Open
access
JTBSRR
Journal
of
Today’s
Biological Sciences :
Research
&
Review
(JTBSRR)
ISSN 2320-1444 (Online)
Published on : 30.12.2012
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Welcome Message from Editor-in-Chief
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Dr. Debashri Mondal
Editor –in- Chief
Journal of Today’s Biological Sciences: Research & Review (JTBSRR)
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Journal of Today’s Biological Sciences: Research & Review (JTBSRR)
ISSN 2320-1444 (Online)
JTBSRR
Vol.1, Issue 1, page 2-15, December 30, 2012
Available on: www.jtbsrr.in
Development of Byproduct and Nutritious Food
Industry Waste-Based Low-Cost Fish Feed
Santosh Zargar, G.V. Mulmuley and Tarun Kanti Ghosh*
National Environmental Engineering Research Institute, Nehru Marg, Nagpur–440 020
Email: tk_ghosh@neeri.res.in; ghoshtk2@rediffmail.com
Abstract
Feed is one of the major inputs in aquaculture, and the success of fish farming depends
primarily on the provision of adequate quantity of nutritionally balanced feed in a form,
acceptable to fish. Under the present investigation, four diets (feeds I, II, III and IV) were
formulated using commonly available grains (maize and soybean), nutritious wastes
(poultry and silkworm) and a by-product (de-oiled rice bran). Experiments were
conducted to evaluate acceptability of different formulated diets by Indian major carp
Catla catla in plastic pools. The values of NWG, PER and FCE were found in the order:
Feed V > Feed II > Feed III > Feed I > Feed IV. Feed V, made of nutritionally rich costly
ingredients, was procured from CIFE. The outcome of a field trial, using feeds II and IV
separately on brood fish in natural ponds, resulted in 100 percent increased growth with
feed II over those fed by conventional feed (feed IV). The cost of the feed II was estimated
to be around two-third of the similar kind of feed, available in the market. The study could
provide the technology to local people for their employment generation, pollution free
environment by utilizing nutritious waste materials in the fish feed and minimization of
incorporating presently practiced grains in fish feed, so that paucity of food grains in
developing countries can be partially solved.
Keywords: Formulation, Byproduct, Nutritious Waste, Fish Feed, Catla catla
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Introduction
Fish, like any other animal, require nutrients to remain healthy and active for
which nutrients play a vital role. Nutrients can be provided by various types of
food items, which are determined both by the feeding behavior or preference of
the animal and the availability of items. Growth and/or net nutrition deposition
is the most accurate and important tool in studying fish feed efficiency and
nutrient requirements (Das et al., 1991). Feed is one of the major inputs in
aquaculture, and the success of fish farming depends primarily on the provision
of adequate quantity of nutritionally balanced feed in a form, which is acceptable
to fish. The traditional feed, presently used in composite fish culture of carps,
comprises of rice bran and groundnut/soybean oilcake in equal proportions.
These are being used only as supplementary feeds and the availability of natural
protein in the form of plankton is essential. The shortcoming of such feeds in
terms of quality becomes obvious when the plankton availability in the pond is
insufficient. Moreover, traditional feed lacks desired levels of protein,
carbohydrate, vitamins and minerals.
Since the livestock, especially poultry, pig and fish compete with humans for
high quality protein; the demand for grains and cereals is high. Developing
countries face nearly 50% deficiency in concentrated feed which is expected to
increase further in forthcoming years (www.tifac.org.in/offer/tlbo/rep/
TMS162.htm). This deficiency in food production can be removed only when
there are alternate feed resources for reducing the composition of crop products
in the animal feed and substituting these with unconventional ingredients like
nutritionally rich waste materials. In view of this, the use of animal waste or its
content in fish feed needs to be enhanced. This will not only reduce the
consumption of grains but also increase the nutritional value of feedstuff. The
animal tissues contain numerous proteolytic enzymes, which help in increasing
the digestibility of the feed.
Fish feed products are mostly based on ingredients derived from crop residues.
On the other hand, vast resources of slaughterhouse/poultry/silkworm byproducts, which are termed as wastes and are rich in proteins and minerals, are
freely available in plenty in most of the countries. Utilization of these so called
waste materials in the production of fish feeds will not only solve the problem of
nutritional needs of fish but also help in reducing environmental pollution,
caused by the discarded animal tissues, scattered all over the rural as well as
urban areas, leading to public nuisance and vector breeding centers.
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The present study aimed at formulating fish feeds, comprising of byproduct and
nutritious food industry waste-based materials and also to improve the quality of
conventional feed, used by pisciculturists. The study could provide a technology
to local people for their employment generation; provide a pollution free
environment by preventing scattered nutritious waste materials; conversion of
waste to wealth and a solution to minimize incorporation of grains in fish feed
by replacing these with waste materials, thereby partially solving the problem of
grain paucity in developing countries.
Materials & Methods
Fish Feed Formulation& Preparation
Feed formulation is the process of designing a mixture that will meet the
nutritional requirement of the animal while taking into account certain practical
consideration (acceptability of mixture to the animal, ability of the mixture to be
pelleted, cost, etc.). The four prepared diets (feeds I, II, III, IV) were formulated
using locally available ingredients and nutritious wastes as shown in Table 1.
Table 1. Percent composition of ingredients used for preparing feeds
Ingredients
Feed I
Feed II
Feed III
(%)
(%)
(%)
Feed IV
(%)
Soybean
35.0
45.0
35.0
-
Rice bran
30.0
15.0
30.0
50.0
DOC Soybean
-
-
-
50.0
Maize Powder
10.0
10.0
10.0
-
Bakery waste
14.0
14.0
14.0
-
Poultry waste
-
5.0
-
-
Silk-worm waste
-
5.0
5.0
-
0.1
0.1
0.1
-
5.0
5.0
5.0
-
Vitamin
mineral
mixture
Starch (binder)
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For preparation of pelleted fish feed, the equipments, like Pulverizer, Extruder
and Packaging machine (Fig. 1 to 3) were used. Corn starch (50 g) was boiled at
100oC with 250 ml water and was mixed with grinded ingredients. Grinding
increases the nutrient availability of feed, and reduced particle size augments
activity of proteolytic enzyme released during tissue grinding. As a pretreatment, the soybean seeds were soaked (overnight: 6 hours) in water,
blanched (boiling water: 20 minutes), de-husked and sun-dried. The treatment
destroyed trypsin inhibitors of soybean meal, thus increasing its nutritional
value. Nutritious wastes, viz. poultry, slaughter house and silkworm, were
cleaned and sun-dried before processing. Dough was prepared and extruded
through 2 mm diameter die of a hand operated Extruder. The extruded moist
noodles were sun-dried for a day, pelletized manually, graded by using sieves,
and then stored in airtight jars/polythene bags at room temperature. Proximate
analysis of major ingredients was undertaken in the laboratory following the
method of Association of Official Analytical Chemists (Anonymous, 1980).
A balanced feed (Feed V) comprising of soybean, fish meal, corn meal, coconut
oil, wheat flour, cod liver oil, sunflower seed, CMC binder and vitamin C at 44,
10, 4.5, 9.5, 4.5, 2.0, 2.0, 2.0 and 0.1 percent respectively was procured from
Central Institute of Fisheries Education (CIFE), a deemed University in Mumbai.
The crude fat levels in feeds I through V were 12.2, 12.8, 13.7, 9.7 and 9.0 percent
respectively.
Fig. 1. Pulveriser used for
grinding the feed ingredients
Fig. 2. Hand operated Extruder
used to make pellets
Fig. 3. Packaging machine
for sealing the packed feed
Design of experiment
Experiment was designed to determine the acceptability of different formulated
diets by select fish (Catla catla). The fish (average weight 20+ 2.0 g) were procured
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from Mansar hatchery farm, Nagpur (Maharashtra, India) and were acclimatised
in experimental condition for a week. Experiment was conducted in triplicate in
plastic tanks of 500 L capacity. Each tank was stocked with fifteen fishes. Prior to
the commencement of experiment, the pools were thoroughly cleaned, filled with
water and stocked with pre weighed acclimatized fish fingerlings. Special care
was taken to avoid escape of the fish during the experiment. The feeding
experiment was conducted for 30 days. In order to maintain the water quality as
good as possible, about one third water from each tank was replaced by
dechlorinated tap water every morning. Since feeding frequency plays an
important role on growth, the fish were fed once a day at the rate of 2% of body
weight throughout the experiment. Any uneaten food and fecal matter from each
pool were removed daily by siphoning method.
Dietary Performance Evaluation
Relevant water quality parameters, viz. temperature, pH, hardness, alkalinity
and DO were analysed (APHA, 2005) during the experiment on alternate days.
Dietary performances were evaluated through following nutritional indices
(Rajan et al., 1996).





Net Weight Gain percent (NWG percent) = (Initial weight/ Final weight) x 100
Specific Growth Rate (SGR) = [ (Log final weight - Log initial weight)/ Number
of days] x 100
Feed Conversion Ratio (FCR) = Amount of dry feed provided/ Live weight gain
Protein Efficiency Ratio (PER) = Gain in body weight/ Protein intake
Food Conversion Efficiency (FCE) = Wet weight gain/ Dry weight of feed given
Note: All weights are expressed in gram
Results and Discussion
Water Quality
The water characteristics during the study period were in the ranges: pH 7.8 +
0.2 to 8.0 + 0.2; alkalinity (mgL-1) 256.1 + 9.5 to 276.2 + 3.4; dissolved oxygen
(mgL-1) 5.3 + 0.5 to 6.0 + 0.4; and total hardness (mgL-1) 120 +5.0 to 136 + 6.0.
Since fish are poikilotherm, water temperature plays an important role in energy
partitioning, protein assimilation and growth (Swamy and Devraj, 1994). Water
temperature varied from 23.8 + 4.0 to 26.7 + 0.5oC. The results of water quality
parameters were within the recommended (CIFE, 2003) ranges for aquaculture
JTBSRR 1(1): 2-15, 2012
ISSN 2320-1444
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practices (pH: 6.7-9.5; alkalinity: 50-300 mgL-1; dissolved oxygen: 5-10 mgL-1 and
total hardness: 30-180 mgL-1). It appears that the growth of fish was not
influenced by the variations recorded in abiotic parameters of water during the
experiment.
Dietary Performance
Growth, in terms of net weight gain, was maximum (50.0 ± 5.0 percent) in fish
feed V and least in feed IV (13.3 ± 1.0 percent) during the study period (P< 0.05)
(Table 2). SGR, PER and FCE were also highest in fish feed V (0.61, 2.77 and 83.33
respectively, Table 2). SGR can be considered as an index of growth evaluation of
diets; however, higher FCE signifies better utilization of the consumed matter.
FCR (1.20) and PER (0.85) were found to be less for fish feeds V and IV
respectively as compared to other fish feeds. FCR values were found to vary
from 1.20 (feed V, 30.0 percent protein) to 4.5 (feed IV, 26.2 percent protein) in
different diets. Earlier, Choudhury et al. (2002) reported varying FCRs from 1.29
to 1.95 in different formulated diets and at different feeding frequencies.
Obviously, different authors reported varied FCR values on different fishes, such
as, 1.12 to 1.35 for Cyprinus carpio with formulated diet (38.74% protein level) at
five times feeding (Capper et al., 1982), 3.68 to 3.82 for L. rohita (23.9% protein
level) in cages (Ahmed et al., 1983) and 3.08 in L. rohita for diet containing 33.92%
protein (Rangacharyulu et al., 1991). Even though theses feeds were not wastebased, the values were comparable to the findings of the present study.
A superior growth performance was noted in fish, fed with feed I as compared to
those on feed IV in spite of the lower protein content (23.1 percent) in the former.
This observation draws attention to two essential inferences; viz. the crude
protein percent in feed IV might be having higher content of indigestible protein
and lower crude lipid percentage in feed IV might have played a crucial role in
feed utilization. The values of NWG, PER, and FCE were found in the order:
Feed V > Feed II > Feed III > Feed I > Feed IV. The minimum growth was found
in fish, fed with feed IV, which contained rice bran and DOC soybean (50:50
percent). There was no mortality of fish during the period of experiment
conducted. The results indicated that animal-based fish feeds like poultry and
silkworm (feed II) and silkworm (feed III) wastes were digested efficiently and
were comparable to balanced fish feed (V) than the other feeds (I and IV).
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Table 2. Feed utilization parameters selected for assessment of the feeds
Diet
NWG
SGR
FCR
PER
FCE
26.7
0.33
2.25
1.90
44.44
Feed II
46.7
0.56
1.28
2.8
77.77
Feed III
33.3
0.44
1.80
2.04
55.55
Feed IV
13.3
0.19
4.5
0.85
22.22
Feed V
50.0
0.61
1.20
2.77
83.33
(%)
Feed I
In the present study, one byproduct (rice bran) and three types of nutritious
wastes, viz. bakery, poultry and silkworm were used. Studies revealed that
growth rate of fish, grown on feed II is comparable to those of feed V (P> 0.05).
Considering cost involved in feed V, subsequently field trial was given in natural
ponds. While composition details of the byproduct/wastes are mentioned in
Table 3, proximate and amino acid compositions of low-cost fish feed II (Fig. 4.),
based on byproduct and nutritious industry wastes are presented in Tables 4 and
5. The feed comprises of soybean (45 percent), rice bran (15 percent), maize (10
percent), bakery waste (14 percent), poultry waste (5 percent), silk worm waste (5
percent), vitamin mineral mix (0.1 percent) and starch (5 percent).
Table 3: Nutrient values of byproduct and wastes used for the study
Sl. No. Parameters
1
2
3
4
5
Rice
bran
15.3
15.92
Silkworm
waste
54.2
30.3
Protein (%)
Fat (%)
Crude fibre
8.44 3.9
(%)
Ash (%)
6.7
5.2
Carbohydrate
53.64 6.4
(%)
Poultry
waste
59.9
17.1
Bakery
waste
10.7
12.7
2.1
0.4
15.5
3.8
5.4
72.4
The nutritious rice bran, being rich in protein and having better amino acid
profile, is produced in great quantities as a by-product of the rice milling
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industries. Further, the wastes from poultry/silkworm/bakery are better sources
of proteins and minerals, and are also available at almost freely in developing
countries. Maize is a relatively poor cereal, although it possesses limiting
amounts of two essential amino acids, viz. lysine and tryptophan (Hanifa et al.,
1987).
Fig. 4. Pellets of recommended Fish Feed II
Among the protein sources of plant origin, soybean (Glycine max) is reported to
have greater value as fish feed ingredient because of its easy availability, better
chemical composition, amino acid balance and relatively low cost (Takeshi and
Juadee, 1993). However, soybean was found deficient in two essential amino
acids- lysine and methionine (Viola, 1982), and is also known to have certain
anti-nutritional factor, responsible for growth depression (Wilson and Poe, 1985).
Studies on Essential Amino Acid (EAA) requirement of Indian major carps were
conducted using synthetic diets. Singh et al. (1986) observed that maximum total
EAA level required in the synthetic diet for rohu fry was 27.4 percent, of which
1.58 percent was methionine along with cystine, and 3.83 percent was lysine.
These are very similar to that used by Nose et al. (1974) for common carp, where
total maximum EAA was 28.95 percent having methionine and cystine 1.76
percent and lysine 3.57 percent. This indicated that EAA requirement of rohu
might be very similar to common carp.
Developed feeds possess required vitamins and minerals (Table 4). It may be
added that vitamin requirements of fishes are dependent on size, age and growth
rate of fishes, besides environmental factors and nutrients. However, vitamin E
(Tocopherol) protects highly unsaturated fatty acids in lipids of biological
membranes from oxidation in the presence of molecular oxygen, and both water-
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Table 4. Proximate analysis of recommended fish feed (Feed II)
Sl.
No.
Parameters
Values
1
Moisture, % by wt.
6.0
2
Total ash, % by wt.
5.7
3
Protein, % by wt. (N x 6.25)
29.5
4
Fat,% by wt.
11.3
5
Crude fibre, % by wt.
5.5
6
Carbohydrates (by difference)*, % by wt.
42.0
7
Calorific value*, (K.cals/100gm)
388
9
Vitamin B1 , mg/100gm
0.2
10
Vitamin B2, mg/100gm
0.6
11
Vitamin C, mg/100g
3.1
12
Tocopherols, mg/100gm
13.2
13
Calcium, mg/100gm
35.1
14
Chromium, mg/100gm
Below
detection limit
of 0.05
15
Copper, mg/100gm
1.6
16
Iron, mg/100gm
18.0
17
Magnesium, mg/100gm
484
18
Manganese, mg/100gm
9.6
19
Potassium, mg/100gm
2094
20
Sodium, mg/100gm
87.2
21
Zinc, mg/100gm
18.1
22
Phosphorus, mg/100gm
773
23
Chlorides, % by wt. (as NaCl)
0.9
*
Carbohydrates (by difference) =100 - (moisture + total ash + protein + fat + crude fiber); calorific value (kcal/100gm)
= (4 x protein %) + (4 x carbohydrates %) + (9 x fat %)
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Table 5. Amino acid composition of recommended fish feed (Feed II)
Sl.
Parameters
Values
1
Aspartic acid
8.49 + 0.34
2
Glutamic acid
18.86 + 0.07
3
Serine (Ser)
3.82 + 0.25
4
Glycine(Gly)
4.97 ± 0.05
5
Histidine (His)
2.48+0.05
6
Arginine (Arg)
6.95 + 0.14
7
Threonine (Thr)
3.72 + 0.12
8.
Alanine(Ala)
3.97 + 0.09
9
Proline (Pro)
6.76 + 0.02
10
Tyrosine (Tyr)
0.82 + 0.04
11
Valine (Val)
5.57 + 0.08
12
Isoleucine (He)
5.49 + 0.22
13
Leucine (Leu)
9.05 + 0.12
14
Phenylalanine (Phe)
5.03 + 0.06
15
Lysine (Lys)
5.43 + 0.07
16
Cysteic
6.73 + 0.55
17
Methionine
1.87 + 0.07
No.
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soluble and fat-soluble vitamins are required for healthy growth of mrigal
(Cirrhina mrigala) and rohu (Labeo rohita). The growth rate of rohu fingerlings was
found to be significantly increased on addition of water-soluble and fat-soluble
vitamins (Singh et al., 1986). Four fat-soluble and eleven water-soluble vitamins
and vitamin-like compounds are essential to fish. Minerals play a great role
within the animal body. Minerals are important nutrients as these are required
for normal bone, tissue, blood plasma and hemoglobin formations and also for
many enzymatic reactions. Calcium (Ca) and Phosphorus (P) are required for the
formation of skeletal tissues. Fishes absorb a good quantity of calcium from
water and rest from diet but most of the phosphorus should be provided through
diet. For common carp, the minimum requirement of calcium in diet is about
0.028 percent and that of phosphorus is 0.6–0.7 percent. Trace elements, required
in traces, are growth stimulants. Elements like manganese, copper, iron, cobalt,
iodine and zinc are required in minute quantities in balanced ration, mainly for
improving protein assimilation and survival rates (Das and Kaviraj, 1994)). Since
fish obtain minerals from their diet and also from the environment, it may be
mentioned that all the desired minerals and trace metals at specific levels are
available in recommended fish feed II (Table 4).
Feeding Trial by Fishery Department
In order to assess the quality of the developed feed (II) in natural condition and
also to have views from State Fishery Department on the feed, experiment was
conducted in natural ponds for 52 days by District Fisheries Department,
Bhandara (DFDB, Maharashtra, India). For this purpose, 100 kg feed was
provided to DFDB for conducting experiment on two varieties of brood fish (
Labeo rohita and Catla catla: 10 nos. each) in two natural ponds (area: each of 51 x
26 x 1m) at fish hatchery farm, located in Iteadoh, District Gondia, using two
different types of feed (feed II and conventional feed, that is, mixture of rice bran
and oil cake) in respective ponds. Both the feeds were dispensed into the
respective ponds measuring at three percent of total fish body weight per day
during the experiment. The studies revealed that the increase of growth of fish,
fed by feed II was doubled over those, fed by conventional feed. Further, the
release of eggs, after artificial breeding, was eleven percent more in fish, grown
on feed II. However, hatching rate was 65 percent in both the cases. Considering
better result in natural ponds, it is recommended to adopt the composition of
feed II as ingredients of low-cost feed, and convert waste to fish flesh.
Evaluation of Cost & Quality
It may be mentioned that production cost of the developed feed (Indian Rs. 25/per kg or around US Dollar 0.55 per kg) is about two-third or less of the similar
kind of feed, available in the market. The cost of the feed procured from CIFE
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was higher, since none of the ingredients (soybean 44 percent, fish meal 10
percent, corn meal 4.5 percent, coconut oil 9.5 percent, wheat flour 4.5 percent,
cod liver oil 2.0 percent, sunflower seed 2.0 percent, CMC binder 2.0 percent and
vitamin C 0.1 percent) are wastes. It may be added that a byproduct is a
secondary and incidental product from a manufacturing process and is not the
primary product. A byproduct or waste can be procured at a reduced price or
free of cost, and be used as raw material of other product. The cost of feed for
prawns/shrimps is still higher. The cost of the developed feed was substantially
reduced by incorporating animal wastes and byproducts, instead of costly
ingredients, which are normally used in prevailing feed industry.
In order to avoid exorbitant cost of marketed fish feeds, farmers commonly use
traditional feed, comprising of agro products like rice bran and
groundnut/soybean oilcake. As per normal practice, a mixture (50:50) of the feed
is spread over the pond for feeding the fish. In such cases, most of the feed is
settled at the bottom and are unused. Moreover, by this method fish do not get
balanced feed, required for their growth and reproduction. The large
entrepreneurs often use costly feed, made of agro products, viz. maize, soybean,
corn meal, coconut oil, wheat flour, sunflower seed etc. This enhances the cost of
the feed, resulting in non-acceptance of fish feed pellets by economically poor
farmers. In the present study, agro products/byproduct were supplemented to
improve the quality of the conventional feed. The invented pelletized fish feed
has essential nutrients for proper growth of fish.
Conclusion
Present study dealt with different formulated fish feeds using select byproduct
and nutritious food industry waste based materials. The cost of the developed
feed, comprising of soybean (45 percent), rice bran (15 percent), maize (10
percent), bakery waste (14 percent), poultry waste (5 percent), silk worm waste (5
percent), vitamin mineral mix (0.1 percent) and starch (5 percent) has been
worked out to be about two-third of the similar kind of feed, available in the
market. The study could provide the technology to local people for their
employment generation, pollution free environment due to utilization of
scattered nutritious waste materials in the form of fish feed and solution to
minimize the incorporation of grains in fish feed by replacing these with
nutritious industry waste materials leading to scope in solving the problem of
grain paucity in developing countries.
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Acknowledgements
The authors are grateful to the Director NEERI, Nagpur for infrastructure
facilities, to Dr. T. Chakrabarti, Scientist H, NEERI, Nagpur for helpful
suggestions and Mr. R. L. Lonkar, DFDO, Bhandara for onsite experimental
support during the investigation. We acknowledge the financial support from
Department of Biotechnology(DBT sanction No.: BT/PR4284/SPD/09/333/2003)
New Delhi, Government of India.
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14: 60-66.
Rangachoryulu, P.V., Sarkar, S., Mohanty, S.N. & Mukhopadhyay, P.K. (1991).
Growth and protein utilization in rohu (Labeo rohita) under different feeding
levels. National Symposium on New Horizons in Freshwater. Aquaculture, 95-97.
Singh, B.N., Singh, V.R.P., Kumar, K. & Swamy, D.N. 1986. Observations on the
feed formulations and fortification of conventional fish feed for rohu and mrigal
fingerlings. Int. J. Acad. Ichthyol., 7:31-34.
Swamy Vijaykumar, H.V. & Devraz, K.V. 1994. Growth response to fry of the fish
Catla catla fed on three formulated feeds. Environ. Ecol., 12/3:519-523.
Takeshi, W. & Juadee, P. 1993. Potential of Soybean meal as protein source in
extruded pellets for Rainbow Trout. Nippon Suisan Gakkaishi., 59/8: 1415-1423.
Viola, S. 1982. Partial and complete replacement of fish meal by soybean meal in
feeds for intensive culture of carp. Aquaculture, 26:223-226.
Wilson, R.P. & Poe, W.E. 1985. Effects of feeding soybean meal with varying
trypsin inhibitor activities on growth of fingerling channel catfish. Aquaculture,
46:19-25.
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16
Journal of Today’s Biological Sciences: Research & Review (JTBSRR)
ISSN 2320-1444 (Online)
JTBSRR
Vol.1, Issue 1, page 16-19, December 30, 2012
Available on: www.jtbsrr.in
Ethno-Botanical Investigation on Important Wild
Edible Herbs of Siliguri Subdivision of Darjeeling
District, West Bengal, India
Tamal Mondal
Assistant Professor, Department of Botany, Netaji Mahavidyalaya, Arambagh, Hooghly,
West Bengal, India, Email- tamalmondal1@gmail.com
Abstract
The present study deals with the identification and ethno-botanical investigation with
respect to food value of wild edible herbs from Siliguri Subdivision Area. A total number
of 19 plants belonging to 15 families are found as wild edible herbs. Edible herbs are not
only delicious but also important for their nutritional value as well as their medicinal
poperties.Special steps should be taken for this cheap source of natural food for a better
tomorrow.
Key words: Ethno-botany, wild edible herbs, food source, cheap diet, nutritional value
Introduction
Plants are source of food since ancient times. Wild plants serve an important role
to fulfillment of the diet of local habitat. Wild edible plants are not only
important for their food quality but also a large contribution to the population’s
nutrition throughout the year (Sasi and Rajendran, 2012; Katewa, 2003; Grivetti
and Ogle Britta, 2000; FAO, 1999).Now a days wild edible plants play an
important role as a cheap source of valuable food. To identify the wild edible
herbs, a short investigation has been done in the area of Siliguri Subdivision, the
foot hills of Darjeeling Himalaya. It was found that 19 wild plants belonging to
15 families are used, collected, marketed, and cooked as vegetables by the local
communities.
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Materials & Methods
The study area, Siliguri Subdivision is situated at 26º43´N latitude and 88º25´E
longitude within Darjeeling District of West Bengal,India.The Subdivision
contains 22 gram panchayats(rural area) under four community development
blocks; Matigara,Naxalbari,Phansidewa,and Kharibari, including SMC(Siliguri
Municipal Corporation).During this field trips to the rural area as well as urban
market place the wild edible herbs were collected and enlisted and also their
uses were recorded through questionnaires with the local poor people, and tribes
related with those wild edible herbs, specially older and aged people of this
Subdivision.The collected specimens were preserved as a voucher specimen for
further identification by standard herbarium technique (Jain and Rao,1977).Next
crucial step is to proper identification of the collected herbs, for this authentic
literature (Prain,1963; Bennet,1987) were used to do so.
Results and Discussion
The wild edible herbs of Siliguri Subdivision,their scientific name,family in
which they belongs,their local names along with their edible portion as well as
their medicinal properties are presented in table 1.
Table 1. List of collected wild edible herbs of Siliguri Subdivision
Sl.
No.
1.
2.
3.
4.
5.
6.
7.
Scientific
Name
Amaranthus
tricolor
Amaranthus
viridis L.
Amorphophallu
s muelleri BI.
Bacopa
monnieri (L.)
Pennell
Brassica
Campestris L.
var.cuneifolia
Roxb
Cannabis sativa
L.
Centella asiatica
(L.)
Family
Amaranthaceae
Amaranthaceae
Araceae
Local
Name
Lal sak
Bon
notey
Ool
Edible
parts
Leaves
plant Medicinal
Uses
Shikness,cou
gh and cold
Whole plants
Piles,shikness
Scrophulariacea
e
Bramhi
Leaves,Rhizo
me
Leaves
Cruciferae
Lai sak
Whole plants
Cough
cold
Cannabinaceae
Vang
Leaves
Pain relief
Apiaceae
Thankuni Whole plants
17
Blood
purifier
Memory loss,
ill health
and
Diarrhea and
Dysentery
18
8.
Chenopodium
album L.
Chinopodiaceae
Bethua
sak
Whole plants
Digestive
problems
9.
Coccinia
cordifolia W. &
A.
Colocasia
esculenta
Schott.
Enhydra
fluctuans Lour.
Glinus
oppisitifolius
(L.) A.DC.
Hygrophila
schulli (BuchHam.) M.R.et
S.M.Almeida
Ipomoea
aquatica Forsk.
Cucurbitaceae
Kudru
Fruits
Araceae
Kochu
Whole plants
Digestive
problems,dys
entry
Blood
purifier,
Asteraceae
Helancha
/Hincha
Gima sak
Leaves
Acanthaceae
Kulekhar
a
Whole plants
Shikness,pain
reliever
Convolvulaceae
Kalmi
sak
Whole plants
Blood
purifier,
15.
Luffa cylindrica
(L.) Roem.
Cucurbitaceae
Dhudul
Fruits
Digestive
problems
16.
Marsilea
quadrifolia L.
Mentha
arvensis L.
Marsileaceae
Susni sak
Leaves
Skin troubles
Lamiaceae
Pudina
Whole plants
Digestive
problems
10.
11.
12.
13.
14.
17.
Aizoaceae
Whole plants
Skin diseases,
insomnia
Cough
and
cold, fever
18.
Nymphaea alba Nymphaeaceae
L.
Saluk
Leaf petiole
Bleeding
problems
19.
Typhonium
trilobatum
Schott.
Kharkon
kachu
Leaves
Blood
purifier
Araceae
Conclusion
These types of field trips are extremely necessary for proper documentation of
wild edible plants of natural origin. These plants are gifted by our mother nature,
and a vast research is required towards the proper use of this plants.
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Acknowledgement
I am very much thankful to Prof.B.N.Mondal,(Retd.),Shivmandir,Siliguri,West
bengal for his kind help and suggestion for preparation of this manuscript.
References
Bennet,S.S.R. 1987 .Name Changes in Flowering Plants of India and Adjacent
Regions,Triseas Publishers,Dehra Dun,India.
FAO (1999) .Use and potential of wild plants. (Information Division,Food and
Agricultural Organization of the United Nations,Rome,Italy).
Jain, S.K.and Rao, R.R.1977.A hand book of field and herbarium methods,Today
and Tomorrow’s Printers and Publishers, New Delhi and Calcutta.
L.E.Grivetti and M.Olga Britta (2000).Value of traditional foods in meeting
macro-micronutrient needs: the wild plant connection .Natl Res.Rev. 13:31-46.
Mondal,T. (2012).Survey of some important ethno-medicinal plants of Siliguri
Subdivision,Darjeeling,West Bengal.Life sciences Leaflets 8 :24-27 ,2012.
Prain, D.1996.Bengal Plants (Vol 1 & 2),Bishen Singh Mahendra Pal Singh, Dehra
Dun ,India.
S.S.Katewa (2003).Contribution of some wild food plants from forestry to the diet
of tribal of Southern Rajasthan.Ind.Forest.129 (9): 1117-1131.
Sasi,R. and A.Rajendran (2012).Diversity of wild fruits in Nilgiri hills of the
Southern Westran Ghats –Ethnobotanical aspects.IJABPT ,Vol 3 issue 1,82-87.
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Journal of Today’s Biological Sciences: Research & Review (JTBSRR)
ISSN 2320-1444 (Online)
JTBSRR
Vol.1, Issue 1, page 20-27, December 30, 2012
Available on: www.jtbsrr.in
Application of Solar Energy for Semiprocessing of
Curcuma Longa (Haldi) and Maranta Arundinacea
(Arrowroot) as a Source of Additional Livelihood
and Entrepreneur Activity in the Selected Villages
of Phanda Block of Bhopal District
Padma Harshan, Madhavi Muchrikar, Uttam Nagwanshi and
Manoj Chourasiya
Society for Human Welfare & Environmental Furtherance (SHWEF) Plot No.277, F-4,
A Sector, Sarvadharma Colony, Kolar Road, Bhopal 462 042, Madhya Pradesh, INDIA.
E. mail- shwef267@gmail.com, shwef17@rediffmail.com
Abstract
India has a major agribusiness sector, which has achieved remarkable successes over the
last three and a half decades. The right post harvest practices such as good processing
techniques, and proper packaging; transportation and storage (of even processed foods)
can play a significant role in reducing spoilage and extending shelf life. The Indian
Government has formulated Vision 2015, to triple the size of the food processing
industry, from the current $ 70 b to around $ 210 b, enhancing her global share to 3%,
increasing value addition to 35%, from the current 20% and raising the level of
processing of perishables to 20%. There are about 300 clear sunny days in most parts of
the country. Solar energy4 can supply and/or supplement many farm energy
requirements.Our project catalyzed and supported by the Ministry of Science and
Technology, SSD, New Delhi, on non chemical farming methods of Curcuma longa
(turmeric)1 and Maranta arundinacea (arrowroot) in Dehrikalan village of Phanda block
of Bhopal, M.P. has given a ray of hope that on a very small and pilot scale the
agricultural applications of solar use for semi processing can be demonstrated to the
beneficiary partners by various means to understand the importance of the same thereby
opening new doors to generate employment opportunities with enhanced income to rural
youth both men and woman to learn and take up trainings. More importantly, the
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21
income generated out this enterprise will keep circling within the local economy and open
new marketing channels for the young rural entrepreneurs.
Keywords: Demonstration, solar equipments, semi processing, additional livelihood,
rural entrepreneur.
Introduction
Agriculture is the backbone of Indian economy, contributing about 29.1 per cent
towards GDP, providing livelihood to about 65 to 70 per cent of population and
nearly 20 per cent of export earnings. It should be made a remunerative option.
The vibrant agriculture markets including derivatives markets are the frontline
institutions to provide sign of future prospect of the sector. Under the current
situation it is better that farmers shift to organic farming in large areas. Globally,
organic foods are in great demand under the changing lifestyle. Organic seal
implies no use of pesticides. As the organic quality assurance in India is still at
the nascent stage, it is useful to learn from the International arena.
India's food processing industry is expected to benefit from this and grow to
around $260-billion from the present USD 200-billion in the next 6-years,
according to industry expert. In India, only 6% of total agro output of India is
currently processed as against 80% in some developed countries leaving a large
potential to be tapped in this sector.
India has a major agribusiness sector, which has achieved remarkable successes
over the last three and a half decades. The Indian government has formulated
Vision 2015, to triple the size of the food processing industry. In India, only 6% of
total agro output of is currently processed as against 80% in some developed
countries leaving a large potential to be tapped in this sector.
At a time when a debate is raging on whether non-chemical farming (organic)
can produce enough food to feed the world, compared to chemical farming,
experts point to the fact that non– chemical method of practices definitely bring
down the input costs required for cultivation (though some say they are labour
intensive).
Continuous and unbalanced use of chemical fertilizers6 is leading to decrease in
nutrient uptake efficiency of plants resulting in stagnation of decrease in crop
yield. At the same time, these fertilizers are costlier and also pollute the
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22
environment through the process of de-nitrification, volatilization and ground
water through leaching. Hence, efforts are to be made to tap new source of
nutrients, which will be economical and may cerate a pollution free environment.
Therefore, more attention is being paid towards the use of bio fertilizers in crop
production.
Cultivating is futile without processing and value addition, especially when our
farmers are struggling to find ways to increase their income from farms. Local
value added agricultural food production is an important strategy to sustain
many small, marginal and agricultural laborers. What farmers need at the
moment is a low budget technology that is efficient and proven. With more than
80 per cent of our farmers having small holding (3-4 acres), the need for such
technology is imperative. Along with this a critical challenge is to ensure that the
expanding use of good agricultural practices (GAP) should be taken into account
the interest of small-scale producers both for the safety, economy and
sustainability of domestic production and livelihoods security.
Unfortunately, India is a country with a big rural market left untapped since
many years. As rural people are unwilling to take risks hence a market has to be
adopted in a totally different approach in locating and identifying the target
market and relevant market segment for their products. And recent trends
indicate that the rural markets are coming up in a big way. Effective
communication can be used as a tool to reach them.
The M.P. State Government has introduced organic policy to promote farmers
towards organic farming and processing of products, wanting to give a lift to
environment and soil health. But, all the policy remains ineffective due to lack of
proper support system and coordination between state run departments,
agriculture universities and farmers.
Moreover, lack of desire marketing net and infrastructure in which organic
products could get desired value is another issue to be addressed. Since August
2010, when the State Government introduced its Organic Policy7 2010 not even a
single step towards shaping marketing system for organic farm products could
be taken. Again in 2011, the Krishi Cabinet discussed organic farming at length
in presence of experts at JNAU. They also gave recommendations on making
policy effective but dependency on chemical fertilizers continues in state. In
present trend, marketing channel has become a lifeline. Secondly, the facility of
storage and distribution system plays key role in providing real benefits to
growers. Other factors that affect progress of organic policy include soil health
and organic certification of crops and low literacy amongst farmers.
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23
The organic farming has become a costlier affair for farmers who are already
using chemical fertilizers. As yield rate is much lower than farming with
inorganic inputs, the cost of labour and application of organic insecticides,
pesticides, fungicides turns the organic farm produces much costlier in
comparison to present chemical based farming. Another challenge before organic
farmers is to maintain the quality of produce when chemical based farm inputs
are being openly marketed and sold to the farmers. The mechanization in
farming has changed the whole scene in villages as they lack natural manure
required to fulfill nutrient requirement of soil.
There are about 300 clear sunny days in most parts of the country. Solar 3 energy
applications can be an integral part of our daily lives touching every one of us in
the future thereby helping us on reducing dependence on fossil fuels and other
environmental benefits especially in agricultural applications of Solar Energy.
Solar energy can supply and/or supplement many farm energy requirements. It
has immense benefit for boiling and crop drying with simple, safe and least
expensive use of solar equipments.
The future of solar in India will explode to a level none of us can imagine if
planned with the participatory efforts of all the stakeholders working in the grass
root level specially the rural areas where frequent and long power cuts is a daily
feature.
Specialized training materials for our rural youths can be taken up from existing
courses from the private sectors to meet the requirement of skilled manpower for
initial setup of the field installations and an after-sales service net-work for the
local area according the needs of agricultural/value additional requirements for
the willing entrepreneurs later after the project takes a pace.
Our project initiatives happened by chance during a monitoring work and it was
then deemed to have a baseline survey of Dehrikalan village of Phanda block to
understand the availability of natural resources, employment opportunities and
other means of livelihood with the aim of improving the general living condition
and the standard of life of the people. The study was also meant to understand
the people of the village, their lifestyle and their level of acceptance to change,
for the betterment of the village and the people of the village in general. Valuable
experience gained during the monitoring process has helped us to find the major
lacunae in the process moreover the programme emphasized on creating
awareness through IEC strategies that needed for the delivery of the concept to
the lower rung for the fulfillment of the objectives, Human resource
development, and capacity development activities.
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24
The land of the village is very fertile where wheat, gram, maize and soyabean are
cultivated. As for irrigation the farmers are totally dependant on the rainfall, the
returns from the fields is less than what is expected.
A discussion with the people revealed that they are ready to learn and adopt
activities that will give them an additional income besides their regular earnings
from the crops. It was observed that at the moment the need was of a low budget
technology that is efficient and proven. The choice of the value added products
would give them an easy market for the products within their local market and
neighborhood.
After the baseline details it was deemed appropriate to take up the cultivation of
Curcuma longa and Maranta arundinacea and its semi processing5 through
demonstration. The application of solar energy for the same and
entrepreneurship activity will help us for developing a rural employment
opportunity.
Materials and methods
We have identified 75-85 SC families in the village to form cluster of 10 families
in the village. The selection is based on motivation, desire to participate and
willingness to lend their land for a long period with sustainability for promoting
alternative additional livelihood means.
Realizing the importance of post harvest processing in the areas it is embarked
on establishing rural agro-processing centre to promote processing also, that is
proposed to be designed to fully managed by clusters /women groups/youth
groups or even family as groups for initial period under our guidance and with
some small condition for the sustainability of the infrastructure so established.
The center is proposed with need-based post harvest processing equipment that
addresses the needs of the selected proposed medicinal plants in the village.
Processing facilities is proposed to be established in production and growing
areas that can render enhanced availability of raw materials at reduced costs and
at the same time increase employment to the target partners. Beneficiary lending
their land can be developed as demonstration plot (DP). A layout plan of the
demonstration plot is designed and implemented. Various scientific methods
using bio-fertilizers2 and bio-pesticides are adopted in the cultivation part. Later
on harvesting, semi-processing of the harvest will be demonstrated during
training schedules.
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25
Results and Discussion
Turmeric usage dates back from 3000 B.C. in India. From a significant part in
daily cuisine to treating diseases like cancer, turmeric is beneficial to mankind. It
is impossible to think of Indian food without turmeric. India is the world's
largest producer of turmeric, i.e. nearly 90% of the world's total production. The
productions of Turmeric in major states of India are as follows: Tamilnadu: 18%,
Orissa: 7%, West Bengal: 4%, Karnataka: 4%, Gujarat: 2%, Maharashtra: 2%. With
Major Trading Centers in India as Nizamabad, Dugirala (Andhra Pradesh),
Sangli (Maharashtra), Coimbatore (Tamilnadu), Salem, Erode, Dharma
Uri.Government’s support in the form of financial help /trainings/promotions
/exhibitions has yet to reach farmers of all type at the grass roots. With that
spirit, it is a small effort to bring the cultivation of haldi and arrowroot with non
chemical techniques in Bhopal on a very small scale on pilot basis is being
carried out with the beneficiary partners.
Maranta arundinacea (arrow root) is a perennial, which grows for 6-12 months
before harvest. It is used as an article of diet in the form of biscuits, puddings,
jellies, cakes, hot sauces, etc., It is an easily digestible food for children and
people with dietary restrictions. Used in diets requiring bland, low-salt, and lowprotein foods
On the basis of the information collected, focus group discussions, semistructured interviews revealed that no new agricultural practices /interventions
have been adopted by the villagers as such no source of additional income is
generated for a decent living with capacities of giving higher educational to their
girl children (which they very much long to do), hence we deemed it appropriate
to take up the cultivation of Curcuma longa (Haldi) and Maranta arundinacea
(Arrowroot) and its semi processing methods by establishing small units to be
managed by the groups and plans of its sustainability.
During the focus group discussions approximately 18-20 marginal farmers along
with agricultural laborers have showed the inclination towards the use of solar
equipments for agricultural application specially for dying of their produce. As
we have targeted for a long-term sustenance of both the non-chemical farming
methods through demonstrations and entrepreneurship activity for the rural
woman with commitments will be taken care off.
Both the plants are of great medicinal value and will prove to be a boon to the
growers too. Maranta arundinacea (Arrowroot) also known as Tavaksira is used
externally as well as internally. The powder of rhizomes with honey is applied
25
26
on the mucous membrane of the oral cavity, in stomatitis. It also promotes the
healing of stomach ulcers. Internally it is extremely beneficial in diarrhea,
dysentery and colitis as it is astringent. The rhizome powder cooked in milk is
given along with sugar, in irritable bowel syndrome and ulcerative colitis to
alleviate the irritation and facilitate the healing of ulcers. C. Longa (haldi) is
widely used in Indian foods.
Similar activities are practiced by various national laboratories of the country,
ayurveda institutes, NGO's etc. Therefore the techno viability aspect of the
project is very much assured. And since the project aims the cultivation of
Curcuma longa (Haldi) and Maranta arundinacea (Arrowroot) and its semi
processing through awareness and demonstration a need to explore assured buy
back arrangement will be highly beneficial to the rural beneficiary partners.
The processing of both the plant products has a great market potential, the usage
of solar equipments will add to its value and a new learning for the beneficiary
partners and a remunerative work. Cultivation of Maranta arundinacea
(Arrowroot) is in a declining stage and has high medicinal value and the bye
product in the arrowroot powder form has high market value, the sale of tubers,
as seeds to other potential farmers of their area will also add to their income.
On a pilot scale a self-sustaining (more of a very small cottage industry like)
activity with assured buy back system is anticipated. Further, these types of
project activities will promote the conservation of biodiversity too and revive
cultivation of Maranta arundinacea (Arrowroot) too in the State
Acknowlegements
We highly acknowledge the kind gesture of Science and Technology Programme for
Socio-Economic Development, Ministry of Science and Technology, Technology Bhawan,
New Delhi to fund our dream of year 2000 to bring to grass root on a pilot basis
the benefits of non-chemical farming techniques to beneficiary partners of two
very important plants of medicinal value.Our deep gratitude to the valuable
advice from Dr. Sivasubramaniam Edison, Member, BoT, International Potato
Centre, Lima, Peru and Former Director, Central Tuber Crops Research Institution
,Trivandrum, Mr. Rajan Kuttapan , Administrator, Institute of Social Sciences,8
Nelson Mandela Road, New Delhi for his suggestions extended for the cause of
beneficiary partners. Dr.U. Prakasham, IFS, Additional Director, SFRI for
sponsoring seeds of medicinal value
Thanks for timely support during the project start off, inclusion and deletions of
necessary shortcomings of Mr. D. Manohar, Scientist NIC, Coimbatore and Mr.
Antony.N.J., New Delhi.
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References
Anandaraj, M., Devasahayam, S., Zachariah, T.J., Eapen, S.J., Sasikumar, B., and
Thankamani, C.K., 2001, Turmeric (Extension Pamphlet). Rema, J., and Madan,
M.S., Editors. Indian Institute of Spices Research, Calicut, Kerala. API-Ayurvedic
Pharmacopoeia of India 1989. New Delhi: Government of India-Ministry of
Health and Family Welfare-Department of Health. 45-46.
Chaudhary, D.R., Kisan world 2004, Bio- fertilizers for Improving crop
Productivity, Vol. 31 No.10.
Chandna, H., Sun rises on Solar Products, 2010, Hindustan Times.
Iyengar, I., 2010, Jhabua village to banish darkness with solar lanterns,
Hindustan Times.
Kamble, K.J., Soni, S.B. Karnataka, and Agri, J., 2009, A study of improving
turmeric processing . Sci22(1)137-139).
Rajshree, V., Balakrishanmoorth, and Prabhuram, R., Kisan world 2006, Good
Agricultural Practices, Vol.33 No-5.
Sharma, A., Dwivedi, N., and Khanuja, S.P.S., 2000, Sourcing information on
R&D and trade of medicinal and aromatic plants through web data mining:
Some utility sites. Journal of Medicinal and Aromatic Plant Sciences. 24 (1):82103.
The Hindu, 2009, Crafts Bazaar to focus on sustainable livelihood.
The Hitavada, 2012, Lack of co-ordination hampering organic farming.
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Journal of Today’s Biological Sciences: Research & Review (JTBSRR)
ISSN 2320-1444 (Online)
JTBSRR
Vol.1, Issue 1, page 28-52, December 30, 2012
Available on: www.jtbsrr.in
Studies on Interaction of Argonaute Protein and
Micro RNA of Chlamydomonas reinhardtii
P.A.
Dang
Pritha Bhattacharya(Sasmal)
Netaji Mahavidyalaya, Arambagh,Hooghly, Email-pritha.sasmal@gmail.com
Abstract
RNA interference(RNAi) is an ancient mechanism of gene suppression. Usually RNA
interference is defined as a homology dependent gene silencing mechanism that involves
double stranded RNA directed against a target gene or its promoter region.
Chlamydomonas reinhardtii is a unicellular green alga.Chlamydomonas contains a set of
endogenous microRNAs (miRNAs) that down-regulate their target gene expression
through mRNA cleavage. During RNA silencing mechanism these miRNA can interact
with Argonaute protein.With the help of CHIMERA software it has been found that
miRNAs contain some conserved nucleotide sequences through which they interact with
Argonaute proteins. If we want to make a new antisense miRNA these conserved
sequences must be targeted for successful interaction with the target miRNA.
Key Words: RNAi, Antisence miRNA, Argonaute protein, NMR spectroscopy,
Protein Data Bank (PDB), MC-FOLD MC-SYM, HEX, UCSF CHIMERA
Introduction
RNA interference is an ancient mechanism of gene suppression. Usually RNA
interference is defined as a homology dependent gene silencing mechanism that
involves double stranded RNA directed against a target gene or its promoter
region. RNA interference is abbreviated as RNAi. RNAi is but one aspect of a
larger web of sequence-specific, cellular responses to RNA known collectively as
RNA Silencing.
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29
RNAi was first discovered in Petunia hybrida at around 1990. After that many
works has been done on RNAi & Finally in 2006 Andrew Z.Fire & Craig C.Mello
received the Nobel Prize for their discovery that ds RNA triggers suppression of
gene activity in a homology dependent manner.
It has been found that All RNA silencing pathways are triggered by 21-27 nt
long ‘small RNAs’ –a term that encompasses small interfering RNAs (si RNA),
repeat –associated small interfering RNA (rasi RNAs),micro RNAs (mi RNAs)
etc.
Materials and Methods
Throughout the entire duration of work many computational approaches have
been used to obtain the ultimate results. The key algorithms that have been used,
are briefly explained in the 1st part of the method section which is followed by a
flow chart of the work flow which was followed.
Resources
A. miRBASE : The microRNA Database
The miRBase database is a searchable database of published miRNA sequences
and annotation. Each entry in the miRBase Sequence database represents a
predicted hairpin portion of a miRNA transcript (termed mir in the database),
with information on the location and sequence of the mature miRNA sequence
(termed miR).Both hairpin and mature sequences are available
for searching and browsing, and entries can also be retrieved by name, keyword,
references and annotation. All sequence and annotation data are also available
for download. The miRBase Registry provides miRNA gene hunters with unique
names for novel miRNA genes prior to publication of results. Visit the help
pages for more information about the naming service. The miRBase Targets
database and pipeline has been rebranded as microCosm, and is now hosted at
the EBI. The microCosm resource continues to be maintained by the Enright
group. miRBase currently links miRNAs to targets predicted by
microCosm,TargetScan and Pictar, and aims to provide a more extensive target
prediction aggregation service in the future.
B. PDB : Protein Data Bank
The Protein Data Bank (PDB) is a repository for the 3-D structural data of large
biological molecules, such as proteins and nucleic acids. The data, typically
obtained by X-ray crystallography or NMR spectroscopy and submitted
by biologists and biochemists from around the world, are freely accessible on the
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30
Internet via the websites of its member organisations (PDBe,PDBj, and RCSB).
The PDB is overseen by an organization called the Worldwide Protein Data
Bank, wwPDB.
The PDB is a key resource in areas of structural biology, such as structural
genomics. Most major scientific journals, and some funding agencies, such as
the NIH in the USA, now require scientists to submit their structure data to the
PDB. If the contents of the PDB are thought of as primary data, then there are
hundreds of derived (i.e., secondary) databases that categorize the data
differently. For example, both SCOP and CATH categorize structures according
to type of structure and assumed evolutionary relations; GO categorize
structures based on genes.
C . MC-FOLD MC-SYM
The MC-Fold | MC-Sym pipeline is a web-hosted service for RNA secondary
and tertiary structure prediction. The pipeline means that the input sequence to
MC-Fold outputs secondary structures that are direct input to MC-Sym, which
outputs tertiary structures.The vertical bar '|' is the symbol indicating pipeline
processing in Unix, which was invented by Malcolm D. McIlroy. MC-Fold
predicts secondary structures from sequence (black arrow). Compared to
classical secondary structures that include the AU, CG, and Wobble GU base
pairs that form the stems of the structure, MC-Fold's secondary structures
include the other base pairing patterns as well. These are often called noncanonical base pairs.
D. HEX
Hex is an interactive molecular graphics program for calculating and displaying
feasible docking modes of pairs of protein and DNA molecules. Hex can also
calculate protein-ligand docking, assuming the ligand is rigid, and it can
superpose pairs of molecules using only knowledge of their 3D shapes. Hex has
been available for about 12 years now, but as far as known, it is still the only
docking and super postion program to use spherical polar Fourier (SPF)
correlations to accelerate the calculations, and its still one of the few docking
programs which has built-in graphics to view the results. The graphical nature
of Hex came about largely to visualise the results of such docking calculations in
a natural and seamless way, without having to export unmanageably many
coordinate files to one of the many existing molecular graphics programs. For
this reason, the graphical capabilities in Hex are generally relatively primitive
compared to professional molecular graphics packages, but you're aiming to
use Hex to do docking, not to make publication-quality images.
30
31
E. UCSF Chimera : An Extensible Molecular Modeling System
UCSF Chimera is a highly extensible program for interactive visualization and
analysis of molecular structures and related data, including density maps, supra
molecular assemblies, sequence alignments, docking results, trajectories, and
conformational ensembles. High-quality images and animations can be
generated. Chimera includes complete documentation and several tutorials, and
can be downloaded free of charge for academic, government, non-profit, and
personal use. Chimera is developed the Resource for HYPERLINK
"http://www.rbvi.ucsf.edu/"BiocomputingHYPERLINK
"http://www.rbvi.ucsf.edu/", Visualization, and Informatics and funded by the
NIH National Center for Research Resources.
Method
ANALYSIS WORKFLOW
COLLECTION OF SEQUENCES OF miRNAs FROM miRBASE
GENERATION OF THE miRNA 2D STRUCTURES
DERIVATION OF THE 3D STRUCTURES OF miRNAs
GENERATION OF HOMOLOGY MODEL FOR ARGONAUTE PROTEIN
STIMULATION OF INTERACTION THROUGH miRNA-AGO COMPLEX
USING HEX
STIMULATION OF THE POINT OF INTERACTION OF miRNA-AGO
COMPLEX
31
32
Fig.1. Docking structure of different miRNA & argonaute protein of
Chlamydomonas reinhardtii
Argonaute 1+ miRNA 20.
Argonaute 1+ miRNA 21
32
33
Argonaute 1+miRNA 22
Argonaute 1+miRNA 23.
Argonaute 1+miRNA 24.
33
34
Table 1. Points of interaction between miRNAS & Argonaute protein of
Chlamydomonas reinhardtii
Sl.
No.
Sequence
Argonaute
Name
of
interacting
amino acid
LYS
GLY
SER
Protein
MiRNA
Position of
those amino
acid
852
A 11.A 2HO’
854
A 9.A HO2’
792
A 11.A C1’
1.
AGO+MiRNA 1
2.
AGO+MiRNA 2
LEU
255
#0(Adenine)
3.
AGO+MiRNA 3
THR
PRO
942
941
#0(Guanine)
G 11.A O2’
4.
AGO+MiRNA 5
ASP
SER
ASP
LYS
ALA
PRO
VAL
473
604
472
435
436
586
605
A 10.A
C 12.A 2OH’
#0(Guanine)
,,
,,
C 12.A HO2’
A 10.A C4’C5’
1.527
Angstrom
5.
AGO+MiRNA 6
GLY
PRO
GLY
GLY
SER
LEU
TYR
278
69
270
68
279
280
275
#0(Cytosine)
#0(Guanine)
G12.A 2HO’
#0(Guanine)
C 11.A HO2’
G 12.A HO2’
C 11.A 2HO’
6.
AGO+MiRNA 7
GLY
SER
MET
PRO
LYS
ASN
706
705
332
237
240
623
#0(Uracil)
,,
C 22.A 2HO’
#0(Adenine)
U 1.A
A 3.A HO2’
34
Remarks
35
7.
AGO+MiRNA 8
ASP
563
#0(Uracil)
LYS
531
#0(Adenine)
TYR
560
#0(Uracil)
8.
AGO+MiRNA 9
HIS
LEU
ARG
GLU
PRO
PRO
GLN
818
220
246
247
817
817
816
U 20.A 2HO’
G 21.A HO2’
G 21.A
G 21.A
U 2.A O2’
U 2.A 2HO’
U 2.A HO2’
9.
AGO+MiRNA
10
PRO
167
#0(Adenine)
ALA
MET
ASP
SER
GLU
PRO
165
332
331
239
238
237
,,
,,
,,
A 9.A O4’
C 8.A
U 7.A HO2’
ARG
246
A 21.A
GLU
247
ARG
PRO
GLN
246
817
816
A 21.A C3’03’
1.431
Angstrom
C 20.A
G 2.A 2HO’
G 3.A 2HO’
ASP
232
#0(Adenine)
ASP
SER
ALA
HIS
PRO
232
234
224
818
817
#0(Cytosine)
C 21.A
#0(Adenine)
A 1.A O2’
,,
10.
11.
AGO+MiRNA
11
AGO+MiRNA
12
35
Very
very
close
Interacti
on.
36
12.
13.
14.
15.
16.
AGO+MiRNA
13
AGO+MiRNA
14
AGO+MiRNA
16
AGO+MiRNA
17
AGO+MiRNA
18
VAL
605
A 9.A
SER
ASP
ASP
HIS
GLU
MET
604
472
473
552
553
554
SER
604
G 10.A
#0(Uracil)
,,
A 9.A HO2’
A 9.A C3’
G 10.A C4’C5’
1.510
Angstrom
U 11.A
ARG
681
C 20.A HO2’
LYS
PRO
PRO
710
586
586
LEU
585
SER
707
G 19.A HO2’
G 19.A HO2’
C 20.A C3’C4’
1.601
Angstrom
C 20.A C3’03’
1.432
Angstrom
U 1.A HO2’
ALA
791
C 10.A
GLY
854
C 8.A
ALA
168
#0(Cytosine)
ALA
VAL
171
159
SER
239
,,
C 9.A C1’-04’
1.412
Angstrom
G 8.A HO2’
ASP
594
C 4.A
ASN
667
G 3.A
36
37
17.
18.
19.
20.
AGO+MiRNA
20
AGO+MiRNA
21
AGO+MiRNA
22
AGO+MiRNA
23
ASP
ASN
ASP
VAL
594
667
473
605
G 2.A C4’
U 1.A
#0(Guanine)
#0(Cytosine)
CYS
641
G 20.A O2’
CYS
CYS
LEU
MET
648
648
644
332
U 1.A HO2’
U 2.A
U 1.A 2HO’
#0(Guanine)
SER
705
#0(Uracil)
GLY
VAL
VAL
SER
GLU
PRO
CYS
LEU
MET
706
704
704
239
238
237
648
644
332
,,
U 1.A N1
U1.A O4’
U 1.A
,,
,,
G 20.A 2HO’
C 21.A C3’
#0(Adenine)
ILE
551
#0(Adenine)
HIS
HIS
593
593
GLY
ARG
ILE
SER
458
441
442
665
A 13.A 2HO’
A 13.A 02’C2’
1.41
Angstrom
#0(Adenine)
,,
,,
A 13.A 2HO’
GLY
706
U 11.A
MET
MET
332
332
U 11.A C4’
A 10.A C1’-
37
38
21.
22.
23.
24.
25.
AGO+MiRNA
24
AGO+MiRNA
25
AGO+MiRNA
27
AGO+MiRNA
28
AGO+MiRNA
ALA
687
N9
1.441
Angstrom
A 10.A C3’
PRO
167
#0(Guanine)
PHE
SER
ALA
LEU
PRO
158
705
171
164
237
#0(Uracil)
C 13.A
#0(Uracil)
#0(Guanine)
#0(Cytosine)
ASP
293
A 123.A
ILE
THR
GLY
292
853
854
ARG
LYS
LYS
855
297
299
A 13.A
A 12.A
G 11.A C3’03’
1.427
Angstrom
G 11.A C4’
G 11.A O4’
#0(Guanine)
GLY
926
G 9.A C3’
SER
HIS
PRO
THR
ASN
925
940
941
807
754
G 10.A
C 11.A
G 10.A
#0(Guanine)
,,
TYR
592
C 1.A
GLN
ARG
GLY
SER
GLU
ASP
329
328
327
604
553
472
,,
#0(Cytosine)
#0(Guanine)
,,
G 21.A HO2’
G 21.A
LYS
258
Guanine
38
39
29
26.
27.
28.
29.
AGO+MiRNA
30
AGO+MiRNA
31
AGO+MiRNA
32
AGO+MiRNA
GLY
HIS
TYR
SER
PRO
GLN
GLY
LEU
TYR
ARG
257
230
231
349
348
450
314
318
319
330
,,
,,
,,
Uracil
,,
U 10.A
Uracil
G 9.A
,,
U 8.A 02’
GLY
439
Cytosine
VAL
440
VAL
ARG
HIS
GLU
GLY
440
441
552
553
458
C 12.A c1’c2’1.527
angstrom
uracil
uracil
,,
,,
Cytosine
PHE
469
C 12.A C4’
ASP
ILE
ASP
VAL
472
551
472
605
G 11.A 2HO’
A 10.A O2’
G 11.A C4’
C 9.A 2OH’
GLY
278
G 9.A
SER
LEU
PRO
PRO
GLU
TRP
PHE
279
280
281
281
303
497
872
,,
,,
,,
A 10.A
,,
U 11.A
U 11.A C3’
TRP
497
Adenine
39
40
33
30.
31.
32.
AGO+MiRN
A 34
AGO+MiRN
A 35
AGO+MiRN
A 36
GLY
PHE
278
402
GLU
PHE
ARG
303
872
856
C 21.A
C 21.A C3’03’
1.431
Angstrom
C 21.A HO2’
U 3.A
G 4.A 2HO’
ALA
815
C 2.A 2HO’
ALA
TYR
ASP
ASP
VAL
SER
815
231
232
232
233
234
C 2.A C1’
U 3.A 2HO’
Uracil
Adenine
,,
A
20.A
2HO’
ASP
473
Guanine
GLN
ASN
549
649
GLU
548
G
12.A
HO2’
Guanine
GLU
247
Uracil
ALA
ALA
224
178
VAL
VAL
221
221
LEU
TYR
ASP
PRO
PRO
966
967
968
817
817
U 21.A
U
21.A
HO2’
U 21.A O2’
U
21.A
2HO’
C 20.A O2’
,,
C 20.A C1’
G 19.A O2’
G 19.A
40
,,
41
33.
34.
35.
36.
37.
AGO+MiRN
A 37
Ago+MiRNA
38.
AGO+MiRN
A 39
AGO+MiRN
A 40
AGO+MiRN
GLN
PRO
PRO
816
96
96
Guanine
G 2.A
U 1.A
ASP
472
A 8.A HO2’
ASP
ASP
GLY
GLN
ARG
473
473
606
329
33O
C 9.A C4’
A 10.A
,,
Adenine
,,
ALA
171
ILE
LEU
LEU
700
701
701
VAL
SER
GLU
704
705
238
C
21.A
2HO’
C 21.A C2’
C 21.A C3’
C 21.A C3’03’
1.432
Angstrom
C 21.A
U 20.A
Uracil
ASP
331
A 11.A
SER
LEU
GLY
SER
SER
683
686
706
705
705
,,
C 12.A
A 11.A
G 10.A
Cytosine
ASN
649
G 3.A 2HO’
LEU
686
ILE
GLY
LEU
MET
645
652
686
332
U
20.A
2HO’
,,
C 4.A 2HO’
C 2.A 2HO’
C 2.A
ARG
330
G 12.A
41
42
A 41
38.
39.
40.
AGO+MiRN
A 42
AGO+MiRN
A 43
AGO+MiRN
A 44
SER
SER
VAL
550
550
605
ILE
ASP
ASN
GLY
MET
645
472
649
652
653
MET
653
CYS
1058
MET
MET
VAL
GLY
GLY
ARG
GLN
GLU
1055
1055
1054
854
854
855
904
892
U
20.A
2HO’
G 4.A HO2’
G 4.A
G 4.A C3’
Guanine
U 19.A
,,
G 21.A O3’
U 20.A O2’
ARG
856
U 9.A 2HO’
GLY
GLU
PHE
30
858
872
Guanine
Uracil
G 10.A
CYS
1058
Uracil
ALA
989
U
2HO’
PHE
LYS
900
852
A 21.A
U
19.A
HO2’
42
G 12.A C3’
A 13.A
A
13.A
HO2’
,,
Guanine
G 15.A
,,
G
15.A
HO2’
C 16.A C4’04’
1.453
Angstrom
20.A
43
41.
42.
43.
44.
45.
AGO+MiRN
A 45
AGO+MiRN
A 46
AGO+MiRN
A 47
AGO+MiRN
A 48
AGO+MiRN
A 49
GLY
58
Uracil
PRO
PRO
GLY
GLY
GLY
60
59
32
29
31
Guanine
,,
U 9.A O2’
U 7.A
U 8.A
VAL
440
Adenine
GLY
PHE
439
469
PHE
THR
GLU
ARG
469
600
598
597
Uracil
G
20.A
2HO’
U 1.A HO2’
A 2.A HO2’
A 3.A
A 3.A HO2’
GLY
439
U 11.A
VAL
GLU
ASP
LYS
440
598
594
599
THR
600
A 12.A
U 17.A
A 18.A
C
16.A
HO2’
A
15.A
HO2’
SER
241
ARG
242
ALA
976
THR
GLY
162
709
LEU
701
PHE
469
43
U
14.A
2HO’
U
14.A
HO2’
G
13.A
2HO’
G 13.A O4’
C
11.A
2HO’
A 15.A O2’
G
HO2’
21.A
44
46.
47.
48.
49.
50.
51.
AGO+Mi
RNA 50
AGO+MiRN
A 51
AGO+MiRN
A 52
AGO+MiRN
A 53
AGO+MiRN
A 54
AGO+MiRN
A 55
PRO
ARG
GLN
424
423
549
G 21.A C4’
,,
G 21.A
TYR
231
ASN
ARG
ASN
PHE
245
246
245
158
C
17.A
HO2’
Uracil
,,
Cytosine
C
19.A
HO2’
GLN
329
Uracil
SER
ILE
VAL
SER
550
645
605
654
,,
Cytosine
C 8.A O4’
G 6.A 2HO’
ASN
764
Cytosine
TYR
967
ASN
764
U
19.A
HO2’
Uracil
SER
683
ASP
CYS
SER
331
648
705
G
12.A
HO2’
G 12.A
Guanine
Cytosine
GLU
758
Guanine
GLU
THR
THR
PRO
758
755
942
941
Adenine
,,
A 9.A
A 9.A HO2’
THR
853
C 12.A
44
45
52.
53.
54.
55.
AGO+MiRN
A 56
AGO+MiRN
A 57
AGO+MiRN
A 58
AGO+MiRN
A 59
ILE
ILE
ALA
292
292
791
LYS
297
,,
U 13.A
U
13.A
HO2’
G 11.A C4’
CYS
1058
Guanine
ARG
ARG
THR
PHE
GLU
856
855
853
900
892
Cytosine
,,
C 10.A C1’
Cytosine
,,
CYS
1058
GLU
GLY
892
854
ASP
881
C 9.A HO2’O2’ O.960
Angstrom
C 9.A
U7.A C4’C5’
1.528
Angstrom
C 8.A C3’
PRO
70
LEU
228
SER
THR
LYS
260
259
258
G
19.A
2HO’
C
20.A
2HO’
,,
Cytosine
Adenine
VAL
605
G 18.A
ARG
GLN
ASP
330
329
473
ASP
ASP
473
472
G 19.A
,,
G
19.A
HO2’
A 20.A C3’
A
20.A
HO2’
45
46
56.
57.
58.
59.
60.
61.
62.
AGO+MiRN
A 60
AGO+MiRN
A 61
AGO+MiRN
A 62
AGO+MiRN
A 63
AGO+MiRN
A 64
AGO+MiRN
A 65
AGO+MiRN
A 66
GLY
706
C 11.A C4’
MET
332
Cytosine
MET
653
G 7.A HO2’
ASN
SER
ASN
ASP
ASP
649
604
474
473
473
G 7.A C4’
Adenine
U 1.A
,,
G 2.A
PRO
817
U 9.A
GLN
ALA
PRO
816
815
96
,,
Adenine
C 6.A 2HO’
ALA
208
U 10.A
LYS
GLY
GLU
808
204
771
G 11.A C4’
Uracil
G
13.A
2HO’
LYS
905
LYS
CYS
1052
1058
C 19.A C3’O3’
1.432
Angstrom
G 18.A
C 19.A
LYS
905
LYS
CYS
1052
1058
C 19.A C3’O3’
1.432
Angstrom
G 18.A
C 19.A
ARG
367
G 11.A
46
47
63.
64.
65.
66.
67.
AGO+MiRN
A 67
AGO+MiRN
A 68
AGO+MiRN
A 69
AGO+MiRN
A 70
AGO+MiRN
A 71
PHE
GLN
ARG
364
363
850
A 10.A C3’
A 10.A C4’
U 12.A C4’
SER
550
Cytosine
VAL
ASP
ASP
PHE
LYS
605
473
472
469
599
C 13.A C2’
A 12.A
U 11.A O4’
Adenine
U
14.A
HO2’
ASN
970
ALA
ASN
PRO
ALA
224
245
817
815
G
21.A
2HO’
G 21.A C3’
Guanine
G 20.A O2’
A 1.A O2’
GLY
854
ARG
TYR
855
1057
CYS
1058
GLY
652
ASN
VAL
VAL
649
605
605
ASP
ILE
472
468
SER
705
C 8.A 2HO’
GLY
SER
706
683
Guanine
Adenine
47
A
19.A
HO2’
G 20.A
C
21.A
2HO’
,,
C
14.A
2HO’
C 14.A C3’
C 13.A
G
12.A
2HO’
G 11.A
A
10.A
HO2’
48
68.
69.
70.
71.
72.
73.
AGO+MiRN
A 72
AGO+MiRN
A 73
AGO+MiRN
A 74
AGO+MiRN
A 75
AGO+MiRN
A 76
AGO+MiRN
A 77
ASP
331
A
HO2’
ARG
910
ARG
LYS
906
905
G
22.A
2HO’
Guanine
Adenine
A
10.A
HO2’
SER
683
Uracil
SER
GLY
GLU
SER
707
706
238
239
,,
U 1.A
G 20.A
A 1.A
LYS
LEU
CYS
240
701
648
THR
GLY
651
652
A 1.A O4’
Adenine
G
19.A
2HO’
U 4.A HO2’
U 4.A 2HO’
ASP
472
G 1.A
ASP
MET
473
653
,,
VAL
605
C
20.A
2HO’
Guanine
ASP
473
A 9.A C1’
ASP
VAL
472
605
Adenine
A 8.A C4’
MET
332
THR
682
A
HO2’
G
HO2’
48
10.A
17.A
16.A
49
74.
75.
76.
77.
78.
79.
80.
AGO+MiRN
A 78
AGO+MiRN
A 80
AGO+MiRN
A 81
AGO+MiRN
A 82
AGO+MiRN
A 83
AGO+MiRN
A 84
AGO+MiRN
A 85
MET
332
THR
682
ALA
815
A 2.A O2’
HIS
ASP
VAL
230
232
233
U 3.A 2HO’
Adenine
A
20.A
2HO’
SER
705
Cytosine
HIS
679
C 10.A O4’
GLN
450
Guanine
ASP
449
HIS
GLN
366
363
G
13.A
HO2’
U 11.A
A 9.A
THR
853
U 1.A 2HO’
GLY
854
Adenine
GLY
854
A 12.A
ALA
GLU
LYS
ALA
791
892
905
791
C 11.A
G 10.A C4’
Guanine
Adenine
HIS
783
A
HO2’
49
A
HO2’
G
HO2’
17.A
16.A
10.A
50
Discussion
From the above result it has been found that in Argonaute protein – Ser 239,Val
605,Glu 238, Asp 472,Gln 549,Asn 649,Met 332,Ser 705,Lys 258,ILE 645,PRO
941,Phe 469,amino acid residues are very common as interacting points. So site
specific mutagenesis among these amino acids can be done to regulate the
binding of miRNA with Argonaute proteins & thus can hamper the RNAi
mechinary. Similarly it has also been observed that miRNAs also contain some
conserved nucleotide sequences through which they interact with Argonaute
proteins.So if we want to make a new antisense miRNA these conserved
sequences must be targeted for successful interaction with the target miRNA.
Future prospects
I hope that these effort will be beneficial for future researchers in the
computational field as well as in the wet lab.
Acknowledgement
I would like to extend my thanks to professor Abhijit Datta of Presidency
University,Kolkata,for his help & advice in the formulation & application of the
computer simulation that was used here.
References
Websites
http://www.biomedcentral.com/content/pdf/gb-2008-9-2-210.pdf
http://www.ncbi.nlm.nih.gov/pubmed?term=Brassica%20RNAi
http://www.biomedcentral.com/content/pdf/gb-2008-9-2-210.pdf
www.loria.fr/~ritchied/hex/
http://www.pdb.org/pdb/home/home.do
http://dinamelt.bioinfo.rpi.edu/quickfold.php.
mfold.rna.albany.edu/?q=DINAMelt/Quickfold
http://www.major.iric.ca/MC-pipeline/
http://www.cgl.ucsf.edu/chimera/
http://www.ncbi.nlm.nih.gov/pubmed/
http://www.jbc.org/content/early/2011/05/16/jbc.M111.240259.long
http://pcp.oxfordjournals.org/content/early/2011/05/16/pcp.pcr063.long
http://www.springerlink.com/content/481420138lt30244/
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53
Journal of Today’s Biological Sciences: Research & Review (JTBSRR)
ISSN 2320-1444 (Online)
JTBSRR
Vol.1, Issue 1, page 53-58, December 30, 2012
Available on: www.jtbsrr.in
An investigation on ethno-veterinary medicinal
plants of Siliguri Subdivision of Darjeeling District,
West Bengal, India
Tamal Mondal
Department of Botany, Netaji Mahavidyalaya, Arambagh, Hooghly, West Bengal, India,
Email: tamalmondal1@gmail.com
Abstract
This study and instigation was aimed to find out the ethno-medicinal plants of Siliguri
Sub division of Darjeeling District, West Bengal, used to treat the various veterinary
diseases. In the present study, 36 medicinal plants belonging to 28 families used as
veterinary medicines have been documented. According to this study, documenting the
medicinal plants and associated indigenous knowledge can be used for conservation and
sustainable use of medicinal plants in the area and for effective treatment of various
disease and disorders of domestic animals.
Key words Ethno-veterinary medicine, medicinal plants, documentation, indigenous
knowledge
Introduction
Plants are used for medicinal purposes by local peoples since ancient times. It is
an established fact that plants serve a potent medicine for curing various diseases
of local people as well as livestock’s. Various plant formulations are also used in
folk medicine as well as our traditional medicines like allopathy, homoeopathy,
ayurvadic, siddha and unani medicines. EVM (Ethno-Veterinary Medicine) is a
system based on folk traditional skills, knowledge, and practices for curing
various diseases and disorders and maintaining good health of our domestic
animals (Mathias-Mundy and McCorkle, 1989; Tabuti et al., 2003; Kumari Rita et
al., 2011; Kumari Reshmi et al., 2011; Bhardwaj, Ugala et al., 2011).Darjeeling
53
54
District, West Bengal ,India is famous for its biodiversity as well as for medicinal
plants.Siliguri Subdivision belongs to Darjeeling District and a part of famous
‘tarai’ region the foot hills of Darjeeling Himalaya; well known area for its rich
biodiversity.The study area,Siliguri Subdivision is situated at 26º43´N latitude
and 88º25´E longitude within Darjeeling District of West Bengal,India.The
Subdivision contains 22 gram panchayats(rural area) under four community
development blocks; 1.Matigara,2.Naxalbari,3.Phansidewa,and 4.Kharibari,
including SMC(Siliguri Municipal Corporation).The ethnic groups lives in this
area are Santhals,Munda and Oraon tribes.In this Investigation as well as field
survey a total 36 number of medicinal plants belonging to 28 families are found
which are used successfully by the traditional healers of that area for treatment
of various disease and disorders of domestic animals.
Materials and Methods
This investigation mainly based on botanical field trips in the blocks and rural
areas of the Siliguri subdivision mainly inhabited by ethnic tribal communities
such as Santhals, Oraon and Mundas. The plants used for their healthcare
purpose were recorded through personal interview with the local traditional
healers and also local aged peoples during these field works. a pre-prepared
questioner was used for this purpose. At first step 36 plants belonging to 28
families were selected and collected from those areas. Next step is proper
identification of the collected plant species. Collected specimens were preserved
and worked out by following standard taxonomic methods and authentic
literature (Prain.1903: Bennet 1987). The local older peoples as well as traditional
healers were the primary informants who were interviewed during this field
work and the data have been recorded along with their names, address and the
medicinal uses are recorded from them.
Results and Discussion
The ethno-veterinary medicinal plants of Siliguri Subdivision,their scientific
name,family in which they belongs,their local names along with their important
portion as well as their medicinal properties are presented in table 1.
Table 1. List of ethno-veterinary medicinal plants found in Siliguri
subdivision Darjeeling, West Bengal
Serial Scientific
No.
Name
Acorus
1.
calamus
Local Name
(In Bengali)
Buch
Family
Plant parts used
Areceae
Leaf,roots
54
Veterinary
Medicinal uses
Stomachic,stimu
lant,digestive
problems
55
2.
Adhatoda
vasica
Basak
Acanthaceae
3.
Aegle
marmelos
Bael
Rutaceae
4.
Aloe vera
Ghritakumari
Liliaceae
5.
Alstonia
scholaris
Chatim
Apocynaceae
6.
Amaranthus
oleraceus
Bonnote
Amaranthaceae
7.
Amaranthus Lal sak
tricolor
Andrographis Kalmegh
paniculata
Amaranthaceae
Artemisia
vulgaris
Azadirachta
indica
Bryophyllum
calycinum
Cajanus
cajan
Nagdamoni,
Titapati
Neem
Asteraceae
Leaves,roots
Meliaceae
Leaves,bark,
Pathar kuchi
Crassulaceae
Leaves
Aorhar
Fabaceae
Leaves,seeds
13.
Calotropis
procera
Akando
Asclepiadeceae
Leaves
14.
Cannabis
sativa
Vang
Cannabinaceae
Whole plant
8.
9.
10.
11.
12.
Acanthaceae
55
Whole plants
Cough
and
cold,bronchitis,s
cabis
Fruits
Gastric
problems,
diarrhea
and
dysentery,
respiratory
disorder,
Flashy leaves
Cut
and
burn,skin
diseases,diarrhe
a
Leaves and bark
Jaundice,
Diarrhea
and
dysentery
Leaves
Skin problems,
cough and cold,
eye
problems,
pimples
Whole plants
Blood purifier,
anemia,
Leaves,
whole Liver problems,
plants
bile problems
Ringworm,bliste
r,pimples
Skin problems,
dental problems
Urinary
problems, piles
Jaundice,
bile
problems,
digestive
disorder,
Acidity,flatulanc
e,
Asthma, dental
problems,
Any type of
pain,nausa and
vomiting
56
15.
Centrella
asiatica
16.
Whole plant
Diarrhea
and
dysentery,fever,
blood purifier,
Chenopodium Bethua sak
Album
Citrus
Kagoji labu
aurantifolia
Chenopodiaceae Whole plant
Rutaceae
Fruits,seeds
Curcuma
longa
Datura metel
Holud
Zingiberaceae
Rhizome extracts
Dhuturo
Solanaceae
Leaf,root,seeds
Worms,
blood
purifier, piles
Digestive
problems,collera
,worm
Jaundice
and
wonds
Muscle
pain,worms,won
ds
Emblica
officinalis
Ficus carica
Amlaki
Euphorbiaceae
Fruit,leaves
Dumur
Moraceae
Leaves, fruits
22.
Heliotropium
indicum
Hatisur
Boranginaceae
Leaves
23.
Ipomoea
hederacea
Kalmi sak
Convolvulaceae
Leaves,total plants
24.
Lantena
camara
Chotra
Verbinaceae
Leaves,flower
25.
Luffa
cylindrica
Marsilea
quardrifolia
Dhudul
Cucurbitaceae
Fruit,leaves
Susni sak
Marsileaceae
Leaves,total plants
Mentha
piperita
Mimosa
pudica
Pudina
Lamiaceae
Whole plants
Lajjabati
Mimosaceae
Leaves
17.
18.
19.
20.
21.
26.
27.
28.
Thankuni
Apiaceae
56
Indigestion,gastr
ic problems
Diabetes, gastric
problems
Insomnia,
digestive
problems
Menstruation
problems,
digestive
problems,
constipation
Antibiotic,antise
ptic,skin
problems
Acidity,
bile
problem
Cough
and
cold,bronchitis,e
ye problems
Indigetion,vomit
ing,ear problems
Asthma,nerve
problems
57
29.
Mirabilis
jalapa
Sondhamoni
Nyctaginaceae
30.
Moringa
oleifera
Ocimum
sanctum
Sojina
Moringaceae
Tulsi
Lamiaceae
32.
Paedenia
foetida
Ghandhamadan Rubiaceae
33.
Solanum
nigrum
Kakmachi
Solanaceae
34.
Tagetes
erecta
Gada
Asteraceae
Leaves,Flowers
35.
Vitex
negundo
Nisinda
Verbenaceae
Leaves
36.
Zizyphus
mauritiana
Kul
Rhamnaceae
Fruits
31.
Roots,Leaves,seeds Wounds,antiinflammatory
activity
Flower,fruit
Piles,worm,coug
h,
Leaves,seeds,total Cough
and
plants
cold,fever,skin
problems,insect
bite
Leaves,total plants Diarrhea
and
decentry,gastric
problems
Fruits,seeds
Fever,
cough
and cold, acidity
Cut
and
wounds,burn,ins
ect bite
Muscle & Joint
pain,antiinflama
tory,antibiotic
Acidity,thrust,bi
le problems
Conclusion
Traditional healers collect their plant remedy from local places and conserve
their knowledge among selected peoples, if their knowledge will spread through
out the world it is a great achievement for mankind to make low cost, effective
potential, natural remedies from plants .However for development of rural tribes
and to conserve their knowledge under intellectual property right a vast effort is
needed.
Acknowledgements
I am very much thankful to my teachers, seniors and friends of University of
North Bengal for their valuable advice and suggestions. I am also thankful to
traditional healers of this area for their kind help and suggestions.
57
58
References
Bennet,S.S.R. 1987 .Name Changes in Flowering Plants of India and Adjacent
Regions,Triseas Publishers,Dehra Dun,India.
Bhardwaj, Ujala; Tiwari, B.K.; Prasad, Arun and Ganguly, Subha (2011) Study on
the post-inoculation histopathilogical effect of Tinospora cordifolia extract in skin
of broiler chicks. Indian J.Vet.Pathol. 35(2):225-226.
Kumari, R.; Prasad, A.; Tiwari, B.K. and Ganguly, S. (2011) Oroxylum indicum
possess a potential effect on humoral and cell mediated immune response in
broiler chicks. Indian J. Anim.Sci. 81(12): 22-24.
Kumari, R.; Tiwari, B.K.; Prasad, A. and Ganguly, S. (2011) Immunomodulatory
effect of herbal feed supplement in normal and immunocompromised broiler
chicks. Indian J.Anim.Sci. 81(2):158-161.
Mathias-Mundy, E. and C. M. McCorkle, 1989. Ethnoveterinary medicine: an
annotated bibliography. Bibliographies in Technology and Social Change, No.6,
p.199. Technology and Social Change Program, Iowa State University, Ames,
Iowa 50011. USA.
Mondal, T. and S. Biswas (2012). Documentation of some ethno-veterinary
medicinal plants of Bankura District, West Bengal,India.Life sciences Leaflets 6 : 4246,2012.
Prain, D.1996.Bengal Plants (Vol 1 & 2),Bishen Singh Mahendra Pal Singh, Dehra
Dun ,India.
Tabuti, J.R.S., S.S. Dhillion and K.A. Lye, 2003. Ethno veterinary medicine for
cattle (Bos indicus) in Bulamogi county Uganda: plant species and mode of use.
J.Ethnopharmacol. 88:279-286.
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59
Journal of Today’s Biological Sciences: Research & Review (JTBSRR)
ISSN 2320-1444 (Online)
JTBSRR
Vol.1, Issue 1, page 59-66, December 30, 2012
Available on: www.jtbsrr.in
Induction of Chitinase and Β-1, 3-Glucanase PR
Proteins in Tomato through Liquid Formulated
Bacillus Subtilis EPCO 16 against Fusarium Wilt
SA. Ramyabharathi*, B. Meena and T. Raguchander
Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore – 641
003, Tamil Nadu, India.
* Corresponding author: ramyabharu@gmail.com
Abstract
The effect of biocontrol agent Bacillus subtilis EPCO 16 on the induction of defense
enzymes chitinase and β-1,3-glucanase in tomato plants infected with Fusarium
oxysporum f. sp. lycopersici was investigated. The defense related proteins viz., chitinase
and β-1,3-glucanase were analysed spectrophotometrically. The maximum activity of
these defense enzymes was observed in seedling dip, soil application and foliar spray with
B. subtilis EPCO 16 liquid formulation in tomato plants challenged with the pathogen.
Activities of these defense enzymes reached maximum at the 7th day after challenge
inoculation with pathogen. The results demonstrated that both the defense enzymes
might play a special role in pathogenesis during fungal infection.
Keywords Fusarium wilt, Bacillus subtilis, PR Proteins
Introduction
Tomato (Solanum lycopersicum L.), a crop of high economic importance is the
second most important vegetable in the world after potato. Among the various
biotic factors affecting tomato, soil borne diseases are playing a major role in
drastic yield reduction (Lukyanenko, 1991). In India, the yield loss up to 45 per
59
60
cent was recorded due to Fusarium oxysporum f. sp. lycopersici. Use of chemicals
against soil borne pathogens leads to environmental pollution and toxic effects
on human health and give possibility to pathogens for building-up resistance to
chemicals. Hence biological control agents creating a more long lasting effect is a
necessity besides their antiphytopathogenic potential of soils. The biocontrol
agent, Bacillus subtilis produces several classes of broad spectrum lipopeptides
antibiotics which are effective suppressors of many plant pathogens, including
species of Fusarium, Pythium, Phytophthora, Rhizoctonia, Sclerotinia, Septoria and
Verticillium (Ongena and Jacques, 2008). Manikandan et al. (2010) reported that
the use of liquid bioformulation on tomato plants decreased the Fusarium wilt
incidence due to the induction of defense enzymes. Collective function of several
PR Proteins may be effective in inhibiting pathogen growth, multiplication and
spread of pathogen and be responsible for the state of induced resistance (van
Loon, 1997). Hence present study was undertaken to evaluate the induction of
defense enzymes by the application of bioformulation against Fusarium wilt of
tomato.
Materials and methods
The laboratory and greenhouse experiments were conducted at the Department
of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, India. The
seeds of tomato cv. PKM1 was obtained from the Department of Vegetable
Crops, Horticultural College and Research Institute, Tamil Nadu Agricultural
University, Coimbatore and used throughout the experiment. Authenticated
Bacillus subtilis EPCO 16 strain (Accession Number EF139864) was obtained from
the Culture Collection Centre of the Department of Plant Pathology,
Tamil Nadu Agricultural University, Coimbatore, India and was used for all the
studies conducted in this investigation.
Root priming of tomato seedlings with B. subtilis EPCO 16
Test bacterial strain EPCO 16 was cultured onto conical flasks containing 100 ml
of L.B. broth media and kept in incubator at 35oC for 24 hours with agitation.
After incubation, material was taken out from flask and centrifuged at 4000 rpm.
Supernatant was discarded and bacterial cells were collected from the pellet.
Inoculum of bacterial cells was prepared in liquid and talc formulation. Roots of
seedlings were primed with EPCO 16 in liquid and talc formulation and in
mancozeb by keeping for 30 minutes. After this the seedlings were transferred in
pots.
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61
Pathogen inoculation
The fungi viz., F. oxysporum f.sp. lycopersici was mass multiplied separately in
sand maize medium. Two months old plants maintained in earthenware pot
(two feet diameter) at the rate of three seedlings per pot were inoculated with
pathogens multiplied in sand maize medium. The pathogen F. oxysporum f.sp.
lycopersici was inoculated separately in different pots grown with tomato plants
@ 5 per cent (w/w) around collar region.
Assay of defense-related enzymes
Samples were collected from individual treatments to study the induction of
defense enzymes in response to foliar as well as soil borne pathogens in tomato
plants under glass house conditions. Leaves and roots from treated plants were
collected starting from 1st day upto 9th day at 48 h intervals. The leaves were
collected from treated and control tomato plants and immediately extracted with
2 ml of 0.1 M sodium citrate buffer (pH 5.0) at 4°C. The homogenate was
centrifuged for 20 min at 10,000 rpm. Protein extracts prepared from tomato
tissues were used for estimation of defense enzymes.
Preparation of Colloidal chitin
Colloidal chitin was prepared by treating 1 g of crab-shell chitin powder with
acetone to form a paste and then slowly adding 20 ml of concentrated
hydrochloric acid (HCl) while grinding in a mortar with the temperature
maintained at 5C. The syrupy liquid was filtered through glass wool and
poured into vigorously stirred 50 per cent aqueous ethanol to precipitate the
chitin in a highly dispersed state. The residue was sedimented and resuspended
in distilled water several times to remove excess acid and alcohol and then
dialysed against tap water. Chitin content of the suspension was determined by
drying a sample in vacuo and adjusted with distilled water to a final
concentration of 10 mg ml-1 (dry weight/volume) and stored at 5C
for further use (Berger and Reynolds, 1958).
Preparation of snail gut enzyme
Six hundred mg of the commercial lyophilized snail gut enzyme (Helicase,
Sepracor, France) was dissolved in 10 ml of 20 mM potassium chloride (KCl) and
chromatographed on a Sephadex G-25 column (38 x1.5 cm) using a 10 mM KCl
solution, containing 1 mM EDTA and adjusted to pH 6.8 for equilibration and
elution. The first 20 ml eluted after the void volume was collected (Boller and
Mauch, 1988).
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62
Preparation of p-dimethyl aminobenzaldehyde (DMAB) reagent
The DMAB reagent was prepared by the procedure described by Reissig et al.
(1955). Stock solution of DMAB was prepared by mixing 8 g of DMAB in 70 ml of
glacial acetic acid along with 10 ml of concentrated HCl. One volume of stock
solution was mixed with 9 volumes of glacial acetic acid immediately before use.
Assay
The reaction mixture consisted of 10 µl of 0.1 M sodium acetate buffer (pH 4.0),
0.4 ml enzyme solution and 0.1 ml colloidal chitin. After incubation for 2 h at 37°
C, the reaction was stopped by centrifugation at 5000 rpm for 3 min. An aliquot
of the supernatant (0.3 ml) was pipetted into a glass reagent tube containing 30 µl
of 1 M potassium phosphate buffer (pH 7.0) and incubated with 20 µl of 3%
(w/v) snail gut enzyme for 1 h. After 1 h, the reaction mixture was brought to
pH 8.9 by the addition of 70 µl of 0.1 M sodium borate buffer (pH 9.8). The
mixture was incubated in a boiling water bath for 3 min. and then rapidly cooled
in an ice-water bath. After addition of 2 ml of DMAB, the mixture was incubated
for 20 min. at 37°C. Immediately thereafter, the absorbance was measured at 585
nm. N-acetylglucosamine (GlcNAc) was used as a standard and the enzyme
activity was expressed as nmoles GlcNAc equivalents min-1 g-1 fresh weight.
Assay of β-1, 3 glucanase
One gram of tomato seedling root tissue was extracted in 5 ml of 0.05 M sodium
acetate buffer (pH 5.0). The homogenate was centrifuged at 10,000 rpm for 10
min. at 4C and the supernatant was used as enzyme source. -1,3-glucanase
activity was assayed by the laminarin-dinitrosalicylic acid method (Pan et al.,
1991). The reaction mixture consisted of 62.5 l of 4% laminarin (Sigma) and 62.5
l of enzyme extract. The reaction was carried out at 40C for 10 min. The
reaction was stopped by adding 375µl of dinitrosalicylic acid and heated for 5
min in boiling water, vortexed and its absorbance was measured at 500 nm. The
enzyme activity was expressed as g glucose released min-1 mg-1 of sample.
Statistical Analysis
The data were statistically analyzed using the IRRISTAT version 92 developed by
the International Rice Research Institute Biometrics unit, the Philippines (Gomez
and Gomez, 1984). Data were subjected to analysis of variance (ANOVA) at two
significant levels (P<0.05 and P < 0.01) and means were compared by Duncan’s
Multiple Range Test (DMRT).
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63
Results and Discussion
Synthesis and accumulation of PR proteins have been reported to play an
important role in plant defense mechanisms. Chitinases (PR-3 protein) and -1,3glucanases (PR-2 protein) have been reported to associate with resistance in
plants against pests and diseases (van Loon, 1997). In general, fungal cells
contain chitin and glucan as their cell wall constituents. The main mode of
antagonistic activity of microbes is production of lytic enzymes (chitinases and
-1,3-glucanases) which act on cell walls or organisms which have chitin and
glucan as their cell wall component (Singh et al., 1999) and also through induced
systemic resistance (ISR) in plant system. In the present study, the elevated levels
of chitinase and -1,3-glucanase in plants treated with bioformulation containing
endophytic bacteria against pathogens was observed. Significant increase in 1,3-glucanase activity was observed in treatment with seedling dip, soil
application and foliar spray of EPCO 16 liquid formulation (28.097) challenged
with F. oxysporum followed by the same combination with talc formulation
(23.652). These treatments recorded higher levels of -1,3-glucanase activity upto
seven days challenged with Fusarium wilt pathogen and declined thereafter
throughout the experimental period of nine days (Table 1). Elicitation of ISR in
cotton by B. subtilis EPCO 102 with chitin led to the lowest bacterial blight
incidence due to the induction of chitinase, -1,3-glucanase, peroxidase,
polyphenol oxidase in cotton (Rajendran et al., 2006).
Table 1. Induction of β-1,3-glucanase activity in tomato plants treated with
Bacillus subtilis (EPCO 16) bioformulation challenged with F. oxysporum f. sp.
lycopersici under glass house condition
*Values are mean of three replications
In a column, mean followed by a common letter are not significantly different at the 5% level
by DMRT
DAI – Days after inoculation
LF - B. subtilis EPCO 16 liquid formulation, TF - B. subtilis EPCO 16 Talc formulation
β-1,3-glucanase activity
Treatments
µmol equivalent glucose released/h/g of root tissue*
1 DAI
Seedling dip with LF 6.175ab
Seedling dip with TF 5.971b
Seedling dip with 5.965b
Mancozeb
Seedling dip + soil 6.092ab
application with LF
3 DAI
11.27c
8.270d
12.13b
5 DAI
14.00d
16.071c
13.960d
7 DAI
18.042d
21.276c
17.242d
9 DAI
16.521d
19.376c
15.691d
11.97bc
16.222c
23.392b
21.218b
63
64
Seedling dip + soil
application with TF
Seedling dip + soil
application
with
Mancozeb
Seedling dip + soil
application + foliar
spray with LF
Seedling dip + soil
application + foliar
spray with TF
Seedling dip + soil
application + foliar
spray with Mancozeb
Inoculated Control
Healthy control
6.357ab
12.47b
17.531b
24.650b
22.317b
6.175ab
11.27c
14.000d
18.042d
16.521d
6.523a
13.37a
20.260a
28.097a
24.375a
6.337ab
12.03b
16.532c
23.652b
22.218b
6.063b
12.23b
13.820d
18.232d
15.921d
5.220c
5.000c
5.200e
5.030e
5.223e
5.011e
5.225e
5.010e
5.200e
5.000e
Chitinases are PR-proteins which hydrolyze chitin, a major cell wall component
(3-10%) of higher fungi. Chitinases cleave a bond between C1 and C4 of two
consecutive N-acetyl glucosamine (GlcNAc) either by endolytic or exolytic
mechanisms. The treatment with seedling dip, soil application and foliar spray of
liquid formulation of EPCO 16 led to the enhanced activity of chitinase compared
to other treatments (Table 2). All the treatments were significantly different from
control. In general, fungal cells contain chitin and glucan as their cell wall
constituents. The main mode of antagonistic activity of microbes is production of
lytic enzymes (chitinases and -1,3-glucanases) which act on cell walls or
organisms which have chitin and glucan as their cell wall component (Singh et
al., 1999) and also through induced systemic resistance (ISR) in plant system.
Thus, in conclusion, the current study reveals the potential of liquid based
formulation of B. subtilis EPCO 16 induces the plant’s own defense mechanism to
suppress the Fusarium wilt of tomato.
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65
Table 2. Induction of chitinase activity in tomato plants treated with B. subtilis
(EPCO 16) bioformulation challenged with F. oxysporum f. sp. lycopersici
under glass house condition
*Values
are means of three replications
In a column, mean followed by a common letter are not significantly different at the 5% level by
DMRT.
DAI – Days after inoculation
LF – B. subtilis EPCO 16 liquid formulation, TF - B. subtilis EPCO 16 Talc formulation
Chitinase activity
Treatments
(mol of GlcNAc equivalent/min/g of fresh
tissue)*
1 DAI
3 DAI
5 DAI
7 DAI
9 DAI
d
d
d
a
Seedling dip with LF
59.213
79.300
89.400
98.900
90.100d
Seedling dip with TF
59.010d 69.126e
79.083e
89.100b
90.020d
Seedling dip with Mancozeb
47.213e 57.213f
69.103f
77.766c
72.010e
Seedling dip + soil application with 62.900b 88.620abc 98.300ab
99.333a
91.200cd
LF
Seedling dip + soil application with 60.366cd 87.316bc
93.433c
99.000a
92.216bc
TF
Seedling dip + soil application with 59.800d 89.143a
97.433b
98.500a
92.176bc
Mancozeb
Seedling dip + soil application + 65.900a 89.726a
99.233a
99.456a
96.333a
foliar spray with LF
Seedling dip + soil application + 65.420a 88.866ab
97.816ab
99.233a
93.523b
foliar spray with TF
Seedling dip + soil application + 61.800bc 87.100c
92.300c
99.400a
90.083d
foliar spray with Mancozeb
Inoculated Control
38.333g 39.416h
41.203h
42.400e
45.010g
Healthy control
40.113f
49.226g
58.126g
58.833d
60.033f
References
Berger, L.R. and Reynolds, D.M. 1958. The chitinase system of a strain of
Streptomyces griseus. Biochem. Biophy. Acta., 29: 522-534.
Boller, T. and Mauch, F. 1988. Colorimetric assay for chitinase. Meth. Enzymol.,
161: 430-435.
Gomez, K.A. and Gomez, A.A. 1984. Statistical Procedures for Agricultural
Research. John Wiley and Sons, New York. p.680.
65
66
Lukyanenko, A.N. 1991. Disease resistance. In : Monographs on theoretical and applied
genetics -14. (Ed.G.Kello). Springer Verlag, Berlin Heidelberg, pp.99-119.
Manikandan, R., Saravanakumar, D., Rajendran, L., Raguchander, T. and
Samiyappan, R. 2010. Standardization of liquid formulation of Pseudomonas
fluorescens Pf1 for its efficacy against Fusarium wilt of tomato. Biological Control,
54: 83–89.
Ongena, M. and Jacques, P. 2008. Bacillus lipopeptides : versatile weapons for
plant disease biocontrol. Trends in Microbiology, 16: 115–125.
Pan, S.Q., Ye, X.S. and Kuc, J. 1991. Association of β-1,3-glucanase activity and
isoform pattern with systemic resistance to blue mold in tobacco induced by
stem injection with Peronospora tabacina or leaf inoculation with tobacco mosaic
virus. Physiol. Mol. Plant Pathol., 39: 25-39.
Rajendran, L., Saravanakumar, D., Raguchander, T. and Samiyappan, R. 2006.
Endophytic bacterial induction of defence enzymes against bacterial blight of
cotton. Phytopathol. Mediterr., 45: 203–214.
Reissig, J.L., Strominger, J.L. and Leloir, L.F. 1955. A modified method for the
estimation of N-acetyl amino sugars. J. Biol. Chem., 217: 959-966.
Singh, P.P., Shin, Y.C., Park, C.S. and Chung, Y.R. 1999. Biological control of
Fusarium wilt of cucumber by chitinolytic bacteria. Phytopathology, 89: 92-99.
van Loon, L.C.1997. Induced resistance in plants and the role of pathogenesisrelated proteins. European J. Plant Pathol., 103: 753–765.
66
67
Journal of Today’s Biological Sciences: Research & Review (JTBSRR)
ISSN 2320-1444 (Online)
JTBSRR
Vol.1, Issue 1, page 67-86 December 30, 2012
Available on: www.jtbsrr.in
In vitro regeneration and genetic transformation in
groundnut (Arachis hypogaea L. cv. Smruti) for
abiotic stress tolerance mediated by Agrobacterium
tumefaciens
Kusum Rana and I.C.Mohanty*
Department of Agricultural Biotechnology, College of Agriculture
Orissa University of Agriculture and Technology
Bhubaneswar, E.mail-icmohanty1@rediffmail.com
* Corresponding Author
Abstract
Genetic improvement of Groundnut through genetic engineering of plant for tolerance to
moisture stress could be achieved by the regulated expression of a large number of stress
responsive genes. In plant, one transcription factor DREB1A/CBF3 controls the
expression of many target genes through the specific binding of the transcription factor to
the cis-acting element in promoters of the target genes (Ingram and Bartels, 1996;
Shinozaki et al., 2000). The present research aimed at developing an efficient regeneration
system of Groundnut cv. Smruti, performing Agrobacterium-mediated transformation of
Groundnut with rd29::CBF3 gene and screening of putative transformants by
kanamycin based selection system. Different explants namely, cotyledon, leaf and stem
segments were used to study the in vitro plant regeneration of groundnut (Arachis
hypogaea L.). All the explants were cultured in MS medium with different
concentrations of 2, 4-D, and NAA. Leaf explants showed better performances than
cotyledon and stem. Among different concentrations, 2, 4-D @ 2mg/l was found more
suitable for good callus induction. When enriched with a cytokinin (Kinetin, 0.5 mg/l),
the same medium produced quick, nodular, brownish friable good callus. MS medium
supplemented with different concentrations of BAP produced small shoot bud at different
subcultures. Enriched with NAA (0.5 mg/l), MS basal nutrient medium with BAP (2.0
mg/l) produced high frequency plantlets from callus cultures. Direct organogenesis was
also achieved with the same medium from de-embryonated cotyledonary segments. Shoot
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68
were rooted on MS medium supplemented with 1.5 mg/l NAA. De-embryonated
cotyledon explants of Groundnut were transformed using Agrobacterium tumefacience
strain GV3107 with the binary vector pCAMBIA2300 containing the gene for increasing
tolerance to moisture stress, CBF3 driven by a stress inducible promoter rd29A. For the
selection of transformed plants, lethal dose of kanamycin was optimized and was found to
be 60mg/l. A transformation frequency of 12.5% was achieved after 3rd round of
selection based on nptII selectable marker gene evaluated against the antibiotic
kanamycin at 80mg/l in the in vitro culture medium.
Key words: Groundnut, In vitro regeneration, genetic transformation, Agrobacterium
Introduction
Groundnut (Arachis hypogaea L. ; Family: Fabaceae, 2n=4x=40) is a major oilseed
crop cultivated largely in many tropical, sub-tropical and warm temperate
regions of the world. Groundnut is grown mainly for human consumption in the
form of seeds and for vegetable oil and a rich source of protein, lipids, and
dietary energy. The global production of peanut averages nearly 26.38 million
metric tones from almost 20 million hectare, with the annual production of 36.06
million tons of nuts-in-shells. The total area under groundnut cultivation in India
is 8.0 million hectares which accounts for the total production of 7.5 million tons
with the productivity of 937.5 Kg ha-1 (FAO Database, 2008). Its yield is
vulnerable to a variety of biotic and abiotic constraints (Nageshwara Rao and
Nigam, 2001) & (Ghewande et al. 2002a). Among the abiotic stresses, soil
moisture deficit at various stages of crop growth during rainy season and low
temperature during germination and vegetative stage but high temperature
during the pod filling and maturation stage during summer hampers the
productivity.
The procedures and objectives for peanut improvement programs largely
depend on the use of the crop, whether for oil or food, and on the amount of the
inputs used in production. When grown as a subsistence crop by low-resource
farmers, measures to manipulate stresses are no longer cost effective. Under
these conditions, the crop by itself must be tolerant to a range of biotic and
abiotic stresses if adequate yields are desired. Conventional breeding has led to
the improvement of few peanut traits like seed yield and drought tolerance.
However, due to its limited applicability, many of the important agronomic traits
have yet to be improved. Although the genetic variability within wild species
includes many of these valuable traits, wide hybridization in peanut has limited
applicability due to cross incompatibility, low frequency to produce hybrid seeds
and linkages with undesired traits. Genetic transformation offers a solution by
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69
making the transfer of genes from alien sources feasible for generating transgenic
plants possessing resistance to biotic & abiotic stresses. Initial attempts to
develop transgenics for abiotic stress tolerance involved single action genes i.e.
genes responsible for modification of a single metabolite that would confer
increased tolerance to salt or drought stress. But abiotic stress tolerance is likely
to involve many genes at a time and single gene tolerance is unlikely to be
sustainable. Therefore, genetic engineering with regulatory proteins/
transcription factors has emerged as a new tool for controlling the expression of
many stress-responsive genes. Through these proteins, many genes involved in
stress response can be simultaneously regulated by a single gene encoding stress
inducible transcription factor (Kasuga et al. 1999), thus offering possibility of
enhancing tolerance towards multiple stresses including drought, salinity and
freezing. In plant, one transcription factor can control the expression of many
target genes through the specific binding of the transcription factor to the cisacting element in promoters of the target genes (Ingram and Bartels, 1996;
Shinozaki et al., 2000). Several cDNAs encoding the dehydration responsive
elements (DRE) binding proteins, DREB1A and DREB2A have been isolated from
A. thaliana and shown to specifically bind and activate the transcription of genes
containing DRE sequences (Liu et al.1998). Over expression of DREB1A/CBF3 in
transgenic Arabidopsis plant showed increased tolerance to freezing, drought
and high salt concentration, suggesting that the DREB 1A/CBF3 proteins
function without modification of the proteins in the development of stress
tolerance.
Strong and constitutive promoters are beneficial for high level expression of
selectable marker genes which is necessary for efficient selection and generation
of transgenic plants. Constitutively active promoters are not always desirable for
plant genetic engineering because over expression of a transgene may compete
for energy and building blocks for synthesis of proteins or RNA that are also
required for plant growth under normal conditions. Over expression of
Arabidopsis DREB1/CBF3 gene also causes severe growth retardation under
normal growth conditions. Use of a stress inducible rd29A promoter instead of
the constitutive CaMV35S promoter for over expression DREB1A/CBF3
minimizes negative effects on plant growth (Kasuga et al., 1999).
A variety of techniques for plant transformation are available, these techniques
can be split into two groups: Agrobacterium mediated transformation and direct
gene transfer methods such as particle bombardment, electroporation,
microinjection etc. Agrobacterium mediated transformation is widely used with
the dicotyledonous crops, which reflects the natural host range of members of
the genus Agrobacterium. Direct gene transfer methods are most commonly used
to transform monocotyledonous crops such as cereals. Successful genetic
transformation of plants generally requires a well-established tissue culture
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70
system to regenerate whole plants from single cells. Gene transfer through single
cell-originated somatic embryogenesis yielded gene transfer into each cells of
regenerated plants with higher transformation frequencies without chimeric
variations (Wilkins et al., 2004. Hence, the present research aimed at developing
an efficient in vitro regeneration and transformation system in groundnut with
rd29A::DREB1A gene construct.
Materials and methods
Materials used
The high yielding popular groundnut variety Smruti collected from the Breeder,
Groundnut, Dean of Research, Orissa University of Agriculture and Technology,
Bhubaneswar was used as the source of explants and Agrobacterium tumefaciens
strain GV 3107 harboring binary vector of pCAMBIA2300 with the gene
construct (IARI, New Delhi) (Fig.1) under MOU were used in the investigation.
Fig.1. Gene Construct and Restriction Map of DREB1a gene
Culture media and Growth regulators
The culture medium used in the plant tissue culture was MS medium
(Murashige and Skoog, 1962).
Benzyl Amino Purine (BAP), Naphthalene Acetic Acid (NAA), Kinetin and 2, 4-D
all from HIMEDIA were used as growth regulators. Agrobacterium strain GV3107
harboring DREB1a gene was cultured in liquid YEMA (Yeast Extract Mannitol
Agar) medium supplemented with 10mg/l Kanamycin, 50mg/l Rifampicin. The
YEMA culture was incubated at 28 º C for optimum growth of bacteria.
Restriction Enzymes like BamH1 and Sac1 from GENEI, Bangalore were used for
restriction digestion analysis.
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71
Callus induction
Three different explants (cotyledons, stem, & leaf) were excised from 10-day-old
aseptically grown seedlings and were cultured on MS medium which was
subjected to different concentrations of 2, 4-D (0.5, 1.0, 1.5, 2.0mg/l) and 0.5mg/l
KIN for callus induction. The explants were begun to enlarge within 13th day of
culture initiation.
Shoot bud regeneration
Calli from cotyledon were cultured on MS-medium supplemented with varying
concentrations of BAP (1.0, 1.5, 2.0, 2.5mg/l) or Kinetin (1.0, 2.0mg/l) in
combination with NAA (0.5mg/l) individually for shoot bud differentiation and
plant regeneration. De-embryonated cotyledonary segments were also used for
direct regeneration. Shoots and buds formed in a cluster from callus as well as
directly from cotyledons were separated into smaller clumps and sub cultured
on MS- medium containing BAP (1.0, 1.5, 2.0, 2.5mg/l) or Kinetin (1.0, 2.0mg/l)
in combination with NAA, IAA for further growth and shoot multiplication.
Later, shoots were separated and transferred to fresh medium for micro
propagation and production of uniform sized plantlets.
Root induction and hardening
The regenerated elongated micro shoots (>3 cm in length) were excised and
planted on MS-medium supplemented with NAA (0.5, 1.0mg/l) for root
induction. Plantlets with well developed roots were removed from the culture
tubes and, after washing roots in running tap water were transferred to beakers
containing water for pre hardening. After 1 day the rooted shoots were
transferred to plastic cups containing soil, sand and compost in the ratio of 1:1:1
in a growth chamber. Subsequently, the plantlets were transferred to field
conditions, where they flowered and set viable seeds.
Optimization of a kanamycin based selection system
In order to determine the lethal dose of the antibiotic, kanamycin on shoot
proliferation and plant regeneration, the primary shoot obtained after 2 weeks of
culture on shoot induction media described as above were transferred to fresh
shoot induction media supplemented with different concentrations of kanamycin
(20, 40, 60 and 80 mg/l). The viability of regenerated shoots was observed after 1,
2 and 3weeks.
Validation of DREB1A gene in recombinant pCambia2300 vector
Isolation of plasmid from Agrobacterium tumefeciencs strain GV 3107
Alkali lysis method was used for isolation of plasmid from GV3107 cells as was
described by Sambrook et al.(1989). Single colonies were picked and grown in
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3ml of LB media with kanamycin (50mg/l), rifamycin (10mg/l) for 16h at 37˚C
and the culture was centrifuged at 10000 rpm for 2 min a 4˚C.
Restriction digestion of DNA
Restriction digestion of plasmid DNA isolated from the bacterial colonies was
done as per the standard procedure (Sambrook et al., 1989) using restriction
endonuclease BamH1 and Sacl (Genei, Bangalore ) in an appropriate buffer at
37˚C for 1h.The digested products were analyzed on a 1.2% Agarose gel.
Fig.2. Schematic map of vector of pCAMBIA2300
Agarose gel electrophoresis
Required amount of Agarose was weighed (1.2%w/v) and melted in TBE buffer
(0.9 M Tris borate, 0.002M EDTA, pH 8.2). Ethidium bromide was added at a
final concentration of 0.5mg/ml. After cooling to 50-55˚C, the mixture was
poured onto a preset template with an appropriate comb. The comb was
removed after solidification and gel with template was placed in an
electrophoresis chamber containing the running buffer (1X TBE). DNA to be
analyzed was mixed with the gel loading buffer (6X buffer contain 0.25%
bromophenol blue, 30% glycerol in water) at 5:1 ratio and loaded into the well.
Electrophoresis was carried out at 50V (Sambrook et al., 1989).
Agrobacterium-mediated transformation
The genetic transformation of the explants was done by co-culturing with the
Agrobacterium cultures. The cotyledons without embryogenic axis were excised
aseptically and treated with Agrobacterium suspension for 30 minutes. After
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immersion, excess bacterial suspension was removed by placing cotyledons on
sterile tissue papers and the cotyledons were transferred and placed adaxial side
in 30mmX25mm Petri plates having MS-medium supplemented with 30g/l
sucrose, 2.0mg/l BAP, 0.5mg/l NAA, 6g/l agar: (pH5.8), overlaid with What
man No.1 filter paper (wetted with 1.0 ml of MS-medium containing 10mg/l
kanamycin) sealed with Para film and incubated at 25˚C for 3 days. The heavily
infected explants were washed with 500mg/l cefotaxime solution and
transferred to fresh multiple shoot induction medium containing 2.0mg/l BAP,
0.5mg/lNAA and 500mg/l cefotaxime. The explants exhibiting multiple shoot
buds after 20 days were sub-cultured on MS medium containing 2.0mg/l BAP,
0.5mg/l NAA and 500mg/l cefotaxime. The well developed healthy shoots were
transferred to MS medium containing 2.0mg/l BAP, 0.5mg/l NAA, 500mg/l
cefotaxime and 60mg/l Kanamycin for rooting and selection of putative
transgenics. The explants without co-cultured were used as negative control.
The well developed shoots that survived the kanamycin selection were rooted on
MS medium supplemented with 2.0mg/l NAA, 500mg/l cefotaxime and 60mg/l
Kanamycin.
Results and Discussion
Morphogenetic responses of different explants on callus induction
All the explants (Cotyledon, stem & leaf) produced callus when cultured on MS
medium supplemented with different concentrations of 2, 4-D and KIN (0.5mg/l).
The effect of different concentrations of 2, 4-D and Kinetin on callus induction of
peanut are presented in Table 1. Friable, compact, light green, brownish and
watery types of callus were observed irrespective of explants. These were
maintained on the same medium by repeated sub culturing for further
proliferation. Callus obtained from stem explants was brown, watery and friable
(Fig. 3). Calli from leaf and cotyledon explants proliferated profusely and turned
deep green and friable (Fig.3b). The highest frequency of callus induction
observed was 90%, 90% and 100% in cotyledon, stem & leaf explants respectively.
The best callus growth was obtained when 2, 4-D (2.0 mg/l) and 0.5 mg/l kinetin
were used in the medium.
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74
Table 1. Effect of phytohormones and explants on Callus induction in
Groundnut (cv.SMRUTI, 10 days old seedling).
Treatment Particulars of treatments
Observation
Explant Growth
Days to % of
medium
callus
callusing
(MS+
induction
Phytohormones)
2,4-D
Kinetin
Nature
of
callusing
Callus type
T-1
Leaf
0.5
0
28
25
(+)*
T-2
Leaf
1.0
0
21
25
(++)
T-3
Leaf
1.5
0
21
50
(++)
T-4
Leaf
2.0
0
14
100
(++++)
T-5
Leaf
0.5
0.5
20
30
(+)
T-6
Leaf
1.0
0.5
20
40
(++)
T-7
Leaf
1.5
0.5
15
40
(+++)
T-8
Leaf
2.0
0.5
12
100
(++++)
T-9
Cotyle
don
Cotyle
0.5
0
35
20
(+)
1.0
0
36
45
(++)
Poor
response,
Poor
growth
Compact,
greenish
Unsatisfacto
ry growth
Light green,
Compact
Optimum
growth
Optimum
growth
Greenish,Po
or growth
Light
green,comp
act
in
middle
&
friable
outside
Green,Opti
mum
growth
Brown,
friable,loos
e
Optimum
growth
Poor
growth
Unsatisfacto
T-10
74
75
T-11
don
Cotyle
don
Cotyle
don
Cotyle
don
1.5
0
28
45
(+++)
2.0
0
21
70
(+++)
0.5
0.5
35
55
(+)
Cotyle
don
Cotyle
don
Cotyle
don
1.0
0.5
34
55
(+)
1.5
0.5
20
85
(++++)
2.0
0.5
16
90
(+++)
T-17
Stem
0.5
0
28
15
(+)
T-18
Stem
1.0
0
28
75
(+)
T-19
Stem
1.5
0
20
75
(+)
T-20
Stem
2.0
0
18
90
(++)
T-21
Stem
0.5
0.5
30
70
(+)
T-22
Stem
1.0
0.5
24
70
(+)
T-23
Stem
1.5
0.5
24
70
(++)
T-24
Stem
2.0
0.5
18
85
(++)
T-12
T-13
T-14
T-15
T-16
CV%
CD (0.5)
*+ : Poor
++ : Good
11.86
1.46
+++: Very good
75
++++ : Excellent
ry growth
Optimum
growth
Optimum
growth
Compact,
poor
growth
Non-friable,
greenish
White,
friable
White,
friable,
quick
proliferatin
g
Compact,
whitish
Compact,
Watery
Compact,
whitish
Yellowish,
friable
Slow
growing
Brownish,
watery
Non-friable,
green
Yellowish,
friable
76
a
b
c
d
Fig. 3. Effect of 2, 4-D on callus induction- a. sterile seedling,
b. leaf ; c. cotyledon ; d. stem
Shoot-bud regeneration and elongation
Indirect Organogenesis
After 4 weeks of culture, the well proliferated nodular, compact calli were
subcultured two to three times in a reduced auxin containing medium.
Afterward, these were transferred to a shoot induction medium containing
different concentrations of BAP (1.0-2.5mg/l) in combination with graded doses
of either NAA (0-1.0 mg/l) or IAA (0-1.0 mg/l) for shoot bud regeneration. The
maximum percentage of calli having shoot bud was observed from the treatment
of BAP (2.0 mg/l) and NAA (0.5 mg/l) (87.5%) followed by BAP (2.5 mg/l) and
NAA (0.5 mg/l) (82.5% ) (Table 2). The highest mean number of shoots (22±1.5)
obtained while using BAP (2.0 mg/l) and NAA (0.5 mg/l) (Table 2) followed by
18±1.4 no of shoots per culture in BAP (1.5 mg/l) and NAA (0.5 mg/l).
76
77
Table 2. Effect of phytohormones on shoot bud differentiation and elongation
T3
2.0
0
_
30
24
80.0
12±1.1
T4
2.5
0
_
30
24
80.0
11±0.9
T5
1.0
0.5
_
40
31
77.5
16±1.2
T6
1.5
0.5
_
40
32
80.0
18±1.4
T7
2.0
0.5
_
40
35
87.5
22±1.5
T8
2.5
0.5
_
40
33
82.5
16±1.0
T9
1.0
1.0
_
30
21
70.0
10±0.8
T10
1.5
1.0
_
30
23
76.7
11±0.8
T11
2.0
1.0
_
30
22
73.3
10±1.0
T12
2.5
1.0
_
30
20
66.7
13±1.1
T13
1.0
_
0.5
40
30
75.0
14±1.2
T14
1.5
_
0.5
40
31
77.5
16±1.4
T15
2.0
_
0.5
40
28
70.0
16±0.9
T16
2.5
_
0.5
40
26
65.0
18±1.2
T17
1.0
_
1.0
30
19
63.3
06±0.5
T18
1.5
_
1.0
30
19
63.3
10±0.8
T19
2.0
_
1.0
30
20
66.7
11±0.8
T20
2.5
_
1.0
30
21
70.0
17±1.0
Treatment
No.of
shoots
culture
-
T2
Treatment particulars Biometrical observations
(MS+Phytohormones)
BAP
NAA
IAA
No.of
No.of
Rate
of
explants explants
Regeneration
cultured regenerated (%)
1.0
0
_
30
No
response
1.5
0
_
30
21
70.0
T1
77
8±0.7
/
78
Direct Organogenesis
Three different phytohormones were tested independently for their ability to
induce organogenesis directly. Complete plants were regenerated from in vitro
cultured sectioned de-embryonated cotyledonary segments. Multiple shoots
arose on 6-benzylaminopurine (BAP) (1.0 – 2.50 mg/l) supplemented Murashige
and Skoog’s medium with maximum production occurring at 2.0 mg/ l of BAP
(Table-3a &b). This medium produced highest number of shoots per culture
(12.1± 1.2) when enriched with NAA ( 0.5 mg/l) (Table 3b). Good callus growth
with bud primordia was observed in MS medium supplemented with 2, 4-D (2.0
mg/l). Callus growth as well as shoot development was observed in case of BAP
depending on the concentration. Slow callus growth was found at different
concentrations of NAA. So, from the above study we can assume that among the
different growth hormones tested for organogenesis, BAP was found to be more
suitable for direct regeneration compared to 2,4-D and NAA. When cultured on
only MS medium without hormone, none of the explants did show any response
(callusing or shoot bud differentiation). When higher concentrations of BAP (2.0
to 2.5 mg/l) were used, the explants directly developed shoot bud. No callusing
was observed in higher concentration of BAP.
Table 3a. Effect of Phyto-hormones in MS medium on plant let differentiation
in different explants of Groundnut (Direct Organogenesis)
2,4-D
mg/l
BAP
mg/l
0.5
0
1.0
0
1.5
0
2.0
0
0
1.0
0
1.5
0
2.0
0
2.5
NAA Morphogenic response
mg/l
De-embryonated
Leaf
Cotyledonary
segment
0
Slow callus growth
Slow
callus
growth
0
Good callus growth
Good
callus
growth
0
Good callus growth
Quick,
Good
callus growth
0
Good callus growth
Quick,
Good
callus growth
0
Swelling
of
the No. Response
explants
0
Growth with shoot No. Response
bud primordial
0
Only shoot developed Slow
callus
growth
0
Only shoot developed Slow
callus
78
Stem
Slow
callus
growth
Good
callus
growth
Good
callus
growth
Quick,
Good
callus growth
No. Response
No. Response
Slow
growth
Slow
callus
callus
79
0
0
0
0
0.5
1.0
No. Response
Callus growth slow
0
0
1.5
Callus growth slow
0
0
2.0
Callus growth slow
growth
No. Response
Poor response
Callus
slow
Callus
slow
growth
No. Response
Poor response
growth Callus
slow
growth Callus
slow
growth
growth
Treatm
ent
Table 3b. Effect of different concentrations of BAP and NAA on shoot
regeneration from cotyledonal explants of Groundnut on MS
medium
(Direct Organogenesis)
Combinations of
Phytohormones (mg/l)
BAP
NAA
Observation on shoot regeneration
1
0.5
0
Regeneratio
n rate (%)
No response
2
3
4
5
6
7
8
1.0
1.5
2.0
2.5
0.5
1.0
1.5
2.0
2.5
0
0
0
0
0.5
0.5
0.5
0.5
0.5
47
64
72
75
38
53
61
87
75
9
10
No.of shoots/ Shoot
culture*
length*(cm)
2.3±0.3
2.5±0.7
5.7±0.8
4.8±0.2
1.6±0.2
2.6±0.3
3.7±0.4
0.4±0.1
0.6±0.1
1.0±0.1
0.8±0.2
0.8±0.1
1.1±0.1
1.1±0.1
12.1±1.2
8.8±0.2
1.6±0.3
0.8±0.1
* Values represent the mean ± standard deviation
Rooting and hardening
The healthy shoots were transferred to rooting MS medium containing NAA
(0.5-2.0mg/l). The development of small primary roots was observed within 4-5
days of transfer into rooting medium. After 15 days all the healthy primary roots
showed formation of secondary roots. All the shoots successfully produced roots
without any deformity. In earlier reports only 0.5 mg/l was used for rooting
(Sharma and Anjaiah, 2000). Hundred percent rooting was observed in the
present study as all the plants rooted successfully, this could be due to the high
concentration of NAA (1.50 mg/l) used (Table 4).The rooted plantlets were
removed from the in vitro system and were maintained in the pots containing
79
80
sterile soil substrate. The sand and soil ratio was maintained properly for the
mixture used in pots; which helped in the proper seepage and no stagnation of
water in pots. The sand soil mixture was autoclaved before use so as to eliminate
any pathogen present in soil. The Hoagland nutrient solution was used for
watering the plants taken for hardening which nourished the plants in the early
days of plant establishment (Radhakrishnan et al., 1999).
Table 4. Effect of PGRs on rhizogenesis
Treatment
Days to root induction
MS-media + 0.5 mg/l NAA
14-16
MS- media + 1.0 mg/l NAA
15-19
MS- media + 1.5 mg/l NAA
12-20
MS- media + 2.0 mg/l NAA
13-17
Percentage of Response
43.16
70.59
84.72
67.30
Optimization of kanamycin based selection system
Different graded doses of kanamycin were taken in the regeneration medium to
find out the toxic level that would suppress the normal growth of the plantlets in
vitro. The cotyledons were first stabilized in MS-medium containing 2.0mg/l
BAP +NAA 0.5mg/l without kanamycin for 7 days and then transferred to the
MS-medium + 2.0 mg/l BAP +NAA 0.5mg/l medium containing various
concentrations of kanamycin for the optimization of the concentration of
kanamycin capable of inihibiting the plant growth. The effect of kanamycin was
only observed at the concentrations above 30 mg/l on the shootlets with yellow
sectorial patches on leaves and at the concentration of 60 mg/l, the growth was
suspended and caused drastic decrease in the frequency of regeneration from
100% to 60% as well as number of shoots per explants and caused more than
50% necrosis in the regenerated shoots (Table 5). Thus, 60 mg/l kanamycin was
determined as the minimum concentration suitable for selection of putative
transformed shoots. This optimized antibiotic selection scheme eliminates the
regeneration of non-transformed escapes and discriminates between resistant
and non-resistant plantlets.
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81
Table 5. Effect of different concentrations of kanamycin on in vitro shoot
regeneration of groundnut cv. SMRUTI after 2weeks of shoot initiation.
Sl.
No.
Kanamycin
( mg/l)
No.
of
explants
cultured in
kanamycin
media
No.
of
explants
inducing
green
shoots
Regeneration
rate (%)
No.
of
shoots/explant
Green & Yellow &
Healthy
necrotic
shoots(a) shoots(b)
Kanamycin
susceptible
shoots
%
(b/a+b)*100
1
0
10
10
100
12
0
0
2
20
10
10
100
13
0
0
3
30
10
8
80
06
04
40
4
40
10
5
50
06
05
45
5
50
10
6
60
04
03
43
6
60
10
6
60
05
08
61
7
70
10
3
30
0
05
100
8
80
10
1
10
0
02
100
Validation of DREB1A gene in recombinant pCambia2300 vector
Double digestion released AtDREB1A gene (642bp) insert from pCAMBIA 2300
vector .
a
c
b
d
e
Fig. 4. Organogenesis(a-b: indirect; c-d: direct from cotyledon explants; e:
rhyzogenesis)
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82
Transformation
The de-embryonated cotyledon segments which were taken for co-cultivation
with strain GV 3107 containing the gene construct were treated by dipping in the
bacterial suspension for 5 minutes , then cultured for 3 days in shooting medium
without selection pressure and subsequently were grown on culture medium
containing 30mg/l kanamycin and 500 mg/l cefotaxime. Multiple shoots were
initiated in the cotyledon after 3 weeks of culture. Multiple shoots were
subcultured after every 2 weeks on selection medium by increasing
concentration of kanamycin from 30 to 60 mg/l and then to 80mg/l. The growth
of kanamycin resistance shoots was rapid. Kanamycin resistant shoots were
subjected to further high selection pressure and were maintained for evaluation
in transgenic green house.
b
a
Fig. 5. Hardening(a: pre-hardening; b: post-hardening)
Table 6. Agrobacterium-mediated transformation of groundnut cultivar
Smruti using de-embryonated cotyledon.
Gene construct
Used
No.
of
explant cocultivated
No.
of
explants
regenerated
No of
shoot
buds
No. of lines
survived after
third selection
Transformation
efficiency
(%)
based
on
kanamycin
selection (80mg/l)
pCambia2300
120
84
327
41
12.53
(DREB1a)
82
83
a
b
c
d
Fig. 6. Normal growth inhibition due to Kanamycin
( a-0mg/l ;b-30mg/l ; c- 60mg/l ; d-100mg/)
Development of an efficient plant regeneration system
The success of transgenic research in crop improvement demands an efficient in
vitro culture system for obtaining high frequency plant regeneration from the
target tissue of the genotype concerned. In vitro plant regeneration can be
accomplished through somatic embryogenesis or organogenesis. During last
decade, peanut regeneration through organogenesis/somatic embryogenesis has
vastly increased by using different explants. The frequency of plantlet
regeneration in all these protocols being quite low has deferred their usage for
genetic transformation studies. Besides, the in vitro regeneration in groundnut is
found to be dependent on particular genotype and type of explants. Even though
there are several reports presented for direct and indirect regeneration, there are
no reports presented for the comparative studies of groundnut regeneration.
Hence, this present research work was aimed to standardize the simple reliable
protocol for direct and indirect regeneration of groundnut in the high yielding
cultivar Smruti adaptable to various agroclimatic situations of the state.
In vitro regeneration following an interveining callus phase was first tried in this
crop. A combination of higher auxins and lower level of cytokinin results in
effective callus formation. Therefore, kinetin at 0.5 mg/l was added to increase
the frequency of callusing. These results are in agreement with the findings of
Bajaj et al (1981), Narasimhulu and Reddy (1983), and Venkatachalam et al.
(1994) in groundnut. Callus initiation from cotyledon explant took longer in
comparison to that in leaf and stem. Results obtained from this experiment
revealed that groundnut leaf explants followed by cotyledon were found to be
best suitable for callus induction and 2, 4-D @ 2.0mg/l was found to be the best
concentration for maximum frequency of callus induction. The well proliferated
nodular, compact calli obtained after frequent subculture were transfered to a
shoot induction medium and the basal salts of MS fortified with BAP (2.0 mg/l)
and NAA (0.5 mg/l) was found to be the best combination for the purpose.
However direct organogenesis reduces the duration of exposure of the explants
to the culture environment and therefore reduces the risk of occurrences of
83
84
somaclonal variation in regenerated plantlets which may scramble up the gene
construct used for genetic transformation. It was designed to assess the direct
regeneration response in vitro of different explants like leaf, stem and deembryonated cotyledonary segments without an intervening callus phase in the
MS nutrient medium fortified with different concentrations of cytokinin (BAP)
and auxins (2,4-D and NAA) separately and later BAP with NAA in combination
(Table-3a&b). Again, the MS basal salts supplemented with BAP (2.0mg/l) and
NAA (0.5 mg/l) was reported to be the best combination for direct
organogenesis.
The classical findings of Skoog and Miller (1957) that organogenesis in tissue
cultures is governed by the balance of auxin and cytokinin in the medium could
be well demonstrated. The cytokinin (BAP) enhanced shoot bud formation in
cultured callus of Arachis hypogaea is in accordance with previous reports on
groundnut (Mrogniski et al., 1981; Banerjee et al., 1988; McKently et al., 1990;
Cheng et al., 1992; Eapen and George, 1993). The combinations of auxins and
cytokinins at definite proportions are very critical and found to be essential for
the induction of shoot bud in groundnut (Mroginski et al., 1981; Narasimhulu
and Reddy. 1983; Banerjee et aL, 1988; McKently et aL, 1990; Cheng et aL, 1992;
Eapen and George, 1993; Venkatachalam et al., 1994). However, the procedure
and efficiency of Groundnut plant regeneration was complicated and even not
easily available to the scientists yet. A simple and efficient method for the
regeneration of autonomous plants from tissue culture is essential to establish a
genetic transformation protocol for Groundnut.
Agrobacterium tumefaciens mediated genetic transformation of groundnut has
been reported by several groups (Mc Kently et al., 1995; Cheng et al., 1996; Li et
al., 1997; Tiwari et al., 2008). However, in the present study, a transformation
frequency of 12.5% was achieved after 3rd round of selection at kanamycin
80mg/l (Table 6). Here, the kanamycin based selection system was used for the
screening of the putative transformants where some false possitives might have
escaped. However, these putative transformants need to be screened further by
employing other powerful techniques like PCR and southern blotting and
subsequently bioassay in the transgenic green house to obtain the groundnut
plants showing tolerance to moisture stress.
Acknowledgements
We sincerely acknowledge the DBT for making financial provision for this
research. We thankfully acknowledge the help rendered by IARI, New Delhi in
providing the gene construct for the research.
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85
References
Cheng M, Hsi DC, Phillips GC (1992) In vitro regeneration of valencia type
peanut (Arachis hypogaea L.) from cultured petioles, epicotyle section and other
seedling explants. Peanut Science. 19: 82-87.
Cheng M, Jarret RL, Li Z, Xing A, Demski JN (1996) Production of fertile
transgenic peanut plants generated by Agrobacterium tumefaciens. Plant cell Report.
15: 653-657.
Eapen S, George L (1994) Agrobacterium tumefaciens-mediated gene
transformation in Peanut (Arachis hypogaea L.). Plant cell Report. 13: 582-586.
Egnin M, Mora A, Prakash CS (1998) Factors enhancing Agrobacterium tumefaciens
mediated gene transfer in peanut (Arachis hypogaea L.). In Vitro Cellular and
Developmental Biology Plant. 34: 4, 310-318.
Grant JE, Cooper PA, Gilpin BJ, Hoglund SJ , Reader JK, PitherJoyee MD,
Timmerman-Vaughan GM (1998) Kanamycin is effective for selecting
transformed peas. Plant Sci. 139:159-164.
McKently AH, Moore GA, Doostdar H, Neidz RP (1995) Agrobacterium mediated
transformation of peanut (Arachis hypogaea L.) embryo axes and the development
of transgenic plants. Plant Cell Report. 14: 699-703.
Nageswararao RC, Nigam SN (2001) Genetic options for drought management in
groundnut. In “Management of Agricultural Drought; Agronomic and genetic options”
(N. P. Saxena ed.). Oxford and IBH Publishing Co., New Delhi.
Radhakrishnan T, Murthy TGK, Bandyopadhyay A (1999) Multiple shoot
induction in groundnut (Arachis hypogaea L.). In: Plant Physiology for Agriculture
(Eds. Srivastava, G. C., Singh,K. and Pal, M.). Ponter Publishers, Jaipur, India. pp.
433-439.
Sharma KK, Anjaiah V (2000) An efficient method for the production of
transgenic plants of peanut through Agrobacterium tumefaciens mediated genetic
transformation. Plant Science. 159: 7-19.
Sharma KK, Lavanya M (2002) Recent developments in transgenics for abiotic
stress in legumes of the semi-arid tropics. Genetic engineering of crop plants for
abiotic stress. Proceedings of an APECJIRCAS joint symposium and workshop,
Bangkok, Thailand, 3-7 September 2001. JIRCAS Working Report. 23:61-73.
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Venkatachalam P, Geetha N, Jayabalan N, Sita L (1998) Agrobacterium-mediated
genetic transformation of groundnut (Arachis hypogaea L.): an assessment of
factors affecting regeneration of transgenic plants. Journal of Plant Research. 111:
1104, 565-572.
Venkatachalam P, Geetha N, Khandelwal A, Shaila MS, Lakshmisita G (2000)
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embryogenesis. Current Science. 78 (9): 1130-1136.
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Journal of Today’s Biological Sciences: Research & Review (JTBSRR)
ISSN 2320-1444 (Online)
JTBSRR
Vol.1, Issue 1, page 87-95, December 30, 2012
Available on: www.jtbsrr.in
“Groundnut (Arachis Hypogaea L.) Germplasm
Cultivation”
R. P. Bansode* and P. R. Shingare
*Department
of Botany, ASP College, Devrukh- 415804, Ratnagiri,
Department of Botany, Dr. Babasaheb Ambedkar Marathwada University,
Aurangabad - 431004 (MS) India.
*Corresponding author email: drranjitbansode@gmail.com
Abstract
Ten varieties of groundnut seeds were collected from “Agricultural College Latur (MS)
India” were sown in randomized block design. The germination percentage was observed
as maximum in LGN-2 and K-411 (98.14%) and minimum in TAG-24(85.18%).
Highest numbers of leaves were measured in TKG-19A at 75 days (205) and minimum
in TLG-45 at 15days (2). Maximum height was measured in K-411 at 105 days (31.7cm)
and minimum in TLG-45 at 15 days (1.0cm). Rhizosphere microflora of these varieties
shows difference in colony number of fungi, bacteria and actinomycetes. But the age of
groundnut plants are directly proportional to colonies of fungi, bacteria and
actinomycetes. The early flowering varieties were observed as K-411, TKG-19A, LGN-1
and TAG-24.The variety K- 411 shows maximum biomass among 10 varieties and Phule
Unap give maximum yield.
Keywords Arachis hypogaea; ten varieties; comparative study
Introduction
Groundnut is named as Arachis hypogaea Linn. and belonging to the family
Fabaceae. It is native of “Brazil”. The first written account of this crop is found
87
88
with the Spanish entry Hispanlola in 1502 where the “Arawak” cultivated under
the name of Mani. Records from the Brazil around 1550 showed the crop was
known there with the name “mandubi”. At present groundnut is grown widely
in almost all the tropical and sub-tropical countries of the world and also in the
warm temperate region for its beneficial fruits used in several ways (Buting A. H.
et al.1985).
It is cultivated through the India in “Kharif” season from April to July. It grows
best in warm region in India where temperature varies between 27 to 30 and
annual rainfall is between 50 and125 cm. well drained sandy looms are the most
favorable soils for groundnut growing (Ohu J. O. et al. 2006). In India groundnut
is grown over 6.4 million hectors with yield of over 6.9 million tons during
2001-2002 .The crop is largely in Andhra Pradesh ,Gujarat ,Tamil Nadu,
Karnataka, U. P., M. P., Punjab and Rajasthan (Shankarappa Talawar 2004).
Groundnut oil is non-drying vegetable oil cold pressed oil is golden yellow in
colour and possesses a faint agreeable smell. Principal fatty acids present in the
oil are oleic acids (56%) linolic acid (25%) and palmitic acid (6-12%). A little
amount of stearic acid arachidic and higher saturated acid is also present. The oil
is rich in phosphorus and vitamins (Thiamine riboflavin and niacin). (Wrenshell,
C. L. 1949). It is predominantly used for caulinary purposes. It is extensively
used for the manufacture of “vegetable ghee” by hydrogenation .It is also used as
lubricant and blends with mineral oil have been developed. Medicinally, the oil
is used as a laxative and emollient, soap-making, shaving creams, cold creams
pomades, candles, glycerin tallow substitutes. Emulsion is used for control of
many insect pests of plants Oil cake is used as feed for cattle and other farm
animals and as manure. The residue after uprooting the groundnut plants also
useful for cattle as fodder. The best quality cake is grounded into flour for
human consumption as a protein-rich food supplement (Singh and Singh 1991).
Materials and Methods
A) Material used for comparative study of Groundnut Germplasm cultivation
The material used for comparative study of groundnut germplasm was of 10different varieties of groundnut. The groundnut germplasm were collected from
“Agricultural College Latur”. These varieties were as follows:
1) LGN – 2
2) AK -320
3) TKG -19A
4) PHULE UNAP
5) M – 13
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89
6) LGN – 1
7) K – 1341
8) K – 411
9) TLG – 45
10) TAG – 24
Out of the total varieties of groundnut, these 10-varieties were selected for the
comparative study.
B) Following methods were used for experimentation
Before sowing, the field was cleared, ploughed 3-4 times, ordinarily no
manuring was required. The seeds of these ten varieties were sown. Before
sowing the plot were wetted with the help of running water. The sowing was
done by hand method in June. The seeds show “epigeal type” of germination
that is cotyledons raises along the growth of seedling. The percentage of
germination of these varieties was calculated with the help of total seed sown
and number of seed germinated. Each variety showed the variation in the leaf
count after 15, 30 45… 120 days. The difference in number of leaf was counted
from sowing to 15 days interval. Height was also noted after 15 days interval
from sowing up to 120 days. After 15 days interval from sowing the microflora of
root was observed on the selective media for fungi, bacteria and actinomycetes.
The media used for fungi was “Rose Bengal”, for bacteria Thornton’s agar
medium and for actinomycetes Jensen’s media. The fungi were observed and
identified. Bacterial and actinomycetes colonies were counted and result was
tabulated.
Three plants of each variety was selected as (small, medium and large.) a sample
for calculation of biomass. The fresh weights of such three samples were taken as
“fresh weight”. After sun drying, the dry weights of sample have measured as a
biomass for each groundnut variety. Firstly, number of pods per plant was
measured and then number of grains and weight of grains was calculated. In this
way, the total yield per variety was calculated.
Results and Discussion
Under the observation germination percentage was found maximum in LGN-2
and K-411 (98.14%). While the minimum was reported in TAG-24 (85.18%)
(Table 1). The maximum number of leaves was noted after 75 days in variety
TKG-19A and minimum in variety TAG-24 after 75 days (Table 2). The height
of the plant was measured at the interval of 15 days. Maximum height in K-411
at 105 days (31.7cm) minimum in TAG-24 (10.0cm) (Table 3). Rhizosphere
microflora of these varieties shows difference in colony number of fungi,
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90
bacteria and actinomycetes (Table 4). However, the age of groundnut plants is
directly proportional to colonies of fungi, bacteria and actinomycetes (Table 5).
Table 1. Percentage of seed germination in groundnut varieties
Sr. No.
Name
varieties
1
2
3
4
LGN-2
AK-320
TKG-19A
PHULE
UNAP
M-13
LGN-1
K-1341
K-411
TLG-45
54
54
54
54
Number
of Percentage of
seed
germination
germinate
53
98.14
49
90.74
52
96.29
48
88.89
54
54
54
54
54
48
49
47
53
51
88.89
90.74
87.03
98.14
94.44
TAG-24
54
46
85.18
5
6
7
8
9
10
of Total Seed
Table 2. Number of Leaves from sowing to 15 days intervals
Sr.
No.
1
Name of 15
Varieties Days
LGN-2
8
30
Days
23
45
Days
72
60
Days
68
75
Days
99
90
Days
102
105
Days
47
120
Days
35
2
3
AK-320
TKG19A
PHULE
UNAP
M-13
LGN-1
K-1341
K-411
TLG-45
TAG-24
7
7
17
13
53
67
53
142
99
205
78
201
27
76
25
75
7
21
52
100
109
100
25
74
5
5
7
13
2
7
32
19
25
32
17
20
66
50
59
180
34
36
73
67
61
198
71
54
105
85
99
200
72
56
106
74
80
119
27
23
75
26
26
68
12
72
35
35
38
-
4
5
6
7
8
9
10
90
91
Table 3. Height of groundnut varieties (cm) from sowing to 15 days intervals
Sr. No.
1
2
3
4
5
6
7
8
9
10
Name of
Varieties
LGN-2
AK-320
TKG-19A
PHULE
UNAP
M-13
LGN-1
K-1341
K-411
TLG-45
TAG-24
15
Days
8
7
7
7
30
Days
23
17
13
21
45
Days
72
53
67
52
60
Days
68
53
142
100
75
Days
99
99
205
109
90
Days
102
78
201
100
105
Days
47
27
76
25
120
Days
35
25
75
74
5
5
7
13
2
7
32
19
25
32
17
20
66
50
59
180
34
36
73
67
61
198
71
54
105
85
99
200
72
56
106
74
80
119
27
23
75
26
26
68
12
72
35
35
38
-
Table 4. Rhizosphere study of groundnut from sowing to 15 days intervals
Sr. No.
Days
1
2
3
4
5
6
7
8
15 Days
30 Days
45 Days
60 Days
75 Days
90 Days
105 Days
120 Days
Number of Colonies
Fungi
Bacteria
37
892
47
1133
54
1176
58
1187
68
1308
65
1370
69
1448
78
1485
Actinomycetes
87
105
114
115
208
285
402
447
Table No. 5 Identified Fungi from Rhizosphere of groundnut
Fungi
Aspergillus sp.
Penicillium sp.
Rhizopus sp.
Fusarium sp.
Trichoderma sp.
Macrophamina sp.
Regarding the flowering time the comparative study was made, it was found
that TKG-19A, LGN-1, K-411, TAG-24 were early flowering varieties of
groundnut; the flowers were appeared at 52 days. Biomass of the varieties was
measured by taking the difference between fresh weight and dry weight and it
was found that TKG-19A have shown maximum biomass (Table 6). It was
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92
recorded variety wise as follows by taking the No. of pods and grain weight.
Variety PHULE UNAP has shown high yield (5.93gm) (Table 7). Groundnut
thrives best in well - drained sandy loam soil, as light soils helps in easy
penetration of pegs and their development and their harvesting. However, soils
in plots are cotton black soil and having high water holding capacity.
Table 6. Biomass of ten varieties of groundnut
Sr. no.
1)
2)
3)
4)
5)
6)
7)
8)
9)
10)
Name of variety
LGN – 2
AK -320
TKG – 19A
PHULE UNAP
M - 13
LGN – 1
K-1341
K- 411
TLG-45
TAG -24
Fresh weight / Dry weight
11.66gm
21.67gm
46.67gm
11.66gm
13.0gm
7.33gm
9.67gm
23.0gm
-
Table 7. Yield of ten varieties of groundnut
Sr. No.
Name of variety
No. of pods
(weight)
1
LGN-2
30(13.36gm)
2
AK-320
20(7.78gm)
3
TKG-19A
10(8.32gm)
4
PHULE UNAP
2817.78gm)
5
M-13
11(13.33gm)
6
LGN-1
17(15.67gm)
7
K-1341
16(10.03gm
8
K-411
21(14.2gm)
Grain weight
4.45 gm
2.49gm
2.77gm
5.93gm
4.44gm
5.22gm
3.34gm
4.74gm
Rhizosphere microflora also affected on growth on development on groundnut
plant it is due to the contact of microorganism with roots, rich microflora can be
also promotes the groundnut plants (Louice M. Nelson 2002 and Sarode P. P. et
al. 2007). It is due to the nitrogen fixing bacteria in nodules provides nitrogen in
the form of nitrate and nitrites. Mycorhiza like Trichodrma spp. also act as anti
aflatoxin again some harmful fungi present in the groundnut growing soil
(Louice M. Nelson 2002).
The early flowering varieties of groundnut escape from groundnut pathogenic
effect (Somewhat less extent). Due to the early flowering and attain early
maturity. These varieties are TKG-19A, LGN-1, K-411 and TAG-24.The variety K411 shows maximum biomass and Phule Unap give maximum yield. (5.93gm /
plant).
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93
Acknowledgements
Authors are thankful to Prof. V. S. Kothekar, Head, Department of Botany, Dr.
Babasaheb Ambedkar Marathwada University, Aurangabad, for providing all
the necessary facilities and encouragement.
Anthesis in early flowering varieties of Arachis hypogaea Linn.
A
B
C
D
Plate: A, B, C and D are Fungi associated with Groundnut varieties.
93
94
References
Buting, A. H. Gibbons, R.W. and Wynne, J.C., 1985. Groundnut (Arachis Hypogaea
L). World geography of peanut.
Mamman, J. O. and Muni, U. B., 2006. Influence of vehicular tropic on air
permeability and groundnut production in asemi-arid loam soil. International
Agrophysiscs, 20:309-315.
Karajikat, P. N., Jadhav, G. S., and Wakle, P. K., 2004. Ecophysiology of yield
expression in groundnut genotype during post monsoon season. Journal of oilseed
research 21:39-41.
Louice M. Nelson, 2002. Plant growth promoting Rhizobacteria prospect for new
inoculants. Plant Management Network.
Nwokolo, E. and Smortt, J., 1996. Food and free from Legumes and oil seeds.
Chapman and Hall New York, pp 49-63.
Patel, L. R., Patel, R.H., Patel, J. K., 1991. Response of groundnut varieties of
different dates of sowing and row spacing. Journal of oil seed research 8:263-266.
Pathi, A. K., 1994. Response of groundnut varieties to time sowing under rainfied
conditions. Journal of oilseeds research 11:132-133.
Sahu, D. D. and Ptoliya, B. M., 2005. Assessment of efficient groundnut cropping
zone in Gujarat, India. International Arachis Newsletter 25:48-51.
Sarode, P. P., Rane, M. P., Chaudhari, B. L. and Chincholkar, S. B., 2007.
Screening for siderophore producing PGPR from black cotton soils of North
Maharashtra. Current trends in Biotechnology Pharmacy. Vol. 1. (1) 96-105.
Satish Kumar, G. D. and Popat, M. N., 2007. Knowledge and adaptation of
aflatoxin management practices in groundnut forming in Junagad, Gujarat,
India. E- journal by ICRISAT volume 3.
Shankarappa Talawar, 2004. Peanut in India, history, production and utilization
of peanut in local and global food system report no. 5(pp 3:4).
Singh, B. and Singh, U., 1991. Peanut as a source of protein for human food. Plant
food for human nutrition 41: 165-177.
94
95
Weiss, E. A., 2000. Oilseed crops. Black well science London.
Wrenshell, C. L. 1949. American Peanut industry. Economic Botany, 3:159-169.
95
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Journal of Today’s Biological Sciences: Research & Review (JTBSRR)
ISSN 2320-1444 (Online)
JTBSRR
Vol.1, Issue 1, page 96-100, December 30, 2012
Available on: www.jtbsrr.in
Effect of Fungicides and Biocontrol Agents in the
Management of Sugarcane Smut Disease
B. Meena* and SA. Ramyabharathi
Department of Plant Pathology, Tamil Nadu Agricultural University,
Coimbatore,TamilNadu,India,Email:meepath@rediffmail.com
*Corresponding author
Abstract
Smut disease caused by Ustilago scitaminea is a dreadful disease of sugarcane
and is endemic in most of the tropical regions. Results are described of field
trials in which attempts were made to control sugarcane smut with fungicides
and biocontrol agents. Smut disease of sugarcane was successfully controlled in
the plant crop when seed cane was treated with the solution of fungicide,
Triademefon. The lowest smut infection (4.4%) and the highest yield of 153 t/ha
was recorded in the sett treatment and foliar spray of Triadimefon 0.1% at 30,
45 and 60 DAP. The highest smut infection of 20.0% and the lowest yield of
113 t/ha was observed in the control.
Key words: Fungicides, biocontrol agents, smut, sugarcane
Introduction
Sugarcane is not only cash crop for the growers, but it is main source of
white crystal sugar. Sugarcane smut caused by the fungus Ustilago
scitanninea Sydow is becoming a more important problem in many cane
growing areas of the world. The disease is referred to as `culmicolous'
smut of sugarcane because it affects the stalk of the cane. Smut may
remain unnoticed for years, then quickly devastate large areas of
susceptible varieties. Hence, the disease has been called the `dread
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97
disease of sugarcane' by some and a `trivial disease with exaggerated
yield losses' by others. Infection ranges from 30-40% in plant crops and
even up to 70% in ratoons. Sucrose content of infected cane is reduced to
3-7%.
The smut pathogen usually infects the cane plant through the buds (Bock,
1964) and the infection of the buds of seed cane at or shortly after planting
is likely to be an important factor in the development of epidemics. The
affected canes produce long, black whip-like and coiled or curved shoots,
which are covered with a thin silvery membrane, containing masses of
chlamydospores of the fungus. Later on that membrane ruptures and
releases a multitude of spores, which contaminate soil and the standing
crop. Whips begin emerging from infected cane by 2-4 months of age with
peak whip growth occurring at the 6th or 7th month. The diseased plants
are unfit for use. Primary spread of the disease is through infected setts
and the secondary spread is through wind borne teliospores. Spores of
sugarcane smut, U. scitaminea are dilute brown, smooth and 5.5 to 7.5 u in
dia. The period from infection to whip production is about 6 months
under field conditions (Waller, 1969). Application to seed cane at the time
of planting is also likely to be the simplest and cheapest method by which
chemicals could be used for disease control in sugarcane. Little work has
been reported on the testing of fungicides applied to healthy or diseased
setts that have been planted in the field under severely smutcontaminated conditions.
Materials and methods
Field experiment was conducted during 2010-2011 for managing smut disease
of sugarcane using systemic fungicides and biocontrol agents. The smut whips
were collected from infected clumps and from these a suspension of
teliospores was prepared with water. Sugarcane setts were dipped for five
minutes in this spore suspension (10 6 spores/ml). Then the setts were treated
with respective fungicides and other microbial treatments for 15 minutes
before planting. The variety Co-Si (SC) 6 was used for the field experiment.
Observations were made on germination percentage, number of tillers per
hectare, smut disease incidence and cane yield (t/ha). The percentage of
smut infection was recorded at monthly intervals.
Results and Discussion
The results presented in Table 1 revealed that germination percentage varied
from 71.3 to 82.0% among the treatments. The number of tillers per hectare
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varied from 68,000 to 92,000 in the various treatments. The plant height
ranged from 7.3 to 9.6 m among the treatments (Table 1). Sett treatment and
foliar spray with Triademefon at a concentration of 0.1% effectively reduced
smut infection which recorded smut disease incidence of 4.4%. This was
followed by sett treatment and foliar spray with Propiconazole 0.1% which
showed disease incidence of 5.0%. The biocontrol agents were not effective
in reducing the smut infection which recorded disease incidence of 8.1%. In
the control, maximum disease incidence of 20% was observed (Table 2). The
yield was also found to be the highest (153 t/ha) in the effective treatment
of sett treatment and foliar application of Triadimefon 0.1%; whereas in the
control, the lowest yield of 113 t/ha was recorded (Table 1).
Table 1. Effect of fungicides and biocontrol agents on plant growth and yield
parameters of sugarcane
Treatments
Germina
tion (%)
Number of Plant
tillers/ha
height
(m)
Cane
yield
(t/ha)
Sett treatment + Foliar Spray with
carbendazim 0.1 % at 30, 45 and 60 DAP
Sett treatment + Foliar Spray with
Triadimefon 0.1% at 30, 45 and 60 DAP
Sett treatment + Foliar Spray with
Propiconazole 0.1% at 30, 45 and 60 DAP
Sett treatment + Foliar Spray with Copper
hydroxide 0.1% at 30, 45 and 60 DAP
Sett treatment + Foliar Spray with
Chlorothalonil 0.1% at 30, 45 and 60 DAP
82.0
86,000
9.6
129
78.0
82,000
8.9
153
77.5
86,000
8.7
145
76.0
72,000
9.2
136
79.0
92,000
9.4
138
Sett treatment + Foliar Spray with
Kresoxinmethyl 40% + Hexaconazole
8% (0.1%) at 30, 45 and 60 DAP
Sett treatment with Pseudomonas fluorescens @
20 g /litre + Foliar Spray with P. fluorescens
0.1% at 30, 45 and 60 DAP
Sett treatment with microbial consortia
(Trichoderma viride + P. fluorescens + Bacillus
subtilis) each @ 10 g/litre + Foliar Spray with
microbial consortia 0.1% at 30, 45 and 60
DAP
Control
80.0
88,000
8.9
145
78.8
92,000
9.4
124
77.5
88,000
9.2
126
71.3
68,000
7.3
113
7.2
12740
0.9
6.4
CD (P=0.05)
Mean of three replications
A successful fungicide treatment that aided in the production of healthy
seed cane and that protected seed cane from infection at planting could
make a useful contribution to the control of smut. Investigations of the use
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99
of fungicides for the control of smut have been carried out with setts that
were inoculated by dipping in spore suspensions, either before or after
treatment with fungicides (Muthusamy, 1973; James, 1976; Atienza and
Reyes (1977). Bharathi (2010) reported that sett treatment with fungicides
had shown radical reduction in smut incidence and fungicidal sett treatment
did not exhibit any influence on germination and shoot production.
Table 2. Management sugarcane smut disease using fungicides and biocontrol
agents
Treatments
Nov
2010
Smut disease incidence
Dec
Jan
Feb
2010
2011
2011
March
2011
Sett treatment + Foliar Spray with
carbendazim 0.1 % at 30, 45 and 60 DAP
Sett treatment + Foliar Spray with
Triadimefon 0.1% at 30, 45 and 60 DAP
Sett treatment + Foliar Spray with
Propiconazole 0.1% at 30, 45 and 60 DAP
Sett treatment + Foliar Spray with Copper
hydroxide 0.1% at 30, 45 and 60 DAP
Sett treatment + Foliar Spray with
Chlorothalonil 0.1% at 30, 45 and 60 DAP
1.8
3.8
4.4
4.4
7.5
0.6
1.3
1.3
1.3
4.4
1.3
1.3
1.3
2.5
5.0
1.8
3.1
3.8
4.4
6.9
1.8
3.1
3.8
4.4
6.9
Sett treatment + Foliar Spray with
Kresoxinmethyl 40% + Hexaconazole
8% (0.1%) at 30, 45 and 60 DAP
Sett treatment with Pseudomonas fluorescens @
20 g /litre + Foliar Spray with P. fluorescens
0.1% at 30, 45 and 60 DAP
Sett treatment with microbial consortia
(Trichoderma viride + P. fluorescens + Bacillus
subtilis) each @ 10 g/litre + Foliar Spray with
microbial consortia 0.1% at 30, 45 and 60
DAP
Control
1.3
2.5
2.5
3.1
5.6
2.5
4.4
4.a
5.6
8.1
2.5
4.4
4.4
5.0
8.1
3.1
5.6
6.9
8.1
20.0
1.9
1.9
2.4
2.5
3.1
CD (P=0.05)
Mean of three replications
Triadimefon applied to seed cane in the hot water tank consistently
provided complete or excellent protection against smut in the plant cane
crops (Bailey, 1979). This treatment was effective for infected seed cane that
was inoculated or planted in soil containing smut spores. The treatment,
therefore, is both eradicative and protective in action.
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100
References
Atienza, C.S. and Reyes, L.G. 1977. Control of sugarcane smut with
pyracarbolid fungicides. Philsutech Proc., 25: 33-36.
Bailey, R.A. 1979. Possibilities for the control of sugarcane smut (Ustilago
scitaminea) with fungicides. Proceedings of The South African Sugar
Technologist's Association, 137-142.
Bharathi, V. 2010. Chemical control of sugarcane smut through sett
treatment with
fungicides. Int. J. Pl. Protect., 2: 151-153.
Bock, K.R. 1964. Studies on sugarcane smut (Ustilago scitaminea) in Kenya.
Trans. Br. Mycol. Soc., 47: 403-417.
James, G.L. 1976. Preplant fungicidal dips : A long term measure against
smut. Sug. Path. News, 17: 4-5.
Muthusamy, S. 1973. Fungicides in the control of sugarcane smut. Sug. Path.
News, 10: 11 – 13.
Waller, J.M. 1969. Sugarcane smut (Ustilago scitaminea) in Kenya : I.
Epidemiology. Transactions of the British Mycological Society, 52: 139-151.
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Journal of Today’s Biological Sciences: Research & Review (JTBSRR)
ISSN 2320-1444 (Online)
JTBSRR
Vol.1, Issue 1, page 101-121, December 30, 2012
Available on: www.jtbsrr.in
Seasonal Movements and Migration of Birds: Indian
Scenario
Swati Bopinwar1, S.B. Zade1, T.K. Ghosh2*
1P.
G. Department of Zoology, R.T.M. Nagpur University,Campus, Nagpur–
440033(M.S.).
2Ultratech
Environmental Consultancy and Laboratory, Survey no. 87, office 7 and 8,
Bandal Prestige (in front of Siddhi Vinayak Mandir), Azad Nagar, Kothrud, Pune-411
038, E. mail- tkghosh@ultratech.in
Abstract
The exploration of bird-migration has entered a new era with individual-based tracking
during multiple years. Attempt has been made to collate available information pertaining
to migratory patterns of long-distance migrating birds to India in order to analyse the
variations amongst different species. While annual timings of migration vary much less
between repeated journeys of the same individual as compared to different individuals,
there are considerable variations in the routes of different varieties of birds within India.
The necessity of bird’s migration, threats encountered and conservation practices have
been critically assessed.
Key words: Bird migration, routes in India, flyways, diurnal & nocturnal migrants,
threats, conservation
Introduction
Migration is the regular, seasonal movement of populations from one geographic
location to another, and is common among most varieties of birds. It is marked
by the eventual return to the original place of departure and is most evident
among certain bird species that usually follow a yearly cycle. Donald (1952)
studied migration of birds across the Himalaya, and Ali (1962) on Wagtails in
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Kerala. In between 1963 and 1969, Biswas studied bird migration in southern
West Bengal with a view to collect data regarding the pattern of migration,
period of stay in the wintering area, time taken for journey etc. (ZSI 1991).While
George (1964) studied the same in Bihar, Mathew (1971) reviewed the recovery
data obtained by the BNHS’s bird migration study project. Khacher (1978)
studied bird migration across the Himalaya. Rainfall has great influence on the
bird population (Bayliss 1989). Birds migrate for many reasons that include the
need to travel to areas where food resources are at their peak abundance. The
food of the birds varies and is different not only in respect of different birds, but
also in respect of different seasons. The adaptive value of migration with
fluctuating food sources has earlier been well documented (Lack
1968; Alerstam et al. 2003). Survival challenges encountered on these journeys
may be responsible for a majority of annual adult mortality in land birds (Sillett
and Holmes 2002). Newton (2008) reported that there was increased mortality
during long migrations of avifauna.
Why do birds migrate?
Birds migrate for many reasons that include the need to travel to areas where
food resources are at their peak abundance, the climate is milder and there is less
competition for safe nesting places. However, the main environmental trigger for
bird migration seems to be the changing ratio of daylight and darkness. With the
onset of winter, days get shorter reducing activity hours. This triggers the almost
entirely instinctive phenomenon of bird migration. Migration has considerable
ecological significance. It enables birds to exploit peaks of food production and
to settle in areas where they could otherwise not live.
Timing of migration
Timing of migration is a mix of internal stimulus which results in a feeding binge
to put on fat to feel that they have put on enough fat to provide them energy
throughout the journey and then the tendency to aggregate into flocks. Once the
pre-migration flock is gathered, the feeding continues while the birds wait for
suitable weather conditions. Thus, while the birds' internal clock probably
releases the hormonal triggers at a fairly accurate date each year, the availability
of food and the presiding weather conditions decide when the migration starts
and hence when the first spring migrants arrive and the last autumn ones leave.
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103
Migration may be either during the day or night. Larger birds generally migrate
by day and smaller ones by night. Birds of prey, swallows and crows migrate by
day. Wildfowl, pelicans, storks, swifts etc are diurnal migrants. Nocturnal
migrants include water-birds, cuckoo, flycatchers, thrushes, warblers, orioles,
buntings, most songbirds etc. It is believed to be some hormonal stimulus to
migrate, resulting, at least in the spring, in the development of the gonads. Other
stimuli appear to involve temperature, daylight/darkness ratios and an internal
clock. The timing of migration is usually a mixture of internal and external
stimulus.
Types of migration
Birds generally migrate in flocks. Most migrations are latitudinal, i.e. from North
to South; also some migrate from East to West. It is used to describe movements
of bird populations. One way to look at migration is to consider the distances
travelled.



Short-distance migrants: May move only a short distance, as from higher
to lower elevations on a mountainside.
Medium-distance migrants: Some species may cover distances that span
from one to several states.
Long-distance migrants: Birds that typically have ranges that extend from
one continent in the summer to another in the winter.
The pattern of migration can vary within each category, but is most variable in
short and medium distance migrants. The long-distance migrants are mainly
insect-eaters and waders, which follow set migration patterns. Seed-eaters, on
the other hand, have more random movements, while fruit-eating birds are
generally
resident
(http://birding.krugerpark.co.za/birding-in-kruger-migrationroutes.html).
How does the weather affect migration?
Weather is the number one driving factor for migration. Bird’s sensitivity
varies towards temperature and other environmental conditions. There is
indication that a following wind is of major importance. A clear sky also helps,
but is of secondary importance. Birds will take flight in overcast days if the
wind is good. For hawks and other soaring birds, updrafts are of extreme
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importance. They can't go very far flapping those heavy, wind-resistant wings.
That's why they usually fly parallel to north-south mountain ridges that will
produce updrafts with the wind. They gain altitude in one updraft and glide
towards the next one. That's how some raptors will migrate all the way to
Argentina and back. Birds also try to avoid storms and foggy weather
whenever possible. Every year thousands of birds die on severe storms,
disoriented or exhausted, many get lost at sea. To avoid this, northern
migration of great ocean crossers American Golden-Plovers is made inland,
following the rivers in the Amazon Basin and the Mississippi valley. This is
much safer than doing the North Atlantic Ocean cross again, as the weather
there is still in the grip of winter and there's nowhere to land. Migration can be
acquired, abandoned or pro­longed by a species, depending on conditions
along their migratory routes (Able and Beltoff 1998). According to BBC news
South East Wales (July 12, 2012) unpredictable weather had altered bird
migration patterns, with many arriving in Wales weeks earlier than expected.
The report further commented “2012 has been a year of unpredictable weather,
and many birds have already been affected”. Sudden changes in the weather
can be disastrous for birds. Many birds prefer to fly at a higher altitude while
migrating. This is because winds usually prevail at higher altitudes and at the
same time, the cold temperature at these altitudes helps them in diffusing the
body heat, which is generated by their flight muscles.
Flyways
Global
The first natural historian to write about migration as an observable fact was
Aristotle. Though Herodotus described the migration of Cranes from north of the
Black Sea to Central Africa 100 years before, Aristotle was an astute observer and
as well as recording the times of departure of some species from Greece, and
listing Pelicans, Turtle Doves, Swallows, Quail, Swans and Geese correctly as
migrants. He accurately observed that all migrating birds fatten themselves up
before migrating. Birds that migrate from the same geographic region often
follow broadly well-defined routes known as migratory flyways. There are eight
recognized
shorebird
flyways
around
the
world
(http://www.kolkatabirds.com/migration.htm). The Asia-Pacific region, as defined by
the main migratory routes of water birds, is made up of three shorebird flyways the Central Asian Flyway, the East Asian-Australasian Flyway and the Western
(or Central) Pacific Flyway crossing 57 countries and territories in the region. The
East Asian-Australasian Flyway is the best studied and stretches from Siberia
and Alaska southwards through East and SE Asia to Australia and New Zealand,
and supports over five million migratory shorebirds. The Central Asian flyway
spans about 30 countries from the Arctic to the Indian ocean. But these flyways
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105
are just generalizations and bird populations have been known not to strictly
follow it. During migration, birds depend on strategically located staging areas
where they stop to rest and "refuel", by building up fat deposits, before
continuing their migration.
Indian
In India and South Asia, out of over 2000 species and sub-species, about 350 are
migrants. It is estimated that over 100 species of migratory birds fly into India,
either in search of food or to escape severe winter of their native habitat. In
Indian subcontinent, the majority of migratory birds are winter migrants. When
the conditions at their natural habitat become unfavorable due to low
temperatures, migratory birds fly to regions where conditions are comparatively
favorable. However, the physiology and mechanics of migratory bird flights are
not very well known in India. The Bombay Natural History Society (BNHS),
Mumbai has been working since 1926 to rectify this shortcoming. Migratory
routes are not fixed and in some species part of the population follows one route
and parts another. In India, the winter migrants from central Asia and Siberia are
thought to use two main flyways; one in the west along the Indus valley and the
other in the NE along the river Brahmaputra. Some migrants fly very long
distances. The majority of the avian migrants to India are from the north and
beyond the temperate latitudes. These come from Asia Minor, Arabia, Central &
NE Asia, East Asia and Europe. First year birds may migrate unescorted to a
winter home they have never before seen and return the following spring to the
area in which they were born.
Arrival of migratory birds in India
Avian migration is a natural process, whereby different birds fly over distances
of hundreds and thousands of kilometers in order to find the best ecological
conditions and habitats for feeding, breeding and raising their young. During
December several birds from the colder regions are spotted at several places in
India. Depending on species and country of origin, there are different migration
patterns. While small birds like flycatchers arrive in early November, wagtails
usually arrive in mid-October. Ducks turn up by end of October or early
November in huge numbers. It has been observed that extended rainfall, beyond
monsoon, delayed arrival of birds who find difficult to fly in the rain. The bird’s
migration has been categorized into winter migrants, summer migrants, and
passage migrants. Birds visiting the lake between November and February are
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the winter visitors. Some residential and migratory birds breed or remain in the
lake in summer and are called summer visitors. A brief account of the migratory
birds in India is summarized in Table 1. Presently, probably due to the climate
change, departure dates of many of the migratory species are postponed and this
has an alarming significance because food cycles and arrival times of migrants in
the tropics become synchronized. Almost 80 per cent of migratory birds did not
turn up in 2010. At least 15-16 varieties of ducks arrive every year but in 2010
there were hardly any despite of abundance of water. The only species present in
the year were the northern shoveller, a few wigeons, the brahminy shelduck and
pintails. Species abundance and diversity both have reduced. Waders (longlegged wading birds), little-ringed plovers and others like the kentish plovers,
greenshank and redshank were only a few in 2010. Birds of prey, such as the
montagu harrier, pale harrier, hen harrier and the pied harrier that usually come
from Eastern Europe, Central, Northern Asia and Southeast Asia did not arrive
in 2010. The biggest change has been observed in the migratory patterns of water
birds. Due to changing crop pattern, the number of cranes coming to India over
the years has reduced by as much as 75 per cent. As many as 4,000-5,000 barheaded geese were sighted in the past. But in the last two years, flocks of only 4050 of these birds were seen at one time. The number of geese has definitely
reduced by 50 per cent (Agarwal, 2011).
Table 1. Distribution of migratory birds in India
Sr.
Common
No
Name
.
Ashy
1
Minivet
Scientific
name
Type of migration
Pericrocotus
divaricatus
Vagrant
Distribution in India
C
Peninsula,
Mumbai,
Andaman
Himalayas, C India, E Ghats,
Resident & widespread W
Ghats,
Peninsula,
winter visitor
Andaman,
Nicobar,
Meghalaya, Ladakh
2
Asian
Brown
Flycatcher
Muscicapa
dauurica
3
Barheaded
Goose
Anser indicus
Widespread winter visitor Ladakh, Assam valley,
& resident
Assam hills, Kashmir
4
Barn
Swallow
Hirundo rustica
Widespread resident
winter visitor
5
Black
Phoenicurus
S
Arunachal, S Assam hills,
& Lakshadweep,
Andaman,
Nicobar,
Assam
valley,
Kolkata, Manipur
Resident & widespread S Assam hills
106
107
Redstart
ochruros
winter visitor
NW India, Assam valley,
Gujarat, Delhi
NW Himalayas, Kashmir, S
Assam hills (E Meghalaya,
Cachar, Manipur)
6
Black Stork Ciconia nigra
Widespread winter visitor
7
Blackeared Kite
Milvus lineatus
Widespread winter visitor
Grus nigricollis
Winter visitor, breeds in Ladakh, Kashmir, W Bengal,
Ladakh
Arunachal
Podiceps
nigricollis
Sparse winter visitor
Himantopus
himantopus
Resident & widespread Kashmir,
Mumbai,
winter visitor
Andhra, Himalayas
Monticola
solitarius
Resident & widespread
Himalayas
winter visitor
Ladakh, saline lakes ( Punjab
Widespread winter visitor salt range, Sambhar Lake;
& resident
Rajasthan, salt Lakes near
Kolkata), W India
8
9
10
11
12
13
14
15
16
Blacknecked
Crane
Blacknecked
Grebe
Blackwinged
Stilt
Blue Rockthrush
Brownheaded
Gull
Caspian
Gull
Caspian
Plover
Caspian
Tern
Cattle
Egret
Larus
brunnicephalus
Larus
cachinnans
Charadrius
asiaticus
Sparse winter visitor
NW India
Vagrant
W & SE coast of India
Sterna caspia
Widespread winter visitor
Gujarat, NW India
Bubulcus ibis
Widespread resident
Amroha, Uttar Pradesh
Buteo buteo
Widespread winter visitor
Kashmir, Uttar Pradesh
& resident
17
Common
Buzzard
18
Common
Crane
Grus grus
19
Common
Kestrel
Falco
tinnunculus
20
21
Common
Nightingal
e
Common
Pochard
Gujarat,W Gangetic plain, E
Assam valley
Delhi,
NW India (Rajasthan, Gujarat),
Common winter visitor
Kashmir,
Gangetic plain,
Assam valley
Himalayas (Chitral Himachal,
Widespread winter visitor
Kashmir), Lakshadweep, S
& resident
Assam hills
Luscinia
megarhynchos
Vagrant
Uttaranchal
Aythya ferina
Widespread winter visitor
N Plains to Assam valley,
Ladakh,
107
108
22
Common
Quail
Coturnix
coturnix
23
Common
Redshank
Tringa totanus
24
Common
Ringed
Plover
Charadrius
hiaticula
25
Common
Rosefinch
26
Common
Sandpiper
27
Common
Shelduck
28
Common
Stonechat
29
Common
Teal
30
31
32
33
Common
WoodPigeon
Dusky
Thrush
Eastern
Marsh
Harrier
Eurasian
Coot
Kashmir, NW India, C India
& (Bheraghat),
S
Gujarat,
Gangetic plains, S Mumbai,
Cachar, S Maharastra
Himalayas
(Ladakh)
Widespread winter visitor
Lakshadweep, S Andaman,
& resident
Nicobars
Widespread resident
winter visitor
Vagrant
SE India, NW India
Kashmir, Himalayas, Pune (N
Carpodacus
Resident & widespread Maharashtra), Nagpur, Assam,
erythrinus
winter visitor
Ladakh, Arunachal, S India, S
Assam hills
Himalayas to Assam valley, S
Tringa
Widespread winter visitor Assam hills
hypoleucos
& resident
( Manipur & Lushai hills),
Garhwal, Uttaranchal
N plains to Assam valley,
Tadorna tadorna Native (Migrant)
Chilika Lake (Orissa), N
Maharashtra
Himalayas to Arunachal, S
Saxicola
Resident & widespread
Assam
hills
(Assam
&
torquatus
winter visitor
Nagaland), S Andaman
Himalayas, Assam valley,
Anas crecca
Widespread winter visitor Kashmir,
Lakshadweep,
Andaman
Columba
palumbus
Sparse winter visitor
Himalayas
Bengal
Turdus
naumanni
Sparse winter visitor
Khasi hills (E Meghalaya)
Circus
spilonotus
Sparse winter visitor
NE India
Fulica atra
34
Eurasian
Hoopoe
Upupa epops
35
Eurasian
Otus scops
of
Kashmir,
W
Resident & widespread Kashmir, Gujarat, Peninsula,
winter visitor
Kerala, S Assam hills
W
Himalayas,
Kashmir,
Widespread resident & Gangetic plain to Assam
winter visitor
valley,
Peninsula,
Lakshadweep, S Andaman
Winter visitor
W
India,
Maharashtra
108
109
Scops-Owl
36
37
Eurasian
Sparrow
hawk
Eurasian
Spoonbill
(Mumbai,
Pradesh
Accipiter nisus
Platalea
leucorodia
38
Eurasian
Woodcock
Scolopax
rusticola
39
European
Nightjar
Caprimulgus
europaeus
40
European
Roller
Coracias
garrulus
41
Glossy Ibis
Plegadis
falcinellus
42
Great
Cormorant
Phalacrocorax
carbo
Great
Egret
Greater
Flamingo
Greater
Spotted
Eagle
Casmerodius
albus
Phoenicopterus
ruber
43
44
Pune),
W
Uttar
Widespread winter visitor Arunachal, S Assam hills, W &
& resident
E Ghats, S Andaman
Widespread winter visitor
N India to Assam, S India
& resident
Arunachal, S Assam hills
Widespread winter visitor
( Meghalaya), W & E Ghats,
& resident
Karnataka, S India
W India (Kutch, Jodhpur),
Sparse summer visitor
Mumbai,
Garhwal,
Uttaranchal
Kashmir, NW India, Gujarat, C
Resident & sparse winter
& W Peninsula, Andhra
visitor
Pradesh
Widespread winter visitor
S India
& resident
Saurashtra (Gujarat), Gangetic
Widespread winter visitor
plains, Punjab to Assam
& resident
valley, C India, S India
Widespread resident & Himalayas to Assam valley, S
sparse winter visitor
Andaman
Widespread winter visitor S Gujarat (Great Runn of
& resident
Kutch)
Aquila clanga
Widespread winter visitor
N plains to W Assam, C India
& resident
46
Grey
Heron
Ardea cinerea
NW & NE India, Kashmir,
Widespread winter visitor
Lakshadweep,
Andaman,
& resident
Nicobars
47
Grey
Wagtail
Motacilla
cinerea
Resident & widespread
S Andaman, Nicobars
winter visitor
48
Greybacked
Shrike
Lanius
tephronotus
Himalayas,
Ladakh
to
Resident & widespread Arunachal, S Assam hills
winter visitor
(Khasi hills & Manipur),
Kolkata
49
Grey-
Vanellus
Widespread winter visitor
45
109
Eastern plains, Bihar, Assam, S
110
headed
Lapwing
50
Indian
PondHeron
cinereus
Ardeola grayii
51
Indian
Skimmer
Rynchops
albicollis
52
Japanese
Quail
Coturnix
japonica
53
Japanese
Sparrow
hawk
Accipiter
gularis
54
Lesser
Flamingo
Phoenicopterus
minor
55
Little
Ringed
Plover
Charadrius
dubius
56
Little Tern
Sterna albifrons
57
Longbilled
Plover
Charadrius
placidus
58
Northern
Harrier
59
Northern
Shrike
Assam
hills
(Cachar,
Manipur),
SW
Bengal
(Kolkata),
Orissa,
Delhi,
Bharatpur, Kashmir, N & S
Gujarat
Widespread resident
Assam valley, S Assam hills,
Lakshadweep, Andaman
NW
India, Rajasthan to
Madhya Pradesh, W Bihar, SE
Madhya Pradesh, EC Andhra
Widespread resident
Pradesh,
N
Maharashtra
(Dhule), SW Gujarat, NW
coast, W Assam
Bihar, Assam (Dibrugarh),
Sparse winter visitor
Manipur valley
S Andaman, Andhra Pradesh,
Dibrugarh
(E
Assam),
Sparse winter visitor
Nicobars, Mhow (Madhya
Pradesh)
S Gujarat (Great Runn of
Common winter visitor,
Kutch), Rajasthan (Sambhar
some resident
Lake)
Punjab, S Kashmir, S Jammu,
Himalayas to Assam valley, S
Widespread winter visitor
Assam hills ( Manipur valley,
& resident
Cachar), Kolkata, Andaman,
Ganges valley, Delhi to Assam
Uttan Washi (Mumbai), C
Widespread resident
India,
Lakshadweep, East
coast of India, S Andaman
Sparse winter visitor
N Uttar Pradesh to Assam
valley, Manipur, Delhi
Circus cyaneus
Widespread winter visitor
Himalayas to Assam valley, S
Assam hills ( E Meghalaya,
Cachar), S Gangetic plains
Lanius
excubitor
Widespread resident
Kashmir
110
111
60
Oriental
Honeybuzzard
Pernis
ptilorhyncus
Widespread winter visitor
& resident
61
Osprey
Pandion
haliaetus
Widespread winter visitor
& resident
Painted
Stork
Pied
Avocet
Mycteria
leucocephala
Recurvirostra
avosetta
Pied
Harrier
Circus
melanoleucos
Widespread winter visitor
& resident
Ardea purpurea
Widespread winter visitor
& resident
S Assam hills (E Meghalaya, N
Cachar,
S
Nagaland,
Manipur), Andaman, Ladakh,
Mhow & Bhundara (Madhya
Pradesh), Nander ( N Andhra
Pradesh),
Patna
(Bihar),
Mangpu
(W
Bengal),
Charduar (Assam)
S Assam hills, Lakshadweep,
Andaman,
Nicobars,
W
Himalayas
Gangetic plains, Gujarat to
Punjab, NE & S Peninsula
Kutch (S Gujarat), N India, W
coast of India, Assam, Kashmir
Plains of Uttar Pradesh to
Assam valley, S Assam hills
(Cachar,
Meghalaya,
Manipur), S India, W & NE
India
NW India, Andaman, C
Nicobars, N Andhra Pradesh
Lanius collurio
Sparse passage migrant
NW India, S Gujarat
62
63
64
65
66
Purple
Heron
Redbacked
Shrike
Widespread resident
Widespread winter visitor
& resident
Lakshadweep,
Kashmir,
& Himalayas from Murree hills
to
Arunachal,
Andaman,
Nicobar
Assam valley, NE Ghats, W
Resident & sparse winter
Khandesh (Maharashtra), S
visitor
Assam hills, W Himalayas
Widespread winter visitor NW & S India, W Bengal,
& migrant
Andaman
Ladakh, Sub-Himalayan plains
of N India, S Assam hills
Winter visitor
(Khasi
hills,
Cachar,
E
Meghalaya, Manipur)
Keoladeo
Ghana
WLS
Rare winter visitor
(Bharatpur), N India, Bihar,
Ladakh
67
Redrumped
Swallow
Widespread resident
Hirundo daurica
winter visitor
68
Rosy
Minivet
Pericrocotus
roseus
69
Rosy
Starling
Sturnus roseus
70
Shorteared Owl
Asio flammeus
71
Siberian
Crane
Grus
leucogeranus
72
Spoonbill
Eurynorhynchu
s pygmeus
Sparse winter visitor
111
W Bengal, Kerala, Tamil Nadu
112
Sandpiper
73
74
75
76
77
Spot-billed
Duck
Sykes's
Nightjar
White
Stork
White
Wagtail
Whiteeyed
Buzzard
(Point
Calimere),
Orissa
(Chilika Lake), Kolkata, Assam
valley
Anas
poecilorhyncha
Caprimulgus
mahrattensis
Ciconia ciconia
Motacilla alba
Butastur teesa
Widespread resident
S Assam hills
W India, Delhi, N Madhya
Pradesh, Mumbai
Gangetic plains, Gujarat, NW
Widespread winter visitor
India
Resident & widespread Arunachal,
Andaman,
winter visitor
Lakshadweep, S Assam hills
Winter visitor
Widespread resident
78
Wood
Sandpiper
Tringa glareola
Widespread winter visitor
79
Yellow
Wagtail
Motacilla flava
Widespread winter visitor
80
Yellowrumped
Flycatcher
Ficedula
zanthopygia
Vagrant
NW Himalayas to Himachal,
W Bengal, S Assam hills
Himalayas to Assam valley, S
Assam
hills
(Meghalaya,
Cachar),
S
Andaman,
Lakshadweep, W & NW India
N Punjab, Garo hills
(W
Meghalaya),
NW
India,
Andaman, Nicobars, Kashmir,
Ladakh
C & SW India
(Source: Ali 1996, Grimmett and Inskipp 2007, http://www.bnhsenvis.nic.in/ )
Migration Routes of birds within India
The flights of many migrating birds follow specific routes, sometimes quite welldefined, over long distances. Geographic factors, ecological conditions and
meteorological conditions determine such routes. The majority of migrants travel
along broad airways within these flyways with minor changing their flight
direction in response to the direction and force of the wind. It has been
estimated that birds generally fly at heights varying from 500 to 900 m, at speed
ranging between 30 and 100km/h, and often fly continuously for 6 to 11
hours/day with an average of 240 to 970 km, before stopping to eat or rest (CSIR
1990).
112
113
Various migratory birds, having native places throughout the Europe, Asia,
Africa, Arctic region etc., arrive through different migratory routes in India.
Bitterns breeds in the temperate Palaearctic Region throughout Europe and Asia
from Great Britain to Japan. These winter visitors are recorded from Rajasthan
eastwards to Assam and Orissa and southwards to Karnataka (Table 2, Route 1;
Fig 1). White stork is recorded as a winter visitor between September and
October, and March-April from North India eastwards to West Bengal and
southwards to Tamil Nadu. It breeds in Northern Europe, North Africa, and
Western Asia (Table 2, Route 2; Fig 1). White fronted goose is sparse and rare
winter visitor to NW India eastwards to Assam and Manipur (Table 2, Route 4;
Fig 1). Barheaded goose breeds in Ladakh and is winter visitor in Assam and
southwards to Karnataka between October–November and March (Table 2,
Route 7; Fig 1). Accordingly, based on available literature (CSIR 1990, Ali 1979,
ZSI 1991, Ripley 1982, Whistler and Kinnear 1949, Walters 1980, Grimmett and
Inskipp, 2007, Hawkins 1986), the native places elsewhere of commonly visible
avifauna and their migratory routes within India are documented in Table 2 and
depicted in Fig. 1.
In general, migration of birds into India is restricted to a main route. The Kutch,
Banaskantha and Kathiawar Peninsula are on this main route through which
hordes of migratory birds sweep into India from the North and NW in autumn
and out in the reverse direction in spring. This region also forms the eastern
fringe of many Asiatic passage migrants.
113
114
LEGEND
Route no. (as per Table 2)
2* : also route 14
4* : also route 10
11* : also route 12
16* : also route 17
Fig.1: Migratory routes of birds within India
114
115
Table 2. Migratory routes of birds within India
Route Native place
No.
Temperate
palaearctic
region
throughout
1
Europe & Asia from
Great Britain to Japan
2
3
4
5
6
7
8
9
10
11
12
13
Migratory routes within
India
Birds
Rajasthan eastwards to
Assam & Orissa and Bitterns
southwards to Karnataka
North India eastwards to
Northern Europe, North
west Bengal & southwards
Africa & Western Asia
to Tamil Nadu
North India eastwards to
Europe through Russia to
Assam & southwards to
North China
Maharashtra
Arctic coasts of Europe & North west India eastwards
Asia
to Assam & Manipur
Kashmir
eastwards
to
Siberia
Assam & southwards to
Maharashtra
North India eastwards to
Europe, Asia Minor &
Assam & southwards to
Central Asia
Andhra Pradesh
Assam & southwards to
Ladakh
Karnataka
Western Europe, Central Kashmir
eastwards
to
Asia, eastern Siberia, Assam & Manipur and
Mongolia & Tibet
southwards to Maharashtra
Assam and southwards to
Kashmir, Europe, Asia
Maharashtra
North
West
India
Europe,
Central
and eastwards to Assam &
Western Siberia
Manipur and southwards
to Karnataka
Europe & Asia from
North India (Punjab, Delhi)
Scandinavia to Siberia &
eastwards to Assam and
South
to
Volga,
southwards to Gujarat
Turkestan & Amur
Southern
Europe
& North & North West India
Southern Russia
and southwards to Gujarat
North East India viz.
Assam, Manipur & West
Northern Siberia
Bengal
thinly
diffuses
westwards to Rajasthan
115
White stork
Black stork
White
goose
fronted
Lesser
white
fronted goose
Eastern
goose
grayleg
Barheaded goose
Common
shelduck
Mallard
Common pochard
Smew
Imperial eagle
Eastern
plover
golden
116
14
Northern
&
central
Europe, Western Asia etc.
15
Eastern Europe & Russia
16
Central Eurasia
17
Southern Siberia
18
Afghanistan, North Iran,
Turkestan & Transcaspia
19
Central Siberia
20
Transcaspia to the Gobi
desert
21
West China & Tibet
22
Mongolia
23
Tibet & West China
24
Turkey, Lake Baikal,
Manchuria & Kansu
25
26
and southwards to Kerala
North India eastwards to
West
Bengal
and
southwards
through
Peninsula
Western
Himalayan
foothills southwards to
Karnataka
MP eastwards to Nagaland
& Manipur and southwards
to Kanniyakumari
MP eastwards to Assam
and
southwards
to
Kanniyakumari
Delhi
&
Kolkata
southwards
to
Kanniyakumari
Foothills of the Himalayas
southwards to Tamil Nadu
and eastwards to West
Bengal
Northeastern
India
(Haryana) southeastern to
Gujarat
Lower Himalayas from
Naini
Tal
eastwards
through
Sikkim
&
Arunachal Pradesh
Gangetic plains of U.P &
Bihar southwards to West
Bengal
Himalayan foothills from
Himachal
Pradesh
eastwards
to
Assam,
Meghalaya ,Nagaland &
Arunachal Pradesh
Plains of Northwestern
India upto Gujrat and
eastwards to U.P
Black
godwit
tailed
Western
breasted
flycatcher
Blyth’s
warbler
red
reed
Siberian booted
tree warbler
Indian
booted
tree warbler
Siberian
lesser
whitethroat
Small whitethroat
Hodgson’s
redstart
Hodgson’s
chat
bush
Tibetan collared
bush chat
Isabelline chat
Lake Baikal , Manchuria,
Ladakh to Lahul Spiti
Pleschanka’s chat
Iran & the Black sea
Himalayas, Nepal
Himalayas (Kulu valley to Pied
ground
116
117
27
Eastern Russia, Siberian
Taiga & Yenisey
28
Siberia
29
Turkestan
30
Southern Siberia & Japan
31
Scandinavia
Northwestern Siberia
32
Central palaearctic region
from Volga to Baikalia
and Ladakh
33
Russian Turkestan
34
North Eurasia, Central
Russia etc.
35
South eastern Tibet &
western Szechuan
36
Baltic sea to central
Siberia
Altic sea to central Siberia
37
Siberia
&
Arunachal Pradesh)
Himalayas
(Arunachal
Pradesh,
Nagaland,
Manipur,
Assam)
southwards to M.P &
Orissa
Himalayas eastwards to
Arunachal
Pradesh
,Nagaland & Manipur
Along
the
Himalayan
foothills to Dehradun
Along
the
Himalayan
westwards to Shimla
Along the Gangetic plains
southwards
in
Kanniyakumari
and
eastwards in Assam and
also Andaman & Nicobar
Islands
Gangetic plains southwards
to Kerala and eastwards to
Assam & also
Nicobar
Islands
Himalayan
foothills
eastwards in U.P and
southwards in Kerala
Kashmir and eastwards to
Mussoorie
Eastern Himalayas through
Sikkim
to
Arunachal
Pradesh
Indian Peninsula & the
Western
Himalayan
foothills extending to Nepal
Kashmir
eastwards
to
Nepal
thrush
Black
thrush
throated
Red
thrush
throated
Turkestan black
throated accentor
Siberia tree pipit
Grey
headed
yellow wagtail
Blue
headed
yellow wagtail
Turkestan black
headed wagtail
Brambling
Tibetan siskin
Common
finch
rose
Pine bunting
Conservation of birds
There are reports that certain bird species in India have almost become extinct,
and many more are becoming extremely rare and increasingly endangered.
Anthropogenic activities have been primarily affecting the birds. The various
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118
factors which are affecting the natural avian ecosystems are: human population
explosion, growth and developmental activities, such as urbanization, buildings
of roads, construction of dams, deforestation, hunting, trapping and exploitation.
Environmental pollution is another cause endangering birds. Bio-accumulation
of chemical fertilizers, pesticides, insecticides, herbicides etc., which are being
increasingly and indiscriminately used, affects birds. Studies have shown that 65
percent of bird’s extinctions are due to destruction and alteration of habitats in
which birds live. Hunting comes next, accounting for 25 percent of the extinction.
The International Union for Conservation of Nature and natural Resources
(IUCN) is a leading, International non–Govt. organization concerned with
conservation. it co-ordinates selection and management of World Wide Fund
conservation projects around the world, manages the UN Environment
Programme (UNEP) and the secretariat of the convention on International trade
in Endangered species of wild fauna and flora (CITES), and also performs the
bureau duties under the conservation of wetlands of International importance,
especially of waterfall habitat.
World Wide Fund for Nature (WWF) International is the world’s largest
voluntary organization raising funds for promoting conservation. Certain birds
get protection in the form of religious beliefs, superstitions and popular
sentiments. Pelicans are looked upon as sacred and in many parts of the country;
people do not allow them to be killed by anyone. The peafowl or Indian peafowl
is also considered as sacred and has found a place in mythology, literature and
folklore. The White stork, Sarus crane and Siberian crane also enjoy varying
degrees of protection by popular sentiment. Pigeons for certain other regions, get
protection; mosques and some buildings have special steeples, ledges and
windows to enable pigeons to roost and breed.
The Indian Board for Wildlife is the main advisory body of the Govt. on the
subject of Wildlife conservation and their important functions are: to devise ways
and means for the conservation of wildlife through co-ordinated legislative and
practical measures, to sponsor the setting up of National Parks, Wildlife
Sanctuaries and Zoological Parks, to advice the Govt.on policy in respect of
export of living animals, trophies, skins, furs, feathers and other wildlife
products etc. The Indian Wildlife (Protection) Act, 1972 provides legal protection
to endangered species of animals. Protection of wild animals, birds, and forests
has also been included in the Indian Constitution.
Since the right kind of habitat is critical for birds to survive and reproduce, it
makes sense that habitat management is an important focus of bird conservation.
The quality of resources and the protection they provide is important to
understand when trying to discern birds' migration timetables and flight paths.
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evolution and determinants. Oikos 103: 247–260.
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Delhi.
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and density – implications for harvest models in fluctuating environment.
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the Wealth of India, Raw Materials. Vol. 2B. CSIR, India.
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of the Art. Zoological Survey of India (ZSI), Kolkata, 1991
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conservation of migratory birds, in Ralph, C.J., and Rich, T.D., eds., Bird
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20-24, 2002: Albany, Calif., U.S. Department of Agriculture, Forest Service, Pacific
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University Press, Mumbai.
Khacher (1978). referred in Animal resources of India: Protozoa to Mammalia;
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those of Nepal, Sikkim, Bhutan and Ceylon (2nd Edn) Bombay Natural History
Society, Mumbai.
Sillett, T. S. and Holmes, R. T. (2002). Variation in survivorship of a migratory
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R. Greenberg and P. Marra), PP. 199–209, Johns Hopkins University Press,
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Mammalia; State of the Art. ZSI, Kolkata.
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Journal of Today’s Biological Sciences: Research & Review (JTBSRR)
ISSN 2320-1444 (Online)
JTBSRR
Vol.1, Issue 1, page 122-143, December 30, 2012
Available on: www.jtbsrr.in
Flowering Manipulation In Mango: A Science
Comes of Age
J. Shankara Swamy
Department of Horticulture, Junagadh Agricultural University Junagadh -362001, Gujarat
E-mail: shankara.swamy@gmail.com
Abstract
The Mango (Mangifera indica L.), member of family Anacardiaceae, is amongst the most
important tropical fruit of the world. Flowering is the first of several events that set the
stage for mango (Mangifera indica L.) production each year. Given favorable growth
conditions, the timing and intensity of flowering greatly determines when and how much
fruit are produced during a given season. Insight into this phenomenon has been of prime
interest to scientists and growers for over a century. As a consequence of efforts to
elucidate the mechanisms in manipulation of flowering in mango becoming clearer at the
molecular, biochemical, and physiological levels resulting in a better understanding of
how to manipulate flowering in the field is critically reviewed here.
Key words: Flowering manipulation, growth regulators, mango flowering
Introduction
The Mango (Mangifera indica L.), one of the 73 genera of the family
Anacardiaceae in order Sapindales, is amongst the most important tropical fruits
of the world. It is also called as king of the fruits (Purseglove, 1972). It is
originated in the South East Asia or Indo-Burma Region having 41 recognized
species of mango originating as forest trees with fibrous and resinous fruits
((Mukherjee, 1951, 1967).The mango (Mangifera indica L.) is an important fruit
crop of India and other tropical and subtropical regions of the world. It is grown
in more than 111 countries but no where it is as greatly valued as in India where
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40 % of total fruits grown is only mango. In India, mango enjoys supreme place
in fruit production has nearly 1000 varieties and grown in an area of 1.60 million
hectare, which accounts for 58 % of total area under fruit crops (Anon., 2008). In
the year 2006-07, India exported 79,060.88 MT of mangoes worth Rs.141.94 crores
(APEDA, 2007). India is the largest producer of mango in the world with the
production of approximately 14 million tones, contributing more than 57 % share
of the world production (FAO, 2009).
The profitability of growing mango is influenced by two key factors:
• Productivity, which consists of yield and quality.
• Supply and demand which rule market prices.
A better understanding of the nature of flowering induction in mango is
necessary not only for yield sustainability but also for yield increase. Flowering
is the first of several events that set the stage for mango (Mangifera indica L.)
production each year. Given favorable growth conditions, the timing and
intensity of flowering greatly determines when and how much fruits are
produced during a given season. Insight into this phenomenon has been of prime
interest to scientists and growers for over a century. I, therefore have chosen to
discuss advance flowering in mango crop.
As one reflects over nearly a century of work in mango fruit crop production, it
is apparent that the area early/regular flowering induction in mango was
received less attention in the past. Although many significant mileposts have
been reached in our understanding of mango flowering induction in the past 30
years.
Flowering phenomena in mango
Flower initiation is very important because it is the first step towards attaining
fruit and it is very complex phenomena in mango. Flowering in mango trees
make them especially challenging for physiologists, breeders, and growers;
Mango is a terminal bearing species and the factors which determine switching
from vegetative to reproductive mode are poorly understood. In general first,
mango pass through a juvenile stage which lasts for several years following
germination during which flowering does not occur; second, interactions
between vegetative growth, flowers, and fruit of the previous year on floral
initiation in the current year, affect growers through phenomena such as biennial
bearing, and make interpretation of research data difficult for scientists.
Once flowering capacity is attained, mango tree respond to environment cues
such as light (especially relative lengths of light and dark periods), temperature
and nutrition. In mango flowering has been found to be chemically controlled.
Leaves are the sites of control substance synthesis; apices are receptor sites. It has
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been suggested that this control may takes the form of: (a) a single flowering
factor (florigen), (b) a group of flower promoting substances, (c) one or more
flower inhibiting substances, or (d) interaction between flowering promoters and
inhibitors and vegetative growth cycle. These aspects are reviewed as follows.
1. Growth pattern and flushing episodes in relation to mango
flowering
Induction of generative (floral), vegetative or mixed shoots from axillary or
apical buds of mature flushes appears to be governed by several factors reviewed
in details as follows.
Growth of mango is not continuous but it occurs as intermittent, short lasting
flushes of shoots from apical or lateral buds. The flushing refers to the emergence
of new shoots on the terminals of old shoots. Generally a healthy mango shoot
completes four to five flushing episodes per year depending upon cultivars and
growing condition (Davenport and Nunez-Elisea, 1997), while blooming occurs
on a few of them during the following year (Issarakraisila et al., 1991). Terminal
inflorescences or panicles are initiated in dormant apical buds on stems that
developed vegetative from lateral buds following the previous flowering seasons
(Litz, 1997). So studying the different vegetative growth cycles may help the
mango growers to know the most important vegetative growth cycle for
regulation of vegetative growth, bearing flowers, fruits subsequently to increase
yield. In this case the growers can use all methods for inducing trees to produce
their vegetative growth cycles in the time which help to maximize income.
According to the Davenport and Nunez-Elisea (1997) Conceptual flowering
model of mango, individual stems borne on branches of mango trees are in rest
or a quiescent mode most of the time. Stems are resting, vegetative structures
composed of the terminal intercalary unit resulting from the previous flush of
vegetative growth.
Stems are different from shoots, which are growing structures that evoke from
buds of stems. Vegetative shoots bear only leaves, whereas generative shoots
produce inflorescences and mixed shoots produce both leaves and inflorescences
within the same nodes. Initiation of shoot growth in buds of resting stems is the
first event that must occur in order to produce flowering (Davenport and NunezElisea, 1997; Davenport, 2000, 2008). Reece et al. (1946, 1949) recognized that the
fate of mango buds is not determined until their growth is initiated. The
vegetative or reproductive fate of resting apical or lateral mango buds is not
predetermined at the time of shoot initiation (Mustard and Lynch, 1946; NunezElisea and Davenport, 1992).
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New shoots arise mostly as laterals from axillary buds around the stump of the
twigs fruited previous year. Such growth either remains unextended or makes
further extension growth in subsequent months, largely depending on the
variety. Terminal growth is always in the form of an extension of shoots already
produced. Growth occurs in different flushes which vary from variety to variety
and under different environmental conditions. Under north Indian conditions,
March-April and May-June are the most important periods for the emergence of
new shoots. However, stray shoots and sporadic extension growth may emerge
any time between July and October (Singh, 1958}. Under south Indian conditions,
two active flushes occurring from February to June and October to November
were reported. Three main growth flushes in February to March, March to April
and October to November were reported in western India. Only one or rarely
two periods of active growth in the dry zone of Ceylon were reported by Buell
(1954). However, he reported two to six growth flushes in the wet zone which
was attributed to the irregular fruiting found in that zone. Nakasone et al (1955)
reported that under Hawaiian conditions, the vegetative flushes in Pairie mango
are scattered throughout the year. An average of 18 months was considered
necessary by them between vegetative flush and subsequent flowering.
Based on earlier works, it was proposed that early initiation and cessation of
growth, followed by a definite dormant period, will help the shoots to attain
proper physiological maturity essential for fruit bud Initiation that means floral
behaviour of a shoot influences by its physiological position within the canopy.
However, now it is more or less established that growth of shoots in mango is a
varietal characteristic and their fruit bud differentiation in regular bearing
cultivars is an annual feature. In biennial bearing varieties, ‘on’ and ‘off’ year
phases, rather than age and cessation of growth of shoots govern the flower bud
differentiation in the trees. The shoot, depending upon the cultivar may stop
putting forth extension growth after May or continue until September or later
and the potential of this shoot to form flower buds will depend on the floriferous
condition of the tree, which in turn will be determined by the amount of fruit
load carried by the tree in the previous year (Singh, 1971).
2. Flowering manipulation by interrupting vegetative growth cycle
Mangoes are considered alternate bearers, although they are less severely
alternate than avocadoes. There is some lack of clarity about their cropping
habits and often alternation is used as synonyms for poor yield. One of the most
important factors responsible for low yield and inferior orchard efficiency is
biennial bearing, which means that the tree carries optimum load of crop in one
year, but in the following year it fails to flower or/ and produce unsatisfactory
crop. Biennial bearing, alternate bearing or cropping periodicity in mango
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cropping is synonyms, which are different from unfruitfulness and shy cropping.
The most important thing in case of flowering in mango is to produce new
vegetative growth in the ‘on’ year which should also be mature to be ready to
enter into reproduction phase and give out flower in following season. Flushes of
one month may re-flush during the subsequent months. Similarly April flushes,
which are considered to be the more productive may re-grow several times
during the following months or may cease to grow anymore to attain blooming
maturity and thus this becomes essential to determine pattern of growth of this
flush. Most of the vegetative growth produced is from non-flowering shoots and
the shoots, which carried mature fruits, have been reported to have markedly
lower probability of vegetative growth (Issarakraisila & Considine, 1991).
Moreover, induction of early flowering results in early maturity of the mango
fruits which fetch the higher price in the market as compared to late maturing
mango fruits. This can be achieved by various ways reviewed as follows.
2.1 By application of Triazoles group (paclobutrazol (PBZ)) growth inhibitor
compounds
The first report in the use P333 (paclobutrazol) @ 1.25 to 10 g.a.i/tree One of
commercialized method to manipulate flowering by post- harvest application to
the soil significantly promotes flowering and fruiting in the following year in
Dashehari and Banganapalli came from India (Kulkarni, 1988). Davis et al (1986)
Reported that paclobutrazol is substituted triazole, checking vegetative growth
by inhibiting the biosynthesis of gibberellins in plants by blocking the conversion
of kaurene and kaurenoic acid. Burondkar and Gunjate (1993) studied the effect
of Paclobutrazol on Alphonso mango at regional fruit research station, vengurla
and revealed that Paclobutrazol significantly suppressed the emergence of
September-October vegetative flush and length of vegetative shoot in 2
successive cropping years. Tandel, Y. N. and Patel (2011) reported the beneficial
effect of paclobutrazol, irrespective of time of application, reduced the vegetative
growth during October-November under Gujarat condition in India.
Paclobutrazol applied in mid of July, significantly reduced the number of shoots
per terminal in Alphonso, Kesar and Rajapuri. It also checked the growth of new
shoots in Alphonso, Kesar and Rajapuri. An application of paclobutrazol (cultar)
had effectively controlled the emergence of this vegetative flush of OctoberNovember by interrupting the biosynthesis of gibberellins. Because
paclobutrazol is a gibberellins bio-synthesis inhibitor. Application of
paclobutrazol (cultar) in mid of July, August and September under Gujarat
condition in India suppressed the vegetative growth and induced early and
profuse flowering during their investigation as compared to control. The
considerable reduction in vegetative growth in the trees treated with
paclobutrazol had been reported by Hoda et al.(2001); Shinde et al. (2000). In
other words, the flower inductive cycle which is a part of phonological and
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physiological cycle of mango tree may commence earlier in the season, but
flowering is prevented by the inhibitor until the build-up of sufficient promoter
to counteract the inhibitor. Paclobutrazol thus, appears to help in achieving this
stage much earlier because of its inhibitory activity. This hypothesis looks
particularly attractive while considering the flower-inhibitory role of gibberellins
in trees together with the anti-gibberellins activity of paclobutrazol (Kulkarni,
1991).
2.1.1 Disadvantages of paclobutrazol
Although the direct effects of paclobutrazol(PBZ) on early induction of flowering
of mango have been well documented, However, it seemed have average weight
of a fruit reduced without affecting the fruit yield and continuous application of
PBZ may cause soil pollution and its residual effect may increase in fruit.
Residual limit of PBZ accepted by the FAO in stone fruit is 0.05 mg/kg (Singh
and Ram, 2000). According to the Davenport (1993) there are problems with use
of paclobutrazol. Because it inhibits the gibberellin syntheses pathway, levels of
the gibberellin which is responsible for internode elongation, possibly GA1 are
reduced. Although fruit set and yield may be increased, the product produces a
compressed panicle which does not dry out very well and can develop powdery
mildew or anthracnose even after light dew.
Another problem is that when paclobutrazol is applied to soil in excess, under
certain conditions, subsequent growth and normal development can be severely
disrupted. There is a growing amount of literature on the use of paclobutrazol to
get early and more uniform flowering in mangoes. No response was observed in
seven or eight months after applying paclobutrazol to trees in Homestead. The
trees then went through a freeze, their irrigation system failed, and major
scaffolding branches were killed. The trees were severely pruned to remove dead
wood. The ensuing growth lacked normal node elongation. Trees having only 1
gram of active ingredient applied are still severely stunted after over six years.
They investigated the possibility that pruning of the major branches following
application was the cause of the undesirable stunting of growth. We applied
paclobutrazol, in the same concentration, to trees and waited three years before
severely pruning. There was no response to the product until after the trees were
pruned. The resulting growth was as severely stunted as before. We believe that
this material is chromatographing itself up through the xylem of the tree. It is
apparently concentrating itself in main trunks and slowly metering itself out to
the branches. When main branches are cut, forcing buds to grow in the area of
high paclobutrazol concentration, then you see this strong effect. As long as you
do not prune the tree, there appears to be no problem and a many-times limited
effect. Recommendations used in Thailand of 1.5 to 2 g/tree/yr to stimulate
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more uniform flowering may eventually result in this kind of damage if and
when they prune those trees for some reason.
Paclobutrazol is persistent in the soil. If a new tree is planted, it will show the
same symptoms. Therefore, we have to be careful when recommending use of
such a compound. Experiments are being conducted in Central America on
'Tommy Atkins'. They involve applying paclobutrazol sprays at 30 ppm, which is
its solubility in water, to get it to the buds at the proper time to facilitate a
flowering response.
3. Environmental cues in relation to the flowering
In the tropical evergreen tree mango, Mangifera indica L., cool temperature is the
only factor known to induce flowering, but does not ensure floral initiation will
occur because there are important interactions with vegetative growth.
Vegetative activity and the relationship between vegetative and floral growth is
variable, both from tree to tree and between years (Scholefield et al., 1986; Cull,
1987). Environmental induction through low temperature (around 15oC) is
considered as the most important flower trigging element in mango still
biennially bearing mango cultivars usually do not flower during off year even
under low temperature conditions. Mango is a terminal bearing species and the
factors which determine switching from vegetative to reproductive mode are
poorly understood, although a period of low temperature (<18 ºC) during the
pre-flowering period is thought to be involved (Davenport and Nunez-Elisea,
1997). It is commonly accepted that opportunities for flowering are maximized as
the terminals become more mature (Scholefield et al., 1986), possibly due to the
presence of floral inhibitors in young leaves (Kulkarni, 1991).
Sen and Mallik (1941) working under Sabour conditions of Bihar reported that
there was a sharp change in climatic conditions at the end of September
especially with the advent of cold and dry weather appeared to influence fruit
bud differentiation. Singh (1960) reported that neither the high humidity and
rain at the time of bloom nor the late rains appeared to influence fruit-bud
differentiation. However according to Chacko and Randhawa (1971) heavy rains
during the critical time of flower-bud-intiation stimulated vegetative growth at
the expense of flowering. In places like Kerala, where rainfall is heavy, mango
flowered sparsely and erratically. Singh (1961) also observed that the mango
trees in extreme humid place and under mild climatic conditions remained
unfruitful owing to their increased tendency towards vegetative growth. A low
temperature resulting in frost was reported to have effected the fruit-bud
formation indirectly in the cultivars ‘Singharha’ and ‘Vijai Rao Garh’. He further
observed that the regular bearing cultivars remained unaffected and no definite
relation between the temperature and the extent of ‘bud break’. Chacko and
Randhawa (1971) found that unlike many other tropical species, vegetative
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growth in mango was never continuous but exhibited periodical quiescence. The
number of flushes varied greatly depending upon the variety, age of the tree,
climatic conditions and the amount of crop borne in the previous season. They
also reported that although flowering in mango trees generally took place during
short days in the areas fall nearer to the equator, the very fact that off-season
cropping was possible at Kanyakumari in South India suggested that flowering
in mango is certainly under the environmental control, most probably
photoperiod. They also reported that mango trees responded to temperature
variations more critically than to photoperiods as evidenced by the different
times of flowering at different places in India. The flowering is known to be
earlier in areas nearer to the equator and late in North India, where extreme low
temperature prevails during the winter months. In tropical conditions, preflowering rest period is usually achieved by drought at temperature above 15
degree Celsius (Whiley et al., 1989). Nunez-Elisea and Davenport (1992) reported
that production of reproductive shoots requires initiation of growth during
exposure to cool, inductive condition. The resting buds of plants, which had been
exposed to cool temperatures (18 degree Celsius day/10 degree Celsius night) for
more than three weeks and then transferred to a warm temperature regime (30
degree Celsius day/25 degree Celsius night) before initiation occurred, typically
produce vegetative growth. The primary impact of water stress on mango is to
prevent vegetative flushing during stress period. The accumulating age of stems
is greater in water stressed trees than the trees maintained under well watered
condition. (Schaffer et al., 1994). Flowering occurs in the subtropics when resting
buds initiate growth during cool, inductive temperatures (Battern and Mcconchi
1995). Yeshtela et al., (2004) found out that mango cultivar 'Keitt' was more
sensitive towards low temperature floral induction than 'Tommy Atkins’. In the
tropical highlands and sub-tropics, where most of the commercial orchards are
situated, the low temperature during the winter months induced a severe growth
check resulting in profuse flowering (Beal and Newman 1986). Rao (1998)
reported that the minimum temperature of 13ºC for seven days favored FBD in
mango cultivars ‘Neelum’ and ‘Totapuri’ under Dharwad conditions. Chen et al.,
(1999) reported that the temperature is considered to be key environmental
factor, with low temperatures (19 ºC in day and 13 ºC in night) favorable for
fruit-bud-differentiation. Li et al., (2010) recently reported that flower bud
differentiation was delayed by high temperature and superabundant rainfall in
subtropical monsoon climate zone and more easily affected by the overlap of
current shoot growth.
3.1 An alternative flowering manipulation to dependence on Environmental
signals
Biennially bearing mango cultivars usually do not flower during off year even
under low temperature conditions. In such circumstances. An alternative to
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dependence upon environmental signals for flower initiation is the development
of management strategies that can substitute for these signals among them few
methods for manipulating of mango flowering alternative to environmental cues
are reviewed here.
3.1.1 Application of growth inhibitors
In above circumstances, growth retarding chemicals, e.g. triazoles group
(paclobutrazol, PBZ), that can stimulate or mimic the effects of the environmental
factors in checking vegetative growth are some times used to correct such a
situation (Nartvaranant et al., 2000) as reviewed above.
3.1.2 Smudging
Smudging is making the Smokey fire below the tree canopy and allows smoke to
pass through the foliage for several days. To produce heavy smoke, place green
grasses on top of combustible materials such as dry leaves and coconut husks.
Smudging is an early commercial method of inducing mango to flower (Wester,
1920). Gonzales(1923) and Borja and Bautista (1932) considered only mature
shoots of 1 year or older with very brittle, dull grayish green to copper coloured
leaves and plump terminal buds are suitable for smudging.
Smudging of the mango is practiced in certain parts of the Philippines to obtain
earlier and increased flowering of 'Carabao' and 'Pico' mango (Dutcher 1972;
Gonzales 1923; Madamba 1978). Ethylene has been identified as the active agent
responsible for flowering during smudging (Dutcher 1972). Smudging is done
continuously for several days and is stopped if flower buds do not appear within
two weeks. The process may be repeated 1-2 months later, but results are
uncertain. It is not, however, known to be practised in India or in any other
mango tract, or even much widely in the Philippines.
According to Sen and Mallik (1947) Experiments were conducted at the Fruit
Research Station, Bihar, and Sabour India with the Langra Mango in order to
study the effect of smudging treatment on the plant under the local conditions.
Instead of flowering smudging has stimulated vegetative growth. In addition to
normal shoots arising from terminals large number of axillary buds appeared in
clusters to form malformed bunchy growths. It is apparent that smudging has a
stimulating effect on growth, but the nature of growth, reproductive or
vegetative, depends on other factors. One of the previous workers also concludes
that smudging can induce flowering only if the shoot is in condition to flower.
But none of them mention any effect of stimulating excessive vegetative growth
as shown in these experiments. Opinion differs as to whether the smudging is
due to heat of the smudge or due to smoke. It is not considered to be due to the
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heat as the average difference of temperature between the treatment and the
control was only 2°C. According to them Ethylene gas which is one of the chief
constituents of the smudge smoke, produced by burning vegetative matter, are
known to have given similar effect on pineapple. It is, therefore, thought that
stimulating effect of smudging is due to the smoke. So they declared that it is
intended to continue the study in connection with researches on the physiology
of the mango.
3.1.3 Potassium nitrate spray
KNO3 can enhance flowering especially in tropical regions where cold
temperature for floral induction may not be sufficient. That is due to its reported
effect in supplementing nitrogen. It is also suggested that induction by
potassium nitrate spray occur as a result of ethylene synthesis. The overall effect
of potassium nitrate when sprayed at different periods of phonological phases,
concentration and locations as well as the mechanism for its effect is reviewed
here.
Potassium nitrate (KNO3) came into general use in the Philippines in the 1970s. It
too was speculated to stimulate flowering through a wound-ethylene response. It
now is widely used in Mexico as well. Although responses may occur at
concentrations ranging from 1 to 8 percent, Mexican growers generally use 4
percent KNO3 or 2 percent ammonium nitrate. Leaf tip burn also occurs in dry
areas at these concentrations. One must be careful in interpreting such
information. Many have found that if KNO3 is applied too early in the season,
they obtain a vegetative instead of a flowering growth response. The same is true
for spring or summer applications. It is likely that KNO3 is not inducing
flowering directly, but is stimulating initiation of growth. If conditions are
present to induce flowering, then growth will be reproductive. If, on the other
hand, conditions are more favorable for vegetative growth then, that will be the
response. This point is further discussed below.
Subsequent discovery and use of ethephon to replace smudging and stimulate
flowering of mango the first studies to demonstrate that potassium nitrate could
induce flowering of mango trees were from the Philippines (Barba 1974, Bondad
and Linsangan 1979; Bueno and Valmayor 1974). Flowering was evident within
seven days after treatment and was effective on shoots that were between 4.5 and
8.5 months old when treated.
Bondad and Linsangan (1979) reported that concentrations of potassium nitrate
between 1 and 8 percent stimulated flowering of seedling 'Carabao' and
'Pahutan' trees and 'Pico' trees within one week after sprays were applied. The
treatment was effective for stimulating flowering of trees that had remained
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vegetative well beyond normal bearing ages, for advancing the flowering and
fruiting periods, and for breaking the biennial bearing habits of trees. Potassium
nitrate is currently recommended in the Philippines for inducing uniform
flowering and for the production of off-season fruits in the 'Pico' and 'Carabao'
cultivars (Madamba 1978). In India, workers have reported variable results with
potassium nitrate (Pal et. al. 1979). Areas that have reported success with
potassium nitrate include Trinidad with 'Tommy Atkins' (James et al. 1992), the
Ivory Coast with 'Kent' and 'Zill' (Goguey 1992) and Mexico with 'Manila' and
'Haden' (Nunez-Elisea 1985; 1986).
Work in Mexico showed that mango flowering could also be stimulated with
ammonium nitrate sprays (Macias-Gonzales et al. 1992; Nunez-Elisea 1988,
Nunez-Elisea and Caldeira 1992). Concentrations of 2 percent ammonium nitrate
were sufficient to promote early flowering in 'Haden', 'Tommy Atkins', 'Kent',
'Diplomatico' and 'Manila'. The similar results between ammonium and
potassium nitrate indicate that the nitrate ion is the active portion of the
molecule.
Experiments in Hawaii by Mike and Melvin(1990) showed that 2 and 4 percent
potassium nitrate sprays applied to mature seedling trees early in the flowering
season (February, 1986) stimulated flowering. A single application stimulated
flowering within three weeks after treatment, and maximum response was
observed at about four weeks. Off-season flowering was also stimulated when
application was made to seedling trees in May after the flowering season was
completed. Nearly 16 percent of the terminals treated with 4 percent potassium
nitrate flowered by six weeks after treatment. Their results also showed that
terminals that flowered were associated with specific trees; some trees in the test
exhibited no response, while others produced vegetative terminals after
treatment. These results suggest that potassium nitrate did not induce flowering,
but probably stimulated growth of terminal buds. Flowering was determined by
the condition of the terminal bud or the environmental conditions at the time
potassium nitrate application was made. Their results with seedling trees also
showed that genotypic differences among trees exist with regard to flowering
responses to potassium nitrate. Some trees were highly responsive to the
treatment and flowered, while others produced vegetative shoots instead of
panicles.
In Mexico, studies by Nunez-Elisea (1986) have shown that 'Haden' shoots
should be six months of age or older. In the case of 'Manila', shoots could be as
young as 3-4 months of age and be responsive. Leaves should be dark green with
a mature, "woody" texture and well developed terminal buds. Upon treatment
with a 4 percent potassium nitrate solution, slight leaf wilting can be observed
within two days, and at 10 days buds begin to swell. A second application is
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made at 15-20 days after the first application if the response is poor. Application
should be made prior to emergence of the flowers, because flowers are usually
damaged by the potassium nitrate sprays. Harvesting occurs at about five
months after treatment. Advancing the flowering season in Mexico has enabled
growers to get fruits into the market at an earlier date, extend the harvest season,
and harvest crops during the drier periods.
Davenport (2003) reported that bud break was initiated three months later by a
foliar application of KNO3 in weakly inductive condition (during warm
temperature condition).
In Veracruz, Mexico, PBZ was applied in July at 1g. a.i. per meter of canopy
diameter combined with two foliar sprayings of KNO3 at 2% in October, and the
flowering of mango ‘Manila’ took place 80 days ahead from the regular natural
flowering; resulting in a selling price 15 times higher, as compared to the regular
crop value for the season.
Mosqueda (1989) reported that KNO3 was effective in stimulating the emergence
of mango inflorescences more than 30 days in advance in Manila mango. Foliar
application of KNO3 stimulated flowering of mango (Yeshitela et al., 2004). It is
possible that KNO3 increased cell division and enlargement in the meristematic
zone (Protacio, 2000).
3.1.4 Hormonal concept of flowering in mango
Floral evocation and morphogenesis can achieve by the application of plant
growth regulators or phytohormones and plant growth regulators (PGR)
antagonists. PGRs are generally present in most plants, some of these
compounds may be present or absent in sub or supra-optimal levels. Hence each
factor will not necessarily act in the same direction in all plants. Work with
exogenous application of PGRs for mango flowering and floral manipulation
with their application comprehended here.
3.1.4.1 Hormonal concept
Sen (1943) suggested that there might be a special hormone and that a heteroauxin might be discovered for practical use to induce flowering in mango. The
terminal bud in mango was considered to inhibit the formation of axillary flower
bud since the removal of terminal buds helped in producing inflorescence from
axillary buds in the ‘Haden’ mango (Reece et al., 1946). Further, it was also
shown that floral primordial in the axillary buds were promoted by the presence
of leaves and inhibited by the decapitated and ringed shoots could induce
axillary flower buds but when the shoots were defoliated immediately or within
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twenty-four hours after decapitation, only vegetative shoots were produced by
the axillary buds (Reece et al., 1949). On the basis of these observations, Chandler
(1950) proposed a hypothesis that flower induction in mango could occur only
when the cell division had started and that a flower inducing hormone played no
part in the initiation of growth; but when present in sufficient amount at the
beginning of growth, it determined the course of differentiation of tissue in the
axillary buds. He also proposed that if a hormone induced flowering in plants
and the source of hormone was the leaf or some precursor formed in the leaf,
then the leaf surface rather than the accumulation of carbohydrates might have
the dominant influence on flowering. Sen (1951) opined that the problem of floral
initiation in plants is not as simple as the one being controlled by the synthesis
and accumulation of a substance up to a certain concentration but is a complex
one involving a photo-mechanism controlling various growth and
developmental processes. Singh (1961) showed that the newly merged leaves in
the shoots of regular bearing cultivars such as ‘Neelum’ was capable of
synthesizing flower inducing hormone. Chacko and Randhawa (1971) noticed a
situation wherein three-month-old seedlings of ‘Bangalora’ raised by stone
grafting initiated flower buds, while in similar grafts of ‘Langra’ and ‘Alphonso’,
the biennial bearing cultivars, only vegetative growth was produced. after two
months, during December first week, flower bud emerged in case of ‘Bangalora’
grafts, whereas a second vegetative growth flush was observed in ‘Bangalora’
and ‘Alphonso’ grafts, demonstrating the inability of young leaves in biennial
bearing cultivars to synthesize the flower inducing hormone. In the light of the
above observation, it was proponed that the ‘on’ and ‘off’ year conditions in
biennial bearing cultivars are governed by the synthesis (or non-synthesis) of a
flower inducing stimulus which in turn depends upon the age and maturity
conditions of the shoots. In regular bearing cultivars ‘on’ and ‘off’ year
conditions do not exist possibly because of the production of the flower inducing
hormone even in young leaves. Singh (1959) demonstrated that the flower
inducing stimulus could be transmitted from a mature tree of juvenile mango
seedlings through grafting resulting the flowering of young stock, however, he
found that the donating action shoots failed to induce flower in the nondefoliated seedling stock. He proposed that the high level of auxins produced in
the leaves of the acceptor seedling counteracted the action of the flowering
hormone donated by the action, resulting in lack of flowering. The response
flowering in the receptor seedling was the same irrespective of cultivar involved,
indicating that the nature and action of flower inducing hormone was the same
in both regular and biennial cultivars. Found that the donating action shoots
failed to induce flower in the non-defoliated seedling stock. He proposed that the
high level of auxins produced in the leaves of the acceptor seedling counteracted
the action of the flowering hormone donated by the action, resulting in lack of
flowering. The response flowering in the receptor seedling was the same
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irrespective of cultivar involved, indicating that the nature and action of flower
inducing hormone was the same in both regular and biennial cultivars.
3.1.4.1.1 Auxins
Chacko (1968) found a high level of auxin-like substance in the shoots of
‘Dashehari’, which were expected to flower. The work of the same person on the
naturally occurring growth substance in the shoots of ‘Dashehari’ and ‘Totapuri
Red Small’ indicated the presence of a zone on paper chromatograms containing
growth promoting substances, which exhibited biological properties similar to
auxins. The shoots from ‘Dashehari’ ‘on’ year and ‘Totapuri Red Small’ trees,
which initiated flower buds during the experimental period had a higher level of
growth promoting substances during the period of flower-bud initiation than the
shoots of ‘Dashehari’ ‘off’ trees which remained vegetative. Daschowdhary
(1969) observed that in the neutral fraction of ‘Langra’ shoot extract, a growth
promoting substance occurred at Rf 0.7 to 0.8 on paper chromatogram developed
in isopropanol : ammonia : water. It was found that the highest concentration of
the promoter coincided with the ripeness to flower stage.
3.1.4.1.2 Gibberellin-like substance
In many of the cold-requiring biennials and long-day annual plants, Gibberellins
are known to be involved in the production of floral stimulus. A study of Chacko
(1968) showed that the amount of gibberellin-like substance was higher in the
shoot extracts of ‘Dashehari’ ‘off’ season trees as compared with those of ‘on’
trees, which were differentiating fruit of grafted seedling as reported by Singh
(1959) was interpreted by Singh (1971) as owing to its high content of
endogenous gibberellins.
3.1.4.1.3 Cytokinin-like substances
Relationships between mango flowering and the endogenous level of cutokinins
studies were conducted on the endogenous cytokinins in the shoot tips of
Dashehari mango between September and February in 'on' and 'off years
(Agarwal et al, 1980). Eleven cytokinin-like substances were isolated in the 'on'
year, including zeatm riboside and zeatin. Cytokinin levels at the time of flower
bud differentiation (December to February) were higher in the 'on' year than in
the ‘off’ year. These results suggested that flowering in mango shoot tips is
associated with high levels of endogenous cytokinins. Chen (1981, 1983) isolated
zeatin, zeatin riboside and other cytokinin-Iike substances from immature mango
seeds. Cytokinin concentration in panicle and pulp of mango was highest 5 to 10
days after full bloom and decreased rapidly thereafter. Highest total cytokinin-
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136
Iike activity was observed in the xylem sap also at the time of full bloom (Chen,
1987).
3.1.4.1.4 Ethylene
Ethylene is unique in that it is the only gaseous phytohormone. It is usually
present in a minute quantity of about 0.1 ppm and causes marked physiological
effects in the plants. Some of the flower promoting effects in mango reviewed.
3.1.5 Floral manipulation in mango by application of exogenous plant
hormones
3.1.5.1 Ethylene spray
The ethylene-generating agent, ethephon, applied at 125-200 ppm, induced
flowering of 'Carabao' mango in the Philippines within six weeks after treatment
(Dutcher, 1972). Flower induction also occurred at concentrations between 500
and 1,000 ppm; however, defoliation was also experienced at the higher
concentrations (Bondad, 1976). Ethephon has also been successful in India for
increasing flowering of 'Langra and 'Deshehari' during "off' years (Chacko et al.
1972, 1974; Chadha and Pal, 1986) and for inducing earlier production in juvenile
plants (Chacko et al. 1974).
In 10-year-old 'Haden', 500-1,000 ppm applied one month before the normal
flowering date increased flowering by 40-55 percent (Nunez-Elisea et al. 1980).
These results are contrary to those obtained by Pal et al. (1979), who found
ethephon ineffective after five consecutive years of treatment, and by Sen et al.
(1978) who reported an increase in flowering during "on" years but failed to
stimulate flowering during "off years.
3.1.5.2 Cutokinins spray
Exogenous cytokinins cause promotion and inhibition of flower initiation in a
variety of species, although promotive effects are much more frequent than
inhibitory ones (Bernier et al., 1981). Elevated cytokinin levels have been
implicated in breaking dormancy in adventitious and axillary buds (Stafstrom,
1995). Chen (1983) First time extensive study was carried out to clear that
cytokinins are important factors in the regulation of flower bud initiation and
development in mango trees.
3.1.5.3 6-Benzyl amino purine (6-BA)
Chen (1987) described precocious bud break and flowering of mango shoots in
response to an early October application of 100 ppm 6-Benzyl amino purine (6136
137
BA). Full flowering was observed one month following application compared
with three months later on non treated trees. And made conclusion that the
elevated cytokinin level found to prior to and during flowering and the
flowering response to applied BA led to conclusion that cytokinins are involved
in stimulation of bud break.
Conclusion
So far, the literature on floral manipulation in mango plants has been reviewed,
it is apparent that floral initiation in trees is controlled by a range of factors
which may include environmental stimuli, developmental cues, and other
interactions with vegetative growth and PGRs. It is also apparent that rarely can
one factor be considered in isolation. Research in trees is expensive, slow, and
has often been focused on limits to production in perennial trees like mango. So
application/ use of particular practice can be recommended strongly after
through assessment of different methods /practices can be recommended and
continuous Research efforts should be strengthened on flowering physiology
especially in genetic control of flowering in mango.
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Journal of Today’s Biological Sciences: Research & Review (JTBSRR)
ISSN 2320-1444 (Online)
JTBSRR
Vol.1, Issue 1, page 144-152, December 30, 2012
Available on: www.jtbsrr.in
Basic and Applied Research in Soil Organic Matter
Pratap V. Naikwade
Department of Botany, Nya. Tyatyasahe Aathalye Arts, Ved. S. R. Sapre Commerce and
Vid. Dadasaheb Pitre Science College, Devrukh-415804, Maharashtra, India.
E- mail: naikwade.pratap@gmail.com
Abstrct
Plants obtain nutrients from two natural sources: organic matter and minerals. Organic
matter includes any plant or animal material that returns to the soil and goes through the
decomposition process. Majority of the soils in world are with less organic matter.
Intensive tillage and agriculture met food production but resulted in sick infertile soil.
Soil organic matter affects the chemical and physical properties of the soil and its overall
health. Its composition and breakdown rate affect: the soil structure and porosity; the
water infiltration rate and moisture holding capacity of soils; the diversity and biological
activity of soil organisms; and plant nutrient availability.These attributes of organic
matter lead it to have a major influence on the quality of soil material itself. As societies
throughout the world begin to realize the potential value of the soil resource in
contributing to sustainable farming practices, the need to understand the role that
organic matter plays in contributing to soil quality has become more important. The
present study deals with basic and applied research about soil organic matter which is not
only related to agronomy and soil science but also to ecology. biochemistry, microbiology,
biotechnology, biophysics, environmental science etc. It gives details about components,
attributes, properties functions and management of soil organic matter.
Key Words: organic matter, soil, basic, research, fertility
Introduction
As we enter the 21st century, pressure on the world’s ecosystems to provide for
human needs is at an unprecedented level. It was estimated by Oldeman (1994)
that by 1990, some 562 million hectares (38% of the world’s cropland) had been
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145
degraded by poor agricultural practices. Further damage had occurred, with the
annual degradation of 5–6 million hectares, and current trends are not
encouraging. Losses in the organic matter content of soils during the last 100
years have been substantial, and have been associated with changing patterns of
land use that are driven by population increases. The process of cultivation of
native soils is nearly always associated with a loss of organic carbon, as
previously protected organic matter is oxidized following exposure to the
atmosphere (Davidson and Ackerman, 1993; Gregorich et al., 1998). It is likely
that these losses were not evenly distributed across the globe, with
disproportionately large losses from upland, organic and wetland soils. Losses of
soil organic matter are also associated with land use change other than direct
conversion to agriculture, such as deforestation and biomass burning (IPCC,
1996).
Some analyses carried out in parts of Africa show that nutrients are being
depleted at an alarming rate (Smaling et al., 1996). Nutrient budgets can be used
at differing scales and, although associated with a high degree of spatial
heterogeneity, they can be valuable in identifying regional trends. In Nigeria,
Smaling et al. (1998) found that the difference between the input and output of N
leads to an average net annual loss of 27 kg ha−1, in soils that in many cases are
already nutrient poor. Shen et al. (1989) found that soils that had received an
annual addition of 144 kg N ha−1 over 137 years contained more organic matter
than those receiving no fertilizer additions.
Components and Functions of soil Organic Matter
Forms and classification of soil organic matter have been described by Tate
(1987) and Theng (1987). For practical purposes, organic matter may be divided
into aboveground and belowground fractions. Aboveground organic matter
comprises plant residues and animal residues; belowground organic matter
consists of living soil fauna and microflora, partially decomposed plant and
animal residues, and humic substances (Bauer and Black, 1994). Although soil
organic matter can be partitioned conveniently into different fractions, these do
not represent static end products. Instead, the amounts present reflect a dynamic
equilibrium. The total amount and partitioning of organic matter in the soil is
influenced by soil properties and by the quantity of annual inputs of plant and
animal residues to the ecosystem (Bell et al, 1998).
Organic matter within the soil serves several functions. From a practical
agricultural standpoint, it is important for two main reasons: (i) as a “revolving
nutrient fund”; and (ii) as an agent to improve soil structure, maintain tilth and
minimize erosion. Organic matter releases nutrients in a plant-available form
upon decomposition (Hudson, 1994). In order to maintain this nutrient cycling
system, the rate of organic matter addition from crop residues, manure and any
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other sources must equal the rate of decomposition, and take into account the
rate of uptake by plants and losses by leaching and erosion (Prasad and Pawar,
1997). Where the rate of addition is less than the rate of decomposition, soil
organic matter declines. Conversely, where the rate of addition is higher than the
rate of decomposition, soil organic matter increases (Lavelle and Spain, 2001).
In terms of improving soil structure, the active and some of the resistant soil
organic components, together with micro-organisms (especially fungi), are
involved in binding soil particles into larger aggregates. Aggregation is
important for good soil structure, aeration, water infiltration and resistance to
erosion and crusting. Traditionally, soil aggregation has been linked with either
total C (Matson et al., 1997) or organic C levels (Dalal and Mayer, 1986a, 1986b).
More recently, techniques have developed to fractionate C on the basis of lability
(ease of oxidation), recognizing that these subpools of C may have greater effect
on soil physical stability and be more sensitive indicators than total C values of
carbon dynamics in agricultural systems (Blair and Crocker, 2000).
At the agro ecosystem, or ‘field’ scale, organic matter influences many readily
measurable soil functions or processes (Schnitzer, 1991). Organic matter is both a
source and a sink for plant nutrients, and provides an energy substrate for soil
organisms. Soil macro- and micro aggregation that aid the infiltration of air and
water, are promoted and stabilized by soil organic matter (Tisdall, 1996). Organic
matter promotes water retention and influences the efficacy and fate of applied
pesticides (Gregorich et al., 1994, 1997). It also influences certain soil physical
processes such as compactibility (Soane, 1990), friability (Watts and Dexter, 1998)
and the range of soil ‘available’ water for plant growth (Kay, 1998). Overall, the
positive interrelationship between soil organic matter and soil aggregation has
important benefits on both water and air infiltration, soil erodibility and
conservation of organic matter and nutrients (Feller and Beare, 1997).
Soil organic matter properties and attributes
Gregorich et al. (1994) indicated that soil organic matter should be viewed as a set
of fractions rather than a single entity. These fractions are descriptive of the
‘quality’ of soil organic matter. Important fractions of organic matter are the light
fraction, macro organic matter (i.e. particulate carbon), microbial biomass carbon,
mineralizable carbon, carbohydrates and enzymes. These fractions have
biological significance as they are involved in several soil functions and
processes such as aggregation and formation of soil structure, and nutrient
cycling and storage. Chemical characterization of organic matter, that provides
information on chemical structure and functional groups, is also useful to
evaluate the influence of land use changes on organic matter (Monreal et al.,
1995). However, the utility of such measurements in soil quality evaluation is not
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so clear. Measurements of soil biota abundance, diversity or activity are
considered potential indicators of soil quality (Gregorich et al., 1997). The
microbial biomass is the main agent that supports the soil function and
associated processes involved with the storing and cycling of nutrients and
energy (Carter et al., 1999). Mycorrhizal fungi play an important role in
sustainable plant productivity and in the formation and maintenance of soil
structural stability (Tisdall, 1996), while soil fauna are major determinants of soil
processes influencing nutrient cycling, aggregate formation and permeability of
soil (Lavelle et al., 1997).
Managing soil organic matter
The maintenance of organic matter for the sake of maintenance alone is not a
practical approach to farming. It is more realistic to use a management system
that will give sustained, profitable production (caligeri et al, 1998). The greatest
source of soil organic matter is the residue contributed by current crops.
Consequently, crop yield and type, method of handling residues and frequency
of fallow are all important factors. Ultimately, soil organic matter must be
maintained at a level necessary to maintain soil tilth (Paustian, 2002).
Depletion of soil fertility due to less organic matter is a major constraint for
higher crop production in not only India but other parts of world. Most of the
cultivated soils have organic matter of below 1.5 % and on the other hand,
addition of organic matter is very low. Almost all farmers are relying on
chemical fertilizers to remove nutrient deficiency for profitable yields (Tate,
1987). Consequently little or no accumulation of organic matter occurs in soil. A
suitable combination of organic and inorganic sources of nutrients is necessary
for sustainable crop yields. Nambiar (1997) reported that integrated use of
organic manure and chemical fertilizers would be promising not only in
providing greater stability in production, but also maintaining better soil fertility
status. A long-term research revealed that the application of dung manure at 5 t
ha-1 y-1 improved soil resources from degradation (Bhuiyan et al. 1994).
Application of organic materials alone or in combination with inorganic fertilizer
helped in proper nutrition and maintenance of soil fertility (Salim et al., 1988;
Talashiker and Rinal, 1986). Hussain et al. (1988) reported that organic manures
increased the efficiency of chemical fertilizers. Beneficial effects of farm yard
manure on crop production through improved fertility and physical properties
of soil is an established fact (Singh and Sarivastore, 1971).
Soil organic matter, N, sulfur (S), and P generally increase immediately after
compost addition because of an increased supply of organic C (Smith, 1991). This
stimulating effect can last a few months, depending on the quantity and quality
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of the amended compost and nutrient availability in the soil (Perucci, 1990).
Microbial biomassis considered the most active fraction of soil organic matter
and represents a significant source of plant-available nutrients (Smith and Paul,
1991). The increased total microbial biomass and enzyme activity due to compost
amendments can improve soil fertility over a long time. the fertility of soils is
often tied to their organic matter content (Brady, 1974). Buchanan and Gliessman
(1990) concluded that organic matter additions by compost, combined with
inorganic N or P fertilizer. Onion (Allium cepa L.) yield on a sandy loam soil
increased with increasing rate of organic matter application, when the organic
matter was biosolids/straw compost or digested or raw biosolids (Smith et al.,
1992).
Application of organic materials reduced soil acidity and improved organic
matter and available nutrients of the soil. (Sanwal et. al., 2007), Singh et al. (2007)
proved that Sugarcane crop responded well to different organic manures in a
multiple ratooning system with a better economic output and improved soil
quality. The application of farm yard manure, poultry manure and sugarcane
filter cake alone or in combination with chemical fertilizers improved the soil
organic C, total N, P, and K status. (Kulvinder Kaur et al.,2008). An experiment
was carried out to compare organic Manures and Chemical Fertilizers on Saffron
(Crocus sativus L.) cultivation. Application of organic manure by green manure,
compost, vermicompost improved soil fertility (Amiri, 2009).
Results and Discussions
The tension between natural resource and economic sustainability in
agroecosystems, which has important consequences for conservation of soil
organic matter, underlines the need to develop strategies for increasing soil
organic matter by addition of humus rich contents like organic manures. Soil
organic matter is derived from once-living plant or animal matter. It includes
leaves, weeds, and animal waste. Organic matter may be one man’s trash, but to
the nursery, it is a treasure. Soil organic matter can greatly improve the
substrate’s chemical and physical properties necessary for good plant growth. It
provides plant nutrients, improves porosity and water-holding capacity, and
makes the substrate lighter and easier to transport. The functioning of soils is
profoundly influenced by their organic matter content. The abilities of a soil to
supply nutrients, store water, release greenhouse gases, modify pollutants, resist
physical degradation and produce crops within a sustainably managed
framework are all strongly affected by the quality and quantity of the organic
matter that it contains. Basic and applied research about soil organic matter
which is not only related to agronomy and soil science but also to ecology.
biochemistry, microbiology, biotechnology, biophysics, environmental science
etc and hence greatly important.
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Journal of Today’s Biological Sciences: Research & Review (JTBSRR)
ISSN 2320-1444 (Online)
JTBSRR
Vol.1, Issue 1, page 153-159, December 30, 2012
Available on: www.jtbsrr.in
Application of Telemedicine
Overview
in India -- An
Subhasis Bandyopadhyay
Institute of Computer and Information Science, Bankura,West Bengal, India
E-mail: subhasis_ban83@yahoo.co.in
Abstract
India got an opportunity to make its health infrastructure better with its faster growing
information and communication technology. With implementation of Telemedicine, it
can serve weakly served or un served parts of country.
Telemedicine uses some modern information and communication techniques to connect
distant part of world. By application of it countries like India can make drastic change in
their health structure by milking its advantages. They can serve cheap but world class
medical facilities and education to rural parts of country (as most ill served part of India
is villages).But to implement it India may face some challenges. It have to overcome those
challenges and implement it with help of Government bodies (like Indian Space Research
Organization), Govt. executives and private players working together.
Key Words: Telemedicine, Health, Medical facility
Introduction
Telemedicine is a boon of modern information and communication system. It is a
process of connecting healthcare system of one part of world with other parts for
exchanging consultancies, education some other healthcare facilities with a view
of betterment of world’s health infrastructure. Here ‘Tele’ is a Greek word which
means ‘Distance’ and ‘Mederi’ is a Latin word which means ‘to heal’. So Times
magazine has described this system as ‘Healing by wire’. World health
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organization has defined Telemedicine as--- “The delivery of healthcare services,
where distance is a critical factor, by all healthcare professionals using
information and communication technologies for the exchange of valid
information for diagnosis, treatment and prevention of disease and injuries,
research and evaluation, and for continuing education of healthcare providers,
all in the interest of advancing the health of individuals and their communities.”
India also has come up with Telemedicine technology with a plane of connecting
excellent healthcare facilities of specialty hospitals with rural hospitals.
There are several types of Telemedicine facilities. These are –
1. Telenpathology: Images and videos can be transmitted as a computerized
file and thus job of pathologist can be made easy.
2. Telesurgery: by use of multiple high resolution video camera doctors can
get three dimensional views. Medical students also can do dissection
using visuals on internet.
3. Telecardiology: In telecardiology, Electrocardiographs (EGCs0 can be
transmitted using information and communication technologies.
Methodology
Application of Telemedicine in India to cover it’s under developed rural
healthcare facilities is discussed here with some case studies. Reliable secondary
data and some case studies are used to analyze the applications of Telemedicine
with giving a trace on Indian villages.
Technologies used in Telemedicine
Growth of Information and communication technologies is the reason of boom of
Telemedicine world wide. There are several technologies used to transmit image
and video files from one place to other. These are –
1. Stored and forward method: In this process digital image is taken and
stored. Then the image is forwarded to another location. Diagnosis and /
or consultation information are sent back.
2.
Video Conferencing:
through televisions.
Two way interactive communications is made
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3. Integrated services digital Network (ISDN), a high-speed international
communication standard for transmitting video, audio and data over
digital or normal telephone wires.
4. T-1 is another technology used to transmit voice and digital data at 1.554
megabyte per second(mbps)
5. Plain old telephone service also used for audio conferencing, storing and
forwarding data and low bandwidth video conferencing.
6. Internet is also used for telemedicine services. Several hospitals have their
own website providing information and consultancies.
Application of telemedicine in India
Out of total 1210.2 million populations in India, 833.1(68.84%) million population
resides in rural India. This 68.84% of rural population lack the basic medical
facilities. According to a report of rural health statistics (RHS), 2010, there is a
shortage of 19,590 sub centers, 4252 primary health centers and 2115 community
health centers in India. Beside this crude birth rate (defined as the number of live
births per 1000 persons over a period of one year.) in rural India has also
declined from 38.9 per thousand in 1971 to 23.7 per thousand in 2010. According
to Indian Institute of public opinion, that 89% of rural Indian patients have to
travel near about 8 km for accessing basic medical facilities. In another report of
Indian medical society has informed that 75% of qualified doctors are practicing
in urban areas, 23% in semi urban areas and only 2% in rural health centers. This
all shows the poor infrastructure of rural India and the need of introduction of
Information and communication technologies like Telemedicine in India.
Telemedicine can provide world class medical facilities of specialty hospital to
the hospitals of remote areas and thus can reduce the cost of treatment there.
Telemedicine can make medical facilities accessible to all rural Indian families.
In Indian telemedicine reform, Indian space research organization (ISRO) has
played a vital role. It has come up with satellite bandwidth, soft wares and hard
wares. ISRO has set up Health SAT, a satellite, to provide telemedicine facilities
to rural people of India. According to L.S. Sathyamurthy, Program director of
Telemedicine at ISRO “There are 650 district hospitals, 3000 taluk(sub
district)hospitals and more than 23000 primary health centers in the country, we
must aim to connect all these phases. First district hospitals connected to
specialty hospitals in major cities, then taluk level hospitals, and finally primary
health centers, so that nobody, irrespective of his location, is deprived of
lifesaving specialty consultation.” In the goal of doing that, ISRO has run a pilot
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project in 2001 and connected 60 remote hospitals with 20 super specialty
hospitals.
India has efficiently applied Telemedicine in several hospitals of it and has got a
good result. Till now Indian Telemedicine network has done efficiently well by
treating more than 25000 patient and proving Telemedicine facilities to 100 of
hospitals(ISRO has connected 78 rural/remote/district health centers with 22
specialty hospitals of major cities. In an telemedicine project, G.B.Pant hospital at
port blair has connected with shri Ramchandra medical college and research
institute, Chennai. In another project, Narayana Hrudayalaya of Karnataka is
linked with Chamaraynagar and Vivekananda memorial hospital,d district
hospital of Sagar by Telemedicine facilities. According to Amrita Telemedicine
Programme’s report, 13 th January 2003, they has performed first remote
Telesurgery to save a pilgrim using local Telemedicine facility. Besides them
Apollo group also has successfully implemented Telemedicine facilities in India.
A Case Study of Apollo Group
Apollo hospital has introduced Telemedicine with a mission of spreading
standard healthcare facilities in the reach of every individual of India and world.
In voice of Dr. Prathap C. Reddy (Founder and Chairman, Apollo Hospitals
Group), “Our mission is to bring healthcare of international standards within the
reach of every individual. We are committed to the achievement and
maintenance of excellence in education, research and healthcare for the benefit of
humanity”.
In the aim of introducing Telemedicine in India, Apollo group has introduced
Apollo Telemedicine Networking foundation (ATNF), non profit organization.
Today ATNF has 125 peripheral centers including 10 overseas. Besides, ATNF
providing telemedicine facilities in the distance ranging 200 to 75000 km with its
75000 teleconsultation in 25 different disciplines.
Ministry of external affair had selected ATNF for providing teleconsultation and
tele education to 53 countries of African union.
In the 12 year journey of ATNF, It has done remarkably well to cover several
parts of country and 29 African countries.
World healthcare congress, Washington, USA has awarded ATNF with the BEST
POSTER AWARD in April, 2011.
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Challenges Faced
Telemedicine
By
India
in
Introduction
of
Poor infrastructure and lack of knowledge of computer in rural India has made
task of implementation of Telemedicine very challenging. Several type of
problems faced in implementation of Telemedicine in India. These are—
1. There are lack of infrastructure in rural india.Several villages still have no
electricity. Though Rajiv Gandhi Gramin Vidyutikaran Yojana (RGGVY)
has introduced as a remedy of that problem by providing connection to
all villages and free connection to BPL families.
2. There is a fear of losing job if this technology is introduced.
3. Due to lack of knowledge and expertise in handling information
technology and other equipments, people hesitate to use Telemedicine.
4. Some people think that setting up of telemedicine facilities will incur high
initial investment. So it is not financially viable.
5. Doctors even sometimes are not fully convinced or familiar with this
technology.
6. There are only few people who know English. So, Language barrier is a
big challenge in India.
Benefits of using telemedicine
Telemedicine is like a magic to Indian healthcare infrastructure which can make
drastic change in rural healthcare practice by bringing world class medical
facilities to remote and rural villages. People who are suffer of poor rural
healthcare facilities in India, will be able to access those facilities at their door
step. Besides this, Telemedicine also possesses several other benefits. These are -1. Telemedicine provides cost effective medical services.
2. Telemedicine is used in optimization of resources.
3. It can provide specialist advice and counseling via use of information and
communication technologies at patient’s home or nearest possible site
eliminating need of unnecessary travelling.
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4. Telemedicine helps in providing healthcare facilities at remote villages.
5. Telemedicine is very helpful for medical professional in exchanging
information and consultation.
6. Telemedicine can be used to save life of injured people at war or disaster
effected areas.
7. Telemedicine is also help in the education of medical healthcare
professionals
Conclusion
With a bird’s eye view of improving healthcare system in India, Telemedicine
emerging in this subcontinent in jet speed. Govt. body (like ISRO, department of
information technology of ministry of communication and information
technology etc.) and private players have come up with efforts in
implementation of telemedicine. Now India is able to milk their opportunity to
cover its weak health infrastructure with the boon of faster growing information
technology dream of Healthy India will come in reality.
References
Websites
http://www.telemedicineindia.com/
http://www.isro.org/publications/pdf/Telemedicine.pdf
http://telemed.esanjeevani.in/Telemedicine/Report.pdf
Others
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Deb Soumya, 2008, Telemedicine – a new horizon in public health in India,
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Mundrey K. Telemedicine : The new frontier, Information systems computer
world, June 1998
Report of technical working group for telemedicine standardization, department
of information technology(DIT),Ministry of communication and information
technology(MCIT),2003,
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Journal of Today’s Biological Sciences: Research & Review (JTBSRR)
ISSN 2320-1444 (Online)
JTBSRR
Vol.1, Issue 1, page 160-190, December 30, 2012
Available on: www.jtbsrr.in
Eutrophication: Causative factors and remedial
measures
T.K.Ghosh1* and Debashri Mondal2
1Ultratech
Environmental Consultancy and Laboratory, Survey no. 87, office 7 and 8,
Bandal Prestige (in front of Siddhi Vinayak Mandir), Azad Nagar, Kothrud, Pune-411
038, E. mail- tkghosh@ultratech.in
2Dept.of
Zoology, Raiganj Surendranath Mahavidyalaya, University of Gour Banga,
Raiganj- 733134, West Bengal, India ,e. mail- debashri_mondal@rediffmail.com
* Address for correspondence
Abstract
Eutrophication represents the aging process of lakes, whereby external or allochthonous
sources of nutrients and organic matter of terrestrial origin accumulate in a lake basin,
gradually decreasing its depth and increasing autochthonous production, to the point
that the lake begins to take on a marsh-like character and, ultimately, a terrestrial
character. Human influences in a drainage basin can greatly accelerate this enrichment
process. Eutrophic lakes are normally more productive, less deep and exhibit reduced
hypolimnetic oxygen concentrations during summer stratification. Indicator species (e.g.
Chironomus) tends to dominate such waters, mainly because of their ability to tolerate
typically lower oxygen concentrations. Diversity of phytoplankton, zooplankton, insects,
fish and benthos clearly indicate trophic status of the water bodies. Also, Secchi disc
transparency, chlorophyll-a level and P concentration of water denotes degree of
eutrophication. Excessive P loading has been shown to promote potentially toxic nitrogen
fixing genera, while excessive P and N loading can stimulate toxic blooms of non-N2
fixing genera (Microcystis, Lyngbya, Planktothrix). From a supply standpoint, both the
absolute amounts and relative proportions of these nutrients play important roles in
determining the composition, magnitude, and duration of Cyanobacterial Harmful Algal
Blooms. In order to combat eutrophication, various remedial measures, commonly
followed elsewhere, and select success stories around the world have been discussed.
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Key Words: Eutrophication, nutrients, allochthonous, autochthonous, phosphorus,
nitrogen, algal bloom, remedial measures
Introduction
Preamble
The term "eutrophication" actually comes from a Greek word meaning "overfed."
This is essentially what happens to a water body when it begins its journey down
the road of dreadful condition. It is "overfed" to the point that it cannot handle all
of the elements flowing into it, and if the trend is allowed to continue it
eventually becomes eutrophic - a dead ecosystem. According to the Organization
for Economic Cooperation and Development (OECD) 1982, eutrophication refers
to the excessive nutrients enrichment of water which results in the stimulation of
an array of undesirable symptomatic changes, such as nuisance production of
algae and other aquatic macrophytes (plants), deterioration of water quality,
taste & odour problems and fish kills. Each of these changes significantly
interferes with human use of water resources.
Eutrophication involves the enrichment of waters chiefly by increasing the levels
of essential nutrients, such as Phosphates, Nitrates and Silicates (Lee et al. 1980;
Uhllmann 1984). One of the hardest problems to overcome with this is that there
are so many possible sources for these nutrients to get into the aquatic
ecosystems - fertilizers, street runoff, animal excrement, and organic debris (such
as leaves) just to name a few. As a result of this nutrient build-up, plant life
(especially algae) begins to form in abundance. If the build-up of nutrients is
severe enough, the algal blooms will cover the entire surface of the water and not
allow any sunlight to penetrate the water column. This then creates a couple of
consequences: 1) it prevents photosynthesis from occurring below the surface of
the water, lowering the dissolved oxygen content of the water, and 2) stops
oxygen transfer from occurring through surface aeration. Consequently the
water body becomes extremely depleted of oxygen and sunlight, which causes
fish and plant life alike to suffer. When fish and plants begin to die off,
decomposing bacteria use the remaining oxygen to break down the dead organic
compounds. The result is a body of water covered in algae, lacking in aquatic life
and oxygen, both of which are necessary to promote a healthy aquatic ecosystem.
In India, it has been assessed that more than 80 per cent of the total pollution
load arises from domestic sources, such as domestic wastewater, which is
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reported to contain P between 6 and 10 mg l-1 (Horan 1990). Per capita
consumption of detergents in India in 1994 was 2.8 kg per annum, which was
moved up to over 4 kg/capita by 2007. However, in rural areas the use of
detergents is expected to grow 7-8 per cent annually. The figures are alarming
because high quality detergents comprise around 35 per cent sodium tripolyphosphate (STPP). In India majority of detergents are triphosphate based, as
its use enhances the cleaning property by sequestering the ions (Ca2+ and Mg2+)
that cause water hardness. The impact of increased use of phosphate-based
detergents on the growth of aquatic plants and cyanobacteria is well emphasized
by Campbell (1987).
In recent years, there has been an increasing awareness on the part of both
scientists and the general public, of problems associated with excessive growth
of the aquatic plants, particularly in lakes. Lake is a potential source of drinking
water supply and about 4/5th of the total water supply comes from lakes. As
such excessive growth of aquatic plants and algae, even in lakes with protected
catchment area, assumes critical importance. Among the important adverse
effects are health hazards to human and animal populations using such water
bodies as a source of potable water.
Eutrophication is the natural ageing process of lakes. It is characterized by a
geologically slow shift from in-lake biological production driven by
allochthonous (external to the water body) loading of nutrients, to production
driven by autochthonous (in-lake) processes. This shift typically is accompanied
by changes in species and biotic community composition, as an aquatic
ecosystem is ultimately transformed into a terrestrial biome. In the process of
eutrophication by natural aging, a lake will be slowly filled in with soil and other
materials carried by inflowing waters, and eventually become a marsh and
ultimately, a terrestrial system (Fig.1). This process usually takes many hundreds
and thousands of years to occur and is largely irreversible. Lakes undergoing
such natural eutrophication generally have good water quality and exhibit a
diverse biological community throughout much of their existence.
Eutrophication is a worldwide issue. It is often most severe in shallow lakes
which are heavily influenced by large external nutrient loads, frequent sediment
resuspension and resultant high turbidity, highly active sediment-water column
nutrient exchange and nutrient regeneration. In response to nutrient enrichment,
these lakes experience accelerated eutrophication, causing the ecosystem to shift
from macrophyte to phytoplankton-dominated conditions, often culminating in
summer cyanobacterial blooms.
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Fig.1. Fate of organic pollutants in aquatic ecosystem
In the area where there is no human settlement, the growth of algae and other
aquatic plants in a lake in the drainage basin is usually minimal, and generally in
balance with the input of plant nutrients. However, human settlements in a
drainage basin and associated cleaning of forests for development of farms and
cities etc., usually changes the natural eutrophication in a dramatic way. The
runoff of the most materials from the land surface to the water body is greatly
accelerated. An increased input of plant nutrients (mainly Phosphorus and
Nitrogen) to a lake or reservoir can stimulate algal and aquatic plant growth,
which in turn, can stimulate the growth of fish and other higher tropic level
organisms in the aquatic food chain. The latter phenomenon is termed cultural
eutrophication to distinguish it from the natural process (the terms ‘artificial’ and
‘anthropogenic’ or ‘man-made’ are also often used to describe the same
phenomenon). The rate of this cultural eutrophication is enhanced by
human/anthropogenic activities such as discharge of effluent from industries,
domestic wastes, as well as point and non point sources from agricultural fields.
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Consequence of eutrophication
 Excessive growth of algae and aquatic plants, which interfere with the uses
and aesthetic quality of water body.
 Production of taste and odour in drinking water caused by the excessive
growth of algae and aquatic plants.
 As algal populations die and sink to the bottom of a water body, their decay
by bacteria can reduce oxygen concentrations in bottom waters to levels
which are too low to support fish life resulting in fish kills.
 Owing to this oxygen deficit there is an excessive increase of Iron and
Manganese in the water and this can interfere with the drinking water
treatment. Also excess growth of algae results in clogging of filters in the
treatment plants.
 There are also negative potential health effects especially in the tropical
regions related to diseases like schistosomiasis, onchocerchiasis and
malaria, all of which can be aggravated by cultural eutrophication, which
can enhance the appropriate habitats for the causative organisms.
Eutrophication and its effect on water Body
The very large number of criteria used for the trophic state determination has
contributed a lot to the belief that the trophic concept is multidimensional and
involves a variety of parameters as represented in following Table 1.
Table 1: Parameters for Determination of the Trophic State of a Lake
Parameter
Oligotrophic
Eutrophic
Occurrence of algal bloom
Rare
Frequent
Frequency of green and
Low
blue green algae
High
Daily migration of algae
Considerable
Limited
Characteristic algal groups
Bacillariophyceae
e.g.
Pinnularia,
Cymbella,
Cyanophyceae
Chlrophyceae e.g. Volvox,
Microcystis, Nostoc
Chrysophyceae
e.g.
Synura, Chromulina
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e.g.
165
Represented by large size
Characteristic zooplankton
Represented by small size
species
e.g.
Cyclops,
groups
species e.g. protozoans
Daphnids
Density of plankton
Low
High
Characteristic of fish
Finer variety of fish
Course fish
Depth
Deeper
Shallower
Present
Absent
Algae
High species diversity
with low density and
productivity
often
dominated
by
Chlorophyceae
Low species diversity with
high density and the
productivity
often
dominated
by
Cyanophyceae
Blooms
Rare
Frequent
Plant nutrient flux
Low
High
Animal production
Low
High
Fish
Finer variety of flux (e.g. Course fish
carps)
breathers
Summer
oxygen
hypolimnion
in
e.g.
Algal blooms
Although nutrient status of a particular water body remains variable, but,
occasionally a particular species becomes dominant leading to growth of ‘water
bloom.’ A water bloom can be defined as dense growth of microscopic organisms
in water, making water odiferous, unpleasant in taste or may turn it toxic during
their growth cycle. It may be induced suddenly due to heavy rainfall or rise in
solar radiation of temperature. Terrestrial plants, when submerged under water
for long time, die and decompose within the lake making water odiferous. The
microflora like diatoms and other algae form a source of food supply for various
animals, directly or indirectly.
Their occurrence may turn troublesome due to decomposition (Abeliovich and
Shilo 1972). There are instances of coloured water in history which was recorded
due to the colour and high density of the organisms like dinoflagellates,
cyanobacteria or other algae. The ‘Red sea’ named after a marine red bloom (redtide), caused by a cyanobacterium ‘Trichodesmium erythreum’, resulted in massive
fish-kills and varieties of cyanobacteria were also found to cause giddiness,
convulsions and deaths of animals drinking fresh water. Many a time water
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air
166
blooms are harmless, natural and go unnoticed; but when dense and continuous
become catastrophic; if not accompanied by growth predatory species of fish and
tiny crustaceans, e.g., Daphnia. Much interest has been generated on the toxins
produced, especially by cyanobacteria, as they are potentially dangerous to
domestic animals and to public safety. Toxicity studies with certain algal extracts
to vertebrates and mice were found to be apparently possessing harmless
chemicals, e.g., geosmin, which is produced when living matter including algal
sludge decomposes, or by living Anabaena circinalis (Walsby 1975, Oliver 1994).
The blooms occur when ideal conditions for growth of algae exist into aquatic
environment. A correct balance of nutrients, ideal temperature and carbon-dioxide causes good growth of algae. At elevated temperatures nutrients are
released more rapidly and bacteria as well as other microbes become more active
in spring and summer; decomposing organic materials and reproducing faster
than their competitors or predators. So, blooms are abundant in springs; when
algae utilize nutrients and exhibit explosive growth and their number may
increase one thousand times from normal. At later stages of death phase, such
bloom ensues to deficient nutrition and extensive multiplication of their
predators. Alternatively, the absorbed nutrients, organic substances and
photosynthates are released which support secondary bloom formation of
various species of algae. During the process the bloom forming organisms ensure
their supply of oxygen, photosynthesis, adopt buoyancy and form characteristic
scums.
Algal bloom / growth in water often turns harmful, e.g., certain toxic heavy
metals are absorbed or adsorbed on algal cells or consumed. Once such
accumulation reaches to sufficient level in algal cells, it may exhibit toxicity to the
consumers like predators, fishes, birds or other aquatic organisms.
Cyanobacterial blooms are harmful in many ways. One of these is secretion of
toxic compounds which are active against cattles, fishes, fowl and even human
beings (Schiwmmer and Schiwmmer, 1964, 1968). Other effects include:
(a) Unpleasant taste to drinking water,
(b) Mortality of fish,
(c) Products of their decay release substances deleterious to aquatic
animals, and
(d) Release of certain endotoxins (Carmichael et al. 2001).
One of such example was in 1995, when Anabaena circinalis caused odiferous
water in Armidale (Australia) due to production of ‘Geosmin’ when living
matter including algal sludge decomposed on a massive scale. Few bloomforming algae are: Aphanocapsa fusae, Microcystis aeruginosa, Microcystis flos-aquae,
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Gloeotrichia, Oscillatoria, Anabaena, Aphanizomenon flos-aquae, Anabaenopsis flosaquae, dinoflagellate Gymnodinium, Prymnesium parvum etc.
Excessive growth of algae destroys the recreational and aesthetic value of lakes.
A thick mat of green algae Hydrodictyon could be observed in Yamuna river near
water intake wells at Delhi. This algal mat resulted in considerable depletion of
dissolved oxygen level. The example of the same is Dal Lake, which has come
under stress due to anthropogenic influences. In the summer of 1991, placid and
limpid waters of two basins of Dal Lake, viz., Gagribal and Boddal turned red
which spread further and engulfed more adjacent lake areas. The causative
organism and responsible factors for initiation and development of such a
phenomenon in the lake became highly controversial and various theories were
put forth. Investigations have shown that the organism Euglena rubra was
responsible for imparting reddish colour to the waters. Similar types of
euglenoid blooms, recorded in other places (Zafar 1986, Venkateshwarlu et al.
1981), were correlated with influx of higher levels of pollutants to the water body
especially due to domestic sewage. The importance of iron in the distribution of
Euglenoid flagellates has been pointed out by Khan (1993).
Primary production data of phytoplankton are available for a number of water
bodies ranging from small fish ponds to large lakes and rivers. The gross
primary production (GPP) ranges from 37 mgCm-2 day-1 in Ramgarh lake
(Gorakhpur) to above 17.5 gCm-2 day-1 in a sewage pond near Ahmedabad. In
general, shallow lakes, fish ponds and temple tanks are more productive than
deep lakes and reservoirs.
Aquatic macrophytes
Aquatic macrophytes can be efficient indicators of water quality, and their
presence may enhance water quality due to their ability to absorb excessive loads
of nutrients. These properties have been used in wastewater treatment as well as
in biomanipulation of water bodies for enhancing fish production. In deep lakes
and reservoirs, the macrophytes are mostly submerged and are confined to
margins. Pandya and Kane (1976) observed maximum biomass of 126 g m-2 for
submerged macrophytes in Lalpari lake, Rajkot. Kane (1977) reported net annual
production by submerged macrophytes of 970 g m-2 in the Manasbal lake,
Srinagar. The wetlands and epilittoral zones of large lakes dominated by large
emergent vegetation like species of Typha, Phragmites etc. show very high values
of standing crops and annual production. Kane (1977) recorded net annual
production (above ground) from 1010 to 3000 gm-2 in different parts of Dal lake,
Srinagar. Of this, the submerged macrophytes contributed only 292-351 g m-2.
Gopal and Sharma (1978) found that standing crop in several wetlands in
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different parts of Rajasthan ranged from 3300 gm-2 (Typha angustata) to 9730 gm-2
(Phagmites karka). Daily rate of production was estimated up to 30 g m-2 day-1.
Rao and Gupta (1980) found that in Andhra Pradesh the major floating aquatic
weeds were Eichhornia, Ipomoea, Pistia, Nelumbo, Nyphaea, Monochoria and Otella,
while Hydrilla, Vallisnaria, Ulothrix, Spirogyra and Chara were observed to be the
submerged weeds.
Factors affecting algal bloom
The factors required for algal growth include:
 Primary nutrients (C,N,P) as well as hydrogen and oxygen
 Minor and micronutrients
 Sufficient light energy in the water column
 Suitable water temperatures for growth
Algae will continue to grow as long as all the above requirements are met. The
rate of production depends highly on the quantity and quality/ suitability of the
factors listed above. When one or more of the stated requirements are not
available for growth, then algal productivity is said to be limited by that
condition. Limitation of algal growth is best described by Liebig’s Law of the
Minimum, which says that algal productivity will be limited by the element
present in least supply relative to algal requirements (O’Brien 1974; Goldman
and Horne 1983). Changes in temperature and light are the two main causes for
algal production to vary significantly between seasons. In order to understand
trophic status of the water body, preliminary screening can be made on the levels
of parameters like Secchi disc transparency, chlorophyll-a and phosphorus (Table
2). However, nitrogen (Table 3) is also considered while classifying water bodies
as per OECD (Rast et al.1989)
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Table 2: Trophic State of Surface Waters
Status
Secchi
Disc
Depth
(m)
Chlorophyll
-a
(g/L)
Total
Phosphorus
(g/L)
Oligotrophic
>5
<2
< 10
Mesotrophic
1.6 to 5
2 to 10
10 to 30
Eutrophic
0.7 to 1.6
10 to 30
30 to 60
Hypereutrophic
< 0.7
> 30
> 60
Table 3: OECD boundary values for open trophic classification system
Parameter
Oligotrophic Mesotrophic Eutrophic Hypertrophic
(annual mean values)
Total
X
8.0
26.7
84.4
phosphorous x±I SD 4.9-13.3
14.5-49
48-189
(µg P/l)
x±2
2.9-22.1
7.9-90.8
16.8-424
SD
range 3.0-17.7
10.9-95.6
16.2-386
750-1200
n
21
19 (21)
71(72)
2
Total
X
nitrogen (µg x±I SD
N/l)
x±2
SD
range
n
661
371-1180
208-2103
753
485-1170
313-1816
1875
861-4081
395-8913
307-1630
11
361-1387
8
393-6100
37 (38)
Chlorophyll
a (µg /l)
1.7
0.8-3.4
0.4-7.1
4.7
3.0-7.4
1.9-11.6
14.3
6.7-31
3.1-66
0.3-4.5
22
3.0-11
16 (17)
2.7-78
70 (22)
X
x±I SD
x±2
SD
range
n
169
100-150
2
170
Chlorophyll X
a peak value x±I SD
(µg /l)
x±2
SD
range
n
4.2
2.6-7.6
1.5-13
16.1
8.9-29
4.9-52.5
42.6
16.9-107
6.7-270
1.3-10.6
16
4.9-49.5
12
9.5-275
46
Secchi depth X
(m)
x±I SD
x±2
SD
Range
N
9.9
5.9-16.5
3.6-27.5
4.2
2.4-7.4
1.4-13
2.45
1.5-4.0
0.9-6.7
5.4-28.3
13
1.5-8.1
20
0.8-7.0
70(72)
0.4-0.5
l. The geometric means (after being transformed to base 10 logarithms) were
calculated after removing values which were greater than, or less than, two times
the standard deviation obtained (where applicable) in the first calculation.
x= geometric mean.
SD= standard deviation.
( ) = the value in brackets refers to the number of variables (n) used in the first
calculation.
[Source: Modified from Organization for Economic Cooperation and
Development 1982]
Nutrients
The relationship between various nitrogenous components and their use by the
total algal biomass has been the subject in scientific studies. The different forms
of nitrogen can be separated into organic and inorganic, as well as particulate
and dissolved components. Particulate organic nitrogen is found in living
biomass and detritus, while soluble organic nitrogenous materials are released
into the water from excretion, secretion, and decomposition processes (Keeney
1973). Soluble inorganic nitrogen is represented primarily by four different
molecules: nitrate (NO3-), nitrite (NO2), ammonia (NH3 / NH4+), and nitrogen
gas (N2). Ammonia is the preferred form for plant growth because the
incorporation of nitrate requires additional metabolic energy and enzymatic
activity (Goldman and Horne 1983). Both algae and bacteria incorporate
ammonia very rapidly (Sugiyama and Kawai 1979). The incorporation of
nitrogen gas into algal biomass occurs through a process known as nitrogen
fixation.
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Transformations between different forms of nitrogen in water are influenced by
environmental conditions (Keeney 1973) of the aquatic body. For example,
ammonia is the principal nitrogenous by-product or organic decomposition and
experiences different fates depending on where in the pond the ammonia is
produced. In waters containing dissolved oxygen, ammonia not incorporated by
algae, can be oxidised (i.e. add oxygen) through microbial processes. The
oxidation of ammonia first to nitrite and then to nitrate is called nitrification
(NH4+, NO2–, NO3-). In this two-step process, the microbial transformation of
ammonia to nitrite is much slower than the subsequent microbial transformation
of nitrite to nitrate (Cavari 1977, Goldman and Horne 1983). This has two
implications; first, algal uptake of ammonia can be relatively fast, and
competition for ammonia between algae and bacteria is predominantly in favour
of the algal community; second, the relatively rapid oxidation of nitrite to nitrate
that very little accumulates in toxic waters.
Unlike N, phosphorus exists in relatively few dissolved and particulate forms in
natural waters. No gaseous forms of P are common, although under anaerobic
conditions, trace amounts of the unstable gas phosphine (PH3) may be generated.
Overall, the main concern is with dissolved vs. particulate forms of inorganic and
organic P. Dissolved inorganic P (DIP) exists as orthophosphate (PO43-), which is
readily assimilated by all CHAB taxa. Many CHABs can accumulate assimilated
P intracellularly as polyphosphates (Boström 1988). Polyphosphates can serve as
internal stores of P for subsequent use in the event of ambient P depletion (Healy
1982). Dissolved organic P (DOP) can be a significant fraction of the total
dissolved P pool. Based upon the relationship between epilimnetic P
concentration and algal bloom frequency observed in various lakes, a linkage of
empirical models relating summer mean P to summer mean chlorophyll-a
(Carlson, 1977) and summer mean chlorophyll-a to algal bloom frequency
(Walker, 1984; Heiskary and Walker, 1988) was established, and accordingly a P
criterion of 0.02 mg l-1 (20 ppb) was selected (Fig.2).
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Fig.2: Relationship of algal bloom and lake P
Freshwater systems having low molar ratios of both total and soluble
(biologically–available) N to P (<15) are most likely to experience cyanobacterial
dominance (Smith 1983 1990). Conversely, waters having molar N: P ratios in
excess of 20 are more likely to be dominated by eukaryotic algal taxa (Smith
1983). This rule has proven broadly applicable to periodically stratified, long
residence (> 30 days) temperate and tropical freshwater systems (Downing et al.
2001). Excessive P (as orthophosphate) loading has been shown to promote
potentially toxic nitrogen (N2) fixing genera (i.e., Anabaena, Aphanizomenon,
Cylindrospermopsis, Nodularia), while excessive P and N (as dissolved inorganic N;
nitrate and ammonium) loading can stimulate toxic blooms of non-N2 fixing
genera (Microcystis, Lyngbya, Planktothrix). From a supply standpoint, both the
absolute amounts and relative proportions of these nutrients play important
roles in determining the composition, magnitude, and duration of CHABs
(Cyanobacterial Harmful Algal Blooms).
P enrichment (i.e., declining N: P ratios) frequently selects for the establishment
of N2 fixing species (Paerl 1988). Once N2 fixers are established, non–
diazotrophic species can remain a significant fraction of the phytoplankton,
because they are able to utilize fixed N produced and released by N 2 fixing
species (Paerl 1990). Co–existing diazotrophic and N–requiring bloom species are
capable of buoyancy regulation, and thus a near–surface existence, in highly
productive, turbid waters. Typically, Anabaena, Aphanizomenon and Microcystis,
(the notorious trio, “Annie, Fannie and Mike”) co–occur under these
circumstances. In clearer waters where light reaches the bottom, benthic N2
fixing and non–fixing assemblages (e.g., Lyngbya, some Oscillatoria, Microcoleus,
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Scytonema, Phormidium) can predominate. Mixed assemblages often persist as a
bloom “consortium” during summer and fall (Paerl 1983, 1986, 1987), until
unfavourable physical conditions, such as cooling (<15oC) and water column
turnover take place.
There are exceptions to the N: P rule. These include; 1) systems in which both N
and P loadings are very large (i.e., hypereutrophic systems in which N and P
inputs exceed the assimilative capacity of the phytoplankton), and 2) highly–
flushed, short residence time systems, in which the flushing rate exceeds growth
or doubling rates of cyanobacteria (generally >1 d-1). In N and P enriched
systems, N: P ratios may readily exceed 20, but since both N and P are being
supplied at close to non–limiting rates, factors other than nutrient limitation (e.g.,
light, vertical mixing, residence time, salinity, organic matter content) may
control algal community activity, biomass and composition. Under these
conditions, N2 fixation confers little if any advantage, and non–N2 fixing taxa
predominate. Often, these conditions favor high rates of primary production and
biomass accumulation.
Minerals and physical factors
Besides nutrients like CNP, there is increasing interest in the role of trace metals
in some systems. The most notable of these trace metals is iron (Fe) in its soluble
form Fe++. Iron is required for the synthesis and activity of photosynthetic, N2
fixing and N assimilatory enzymes. Unlike N and P, Fe inputs are not strongly
linked to human activities, such as agriculture, urbanization and most industrial
activities. Rather, Fe availability is more often controlled by natural weathering
or rocks, aeolian processes (dust transported by wind), and within-system
oxygen (e.g., hypoxia) and biogeochemical (redox) cycling. There is also evidence
that the production of toxic substances by CHABs is at least in part determined
by the amounts and ratios of nutrients and trace metals supplied to affected
water bodies (Sivonen 1996, Skulberg et al. 1994). Nutrient supply rates strongly
interact with other environmental factors, including light, turbulence and
flushing rates, temperature, pH (and inorganic C availability), salinity, and
grazing pressure to determine; 1) if a specific water body is susceptible to CHAB
formation, 2) the extent (magnitude, duration) to which CHABs may dominate
planktonic and/ or benthic habitats, and, 3) whether an affected water body is
amenable to management steps aimed at minimizing or eliminating CHABs.
If the sewage discharge exceeds the self-purifying capacity of the aquatic
environment, major alterations in the concentrations of dissolved oxygen (DO),
hydrogen ion (pH), carbon dioxide (CO2), ammonical nitrogen (NH3-N),
hydrogen sulphide (H2S), chlorine (Cl), nitrite (NO2) and nitrates (NO3) are
bound to occur. The changes in these physico-chemical conditions of the
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receiving water body would affect its normal ecology and biology in the
following way.

Alkaline water (pH 9.5 to 9.6) associated with supersaturation of dissolved
oxygen (138-141 percent) concentration was found to be the cause of fish
kill in shallow lake.

Discharge of domestic sewage or treated effluents tend to increase the
ammonical nitrogen concentrations in the natural water bodies. A limit of
5 mg/l of ammonical nitrogen has been suggested as the upper limit for
the effluent quality before discharge into the streams and rivers during
summer.

A threshold concentration of 0.25 mg/l of unionized ammonia has been
suggested for the freshwater fish. The amount of ammonia toleration
varies from one species to the other. A great deal of variation (0.25 to 3.8
mg/l) in the lethal concentration of ammonia exists to different fish
species.

Presence of hydrogen sulphide in water bodies is due to the microbial
decomposition of sewage, sludge, and animal and plant proteins.

Different species of hydrogen sulphide toxicity to the fish have been
reviewed earlier and it was noticed that under average water conditions, a
concentration of 0.5 to 1.0 mg/l was critical for many sensitive fishes.
Remedial measures
One must first determine the nature of the eutrophication problem and decide on
the goals of a control programme. The eutrophication problem in a given
situation may be excessive growths of algae and/or macrophytes, decreased
water transparency, hypolimnetic oxygen depletion and related fish kills,
nutrient regeneration or water quality deterioration due to the regeneration of
reduced chemicals, taste and odour problems in drinking water supply
reservoirs, or a combination of these types of problems. In this manner, one can
relate the major water use (or uses) of the lake or reservoir to the necessary water
quality for such a use. Intended lake and reservoir water uses as related to
trophic conditions (Bernhardt 1981) are presented in Table 4. Obviously, if the
existing trophic state of a water body is compatible with the water use, no action
is necessary in regard to phosphorus loading conditions. If not, both point and
non-point phosphorus control measures may be necessary.
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175
Table 4. Intended lake and reservoir water uses as related to trophic conditions
Desired utilization
Drinking water production
Bathing purposes
Low-water improvement
with long distance supply
line
without long distance
supply line
Fish culture
salmonid waterbodies
cyprinid waterbodies
Providing process water
Cooling water production
Water
sports
(without
bathing)
Landscaping in recreation
areas
Irrigation (by means of
channels)
Energy production
1.
2.
3.
Trophic state
Required
oligotrophic
mesotrophic
Still tolerable
Mesotrophic
slightly eutrophic
-
Mesotrophic
-
slightly eutrophic
oligotrophic
mesotrophic
mesotrophic
Mesotrophic
Eutrophic
slightly eutrophic
Eutrophic
Eutrophic
-
slightly eutrophic1
-
strongly eutrophic
-
strongly eutrophic2,3
Within the scope of landscaping, a eutrophic state caused by the natural ageing process,
can even be desirable.
Without consideration of the eventual water quality requirements for the receiving
canal.
Not valid for river power plants, which may be impaired by macrophyte and algal
growths.
[Source: Adapted from Bemhardt 1981]
Options of restoration techniques
First of all, there is a need to specify the purpose of restoration. Accordingly
various options are available in the literature (Rast et al. 1981) as guidelines. The
most important methods, applicable for specific sites worldwide, are listed
below, and a brief description of their application, advantages, and
disadvantages is given for each approach. It may be mentioned that some are
more ecological and sustainable than others.
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176
Diversion of wastewater
Wastewater diversion has been used extensively to rehabilitate lakes, often
replacing wastewater treatment. Discharge of effluents into an ecosystem that is
less susceptible than the one used at present is, as such, a sound principle, which
under all circumstances should be considered. Diversion might reduce the
number of steps in the treatment but cannot replace wastewater treatment
totally, as discharge of effluents should always require at least mechanical
treatment to eliminate suspended matter. Diversion has often been used with a
positive effect when eutrophication of a lake has been the dominant problem.
Canalization, either to the sea or to the lake outlet, has been used as a solution in
many cases of eutrophication.
If canalization is a significant part of the overall cost of handling wastewater, it
might often turn out to be both a better and cheaper solution to have smaller
treatment units with individual discharge points. Although diversion is not
considered an ecotechnological method based on sound ecological principles, a
number of successful applications of diversion has been reported in the
limnological literature. The most frequently cited case of wastewater diversion is
probably the restoration of Lake Washington in Seattle, Washington. Wastewater
was diverted from the lake to coastal Puget Sound in the 1960s, resulting in
immediate improvement in Lake Washington. If diversion is accompanied by
adding low-nutrient water from other sources, the recovery of the lake will, of
course, take place faster.
Removal of superficial sediment
Sediment removal can be used to support the recovery process of very eutrophic
lakes and of areas contaminated by toxic substances. This method can be applied
in small ecosystems only with great care. Sediments have a high concentration of
nutrients and many toxic substances, including trace metals. If a wastewater
treatment scheme is initiated, the storage of nutrients and toxic substances in the
sediment might prevent recovery of the ecosystem due to exchange processes
between sediment and water. Anaerobic conditions might even accelerate these
exchange processes; this is often observed for phosphorus, as iron(III) phosphate
reacts with sulfide and forms iron(II) sulfide by release of phosphate. The
amount of pollutants stored in the sediment is often significant, as it reflects the
discharge of untreated wastewater for the period prior to the introduction of a
treatment scheme. Thus, even though the retention time of the water is moderate,
it might still take a very long time for the ecosystem to recover.
Perhaps the best known case of removal of superficial sediment occurred in Lake
Trummen in Sweden, where 40 cm of the superficial sediment was removed. The
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177
transparency of the lake improved considerably, but it decreased again due to
the phosphorus in overflows from rainwater basins. Treatment of the overflow
after the removal of superficial sediment might give a better result.
Uprooting and removal of macrophytes
Uprooting and removal of macrophytes has been widely used in streams and to a
certain extent in reservoirs. The method can, in principle, be used wherever
macrophytes are a significant result of eutrophication. A mass balance should
always be set up to evaluate the significance of the method compared with the
total nutrient input. Collection of the plant fragments should be considered
under all circumstances. Simultaneous removal of nutrients from the effluent
should also be considered.
Coverage of sediment by an inert material
Covering sediment with inert material is an alternative to removal of superficial
sediment. The idea is to prevent the exchange of nutrients (or perhaps, toxic
substances) between sediment and water. Polyethylene, polypropylene,
fiberglass screen, or clay is used to cover the sediment surface. The general
applicability of the method is limited due to the high cost, even though it might
be more moderate in cost than removal of superficial sediment. It has been used
in only a few cases, and a more general evaluation of the method is still lacking.
Siphoning hypolimnetic water from reservoirs
In reservoirs or large ponds, this approach is feasible for reducing the causes of
epilimnetic eutrophication and can be used over a longer period and thereby
gives a pronounced overall effect. The effect depends on a significant difference
between the nutrient concentrations in the epilimnion and the hypolimnion,
which is often the case if the lake or reservoir has a pronounced thermocline. As
hypolimnetic water is colder and poorer in oxygen, the thermocline will move
downward and the possibility of anaerobic zones will be reduced. This might
have an indirect effect on the release of nutrients from the sediment. If there are
lakes or reservoirs downstream, the method cannot be used, as it removes but
does not solve the problem. A possibility in such cases would be to remove
phosphorus from the hypolimnetic water before it is discharged downstream.
The low concentration of phosphorus in hypolimnetic water (perhaps 0.5 to 1.0
mg L-1) compared with wastewater makes it almost impossible to apply chemical
precipitation.
Several lakes have been restored by this method, mainly in Austria, Slovenia,
and Switzerland, with a significant decrease in the phosphorus concentration as
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178
a result. Generally, the decline in total phosphorus concentration in the
epilimnion is proportional to the amount of total phosphorus removed by
siphoning and to the time the process has been used.
Flocculation of phosphorus
Either aluminum sulfate or iron (III) chloride can be added to lakes or reservoirs
to stimulate the flocculation and subsequent settling of phosphorus from surface
waters. Calcium hydroxide cannot be used, even though it is an excellent
precipitant for wastewater, as its effect is pH-dependent and a pH of 9.5 or
higher is required. The method is not generally recommended, as (1) it is not
certain that all flocs will settle and thereby incorporate the phosphorus in the
sediment, and (2) the phosphorus might again be released from the sediment at a
later stage.
Water circulation and aeration
Circulation of water can be used to break down the thermocline. This might
prevent the formation of anaerobic zones, and thereby the release of phosphorus
from sediment. Aeration of lakes and reservoirs is a more direct way to prevent
anaerobic conditions from occurring. Aeration of highly polluted rivers and
streams has also been used to avoid anaerobic conditions. In the Danish Lake
Hald, pure oxygen has been used instead of air. The water quality of the lake has
been improved permanently since the oxygenation started. In most cases,
however, the effect was not very great nor as permanent as with other
techniques, such as siphoning of hypolimnetic water.
Hydrologic regulation
Regulation of hydrology has been used extensively to prevent floods. More
recently, it has also been considered as a workable method to change the ecology
of lakes, reservoirs, and wetlands. If the retention time in a lake or a reservoir is
reduced with the same annual input of nutrients, eutrophication will decrease
due to decreased nutrient concentrations. The role of the depth, which can be
regulated by use of a dam, is more complex. Increased depth has a positive effect
on the reduction of eutrophication, but if the retention time is increased
simultaneously, the overall effect cannot generally be quantified without the use
of a model. The productivity of wetlands is highly dependent on the water level,
which makes it highly feasible to control a wetland ecosystem by this method.
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179
Fertilizer control
Controlling high nutrient water from even getting into a lake or reservoir is, of
course, the best action to prevent signs of eutrophication. Fertilizer control can be
used in agriculture and forestry to reduce nutrient loss to the environment.
Utilization of nutrients by plants depends on a number of factors [temperature,
humidity of soil, composition, growth rate of plant (which again depends on a
number of factors), chemical speciation of nutrients, etc.].
The occurrence of cyanophyte blooms to a great extent determines the N: P ratio
in the lake water. If the ratio is less than 5, at least 50 percent of the blooms are in
the form of cyanophytes. By very low ratios (e.g., less than 2), an almost 100
percent cyanophyte bloom may be observed. Adjusting the ratio is possible to a
certain extent, as the main source of phosphorus is primarily wastewater. The
phosphorus concentration in treated wastewater can easily be reduced by
chemical precipitation to 1 mg L-1 and even to 0.1 mg L-1. To avoid cyanophyte
blooms, it is important to utilize all the possibilities, for example, fertilizer
control, wastewater treatment, and various restoration methods to obtain the
right N: P ratio, which means > 7.
Calcium hydroxide neutralization
Calcium hydroxide is used widely to neutralize low pH values in streams and
lakes in areas where acidic rain has a significant impact. Sweden spends about
$100 million per year to neutralize acid in streams and lakes.
Algaecides
Chemicals, such as, various copper salts (e.g., copper sulfate) were previously
used widely in relatively small lakes, but are now rarely used due to the general
toxicity of copper, which accumulates in the sediment and can thereby
contaminate a lake for a very long time. The effect of copper on algae varies
substantially from species to species. Blue-green algae are generally most
sensitive to copper ions. Mitsch and Kaltenborn (1980) performed in situ
measurements of metabolism in the euphotic zone of an Illinois lake. Although
few differences were seen in a treated lake compared to a control lake, during
one period about a week after CuSO4 treatments, gross primary productivity
appeared to be depressed. However, by 10 to 14 days after the treatment, the
effects of the treatment on metabolism had disappeared. Obviously copper gets
accumulated in living organisms and there is strong possibility of
biomagnifications in animals at higher trophic levels.
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180
Shoreline vegetation
Shading by use of trees at the shoreline is a cost-effective method that can give an
acceptable result for small lakes, due to their low area/circumference ratio. It is
relatively ineffective in restoring large lakes because of the smaller edge/area
ratio.
Biomanipulation
Biomanipulation can be used as a method of lake restoration if the phosphorus
concentration ranges from about 50 to 150 g L-1, depending on the lake. In this
range two ecological structures are possible. When the phosphorus concentration
initially is low and increases, zooplankton are able to maintain a relatively low
phytoplankton concentration by grazing. carnivorous fish are also able to
maintain a low concentration of planktivorous fish, which implies relatively low
predation on zooplankton. At a certain phosphorus concentration (about 120 to
150 g L-1), zooplankton is no longer able to control the phytoplankton
concentration by grazing, and as carnivorous fish hunt by sight and the turbidity
increases, planktivorous fish become more abundant, which involves more
pronounced predation on zooplankton).
There are two possible ecological structures in the phosphorus range of
approximately 50 to 150 g L-1. Biomanipulation (Giussani and Galanti 1995) can
be used in this range to make a "shortcut" by removal of planktivorous fish and
release of carnivorous fish. If biomanipulation is used at above 150 g-P L-1,
some intermediate improvement of the water quality would be necessary, but
the lake will sooner or later get an ecological structure corresponding to the high
phosphorus concentration (i.e., a structure controlled by phytoplankton and
planktivorous fish). Biomanipulation is a relatively cheap and effective method
provided that it is applied in the phosphorus range where two ecological
structures are possible.
There are a number of cases where biomanipulation has been successful, but only
if the phosphorus loading was reduced simultaneously with total phosphorus
concentrations were made below 150 g L-1. Benndorf (1990) mentioned that
consistent response to biomanipulation can only be foreseen with a loading of
less than about 0.6 to 0.8 g P m-2 yr-1.
The Chinese grass carp or white amur (Ctenopharyngodon idella val) feeds
primarily on submerged plants. It also feeds on small floating plants. It thrives
best under cool waters although it tolerates warm waters. The small fish
consume vegetation several times of their body weight. For every one gram
increase in fish weight it needs to consume 48 g Hydrilla. About 75 fish can
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181
consume a vegetation of one hectare. White amur is now increasingly used for
clearing aquatic vegetation in India. Among the indigenous species of fish
Puntius pulchellus showed great promise (Devraj and Manissery 1979). It was
estimated that 10,000 fingerlings of this fish (each weighing 10 to 14 g) consume
Lemma and Hydrilla weighing 25 to 50 kg/day and 9 to 18 tons of aquatic weeds
per year.
The other fish species found useful for aquatic weed control are Tilapia (T. zilli
and T. quineensis), silver carp (Hypopthalmichthys molitrix), silver dollarfish
(Metynnis roosvelti val.), common carp (Cyprinus carpio L), gold fish (Carassius
auratus), etc.
Among the various diseases of water hyacinth, thread blight caused by
Marasmiellus inoderma (Berk), Singh, and a disease caused by Alternaria eichhorniae
are among the potential biological agents for controlling the growth of this weed.
The fungi which showed promise against water hyacinth are Cercospora rodmanii,
Acromonium zonatum and Ureds eichhornia. The insects found effective are flea
beetle (Agasiches hydrophilla) on water hyacinth and Salvinia, and thrips
(Amgnotthrips andersoni) and moth (Vogtia mallloi) on the weeds. In South
America, a grasshopper, Paulinia acuminate De Geer attacks Salvinia spp. and
Azolla spp.)
In India, an aquatic snail Limnaea acuminata has been observed to be a good
biological agent for control of Salvinia (Ittyavarah et al. 1979).
Location of treatment
Attempt should be made to estimate the nutrient load that the wetland can
sustain and accordingly identify the treatment options for implementation at
desired locations.
Desired nutrient load
Determination of the ‘acceptable’ nutrient load to a lake or reservoir is
increasingly being accomplished through the determination of total maximum
daily loads, using a process known as waste-load allocation. In this process, all
the possible nutrient sources (point and non-point) in a drainage basin are
identified and quantified. This is either by direct measurement or by prescribed
estimation techniques. One example of the latter is regionally relevant unit area
loads (sometimes termed nutrient export coefficients; see Rast and Lee 1983).
This information is then compared with the maximum permissible nutrient load
that will still allow achievement of the ‘desired’ trophic state in a downstream
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lake or reservoir. The term ‘desired’ is based on the trophic condition which
allows a specific human-designed water use(s) to be achieved. If the estimated
nutrient load exceeds the level conducive to a desired water use(s), the required
reduction in the nutrient load to achieve the desired tropic state can be
calculated.
Point sources
Oligotrophic waters often have an N/P ratio greater than or equal to 10, which
means that phosphorus is less abundant than nitrogen relative to the needs of
phytoplankton. If sewage is discharged into the lake, the ratio will decrease,
since the N: P ratio for municipal wastewater is about 3:1, and consequently,
nitrogen will be less abundant than phosphorus relative to the needs of
phytoplankton. Municipal wastewater typically contains 30 mg-N L-1 and 10 mgP L-1. In this situation, however, the best remedy for excessive algal growth is not
necessarily the removal of nitrogen from the sewage, because the mass balance
might show that nitrogen-fixing algae would produce an uncontrollable input of
nitrogen into the lake. Efficient eutrophication control methods can be managed
by eliminating the root-cause / the after effects and the nutrients from polluting
sources (drainage etc.) to check plant and phytoplankton growth into the water
bodies (Lee et al. 1980, Bern Hardt 1981, Rast and Holland 1988, Ryding and Rast
1989). Eutrophication control measures, at least in developed countries, have
focused primarily on the reduction of the external phosphate load to water
bodies. As noted by Ryding and Rast (1989), this is generally thought to be the
most effective, long-term measure for attempting to control cultural
eutrophication. This is typically achieved by removing phosphate from
wastewaters at municipal wastewater treatment plants, via its precipitation from
the wastewater before its release in effluents (usually termed tertiary treatment).
In extreme cases, phosphorus removal can also be applied directly to a lake or
reservoir by applying aluminium or iron salts, or in some instances calcium salts,
directly to the water column (although trivalent cations are generally more
effective in removing phosphorus from the water column). Numerous case
studies from throughout the world illustrating phosphorus removal from
wastewater effluents and/or water
bodies are given in Dunst et al. (1974) and Ryding and Rast (1989).
Non-point sources
The control of nutrients from non-point sources in a drainage basin has lagged
behind the control of nutrients from point sources, both in terms of available
technologies and the legal requirements for implementation. Available
technologies for achieving control of urban non-point source nutrients, metals
and sediments have been reviewed by Stahre and Urbonas (1990). These
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technologies consist primarily of detention, retention and infiltration techniques.
They are based on a system of containment and release that parallels traditional
wastewater or flood control engineering. It relies primarily on the stilling of
storm waters to allow sedimentation of particulates and adsorbed contaminants.
The subsequently
produced supernatant can then either be discharged
downstream with reduced sediment and contaminant load, transferred to a
wastewater treatment facility for final ‘polishing’, or allowed to percolate into the
substratum.
In-lake control
In-lake control of eutrophication, via the application of alum or other multivalent
cation salts directly to a water body, has been undertaken much less frequently
than the control of point sources. Such applications are costly, logistically
difficult and usually only partially effective, compared with reducing the
external nutrient load to a water-body. Examples of the application of this
method are given in Dunst et al. (1974). In fact, this control method is usually
attempted only when point and/or non-point source control measures applied in
a drainage basin have failed to achieve the desired reduction in trophic state, or
in situations in which the nutrient content in the bottom sediments of a lake or
reservoir has accumulated to the extent that internal loading from the sediments
is anticipated. In some water bodies, in fact, the internally generated phosphorus
load can equal or even exceed the external nutrient load (Ryding 1981, 1985).
Such extraordinary internal nutrient loads usually can be moderated by alum
treatments. However, the removal of the contaminated sediments, via dredging,
is the more common alternative. This is especially the case for shallow lakes, in
which alum addition to the water column cannot generally establish a cohesive
flocculent ‘blanket’ at the sediment-water interface to induce nutrient
sedimentation, due to wind-induced turbulent mixing (Ryding 1982).
Rast et al. (1989) has documented certain guidelines towards in-lake control
measures (Table 5) of eutrophication. Decomposing barley straw has successfully
been used to control the growth of cyanobacteria (Microcystis, Anabaena and
Aphanizomenon ) under field condition. The straw was effective for controlling
overall growth of cyanobacteria compared to controls (Rajabi et al. 2010).
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Table 5: Water quality problems treatable by in-lake restoration measures
Odours
Control meseare
Dredging
Hypolimnetic
aeration
Nutrient
inactivation
Altered circulation
Algicides
Biomanipulation
Dilution/flushing
Removal
of
hypolimnetic
waters
Lake drawdown
Harvesting
Covering
sediments
X
Fish
kills
Trophic
algae
X
X
Water quality problem
Interference Reduced
Excessive
with
commercial macrophyte
swimming
fishing
growth
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Excessive
algal
blooms
X
X
X
X
Poor
drinking
water
quality
X
X
X
X
Select success stories
Numerous examples of the assessment and control of eutrophication exist,
although most are overwhelmingly from lakes situated in the northern
hemisphere (Chapman 1992). Perhaps most well-known (and well-documented)
is the case of Lake Washington (USA). This lake rapidly eutrophied in the postSecond World War years as a result of the discharge of wastewater from several
neighbouring municipalities (Edmondson et al. 1956). Subsequent installation of
wastewater treatment plants and the diversion of wastewaters away from the
lake resulted in nearly complete recovery of the water body to its former
(natural) state (Edmondson and Lehman 1981). In contrast, Lake Shagawa (USA)
is probably the antithesis of successful eutrophication management. This lake
also received municipal wastewater, which was subsequently treated to tertiary
standards using a calcium carbonate flocculation technique (Larsen et al. 1979).
Unlike Lake Washington, however, Shagawa Lake failed to respond as predicted
and remained eutrophic. Further investigation determined that the lake was
subject to extremely intense internal loading from anoxic hypolimnetic
sediments. Owing to its shallow nature, much of the nutrient released from the
sediments was mixed into the lake in sufficient amounts to continue to fuel
nuisance levels of aquatic plant and algal growth (Larsen et al. 1981). Although
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185
the reduction in the external load has resulted in some improvement in water
quality, the complete recovery of this lake is expected to take close to a century as
a result of continued leaching of phosphorus from the sediments (Chapra and
Canale 1991).
Blue-green algae dominated the open waters of Lake Victoria during an algal
bloom between February and August 1986. Microcystis sp. accounted for more
than 90% of the bloom organisms with algal counts reaching a high density of
34,000 colonies per ml. Secchi disc visibilities and the 1 % light penetration were
reduced to 0–2 m and 1 m, respectively. The release of nutrients from river
inflows, from upwelling and from sediments into the euphotic zone, coupled
with high temperatures, produced the observed blooms. The blooms
subsequently declined as a result of physical flushing, temperature reduction
associated with the rainy season and nutrient exhaustion (Ochumba and Kibaara
1989).
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Journal of Today’s Biological Sciences: Research & Review (JTBSRR)
ISSN 2320-1444 (Online)
JTBSRR
Vol.1, Issue 1, page 191-196, December 30, 2012
Available on: www.jtbsrr.in
Nanomedicine “A Future Medicine ”
Gurudutt Joshi
Surat medical Institute of medical education and Research (S.M.I.M.E.R.), Surat,
Gujarat, E-mail: joshigurudutt@yahoo.com
Abstract
Nano, as in nanotechnology, nanobiology, nanomedicine, refers to phenomena at the
nanometer or molecular level. A nanometer (nm) is one-billionth of 1 m, which is an
extremely small linear measurement. To put this in perspective, a typical human hair is
≈100,000 nm in diameter. An adenovirus is 90-nm wide.
Evolution of this technology has revolutionalized and opened many new gateways in
medical research, therapeutic applications and newer perspectives. This article is a
compilation of references and is written with a view to provide a basic idea and to expose
scientific personnel to this promising and advance technology in combination with
medicine.
Key Words: Nanomedicine, medical research, nanosystem, nanodevices
Introduction
Nanomedicine has been defined as the monitoring, repair, construction and
control of human biological systems at the molecular levels using engineered
nanodevices and nanostructures [1]. It is the process of diagnosing, treating, and
preventing disease and traumatic injury, of relieving pain, and of preserving and
improving human health, using molecular tools and molecular knowledge of
the human body [2]. Relevant processes of living organisms occur basically at
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nanometer scale, elementary biological units like DNA, proteins or cell
membranes are of this dimension. [3]
In the relatively near term, nanomedicine can address many important medical
problems by using nanoscale-structured materials and simple nanodevices that
can be manufactured today, including the interaction of nanostructured
materials with biological systems. Nanomedicine represents medicine at the
nanotechnology scale with typical examples being nanoparticles and
nanodevices. [4]
Medical Nanomaterials and Nanodevices
Nanopores
One of the simplest medical nanomaterials is a surface perforated with holes, or
Nanopores. It is one of the earliest therapeutically useful nanodevices employing
bulk micromachining to fabricate tiny cell containing chambers filled with single
silicon wafers.[5]
These pores are large enough to allow small molecules such as oxygen, glucose,
and insulin to pass, but are small enough to impede the passage of much larger
immune system molecules such as immunoglobulins and graft-borne virus
particles. Nanopore-based DNA sequencing devices could allow per-pore read
rates potentially up to 1000 bases per second, possibly eventually providing a
low-cost high-throughput method for very rapid genome sequencing.[6]
Artificial Binding Sites and Molecular Imprinting
Molecular imprinting is an existing technique in which a cocktail of
functionalized monomers interacts reversibly with a target molecule using only
noncovalent forces.[7] Molecularly imprinted polymers could be medically useful
in clinical applications such as controlled drug release, drug monitoring devices,
quick biochemical separations and assays, recognition elements in biosensors
and chemosensors, and biological and receptor mimics including artificial
antibodies. [8].
Quantum Dots and Nanocrystals
These dots are tiny particles measuring only a few nanometers across, about the
same size as a protein molecule or a short sequence of DNA. Quantum dots are
useful for studying genes, proteins and drug targets in single cells, tissue
specimens, and living animals.[9][10] Quantum Dots are being investigated as
chemical sensors[11] for cancer cell detection ,gene expression studies[12] ,gene
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mapping [13] ,immunocytochemical probes
screening.[15]
[14]
,medical diagnostics and drug
Fullerenes and Nanotubes
Soluble derivatives of fullerenes such as C60 have shown great utility as
pharmaceutical agents. Fullerene compounds may serve as antiviral agents most
notably against HIV photodynamic antitumor and anticancer therapies,
antioxidants and anti-apoptosis agents which may include treatments for
amyotrophic lateral sclerosis and Parkinson’s disease.[15]
Nanoshells and Magnetic Nanoprobes
They are developed as a platform for nanoscale drug delivery . They are slightly
larger than Fullerenes.Nanoshells are embedded in a drug-containing tumor
targeted hydrogel polymer and injected into the body.They circulate in the body
,when they reach target , the polymer gets melted by laser and drug is released
at specific site , this technique can be useful in Diabetes to release Insulin and
antibodies targeted against specific tumor cells.
Dendrimers and Dendrimer-Based Devices
Dendrimers are tree-shaped synthetic molecules with a regular branching
structure emanating outward from a core that form nanometer by nanometer,
with the number of synthetic steps. Upon encountering a living cell, dendrimers
of a certain size by endocytosis release DNA which migrates to the nucleus
where it becomes part of the cell’s genome.
Radio-Controlled Biomolecules
Tiny Radiofrequency antennas attached to nano sized gold nanocrystals ,by
exposing them to radiofrequency magnetic field can be utilized ,to separate
double stranded DNA and by remote electronic switching of these antennas to
turn genes off and on.
Researchers from the University of Missouri (MO, USA) have designed a new
treatment for prostate tumors using radioactive gold nanoparticles (AuNPs) and
epigallocatechin-gallate (EGCg),a compound found in tea leaves. The team, has
synthesized biocompatible nanoparticles (NPs) utilizing the redox chemistry of
EGCg, which converts gold salt into AuNPs.[19]
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Nanorobots
Nanoscale devices such as artificial R.B.C.or ‘Respirocyte” and artificial
mechanical “W.B.C.” called “Microbivore”. Respirocyte can be used to carry more
oxygen . Primary medical applications of respirocytes would include
transfusable blood substitution; partial treatment for anemia, lung disorders,
enhancement of cardiovascular/neurovascular procedures, tumour therapies
and diagnostics, prevention of asphyxia, artificial breathing. Microbivore, has as
its primary function to destroy microbiological pathogens found in the human
bloodstream using a digest and discharge protocol.
The Microbivore would be 80 times more efficient as phagocytic agents and
would have far larger maximum lifetime capacity for phagocytosis than natural
white blood cells.Microbivores would fully eliminate septicemic infections in
minutes to hours.[18]
Nanozymes
The nanoparticle-based enzyme system . RNA silencing is an integral process in
the normal functioning of cells, carried out in part by the RNA-induced silencing
complex (RISC). This process can be synthetically exploited in the treatment of
HCV infection, since if a key HCV RNA sequence can be targeted and
destroyed.[17]
Nanolipogels
In a recent Nature Materials paper, a group of scientists from Yale University
(CT, USA) has described the development of nanoscale liposomal polymeric gels
termed ‘nanolipogels’, which have shown efficacy in mice models for the
immunotherapeutic treatment of metastatic melanoma.[20]
Many such novel nanoparticles and nanodevices are expected to be used, with an
enormous positive impact on human health. The vision is to improve health by
enhancing the efficacy and safety of nanosystems and nanodevices.
References
P. Webster 2005..World nanobiotechnology market .Frost and Sullivan
Freitas A.R. 2005. Current status of Nanomedicine and Medical nanorobotics,
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Logothetidis S. 2006. Nanotechnology in Medicine:The Medicine of Tomorrow
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Desai T, Chu W, Tu J, Beattie G, Hayek A and Ferrari M. 1998. Microfabricated
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Deamer D and Akeson M.2000. Nanopores and nucleic acids: prospects for
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Shi H and Ratner B. 2000.Template recognition of protein imprinted polymer
surfaces, J. Biomed. Mater. Res. 49: 1-11.
Oshikawa M . 2001. Molecularly imprinted polymeric membranes Bioseparation
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Bruggemann O. 2002. Molecularly imprinted materials –receptors more durable
than nature can provide Adv. Biochem. Eng. Biotechnol. 76, 127-163 .
Wu X, Liu H, Liu J, Haley K, Treadway J, Larson J, Ge N, Peale F. 2003.
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Multiplexed Optical Coding of Biomolecules Nat. Biotechnol.19: 631 .
Gerion D, Parak J, Williams S, Zanchet D, Micheel C, Alivisatos A, Larabel C.
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Schinazi R, Sijbesma R, Srdano G, Hill C, and Wudl F.1993 . Synthesis and
virucidal activity of a water-soluble, configurationally stable, derivatized C60
fulleren Antimicrob. Agents Chemother. 37:1707-1710.
Freitas A.R. 2005. “Microbivores: Artificial Mechanical Phagocytes Using Digest
and Discharge Protocol,” j evolu tech .14(4):56-106
Wang Z, Liub H, Yang S.2012 .Nanoparticle-based artificial RNA silencing
machinery for antiviral therapy. Proc. Natl Acad. Sci. USA. 10:1073
Shukla R, Chanda N, Zambre A .2012 . Laminin receptor specific therapeutic
gold nanoparticles (198AuNP-EGCg) show efficacy in treating prostate cancer.
Proc. Natl Acad. Sci. USA .109(31): 12426–12431.
Park J, Wrzesinski S, Stern E . 2012 .Combination delivery of TGF-b inhibitor and
IL-2 by nanoscale liposomal polymeric gels enhances tumour immunotherapy.
Nat. Mater. 11:895
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Journal of Today’s Biological Sciences: Research & Review (JTBSRR)
ISSN 2320-1444 (Online)
JTBSRR
Vol.1, Issue 1, page 197-209, December 30, 2012
Available on: www.jtbsrr.in
Biofuels: Potential, Current Issues and Future
Trends
Pratap V. Naikwade*, Ranjit P. Bansode* & Sagar T. Sankpal1
*Department
of Botany, Nya. Tyatyasahe Aathalye Arts, Ved. S. R. Sapre Commerce and
Vid. Dadasaheb Pitre Science College, Devrukh-415804, Maharashtra, India.
*Department of Chemistry, Nya. Tyatyasahe Aathalye Arts, Ved. S. R. Sapre Commerce
and Vid. Dadasaheb Pitre Science College, Devrukh-415804, Maharashtra, India.
E- mail of corresponding author: naikwade.pratap@gmail.com
Abstract
Biofuels are renewable liquid fuels coming from biological raw material and have been
proved to be good substitutes for oil in the transportation sector. Biofuels such as ethanol
and biodiesel are gaining worldwide acceptance as a solution to energy security, reducing
imports and improving agricultural economy. Bio fuels are to reduce negative
environmental effects through lower emissions and climatic impacts. Local production of
bio energy is projected to have a broad range of positive economic, social and
environmental implications. India is sixth in the world in energy demand accounting for
3.5% of world commercial energy consumption. Indian petrol reserves are expected to
last for another 20 years plus. There is more potential for biodiesel to be produced on a
smaller scale, it requires the least economies of scale and has the greatest potential to
benefit small farmers and rural development. Europe, Brazil, China and India each have
targets to replace 5% to 20% of total diesel with biodiesel. It is possible that Biodiesel
could represent as much as 20% of all on-road diesel used in Brazil, Europe, China and
India by the year 2020 with the pursuit of second generation, non-food feed stocks.
Present study reveals potential of biofuel as solution of environmental problems, energy
security and many other issues. It also focuses on current issues and bright future trends
of biofuel as it is getting significant importance globally due to growing world energy
demand, the insecurity of long-term supply and the consequences of fossil fuel use for
climate change.
Key words: biofuel, environment, future, global market, potential.
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Introduction
Biofuels are renewable liquid fuels coming from biological raw material and
have been proved to be good substitutes for oil in the transportation sector. As
such biofuels – ethanol and biodiesel- are gaining worldwide acceptance as a
solution to environmental problems, energy security, reducing imports, rural
employment and improving agricultural economy (Wilson et al.,2005). Biofuel as
an alternative fuel is becoming increasingly important due to diminishing
petroleum reserves and the environmental consequences of exhaust gases from
petroleum-fuelled engines. Biodiesel, which is made from renewable sources,
consists of the simple alkyl esters of fatty acids (Wilson et al., 2005). As a future
prospective fuel, biodiesel has to compete economically with petroleum diesel
fuels (Ma and Hanna, 1999). One way of reducing the biodiesel production costs
is to use the less expensive feedstock containing fatty acids such as inedible oils,
animal fats, waste food oil and byproducts of the refining vegetables oils
(Veljkovic et al., 2006). The availability and sustainability of sufficient supplies of
less expensive feedstock will be a crucial determinant delivering a competitive
biodiesel to the commercials filling stations. Fortunately, inedible vegetable oils,
mostly produced by seed-bearing trees and shrubs can provide an alternative
(Shay, 1993).
Biofuels are solution to issues such as sustainable development, energy security
and a reduction of greenhouse gas emissions etc. Biodiesel is a methyl or ethyl
ester of fatty acid made from renewable biological resources such as vegetable
oils recycled waste vegetable oil and animal fats (Demirbas, 2000; Kinney and
Clemente, 2005). The use of vegetable oils as alternative fuels has been around
since 1900 when the inventor of the diesel engine Rudolph Diesel first tested
peanut oil in his compression ignition engine (Shay, 1993). However, due to
cheap petroleum products such non-conventional fuels never took off until
recently. Biodiesel derived from surplus edible oils like soybean, sunflower and
rapeseed oils is already being used in USA and Europe to reduce air pollution, to
reduce dependence on depleting fossil fuel localised in specific regions of the
world and increases in crude oil prices (Berchmans and Hirata, 2008; Foidl et al.,
1996).
The main commodity sources for bio-diesel in India can be non-edible oils
obtained from plant species such as Jatropha curcas, Pongamia pinnata, Calophyllum
inophyllum, Hevca brasiliensis etc. Bio-diesel contains no petroleum, but it can be
blended at any level with petroleum diesel to create a bio-diesel blend or can be
used in its pure form. The use of bio-diesel in conventional diesel engines results
in substantial reduction of un-burnt hydrocarbons, carbon monoxide and
particulate matters. Bio-diesel is considered clean fuel since it has almost no
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sulphur, no aromatics and has about 10 % built- in oxygen, which helps it to burn
fully (Sarin et al., 2007).
The use of edible oil to produce biodiesel in India and other developing countries
is not feasible in view of a huge gap between demand and supply of such oils in
the developing world. In Asia and Africa, which are mostly net importers of
vegetable oil, Jatropha curcas has been recognised as new energy crop for the
countries to grow their own renewable energy source with many promising
benefits (Pramanik, 2003).
Advantages of biodiesel
1. Produced from sustainable / renewable biological sources
2. Ecofriendly and oxygenated fuel
3. Biodiesel has higher flash point for safety
4. It will provide income to rural community
5. Non toxic and safety to handle
6. Fuel properties similar to the conventional fuel
7. Blending of biodiesel with diesel fuel increases engine efficiency.
8. Used in existing unmodified diesel engines
9. Reduce expenditure on oil imports
10. Biodiesel degrades four times faster than diesel.
11. Sulphur free, less CO, HC, particulate matter and aromatic compounds
emissions
12. Biodiesel is carbon neutral because the balance between the amount of
CO2 emissions and the amount of CO2 absorbed by the plants producing
vegetable oil is equal.
Current trends
The first significant large-scale push for the production and use of biofuels
occurred in Brazil and the United States, as a response to the 1973 oil export
embargo imposed by the Arab members of OPEC (Organization of the Petroleum
Exporting Countries) against Japan, the United States and Western European
countries (Walter,2006). The export restriction resulted in a dramatic increase of
oil prices, from $3 to $12 per barrel. The United States invested in biofuels as a
way to address the fuel shortages induced by the embargo and to reduce
dependence on imported oil. Brazil’s objective was to reduce the pressure on its
balance of payments due to the rising cost of fossil fuel imports. At present
biofuels are once again at the centre stage of the debate on energy due to high
and volatile oil prices and oil supply instability. In addition, a strong global
consensus nowadays advocates for reductions in GHG emissions as a crucial step
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to combat rising global temperatures. Governments seeking to curb emissions
are now promoting biofuels because of their potentially cleaner emissions profile
as compared to fossil fuels (Tyner and Taheripour,2007).
Biodiesel is formed chemically by trans-esterification of vegetable oils obtained
by physical and/or chemical separation from oilseed crops (Connor and
Hernandez, 2008). Bioethanol is produced by fermentation of glucose and
fructose, which are easily obtained from sucrose crops such as sugarcane or
sugar beet. Bioethanol can also be made from cellulose, also but with a different
chemical bonding to starch (Badger, 2002). Currently, raw material used for
producing ethanol varies from sugar, cereals, sugar beet to molasses in India.
Brazil uses ethanol as 100 % fuel in about 20 per cent of vehicles and 25% blend
with gasoline in the rest of the vehicles. USA uses 10 % ethanol-gasoline blends
whereas a 5% blend is used in Sweden. Australia uses 10% ethanol- gasoline
blend. Use of 5% ethanol- gasoline blend is already approved by BIS and is in
progressive state of implementation in the country. Biodiesel and bioethanol are
produced from a small range of crops provide essentially all renewable liquid
transport fuels. Other liquid fuels, such as synthetic gasoline and diesel, play
minor roles. However, non-liquid transport fuels, including biogas, hydrogen,
and electricity, can also be produced from biomass.
Maize (USA), and sugarcane (Brazil) provide the bulk of feedstock for bioethanol
production, currently at 1090 PJ per year or 52 billion liters (FAO, 2008). Other
crops (e.g. sugar beet, wheat, barley, cassava, potato, and rice) are also used in
various countries. The dominant crop for biodiesel production, currently 340 PJ
per year or 10 billion liters (FAO, 2008), is rapeseed (i.e. canola) (EU), although
oil palm (Malaysia and Indonesia), soybean (USA and Brazil), and sunflower
(Eastern Europe) are gaining importance. (Liska and Cassman, 2008), Peanut,
cotton, sesame, and coconut are also used as feedstock. Sunflower and rapeseed
are the raw materials used in Europe whereas soybean is used in USA. Thailand
uses palm oil, Ireland uses frying oil and animal fats. It is proposed to use nonedible oil for making biodiesel. The current installed production capacity will not
be sufficient to cover the demand induced (Espey, 1996). For almost all the
countries analyzed, there is a gap between the potential demand generated by
mandatory or voluntary blending targets and their production capacity.
Therefore additional production will be needed to fulfill the mandates and
reduce the pressure on biofuel prices.
Effect on food due to biofuel crop production
India has total geographical area of 328 million hectares out of which around 142
million hectares is used for agriculture. By 2030 Indian population is expected to
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rise to 1.5 billion from around 1.1 billion presently. To feed this much number
even with very conservative estimates will require around 185 Million hectares
of agricultural land with an assumption that the land productivity remains same.
Promotion of biofuels at the cost of rise of food products may have catastrophic
results on the Indian social equity and peace. For food security we should focus
on the increased use of waste land to promote environment friendly biofuels.
High speed diesel is the largest consumed petro-product in India on account of
better mileage, power and lower administered price compared to petrol (Kumar
et al., 2008a). Mass utilization of diesel in India imposes a threat to meeting the
future energy needs, if the unexpected volatilities in the price of petroleum
persists in future and government of India enforces oil marketing companies to
sell diesel at uncapped price. It’s demand is expected to raise up to 100 million
tones with an assumption of 6% per annum growth rate on very conservative
basis by 2020. With an approximate import dependency of 90%, energy security
favours the adoption of 20% blending by Jatropha biodiesel. Production of 31.4
million tonnes of Jatropha biodiesel i.e. is 20% of total diesel requirement will
require about 26 million hectares of land by 2020. When we are considering key
macro-indicators of India, it shows that with the growth in population, per capita
food intake and energy requirement has also increased. Increased population
may result in food vs fuel conflict. Apprehensions have been expressed that the
cultivators may willingly opt for such crops, which may be used for producing
biofuels and may be more remunerative in shorter terms. Farming process itself
is an energy intensive activity requiring power for irrigation, ploughing and
processing of farming produce.
Biofuel and green house gas policies
It is widely believed that the biofuels industry has an exclusive role in climate
policy because it represents a low-carbon alternative to fossil fuels. GHG (green
house gas) policies that create an emissions trading system such as the cap and
trade mechanism can also stimulate the production of biofuels by imposing a cap
on carbon emissions and allowing trade of emissions permits (allowances). In
practice, such a system creates a price for carbon, similarly to the imposition of a
tax on GHG emissions. There are two main approaches to create tradable
emissions reductions (Ellerman, 2000). The first is a cap and trade system in
which a central authority sets a limit or cap on the amount of a pollutant that can
be emitted. Second approach is a baseline and credit system. Polluters not under
an aggregate cap can create credits by reducing their emissions below a baseline
level of emissions. These credits can be purchased by polluters that are under a
regulatory limit.
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The main international agreement currently addressing GHG mitigation is the
Kyoto Protocol (United Nations Framework Convention on Climate Change
(UNFCCC), 1997). Under this agreement, 37 industrialized countries and the
European Community are committed to reduce their overall emissions of
greenhouse gases by at least 5 per cent below 1990 levels during the period 2008–
2012. Biofuel is considered a low-carbon emissions fuel, and therefore a biofuel
production project is a potential candidate for eligibility under the CDM or Joint
Implementation mechanisms of Kyoto Protocol. On the other hand even without
explicit GHG markets that allow for CO2 credits, the demand for biofuels is
likely to expand unless another low-carbon alternative in the transportation
sector emerges.
There are also supplementary benefits from carbon sequestration and emissions
reductions. Emissions reductions by fuel switching may reduce the emissions of
other air pollutants (Matus et al., 2008) and carbon sequestration may reduce soil
erosion and leaching agricultural chemicals, thereby reducing water pollution
(Marland et al., 2001). If biofuels replace row crops or severely degraded grazing
land, this could result in benefits in terms of reduced soil erosion or reduced use
of chemicals pesticides. However, sustained production of biofuels would likely
require fertilizer inputs, generating the negative externality of N2O emissions
from nitrogen fertilizer. The establishment of a carbon dioxide (CO2) price
creates incentives for the development of a global biofuels market either directly
through enticements to substitute biofuels for fossil fuel use in countries with
greenhouse gas (GHG) policy or indirectly through the Clean Development
Mechanism (CDM) of Kyoto protocol. So it is reasonable to assume that
bioenergy production will increase even in the absence of climate policy.
Opportunities for developing countries
In general, developing countries have a larger potential to produce biomass and
biofuel than industrialized countries due to better climate conditions and lower
labour costs. Due to this international trade in biofuels and feedstocks from
developing to developed countries is expected to increase with significant
positive implications for development. This puts them in an ideal position to
fully benefit from a new and dynamic sector of the world economy. Agriculture
is an ecosystem-based activity, the major factor determining agriculture’s
productive capacity is the natural endowment (De La Torre Ugarte, 2007). Global
biomass supply estimates from plantations range from 47 to 238 EJ/year, with
over 80 per cent coming from developing nations (Berndes et al., 2003). Fischer
and Schrattenhozer (2001) estimate that 34 per cent of global bioenergy could
come from developing nation plantations, with 687 Mha in Africa, 400 Mha in
Asia and 307 Mha in Latin America. Developing countries are also significant
sources of forest biomass, with Sorensen (1999) indicating that Latin American
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and sub- Saharan Africa are the greatest potential sources. The expansion of
biofuels is closely linked to the productive capacity of the agricultural sector and
to its ability to provide food, feed, fibre and energy feedstocks simultaneously. A
crucial determinant of the agricultural productive capacity of a country is given
by its endowment of natural resources, while investment in research and
development and in infrastructure has the ability to enhance this potential.
Innovative Biofuels
New and innovative biofuels are expected to contribute to efforts to reduce net
greenhouse gas emissions, improve energy security and aid development, For
development of new biofuels some properties are focused as it can be produced
without harming the environment or local populations, It will not cause negative
effect on food production, It will need minimal resources, such as water and
land, it can be processed efficiently to yield high-quality liquid biofuels; and
deliverable in sufficient quantities. Some of the new biofuels are lignocellulosic
biofuels, algal biofuels, electrofuels (use microoganisms typically bacteria to
directly utilize energy from electricity and do not need solar energy to grow or
produce biofuels), thermochemically generated fuels etc.
Lignocellulosic biofuels
Lignocellulosic biofuels use all of the plant instead of just the starch or sugary
parts. Residue products from arable food agriculture, such as straw, could be
used as feedstocks. In this way, food crop plants could become effectively dualuse, producing both food and fuel (Banerjee et al.,2010). A second option is to use
plants grown solely for the production of lignocellulosic biofuels, such as trees
and grasses (e.g. willow, poplar, switchgrass and miscanthus). In addition to the
greater utilisation of biomass compared with biofuels produced from food crops,
there is significant potential to improve feedstock characteristics such as yields,
water use, and pest and frost resistance using advanced plant breeding strategies
and genetic modification. However, technology in this field is mostly still at the
research and development stage. Moreover, lignocellulosic biofuels require more
sophisticated processing than current biofuels, and this is currently very costly
(Weng et al.,2008). However, given further technological advances, there are
options to improve efficiency and bring down costs significantly. An additional
issue with agricultural residues is their limited supply. As straw can be used to
provide organic amendment to soil to maintain good soil condition, some
suggest that a maximum of only 40 per cent of straw should be used in ethanol
production or other industrial purposes (Lafond et al.,2009)
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Algal biofuels
Algae constitute a diverse group of aquatic photosynthetic organisms that
produce an equally diverse range of chemicals, including an array of oils that can
be used to produce biodiesel, avoiding some of the technical challenges of
converting lignocellulose to liquid fuels. They do not require freshwater and can
be cultivated in wastewater or sea water, and it is expected that under optimal
conditions they will produce high yields. Algae can be cultivated in open ponds
or closed photobioreactors, or in hybrid systems (Chisti, 2007). Currently, the
production of algal biofuels is experimental, and costs are very high. There is
significant potential for improvements of feedstocks and processing, for example
using genetic modification or synthetic biology. The production of algal-based
biofuels (ABBs) sometimes referred to as third generation biofuels Using algal
oils produces algal biodiesel which is similar in chemical and physical properties
to diesel derived from fossil oil and which compares well with the international
biodiesel standard for biodiesel use in vehicles (Brennan and Owende, 2010). In
comparison with diesel, algal biodiesel is non-toxic and has reduced levels of
particulates, carbon monoxide, soot, hydrocarbons and sulphur oxides. It is also
cited as being more suitable for aviation use than first generation biodiesel,
having a low freezing point and high energy density (Greenwell et al.,2010).
Global market of biofuels
With the price of fossil oil surging above the historical mark of US$ 100 a barrel
in the year 2007/2008 the search for alternative energy sources has become more
urgent than ever. The global market for biodiesel is poised for explosive growth
in future years. Although Europe currently represents 80% of global biodiesel
consumption and production, the U.S. is now ramping up production at a faster
rate than Europe and Brazil is expected to surpass U.S. and European biodiesel
production by the year 2015. It is possible that Biodiesel could represent as much
as 20% of all on-road diesel used in Brazil, Europe, China and India by the year
2020 with the pursuit of second generation, non-food feed stocks. Biodiesel
demand and over-capacity in Europe, the US and Asia is driving investment in
the global trade of alternative feeds tocks. In China, India, Brazil and Europe,
economic and environmental security concerns are giving birth to new
government targets and incentives, aimed at reducing petroleum imports and
increasing the consumption and production of renewable fuels.
Europe, Brazil, China and India each have targets to replace 5% to 20% of total
diesel with biodiesel. If governments continue to aggressively pursue targets for
second generation fuels; and continue to promote research and development in
investment in alternative, non-food feedstocks such as grease tallow, Jatropha,
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Castor, algae, and renewable diesel, the prospects for biodiesel will be realized
faster than anticipated. It is estimated that the United States produces 44%, Brazil
41%, the European Union 13% and South East Asia 2% of the world‘s total
supply of biofuel, which is about 16Mtoe (million ton oil equivalent comprising
80% ethanol and 20% biodiesel) (Prota, 2007). The world biofuels market has
been growing at an accelerated pace in the last twenty years, and this trend is
expected to continue in the future.
International trade
The world market of biofuels has been steadily growing in the last years, with an
increasing number of countries participating in it for environmental and security
reasons. In 2002 world production of ethanol reached 21,841 million liters, while
biodiesel production was 1,503 million liters. This production not only provided
an alternative to fossil fuel, but it is also generated large number of employment
since biofuel production requires 100 times more workers per unit of energy
produced than fossil fuels. In 2002, the ethanol industry provided more than
200,000 jobs in the US and ½ million direct jobs in Brazil (IEA, 2004). In recent
years, Jatropha curcas has become the focus of large planting programmes in
several tropical countries on account of its potential as a bio-fuel crop with low
agro-ecological demands. Most of these are still in pilot stage of development,
together probably not exceeding 100,000 ha. India alone may have more than 10
million ha of small-scale and large plantation by 2030, mostly reclaimed
wastelands.
There are many crops that can be used for producing biodiesel, but the choice
normally depends on local availability, affordability and government incentives.
Both India and China have large Jatropha plantations under development. In
addition, China is investigating recycled cooking oil as an option. Since biofuels
can be produced from a diverse set of crops each country is adopting a strategy
that exploits the comparative advantages it holds in certain crops. India has
launched a National Mission on Biofuels, the main strategy of which has been to
promote Jatropha Curcas; a perennial shrub that bears non edible oil seeds that
can be used to produce biodiesel. Many biofuel crops are used for extraction of
biodiesel. In terms of the market size, the biodiesel industry reached 3,524
million liters in 2005, with Western Europe having the largest share of the
market. Although it is still the largest producer, market fragmentation has
decreased Western Europe‘s monopoly in the biodiesel market. Its share which
represented 95% of the market in 2000, had been reduced to approximately 80%
by 2005. This is accounted by new players, such as Asia, entering into the market.
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Conclusion
Use of biofuels has significant importance globally as the world addresses
changing patterns in energy supply and demand. Biofuels have potential to solve
issues as green house effects, carbon sequestration, growing world energy
demand, the insecurity of long-term supply and the consequences of fossil fuel.
Production of biofuels will help developing countries by reducing imports and
improving agricultural economy and subsequently poverty reduction. Many
countries are promoting the production and use of biofuels - energy extracted as
gas, liquid or oil from plants. Biofuel derived from food crops such as corn,
sugarcane, soybean, oil palm and sugarbeet has been on the rise in recent years.
The use of non food crops as Jatropha curcas, Pongamia pinnata etc. is also
promoted. In future innovative biofues such as lignocellulosic biofuels, algal
biofuels, electrofuels will have bright future. The world biofuel market has been
growing at an accelerated pace in the last twenty years and this trend is expected
to continue in the future due to increasing number of countries participating in it
for environmental and security reasons. Biofuels have the potential to meet more
than a quarter of world demand for transportation fuels by 2050. Social
awareness is needed on production and usage of biofuels as an alternative to
petroleum based fuels.
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