Introduce Species
abundance
1 Notopteridae Notopterus notopterus Native +
2 Notopteridae Notopterus chitala Native +
3 Anguillidae Angulla bengulensis bengalenss Native -
4 Clupeidae Hilsa ilisha Native +
5 Cyprinidae Salmostoma navacula Native ++
6 Cyprinidae Cyprinus carpio carpio Native ++
7 Cyprinidae Osteobrama vigorsii Native +++
8 Cyprinidae osteobrama dayi Native -
9 Cyprinidae Thynnichthys sandkhol Native +
10 Cyprinidae Cirrhinus mirgala Native +
11 Cyprinidae Cirrhinus reba Native +
12 Cyprinidae Puntius Vittatus Native -
13 Cyprinidae Puntius ticto Native +
14 Cyprinidae Puntius fraseri Native +
15 Cyprinidae Puntius filamentosus Native -
16 Cyprinidae Catla catla Native ++
17 Cyprinidae Labeo rohita Native +
18 Cyprinidae Labeo calbasu Native ++
19 Cyprinidae Labeo arzia Native ++
20 Cobitidae Lepidocephalus guntea Native ++
21 Heteropneustidae Heteropneustes fossilis Native -
22 Bagiridae Mystus seenghala Native +
23 Bagiridae Mystus bleekeri Native ++
24 Bagiridae Mystus cavasius Native ++
25 Bagiridae Aorichthys aor Native +
26 Bagiridae Rita rita Native +
27 Siluridae Wallago attu Native ++
28 Siluridae Ompak bimaculatus Native +++
29 Pangasiidae Pangasius pangasius Native +
30 Sisoridae Bagarius bagarius Native ++
31 Schilbeidae Silonia children Native +
32 Nandidae nandus nandus Native -
33 Gobiidae Glossogobius giuris giuris Native ++
34 Chanidiae Chanda nama Native ++
35 Chanidiae Parambassis ranga Native ++
36 Channidae Channa marulius Native ++
37 Channidae channa striatus Native ++
38 Channidae channa punctatus Native ++
39 Channidae channa orentalis Native ++
40 Cichlidae Etroplus suratensis Native +
41 Cichlidae Oreochromis mossambica introduced +
42 Anabantidae Anabus testudineus Native -
43 Mastacembelidae Mastacembelus armatus Native +
44 Mastacembelidae Macrognathus pancalus Native +++
45 Mastacembelidae Macrognathus aculeateda Native +
46 Beonidae Xanthodon cansula Native ++
47 Mugilidae Rhinomugil corsula Native +
important tributary of the river Chenab J. and K Sakhare VB and Joshi PK. 2002. Ecology of
state. J. Aqua. Biol., 18(2):61-68. Palas NI Legaon reservoir in Osmanabad district
Maharashtra. J. Aqua. Biol., 18(2):17-22.
Ehrlich PR and Wilson EO. 1991. Biodiversity
studies science and policy. Science 253:758-762. Sakhare VB and Joshi PK. 2004. Present status of
reservoir fishery in Maharashtra. Fishing Chimes 24
Hamilton-Buchanan. 1822. An account of the (8):56-60.
fishes found in the river Ganges and its branches,
Edinburg and Landon. Viii (405):39. Sakhare VB and Joshi PK. 2003. Water quality of
Migni (Pangaon) Reservoir and its significance to
Jerdon TC. 1849. On the freshwater fishes of fisheries ABN-008. Nat. Conf. Recent Trends
southern India, Madras. J. Lit. sci., 15:302-346. Aquat. Biol., 56.
Jayaram KC. 1995. The Krishna River system: A Sugunan VV and Yadava YS. 1992. Hirakhud
bio resource study. Rec. 2001.surv.India occ papers, reservoir strategies for fisheries development.
No-160:167, 8 pls with 30 Col. Photographs. Bulletin CIFRI, Barrackpore, India. 66.
Jayaram KC. 1999. The fresh water fishes of the Talwar PK and Jhingran A. 1991. In land fishes
Indian Region, Narendra Publishing house. Delhi- of India and adjacent countries oxford and I B H
551. publishing co. New Delhi. 1, 2.
Klinger RC. 2003 The proceeding book turning Yadav BE. 2005. Pisces, Fauna of Nathsagar
tide: the eradication of invasive species 141-154 pp. Wetland, Wetland Ecosystem Series. Zool. Surv.
India. 7:137-143.
McClelland J. 1839 Indian cyprinidae 19, Asiatic
Researches, Calcutta, Bishop College Press. 217- http://www.physorg.com/news140269006.html
468. Maths model helps to unravel relationship between
nutrients and biodiversity.
Mahapatra DK. 2003. Present status of fisheries of
Hirakund reservoirs, Orissa. Fishing chimes. 22 http://www.Fish base organization (2004).
(10&11):76-79.
Authors: ABSTRACT:
Godishala Vikram1,2,
Kairamkonda An efficient and reproducible protocol for organogenesis from cotyledon
Madhusudhan2, explants in cultivated tomato (Solanum lycopersicum L.) cv S-22 is reported. The
Kagithoju Srikanth2,
cotyledon explants of 10-12 days old excised from in vitro grown seedlings were
Mangamoori
cultured on MS medium supplemented with 0.5-5.0 mg/L BAP as a sole growth
Laxminarasu 1 and
Nanna Rama Swamy 2. regulator and also in combination with 0.1 mg/L IAA (Indole-3-acetic acid). Highest
percentage of response for callus induction was recorded in cotyledon explants at 3.0
mg/L BAP where as multiple adventitious shoots were formed at 0.1 mg/L IAA + 2.5-
5.0 mg/L BAP containing medium. Shoots obtained were transferred on to MS
Institution: medium augmented with 0.2-1.0 mg/L GA3 + 3.5 mg/L BAP for shoot elongation. The
1. Center for Biotechnology, medium supplemented with 0.6 mg/L GA 3 in combination with 3.5 mg/L BAP showed
Jawaharlal Nehru the maximum percentage of enhancement of shoot elongation. For in vitro rooting,
Technological University, elongated micro-shoots were transferred onto MS medium supplemented with
Hyderabad, India. 0.5mg/L Indole-3-acetic acid (IAA) / Indole butyric acid (IBA) / Napthalene acetic acid
2. Department of (NAA). Profuse rhizogenesis was observed at 0.5 mg/L IAA compared to NAA / IBA. The
Biotechnology, Kakatiya regenerated plants were acclimatized in the culture room and maintained in the green
University, Warangal 506 house and transferred to the field. These plants were found to be normal and similar
009, India. to the donar plant. Thus, an efficient and reproducible protocol has been developed in
cultivated tomato cv S-22 which is genotype dependent. This protocol can be used for
Agrobacterium tumefaciens mediated genetic transformation in tomato cv S-22 .
Corresponding author:
Nanna Rama Swamy
Keywords:
Solanum lycopersicum, cotyledon explants, organogenesis, in vitro rooting.
Email: Abbreviations:
swamynr.dr@gmail.com IAA-Indole-3-acetic acid ; IBA-Indole-3-butyric acid; NAA--naphthalene
acetic acid; GA3 -Gibberelic acid.
Article Citation:
Web Address: Godishala Vikram, Kairamkonda Madhusudhan, Kagithoju Srikanth, Mangamoori
http://jresearchbiology.com/
Documents/RA0076.pdf.
Laxminarasu and Nanna Rama Swamy.
Effect of plant growth regulators on in vitro organogenesis in cultivated tomato
Journal of research in Biology (2011) 4: 263-268
Dates:
Received: 04 Aug 2011 /Accepted: 11 Aug 2011 /Published: 16 Aug 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.
Table.1. Effect of various concentrations of BAP on shoot buds proliferation efficiency with more
morphogenesis from cotyledon explants in tomato cv S-22 number of multiple shoots formation in tomato cv S
-22.While.
Concentratio % of Morphogenic
n of PGR Response Response Gunay and Rao (1980) have found more
(mg/L) BAP multiple shoots induction on MS medium
augmented with 2.0 mg/L BAP + 0.2 mg/L IAA of
0.5 40 Callusing
tomato cv Rio Grande. According to our
1.0 45 Callusing
1.5 45 Callusing
observation, BAP alone in the medium supported
2.0 50 Nodular Callus for the callus induction and addition of 0.1 mg/L
2.5 60 Green Nodular Callus IAA to the medium along with 2.5-5.0 mg/L BAP
3.0 75 Green Nodular Callus induced multiple adventitious shoots. Similar
3.5 70 Green Nodular Callus results were also obtained from the cotyledon
4.0 65 Brown Callus explants on MS + BAP with IAA in tomato (Oktem
4.5 50 Brown Callus et al., 1999, Fariduddin et al., 2004).
5.0 50 Brown Callus The regenerated micro-shoots when sub-
cultured on strength MS, MS basal media and
medium supplemented with different concentration also the same plant growth regulators combination
of BAP used in combination with 0.1 mg/L IAA and concentration did not support elongation.
(Table 2) .Multiple shoots were induced directly Elongation of micro-shoots was found on all the
from the explants in all the concentrations of BAP concentrations of GA3+3.5 mg/L BAP used (Table
in combination with 0.1 mg/L IAA (Fig.1b) except 3). Maximum percentage of shoot elongation and
at 0.5-2.0 mg/L BAP. Maximum percentage of also longer shoots were recorded at 0.6 mg/L GA3 +
response in cultures with more number of multiple 3.5 mg/L BAP (Fig.1c) without callus formation.
shoots formation was recorded at 3.0-3.5 mg/L For in vitro rooting the elongated shoots
BAP in combination with 0.1 mg/L IAA. But were cultured on strength MS, MSO and MS
multiple shoots induction was found to be reduced medium supplemented with 0.5 mg/L IBA / IAA/
when the concentration of BAP increased beyond NAA (Table 4). Rooting was absent on strength
to 3.5 mg/L BAP (Table 2). MS and MSO media and callus was formed without
Various studies demonstrated that 8-10 days rhizogenesis at the basal region of the shoots. Root
old cotyledons of tomato were superior to other formation was initiated within 2 weeks of
source of explants, including hypocotyls, stems and incubation in all the auxins used.100 % rooting was
leaves for promoting shoot organogenesis of tomato observed on MS medium supplemented with 0.5
(Hamza and Chupeau1993, Van Roekel et al., 1993, mg/L IAA with profuse rhizogenesis (Fig.1d). In
Ling et al., 1998). Where as in the present vitro rooting was reported without PGRS in tomato
investigation 10-12 day-old cotyledons were cv UC 105 (Mensuali-Sodi et al., 1995). However,
selected as the source of explants which were found the current study could find that cultivation of the
to be superior and showed maximum percentage of micro-shoots on MS medium substituted with
Table.2. Effect of various concentrations of BAP + 0.1 mg/L IAA on organogenesis from
cotyledon explants of tomato cv S-22
Concentration % of Morphogenic Average number Average length
of PGRS (mg/L) Response Response of shoots/ explant (in cm) of shoots
IAA + BAP (SE)a (SE)a
0.1+0.5 45 White friable Callus
0.1+1.0 45 White friable Callus
0.1+1.5 50 White friable Callus
0.1+2.0 55 White friable Callus
0.1+2.5 65 Multiple Shoots 3.40.30 0.80.22
0.1+3.0 80 Multiple Shoots 3.80.12 1.20.32
0.1+3.5 80 Multiple Shoots 4.00.20 1.40.36
0.1+4.0 75 Multiple Shoots 3.60.36 1.60.42
0.1+4.5 65 Multiple Shoots 3.50.30 1.00.33
0.1+5.0 60 Multiple Shoots 3.30.26 0.60.40
a
Mean Standard Error
different auxins resulted an effective rooting than based direct regeneration protocol is a pre-requisite
culturing on an auxin-free medium. for Agrobacterium tumefaciens mediated genetic
transformation in the cultivar S-22 for producing
Table.3. Effect of GA3 + 3.5 mg/L BAP on elongation agronomically useful transgenic plants.
of the micro-shoots in tomato cv S-22.
Medium + % of Average length of CONCLUSION.
Growth Response shoots From the above study, it is concluded that
regulators (in cm) (SE)a the plantlet development was established through
(mg/L) direct organogenesis form cotyledon explants in
MS+0.2 GA3 +
55 2.60.30 cultivated tomato cv S 22 on MS medium
BAP
supplemented with BAP(2.5-5.0 mg/L) + 0.1 mg/L
MS+0.4 GA3 +
70 3.00.12 IAA. This protocol is simple and reproducible,
BAP
MS+0.6 GA3 + which can be used for Agrobacterium tumefaciens
95 3.80.26 mediated genetic transformation in cultivated
BAP
MS+0.8 GA3 + tomato cv S-22.
80 3.60.41
BAP
MS+1.0 GA3 + ACKNOWLEDGEMENTS:
66 2.90.36
BAP Author is grateful to Sri Ch.Devender
a
Mean Standard Error Reddy, Secretary cum Correspondent, Vaagdevi
Institutions and Mr.A.Sheshachalam Principal,
The in vitro regenerated plants were taken Vaagdevi Degree and PG College for their
for hardening by removing the residues of agar encouragement during the research work.
followed by washing with sterile distilled water
under aseptic conditions. Later these were shifted to REFERENCES:
plastic pots containing sterile vermiculite: soil (1:1) Bhatia P, Nanjappa A, Tissa S and David M.
covered with polythene bags for four weeks 2004. Tissue Culture studies in tomato
(Fig.1e) followed by shifting to earthenware pots (Lycoperiscon esculentum) Plant Cell, Tiss. Org.
containing garden soil and maintained in the green Cult., 78:1-21.
house (Fig.1f). Later these were transferred to
research field and maintained under shady place. Chen H, Zhang J, Zhuang T and Zhou G. 1999.
The survival rate was found to be 70 % and the Studies on optimum hormone levels for tomato
plants were similar to donor plants. Thus, the plant regeneration from hypocotyls explants
results presented here describe an efficient protocol cultured in vitro. Acta Agric. Shanghai 15:26-29.
for multiple shoots induction and plantlet
establishment from cotyledon explants of tomato cv Evans DA. 1989. Somaclonal variation genetic
S-22. Since cotyledon is a favoured source of basis and breeding applications Trends Genet 5:46-
explant for transformation studies, the cotyledon 50.
Table 4: Effect of IBA, IAA and NAA on in vtiro Fariduddin M, Taher A, Islam SMA and
rooting of cultivated tomato cv S-22 Hossain MZ. 2004. Effect of Variety and Plant
Type of % of Average Growth Regulators in MS Medium on Shoot
Response Rooting number of Induction from Virus Infected Calli of Tomato.
roots/shoot Journal of Biological Sciences 4(4):52-526.
(SE)a
Geetha N, Venkatachalam P, Reddy PS and
MSO Callusing
Rajaseger G. 1998. In vitro plant regeneration
MSO Callusing from leaf callus cultures of tomato (Lycopersicon
MS + IBA (0.5) Rooting 70 200.16 esculentum Mill) Adv. Plant Sci., 11:253-257.
MS + IAA (0.5) Rooting 100 260.08
Rooting* 40 160.21 Godishala V, Mangamoori L and Nanna R.
a
2011. Plant regeneration via Somatic
Mean Standard Error embryogenesis in cultiveted tomato (Solanum
*With Callusing lycopersicum L.). J Cell & Tiss Res 11:2521-2528.
266 Journal of Research in Biology (2011) 4: 263-268
Vikram et al.,2011
Gubis J, Lajchova Z, Farago J and Jurekova, Z. Praveen Mamidala and Rama Swamy Nanna.
2003. Effect of genotype and explant type on shoot 2011. Effect of genotype, explant source and
regeneration in tomato (Lycopersicon esculentum medium on invitro regeneration of tomato
Mill.) in vitro Czech J.Genet. Plant Breed 39:9-14. International Journal of Genetics and Molecular
Biology 3(3):45-50.
Gunay AL and Rao PS. 1980. In vitro propagation
of hybrid tomato plants (Lycopersicon esculentum Van Roekel JSC, Damm B, Melchers LS and
L.) using hypocotyl and cotyledon explants. Ann. Hoekema A. 1993.Factors influencing
Bot., 45:205-207. transformation frequency of tomato (Lycopersicon
esculentum).Plant Cell Rep 12:644-647.
Hamza SY and Chupeau. 1993. Re-evolution of
conditions for plant regeneration and Wing AR, Zhang BH and Tanksley DS. 1994.
Agrobacterium-mediated transformation from Map-based cloning in crop plants: tomato as a
tomato (Lycopersicon esculentum). J Exp Bot model system.I. Genetic and physical mapping of
44:1837-1845. joint less Mol., Gen. Gene 242:681-688.
Ling HQ, Kriseleit D and Ganal MG. 1998. Ye Li HX and Zhou GL. 1994. In vitro culture of
Effect of ticarcillin /potassium clavulanate on callus tomato cotyledons and regenerated plants J
growth and shoot regeneration in Agrobacterium Huazhong Agric Uni 13:291-295.
mediated transformation of tomato (Lycopersicon
esculentum Mill) Plant Cell Rep 17:843-847. Zelcer A, Soferman O and Izhar S. 1984. An in
vitro screening for tomato genotypes exhibiting
McCormick S, Niedermeyer J, Fry J, Barnason efficient shoot regeneration. J Plant Physiol.,
A, Horsch R and Fraley R. 1986. Leaf disc 115:211-215.
transformation of cultivated tomato (L. esculentum)
using Agrobacterium tumefaciens . Plant Cell Rep
5:81-84.
Fig 1: Callus induction and organogenesis from cotyledon explants in cultivated tomato cv S-22.
a: Callus induction on MS + 3.0 mg/L BAP (after 4 weeks of culture )
b: Multiple shoots induction from cotyledon explant on MS + 0.1 mg/L IAA + 3.5 mg/L BAP.
c: Shoot elongation on MS + 0.6 mg/L GA3 + 3.5mg/L BAP.
d: Profuse rhizogenesis on MS+ 0.5mg/L IAA.
e: Acclimatization of regenerated plant.
f: Regenerated plant growing in the research field.
Authors: ABSTRACT:
Sonia R and Ramanibai R.
Institution:
Aquatic Biodiversity Unit,
Department of Zoology,
University of Madras, Hydra viridissima Pallas, 1766, a green colour fresh water polyp is well known
Guindy Campus, for over 200 years for its remarkable regenerative capacity. New informations on the
Chennai - 25. Tamil Nadu. genus Hydra collected from Kolavoi Lake, Chingleput is reported here with its
morphometric features.This is the first report on the occurrence of Hydra viridissima
(Pallas, 1766) from the Kolavoi Lake, Chingleput.Tamil Nadu - India.
Corresponding author:
Ramanibai R.
Email:
rramani8@ hotmail.com,
soniadaisy7@gmail.com
Article Citation:
Web Address:
http://jresearchbiology.com/ Sonia R and Ramanibai R.
Documents/RA0071.pdf. Hydra viridissima Pallas, 1766 (Cnidaria, Hydrozoa, Hydridae) from Chingleput Lake,
Tamil Nadu- India.
Journal of research in Biology (2011) 4: 276-278
Dates:
Received: 01 Aug 2011 /Accepted: 11 Aug 2011 /Published: 22 Aug 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.
Materials Examined:
Kolavoi Lake, Chingleput:
Kolavoi Lake is located in the Chingleput
district and it is 58 Km away from the Chennai
City. It is one of the largest lakes of Chingleput A: Hydra Single Polyp B: Hydra Polyp with bud.
district. People use this lake water for agriculture,
278 Journal of Research in Biology (2011) 4: 276-278
Sonia et al.,2011
the tentacles. Two headed hydra were also seen P (COORDS). Ssswasserfauna von Mitteleuropa.
(Fig. D & Fig. E). Stuttgart: Gustav Fischer Verlag. 1-110.
Hydra viridissima are hermaphroditic in Simona Chera , Wanda Buzgariu , Luiza Ghila
nature. But sexual hydras were not reported from and Brigitte Galliot. 2009. Autophagy in Hydra: A
our sample collections. These hydras are response to starvation and stress in early animal
phototactic and often crowd into one area within a evolution. Biochimica et Biophysica Acta.
culture tank. 16057:12 , 4C:2,3,5,6,8,9,11.
Based on literature records, we conclude that
our report is the first on the occurrence of Hydra
viridissima (Pallas, 1766) from the Kolavoi Lake,
Chingleput.
ACKNOWLEDGEMENT:
We thank UGC - New Delhi for financial
support.
REFERENCES:
Bharathi D Limnological studies of few fresh water
habitats in and around Chennai City. 2003; Ph.D
Thesis submitted to University of Madras, Chennai.
Authors: ABSTRACT:
*MohanaSundaram,
Sivakumar, Karthikeyan,
Magesh, Gopinath, In this investigation, Lamprachaenium microcephalum was analysed for its
Sheeladevi, Thirumalai,
phytochemical constituents qualitatively and quantitatively. The antibacterial property
Pennarasi and Prasanna.
of aqueous, ethanolic and hexane extracts of Lamprachaenium microcephalum was
studied against different bacteria include Escherichia coli, Proteus mirabilis,
Salmonella typhi, Enterobacter aerogenes, and Shigella flexineri and showed growth
Institution: inhibition activity at concentrations in the range of 300mg to 500mg. The antioxidant
Department of effect of those extracts was also studied against -tocopherol as a control. From the
Biotechnology, results, Alkaloids, flavonoids, saponins, and tannins were revealed to be present in
Karpaga Vinayaga College Lamprachaenium microcephalum. Ethanol extract at the concentration of 500g/ml
of Engineering and showed 61.16% antioxidant activity against 500 g/ml of -tocopherol which showed
Technology, 76.86% as a standard reference.
Maduranthagam,
TamilNadu-603308.
*Lecturer, Department of
Biotechnology,
Karpaga Vinayaga College
of Engineering and
Technology, Keywords:
Maduranthagam, Lamprachaenium microcephalum , phytochemicals, Antibacterial, Antioxidant
Kanchipuram District,
TamilNadu-603308, India.
Corresponding author:
MohanaSundaram
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.
ml of 45% ethanol was added and boiled for 5 min. of methanol and the content poured at once into the
The mixture was cooled and filtered. 1 ml of the funnel. The filtrate was mixed with 50 ml of water
filtrate was added 3 drops of lead sub acetate and analyzed within an hour. For aqueous
solution. A gelatinous precipitates were observed extractions, 5 ml of water was used for the
which indicates the presence of Tannins. Another 1 extraction and for the rinse and the filtrate was
ml of the filtrate was added 0.5 ml of bromine added to 50 ml of water. 3 ml of 0.1 ml FeCl3 in 0.1
water. A pale brown precipitates were observed NH4Cl was added to 5 ml of the extract and
indicating the presence of Tannins (Trease and followed immediately by timed addition of 3 ml of
Evans, 1989). 0.008 ml K2Fe(CN)6. The absorbance was taken at
Test for glycosides: 2 g of the sample was mixed 720 nm spectrophotometrically (Onwuka, 2005).
with 30 ml of distilled water and it was heated for 5 Antibacterial effect:
min on a water bath, filtered and used as follows: The antibacterial activity of
five ml of the filtrate was added to 0.2 ml of fehling Lamprachaenium microcephalum was evaluated by
solution A and fehling solution B until it turns agar well diffusion method (Chung et al., 1990).
alkaline and heated in a water bath for 2 min. A Muller Hinton agar medium was prepared and
lightish blue colouration was observed (instead of poured into the petridishes. Then it was inoculated
brick red precipitate) which indicates the absence of with a swab of bacterial culture (mid log phase) and
glycosides (Oloyed, 2005). spread throughout the medium uniformly with a
Quantitative Analysis of Phytochemical sterile cotton swab. Using a sterile cork borer
Constituents (10mm diameter) wells were made in the agar
Estimation of alkaloids: 0.5 g of the sample was medium. The test compound was introduced into
dissolved in 96% ethanol-20% H2SO4 (1:1) the wells and all the plates were incubated at 37C
mixture. 1 ml of the filtrate was added to 5 ml of for 24 h. The experiment was performed five times
60% tetraoxosulphate (VI), and allowed to stand for under strict aseptic conditions. Sensitivity of the
5 min. Then, 5 ml of 0.5% formaldehyde was added organism was determined by measuring the
and allowed to stand for 3 h. The absorbance was diameter of the zone of inhibition. Each assay was
measured at 565 nm (Harborne, 1976). repeated for five times and the mean value was
Estimation of flavonoids: Flavonoid in the test taken for analyses. The control experiment was
sample was determined by the acid hydrolysis of carried out with the antibiotics such as streptomycin
spectrophotometric method. 0.5 g of processed and chloramphenical (S.Shobana et al, 2009).
plant sample was mixed with 5 ml of dilute HCl Antioxidant effect:
and boiled for 30 min. The boiled extract was The antioxidant activity of aqueous,
allowed to cool and filtered. 1 ml of the filtrate was ethanolic and hexane extracts of Lamprachaenium
added to 5 ml of ethyl acetate and 5 ml of 1% NH3 . microcephalum were determined by ferric
This was then scanned 3 from 420nm-520nm for thiocynate method (Mistuda et al., 1996). 10 mg of
the absorbance. (Harborne,1976). each extract was dissolved separately in 99.5% of
Estimation of saponins: 0.5 g of the sample was ethanol and various concentrations (100, 200, 300,
added to 20 ml of 1N HCl and was boiled for 4 h. 400 & 500g/ml) were prepared. A mixture of a 2
After cooling it was filtered and 50 ml of petroleum ml of sample in 99.5% ethanol, 2.052 ml of 2.51%
ether was added to the filtrate for ether layer and linoleic acid in 99.5% ethanol, 4 ml of 0.05 M
evaporated to dryness. 5 ml of acetone ethanol was phosphate buffer (pH 7.0) and 1.948 ml of water
added to the residue. 0.4 ml of each was taken into was placed in a vial with a screw cap and placed in
3 different test tubes. 6 ml of Ferrous sulphate an oven at 60oC in the dark. To 0.1 ml of this
reagent was added into them followed by 2 ml of sample solution 9.7 ml of 75% ethanol and 0.1 ml
concentrated H2SO4. It was thoroughly mixed after of 30% ammonium thiocynate was added. After the
10 min and the absorbance was taken at 490 nm addition of 0.1 ml of 2 x 10-2 M ferrous chloride in
(Oloyed, 2005). 3.5% hydrochloric acid to the reaction mixture, the
Estimation of tannins: 5 g of the ground sample absorbance of the red color developed was
was shaken constantly for 1 min with 3 ml of measured in 3 min at 500 nm (Matook and
methanol in a test tube and then poured into a Hashinaga, 2005). The control and standard were
Buchner funnel with the suction already turned on. subjected to the same procedures as the sample,
The tube was quickly rinsed with an additional 3 ml except that for the control, only solvent was added,
and for the standard, sample was replaced with the Table 1.2: Quantitative analysis of Lamprachaenium
same amount of -tocopherol (reference microcephalum for phytochemicals
compound) (Ali Yildirim et al., 2001). The Lamprachaenium
inhibition of lipid peroxidation in percentage (Table S. Name of the microcephalum
3.0) was calculated by following equation: No phytochemical
Dry sample Wet sample
% Inhibition = 1 - (A1/A2) x100 Alkaloids
1. 46.68 3.21 21.39 2.17
(mg/100g)
Where, Flavonoids
A1 - absorbance of the test sample 2. 69.33 4.14 56.18 3.19
(mg/100g)
A2 - absorbance control reaction Saponins
3. 03.77 0.54 01.21 0.84
(mg/100g)
RESULTS & DISCUSSION Tannins
Phytochemical analysis is very useful in the 4. 02.37 0.76 01.67 0.46
(mg/100g)
evaluation of some active biological components of
medicinal plants. The qualitative and quantitative Antioxidants are compounds that protect
analyses were carried out in both dry and wet cells against the damaging effects of reactive
samples. Alkaloids, flavonoids, saponins, tannins, oxygen species, such as singlet oxygen, super
were revealed to be present in Lamprachaenium oxide, peroxyl radicals, hydroxyl radicals and
microcephalum (Table 1.1). This shows high level peroxynitrile. An imbalance between antioxidants
of its possible medicinal and dietary values and reactive oxygen species results in oxidative
(Oloyed, 2005). Although, some of these analyzed stress, leading to cellular damage (Burlon and
constituents of the vegetable species may be Ingold, 1984). Oxidative stresses have been linked
completely harmful to both man and farm animals to cancer, aging, atherosclerosis, inflammation,
and some are species specific as observed in the ischemic injury and neuro degenerative diseases
case of tannins (Odebiyi and Sofowora, 1979). (Parkinsons and Alzheiners) (Palozza, 1998).
Some of these active components have been Flavonoid may help provide protection against
demonstrated to possess anti nutritional effects, these diseases by contributing along with
following their ability to reduce palatability and antioxidant vitamins and enzymes, to the total
digestibility of feedstuff (Odebiyi and Sofowora, antioxidant defense system to the human body.
1979). Epidemiological studies have shown that flavonoids
In Table 1.2, the levels of these and carotenoids intake are inversely related to
phytochemicals (bioactive compounds) were mortality from coronary heart diseases and to the
shown. Generally, the dry sample showed higher incidence of heart attacks (Donald and Cristobal,
levels of these bioactive compounds than the wet 2006).
sample. The reason may be that the bioactive Antibacterial Property
compounds are not volatile compounds and hence From Table 2.0, it is very clear that the
have a high dried weight. High levels of flavonoids aqueous, ethanolic and hexane extracts of
(69.33 4.14 and 56.18 3.19) in Table 1.2 Lamprachaenium microcephalum showed growth
showed that the vegetable is good for the inhibition activity only at higher concentrations in
management of cardiovascular diseases and the range of 300mg to 500mg. E.aerogens and
oxidative stress, since flavonoids are biologic P.mirabilis were sensitive to aqueous extracts of
antioxidants. Lamprachaenium microcephalum rather than
Table 1.1: Qualitative analysis of different extracts of Lamprachaenium microcephalum for phytochemicals
Name of the Different extracts of Lamprachaenium microcephalum
S.No
phytochemical Aqueous extract Ethanolic extract Hexane extract
1. Alkaloids +ve +ve +ve
2. Flavonoids +ve +ve +ve
3. Saponins +ve +ve +ve
4. Tannins +ve +ve +ve
5. Glycosides -ve -ve -ve
+ve presence -ve - absence
282 Journal of Research in Biology (2011) 4: 279-284
MohanaSundaram et al.,2011
E.aerogenes
moderate resistance to all the extracts when
10.3
11.9
14.2
21.4
20.1
NS
NS
compared to others (Table 2.0). It was observed that
in both ethanolic and hexane extracts of
Lamprachaenium microcephalum, bacterial strains
Hexane extract
S.typhi
18 .9
20 .1
are not highly susceptible even at high
10.8
12.3
15.7
NS
NI concentration than aqueous extract.
The antioxidant activity of the aqueous,
P.mirabilis
Table 2.0: Antibacterial activity of aqueous, ethanolic and hexane extracts of Lamprachaenium microcephalum
11.5
13.1
16.2
19.9
19.1
NS
10.2
11.6
14.4
18.9
19.3
12.7
14.6
15.3
20.8
20.1
NS
NS
CONCLUSION
Based on the results of the present study, it
Aqueous extract
11.8
15.6
19.1
20.7
NS
NS
NI
10.1
12.6
16.1
19.3
20.8
19.6
interesting to learn.
NS
ACKNOWLEDGEMENT
The authors are grateful to Mrs.Meenakshi
E.coli
10.3
13.1
16.5
21.6
22.3
NS
NS
microcephalum
Chloramphenic
Lamprachaeni
Streptomycin
study
um
al
Table : 3.0 Antioxidant activity of aqueous, ethanolic and hexane extracts of Lamprachaenium microcephalum
% of inhibition of lipid peroxidation
S.No Extract
100g/ml 200g/ml 300g/ml 400g/ml 500g/ml
1. Water 12.31 21.12 35.73 45.34 57.75
2. Ethanol 18.17 24.63 43.08 50.67 61.16
3. Hexane 14.71 20.32 28.63 39.34 48.55
4. -Tocopherol 27.43 39.71 47.87 62.78 76.86
Authors: ABSTRACT:
Govindappa M1, Anil
Kumar NV2 and Gustavo
Santoyo3.
Fax:
+91-816-2212629 Ficus Publishers.
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Web Address: creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
http://jresearchbiology.com/ commercial, distribution, and reproduction in all medium, provided the original work is properly
Documents/RA0069.pdf. cited.
(ethanol and methanol) up to the volume of 2ml for extracts of C. pallida, the alkaloids, flavonoids,
two cycles of 5 min individually. The resulting terpenoids, saponins, phenols, cardiac glycosides,
extracts were filtered through Whatman filter No.2. steroids, coumarin and tannins were present in all
Clarified supernatant was kept in refrigeration for the solvent extracts. The methanol extract yielded
further studies (phytochemical analysis, coumarin strongly all the phytochemicals compared to
identification and HIV protease inhibition). ethanol (Table 1). In ethanol leaf extracts, it
Phytochemical analysis
Phytochemical analysis was carried out for
Anthraquinones
saponins, flavonoids, terpenoids, steroids, phenol,
alkaloids (Obdoni and Ochuko, 2001), tannins
(Kaur and Arora, 2009) and coumarin
-
-
-
-
-
-
spectrophotometrically as described by Yao et al.
(2008). Wagners and Hegers reagents were used
for alkaloid foam test for saponins, Mg-HCl and Zn
-HCl for flavonoids, Keller-Killani test for cardiac
Coumarins
glycosides, Salkonoski test for terpenoids, acetic
+
+
+
+
+
+
and gelatin for tannins, ferric chloride for phenol,
hexane and diluted ammonia for anthraquinones
test. All these experiments were carried out for each
Saponins
solvent extracts separately.
+
+
+
+
+
+
Enzyme pepsin inhibition assay
Pepsin has a quite close resemblance in
proteolytic activity with HIV-1 protease one key
enzyme of HIV-1 life cycle as both of them belong Tannin Steroids
+
+
+
+
+
+
to same aspartate enzyme family (Maria et al.,
Phytochemicals
+
+
+
2010).
We followed the method of Aoyagi (1978)
and Singh et al. (2010), for this assay, 50 g pepsin,
glycosides
+
+
+
were taken in 500 l of reaction mixture. The
mixture was allowed to incubate at 370C, after 20
min, 700 l of 5% TCA was added to stop the
reaction. It was then centrifuged at 14, 000 g for 5
Flavonoids Terpenoids Phenol
+
+
-
-
-
+
+
+
+
+
-
Methanol extract
Ethanol extract
RESULTS
Phytochemical analysis
Sample
Flower
Stem
Stem
Leaf
Leaf
showed the absence of flavonoids, the ethanol stem and protease inhibitors have experienced drug
extract showed absence of phenol and ethanol resistance by HIV strains. This imposes the demand
flower and stem extracts showed absence of tannin. for the development of new drugs particularly of
The ethanol stem extract exhibited the absence of plant origin owing to their success as sources of anti
steroids. Methanol leaf extracts showed the absence -HIV drugs. The use of plants for management of
of flavonoids and methanol stem extract exhibited different diseases has become a common practice
the absence of phenol. since olden days in developing countries. Especially
The ethanolic flower and stem extracts in India, most of the people are depending on
exhibited highest amount of coumarins in tested traditional medicines for their primary health care
methods where as methanol flower and stem including the management of HIV/AIDS.
extracts exhibited highest amount of coumarins. Our results have showed protease inhibitor
The ethanol and methanol stem and flower activity and phytochemical screening showed the
extracts exhibited strong inhibition of pepsin presence of many active compounds. The various
enzyme. Strong inhibition was noticed in methanol plant active molecules exhibited as anti-HIV
stem extract followed by ethanol stem extract, properties, such as flavonoids (Mantas et al., 2000),
methanol flower extract and ethanol flower extract terpenoids (Sun et al., 2003), cardiac glyucosides
while other extracts with negligible toxic effect (Prinsloo et al., 2010), tannin (Sakagami et al.,
(Table 2). 1999), steroids (You et al., 2003), saponins
(Konoshima et al., 1995), coumarins (Zhou et al.,
Table 2. Effect of Crotalaria pallida constituents on 2000) and athraquinones are under study (Schinazi
HIV protease inhibition
et al., 1990).
Sample Absorbance at 280 The structure of the dimeric enzyme of HIV-
nm 1-PR superficially resembles that of other aspartic
Control (without any extract/ proteases such as pepsin (Hutchins et al., 1991;
0.4212+0.0085a
inhibitor) Wlodawer and Erickson, 1993). However, whereas
Control with Pepstatin A 0.0016+0.0085 g pepsin exists as a 326-residue monomer, with two
differing domains forming a cleft containing the
Control with methanol leaf
0.2430+0.0085c active site, HIV-1-PR forms a similar groove in the
extract
interface between its two 99-residue subunits. As in
Control with methanol flower
0.0046+0.0085d pepsin, HIV-1-PR has two highly mobile arms of
extract
Control with methanol stem about 10 residues each, which surround and anchor
0.0029+0.0085f the substrate in the region of the active site. The
extract
Control with ethanol leaf flaps themselves are not necessary for enzyme
0.3568+0.0085b activity, although the absence of flaps reduces
extract
Control with ethanol flower d enzymatic activity (Chatfield and Brooks, 1995;
0.0051+0.0085
extract Silva et al., 1996).
Control with ethanol stem e The main aim of our present investigation is
0.0036+0.0085
extract the screening of Crotalaria pallida phytochemical
Repeated the each experiments thrice, +: presence, -: extracts for HIV protease inhibition. The plant has
absent, According to Duncans Multiple Range Test potential antimicrobial compounds, two solvent
(DMRT), values followed by different subscripts are extracts of Crotalaria pallida showed a very
significantly different at P<0.05, SE-standard error of
significant inhibition of pepsin enzymatic activity.
the mean.
The previous reports suggested that there are close
structural and functional similarities between pepsin
DISCUSSION and HIV protease. The plant extracts have showed
AIDS-related diseases remain one of the inhibitory activity of pepsin enzyme, may be these
leading causes of death globally. The declines in extracts inhibit the activity of HIV protease. Our
new infections and AIDS-deaths may be attributed results, the methanol and ethanol extracts of stem
to the scale-up of anti-retroviral therapy (ART) and flower have proven potential inhibition of the
programmes, especially in the developing world. pepsin enzyme activity due to the different
Presently, no reliable and friendly treatment can be phytochemical constituents, may be our extracts
claimed to combat this disease. The current anti- have strong inhibitory activity of HIV protease.
HIV drugs have focused on reverse transcriptase Many similar works has been done with plant
288 Journal of Research in Biology (2011) 4: 285-291
Govindappa et al.,2011
extracts (Cos et al., 2004; Debouck, 1992; Aiken Scientific Research 5(3):212-215.
and Chen, 2005; Polya, 2003). The current study
can append one more alternative HIV protease Chinsembu KC and Hedimbi M. 2010.
inhibitor to solve the problem especially arresting Ethanomedical plants and other natural products
the HIV replication. But, it needs further with anti-HIV active compounds and their putative
characterization of active molecules in the extracts, modes of action. International Journal of
purification and mode of action on HIV replication Biotechnology and Molecular Biology Research 1
is needed. (6):74-91.
Lipsky JJ. 1996. Antiretroviral drugs for AIDS. Polya GM. 2003. Protein and non-protein protease
Lancet 348:800-803. inhibitors from plants. Studies in Natural Product
Chemistry 29(10):567-641.
Mantas A, Deretey E, Ferretti FH, Estrada MR
and Csizmadia IG. 2000. Structural analysis of Prinsloo G, Meyer JJ, Hussein AA, Munoz E
flavonoids with anti-HIV activity. Journal of and Sanchez R. 2010. A cardiac glycoside with in
Molecular Structure : Theochem 504(1-3):171-179. vitro anti-HIV activity isolated from Elaeodendron
croceum. Natural Product Research 24(8):1743-
Martino E, Ramaiola I, Urbano M, Bracco F and 1746.
Collina S. 2006. Microwave-assisted extraction of
coumarin and related compounds from Melilotus Rao MS and Narukulla R. 2007. A new
officinalis (L.) Pallas as an alternative to Soxhlet trimethoxychalcone from Crotalaria ramosissima.
and ultrasound-assisted extraction. Journal of Fitoterapia 78(6):446-447.
Chromatography 1125:147-151.
Rego EJ, De Carvalho DD, Maragoni S, de
Matsuse IT, Lim Y, Hattori M, Correa M and Oliveira B and Novello JC. 2002. Lectins from
Gupta MP. 1999. A search for anti-viral properties seeds of Crotalaria pallida (smooth rattlebox).
in Panamanian medicinal plants the effect of HIV Phytochemisrty 60(5):441-446.
and its essential enzymes. Journal of
Ethanopharmacology 64:15-22. Ricardo RC, Elizabet EM, Teresa RA, Badia A,
Andre A, Christopher KJ and Mario VT. 2004.
Mekkawy SE, Meselhy MR and Nakamura Cytotoxic effect of Mammea type coumarins from
N.1998. Anti-HIV-1 and anti-HIV- 1 protease Calophyllum brasiliennse. Life Sciences 75(13):
substances fr om Ganoderma lucidum. 1635-1647.
Phytochemistry 49:1651-1657.
Sakagami H, Satoh K, Ide Y, Koyama N,
Notka F, Meier G and Wagner R. 2004. Premanathan M, Arakaki R, Nakashima H,
Concentrated inhibitory activities of Phyllanthus Hatano T and Yoshida T. 1999. Induction of
amarus on HIV replication in vitro and ex vitro. apoptosis and anti-HIV activity by tannin and lignin
Antiviral Research 64:93-102. related substances. Basic Life Sciences 66: 595-611.
Maria MY, Justo P, Cristina M, Julio G, Manuel Scinzi R, Mead J and Feorino P. 1992. Insights
A, Francisco M and Javier V. 2004. Rapeseed into HIV chemotherapy. AIDS Research Human
protein hydrolysates: a source of HIV protease Retroviruses 8:963.
peptide inhibitors. Food Chemistry 87:387-392.
Schinazi RF, Chu CK, Babu JR, Oswald BJ,
Min BS, Jung HJ and Lee JS. 1999. Inhibitory Saalmann V, Cannon DL, Erikkson BFH and
effect of triterpenes from Crataegus pinatifida on Nasr M. 1990. Anthraquinones as new class of
HIV-1 protease. Planta Medica 65:374-375. antiviral agents human immunodeficiency virus.
Antiviral Research 13(5):265-272.
Obdoni BO and Ochuko PO. 2001.
Phytochemical studies and comparative efficacy of Silva AM, Cachau RE, Sham HL and Erickson
the crude extract of some homostatic plants in Edo JW. 1996. Inhibition and catalytic mechanism of
and Delta States of Nigeria. Glob. J. Pure Appl. Sci. HIV-1 aspartic protease. J. Mol. Biol. 255(2):321-
8b:203-208. 346.
Pelyrini PB, Farias LR, Saude AC, Costa FT, Singh KP, Upadhyay B, Prasad R and Kumar A.
Bloch C Jr, Silva LP, Oliveira AS, Gomes LE, 2010. Screening of Adhatoda vasica Nees as a
Sales MP and Franco OL. 2009. A novel putative HIV-protease inhibitor. Journal of
antimicrobial peptide from Crotalaria pallida seeds Phytology 2(4):78-82.
with activity against human and phytopathogens.
Current Microbiology 59(4):400-404. Sun FC, Kashiwada Y, Morris-Natshke SC and
Lee KH. 2003. Plant derived terpenoids and
290 Journal of Research in Biology (2011) 4: 285-291
Govindappa et al.,2011
Authors: ABSTRACT:
Tambekar DH and
Tambekar SD.
Lonar Lake, an impact crater located in the Buldhana district of Maharashtra
State, India is occupied by saline water and harbors various unidentified, unique
haloalkaliphilic bacterial bacillus species which produces thermo-halo-alkaliphilic
Institution: proteases. The present study deals with the isolation, production dynamics,
Post Graduate Department purification, characterization and optimization of a protease from Cohnella
of Microbiology thermotolerans isolated and identified by 16s RNA ribotyping from the Alkaline Lonar
S.G.B. Amravati University Lake. The C. thermotolerans produced protease at maximum rate after 72 h of
Amravati 444604 (India). incubation at 370C with the agitation speed of 120 rpm and 5% of starter culture. The
best carbon sources for this C. thermotolerans were fructose and maltose and best
nitrogen source was yeast extract, while the most effective inorganic nitrogen source
was ammonium carbonate. Supplementation of the culture medium with amino acid L
-glutamic acid and metal ion Mg2+ improved the protease production substantially.
Under these conditions, newly isolated Cohnella thermotolerans strain was found to
Corresponding author: produce alkaline protease at a maximum rate of optimum pH 10 and temperature at
Tambekar DH 750C.
Keywords:
Email: Alkaline Protease, Cohnella thermotolerans, Environmental factors,
diliptambekar@rediffmail.com Nutritional conditions.
Article Citation:
Web Address:
http://jresearchbiology.com/ Tambekar DH and Tambekar SD.
Documents/RA0073.pdf. Partial characterization and optimization of protease production from newly isolated
Cohnella thermotolerans from Lonar Lake
Journal of research in Biology (2011) 4: 292-298
Dates:
Received: 02 Aug 2011 /Accepted: 11 Aug 2011 /Published: 30 Aug 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.
phylogenetic analyses were performed by the read at 650 nm (Lalitha et al, 2010).
maximum-likelihood method, using 1,292 Determination of proteolytic activity: Proteases
nucleotide positions. The functional genes were activity was determined by a slightly modified
translated into amino acid sequences, and these method of Yang et al, 2001. The reaction mixture
were included in phylogenetic analyses using the containing 1 ml of 1.0 % casein solution in 0.2 M
neighbor-joining method (Dayhoff PAM model). Glycine-NaOH buffer having pH 10.5 and 1 ml of a
Preparation of crude enzyme extracts: The 100 given enzyme solution was incubated at 40oC for 10
ml Yeast extract casein medium (Glucose 1%, minutes and the reaction was then stopped with 2
Casein 0.5%, yeast extract 0.5%, KH2PO4 0.2%, ml of 10 % tri-chloroacetic acid (TCA). The
K2HPO4 0.2%, MgSO4 0.1%, pH.10.5) was amount of tyrosine liberated was determined as per
dispensed (50 ml each) into two 250 ml capacity tyrosine assay procedure at 650 nm. The proteolytic
conical flasks, after adjusting the pH to 10.5 and unit was defined as the amount of the enzyme that
sterilized in autoclave. After cooling, the broth was released 1ug of tyrosine per minute under the assay
inoculated with Cohnella thermotolerans cultures conditions.
and incubated for 72 h at 370C in shaking incubator. Partial characterization of protease: Partial
After 72h incubation, centrifuged the broth at 5000- characterization of protease was carried out using
8000 rpm for 15 min. The supernatant served as methodology. (Joo et al, 2002).
crude enzyme source. Effect of pH on alkaline protease activity: The
Optimization of crude enzyme protease: The effect of pH on alkaline protease from Bacillus spp.
standard graph of tyrosine was prepared by adding was determined by assaying the enzyme activity at
different concentration of standard tyrosine (1 mg/ different pH values ranging from 7.0 to 10.5 using
ml) into a series of test tubes and made the final the following buffer systems with concentration of
volume in each test tube to 1 ml with distilled each buffer at 0.2 M: phosphate (pH 6-7) trisHCl
water. Estimation of proteases was carried out with (pH 8-9) and Glycine-NaOH (pH 10-12).
1 ml of casein in a test tube; 1 ml of enzyme source Effect of temperature on alkaline protease
was added and incubated for 10min at room activity: The effect of temperature on alkaline
temperature. After incubation 2 ml of TCA was protease activity was determined by incubating the
added to stop the reaction and centrifuged the reaction mixture (pH 10.5) for 20 minutes at
reaction mixture at 5000-8000 rpm for 15 min. different temperature ranging from 55oC to 90oC.
Supernatant was separated and 1ml of Folin- Effect of substrate on alkaline protease activity:
Ciacalteau reagent and 2 ml of Na2CO3 were added The effect of substrate concentration on alkaline
in 1 ml of supernatant. The reaction mixture was protease activity is determined by incubating the
boiled for 1 min in a boiling water bath and 6 ml of reaction mixture (pH 10.5) for 20 minutes with
distilled water was added to make a final solution to different substrate concentration, ranging from 5
10 ml. In control tube, the reaction was terminated mg/ml to 40 mg/ml.
the reaction at zero time and the absorbance was
120
140
100
Tyrosine (g/ml)
Tyrosine (g/m)l
80
120
60
40 100
20
0 80
70 70
Tyrosine (ug/ml)
Tyrosine (ug/ml)
60
60
50
50
40
40
30
20 30
7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 55 60 65 70 75 80 85 90
pH Temerature in degree centigrade
1999). Various sources of carbon such as glucose, nitrogen. Despite the luxurious bacterial growth the
fructose, sucrose, maltose, starch and lactose were presence of beef extract, peptone and tryptone
used in enhancing the production of alkaline resulted in low protease production. This
proteases. Results obtained showed that starch observation contradicted (Phadatare et al, 1993)
instigated highest protease production compared to which reported that protease production in
other carbon sources at 72 h of incubation due to Conidiobolus coronatus was enhanced by organic
the prolonged incubation time perhaps led to auto nitrogen sources like yeast extract, peptone and
digestion of proteases and proteolytic attack by tryptone.
other proteases. Fructose, maltose and lactose Effects of inorganic nitrogen sources on protease
caused slightly low protease production. production: Inorganic nitrogen sources like
Effect of nitrogen sources on protease ammonium carbonate, ammonium nitrate and urea
production: were tested on the growth and protease production.
Effect of organic nitrogen sources on protease Among them, ammonium carbonate led to high
production: In this study, sources of organic protease activity at 48 and 72 h. Urea did not
nitrogen like soy ton, soya bean cake, beef extract, enhance protease production at early stages of
yeast extract and peptone were used. It was showed incubation but at later stages (48 and 72 h) protease
that soy ton resulted in the highest level of protease production increased. Even though growth was
activity compared to other sources of organic stimulated, only moderate levels of enzyme
100
Tyrosine (g/ml)
60 80
60
40
30 20
0
Ammonium nitrate
Peptone
Ammonium
Soy tone
Yeast extract
Urea
Beef extract
carbonate
0
Glucose
Fructose
Maltose
Lactose
Sucrose
Starch
activities were obtained when ammonium nitrate protease at pH 9 and temperature at 800C.
was used as a nitrogen source. This was perhaps In su m mar y, i sol a t ed Cohn el l a
due to the inability of bacteria to utilize ammonia in thermotolerans species from Lonar Lake produce
the media (Ellaiah et al, 2005). alkaline protease and maximum growth at pH 9-
Effects of amino acid on protease production: 10.5. The isolated bacterial C. thermotolerans strain
This observation was corroborated by L- produces the proteases enzyme which was
aspartic acid; glutamic acid and glycine were also theomorphic, alkaliphilic and has potential to
tested as sources of amino acids for protease produce good quality proteases which can use in the
production in Cohnella strain 146. In the presence industry. The C. thermotolerans species were most
of Glycine, protease production was observed to be efficient for protease producing at pH 10.5
high. incubated at 370C for 72h. The protease produced
Effects of metal ions on protease production: The from this species were highly efficient at high
highest level of protease activity was observed in temperature, high salt concentration and tolerate the
the presence of Mg2+ at 72 h incubation. Addition other environmental conditions. This bacterial
of Ca2+, Cu2+ and Mn2+ resulted in high protease species is ubiquitous and non-pathogenic, not
production only at 48 h incubation. It was suggested causing any diseases to human beings and most
that these metal ions increased stability of efficient for protease production among all isolated
proteases, even though effects of the different metal protease-producing bacteria. Protease enzymes have
cations on protease production vary, their presence importance in various industries. Bacillus species
in the culture medium improved the growth of particularly Cohnella thermotolerans, were known
Cohnella strain. Supplementation of culture for their ability to produce proteolytic enzymes with
medium with metal cations improved substantially potential use in industries (Kampfer et al, 2006). In
the protease production of Cohnella addition to the limited number of reports, protease
thermotolerans. This observation strongly production by this microorganism also was shown
suggested the requirement of some metal ions for to be affected by various environmental and
protease production by this organism. These results nutritional conditions. In the present investigation,
were in agreement with the earlier findings which we have determined the optimum parameters for
showed enhancement of protease activity in the maximum production of alkaline protease by the
presence of metal ions and it was suggested that newly isolated thermophilic bacterium Cohnella
these metal ions increased stability of proteases. thermotolerans. This information has enabled the
(Banerjee et al, 1999). ideal formulation of media composition for
Effect of pH and temperature on protease maximum protease production by this organism.
production: This observation was corroborated by
different pH and Temperature range. Cohnella
thermotolerans strain produced maximum alkaline
297 Journal of Research in Biology (2011) 4: 292-298
Tambekar et al.,2011
Ismail AMS and Abdel-Fattah AF. 1999. Srinivasan TR, Das Soumen, Bal Krishnan V,
Optimization of alkaline protease productivity by Philip R, Kannan N. 2009. Isolation and
Bacillus licheniformis ATCC 21415. Bioresource characterization of thermostable protease producing
Technol., 69:155-159. bacteria from tannery industry effluent. Recent
research in science and technology 1(2):063-066.
Joo HS, Kuma CG, Park CG, Paik SR, Chang
CS. 2002. Optimization of the production of an Yang SS and Lee CM. 2001. Effect of culture
extracellular alkaline protease from bacillus media on protease and oxytetracycline production
horikoshii. Process Biochem. 38:155-159. with mycelium and protoplasts of Streptomyces
rimosus. World J. Microbiol Biotech., 17:403-410.
Joshi AA, Kanekar PP, Kelkar AS, Shouche YS,
Vani AA, Borgave SB and Sarnaik SS. 2008.
Cultivable bacterial diversity of alkaline Lonar
Lake, India. Microb Ecol., 55(2):163-72.
Authors: ABSTRACT:
Sunukumar SS, Harish SR,
Manoj GS, Remya Salinity is currently the major factor which reduces crop yields. One of the
Krishnan and biological approaches is to use salt tolerant plants. Amaranthus tricolor L. has been
Murugan K.
used as a promising plant to ameliorate the salt affected area. The objective of the
study is to evaluate the effect of NaCl stress on synthesis and catabolism of proline,
soluble proteins, carbohydrates and Na+/K+ ratio in A. tricolor and to compare with the
salt-sensitive species, Phaseolus vulgaris L. The experiments were designed with six
Institution: replications. Seedlings of both species were grown hydroponically with 0, 50, 100,
Plant Biochemistry and 150, 200, 250 and 300 mM NaCl. The in vitro activity of the enzyme pyrroline 5-
Molecular biology Lab, carboxylate synthetase under NaCl stress was higher, while, the activity of proline
Department of Botany, degrading enzyme - proline oxidase showed a reverse trend i.e., low activity in high
University College, NaCl concentrations. Soluble protein content was increased in the shoot of A.tricolor
Thiruvananthapuram, but decreased in P.vulgaris. Roots of both the species showed variation in the protein
Kerala-695 034, India. content. Proline content of shoot and roots increased significantly in all the
treatments in the plants. However, A.tricolor showed a higher level. The total
carbohydrate also showed a similar trend. High level of NaCl decreased the reduced
sugar in shoots and roots of the species. Salt stress increased Na + significantly and
decreased the K+ content in both species. The biochemical variation may be
Corresponding author: interpreted as differential response of the plants to NaCl stress.
Murugan K
Email: Keywords:
harimurukan@gmail.com Amaranthus tricolor, ions, proline, salt stress, soluble proteins.
Dates:
Received: 12 Aug 2011 /Accepted: 27 Aug 2011 /Published: 30 Aug 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.
Shoots and root were dried at 70oC for 24 h and extracted with 5 mL of Tris-HCl buffer (pH 8.5)
weighed. Sodium, chloride and potassium were grinding medium and centrifuged at 10,000 g for 10
extracted from each sample in a mixture of HNO3, min at 4C. The supernatant was again centrifuged
HClO4, and distilled water (1: 5: 2.5). The solution at 25,000 g at 20 min at 4C. A 3-mL assay mixture
was kept for 12 h at 100C, diluted with 100 mM was prepared by taking 0.1 mL of extract, 1.2 mL
HC1, and analyzed by atomic emission of 50 mM Tris HCl buffer (pH 8.5), 1.2 mL of 5
spectrometry. Soluble proteins were extracted from mM MgCl2, 0.1 mL of 0.5 mM NADP, 0.1 mL of 1
young shoots and roots in an extraction buffer (0.01 mM KCN, 2 0.1 mL of 1 mM phenazine
M Tris-HCl, 10% glycerol, 5% PVP, 1% Triton X methosulphate (PMS), 0.1 mL of 0.06 mM 2, 6-
100, pH = 6.8) and protein assay was carried out dichlorophenol indophenol (DCPIP) and 0.1 mL
according to the method of Bradford, (1976). distilled water instead of PRO. The reaction was
For proline determination, 10 mL of 3% (W/ monitored at 600 nm at 25C using PRO to initiate
V) aqueous sulfosalicylic acid solution was added reaction; the increase in OD values increase were
to 1 g of fresh shoot and root samples and was noted at 0, 1, 2, 3, 4 and 5 min. PO activity was
homogenized, filtered through one layer of expressed in U (one U = mM DCPIP reduced min-1
Whatmann, No. 1, filter paper, then proline was mg-1 protein).
assayed according to the method of Bates et Five independent determinants from
al ,1973. Total free amino acids were extracted and individual plants were used for statistical analysis.
estimated as per the method of Walter Troll and Students t test and analysis of variance (ANOVA)
Keith Cannan (1952). were used for analyzing significant differences
Carbohydrates were extracted from dry between the control and treated plants (P < 0.05).
shoots and roots of plantlets in warm water.
Concentration of total and reduced sugars were RESULTS AND DISCUSSION
determined based on the methods of Dubois, (1956) Total protein and proline content of
and Jeffries,(1998) respectively. A.tricolor and P. vulgaris shoots were increased
Enzyme extraction was based on the method moderately by salt treatment up to 150 mM NaCl
of Chen et al, (2001). Seedlings were homogenized and thereafter declined sharply at higher NaCl
in prechilled mortar and pestle in the extraction concentrations (Data not shown). In the roots the
medium at 4 C. The extraction medium contained protein and proline content increased at all NaCl
100 mM potassium phosphate buffer (pH 7.4), 1 concentrations tested, although a decline was
mM EDTA, 10 mM 2-mercaptoethanol, 1% (w/v) apparent at 300 mM (Table 1 a and b).
PVP, 5 mM MgCl2, and 0.6 M KCl. The Nevertheless, the protein and proline contents of
homogenate was centrifuged at 12000 g for 20 min severely stressed roots were lower than those of
at 4C, the resulting supernatant was kept at 20C moderately stressed ones, but still higher than the
and used for enzyme assaying. control. However, seedlings of P. vulgaris wilted or
P5CS activity was assayed according to the died at higher NaCl concentrations (100 mM and
method of modified Vogel and Kopac, (1960) as above). Significant positive relationship was
follows. 0.7 mL reaction medium containing 50 examined between salinity and total protein and
mM Tris (pH 7.5), 2 mM MgCl2, 10 mM ATP, 1.0 proline content (P<0.05).
mM NADH, 50 mM glutamic acid, and 0.1 mL The amount of soluble amino acids
enzyme extract was incubated at 37C for 30 min. increased in both plants with increasing NaCl
The reaction was then stopped by adding 0.3 mL of concentration. At the highest concentration,
10% (w/v) trichloroacetic acid. Color reaction was although the total amino acid was clearly built up in
developed by incubating with 0.1 mL of 0.5% (w/v) both the shoots and roots of A. tricolor, their
o-aminobenzaldehyde for 1 h. After centrifugation accumulation was more obvious in shoots (Table 1a
at 12000 g for 10 min, the clear supernatant fraction and b). Similarly, the free amino acids
was taken to measure the absorbance at 440 nm. accumulation was also found in P. vulgaris at the
Enzyme activity was calculated using the extinction higher concentration (100 and 150 mM). The data
coefficient of 2.68 / (mM cm). was statistically significant at 1% level.
Proline oxidase (PO) activity was determined We found significant difference in total
according to the method outlined by Huang and carbohydrate and reduced sugar between the two
Cavalieri, (1979). Plant samples (1 g) were species under NaCl treatment. For example, at 150
Table 1a and b. Amino acid contents (mg/g DW) in NaCl treated A.tricolor and P. vulgaris 14 days after starting
NaCl treatment.
Table 1a
NaCl Roots Shoots
(Mm) Free aminoacids Total proteins Proline Free aminoacids Total proteins Proline
0 2.5 0.9 3.60.5 0.41 3.8.21.9 4.10.3 0.530.01
50 5.90.29 4.00.4 0.520.29 12.72 5.20.09 1.20.03
100 9.20.35 4.80.25 1.50.38 16.93.2 6.50.26 1.51.9
150 11.42.9 5.70.11 2.20.7 230.99 7.80.38 2.92.3
200 122.6 6.30.23 2.20.5 27.80.65 8.50.4 3.33.9
Table 1b
Roots Shoots
NaCl
Total Total
(Mm) Free aminoacids Proline Free aminoacids Proline
proteins proteins
0 3.5 0.91 4.00.5 0.20.45 8.81 4.82.1 0.50.2
50 8.30.8 5.41.5 0.30.12 11.32.1 5.60.7 0.80.34
100 13.10.45 6.92 0.810.33 19.22.7 7.00.9 0.60.5
150 22.40.78 7.21.7 0.60.65 232.9 8.02.8 0.50.8
The data are the mean of six replicates S.E. Significant at 0.01 levels. P < 0.05.
mM NaCl total carbohydrate in A.tricolor 18.6 mg observed in P. vulgaris, while it slightly reduced in
g-1 DW while in P. vulgaris was only 8.4 mg g-1 A.tricolor (Fig. 5). The ion analyses in individual
DW. A similar but moderate pattern of leaf showed that Na+ were tended to accumulate in
carbohydrate changes was observed in roots. the older leaves with higher concentration, but roots
Reduced sugar of shoot and root in both the species in P. vulgaris (Fig. 6 a and b).
was decreased when plants are exposed to NaCl 1-Pyrroline-5- carboxylate synthetase (P5CS)
stress (Fig. 1 and 2). shows a positive correlation with NaCl
Na+ and Cl contents were enhanced with concentrations up to 150 mM in shoot and root of
increasing salinity in roots and shoot of A.tricolor A.tricolor, but P. vulgaris showed decrease in the
and P. vulgaris (Fig. 3 and 4) However, their activity of P5CS at 100 mM NaCl concentration
accumulation was greater in shoots than in roots in (P< 0.05) (Fig. 7). The trend of change in the
A.tricolor. In the case of P. vulgaris the pattern of activity of P5CS corroborates with the proline
accumulation was different i.e. higher in roots than accumulation in different NaCl treatments in the
in shoots. K+ content on the other hand has been species. The level of proline degrading enzyme,
reduced markedly in both the species. Instability of proline oxidase (PO) activity was inhibited to a
Potassium (K+) content in shoots and roots was large extent in both shoot and roots of A.tricolor by
Shoot Tot. Carbo Shoot Tot. Carbo
Total carbohydrates & Reduced
25 10
Reduced sugar (mg/g DW)
7 Root Red.Sugar
Root Red.Sugar
15 6
5
10 4
3
5 2
1
0 0
0 50 100 150 200 0 50 100 150
NaCl concentrations (mM) NaCl concentrations (m M)
Figure 1. Total carbohydrates and reduced sugar in Figure 2. Total carbohydrates and reduced sugar in
shoots and roots from NaCl-treated A.tricolor 18 days shoots and roots from NaCl-treated P.vulgaris 18 days
after starting NaCl treatment. after starting NaCl treatment.
302 Journal of Research in Biology (2011) 4: 299-306
Sunukumar et al.,2011
1200
Na+ and Cl- content
1000
800
600
400
200
0
0 50 100 150 200
NaCl concentration (mM)
+
Figure 3. Ions distribution (Na+ and Cl- content mol/g Figure 5. Ions distribution (K mol/g DW) in NaCl-
DW) in NaCl-treated A.tricolor 18 days after starting treated A.tricolor and P.vulgaris 18 days after starting
NaCl treatment. NaCl treatment.
NaCl stress (Figure 8), with the lowest activity was vacuole and balancing by compatible solutes within
recorded at 150 mM of NaCl concentrations (50 the cytoplasm was reported as the important salt
mM in P.vulgaris) compared with control. tolerant mechanisms at cellular level (Bremberger
In plants, higher accumulation of Na+ and and Luttge 1992). It may likely indicate that
Cl requires compatible solutes for osmotic A.tricolor has an ability to translocate the ions and
adjustment under salinity stress (Kavi Kishor et al., hold them in the leaves. This physiological process
2005). In this study, accumulation of Na+ and Cl may be important to reduce the salt toxicity in and
found in the species as a response to changing away from the root cells. As a result the plant could
salinity stress levels. However, a different pattern of
Na+ and Cl distribution in each part of the two
species was clearly seen with increasing salinity.
A.tricolor has a higher accumulation of the ions in
the shoots, but P. vulgaris in roots. The ability to
translocate Na+ and Clfrom roots and transfer them
to shoots is considered to be one of the mechanisms
of salt tolerance in A.tricolor. High accumulation of
the excessive salts in shoots indicates there is some
mechanism to reduce detrimental effect of the ions
in the shoot cells. Localizing Na+ and Clin the
Shoot (Na+) Root(Na+)
1400 Shoot(Cl-) Root(Cl-)
1200
Na+ and Cl- content
1000
800
600
400
200
0
0 50 100 150 200
Figure 6 a and b. Distribution of Na + and Cl- in leaves
NaCl concentrations (mM)
(a and b), and 1st 6th leaves of A.tricolor 14 days after
Figure 4. Ions distribution (Na+ and Cl- content mol/g starting NaCl treatment. 0 mM, 50 mM, 100
DW) in NaCl-treated P.vulgaris 18 days after starting mM, 150 mM, 200 mM of NaCl). Vertical bars
NaCl treatment. indicate standard errors (n = 3).
Huang AHC, Cavalieri A.1979. Proline oxidase Walter Troll, Keith Cannan R. 1952. A modified
and water stress induced proline accumulation in photometric ninhydrin method for the analysis of
Spinach leaves. Plant Physiol., 63:531-535 amino and imino acids. J of Biol. Chem., 23:803-
811.
Jaleel CA, Manivannan P, Lakshmanan GMA,
Sridharan R, Panneerselvam R. 2007e. NaCl as a
physiological modulator of proline metabolism and
antioxidant potential in Phyllanthus amarus.
Comptes Rendus Biologies. 330:806-813.