Volume 2, Issue 3 (2015) Tropical Plant Research

ISSN (E): 2349 1183
ISSN (P): 2349 9265

2(3): 168171, 2015
Research article
Morpho-taxonomy of Hydrodictyon reticulatum (L.) Lagerheim and

Pediastrum tetras var. tetraodon (Corda) Hansgirg,
Hooghly, West Bengal, India
Nilu Halder*
Department of Botany, Raja Peary Mohan College, Uttarpara-712258, Hooghly, West Bengal, India
*Corresponding Author: niluhalder1@gmail.com [Accepted: 25 September 2015]
Abstract: The present paper was communicated with the morpho-taxonomic descriptions of two
freshwater members viz. Hydrodictyon reticulatum (L.) Lagerheim and Pediastrum tetras var.
tetraodon (Corda) Hansgirg belonging to the family Hydrodictyaceae of the class Chlorophyceae.
The two taxa were collected from aquatic ecosystems in Hooghly district, West Bengal, India. The
limnological characteristics of the water bodies where they occurred were recorded. The above
stated taxa were new taxonomic reports from this district and Pediastrum tetras var. tetraodon
(Corda) Hansgirg was new report from West Bengal, India.
Keywords: Morpho-taxonomy - New report - Hydrodictyaceae - Limnology - West Bengal.
[Cite as: Halder N (2015) Morpho-taxonomy of Hydrodictyon reticulatum (L.) Lagerheim and Pediastrum
tetras var. tetraodon (Corda) Hansgirg, Hooghly, West Bengal, India. Tropical Plant Research 2(3): 168171]
INTRODUCTION
Algae are photoautotroph, replenish oxygen content in water, dominant primary producer and contribute
much to the productivity of freshwater ecosystems. On the other hand, physico-chemical parameters affect the
composition and diversity of algal flora in aquatic bodies. Therefore, morpho-taxonomic study for
documentation of algae and assessment of water quality are of utmost importance in taxonomic and ecological
investigation.
Hydrodictyon reticulatum known as "water net" is widely distributed in Asia, North Africa, Europe, America
and New Zealand. This alga is characteristic of the northern warm-temperate zone (Pocock 1960). It is a
macroscopic, free-floating and filamentous green alga that forms a closed, pentagonal or hexagonal net of
coenocytic cells in water bodies. At maturity, the alga turns into slightly yellowish colour and the coenocytic
cells are detached from each other or the coenobium is disintegrated and can form vegetative daughter nets.
Whereas, the genus Pediastrum was established by Meyen in 1829 and it is an interesting coenobial
chlorococcalean algal genus which grows commonly as planktonic form or in periphytic association (grows on
submerged plants or other water logged objects) of freshwater habitats. This is a microscopic green alga and
occurs commonly in natural freshwater bodies like ponds, lakes, moats, rivers and other aquatic reservoirs. This
alga was found generally in post monsoon season in the district Hooghly, West Bengal. It should be
mentionable that after the publication of the monograph "Chlorococcales" by Philipose (1967), several authors
added many algal taxa in the order Chlorococcales from the Indian sub-continent. Singh (1973), Patel & George
(1982), Pal & Santra (1984), Sharma et al. (1985), Pal et al. (1986), Banerjee & Santra (2001), Jena & Adhikary
(2007), Mallick & Keshri (2008, 2009) and Sau & Gupta (2008), Kumar et al. (2012), Rai & Misra (2012) and
Keshri & Mallick (2013) were some contributors who had worked earlier on the taxonomy of these algae.
MATERIALS AND METHODS

The algae had been collected in glass containers from different places viz. Tribeni (N 2299' E 8840'),
Chinsurah (N 2290' E 8839'), Khamargachi (N 2305' E 8843'), Dumurdaha (N 2303' E 8843'), Magra (N
2312' E 8828'), Kamarkundu (N 2383' E 8820') and Hooghly river at Kalichar Ghat (N 2303' E 8826') of
Hooghly district, West Bengal. Detailed study was made by examining specimens under Olympus microscope
(Model-CH20i) for identification of species. Samples were preserved in 4% formalin. Identifications of these
www.tropicalplantresearch.com 168
Received: 16 June 2015 Published online: 31 October 2015
Halder (2015) 2(3): 168171
.
taxa were accomplished with the help of authentic literatures viz. Phlipose (1967), Singh (1973), Coffey& Miller
(1988), Sharma et al. (1985), Kumar et al. (2012) and Rai & Misra (2012).The pH and temperature of the water
bodies were determined at the site immediately after collection with the help of portable pH meter (Model No.
PP9046 Philips, India) and Zeals mercury thermometers (UK).The other limnological parameters such as
nitrate-nitrogen (NO3-N), phosphate (PO43-), dissolved oxygen (DO), biochemical oxygen demand (BOD),
chemical oxygen demand (COD), total suspended solids (TDS)and sulphate (SO42-) of waters were estimated by
UV-VIS Spectrophotometry (CECIL CE- 7200) following the standard method (APHA 2005). All the physico-
chemical parameters in ecological notes are expressed in mg/l except pH and temperature.
RESULTS AND DISCUSSION

A total number of two algal species viz. Hydrodictyon reticulatum (L.) Lagerheim & Pediastrum tetras var.
tetraodon (Corda) Hansgirg under the genera Hydrodictyon Roth and Pediastrum Meyen of the family
Hydrodictyaceae belonging to the order Chlorococcales of the class Chlorophyceae were recorded for the first
time from different aquatic ecosystems in Hooghly district of West Bengal, India. Each currently accepted name
has been provided with its author (s) name. They were described below:
Morpho-taxonomic description
1. Hydrodictyon reticulatum (L.) Lagerheim in K. Svenska.Vetenskakad. Frhandl. 40: 71, 1883; Biswas,
Rec. Bot. Surv. India 15 (1): 68. Pl. 3. fig. 29, 1949; Philipose, Chlorococcales 134. fig.48, 1967; Coffey &
Miller, New Zealand Journal of Botany, 26: 319.figs. 2-5, 1988; Anand, Indian freshwater microalgae 32.fig.
90, 1998; Kant & Gupta, Algal Flora of Ladakh 81.pl.19. fig. 6, 1998. (Figs. 1 A-B)
Conferva reticulate L. 1753; Hydrodictyon utriculatum Roth 1800.
Plant macroscopic, grass green; free floating, saccate reticulum, colonial; colonies reticulate; 6 cells adjoined
together end to end walls repeatedly forming hexagonal mesh and whole structure of the alga appears as
cylindrical net; net may vary in size; cells coenocytic, elongate, cylindrical, 51.254.8 m long, 9.110.8m
broad; cell wall smooth, double layered; chloroplast reticulate; pyrenoids many; asexual reproduction by auto
colony formation.
Habitat: Ponds water at Tribeni and Chinsurah; canal water at Khamargachi, moat water at Dumurdaha and
rice fields in Magra.
Collection No: 138, 808; Dated: 20.03.06, 03.01.11
Ecological Notes: Grows as weed & forming net in pond at Tribeni; water temperature: 20C; pH: 7.6; NO 3-
N: 0.17; PO43-: 0.20; DO: 8.0; BOD: 7.2; COD: 68.0; TDS: 124.0; SO42-: 7.0
Significance: Good source of nutrients in rice fields after decomposition; provides shelter to aquatic
zooplanktons and used as food by grass carp fishes when grows in ponds.
Figure 1. A-B, Hydrodictyon reticulatum (L.) Lagerheim; C, Pediastrum tetras var. tetraodon (Corda) Hansgirg
2. Pediastrum tetras var. tetraodon (Corda) Hansgirg in Prodr. Alg. Bhmen 1: 112, 1888; Phlipose,
Chlorococcales pl. 129.figs. 45 d, e, g, 1967; Kumar, Seth and Suseela, Phykos 42 (2): 38, fig. 18, 2012; Rai and
Misra, Our Nature 10: 172-173, fig. 4, 2012. (Fig. 1C)
Euastrum tetraodon Corda 1839.
Planktonic, colonial; colony entire, discoid to slightly rectangular of 8 cells; cells more or less straight;
peripheral cells crenate or angular, outer margins of peripheral cells with deep incision and pronounced
Halder (2015) 2(3): 168171
.
projections; cell wall smooth; diameter of 8 celled colony 32.034.0 m; inner cell also with straight sides but
one margin deeply incised; lateral margins of peripheral cells adjoined along their length; vegetative cells 7.2
14.0 m long, 2.413.0 m broad.
Habitat: Pond water at Kamarkundu, canal water at Khamargachi and Hooghly River water.
Collection No: 349, 1194; Dated: 15.07.06, 25.11.11
Ecological Notes: Planktonic in a canal at Khamargachi; water temperature: 23C; pH: 7.5; NO3-N: 0.10;
PO43-: 0.18; DO: 6.6; BOD: 3.8; COD: 90.0; TDS: 72.0; SO42-: 6.0
Significance: Primary producer & a component of aquatic food chain in freshwater ecosystems.
Documentation of species and varieties as new records or their recollection from a particular habitat have a
significant importance from the taxonomical point of view in the floristic study of algal flora (Bajpai et al. 2013;
Singh et al. 2014; Srivastava et al. 2014; Halder 2015). In the present study, two freshwater green algal
members of the family Hydrodictyaceae under the class Chlorophyceae had been morpho-taxonomically
described first time from Hooghly district, West Bengal, India. Among them, Pediastrum tetras var. tetraodon
(Corda) Hansgirg was the new report from this state. The results of the analyses of physico-chemical
characteristics of studied water bodies were indicated that water was alkaline (pH: 7.57.6), although the range
of pH of different water bodies was reported from 5.57.0 in Bankura district by Mallick & Keshri (2009) which
was slightly lower than the present investigation. The values of the different physico-chemical parameters like
NO3-N, PO43-, DO, BOD, COD, TDS and SO42- were found within the permissible limit prescribed by WHO
(2011) and favoured algal growth in these studied water bodies. The morpho-taxonomic study and
documentation of algal species or new records will explore algal biodiversity of an area and physico-chemical
characterization of water will reveal water quality status in respect of pollution and species evenness (E) of a
particular aquatic body. Moreover, such kind of work will provide baseline information and sufficient
knowledge for the future studies on algal taxonomy and freshwater ecology.
ACKNOWLEDGEMENTS
The author is grateful to Dr. S. N. Sinha, Dept. of Botany, University of Kalyani, Nadia, West Bengal for
providing opportunity to work under his guidance. The author is also thankful to Dr. R.K. Gupta, BSI, Howrah
for his kind co-operation.
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Anand N (1998) Indian freshwater microalgae. Bishen Singh Mahendra Pal Singh Publishers, Dehra Dun. pp. 1
200.
APHA (2005) Standard methods for the examination of water and waste water (21sted.). American Public Health
Association, Washington, DC. New York.
Bajpai O, Mishra S, Mohan N, Mohan J & Gupta RK (2013) Physico chemical charecteristics of Lakhna Devi
temple water tank, Lakhna, Bakewar, Etawah, U.P. with reference to Cyanobacterial diversity. International
Journal of Environment 1(1): 2028.
Banerjee A & Santra SC (2001) Phytoplankton on the rivers of Indian Sunderban Mangrove estuary. Indian
Biologists 33(1): 6771.
Coffey BT & Miller ST (1988) Hydrodictyon reticulatum L. Lagerheim (Chlorophyta) a new genus record from
New Zealand. New Zealand Journal of Botany 26: 317320.
Halder N (2015) Two species of Zygnemopsis (Skuja) Transeau from West Bengal, India. Tropical Plant
Research 2(2): 8284.
Jena M & Adhikary SP (2007) Chlorococcales (Chlorophyceae) of Eastern and North-eastern states of India.
Algae 22(3): 167183.
Kant S & Gupta P (1998) Algal flora of Ladakh. Scientic publishers, Jodhpur, India. pp.1341.
Keshri JP and Mallick P (2013) On the occurrence of the genera Pediastrum Meyen & Stauridium (Ehrenberg)
E. Hegewald (Sphaeropleales, Chlorophyta) in West Bengal, India with the description of four new taxa.
Phycological Society of India 43 (2): 917.
Kumar R, Seth MK & Suseela MR (2012) Chlorophyceae of district Kangra of Himachal Pradesh. Phycological
Society of India 42 (2): 3538.
Halder (2015) 2(3): 168171
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Mallick P & Keshri JP (2008) New record of Pediastrum Meyen from West Bengal, India. Journal of Applied
BioSciences 34(1): 8386.
Mallick P & Keshri JP (2009) A study on Chlorococcalean algae and their associate plants in Bankura district,
West Bengal. International Journal of Plant Sciences 4(1): 285288.
Pal TK&Santra SC (1984) New additions to algal flora of Murshidabad, West Bengal. Phycological Society of
India 23(1&2): 139141.
Pal TK, Adhya TK & Santra SC (1986) Algal flora of Murshidabad district, West Bengal I. A survey from
Berhampore and adjoining areas. Bulletin of Botanical Society of Bengal 40: 3343.
Patel RJ & George I (1982) A new variety of Pediastrun- P. integrum Ng.var. undulatum var. nov.
Phycological Society of India 21: 129130.
Philipose MT (1967) Chlorococcales. Indian Council of Agricultural Research, New Delhi. pp. 1365.
Pocock MA (1960) Hydrodictyon: a comparative biological study. Journal of South African botany 26: 167
319.
Rai SK & Misra PK (2012) Taxonomy and diversity of genus Pediastrum Meyen (Chlorophyceae, Algae) in
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Sau A & Gupta RK (2008) A contribution to some fresh water Chlorococcales of Howrah district, West Bengal,
India. Journal of Economic & Taxonomic Botany 32(1): 186191.
Sharma SP, Saxsena DN & Agarkar MS (1985) A note on two new species of Pediastrum from Gwalior India.
Phycological Society of India 24: 13.
Singh PK (1973) Occurrence of green algae Pithophora sp. and Hydrodyctyon reticulatum as weed in Rice
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Singh A, Tiwari V & Mohan J (2014) Chroococcales in River Ganga at JajmauGhat, Kanpur. Tropical Plant
Research 1(1): 2830.
Srivastava N, Suseela MR &Toppo K (2014) Fresh water cyanobacteria of Sai River near Lucknow, Uttar
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WHO (2011) Guidelines for drinking water quality. 4th edition. World Health Organization, Geneva,
Switzerland.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(3): 172174, 2015
Research article
Effect of red laterite soil and vermicompost on growth and

development of chilli and brinjal grown under polypot conditions
Hruda Ranjan Sahoo, Madhuchhanda Sahoo, Mayeetreyee Baboo and Nibha Gupta*
Division of Plant Pathology and Microbiology, Regional Plant Resource Centre,
Bhubaneswar-751015, Odisha, India
*Corresponding Author: nguc2003@yahoo.co.in [Accepted: 29 September 2015]
Abstract: The effect of vermicompost on growth enhancement and productivity of vegetable

crops like chilli and brinjal grown in polypots was observed under greenhouse conditions and
compared with red laterite soil treated as control. Periodical observations noted at 30 days interval
up to 120 days exhibited increasing order of growth enhancement in both the crop plants with
vermicompost mixed soil. Although the effect of vermicompost on growth of chilli could not be
significant; its impact on plant productivity is quite evident. However, significant difference could
be observed in Brinjal plants grown in both the normal soil and vermicompost treated soil.
Enhancement in growth parameters such as leaf area, fresh weight and shoot fresh weight was
noticed but no direct effect of vermicompost in fruit productivity and total weight was observed.
Keywords: Vegetable crops - Growth parameters - Productivity - Vermicompost.
[Cite as: Sahoo HR, Sahoo M, Baboo M & Gupta N (2015) Effect of red laterite soil and vermicompost on
growth and development of chilli and brinjal grown under polypot conditions. Tropical Plant Research 2(3):
172174]
INTRODUCTION
Vegetables are important food components of dietary systems as they provide essential nutrients for human
health. However, they require high amounts of nutrients for its luxuriant growth and development. Brinjal and
chilli are widely cultivated vegetable crops. Brinjal (Solanum melongena L.) is most popular vegetable crop
grown in the world and Chilli (Capsicum annuum L.) is also an important vegetable crop with high consumption
rate (Ahmed et al. 2000, Datta et al. 2011). The economic, nutritious and pharmacological significance is
responsible for its high demand. Although India is the largest producer of chilli in the world but lower yield in
terms of area used for cultivation (Bharathi et al. 2004, Khan & Raj 2006). As a result of which large amount of
chemical fertilizer is applied to enhance productivity of vegetable crops. There is enormous use of fertilizers that
has led to major environmental and health concerns due to its deleterious effect on aquatic ecosystem.
Mesophilic processes such as vermicomposting better known as vermiculture biotechnology refers to the
breeding and propagation of earthworms for cost-effective and eco-friendly organic manure (Beffa et al. 1998,
Masciandaro et al. 2000, Aalok et al. 2008, Perera & Nanthakumaran 2015).Vermicompost can be used to
improve soil health and enhance plant growth without causing damage to the environment. Vermicompost plays
a major role in improving growth and yield of different field crops, vegetables, flower and fruit crops such as
sorghum (Patil & Sheelavantar 2000), sunflower (Devi & Agarwal 1998), coriander (Vadiraj et al. 1998), brinjal
(Babu et al. 2010) etc. The present investigation was carried out to observe the effect of vermicompost on
vegetative growth and fruiting of chilli and brinjal under the pot culture conditions.

In order to study the impact of vermicompost on growth of vegetable crops, a pot culture experiment was set
up at the green house in Polybags of size 1010" capacity containing 5kg of red laterite soil. Only soil was
treated as control whereas test experimental sets were supplemented with vermicompost at the rate of 250g/pot.
Seeds of chilli and Brinjal were sown into polypots containing control soil and soil supplemented with
vermicompost. The pots were maintained in the green house at an adequate temperature and water was supplied
Received: 25 July 2015 Published online: 31 October 2015
Sahoo et al. (2015) 2(3): 172174
.
daily to maintain the moisture level of the soil. Data on growth parameters of both the vegetable crops was
recorded for Plant height, fruiting pattern and biomass periodically. The fruit development and harvest was
recorded after 120 days.

Data recorded on leaf number and plant height of chilli is presented in figure 1. Periodical observations
noted at 30 days interval up to 120 days exhibited the increasing order of growth enhancement. Vermicompost
treated plants showed better growth than simple soil. 38.9% enhancement in shoot height was observed at 90
days of growth in vermicompost added plants of chilli. Data recorded on fruit harvest of chilli at 120 days has
been depicted in figure 2. Effect of vermicompost on plant growth could not be significant; its impact on plant
Figure 1. Effect of Vermicompost on Chilli: A, Leaf number; B, Shoot height.
Figure 2. Effect of vermicompost on Chilli fruit at 120 days: A, Number, weight & length; B, Total weight.
productivity is quite evident. Four times more number of fruits and their weight was observed in vermicompost
treated plants as compared to the untreated plants in first harvest. Fruit length and weight per fruit was also
observed in the chilli plants grown in vermicompost added soil. Second harvest of the vegetable product showed
similar pattern of growth enhancement in plants under treated condition. Data recorded on growth of Brinjal has
been presented in figure 3. Very significant difference could be observed in plants of both the normal soil and
Figure 3. Effect of Vermicompost on Brinjal: A, Leaf number; B, Shoot height.
Sahoo et al. (2015) 2(3): 172174
.
vermicompost treated soil. The growth performance of brinjal under polypot conditions was influenced by the
vermicompost treatment. Enhancement in leaf area, fresh weight and shoot fresh weight as presented in figure 4
is clearly evident from the present study. No direct effect of vermicompost in fruit productivity and total weight
Figure 4. Effect of Vermicompost on Brinjal: A, Leaf area; B, Leaf fresh weight; C, Shoot fresh weight.
was observed. However, (40.3720.69) and (51.8318.53) g/fruit was observed in normal soil and
vermicompost treated soil, respectively. The effect of vermicompost has been clearly demonstrated in the
present study. To formularize the general usage of vermicompost for these crops more in depth study is required
as crop productivity and improvement is also dependent upon agro climatic zones and its microhabitats.
ACKNOWLEDGEMENTS
The financial assistance obtained through Forest and Environment Department, Govt. of Odisha (State Plan
Project 2014-15) and INSPIRE programme (No. DST/INSPIRE Fellowship/2013/506) DST, Govt. of India is
gratefully acknowledged.
REFERENCES
Aalok A, Tripathi AK & Soni P (2008) Vermicomposting: A better option for organic solid waste management.
Journal of Human Ecology 24: 5964.
Ahmed J, Shivhare US & Raghavan GSV (2000) Rheological characteristics and kinetics of colour degradations
of green chilli puree. Journal of Food Engineering 44: 239244.
Babu NS, Panicker S & Clarson D (2010) The effect of Vermicompost with different microbial fertilizers on the
growth and yield of Brinjal. Baselius Researcher 11(2): 168177.
Beffa T, Staib F, Fisher JL, Lyon PF, Gumowski P, Marfenina OE, Dunoyergeindre S, Georgen F, Roch-Susuki
R, Gallaz L & Latge P (1998) Mycological control and surveillance of biological waste and compost.
Medical Mycology 36:137145.
Bharathi R, Vivekananthan R, Harish S, Ramanathan A & Samiyappan R (2004) Rhizobacteria-based
bioformulations for the management of fruit rot infection in chillies. Crop protection 23: 835843.
Datta M, Palit R, Sengupta C, Pandit MK & Banerjee S (2011) Plant growth promoting rhizobacteria enhance
growth and yield of chilli (Capsicum annuum L.) under field conditions. Australian Journal of Crop Science
5(5): 531536.
Devi D & Agarwal SK (1998) Performance of sunflower hybrids as influenced by organic manure and fertilizer.
Journal of Oilseeds Research 15(2): 272279.
Khan MS & Raj SK (2006) First report of molecular detection of an Aster yellows phytoplasma (Candidatus
Phytoplasma asteris) isolate infecting chilli (Capsicum annum) in India. Plant Pathology 5: 822.
Masciandaro G, Ceccanti B & Garcia C (2000) In situ vermicomposting of biological sludges and impacts on
soil quality. Soil Biology and Biochemistry 32: 10151024.
Patil SL & Sheelavantar MN (2000) Effect of moisture conservation practices, organic sources and nitrogen
levels on yield, water use and root development of rabi sorghum [Sorghum bicolor (L.)] in the vertisols of
semiarid tropics. Annals of Agricultural Research 21(21): 3236.
Perera KIM & Nanthakumaran A (2015) Technical feasibility and effectiveness of vermicomposting at
Household level. Tropical Plant Research 2(1): 5157.
Vadiraj BA, Siddagangaiah D & Potty SN (1998) Response of coriander (Coriandrum sativum L.) cultivars to
graded levels of vermicompost. Journal of Spices and Aromatic Crops 7(2): 141143.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(3): 175179, 2015
Research article
Ethnopharmacological investigation and antibacterial evaluation of

the methanolic extract of Asparagus racemosus (Shatavari)
T. Kumaran1, 2* and T. Citarasu1
1
Centre for Marine Science and Technology, Manonmaniam sundaranar University, Rajakkamangalam,
Kanyakumari, Tamilnadu
2
PG and Research Department of Zoology, Muslim Arts College, Thiruvithancode, Kanyakumari, Tamilnadu
*Corresponding Author: kumaranmac@gmail.com [Accepted: 02 October 2015]
Abstract: Plants are the base of sophisticated traditional medicine systems including Ayurvedic,
Unani and Siddha. Phytochemical analysis of Asparagus racemosus revealed that numerous
compounds of plants traditionally used for medicinal purposes have several therapeutical
properties. The result of the phytochemical studies revealed the presence of saponins, tannins,
alkaloids, steroids and other biochemicals. Saponins, tannins and alkaloids are well known for
their antibacterial properties. Experiments demonstrating the pharmacological properties of
saponins have aroused considerable clinical interest in these substances. The concentrations of the
plant used were 25 mg ml-1, 50 mg ml-1 and 100 mg ml-1 respectively. At these concentrations, the
extract inhibited the growth of Escherichia coli, Pseudomonas aeruginosa and Vibrio
parahaemolyticus and produced percentage inhibition ranging from 72.486.5 %. The anti-
bacterial activity demonstrated by the plant extract may due to the presence of the phytochemicals
present in the plant.
Keywords: Asparagus racemosus - Phytochemical - Saponins - Antibacterial - Pharmacology.
[Cite as: Kumaran T & Citarasu T (2015) Ethnopharmacological investigation and antibacterial evaluation of
the methanolic extract of Asparagus racemosus (Shatavari). Tropical Plant Research 2(3): 175179]
INTRODUCTION
Herbal medicine is a powerful method of disease treatment. Western drugs are usually used to control
symptoms, but do not alter the disease process Citarasu (2010). Many infectious diseases have been known to
be treated with herbal remedies throughout the history of mankind. Natural products, either as pure compounds
or as standardized plant extracts, provide unlimited opportunities for new drug leads because of the unmatched
availability of chemical diversity Harbone (1973). There is a continuous and urgent need to discover new
antimicrobial compounds with diverse chemical structures and novel mechanisms of action for new and re-
emerging infectious disease.
Plants have formed the base of sophisticated traditional medicine systems that have been in existence for
thousands of years and continue to provide mankind with new remedies. Green plants are the indispensable
storehouse of many chemical metabolites which are grouped into two categories namely: primary and secondary
metabolites. Secondary metabolites are the substances produced by plants as defence chemicals. They include
alkaloids, flavonoids, essential oils, phenols, saponins etc. In India, different regions have specific features
according to the climatic conditions (Kumaran & Citarasu 2015). These plants including medicinal plants are
also used as a feeding for animals. They are indirectly shown by their effects by which animals do not suffer by
any types of diseases. Growing plants are one of the cheapest sources of feeding for animals having crude
proteins of 1425% (Babu et al. 2011).
Saponins are secondary metabolites and play a role in the protection of plants against microorganism. Many
saponins show strong antibacterial activities. As saponins are probably a part of plants defence systems, they
have been included in a group of protective molecules in plants called phyto-protectant (Francis et al. 2002).
Saponins are used antioxidant, antimicrobial, and anti-inflammatory etc. according to medical field. It is a
Kumaran & Citarasu (2015) 2(3): 175179
.
bioactive antibacterial agent of plants (Yoshiki 1998). The present study was designed to evaluate the
fundamental phytochemical constituents and antimicrobial activities of the Asparagus racemosus (Shatavari).

Collection and Extraction
Asparagus racemosus Willd. (Shatavari) roots were purchased from the commercial market at Nagercoil,
kanayakumari district, Tamilnadu, India. Dried powder plant materials were boiled at above 100C with two
hour. After filtered the extracts, the supernatant was collected and the residue were discarded. The supernatant
was condensed in the water bath and the condensate was extracted again by methanol. The methanolic extract
was concentrated in rotatory evaporator under reduced pressure at the room temperature of 45C to 50C in
order to avoid the evaporation of plant materials. Aqueous extract was concentrated using Lyophilizer and
stored at 4C as described by Andrea et al. (2012).
Phytochemical screening
The Phytochemical screening was determined by the method (Sofowora 1993; Trease 1989). This screening
was carried out with the methanolic extracts using chemical methods and thin-layer chromatography (TLC) as
per standard protocol (Wagner & Bladt 1996).
Saponin Estimation Procedure
Weigh accurately 1.5 to 2 gm of the material in a beaker add 50 ml of petroleum ether and gently heat to
40C on a water bath for 5 minutes with regular shaking. Filter the petroleum ether repeat the operation with
further 250 ml of petroleum ether. Discard petroleum ether and preserve the marc. Extract the marc obtained in
the previous test with 460 ml of methanol with mild heating. Filter the methanol layer to another beaker.
Concentrate the combined methanol layer to about 25 ml. Add 150 ml of dry acetone to precipitate the saponins.
Filter the saponins through a filter paper and dry at 100C for constant weight.
Calculation
Bioautography
A TLC Bioautographic method was used to detect active components. After application of the extract on a
silica gel plate, thin layer chromatography (TLC) was developed using ethylacetate : methanol (9:1) as the
eluent system for Asparagus racemosus. Observe the bands- the TLC plates were dried for complete removal of
solvents. Then the fractions of TLC were spotted on already swabbed agar plates by bioautography method to
evaluate the activity of the different essential compounds, and the plates were incubated at 35C for 24 hours.
The activity of compound can detect by its zone formation.
Antibacterial Screening
Test Organisms
The test organisms were standard laboratory strains of Escherichia coli, Pseudomonas aeruginosa and
Vibrio parahaemolyticus. The organisms were obtained from the Department of marine Science (CMST),
Manonmaniam Sundaranar University, Rajakkamangalam, Kanyakumari district, Tamilnadu, India.
Antibacterial activity
Muller-Hinton agar was poured on to sterile Petri plates. When the media solidified, 0.1ml of inoculums
with 0.5 OD was poured over feeder layer and spread evenly with a sterile spreader. A well of 6mm diameter
was made by using a sterile cark borer. Each well-received the extract was tested in a different concentration (25
mg ml-1, 50 mg ml-1 and 100 mg ml-1. Distilled water was used as negative control while ampicillin was used as
positive control. And the commercial antibiotics like as Ampicillin and Tetracycline tested against pathogens.
They were incubated at 37C for 24 hours. After incubation, the diameter of the inhibition zone was measured.
RESULTS
Phytochemical Screening
The phytochemical screening of methanolic extracts showed the presence of different types of active
constituents, namely alkaloids, anthraquinones, cardiac glycosides, flavonoids, terpenoids, tannins, saponins,
sterols and triterpenes. These compounds were present in almost all the plants extracts. The details were given in
.
the (Table 1). The total percentage of saponin was estimated from the Asparagus racemosus, and it was found
that 2 g of Asparagus racemosus contains 40% of saponin molecule.
Table 1. Summary of The Results Phytochemical Analysis of Asparagus racemosus.
S.No. Phytochemical group Result
1. Alkaloids +
2. Saponins +
3. Flavonoids +
4. Tannins +
5. Steroids +
6. Terpenoides +
7. Titerpenoides +
8. Anthraquinones +
9. Cardiac glycosides +
Note: +, Present.
TLC Studies on Asparagus racemosus
On TLC analysis for the hot water extract Asparagus racemosus was revealed that, the single spot were
obtained, and it observed under UV-illuminator. The fraction obtained having the Rf values of 0.86 and it shows
on figure 1.
0.86
Figure 1. Thin layer Chromatography (TLC) for the steroidal saponin from Asparagus racemosus.
Bioautography
Bioautography method was used to detect active components by its zone formation. The maximum zone of
inhibition is measured in 6.1 mm in dm. The minimum zone of inhibition for the fraction of Asparagus
racemosus is 1.8 mm against Vibrio parahaemolyticus (Table 2).
Table 2. Bioautography of the saponin activities of Asparagus racemosus against some pathogenic bacteria.
S. No. Concentration Zone of Inhibition in pathogenic bacteria (mm)
(g ml-1) Escherichia coli Pseudomonas aeruginosa Vibrio parahaemolyticus
1 1.0 4.2 3.8 4.5
2 2.0 5.9 5.4 6.1
3 3.0 2.7 1.4 1.8
Antimicrobial activity
The antimicrobial activities of the plant extracts against the three bacteria strains examined were assessed by
the presence or absence of inhibition zones. The aqueous extract of Asparagus racemosus exhibited moderate
level antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa and Vibrio parahaemolyticus the
test organisms. Methanol extract of Asparagus racemosus was active against all the test organisms except
Pseudomonas aeruginosa. On the other hand, it was found that the methanol extract of Asparagus racemosus
exhibited high activity against Escherichia coli and Vibrio parahaemolyticus (Fig. 2)
.
8
Methanol Aqueous
7
Zone of inhibition (mm)

6
5
4
3
2
1
0
E.coli P.aerug V.para
Figure 2. Antibacterial activity Asparagus racemosus against pathogenic microorganism (E. coli
= Escherichia coli; P. aerug = Pseudomonas aeruginosa; V. para = Vibrio parahaemolyticus).
To screen the antibacterial activity against tested organisms, ampicillin and tetracycline were used as a
standard. It was found that tetracycline (5 g ml-1) standard showed higher activity than ampicillin (30 g ml-1)
standard against tested microorganisms (Fig. 3).
Ampicillin Tetracycline
7
6
Zone of inhibition (mm)
5
4
3
2
1
0
E.coli P.aerug V.para
Figure 3. Antibacterial activity Ampicillin and Tetracycline against pathogenic microorganism.
DISCUSSION
Saponins may beconsidered a part of plants defence systems, and as such have been included in a large
group of protective molecules found in plants (Morrissey & Osbourn 1999). The present study focuses on both the
phytochemical analysis and antimicrobial potential of Asparagus racemosus. In the present investigation, different
extracts of Asparagus racemosus was evaluated for exploration of their antimicrobial activity against certain
bacteria, which was regarded a pathogenic microorganism. Susceptibility of plant extract was tested by agar
well diffusion method was determined.
The results of our studies have shown that Asparagus racemosus contains saponins, tannins, flavonoids,
steroids, alkaloids and cardiac glycosides. The plant extract also showed antibacterial activity at concentrations
of 25 mg ml-1, 50 mg ml-1 and 100 mg ml-1 respectively. At these concentrations, the extract inhibited the
growth of Escherichia coli, Pseudomonas aeruginosa and Vibrio parahaemolyticus and produced percentage
inhibition ranging from 72.4% to 86.5%. Therefore, the ethnomedical application of the plant in the treatment of
bacterial infections is justified.
The antibacterial activity of aqueous extract (25 mg ml-1, 50 mg ml-1 and 100 mg ml-1) showed that the
extract has activity against Escherichia coli, Pseudomonas aeruginosa and Vibrio parahaemolyticus. At 100 mg
ml-1, it produced the highest percentage inhibition of 80.8% on Vibrio parahaemolyticus; at 25 mg ml-1 and least
percentage inhibition of 72.4% on Vibrio parahaemolyticus. The percentage inhibition of growth produced by
the extract on the other bacterial strains also tested ranges from 65% to 75% (Table 3). It may be deduced that
Asparagus racemosus showed a remarkable antibacterial activity (Table 2) when compared with Ampicillin
.
(positive control) which produced a percentage inhibition of 98.8% (Table 3). Therefore, the antibacterial
activity exhibited by this plant extract may be due to the presence tannins, saponins and flavonoids in plant
which have been reported to have antibacterial properties. In conclusion, of the present investigation Asparagus
racemosus contain potential antimicrobial components that may be of great use for the development of
pharmaceutical industries as a therapy against various diseases.
Table 3. Summary of results of antibacterial activities of the aqueous extract of Asparagus racemosus.
Percentage inhibition of growth (%)
Test organisms Asparagus racemosus Ampicillin Distilled
(25 mg ml-1) (50 mg ml-1) (100 mg ml-1) (100 mg ml-1) water
Escherichia coli 65.0 70.6 73.0 98.8 0.0
Pseudomonas aeruginosa 65.0 65.2 75.0 98.7 0.0
Vibrio parahaemolyticus 72.4 68.0 86.5 98.8 0.0
Note: Results are means of triplicate values.
ACKNOWLEDGEMENTS
The authors are thankful to Centre for Marine Science and Technology, Manonmaniam Sundaranar
University, Tamilnadu, India for providing necessary facilities.
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Harbone JB (1973) Phytochemical Methods: A guide to modern techniques of plant analysis. Chapman and Hall
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methanolic extract of glycine max (soybean). Global journal of medicine and public health 4(3): 17.
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Microbiological and Molecular Biological Reviews 63: 708724.
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Trease GE & Evans WC (1989) Pharmacognosy, 13th edition. Bailliere Tindall, London, pp. 176180.
Wagner H & Bladt S (1996) Plants Drug Analysis: A Thin Layer Chromatography Atlas, 2nd edition. Springer,
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(3): 180191, 2015
Research article
Bryophyte diversity in Terai regions of Uttar Pradesh, India with

some new additions to the state
Vinay Sahu and A. K. Asthana*
Bryology Laboratory, CSIR-National Botanical Research Institute Lucknow- 226 001, India
*Corresponding Author: drakasthana@rediffmail.com [Accepted: 17 October 2015]
Abstract: An investigation of the Bryophytes from Terai region of Uttar Pradesh has revealed the
occurrence of 29 species of bryophytes: 21 species belonging to 16 genera of 11 Families of
Mosses; 6 species belonging to 3 genera of 3 families of Liverworts and 2 species belonging to 1
genus of 1 family of Hornworts. This includes several new reports viz., Notothylas kashyapii D.K.
Singh from Gangetic Plains, Dicranella macrospora Gangulee, Entodontopsis tavoyense (Hook. ex
Harv.) W.R. Buck & R.R. Ireland, Trachyphyllum inflexum (Harv.) Gepp., Weissia controversa
Hedw., Fissidens crenulatus Mitt., Fissidens flaccidus Mitt. and Fissidens zollingeri Mont. from
Uttar Pradesh.
Keywords: Uttar Pradesh - Terai region - Liverworts - Hornworts - New record.
[Cite as: Sahu V & Asthana AK (2015) Bryophyte diversity in Terai regions of Uttar Pradesh, India with some
new additions to the state. Tropical Plant Research 2(3): 180191]
INTRODUCTION
Uttar Pradesh (U.P.) can be divided into three topographical zones (i) The sub Himalayan Terai region in the
North (ii) The Gangetic Plain in the centre (iii) The Vindhya Hills and plateau in the South. In the state average
temperature ranges from 0o to 46 C and average annual rainfall is around 65 to 70 cm
(https://en.wikipedia.org/wiki/Climate_of_Uttar_Pradesh). A total of 9.26% of the state's geographic area is
under forest/tree cover in U.P. Thus area is not much favourable for the growth of bryophytes. Various workers
from time to time made an attempt to provide consolidated account of bryophytes of Uttar Pradesh (Ahmad
1942, Pande & Ahmad 1944, Pande et al. 1954, Sahai 1962, Sahai & Sinha 1972, Srivastava 1964, Sinha et al.
1990, Singh & Kumar 2003, Singh et al. 2005, Lal 2007, Nath et al. 2010, Kumar & Kazmi 2004, 2006). Singh
(2013) has listed 27 species of liverworts, 3 species of hornworts and 24 species of mosses from Uttar Pradesh.
During the course of bryological exploration in that region, the authors come across some interesting taxa of
bryophytes. In the present study, an attempt has been made to provide an enumeration of bryophytes of this area
with ecology and distribution, range in India and abroad along with details of specimens examined.

Plant exploration trip has been undertaken to Lakhimpur, Pilibhit and their neighbouring areas in Uttar
Pradesh. Bryophyte plant samples has been collected from Gola forest, Salempur Forest Division, Malani
Range, Lalapur, near forest nursery, Lakhimpur-Kheri and Pilibhit Tiger Reserve, Barahi range. Collected
specimens have been deposited in Herbarium, CSIR-National Botanical Research Institute, Lucknow (LWG).

During the present investigation on the bryophytes of Pilibhit Tiger Reserve and its neighbouring areas
(Lakhimpur-Kheri, Gola, Shahjahanpur) about 30 taxa has been identified and enumerated. It includes an
account of 22 species belonging to 17 genera of 11 Families of Mosses, 6 species belonging to 3 genera of 3
families of Liverworts and 2 species belonging to 1 genus of 1 family of Hornworts. During the study one
Hornwort, Notothylas kashyapii D.K. Singh from Gangetic plains; and 8 mosses, Dicranella macrospora
Gangulee, Entodontopsis tavoyense (Hook. ex Harv.) W.R. Buck & R.R. Ireland, Trachyphyllum inflexum
(Harv.) Gepp., Weissia controversa Hedw., Fissidens crenulatus Mitt., Fissidens flaccidus Mitt. and Fissidens
zollingeri Mont. are reported from Uttar Pradesh for the first time.
Received: 02 August 2015 Published online: 31 October 2015
Sahu & Asthana (2015) 2(3): 180191
.
It has been observed that terricolous forms are dominant than epiphytic ones and out of 30 taxa investigated
from the area, mosses viz., Barbula indica, Philonotis mollis, Fissidens zollingeri and Hyophila nymaniana
were more frequent in occurrence. Among liverworts Riccia billardieri and Cyathodium cavernarum have been
frequently found in the area. Among the mosses, family Pottiaceae seems to be more dominant in the region
with 5 taxa and Genus Fissidens has maximum number (5) of species. As far as the liverworts are concerned,
family Ricciaceae exhibits maximum number of 4 species.
Enumeration
Liverworts
Family - Aytoniaceae
1. Plagiochasma appendiculatum Lehm. et Lindenb; Pag. IV. 14 (1832). (Fig. 1A)
Habitat: On stony wall, soil; Altitude- 452536 ft.
Distribution: Central India (Amarkantak, Pachmarhi), Eastern Himalaya (Assam, Darjeeling, Sikkim), Gangetic
Plains (Uttar Pradesh), Punjab and West Rajasthan plain, South India (Maharashtra, Munnar, Tamil Nadu),
Western Himalaya (Corbett National Park, Dhanolti, Himachal Pradesh- GHNP, Kangra, Kullu, Simla, Nainital,
Mussoorie, Ranikhet); Afghanistan, Africa, China, Myanmar, Nepal, Pakistan, Philippines, Vietnam.
Specimens examined: INDIA, Uttar Pradesh, Lakhimpur-Kheri Dist., Near Radha Swami Inter College,
Kevalpurva, N 2753.194' E 8056.046', 08.09.2014, Vinay Sahu 257465A (LWG); Pilibhit Dist., Chimtiya
Puranpur, Barahi range, N 2837.006' E 08011.5', 11.09.2014, Vinay Sahu 258207A (LWG).
Family - Cyathodiaceae
2. Cyathodium cavernarum Kunze in Lehm., Pugillus 6: 17 (1834) Mont. in Ramon de la Sagra, Hist. de Cuba;
Crypyt. 491, tab. 19, Syn. Hepat. 577 (1846). (Fig. 1B)
Distribution: Central India (Gujarat, Madhya Pradesh), Eastern Himalaya (Darjeeling, Khasi Jaintia Hills,
Shillong), Gangetic Plains (Uttar Pradesh Bareilly, Lucknow), South India (Mumbai, Elephanta caves,
Khandala, Mahabaleshwar, Malabar Hills, Panchagani, Pratabgarh), Western Himalaya (Dehradun, Gumkhal,
Karn Prayag, Mussoorie, Salkuli); Africa, America, Myanmar, Java.
Specimens examined: INDIA, Uttar Pradesh, Lakhimpur-Kheri Dist., Near Radha Swami Inter College,
Kevalpurva, N 2753.194' E 8056.046', 08.09.2014, Vinay Sahu 257464A, 257465D (LWG); Gola Forest, N
2803.533' E 8029.538', 09.09.2014, Vinay Sahu 257470A (LWG); Salempur Forest Division, Malani Range,
N 2827.337' E 8029.556', 09.09.2014, Vinay Sahu 257481A (LWG); Pilibhit Dist., Near Forest Office, Mala
range, Rechaula, N 2837.070' E 7954.894', 11.09.2014, Vinay Sahu 257495A (LWG); Barahi Range 72, N
2837.006' E 08011.5', 11.09.2014, Vinay Sahu 258204 A (LWG); Chimtiya Puranpur, Barahi range, N
2837.006' E 08011.5', 11.09.2014, Vinay Sahu 258213A (LWG).
Family - Ricciaceae
3. Riccia billardieri Mont. et Nees in Gottsche, Lindenberg & Nees, Syn. Hepat. 4: 602. (Fig. 1C)
Habitat: On soil; Altitude- 485547ft.
Distribution: Central India (Gujarat, Jharkhand, Madhya Pradesh, Rajasthan), Eastern Himalaya (Assam,
Manipur, Sikkim), Gangetic Plains (Uttar Pradesh, West Bengal), South India (Andaman, Karnataka, Kerala,
Maharashtra, Tamil Nadu), Western Himalaya (Himachal Pradesh, Uttarakhand); Australia, Bangladesh,
Indonesia, Nepal, Philippines, Sri Lanka, Thailand.
Specimens examined: INDIA, Uttar Pradesh, Lakhimpur-Kheri Dist., Gola Forest, N 2803.533' E 8029.538',
09.09.2014, Vinay Sahu 257467B, 257468B, 257471A (LWG); Salempur Forest Division, Malani Range, N
2827.337' E 8029.556', 09.09.2014, Vinay Sahu 257476A, 257477E (LWG); Pilibhit Dist., Barahi Range 72, N
2837.006' E 08011.5', 11.09.2014, Vinay Sahu 258203A, 258210A (LWG).
4. Riccia gangetica Ahmad Ahmad ex L.Sderstr., A. Hagborg et von Konrat, Phytotaxa 65: 57 (2012). (Fig.
1D)
Habitat: On soil; Altitude- 530600 ft.
Distribution: Central India (Gujarat, Madhya Pradesh Rajasthan), Eastern Himalaya (Meghalaya), Gangetic
Plains (Uttar Pradesh, West Bengal), Western Himalaya (Himachal Pradesh, Uttarakhand), South India (Kerala,
Maharashtra, Tamil Nadu); Australia, Bangladesh, Indonesia.
Sahu & Asthana (2015) 2(3): 180191
.
Specimens examined: INDIA, Uttar Pradesh, Pilibhit Dist., Pipariya, N 2832.471' E 7954.905', 10.09.2014,
Vinay Sahu 257493A (LWG); Barahi Range 69, N 2838.207' E 8010.465', 11.09.2014, Vinay Sahu 258201A
(LWG).
Figure 1. A, Plagiochasma appendiculatum; B, Cyathodium cavernarum; C, Riccia billardieri; D, Riccia gangetica; E,

Riccia discolor; F, Riccia stricta; G, Notothylas indica; HI, Archidium birmannicum; JK, Philonotis mollis.
Sahu & Asthana (2015) 2(3): 180191
.
5. Riccia discolor Lehm. et Lindenberg. in Lehmann, Nov. Stirp. Pug. 4: 1. (1832). (Fig. 1E)
R. himalayensis Kashyap, J. Bombay Nat. Hist. Soc. 24: 349. (1916).
Distribution: Central India (Chattishgarh, Gujarat, Madhya Pradesh, Punjab, Rajasthan) Eastern Himalaya
(Assam, Sikkim, Meghalaya), Gangetic Plains (Uttarakhand, Uttar Pradesh, West Bengal), Western Himalaya
(Himachal Pradesh, Jammu & Kashmir), South India (Karnataka, Kerala, Maharashtra, Tamil Nadu); Africa,
Australia, Bangladesh, Myanmar, Nepal, Pakistan, Sri Lanka.
Specimens examined: INDIA, Uttar Pradesh, Lakhimpur-Kheri Dist., Barahai range 69, N 2838.207' E
8010.465', 11.09.2014, Vinay Sahu 257497A, 257500A (LWG).
6. Riccia stricta (Gottsche, Lindenb. & Nees) Perold, Bothalia 20: 197 (1990). (Fig. 1F)
Distribution: Central India (Madhya Pradesh), Eastern Himalaya (Sikkim), South India (Kerala, Tamil Nadu),
Gangetic Plains (West Bengal); Africa.
Specimens examined: INDIA, Uttar Pradesh, Shahjahanpur Dist., Near Khutar Forest, N 2818.768' E
8013.827', 12.09.2014, Vinay Sahu 258220E (LWG).
Family - Archidiaceae
7. Archidium birmannicum Mitt. ex Dix. in J. Ind. Bot., 2: 175 (1921). (Fig. 1H, I)
Distribution: Eastern Himalaya (Assam), Gangetic Plains (West Bengal Plains-Ramnagar, Hooghly), South
India (Nilgiri and Palni Hills); Myanmar, Nepal.
Specimens examined: INDIA , Uttar Pradesh, Lakhimpur-Kheri Dist., Gola Forest, N 2803.533' E 8029.538',
09.09.2014, Vinay Sahu 257467A, 257469A (LWG); Pilibhit Dist., Barahi Range 69, N 2838.207' E
8010.465', 11.09.2014, Vinay Sahu 257498A, 257500B, 258201B (LWG); Shahjahanpur Dist, Near Khutar
Forest, N 2818.768' E 8013.827', 12.09.2014, Vinay Sahu 258220C (LWG).
Family - Bartramiaceae
8. Philonotis mollis (Dozy & Molk.) Mitt. in Musci Ind. Or.: 60 (1859). (Fig. 1J, K)
Distribution: Eastern Himalaya (Sikkim), South India (Coorg, Kanara, Andaman Is.), Bhutan, Borneo, Celebes,
Japan, Java, Madagascar, Philippines, Sri Lanka, Sumatra, Taiwan, Tonkin.
Specimens examined: INDIA, Uttar Pradesh, Lakhimpur-Kheri Dist., Salempur Forest Division, Malani Range,
N 28 27.337' E 8029.556', 09.09.2014, Vinay Sahu 257480A (LWG); Lalapur, near forest nursery, N 2810.254'
E 8019.395', 10.09.2014, Vinay Sahu 257485A, 257491A (LWG); Pilibhit Dist., Barahi Range 69, N
2838.207' E 8010.465', 11.09.2014, Vinay Sahu 257499A (LWG); Chimtiya, Puranpur, Barahi range, N
2837.006' E 08011.5', 11.09.2014, Vinay Sahu 258208A, 258210B, 258211B, 258212A, 258214B (LWG);
Shahjahanpur Dist., Near Khutar Forest, N 2818.768' E 8013.827', 12.09.2014, Vinay Sahu 258219A (LWG).
Family - Dicranaceae
9. Dicranella macrospora Gangulee in Nov. Hedwigia, 8: 145 (1964). (Fig. 2A)
Distribution: Assam, Uttar Pradesh (Lakhimpur Kheri).
N 2827.337' E 8029.556', 09.09.2014, Vinay Sahu 257484A (LWG); Lalapur, near forest nursery, N
2810.254' E 8019.395', 10.09.2014, Vinay Sahu 257491B (LWG). New record to Uttar Pradesh.
Family - Ditricaceae
10. Ceratodon purpureus (Hedw.) Brid. in Bryol. Univ., 1: 480 (1826). (Fig. 2D)
Distribution: Eastern Himalaya (Assam, Darjeeling), Gangetic plains (Raebareli), South India, Western
Himalaya (Kashmir); Antarctica, Australia, Brazil, China, Chili, East Nepal, Europe, Japan, Java, Madagascar,
New Zealand, North America (including Green Land, Alaska) North, Central and South Africa, Oceania,
Siberia, Sri Lanka, Tajikistan, Thailand.
Sahu & Asthana (2015) 2(3): 180191
.
Specimens examined: INDIA, Uttar Pradesh, Pilibhit Dist., Barahi Range 72, N 2837.006' E 08011.5',
11.09.2014, Vinay Sahu 258202A (LWG).
Figure 2. A, Dicranella macrospora; BC, Erpodium mangiferae; D, Ceratodon purpureus; E, Fissidens bryoides; F,
Fissidens crenulatus; G, Fissidens flaccidus; HI, Fissidens involutus; JK, Fissidens zollingeri; L, Physcomitrium
eurystomum.
Sahu & Asthana (2015) 2(3): 180191
.
Family - Erpodiaceae
11. Erpodium mangiferae C. Muell in Linnaea 37: 178. (1873). (Fig. 2B, C)
Habitat: Epiphytic; Altitude- 452 ft.
Distribution: Central India (Gujarat), Eastern Himalaya (Assam), Gangetic plains (U.P.-Allahabad, Saharanpur;
W. Bengal-Kolkata, Hooghly), Western Himalaya (Uttarakhand), South India (W. Ghats of Karnataka,
Maharashtra and Tamil Nadu); Bangladesh, Nepal.
Specimens examined: INDIA, Uttar Pradesh, Lakhimpur-Kheri Dist., Sankarpur police station, N 2754.352' E
8054.259', 08.09.2014, Vinay Sahu 257466A (LWG); Bira Salempur Forest Division, N 2827.337' E
8029.556', 09.09.2014, Vinay Sahu 257473A (LWG).
Family - Fissidentaceae
12. Fissidens bryoides Hedw. in Sp. Musc.: 153 (1801). (Fig. 2E)
Distribution: Eastern Himalaya (Khasi Hills), Gangetic Plains (Lower Bengal); South India (Coonoor, Nilgiri
Hills, Western Ghats), Western Himalaya (Nainital, Ranikhet, Simla), Africa, Caucasus, China, East Nepal,
Europe, Japan, Java and Philippines, Malay, N. & S. America, Sri Lanka, Siberia, Taiwan.
Specimens examined: INDIA, Uttar Pradesh, Pilibhit Dist., Chimtiya Puranpur, Barahi range, N 2837.006' E
08011.5', 11.09.2014, Vinay Sahu 258214A (LWG).
13. Fissidens crenulatus Mitt. in Musc. Ind. Or.: 140 (1859). (Fig. 2F)
Distribution: Gangetic Plains (Orissa), South India; East Nepal, Upper Burma.
Specimens examined: INDIA, Uttar Pradesh, Pilibhit Dist., Salempur Forest Division, Malani Range, N
2827.337' E 8029.556', 09.09.2014, Vinay Sahu 257474B, 257482A, 257483A, 257484B (LWG); Lalapur,
near forest nursery, N 2810.254' E 8019.395', 10.09.2014, Vinay Sahu 257490A, 257491C (LWG); Pilibhit
Dist., Near Gajraula, N 2831.307' E 7956.937', 12.09.2014, Vinay Sahu 258215A (LWG); Shahjahanpur Dist.,
Near Khutar Forest, N 2818.768' E 8013.827', 12.09.2014, Vinay Sahu 258219B (LWG). New record to Uttar
Pradesh.
14. Fissidens flaccidus Mitt. in Transactions of the Linnean Society of London 23: 56. 6 f. 18 (1860). (Fig. 2G)
Fissidens splachnobryoides Broth. in Schum. et Lauterb in Fl. Deutsch Schutzgeb. Sdsee 81 (1900).
Distribution: Central India (Pachmarhi), Gangetic Plains (Lower Bengal), South India (Bombay, Khandala),
Western Himalaya (Kalka); Africa, Brazil, Borneo, Burma, China, Japan, Java, Nepal, New Guinea, Philippines,
Sri Lanka, Vietnam.
Specimens examined: INDIA, Uttar Pradesh, Lakhimpur Kheri Dist., Salempur Forest Division, Malani Range,
N 2827.337' E 8029.556', 09.09.2014, Vinay Sahu 257478A, 257480B (LWG); Lalapur, near forest nursery, N
2810.254' E 8019.395', 10.09.2014, Vinay Sahu 257488B (LWG); Pilibhit Dist., Barahi Range 72, N
2837.006' E 08011.5', 11.09.2014, Vinay Sahu 258204C (LWG); Chimtiya Puranpur, Barahi range, N
2837.006' E 08011.5', 11.09.2014, Vinay Sahu 258213C LWG). New record to Uttar Pradesh.
15. Fissidens involutus Wilson ex Mitt. in J. Proc. Linn. Soc. Botany, Supplement 2: 138. 1859. (Fig. 2H, I)
Distribution: Central India (Bastar, Chhotanagpur), Eastern Himalaya (Darjeeling, Sikkim), Gangetic Plains
(Saharanpur), South India (Bombay, Khandala), Western Himalaya; Borneo, Burma, China, Nepal, Thailand,
Vietnam.
Specimens examined: INDIA, Uttar Pradesh, Pilibhit Dist., Chimtiya Puranpur, Barahi range, N 2837.006' E
08011.5', 11.09.2014, Vinay Sahu 258209A (LWG).
16. Fissidens zollingeri Mont. in Ann. Sci. Nat. ser.3, 4: 114 (1845). (Fig. 2J, K)
Fissidens xiphioides Fleisch. in Hedwigia 38: 125 (1899).
Distribution: Central India (Pachmarhi), Gangetic Plains (Lower Bengal plains), South India (Bombay,
Khandala, Western Ghats, Kanara, Andamani Is.), Western Himalaya (Nainital, Simla); widely distributed in
Southwest Asia and Oceania, Borneo, Burma, China, Fiji, Indonesia, Nepal, Thailand, New Guinea, New
Sahu & Asthana (2015) 2(3): 180191
.
Zealand, North and Central Vietnam, Philippines, Ryuku Is., Sri Lanka, Samoa, South and North America,
Tahiti.
N 2827.337' E 8029.556', 09.09.2014, Vinay Sahu 257474D, 257475A, 257477B, 257478B, 257483B,
257485B, 257486B (LWG); Lalapur, near forest nursery, N 2810.254' E 8019.395', 10.09.2014, Vinay Sahu
257490B, 257491D (LWG); Pilibhit Dist., Chimtiya Puranpur, Barahi range, N 2837.006' E 08011.5',
11.09.2014, Vinay Sahu 258208C (LWG); Shahjahanpur Dist., Near Khutar Forest, N 2818.768' E 8013.827',
12.09.2014, Vinay Sahu 258217B, 258218A, 258220B (LWG). New record to Uttar Pradesh.
Family - Funariaceae
17. Physcomitrium eurystomum Sendtn. in Denkschr. Bayer Bot. Ges. Regensb., 3: 142 (1841). (Fig. 2L)
Distribution: Gangetic plains (Lower Bengal, Hooghly, Allahabad), Western Himalaya (Kumaon); Britain,
Central and South Africa, China, France, North Vietnam, Taiwan.
N 2827.337' E 8029.556', 09.09.2014, Vinay Sahu 257474C (LWG); Pilibhit Dist., Pipariya, N 2832.471' E
7954.905', 10.09.2014, Vinay Sahu 257493B (LWG).
18. Entosthodon wichurae M. Fleisch. in Musci Fl. Buitenz., 2: 481 (1904). (Fig. 3C)
Distribution: Eastern Himalaya (Meghalaya- Khasi and Jaintia Hills), Western Himalaya (Ranikhet); Burma,
Java, Sri Lanka.
09.09.2014, Vinay Sahu 257496B (LWG); Chimtiya Puranpur, Barahi range, N 2837.006' E 08011.5',
11.09.2014, Vinay Sahu 258212B (LWG). New record to Uttar Pradesh.
Family - Pottiaceae
19. Barbula indica (Hook.) Spreng. Steud. Nomencl., 2: 72 (1824). Saito J. Hattori Bot. Lab., 39: 488 (1975).
(Fig. 3 A, B)
Semibarbula orientalis (Web.) Wijk et Marg., Taxon, 8: 75 (1959); Gangulee, Moss. E. Ind., 3: 717 (1972).
Habitat: On soil, stony wall; Altitude- 353547 ft.
Distribution: Widely distributed plain area in the country and up to 1000 m in the Himalayas; Nepal, Southeast
Asia, Japan, South Africa, New Guinea.
Specimens examined: INDIA, Uttar Pradesh, Lakhimpur-Kheri Dist., Sankarpur police station, Near Radha
Swami Inter College, Kevalpurva, N 2754.352' E 8054.259', 09.09.2014, Vinay Sahu 257460A, 257462A,
257463A, 257465B (LWG); Gola Forest, N 2803.533' E 8029.538', 09.09.2014, Vinay Sahu 257470B,
257472B (LWG); Khalsa Public School, N2824.987' E 8011.207', 10.09.2014, Vinay Sahu 257492A (LWG);
Salempur Forest Division, Malani Range, N 2827.337' E 8029.556', 09.09.2014, Vinay Sahu 257477C,
257481B, 257486A, (LWG); Near Forest Office, Mala range, Rechaula, N 2837.070' E 7954.894', 11.09.2014,
Vinay Sahu 257494B, 257495C (LWG); Pilibhit Dist., Barahi Range 72, N 2837.006' E 08011.5', 11.09.2014,
Vinay Sahu 258204B (LWG); Chimtiya Puranpur, Barahi range, N 2837.006' E 08011.5', 11.09.2014, Vinay
Sahu 258211A (LWG); Near Amritsar dhabha, Ashram road, 12.09.2014, Vinay Sahu 258216A (LWG).
20. Gymnostomum calcareum Nees et Hornsch., Bryol. Germ., 1: 153 10f.15 (1823); Gangulee, Moss. E. Ind.,
3: 641 (1972). (Fig. 3D)
Habitat: On soil, Altitude- 547 ft.
Distribution: Eastern Himalaya, Western Himalaya (Uttarakhand-Mussoorie, Corbett National Park); Africa,
Australia, China, Europe, Japan, New Zealand, North Central and South America.
11.09.2014, Vinay Sahu 258202A (LWG); Near Gajraula, N 2831.307' E 7956.937' 12.09.2014, Vinay Sahu
258215B (LWG). New record to Uttar Pradesh.
21. Hydrogonium arcuatum (Griff.) Wijk et Marg., Taxon, 7: 289 (1958); Gangulee, Moss. E. Ind., 3: 725
(1972).
Habitat: On soil, Altitude- 353 ft.
Sahu & Asthana (2015) 2(3): 180191
.
Figure 3. AB, Barbula indica; C, Entosthodon wichurae; D, Gymnostomum calcareum; E, Hyophila spathulata; FG,
Hyophila nymaniana; H, Weissia controversa; I, Splachnobryum obtusum; J, Trachyphyllum inflexum; KL, Entodontopsis
tavoyense.
Sahu & Asthana (2015) 2(3): 180191
.
Distribution: Central India (Madhya Pradesh, Jharkhand, Orissa), Eastern Himalaya (Assam, Arunachal
Pradesh, Meghalaya West Bengal), South India (Tamil Nadu), Western Himalaya (Himachal Pradesh, Kashmir,
Uttarakhand); Bhutan, China, East Nepal, Japan, Java, Malaya, Moluca, Myanmar, New Guinea, Philippines,
Oceania.
Specimens examined: INDIA, Uttar Pradesh, Lakhimpur-Kheri Dist., Sankarpur police station, N 2754.352' E
8054.259', 08.09.2014, Vinay Sahu 257461A (LWG).
22. Hyophila nymaniana (Fleisch.) Menzel 22: 198 (1992); Nair M. C. et al., Moss Waynad in W. Ghats 117
(2005). (Fig. 3F, G)
Hyophila rosea Williams, Bull. N.Y Bot. Gard., 8: 341 (1914); H. comosa Dix. et P. Verd., Arch. Bot., 1:
166 (1927).
Habitat: On soil, stony wall; Altitude- 452600 ft.
Distribution: Central India (Gujarat), Eastern Himalaya (Meghalaya), Gangetic Plain (Allahabad), Western
Himalaya (Uttarakhand, Corbett National Park), South India (Karnataka, Kerala, Tamil Nadu); Nepal,
Philippines.
Specimens examined: INDIA, Uttar Pradesh, Lakhimpur Kheri Dist., Near Radha Swami Inter College,
Kevalpurva, N 2753.194' E 8056.046', 08.09.2014, Vinay Sahu 257463B, 257465C (LWG); Salempur Forest
Division, Malani Range, N 2827.337' E 8029.556', 09.09.2014, Vinay Sahu 257479A (LWG); Lalapur, near
forest nursery, N 2810.254' E 8019.395', 10.09.2014, Vinay Sahu 257487A, 257489A (LWG); Pilibhit Dist.,
Khalsa Public School, Ruria Puranpur, N 2824.987' E 8011.207', 10.09.2014, Vinay Sahu 257492B (LWG);
Near Forest Office, Mala range, Rechaula, N 2837.070' E 7954.894', 11.09.2014, Vinay Sahu 257494A
(LWG); Barahi Range 69, N 2838.207' E 8010.465', 11.09.2014, Vinay Sahu 257498B (LWG).
23. Hyophila spathulata (Harv.) A. Jaeger Ber. S. Gall. Naturw. Ges., 1871-72: 353 (1873). Gangulee, Moss. E.
Ind., 3: 687 (1972). (Fig. 3E)
Distribution: Gangetic Plain (Allahabad, Delhi), Western Himalaya (Uttarakhand) South India (Tamil Nadu);
China, Japan, East and West Nepal, Myanmar, Sri Lanka.
N 2827.337' E 8029.556', 09.09.2014, Vinay Sahu 227477D (LWG); Shahjahanpur Dist., Near Khutar Forest,
N 2818.768' E 8013.827', 12.09.2014, Vinay Sahu 258217A, 258219C, 258220A (LWG).
24. Weissia controversa Hedw., Spec. Musc., 67 (1801). Saito J. Hattori Bot. Lab., 39: 426 (1975). (Fig. 3H)
Distribution: Western Himalaya (Kashmir, Uttarakhand), South India (Tamil Nadu); Australia, China, Europe,
Japan, New Zealand, North Central and South America, Pakistan, Sri Lanka, West Indies.
Specimens examined: INDIA, Uttar Pradesh, Shahjahanpur Dist., Near Khutar Forest, N 2818.768' E
8013.827', 12.09.2014, Vinay Sahu 258220D (LWG). New record to Uttar Pradesh.
Family - Splachnaceae
25. Splachnobryum obtusum (Brid.) Mull. Hal., Verh. K. K. Zool. Bot. Ges. Wien 19: 503 (1869). (Fig. 3I)
S. indicum Hamp. et C. Muell. In Linnaea 37: 174 (1872).
Habitat: On rock, stony wall; Altitude- 485 ft.
Distribution: Gangetic plains (Gangetic Southern Bengal, Howrah, Hooghly, Kolkata, Delhi, Allahabad,
Orissa), Western Himalayas (Tehri), South India (Western Ghats); Java, Africa, America, Australia, Burma,
Europe, Indonesia, Macaronesia, Philippines, Papua New Guinea. Thailand.
09.09.2014, Vinay Sahu 257467C (LWG); Pilibhit Dist., Near Gajraula, N 2831.307' E 7956.937', 11.09.2014,
Vinay Sahu 258216B (LWG).
Family - Thuidiaceae
26. Trachyphyllum inflexum (Harvey) Gepp. in Hiern: Cat. Weln. Afr. Pl. 2, 21: 299 (1901). (Fig. 3J)
Distribution: Central India (Madhya Pradesh-Amakantak), Eastern Himalaya (Darjeeling, Khasi Hills, Sikkim),
Gangetic plains (Orissa), South India (Palni Hills, Kanara), Western Himalaya (Valley of Flowers, Corbett
Sahu & Asthana (2015) 2(3): 180191
.
Figure 4. Notothylas kashyapii: AC, Thalli; D, Sporophytes in the apical region; E, Lamellae present on involucres; F,
Capsule wall; G, chloroplast; HI, Spores.
Sahu & Asthana (2015) 2(3): 180191
.
National Park); Australia, Bangladesh, Burma, Central Vietnam, China, Combodia, Java, Madagascar,
Moluccas, Nepal, New Caledonia, Philippines, Thailand.
Specimens examined: INDIA, Uttar Pradesh, Pilibhit Dist., Barahi range 72, N 2837.006' E 08011.5',
11.09.2014, Vinay Sahu 258206B (LWG). New record to Uttar Pradesh.
Family - Stereophyllaceae
27. Entodontopsis tavoyense (Hook. ex Harv.) W.R. Buck. & R.R. Ireland, Nova Hedwigia 41: 105 (1985).
(Fig. 3K, L)
Sematophyllum tavoyense (Hook.) Jaeg.
Distribution: Gangetic plain (Bihar), Western Himalaya (Dehradun, Corbett National Park); South India
(Kerala); Bangladesh, East Nepal, Moulmein, Penang, Tavoy.
Specimens Examined: INDIA, Uttar Pradesh, Pilibhit Dist., Barahi range 72, N 2837.006' E 08011.5',
11.09.2014, Vinay Sahu 258205A (LWG). New record to Uttar Pradesh.
Hornworts
Family - Notothylaceae
28. Notothylas indica Kash. in Kashyap and Dutt in Proc. Lahore Phill. Soc. 4: 49-56 (1925); Asthana &
Srivastava in Bryophyt. Biblioth. 42:94 (1991). (Fig. 1G)
Distribution: Gangetic plain (Lucknow, Allahabad), Central India (Pachmarhi, Tikamgarh), South India
(Mumbai, Nagpur), Western Himalaya (Dehradun, Mussoorie); Pakistan (Parachhinar), Myanamar (Yangong).
09.09.2014, Vinay Sahu 257468A, 257471B, 257472A (LWG).
29. Notothylas kashyapii D.K. Singh, in Singh and Semwal in India J. For. 23(4): 386 (2000).
Habitat: On soil; Altitude- 418600 ft. (Fig. 4A-I)
Distribution: Western Himalaya (Uttarakhand-Dehradun).
N 2827.337' E 8029.556', 09.09.2014, Vinay Sahu 257474A, 257477A, 257478C (LWG); Lalapur, near forest
nursery, N 2810.254' E 8019.395', 10.09.2014, Vinay Sahu 257488A (LWG), Pilibhit Dist., Barahi Range 69,
N 2838.207' E 8010.465', 11.09.2014, Vinay Sahu 257496A, 257497B (LWG); Barahi Range 72, N 2837.006'
E 08011.5', 11.09.2014, Vinay Sahu 258203B (LWG); Chimtiya Puranpur, Barahi range, N 2837.006' E
08011.5', 11.09.2014, Vinay Sahu 258213B (LWG); Shahjahanpur Dist., Near Khutar Forest, N 2818.768' E
8013.827', 12.09.2014, Vinay Sahu 258218B (LWG). New record to Uttar Pradesh.
ACKNOWLEDGEMENTS
Authors are grateful to the Director, CSIR-National Botanical Research Institute, Lucknow for
encouragement and providing the facilities and work has been carried out under in house project OLP-0083.
REFERENCES
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Kumar A & Kazmi S (2006) Leaf area indices of mosses from Unchahar, Raebareli, U. P. Geophytology 36: 23
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Lal J (2007) Mosses of Gangetic plains a neglected Biogeographic Zone of India. In: Nath V & Asthana AK
(eds.) Current trends in Bryology. Bishen Singh Mahendra Pal Singh, Dehradun, India, pp.131147.
Nath V, Sinha S, Sahu V, Govind G, Srivastava M & Asthana AK (2010) A study on metal accumulation in two
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Pande SK & Ahmad S (1944) Liverworts of Lucknow and its neighbourhood. Proceedings of Indian Science
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Pande SK, Misra KC & Srivastava KP (1954) A species of Riella Mont., R. vishwanathii Pande, Misra et
Srivastav, sp. nov., from India. Revue Bryologique et Lichenologique 23:165172.
Sahai R (1962) Occurrence of Anthoceros crispulus (Mont.) Douin in Gorakhpur. Current Science 31: 519520.
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Sahai R & Sinha AB (1972) Riccia perssonii Khan - a new record from India. Current Science 41: 226227.
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Singh SK (2013) A Checklist of liverworts, hornworts and mosses of Uttar Pradesh, India. Geophytology 42(2):
163167.
Singh SK & Kumar S (2003) A note on bryophytes of Ram Nagari (Ayodhya, Faizabad, Uttar Pradesh, India.
Phytotaxonomy 3: 108111.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(3): 192203, 2015
Research article
Contribution of environmental factors

on in vitro culture of an endangered and endemic mangroves
Heritiera fomes Buch.-Ham. and Bruguiera gymnorhiza (L.) Lam.
Abdul Kader1, Sankar Narayan Sinha2, Parthadeb Ghosh1*
1
Cytogenetics & Plant Biotechnology Research Unit, Department of Botany, University of Kalyani, West
Bengal, India
2
Environmental Microbiology Research Laboratory, Department of Botany, University of Kalyani, West
Bengal, India
*Corresponding Author: pdggene@rediffmail.com [Accepted: 19 October 2015]
Abstract: Importance and destruction of mangroves have appeared in some recent surveys. So
their restoration through tissue culture study is urgently required because in vivo propagation is
plagued with unforeseen obstacles. This study describes for the first time in vitro approach for
threatened species Heritiera fomes and Bruguiera gymnorhiza through callus. For initiation of
callus modified MS medium was formulated for each species which correlated with soil conditions
of Sundarban mangrove forest. For both species the auxin NAA, nodal or shoot tip explants and
rainy season were found to be most suitable for callusing. NaCl at the concentration of 20 mM and
60 mM promoted growth for H. fomes and B. gymnorhiza callus respectively which was found to
be comparative for their growth in vivo as in Sundarban. Histological study indicated
morphogenicity of callus. Previous in vitro studies on mangroves were mostly based on the effect
of variety of hormones and different sea salts. However this present study clearly indicates that the
in vitro studies of mangroves not only depend on these factors but greatly influence by soil
condition of their habitual environment, seasonal condition etc. From this study it seems that more
and more in vitro studies of mangroves are possible if researchers focus on their habitual
environmental conditions as many mangrove species remains recalcitrant for in vitro study. The
present research clearly indicated that the species may be restored in low saline or non-saline land
as land destruction is another vital reason for mangrove extinction.
Keywords: Bruguiera gymnorhiza - Heritiera fomes - Mangrove - Callus culture - Seasonal
influence.
[Cite as: Kader A, Sinha SN & Ghosh P (2015) Contribution of environmental factors on in vitro culture of an
endangered and endemic mangroves Heritiera fomes Buch.-Ham. and Bruguiera gymnorhiza (L.) Lam. Tropical
Plant Research 2(3): 192203]
INTRODUCTION
Mangrove ecosystems are found in tropical and subtropical muddy beaches worldwide. The importance and
threats to mangrove ecosystem have been discussed by various authors (Al-Bahrany & Al-Khayri 2003, Ren et
al. 2009). Because of their importance and destruction, mangroves have attracted attention for their conservation
and preservation (Al-Bahrany & Al-Khayri 2003). Problems for restoration of mangroves arise mostly in the
form of shortage of seeds or viviparous seedlings and the disturbed soil conditions (Ohnishi & Komiyama 1998,
Feller et al. 2003). Mangrove species are physiologically unique in their adaptations to such water logged and
saline condition. Crop scientists, studying the unique adaptation pattern of mangroves, are keen to impart these
unique characters in food crops by breeding or biotechnological means (Fukomoto et al. 2004) as salinity and
water logging are among the major environmental threats with serious implication on food, fuel and fibre
production, especially in arid and semiarid regions (Dagar 2005). Besides, about one-third of all agricultural
lands are becoming saline (Dagar 2005). To understand the salt and water logging tolerance theoretically or
biochemically, callus or cell culture of mangroves may provide promising result. However, detailed knowledge
of the plant material and its requirements for callus initiation is necessary before mass in vitro propagation can
Kader et al. (2015) 2(3): 192203
.
become a reality. However scanty literature is available for few mangrove species because it is recalcitrant to
tissue culture study (Al-Bahrany & Al-Khayri 2003, Fukomoto et al. 2004, Kawana & Sasamoto 2008). During
in vitro culture of mangroves explants frequently turn brown or black and eventually die shortly after
inoculation (Kawana & Sasamoto 2008, Arumugam & Panneerselvam 2012) as it excretes high tannin and
phenol or phenolic compound. Besides, it is very difficult to maintain their habitual environment. Moreover,
success of in vitro response does depend not only on the plant genotype but also is strongly affected by
environmental conditions.
Preventing or avoiding microbial contamination is the basis of successful plant tissue cultures. Endogenous
microbial contamination is known to be one of the most serious threats in plant tissue culture, especially in
tropical species (Kneifel & Leonhardt 1992). Various literatures are available indicating the association of
microbes and mangroves in root, bark, leaves etc (Gupta et al. 2009). Among them Uchino et al. (1984)
identified some entophytic microbes in Bruguiera gymnorhiza species from its aerial parts.
Bruguiera gymnorhiza (L.) Lam. (Rhizophoraceae) is a multipurpose true mangrove species found in all
over the world. The fruits and bark of the whole plant have been used for treating diarrhoea, fever, malaria,
shingles and eye diseases (Naskar & Bakshi 1987, Bandaranayake 1998). The durable wood is used for making
boat, house, poles, beams etc (Naskar & Bakshi 1987). Natural products of this plant have anti-tumor activity
and antibacterial activity (Naskar & Bakshi 1987, Bandaranayake 2002).
Heritiera fomes Buch.-Ham. is a true mangrove tree from family Sterculiaceae, known as Sundari in
Bengali, found mainly in Southeast Asia (Naskar & Bakshi 1987, Ali et al. 2011). The largest deltaic mangrove
forest, Sundarban is derived from its Bengali name (Naskar & Bakshi 1987, Gopal & Chauhan 2006). The wood
of this species is used for making boat, raft, house and charcoal (Naskar & Bakshi 1987, Ali et al. 2011).
Besides, various parts of the tree are used as folk medicine for heart diseases, diabetes, pain, diarrhoea, skin
disorders, hepatic disorders, and goiter (Ali et al. 2011). Ethanolic extract of stem bark showed antioxidant,
lipoxygenase inhibitory, antihyperglycemic, antinociceptive effects and antibacterial activities (Wangensteen et
al. 2009, Ali et al. 2011). Due to its medicinal and economical values and increasing environmental stress
(Various salt concentrations, global warming etc), this species is being exploited indiscriminately since a very
long time and it is considered as a threatened plant according to IUCN red list 2013 (Naskar & Bakshi 1987,
Gopal & Chauhan 2006).
Keeping the deforestation, tissue culture problem and multiple utility of these two species in mind, we
describe here a preliminary study of micropropagation through callus culture for preservation and production of
micropropagated plant for future restoration of degraded mangrove forest areas. To our knowledge, this is the
first report of callus initiation as well as in vitro investigation of Heritiera fomes and Bruguiera gymnorhiza.

Plant materials
Different explants were collected from an respective 810 years old tree at various seasons all over the year
from Gosaba region (88 3946" East and 22 1545" North) of Indian Sundarban Mangrove forest.
Preparation of explants
Firstly explants were washed with running tap water, then dipped in 2% teepol solution for 8 min and
washed two to four times with sterile distilled water. The explants were then surface sterilized with 0.10.2%
HgCl2 (w/v) solution for different time duration to standardize the surface sterilization protocol. Thereafter they
were dipped in 70% ethanol for 12 minute and finally they washed three times with sterile distilled water to
remove any traces of the HgCl2 and ethanol.
Culture Media and Conditions
For Heritiera fomes surface sterilized segments (1.01.5 cm long) were cultured on modified MS medium
(Murashige & Skoog 1962) having complete omission of ammonium nitrate and half concentration of potassium
nitrate with 3% (w/v) sucrose for callus initiation and further experiments. On the other hand for Bruguiera
gymnorhiza surface sterilized segments (1.01.5 cm long) were cultured on another type of modified MS
medium having slight modifications including thrice addition of micro salts and addition of beef extracts, yeast
extracts and casein hydrolysates into medium at the concentration of 50 mg l-1 each with 3% (w/v) sucrose for
callus initiation. For further experiments the sucrose concentration were altered to design the experiment. The
Kader et al. (2015) 2(3): 192203
.
pH of the medium was adjusted to 5.65.8 before autoclaving. To eliminate browning problem polyvinyl
pyrrolidone (PVP) was used to treat explants at the concentrations of 1gm l-1. The explants initially were
implanted vertically on the culture medium and plugged tightly with non-absorbent cotton. All the cultures were
kept under cool fluorescent light (16 h photo period 40 mol m2 s1 at 252 C) and 6070 % relative humidity.
For this study 2, 4-D and NAA in combination with BAP were used. For induction of callus and determining the
degree of salt tolerant, NaCl was added in the medium at various concentration of this experiment. The callus
initiation rate (the ratio of the number of explants pieces having calli to total number of explants pieces planted
in the same culture) was scored about one month after planting. Callus growth was measured by increment in
fresh weight. In the present study the callus growth was measured after 2 months of inoculation. Culture tube
containing medium was weighed just before and after inoculation. The difference in weight gave the fresh
weight of the inoculated tissue. In successive cultures the total amount of tissues were transferred to pre-
weighed fresh medium. 50% excess was taken for sacrificing due to contamination though all the experiments
were done under aseptic condition in laminar air flow chamber.
Figure 1. Callus initiation, in vitro sprouting of different explants, histological section and media discoloration of Heritiera
fomes: A, Callus initiation site after 10 days of explants inoculation using NAA and BAP combination of H. fomes; B,
Yellow and light brown callus formation after 2 weeks of inoculation of explants of H. fomes; C, General view of 3 week old
protuberance produced at the proximal part of the explant (Vetical section), VC- Large vaculated cells, Black arrow indicates
calli at inner region containing both small meristematic cells with highly-stained nucleus in mitotic cells zone (MCZ), Green
arrow indicates embyogenic cells where red arrow indicates non embryogenic cells; D, Close view of embryogenic cells on
callus where black arrow shows the embryogenic cells; E, Media discoloration caused by secretion of tannin or phenolic
compounds after five days of inoculation; F, In vitro sprouting of leaf using NAA and BAP in combination; G, Deep brown
small callus formation in combination of 2, 4-D and BAP.
Kader et al. (2015) 2(3): 192203
.
Histological preparation
For histological studies, the explants were fixed in FAA (formaldehyde: acetic acid: ethanol; 100:50:50)
solution for 10 days. The fixed samples were washed for 40 min, twice with double distilled water. After
washing, the fixed samples were dehydrated through the ethanol series (30%, 50%, 70%, 80% and 90%) for 30
min at each stage. The samples were embedded in paraffin wax (melting point 58C) and section vertically at
10M thickness on a rotary microtome. The sections were mounted onto slides and allowed to dry for at least 10
min before staining. The specimens were stained with hematoxylin-eosin and counter stained with safranin
solutions. The sections were then examined under phase contrast microscope.
Statistical Analysis
Experiments were set up in completely randomized design. Each experiment was repeated three times with
10 - 13 replicates. Data were analyzed by one way analysis of variance (ANOVA) and the difference between
means were scored using Duncans Multiple Range Test P 0.05 (Duncan 1955) on the statistical package of
SPSS (Version 10).
RESULTS
Selection of explants for callus initiation
Among the different explants used, leaves were not found to be suitable for callusing as it showed only in
vitro sprouting for both the species. For Heritiera fomes callus was obtained from nodal and internodal segments
only (Fig. 1) whereas for Bruguiera gymnorhiza, the callus formation was best observed with shoot tip (Fig. 2).
Figure 2. Callus initiation, histological section and media discoloration of Bruguiera gymnorhiza: A, Media browning by
secretion of phenolic compounds of explants of B. gymnorhiza; B, Showing fungal contamination by explants; C, Showing
bacterial contamination; D, White callus formation after 2 weeks of inoculation of explants; E, General view of 4-week-old
protuberance produced at the proximal part of the explants (Vertical section) with wound callus formation; F, Showing
extensive enlargement of scutellar parenchyma cells and the formation of peripheral pockets of dividing scutellar cells. Calli
inner region containing both small meristematic cells with highly-stained nucleus in mitotic cells zone (MCZ) and
vacuolated large cells; G, Higher magnification of the MCZ: Blue arrow indicates accumulation of soluble carbohydrate
(sucrose and/or glucose) or tannin and Black arrow indicates 4 thick-walled proembryonic cells.
Kader et al. (2015) 2(3): 192203
.
Standardisation of surface sterilization protocol for explants
Approximately 70% of successful sterilization was achieved from 0.1% HgCl2 (w/v) solution treatment for
10- 15 minute incubation period for H. fomes. However for surface sterilization of B. gymnorhiza 0.2% HgCl2
(w/v) solution treatment for 15-20 minute incubation period were required (Table 1) for efficient sterilization.
Although initially some other treatments showed promising result however after 2 to 3 weeks of incubation,
some cultures that had showed no contamination was found to be contaminated (Fig. 2).
Table 1. The effect of different concentration of HgCl2 solution for different periods of time for removal of microbial
contamination for Bruguiera gymnorhiza.
Percentage of HgCl2 Incubation Period (minutes) Aseptic inoculation (%)
Solution (w/v)
0.10 10 5.580.00d
0.10 15 7.684.43cd
0.10 20 30.255.12cd
0.15 10 25.384.43cd
0.15 15 30.502.56c
0.15 20 51.022.56b
0.20 10 46.157.69b
0.20 15 76.666.78a
0.20 20 76.666.78a
Note: Values in the last column are Mean SE of Mean followed by the letters within the column indicating the
level of significance at P0.05 by Duncans Multiple Range Test (same letter within the column of the
treatments indicates the absence of difference; different letters indicate the significant difference; and
combination of letters indicate no significant difference).
Elimination of browning problem

Generally media browning is caused by the secretion of phenolic compounds and its callus inhibition activity
was discussed by various authors (Gill et al. 2004). Before inoculation and after sterilization the explants were
treated with PVP solution at the concentration of 1gm/L (W/V) for 45 min and kept the culture after inoculation
was kept in darkness for seven days which was found to remove media discoloration sufficiently (Figs. 1 & 2).
Effect of sucrose on callus initiation
To check the effect of sucrose concentration on callus initiation, we incubated the cultures on different
sucrose concentration containing medium like 1%, 2%, and 3%. Among them, 3% (w/v) sucrose containing
medium gave best results for H. fomes while for B. gymnorhiza gave best result on 2% (w/v) sucrose containing
(Fig. 3).
Figure 3. Effect of sucrose concentration on callus response of Bruguiera gymnorhiza.

Callus initiation rate between different hormone concentrations
For both the species, callus initiation was observed within 2 to 3 weeks after inoculation. Higher rates of
callus initiation were obtained at combinations of 2 mg l-1 and 0.5 mg l-1 BAP and NAA respectively for H.
Kader et al. (2015) 2(3): 192203
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fomes (Table 2) and for B. gymnorhiza it was 1.5 mg l-1 and 0.5 mg l-1 NAA and BAP respectively (Table 3).
Before fixing in FAA for histological preparation, the B. gymnorhiza callus showed white, compact and almost
smooth nature while the H. fomes callus was yellow or light brown in colour and compact and nodular in nature
in respective hormone combination (Figs. 1 & 2). It was observed that 2, 4-D failed to initiate callus on B.
gymnorhiza. The action of 2, 4-D on callus initiation showed slow growth rate for H. fomes (Table 3), the callus
being deep brown in colour (Fig. 1) and dormant in nature. From these result it seems that 2, 4-D was not
suitable for callus culture for these species. During this study it was observed that the rate of callusing response
of mangroves were very low and generally initiated callus showed slow growth than other territorial plants and
it may be because of their fluctuating and extreme environment of their habitat. Similar results were obtained by
Mimura et al. (1997).
Table 2. Rate of callus initiation of Heritiera fomes in modified MS medium at different concentrations of plant
hormones (mg l-1).
Hormone Concentrations (mg l-1) Callus response (%) Nature of Callus
NAA 2, 4-D BAP
0 0 0 0.000.00h -
defg
0.5 - 0.5 17.945.12 Soft, Granular, Yellow
1.0 23.076.28def Soft, Granular, Yellow
2.0 69.254.44a Soft, Granular, Yellow
1.0 - 0.5 23.224.30def Soft, Granular, Yellow
1.0 25.632.56cde Soft, Granular, Yellow
2.0 15.384.43efgh Soft, Granular, Yellow
cde
1.0 41.026.78bc Soft, Granular, Yellow
cd
1.0 17.942.56defg Soft, Granular, Yellow
2.0 28.206.78cde Soft, Granular, Yellow
2.5 - 0.5 28.205.13cde Soft, Granular, Yellow
1.0 51.272.56b Soft, Granular, Yellow
h
- 0.5 0.5 0.000.00 -
1.0 7.694.43fgh Hard, Deep brown
2.0 2.562.56gh Hard, Deep brown
defg
- 1.0 0.5 17.946.78 Hard, Deep brown
1.0 23.074.43def Hard, Deep brown
2.0 15.384.43efgh Hard, Deep brown
- 1.5 0.5 28.205.13cde Hard, Deep brown
1.0 41.026.78bc Hard, Deep brown
def
- 2.0 0.5 20.519.24 Hard, Deep brown
1.0 28.205.13cde Hard, Deep brown
- 2.5 0.5 19.223.84defg Hard, Deep brown
1.0 28.205.13cde Hard, Deep brown
Note: Values in the second last column are Mean SE of Mean followed by the letters within the
column indicating the level of significance at P0.05 by Duncans Multiple Range Test (same letter
within the column of the respective hormone indicates the absence of difference; different letters
indicate the significant difference; and combination of letters indicate no significant difference).
We also tried to initiate callus on different media like MS, Woody Plant medium (WPM, Lloyd & McCown
1981), Linsmaier and Skoog (LS) medium (Linsmaier & Skoog 1965), X medium (Rao et al. 1998) and Amino
Acid (AA) medium (Thompson et al. 1986) but there were no response found for these species.
Kader et al. (2015) 2(3): 192203
.
Histological study of initiated callus
The histological study showed the production of protuberance at the proximal part of the explant (horizontal
section) of H. fomes. This study of both species showed the formation of mitotic cells zone (MCZ) i.e.,
parenchymatous cells with nucleus and dense cytoplasm dividing actively (Figs. 1& 2). It also noted that in the
intercellular spaces of callus tissue there was a more or less dense fibrillar or reticular network which is
complexes of polysaccharide polymers having a microfibrillar network texture (Fig. 2) or accumulation of
tannin. B. gymnorhiza callus also indicated extensive enlargement of scutellar parenchyma cells and the
formation of peripheral pockets of dividing scutellar cells. Two types of cells present in callus might be
distinguished: embryogenic and nonembryogenic (Figs. 1 & 2).
Table 3. Rate of callus initiation of Bruguiera gymnorhiza in modified MS medium at different concentrations of plant
hormones (mg l-1).
NAA BAP Callus Response (%) Nature of Callus
(mg l-1) (mg l-1)
0 0 0.000.00f -
e
0.5 0.5 12.815.12 Hard, Compact, White
1.0 5.122.56bcef Hard, Compact, White
1.0 0.5 53.847.69b Hard, Compact, White
1.0 28.205.13d Hard, Compact, White
a
1.0 56.405.12b Hard, Compact, White
2.0 0.5 56.402.56b Hard, Compact, White
1.0 38.882.77cd Hard, Compact, White
bc
1.0 43.582.56bc Hard, Compact, White
Note: Values in the second last column are Mean SE of Mean followed by the letters within the column
indicating the level of significance at P0.05 by Duncans Multiple Range Test (same letter within the column
of the respective hormone indicates the absence of difference; different letters indicate the significant
difference; and combination of letters indicate no significant difference).
Effect of salt concentration on callus initiation and growth

Bruguiera gymnorhiza gave maximum callus response and growth at the concentrations of 60 mM NaCl
(Fig. 4) concentrations whereas endangered and endemic H. fomes gave best callus response and growth at the
concentration of 20 mM NaCl (Fig. 5).
Figure 4. Effect of salt concentration on callus initiation and growth of Bruguiera gymnorhiza.
Kader et al. (2015) 2(3): 192203
.
Seasonal effect on callus formation
This investigation was carried out in different seasons viz., rainy season, winter season and summer season
to check the seasonal effect for callus formation. From this experiment it was found that for callus culture of this
two species rainy season was best time (Fig. 6) as compared to other seasons which generally showed explants
dormancy and excretion of phenolic compound vigorously.
Figure 5. Effect of salt concentration on callus initiation and growth of Heritiera fomes.
Figure 6. Effect of various seasons on callus initiation in modified MS medium: A, Bruguiera gymnorhiza; B, Heritiera
fomes.
DISCUSSIONS
Literature studies indicated that the leaves of mangrove species are excellent source for callusing or cell
culture studies (Mimura et al. 1997, Hayashi et al. 2009). However in this study both the species failed to
produce callus from leaf. They gave callusing response either on shoot tip or nodal and intermodal segments
which was not previously reported for any mangrove species. This may be due to the selection of explants, their
size, age and ecological factors, which greatly influence the success of the in vitro culture which varies widely
from plant to plant (George 1993).
In this study mercuric chloride was used as surface sterilant as it is the mostly used surface sterilant for
tissue culture. Here for surface sterilization of Bruguiera gymnorhiza 0.2% HgCl2 (w/v) solution treatment for
Kader et al. (2015) 2(3): 192203
.
15- 20 minute incubation periods was required.
In vitro response of most of the mangrove species till now reported maximum were done with MS medium
viz Yasumoto et al. (1999) reported for Sonneratia alba; Al-Bahrany & Al-Khayri (2003) reported for Avicennia
marina; Ogita et al. (2004) reported for Kandelia candel; Kawana & Sasamoto (2008) reported for Sonneratia
alba and Bruguiera sexangula; Hayashi et al. (2009) reported for Avicennia alba, Avicennia marina, Sonneratia
alba and Bruguiera sexangula. In this present work for callus culture of Bruguiera gymnorhiza and Heritiera
fomes, the MS medium was modified. Similar findings of use of modified MS medium for cultivation of
mangroves were also reported by Rao et al. (1998), formulated a new medium named X for Excoecaria
agallocha; Vartak & Shindikar (2008) for Bruguiera cylindrica. Besides for optimum growth of B. gymnorhiza
yeast extract and casein hydrolysates were added. It may happen because mangroves are unique in their
physiological adaptations. They can tolerate various nutrient statuses, water logging and various levels of sea
salts (Feller et al. 2002). Similarly plant tissue culture depends on media composition with special emphasis to
growth regulators, carbon source and organic additives, the genotype and the source of explants. In this study
we found that B. gymnorhiza gave no callus response in MS media but when the media was formulated with
thrice micro nutrients and addition of organic substances i.e., yeast extracts and casein hydrolysate the callus
initiation occurred. It may have happened because they are naturally adapted to higher micronutrients and
organic matter, particularly for Sundarban forest (Naskar & GuhaBakshi 1987, Ramanathan et al. 2008). In
Sundarban mangrove region sulphates are higher (Naskar & GuhaBakshi 1987, Ramanathan et al. 2008). In our
study, addition of thrice concentration of micro nutrient was actually the addition of higher sulphates as
micronutrient to MS. It is well known that in plant tissue culture media, casein hydrolysate is a rich source of
different amino acids (Shahsavari 2010, Talapatra & Raychaudhuri 2012). Addition of casein hydrolysate in the
medium is required because Mimura et al. (1997) showed that the Amino Acid medium (Thompson et al. 1986)
enhanced the rate of callus initiation of Bruguiera sexangula which is another species of the Bruguiera genus.
From this study and from the study of Mimura et al. (1997) it seems that somehow amino acids play a crucial
role for in vitro response particularly for this genus and casein hydrolysate was found to be fit for this type of
study.
The physiological state of the donor plants on growth depends on environment, which affects the response of
explants under in vitro conditions (Jahne & Lorz 1995). The MS medium presented here for H. fomes had low
nitrogen level as compared to other plant medium which supports the low nitrogen level in mangrove region
(Feller et al. 2002, Feller et al. 2003) as well as Sundarban mangrove forest as there is no humus deposition in
the soil (Naskar & Bakshi 1987, Ramanathan et al. 2008). So it seems that mangrove species can grow in vivo in
low level of nitrogen, which was also retained in tissue culture conditions. Similarly in vitro response to low
level of nitrogen was also reported by Mimura et al. (1997), Rao et al. (1998) and Arumugam & Panneerselvam
(2012). The complete omission of ammonium nitrate may be essential as it has toxic effect in many higher plant
species which inhibit plant growth (Britto & Kronzucker 2002, Shanjani 2003).
In this study B. gymnorhiza respond and grew better in 2% sucrose concentration in respective medium. In
mangrove ecosystem carbon source are varied and in Sundarban the parcentage of organic carbon source was
too low (Naskar & GuhaBakshi 1987). In the present study with B. gymnorhiza we found that callus initiation
rate was highest in 2% (w/v) sucrose containing medium which correlate with nutrient status of Sundarban
mangrove ecosystem.
From this experiment it was found that the NAA could alone or in combination with BAP initiate callus
from mangrove species. However in case of 2,4-D it failed to initiate callus alone or in combination with with
BAP from Bruguiera gymnorhiza. It may be because recent study showed that 2,4-D has toxic effect on plants
i.e., it alters the chlorophyll, protein and phenol content (Peixoto et al. 2007). Generally high concentrations of
cytokinin and low concentrations of auxins favour shoot response. Here in case of Heritiera fomes this type of
ratio too favoured callus initiation from this species.
In this experiment two different mangroves showed different pattern of NaCl tolerance. Based on this study
it was found that the Bruguiera gymnorhiza gave best callus initiation and growth at 60 mM NaCl
concentration. Mimura et al. (1997) found that seedling callus of Bruguiera sexangula gave highest growth at
100 mM NaCl. Sundarban this species is very common on the side of creeks and river beds and plays a
dominant role for its better adaptation to the higher degree of salinity and tidal influences (Naskar &
GuhaBakshi 1987). Heritiera fomes calli showed better response and growth at 20 mM NaCl concentration.
Kader et al. (2015) 2(3): 192203
.
Hossain et al. (2014) showed that Heritiera fomes prefers extremely low saline condition for their survival and
growth. Recent studies on palynological evidence and salinity influences have clearly showed that Heritiera
fomes has declined relatively recently as the salinity has increased in Indian Sundarban regions which are also
retained in this experiment (Naskar & GuhaBakshi 1987, Gopal & Chauhan 2006, Mitra & Banerjee 2010).
Under natural conditions, mangroves exhibit clear tolerance of salts differences among species. Findings of the
present study are comparable with naturally relative tolerance levels of two different mangrove species.
From this experiment it was found that, for tissue culture of these species rainy season was best time as
compared to other seasons which generally showed explants dormancy and excretion of phenolic compound
vigorously. Many tree species which are collected during rainy season (active growth time) shows tremendous
growth in in vitro conditions because physiological state of tissue of tree species varies due to variation of
season (El-Morsey & Millet 1996). During winter season the explants showed low viability i.e., dormant in
nature and exuded maximum phenolic compounds. This may be because the cytosolic ribosome contents are
altered in winter metabolism at cellular level in tree species (Haggman 1986).
Land destruction in coastal region is another important factor for mangrove extinction (Ohnishi &
Komiyama 1998). In Indian Sundarban region approximately 50% of land has been destroyed for human
habitation and settlement, agricultural development and brackish water fisheries (Ramanathan et al. 2008). The
present study clearly indicates that these species may be restored in low saline or none of saline land as callus as
it is being extensively used for afforestation programmes (Ahuja 1991) and tapping useful compounds from
plants. The present investigation is a primilary study for micropropagation of mangrove species as most
mangrove species remain very hard to be established through cell culture systems or callus culture (Kawana &
Sasamoto 2008). The totipotent character of plant cells and tissues can be expressed by their ability to
regenerate into plants via embryogenesis or organogenesis and for this histological study of callus is very much
needed. Both processes lead to in vitro regeneration and are a major prerequisite for genetic transformation
study. However, the widespread application of gene transfer techniques for crop improvement cannot be
successfully achieved if the processes leading to morphogenesis are not well understood. Callus culture give
tools for genetic cell transformation by somaclonal variation, induced mutagenesis and genetic engineering
which are not only much more rapid than conventional breeding but can also give rise to novel genes and
genotypes rather than other traditional methods like mass selection, inbreeding and hybridization which is
laborious and time consuming depending on environmental conditions and existing gene pool(s) for plant
development (Ahmad et al. 2010). This study can thus provide opportunities of micropropagation of these
multipurpose mangrove plants.
According to UNESCO there are approximately 50 different types of true mangrove species are found
worldwide. Among them less than 10 true mangrove species were reported for in vitro study. It seems from this
study that different environmental condition or stress which promotes the growth of mangroves in vivo greatly
influences the in vitro culture of mangroves. Previous in vitro studies on mangroves were mostly based on the
effect of variety of hormones with their different concentration taken and different effect of sea salts taken with
their different concentration. However this present study clearly indicates that the in vitro studies of mangroves
not only depend on variety hormones or different sea salts but greatly influence by soil condition of their
habitual environment, seasonal condition etc. From this study it also seems that more and more in vitro studies
of mangroves are possible if researchers focus on their habitual environmental conditions. The results presented
here give an insight into the in vitro studies suitable for mangrove species which is correlated with various
environmental factors of mangrove ecosystems. Potentially, higher callus efficiency may be achieved through
investigating medium components other than this study, hormones other than used in this study and use of
various sea salts other than used in this study.
ACKNOWLEDGEMENTS
The authors are grateful to University Grant Commission (UGC), New Delhi, for providing financial
assistance to the first author. The authors extended their thanks to the head of the department of botany,
University of Kalyani for providing DST-FIST central equipment facility. The authors are thankful to Dr.
Soumen Saha for help in statistical analysis. They are also thankful to Subhas Bhaumik for help in histological
analysis.
Kader et al. (2015) 2(3): 192203
.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(3): 204214, 2015
Research article
Plant community structure and composition in secondary

succession following wildfire from Nues Ardentes of
mount Merapi, Indonesia
Sutomo1, 2*, Richard J. Hobbs1 and Viki A. Cramer1
1
School of Plant Biology the University of Western Australia, 35 Stirling Hwy, Crawley, Perth, Western Australia
2
Bali Botanic Garden, Indonesian Institute of Sciences (LIPI), Candikuning, Baturiti, Tabanan, Bali, Indonesia
*Corresponding Author: sutomo.uwa@gmail.com [Accepted: 20 October 2015]
Abstract: Patterns of plant community structure and composition during secondary succession
following volcanic-fire induced disturbance of nues ardentes was examined in Mount Merapi
National Park, Indonesia. Five sites with different age (time since fire) and one undisturbed site
were sampled. Species richness, diversity, turnover and importance value index (IVI) were
calculated. Sixty one species belonging to 29 families were recorded in the study sites. The highest
number of species belonged to the Poaceae (10), followed by Fabaceae (9) and then Asteraceae
(6). The number of species present varied as time progressed with a rising trend of species richness
and diversity over time and significant differences in species richness and diversity among sites
(ANOVA, p = 0.05). Species turnover was highest between the 2006 and 1998 sites, and then
between the 1997 and 1994 sites. Species turnover between the 1998-1997 sites was similar to the
turnover between the 1994 site and the reference site. In terms of vertical structure, four strata
were identified in the fire sites whereas in the reference site, all five stratums (A, B, C, D, and E)
were present. In terms of quantitative structure based on IVI, each site had different dominating
species for tree, groundcover and seedling layers. Non metric multidimensional scaling (NMDS)
ordination of plots and analysis of similarity (ANOSIM) test results showed that there were
significant differences in species composition between sites (Global RANOSIM = 0.93, P < 0.001). In
the Mount Merapi succession, the changes in abundance of some invasive species such as I.
cylindrica, Brachiaria spp., and Eupatorium spp. are important to note. These invasive species
have different timing in entering the system, but Imperata cylindrica was noted almost constantly
in every stage of succession except in the undisturbed site.
Keywords: Plant community - Volcanic fire - Disturbance - Secondary succession.
[Cite as: Sutomo, Hobbs RJ & Cramer VA (2015) Plant community structure and composition in secondary
succession following wildfire from Nues Ardentes of mount Merapi, Indonesia. Tropical Plant Research 2(3):
204214]
INTRODUCTION
In active volcanoes, volcanic activity remains the most significant threat to forest vegetation (Lavigne &
Gunnell 2006, Whitten et al. 1996). Fire is an integral part of volcanic disturbance and has shaped community
composition in montane forests of Java (van Steenis 1972, Whitten et al. 1996). On Mt. Merapi, the intense heat
(often more than 700 C) released from nues ardentes ignites wildfires (Bardintzeff 1984). A nue ardente
(French for glowing cloud) is the rapid movement of extremely hot (often more than 700 C) turbulent gases
and fragmental material across a land surface from a volcanic vent. The denser part of a pyroclastic flow, hugs
the ground and follows topography and moving with great force and speed (up to 200 km h-1) (Dale et al.
2005a).
The montane forests of Java and Bali are not resistant to fire (Marrinan et al. 2005). The forests are easily
ignited under conditions of prolonged drought, and when lightning strikes oil-rich species such as Vaccinium
spp. On Mt. Merapi, nues ardentes are the primary cause of forest fire (Simon 1998, Whitten et al. 1996).
Recovery of the montane forest following fire is usually slow (Horn et al. 2001). Fire destroys the aboveground
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part of shrubs and some surviving species may be covered with ash, which could slow the rate of the secondary
succession (Antos & Zobel 2005, Whitten et al. 1996). Severely burned areas on mountains in Java and Bali are
usually characterized by the increase in abundance of invasive species, such as alang-alang grass (Imperata
cylindrica), and also white-leaved edelweis (Anaphalis longifolia) and bracken fern (Pteridium aquilinum)
(Whitten et al. 1996). Homalanthus giganteus is also a common pioneer tree species that occurs during
secondary succession in these areas (van Steenis 1972).
Li et al. (1999) stated that many succession theories were based on intensive work in temperate forests.
Gomez-Pompa and Vasquez-Yanes (1981) and Chazdon et al. (2007) studied secondary succession that occurs
in the tropics; however their findings were based on work on old fields or in lowland tropical forests. Other
forest types such as volcanic tropical montane forest have received little attention (Tsuyuzaki & Hase 2005,
Whittaker et al. 1999). Furthermore, it is increasingly acknowledged that one model fits all is not appropriate
for all communities and ecosystems due to the complexity of each system (Hobbs et al. 2007).
The objective of this study was to describe plant community structure and composition in secondary
succession using a chronosequence of sites that had been burnt by fires caused by nues ardentes in the tropical
montane forest of the Merapi Volcano National Park. We were particularly interested in whether there are any
differences in species diversity, turnover, and community structure and composition across sites of different
ages, and which species contributed the most to these differences.

Site description
Mt. Merapi (7 35' S and 110 24' E) is administratively located in two provinces, Central Java (Magelang,
Boyolali and Klaten Districts) and Yogyakarta (Sleman District). In Yogyakarta Province, Mt. Merapi ( 2,900
m asl) is located approximately 30 kilometres north of Yogyakarta. Mt. Merapi is representative of the
landforms, soils and vegetation on a volcanic mountain that typify a large portion of montane ecosystems in
Java (Whitten et al. 1996). Based on Schmidt and Fergussons climate classification (1951), the Merapi area is
classified as type B - tropical monsoon area, which is characterized by a high intensity of rainfall in the wet
season (NovemberMarch) with a dry season that can often be very dry without any rainfall (AprilOctober).
Annual precipitation varies from 2,5003,500 millimetres (Anonym 2004). The variation of rainfall on Mt.
Merapis slope is influenced by orographic precipitation. As in many other tropical monsoon areas, there are
minor temperature and humidity variations during the year. Relative humidity on Mt. Merapi varies from 70%
90%, with daily average temperatures from 19 to 30 C (Dinas Kehutanan DIY 1999). Soils of the study area
are mainly of young volcanic-ash origin (regosol) with shallow and/or deep, low to medium fertility solums with
a profile not yet developed (Anonym 2004, Darmawijaya 1990). The soil textures are granulated, whereas the
structures are crumbly (Anonym 2004, Dinas Kehutanan DIY 1999).
On Mt. Merapi, areas which have been completely buried by the nues ardentes deposits undergo primary
succession. These areas usually occur along the streams, channels or valleys created by the solid material flow
paths of nues ardentes. The secondary succession areas were located adjacent to the primary succession areas.
These areas are the adjacent forest on either side of the valley or deposit channel which escapes burial and is
mainly scorched by the extreme temperature of the nues ardentes. Sites or areas of different ages (years since
last nue ardente) were selected to obtain a chronosequence. Identification of site age was conducted by
studying aerial photographs, topography maps, and nue ardente history maps (obtained from the Merapi
Volcanology Observatory) to date sites affected by recent nues ardentes. Identification was also conducted by
reconnaissance study, interviewing long-term residents of the surrounding villages, personal communication
with the national parks ranger and managers, and also field site visits. Sites also had to show no obvious signs
of human disturbance and be at least 50 metres from any human activities or structures. Based on these, we
chose four sites that were affected by nues ardentes at different times (2006, 1998, 1997 and 1994) and one
forest area that was mostly undisturbed and had not been affected by nues ardentes for at least 50 yr as a
reference site (Fig. 1). The five sites were located in a lower montane zone and were located at a range of
altitudes from 1,000 to 1,600 metres. Chronosequence assumptions were met within these sites as they had
similar environmental conditions such as climate, substrate, topography and geomorphology, although we
acknowledge the limitations of the chronosequence approach and the potential for site-specific factors to be
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important. The fieldwork was conducted from March to August 2008. Average environmental conditions in
each site are summarized in table 1.
Figure 1. Map of mount Merapi National Parks eruption deposit sites (Circular symbols refer to the position of sampling
sites in each deposit. The rectangle refers to the site position of an undisturbed forest in Kaliurang).
Table 1. Site location, nues ardentes history and environmental information in each study sites at Mt. Merapi National Park.
Location Year of nue ardente Site age (years) Soil type Elevation (m)
Kaliadem 2006 2 Regosol 1,220
Kalilamat 1998 10 Regosol 1,579
Kalibedog 1997 11 Regosol 1,207
Kalikuning 1994 14 Regosol 1,180
Kaliurang - - Regosol 1,000
Vegetation sampling
In April 2008, vegetation was sampled in each of the four sites burnt by fire from nues ardentes in 1994,
1997, 1998 and 2006. One area of unburnt forest (the reference site) was also sampled. The position and altitude
of each site were recorded using a GPS, and slope was measured using a clinometer. At each site, an area of
approximately 2.5 hectares was chosen and five circular plots (diameter range approximately 60 metres) were
randomly placed in the chosen area. In each of these larger plots, three sets of circular plots of 10, 5 and 2
metres diameter were nested within each other to measure trees (10 metre plots), groundcover (5 meter plots)
and seedlings (2 metre plots) (Isango 2007, Supriyadi & Marsono 2001). The species name, height and
diameter of trees (dbh 10 cm) and young trees (dbh 2.09.9 cm, height 1.3 m) were recorded. Understorey
plants and seedlings were counted (Kent & Coker 1992). All plants were identified to species level when
possible. Identification was conducted at the dendrology laboratory, Faculty of Forestry, Gadjah Mada
University Yogyakarta, Indonesia. Identification was done using flora books such as The Flora of Java
(Backer & van den Brink 1963) and Mountain Flora of Java and the results were confirmed by a botanist in
the Faculty.
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Data analysis
Species diversity at each site was calculated using the Shannon-Wiener diversity index. Differences in
diversity between sites were tested for significance using a one-way ANOVA in SPSS package V.11.5. To
examine short term species turnover (beta diversity), a modified Sorensens community correspondence index
or CCI was used (Barbour et al. 1980, Cook et al. 2005) with the formula as follows:
2c
CCI
ab
Where, a = the number of species present in the first community, b = the number of species present in the
second community, and c = the total number of species found in both communities.
I then calculated D, which is an index of how much a species list changes across sites with the formula as
follows (Cook et al. 2005):
D 1 CCI
This index ranges from 0 to 1, and a low value indicates little change in the species composition between
sites whereas a high value indicates the opposite.
In order to examine the vertical structure, forest vegetation was divided into five strata (A, B, C, D and E), as
recognized for humid tropical forests (Simon 1996). Stratum A consisted of emergent trees more than 35 metres
tall. Stratum B was the main canopy layer with trees 1835 metres in height. Stratum C consisted of young trees
818 metres tall. Stratum D consisted of shrubs and sapling (of trees) with height ranges from 1.55.0 metres.
Stratum E was the groundcover layer, including grasses, herbs, tree seedlings and fern allies (Simon 1996). The
number of trees, young trees (poles), sapling and shrubs that have the characteristics of stratum A, B, C and D
were noted, while the number of groundcover species was noted for the E stratum in each of the study sites.
Importance Value Index (IVI) (Curtis & McIntosh 1950, Kent & Coker 1992) was used to describe the
quantitative structure of the community. This statistic represents the contribution that a species makes to the
community in terms of the number of plants within the plots (density), its contribution to the community
through its distribution (frequency), and its influence on the other species through its dominance. Importance
Value Index was calculated for each species of tree and groundcover in each of the study sites. The formula for
tree IVI is as follow:
IVI = RD + RF + RDm
Where, RD = relative density of a species, RF = relative frequency of a species and RDom = relative
dominance of a species.
Number of individual of A species
Relative Density of species A = 100%
Total number individual of all species
Frequency value of A species

Relative Frequency of species A = 100%
Total frequency value of all species
Dominance value of A species

Relative Dominance of species A = 100%
Total dominance value of all species
Dominance values for a tree species were obtained by dividing the basal area of the tree with the size of the
plot (Simon 1996, Supriyadi & Marsono 2001). The IVI formula for groundcover species (including seedlings)
was similar to the tree layer but without the calculation of relative dominance (Kusmana 1995), and so the
formula is as follow:
IVI = RD + RF
Where, RD = relative density of a species, and RF = is relative frequency of a species.
Species abundance data were square root transformed prior to all multivariate analyses. A resemblance
matrix based on a Bray-Curtis similarity index was generated as a basis for the subsequent ordination and cluster
analyses. Plant species composition and abundance at each site were compared using non-metric
multidimensional scaling ordination (NMDS) (Clarke 1993). Statistically significant differences in species
composition and abundance between the sites were determined by analysis of similarity (ANOSIM), which tests
the null hypothesis that there is no difference in species composition and abundance among groups (Clarke
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1993). SIMPER, an analysis that calculates the average Bray-Curtis dissimilarity between all samples, was used
to identify the species that differentiate sites (Clarke 1993). These analyses were done using PRIMER V.6
(Clarke & Gorley 2005).
RESULTS
Sixty one species belonging to 29 families were recorded in the study sites. The highest number of species
belonged to the Poaceae (10), followed by Fabaceae (9) and then Asteraceae (6). There were significant
differences in species richness between sites (ANOVA P = 0.05, table 2). Species richness was lowest at the
2006 site and highest at the 1994 site. Species richness in the reference site (undisturbed site) was much lower
when compared to the 1994 site. Species richness in the reference site was significantly lower than in all but the
2006 site.
Table 2. Species richness and diversity in the burnt sites and reference site in mount Merapi National Park. Superscript
letters (a-c) after mean values (SD) indicates significant difference between sites as assessed with Tukeys HSD test. Dates
are those in which the site was burnt by fire generated by nues ardentes.
ANOVA group/years since fire Species richness Species diversity
2006 site 9.20 (1.48)a 1.91 ( 0.19)a
1998 site 14.0 (3.39)b 2.13 ( 0.27)ab
1997 site 15.4 (1.51)b 2.41 ( 0.19)bc
1994 site 19.4 (2.96)c 2.7 ( 0.21)c
Reference site 10.6 (1.67)a 2.21 ( 0.27)ab
The changes in species diversity are not as distinct as the changes in species richness over time (ANOVA P
= 0.05). The reference site is significantly different to the 1994 site, but not significantly different from 2006,
1998, and 1997. The 1998 site is not significantly different from 2006 and 1997 sites and also the 1997 site is
not significantly different from the 1994 site.
Species turnover was highest (lowest species similarities) between the 2006 and the 1998 sites, and then
between the 1997 and 1994 sites (Table 3). Species turnover between the 19981997 sites was similar to the
turnover between the 1994 site and the reference site.
Table 3. Species turnover rates (D) between pairs of sites in the chronosequence on mount Merapi.
2006-1998 1998-1997 1997-1994 1994-Ref site
D 0.89 0.63 0.83 0.66
Sorenson Index 0.11 0.37 0.17 0.34
In terms of vertical structure, the number of individuals found in each stratum indicates the presence of the
particular stratum in each site (Table 4). In the 2006 site, stratums B, C, D, and E were recorded. The 1998,
1997 and 1994 sites also had four stratums (B, C, D, and E) whereas in the reference site, all five stratums (A,
B, C, D, and E) were present.
Table 4. Number of individuals in each stratum for each site of secondary succession at mount Merapi. Stratum A refers to
the number of trees that are more than 35 m in height. Stratum B is number of trees that are 18 to 35 m in height. Stratum C
comprises of trees that are 8 to 18 m tall. Stratum D is the total number of saplings and Stratum E is the total number of
groundcover species.
Stratum 2006 site 1998 site 1997 site 1994 site Ref site
A - - - - 41
B 3 5 16 25 28
C 5 45 18 17 1
D 4 10 15 6 11
E 12 20 23 25 16
In terms of quantitative structure, tree and groundcover species in the sites were compared on the basis of the
Importance Value Index, (IVI) (Table 5). In the 2006 site, the tree layer was dominated by Pinus merkusii,
whereas in the 1998 and 1997 site, Homalanthus giganteus and Paraserianthes falcataria were the most
important tree species. In the oldest site (1994), Schima wallichii and P. merkusii were the most important tree
species whereas in the reference site, Altingia excelsa was the most important tree species. In the groundcover
layer, the 2006 site was dominated by Imperata cylindrica, whereas Eupatorium riparium was the most
important species in the 1998 and 1997 sites. In the oldest site (1994) Brachiaria reptans was the most
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important species, while in the reference site, Selaginella doederleinii was the most important species in the
groundcover layer. In the tree seedling layer, Acacia decurrens was the most dominant tree seedlings species in
the 2006 site, followed by P. merkusii. Albizia lopantha dominated the seedling layer in the 1998 site, while in
the 1997 and 1994 sites Calliandra callothyrsus was the most important seedling. In the reference site, A.
excelsa was the dominant seedling.
Table 5. Importance Value Index (IVI), and shade tolerance for the most important species in each stratum at each of the
study sites. Asterisks indicate exotic species.
Species Shading tolerance 2006 site 1998 site 1997 site 1994 site Reference site
Trees
Acacia decurrens* Intolerant 17.27 6.17 - 58.88 -
Albizia lopantha Intolerant - 33.68 - - -
Altingia excelsa Tolerant 60.63 - - 8.44 217.51
Erythrina sp Intolerant - 12.51 - - -
Homalanthus giganteus Intolerant - 148.65 - - -
Macaranga javanica Intolerant - 31.55 - - -
Paraserianthes falcataria Intermediate - - 116.20 46.83 -
Parkia sp Intolerant - 6.53 29.32 - -
Pinus merkusii Intermediate 222.09 - - 87.85 54.05
Schima wallichii Intermediate - - 104.92 89.0 21.80
Groundcover
Brachiaria reptans* Intermediate - 2.19 19.90 16.54 2.23
Eleusine indica Intermediate - - 8.82 14.49 3.11
Eupatorium riparium* Intermediate - 55.35 57.03 - 13.43
Eupatorium odoratum* Intermediate - 8.69 4.70 1.95 -
Imperata cylindrica Intolerant 73.77 14.18 23.51 9.55 -
Polygala paniculata* Intermediate 26.83 - 2.35 7.60 -
Selaginella doederleinii Tolerant - - 0.87 0.56 61.80
Seedling
Acacia decurrens* Intolerant 99.04 13.88 - 13.57 -
Albizia lopantha Intolerant - 77.77 - - -
Altingia excelsa Tolerant 9.52 - 11.05 7.46 33.35
Calliandra callothyrsus Intermediate - - 146.56 84.04 -
Pinus merkusii Intermediate 62.85 - - 41.31 -
Schima wallichii Intermediate - - 17.10 19.69 22.40
In addition to the IVI, table 5 also shows the presence and absence of the most important (dominant) species
in each layer throughout the succession. In the tree layer, A. excelsa and P. merkusii were present at the
youngest site (2006) and then absent in the next two older sites (1998 and 1997), and then reappeared in the
oldest (1994) and the reference site. Erythrina sp., H. giganteus, Albizia lopantha and Macaranga javanica were
only present at the 1998 site. In the groundcover layer, I. cylindrica was recorded in all four of the burnt sites,
but was more dominant in 2006, 1998 and 1997 sites than in the 1994 site, and was absent in the reference site.
In contrast, Brachiaria reptans was absent in the 2006 site and then present throughout the rest of the
chronosequence and was at a very low abundance in the reference site. Selaginella doederleinii started to appear
in the 1997 and, 1994 sites and became dominant in the reference site.
There was clear separation between the sites as shown by the NMDS ordination result (Fig. 2). Plots from
the youngest site (2006) were separated from plots from the older sites (1998, 1997 and 1994), and from the
undisturbed site. An analysis of similarity (ANOSIM) test confirmed that there were significant differences in
Bray-Curtis species similarities between sites (Global RANOSIM = 0.93, P < 0.001).
Six pairwise comparison tests between sites (2006 and 1998, 2006 and 1997, 2006 and 1994, 2006 and
reference site, 1998 and 1994, and 1994 and reference site) had an R value of 1.0 (Table 6). The comparison
between the 1997-1998 sites and 1997-1994 sites had R-values of 0.72 and 0.86 and also, the comparison
between 1997-undisturbed and 1998 undisturbed had R-value of 0.98 (Table 6).
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Figure 2. NMDS of sites based on vegetation composition and abundance: 2006 site (triangles), 1998 site
(inverse triangles), 1997 site (squares), 1994 site (diamonds) and reference site (circles).
Table 6. ANOSIM pairwise test of NMDS vegetation plots ordination. Sample statistic
(Global R): 0.93, significance level of sample statistic P < 0.001, number of permutation: 999.
Groups R Statistic
2006, 1998 1
2006, 1997 1
2006, 1994 1
2006, Reference site 1
1998, 1997 0.72
1998, 1994 1
1998, Reference site 0.98
1997, 1994 0.86
1997, Reference site 0.98
1994, Reference site 1
In table 7, Eupatorium riparium contributed most to the dissimilarity between the 2006 and 1998 sites
(21.27%), 2006 and 1997 sites (20.96%), 1998 and 1994 sites (16.31%), and 1997 and 1994 sites (21.39%).
Brachiaria reptans contributed most to the dissimilarity between the 2006 and 1998 sites (9.96%) and 1998 and
1997 sites (9.89%). Dichantium caricosum contributed most to the dissimilarity between the 2006 and 1994
sites (10.60%). Selaginella doederleinii was the most important species contributing to dissimilarities between
the reference site and the burnt sites. Imperata cylindrica was the second most important species in the
comparison between 2006 and 1998 sites and 2006 and the reference sites.
DISCUSSION
In the first decade after disturbance by fire there was a rapid recovery at the sites, with 54 species belonging
to 23 families recorded in the secondary forest at the study sites. The highest number of species belonged to the
Poaceae (10), followed by Fabaceae (9) and then Asteraceae (6). Species richness and diversity increased with
time since the fire, however species richness and diversity in the reference site was not significantly different
from the youngest (2006) site. This pattern was similar to that reported in other studies where species diversity
reached its peak in older succession sites after most of the climax species had entered the system, and then
decreased with the loss of the species present in early successional stages (Magurran 1988, Peet 1992, Zhu et al.
2009). The results support the hypothesis of Aubert et al. (2003) that species diversity will increase during the
early succession stage, reach a maximum in the mid-succession stage and decrease in the late succession stage.
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.
Table 7. SIMPER result. Percentage contribution of species to average BrayCurtis dissimilarities in all pairs of sites. Only
those species with a contribution to average dissimilarity of >5% are included. The average dissimilarity value (%) is also
shown for each pair of the sites. Asterixis indicates exotic species.
Site comparison
2006 and Ref site
1998 and Ref site
1997 and Ref site
1994 and Ref site

1998 and 1994
2006 and 1998
2006 and 1997
1998 and 1997
2006 and 1994
1997 and 1994

Species
Brachiaria paspaloides* 6.21 - 6.51 - 5.07 - - 5.98 - -

Brachiaria reptans* - 9.96 9.89 8.01 5.42 8.45 - - 8.30 5.89
Calliandra callothyrsus - 8.01 8.68 - - 6.31 - - 6.80 -
Dichantium caricosum* - - - 10.60 7.81 7.22 - - - 8.08
Eleusine indica - - - 8.96 6.53 - - - - 5.97
Eupatorium odoratum* - - - 9.84 5.29 6.42 - - - 7.42
Eupatorium riparium* 21.27 20.96 5.76 - 16.31 21.39 6.15 13.94 13.60 -
Imperata cylindrica 9.89 - 7.41 9.02 - 5.63 15.14 5.83 9.89 5.13
Polygala paniculata* 6.82 - - - - - 6.81 - - -
Selaginella doederleinii - - - - - - 18.45 16.94 13.77 14.33
Average dissimilarity (%) 88.98 79.38 61.51 75.50 85.38 60.75 95.56 83.35 80.67 87.82
A decrease in the light availability at the forest floor as the succession proceeds might be the cause of the
decline of species diversity in the reference site (Gomez-Pompa & Vazquez-Yanes 1981). Direct shading of
overstorey species inhibits the existence and regeneration or growth of less tolerant and intolerant understorey
species in the reference site (Lep 1990).
There was progressive development of forest structure over time. Although all of the burnt sites had four
strata (B, C, D, E), the number of individuals in each stratum differed. The number of individuals of stratum B
(tall trees 1835 m) was lowest in the 2006 site, greater in the older sites, but was the greatest in the reference
site. The reference site had five strata (A, B, C, D, and E) with the lowest number of individuals of stratum E
compared with the proportion of stratum E in the burnt sites. There were also differences in the patterns of
abundance of the most important species with different light requirement characteristics (shade
tolerant/intolerant) in the groundcover layer. The gradual decrease in Imperata cylindrica (shade-intolerant
species) abundance over time contrasted with the gradual increase in the abundance of Selaginella doederleinii
(shade-tolerant species), suggesting that there was a decrease in the light availability at the forest floor as the
canopy developed and the succession proceeded.
Over the course of succession, the characteristics of species found at a site will change (Wills 2002). In the
Mt. Merapi sites, the younger sites were characterized by shade-intolerant colonizer species with good dispersal
capability. I. cylindrica is a widely distributed invader grass that has a long record of colonising cleared lands in
Indonesia (Eussen & Soerjani 1975, Soerjani et al. 1983). I. cylindrica has wide-spread rhizomes and its seeds
are wind-dispersed (Jonathan & Hariadi 1999), making it an effective colonizer following fire (Murniati 2002).
Acacia decurrens, however, is a nitrogen-fixing shrub that is usually recruited after fire. At Mt. Merapi, it may
have regenerated following the nues ardentes fire from a soil seed bank (Hardiwinoto et al. 1998, Spurr &
Barnes 1980). I. cylindrica and A. decurrens can also be found in other degraded areas on volcanoes in Java,
such as in Mt. Bromo-Tengger and Mt. Semeru (Anonym 2009, Whitten et al. 1996). The species that occurred
in the older sites and reference site on Mt. Merapi were characterized as intermediate to shade-tolerant species
with greater longevity. In the older sites, A. decurrens was replaced by the leguminous tree, Calliandra
callothyrsus, which occurred with other tree species such as Altingia excelsa. A. excelsa is a native emergent
tree species and its seedlings are shade tolerant. Older sites were also characterized by the presence of the fern
Selaginella doederleinii and the exotic invasive Eupatorium spp. in the groundcover layer. Eupatorium spp. is a
fast growing species, usually found on steep slopes in a wide range of soil conditions and light availability
(Heyne 1987).
Many studies have shown that generally species composition changes with time after a fire (Clearly et al.
2006, Reilly et al. 2006, Ross et al. 2002, Spencer & Gregory 2006). The result of NMDS ordination was
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.
notable in that species composition differed among all sites, suggesting that the species composition changes
with time after a fire. The 2006 and 1998 sites were different in terms of floristic composition and abundance
with highest species replacement rate when compared with the replacement rate in the other sites (D = 0.89).
Altingia excelsa, Pinus merkusii, and Polygala paniculata which were present in the 2006 site, dropped out in
the 1998 site whereas there was an increase in the number of species from the Fabaceae family in the 1998 site
with the colonization of Albizia lopantha, Erythrina sp and Parkia sp. The differences in species composition
between the 1998 and 1997 sites (short interval) was the lowest in all of the site pair-wise comparisons, but they
were still significantly different from each other. Consistent with this, the species replacement rate was also
lower (D = 0.63) when compared with the replacement rate in the other site comparisons. Although ANOSIM
showed that the reference site and the 1994 site were significantly different, the turnover rate between these sites
was more or less the same as the rate in the 1998-1997 sites (D = 0.66). This result indicates that some of the
species that characterized the reference site, such as Altingia excelsa, Schima wallichii and Selaginella
doederleinii, had appeared earlier in the 1994 site and thus suggested convergence of floristic composition in
these sites.
In the Mt. Merapi succession, the changes in abundance of some invasive species such as I. cylindrica,
Brachiaria spp., and Eupatorium spp. are important to note. I. cylindrica is an invasive native of south-east
Asia. I. cylindrica dominated the early succession sites, but was not recorded in older sites as it was most likely
shaded out by increasing canopy cover. In contrast, the invasive exotic species Eupatorium spp. remained in the
system long after the fire had occurred and forest structures had developed. Eupatorium is native to South
America, and this noxious and highly competitive species has become a problem elsewhere in Asia, such as in
Nepal (Kunwar 2003). In the longer term, domination of invasive exotic species may limit the chance of
recruitment of other native species including seedlings of woody species, thereby reducing diversity and even
changing the successional trajectory and ecosystem functioning (Dale et al. 2005b, Hobbs & Huenneke 1992,
Raghubanshi & Tripathi 2009, Standish et al. 2009).
This study suggested that the Merapi forest exhibited a high resilience for site recovery following nues
ardentes-induced wildfire with the rapid re-colonisation of plant species. It is also important to consider the
potential problems of invasive species Eupatorium spp. as weeds, as these species remain abundant even in the
much more developed sites. These findings may have important consequences for forest management as there is
still much to learn about the capability of alien invasive species to change soil chemical properties, which can be
crucial factors in driving the successional trajectory.
ACKNOWLEDGEMENTS
We would like to thank AusAid for funding this research. We are very grateful to Kuspriyadi Sulistyo from
the Merapi National Park for granting the research permit to our project. Gratitude also goes to Mbah Maridjan
the gatekeeper and caretaker of Mt. Merapi and also for kind helps of the fieldwork team, Gunawan, Ali, Indri
and Iqbal.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(3): 215223, 2015
Review article
Application of information technology and GIS in agroforestry

Rajesh Kumar Mishra1* and Rekha Agarwal2
1
Tropical Forest Research Institute, P.O. RFRC, Mandla Road, Jabalpur - 482021, Madhya Pradesh, India
2
Department of Physics, Government Model Science College, Jabalpur - 482021, Madhya Pradesh, India
*Corresponding Author: rajeshkmishra20@gmail.com [Accepted: 22 October 2015]
Abstract: Computer-based Decision Support Tools (DST) helps to integrate information to

facilitate the decision-making process that directs development, acceptance, adoption, and
management aspects in agroforestry. Computer-based DSTs include databases, geographical
information systems, models, knowledge-base or expert systems, and hybrid decision support
systems. Although agroforestry lacks the large research foundation of its agriculture and forestry
counterparts, the development and use of computer-based tools in agroforestry have been
substantial and are projected to increase as the recognition of the productive and protective
(service) roles of these tree-based practices expands. The utility of these and future tools for
decision-support in agroforestry must take into account the limits of our current scientific
information, the diversity of aspects (i.e. economic, social, and biophysical) that must be
incorporated into the planning and design process, and, most importantly, who the end-user of the
tools will be. Incorporating these tools into the design and planning process will enhance the
capability of agroforestry to simultaneously achieve environmental protection and agricultural
production goals. This paper highlights the relevance of information technology (IT) in
Agroforestry. Existing areas of applications such as forest and environmental management, specie
identification and research publications are identified. The paper also looked into future possible
usage of information technology and concludes that while the application of information
technology to Agroforestry practices nowadays is of tremendous importance it is important to
know that there are still more areas where information technology would be applicable in
Agroforestry which are yet to be discovered.
Keywords: Forest management - Information technology - Agroforestry education.
[Cite as: Mishra RK & Agarwal R (2015) Application of information technology and GIS in agroforestry.
Tropical Plant Research 2(3): 215223]
INTRODUCTION
Within half a century, computers and information technology have changed the world and affected millions
of lives in ways that no one could have foreseen (Heathcote 2000). The great impacts, contributions to
knowledge, importance and economic achievements that have emerged from the fields of computer science
(information science) and electronic engineering, in the 21st century, are revolutionary and mind boggling
(Bamgbade 2011). The extent to which IT applications have improved agro-forestry practices in recent times
cannot be over emphasized. Area of application includes:
Forestry and environmental management, species identification, research publication, Information
Communication Technology ICT in agro forestry education, plant pathology studies, wood anatomy, biometrics,
Data management, modelling, analysis and mining. The list is infinite; however some of these applications
would be discussed in the present paper.
Advances in information and communications technology (ICT) and knowledge management (KM) have
changed the way people learn and e-learning is increasingly recognized as a viable and learner-friendly
approach that can complement, or even replace, more traditional training and education approaches. Agriculture
however is a very practical subject and not all of it can be generalized at a global level since local context will
largely determine success or failure of agricultural and natural resources management innovation. The
management of plant genetic resources for example involves practices that are almost impossible to teach in an
Mishra & Agarwal (2015) 2(3): 215223
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online environment and transmission of knowledge is often better achieved through peer-to-peer learning
(Baena et al. 2007). Likewise, many agricultural practices need to be adapted to local biophysical and
socioeconomic conditions if they are to be successfully adopted by those they intend to serve. Blended learning,
combining an online, more general learning experience with more practical face-to-face problem solving
activities, has the potential to include more learners while dealing with the issues of practicality and
contextualization.
Agroforestry, the deliberate integration of trees into crop and livestock operations, has the potential to
achieve many of the environmental, economic, and social objectives being demanded from working landscapes
by landowners and society (Fig. 1). By adding structural and functional diversity to the landscape, these tree-
based plantings can perform ecological functions that have significance far greater than the relatively small
amount of land they occupy (Guo 2000, Nair 2001, Ruark et al. 2003). Realizing this potential is, however, a
complex task of determining what opportunities, limitations, and trade-offs exist in each situation, and of
designing an agroforestry practice that achieves the best balance among them. There are numerous impacts
created by agroforestry plantings, ranging from intended to non-intended and, therefore, ranging from
detrimental to advantageous, occurring both on- and off-site, and varying over time. Consequently, if
agroforestry is to be a viable strategy in promoting agro-ecosystem sustainability, the decision making process
must incorporate many considerations, not only at the practice scale but also at the larger scales of farm,
landscape, and watershed (Schoeneberger et al. 1994). Simply putting, agroforestry creates a complex system of
interactions that must be managed for multiple objectives, multiple alternatives and multiple social interests and
preferences, while being applied over a wide range of landscapes and landscape features.
A B
Figure 1. Wheat based agro-forestry system with: A, Walnut; B, Salix.
The decision-making process involved in agroforestry research, development and application is composed of
several components: the person or group making the decision, the problem, the approach or method to solve the
problem, and the decision. Decision support tools (DST) are a wide variety of technologies that can be used to
help integrate diverse and large sets of information. DSTs do not replace the decision-making by the landowner
or natural resource manager, but they do facilitate the decision-making process by making the planning process
more informed and more objective (Grabaum & Meyer 1998). Although agroforestry, like most natural-resource
management sciences, is characterized by high complexity of which we have limited understanding and data
(Sanchez 1995, Nair 1998), the science and application of agroforestry can be greatly enhanced through the use
of these tools.
FOREST AND ENVIRONMENTAL MANAGEMENT
Mathematical and computational programming in Forest Management
Mathematical and computational programming remains a viable approach for strategic planning in forestry
and agriculture worldwide. One of such computational based system is the SPECTRUM used by the United
State government to carry out strategic planning in agroforestry. The system which evolved from an earlier
system (FORPLAN) has the following key attributes:
1. Multi-resource modeling
The system provides a generic framework for modelling any resource. A basic configuration depends on
user-defined analysis units, management actions, activities and outputs, resource coefficients, and economic
information.
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2. Spatial and temporal scales
Spectrum applications are not scale-specific. Up to 90 time periods of any length may be used to support
analysis at relevant spatial and temporal scales.
3. Multiple options for mathematical programming
Spectrum supports numerous combinations of optimization techniques and objective functions. Optimization
techniques includes,
Linear programming (optimization of a single criterion).
Mixed-integer programming (optimization with categorical outcomes).
Multi-objective goal programming (simultaneous optimization of multiple goals).
Stochastic programming to account for random events such as fires, pest epidemics, and uncertainty about
data.
Specifications for objective functions
Options for objective functions in traditional linear programming include maximizing or minimizing a single
outcome or measure of performance. Objective Functions for goal programming include minimizing under-
achievement of goals, minimizing over- achievement of goals beyond thresholds, or minimizing both.
Two additional options for objective functions are MAXIMIN (maximizing the minimum level of
occurrence for a critical resource) and MIN/MAX (minimizing the highest level of occurrence of an undesirable
outcome).
Simulation of ecological processes and modelling natural disturbance
Spectrum allows embedding simulation of ecological processes and modelling of natural disturbances by
means of state, flow, and accessory variables in dynamic equations.
The Regional Ecosystems and Land Management (RELM) system extends the utility of Spectrum solutions
by apportioning forest-wide, strategic planning solutions to tactical sub-units of the forest such as watersheds.
Cumulative effects and connected actions can be analysed both within and between sub-units, allowing
planners to evaluate how alternative management scenarios affect neighbouring units.
Research publication
Information dissemination is a prominent activity in any research institute as it is the means through which it
could be adjudged whether it is living up to the mandate and purpose for which, it was established. Also,
research publications play a pivotal role in any academics environment. Paper publication is a useful instrument
through which research discoveries and breakthroughs are disseminated to the stakeholders. However,
publishing research papers in a manual format is attached with great difficulties and problems, which includes
ineffective and inefficient delivery system of the journal as at when due, prone to natural disasters, lost, theft,
mutilation. Sequel to the aforementioned problems, a software (FRIN eJOURNAL: An Electronic Submission
Platform for Research papers) has been developed in FRIN which would allow for easy electronic retrieval,
storage and efficient research information delivery system. It will go a long way to automate the existing manual
journal with easy search tool and navigation properties, providing researchers, administrator and FRIN editors
with separate interface with hands on functionality and notification capability also creating a proper record of
subscribers and records of subscription. It is cost effective and is not regional bound.
Species identification
Although automated species identification for many reasons is not yet widely employed, efforts towards the
development of automated species identification systems within the last decade is extremely encouraging; that
such an approach has the potential to make valuable contribution towards reducing the burden of routine
identification.
There are many factors influencing the taxonomic impediment to the study of biodiversity. A major one
being that the demand for routine identification in biodiversity studies extends beyond the available resources.
In many spheres the volumes of plant or animal specimens that can usefully be obtained, particularly using
modern sampling methods, vastly outstrip any capacity to identify this material. This has limited the progress in
some aspect of biodiversity research. These demands are likely to steadily increase as the proportion of
previously un-described species in local, national or regional floras and fauna declines and as requirement or
desirability of biodiversity inventories and other such survey grows.
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This has led to several solutions being preffered to reduce the burden of routine identification. One of the
preffered solutions is automating the identification process in some way. This is generally referred to as
Computer Assisted Taxonomy (CAT). However, the development and application of an automated approach to
taxonomic identification has remained a minority interest till date.
Among reasons for this are the notions that it is too difficult, too threatening, too different or too costly. It is
most encouraging to know that despite these limitations, efforts towards the development of automated species
identification systems have been progressive.
From the evidences witnessed in this area, it buttresses the present minority notion that the automation of
species identification process is possible and achievable. A system that uses binary codes generated based on the
morphological characters of trees to uniquely identify tree species has been developed. Though this is not the
first time an attempt is made to automate species identification using their morphological characters, our
approach is far simpler and less expensive to implement. For instance while previous approaches are centred
round the need for a computerized pattern recognition system, ours does not require such. We were able to
easily prove the effectiveness of the system by restricting our study to the over one thousand Nigerian Trees
species. All the user need is a functional computer system, a ruler and personal ability to supply answers to the
questions asked by the system and the tree identification process is complete.
INFORMATION TECHNOLOGY IN AGRO FORESTRY EDUCATION
Information technologies (ITs) have the potential to enhance access, quality, and effectiveness in education
in general and to enable the development of more and better teachers (Fig. 2). As computer hardware becomes
available to an increasing number of schools, more attention needs to be given to the capacity building of the
key transformers in this process, namely, teachers.
While societies undergo rapid changes as a result of increased access to information, the majority of the
school going youth continues to undergo traditional rote learning. Very little is done to take advantage of the
wealth of information available on the Internet. Whereas the processing of information to build knowledge is
one of the essential literacy skills vital for the workforce in the 21st century, it is often overlooked in current
educational practices. The Computers for Schools Program appears to be doing valuable work and in the process
has become an unwitting champion of ITs in education. Its experiences are real, its challenges huge, and the
lessons valuable for the future resource for poor countries. In order to function in the new world economy,
students and their teachers have to learn to navigate large amounts of information, to analyse and make
decisions, and to master new knowledge and to accomplish complex tasks collaboratively. Overloaded with
information, one key outcome of any learning experience should be for learners to critically challenge the
material collected in order to decide whether it can be considered useful input in any educational activity.
This is the basis for the construction of knowledge. The use of ITs as part of the learning process can be
subdivided into three different forms: as object, aspect or medium.
As object, one refers to learning about ICTs as specific courses such as 'computer education.' Learners
familiarize themselves with hardware and software including packages such as Microsoft Word, Microsoft
Excel, and others. The aim is computer literacy.
As aspect, one refers to applications of ICTs in education similar to what obtained in industry. The use of
ITs in education, such as in computer-aided design and computer aided agroforestry technology, are examples.
ITs are considered as a medium whenever they are used to support teaching and learning.
The use of IT as a medium is rare where the availability of resources is a major obstacle to the widespread
integration of ITs in education. In order to sustain what has already been done and expand into areas still
unreached. Sequel to this is the need to explore the use of ITs in education, such as in computer-aided design
and computer-aided agroforestry technology, are examples.
Need of applications of ITs in Agroforestry Education
With the advent of IT, it is found that IT forms the "backbone" of several industries and is today a dominant
force in enabling companies to exploit new distribution channels, create new products, and deliver differentiated
value added services to customers. IT is also an important catalyst for social transformation and national
progress. Disparities in levels of IT readiness and usage could translate into disparities in levels of productivity
and, hence, different rates of economic growth. It is also important to observe that ICTs can lead to economic
growth but not development. The social exclusion of large groups of persons, especially women, children, and
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people living in rural areas, is a common phenomenon when adequate planning has not accompanied IT
exploitation. Education faces a number of problems. These problems include the shortage of qualified teachers,
very large student populations, high drop-out rates of students and teachers, and weak curricula.
All of these negative aspects result in poor delivery of education. The education crisis is worsened by the
devastating effects of increasing poverty, a brain drain in the teaching community, budgetary constraints, poor
communication, and inadequate infrastructure. Technology is not new to education. However, contemporary
computer technologies, such as the Internet, allow new types of teaching and learning experiences to flourish.
Many new technologies are interactive, making it easier to create environments in which students can learn by
doing, receive feedback, and continually refine their understanding and build new knowledge.
Access to the Internet gives unprecedented opportunities in terms of the availability of research material and
information in general. This availability of research material and information happens to both inspire and
threaten teachers. ITs are one of the major contemporary factors shaping the global economy and producing
rapid changes in society. They have fundamentally changed the way people learn, communicate, and do
business. They can transform the nature of education where and how learning takes place and the roles of
students and teachers in the learning process.
Figure 2. Application of information technology in agro forestry.

IT application in agroforestry
Diagnosis and Design methodology is a methodology for the diagnosis of land-management problems and
the design of agro forestry solutions. There is a need to develop programmes to assist agroforestry researchers
and fieldworkers to plan and implement effective research and development projects. From on-farm research
trials, more rigidly controlled on-station investigations, and eventual extension trials in an expanded range of
sites. It provides a basis for prompt feedback and complementarities between different project components. In
an integrated agroforestry research and extension program, pivotal decisions can be made in periodic meetings
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of the various project personnel who evaluate new results and revise the action plan accordingly. The process
should be continue until the design is optimal and further refinement is deemed unnecessary.
Research and development
To advance agro forestry, research is needed both on basic, process-level questions and on applied
management techniques that are appropriate for commercial farm or forest operations. While basic research
may, for example, investigate the long-term biological interactions between the components of an agro forestry
practice, applied research should seek to maximize the tangible short and intermediate term benefits. Agro
forestry practices should be tailored to readily integrate into existing farming or forestry enterprises, minimize
the displacement of existing crops, use equipment and technical skills that are readily available, and allow some
harvesting of products within conservation agro forestry practices (e.g. hardwood timber from riparian buffer
strips). There is the potential to expand the participation of state, community and institutions, through their
agriculture and forestry programs, in agro forestry research.
The greatest research need is to develop farm-level analyses of the potential economic costs, benefits, and
risks associated with agro forestry practices. This information is a vital prerequisite to the objective comparison
of both production-and conservation-driven agro forestry practices with alternative land use options.
Furthermore, attention should be given to evaluations of future price trends in regional, national and
international markets for commodities that can be produced using agro forestry (e.g. hardwood lumber or high-
value, wind-sensitive crops). Research on tree-crop-animal-environment interactions should be pursued to
provide a scientific basis for optimizing agro forestry designs.
GIS and Remote Sensing in Environmental Management
Use of geographic information systems (GIS), a collection of computer hardware and software used to
analyse and display geographically referenced information, can facilitate planning process. A GIS can be
defined as a data management system designed to input, store, retrieve, manipulate, analyse, and display spatial
data for the purposes of research and decision-making (DeMers 1997). In a GIS, a database is associated with
map features, and data values are geographically referenced, so resource managers can spatially represent
information such as soil types or plant communities. Since land use and a diversity of related disciplines (i.e.
agriculture, forestry, rural planning, and conservation) all deal with spatial characteristics of landscapes (Lacher
1998), GIS has gained considerable use in land use planning and natural-resource management, providing a
spatial framework to aid in the decision-making process (Zeiler 1999).
Additional technologies are often associated with GIS, such as Global Positioning Systems (GPS) and
remote sensing. GPS is a means for inputting spatial data with real world coordinates into a GIS and has become
an important tool for researchers locating and recording information in the field. Remote sensing involves using
spatial data from photographic and satellite images, and software tools to analyse and interpret these data. Rhind
(1988) defined GIS as a computer system for collecting, checking, integrating and analysing information
related to the surface of the earth. As it were, there is an ever increasing recognition of the need to perform
large scale mapping and map analysis operations for a wide variety of traditionally manual tasks. Furthermore,
forests see GIS (a computer based application) as an efficient management tool for their day to-day operations.
A wide variety of software applications are available to support decision making in forest management,
including databases, growth and yield models, wildlife models, silviculture expert systems, financial models,
geographical information systems (GIS), and visualization tools (Schuster et al. 1993). Typically, each
application has its own interface and data format, so managers must learn each interface and manually convert
data from one format to another to use combinations of tools. Considering the scope of topics that may need to
be addressed in a typical ecosystem management problem, and consequently the need to run several to many
applications, manual orchestration of the entire analysis process can quickly become a significant impediment.
Learning Management System (LMS) relieves this problem by managing the flow of information through
predefined pathways that are programmed into its core component. LMS integrates landscape-level spatial
information, stand-level inventory data, and distance independent individual tree-growth models to project
changes on forested landscapes over time.
Spatial data layers like soil type, slope, and land cover can be used to develop suitability assessments that
can identify optimal locations for agroforestry practices to solve landowner and community concerns. By
selecting data with the appropriate spatial resolution, this assessment process can be used at any scale for
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planning agroforestry practices. The most significant benefit of using GIS-guided suitability assessments is the
ability to combine different assessments to determine locations where multiple objectives can be achieved.
Suitability assessments have been used for several decades to identify locations for different land uses such
as landfills, wildlife reserves, and residential development (McHarg 1995). Some of the first examples of
suitability assessments in the United States were prepared by the Natural Resources Conservation Service
(previously the Soil Conservation Service), which ranked soil types based on suitability for different engineering
and agricultural functions (Soil Survey 1993). Although GIS and the suitability process have been used for
many environmental protection applications, this technology has yet to be used extensively in agroforestry (Ellis
et al. 2000, Bentrup & Leininger 2002).
Considering that GIS technology is widely available and affordable today and the fact that agroforestry is
directly dependent upon spatial characteristics, it is logical to expect to have several agroforestry-specific GIS
DSTs; but the reality is that only a few are available. An early GIS application compiled information on 173
species including their descriptions, soil and climate preferences, and management characteristics for Africa
(Booth et al. 1989). This application allowed users to query the database and generate maps showing the
climatic suitability for different species. At a regional scale, Booth et al. (1990) created a similar application for
Zimbabwe, demonstrating how GIS applications can be done at many scales. Unruh & Lefebvre (1995)
performed a similar GIS application for sub-Saharan Africa to determine areas suitable for different agroforestry
systems. Integrating ICRAFs agroforestry database with spatial data on geographic regions, climate and land
uses in the region, their application was able to map out potential regions for 21 specific types of agroforestry
systems.
Most of the past agroforestry GIS applications mentioned above have been research-oriented. The
Southeastern Agroforestry Decision Support System (SEADSS), developed recently by the Center for
Subtropical Agroforestry (CSTAF) at the University of Florida brings on-line GIS capabilities directly to
extension agents and landowners; it offers county soils, land use and other spatial data for selecting suitable tree
and shrub species in a specified location (Ellis et al. 2003). The USDA National Agroforestry Center (NAC) is
currently using GIS to facilitate conservation buffer planning in the Western Corn Belt eco-region in the central
United States (Bentrup et al. 2000). GIS guided assessments, derived from publicly available datasets, are being
used to evaluate four key issues of the Western Cornbelt: biodiversity, soil protection, water quality, and
agroforestry products. By combining these assessments, information is generated for use in identifying
opportunities and constraints on the landscape where multiple benefits from conservation buffers, especially
agroforestry plantings, can be achieved (Bentrup et al. 2000). Utilizing the agroforestry product assessments
(Bentrup & Leininger 2002) in conjunction with the riparian buffer connectivity assessments, areas were
identified where riparian forest buffers could be located to improve habitat connectivity while offering
landowners the option to grow woody floral for profit (Bentrup & Kellerman 2003). GIS-guided agroforestry
suitability analysis will only improve as spatial data and computer resources become more accessible. Many
states and countries already are assembling internet-accessible GIS data clearinghouses to facilitate the use of
spatial information.
Information and technology transfer
Technical information must be developed locally or regionally for application within that region. Information
which is too general or which is based on studies conducted in dissimilar regions or climate zones is not likely
to convince landowners to adopt agro forestry practices, or provide relevant skills and knowledge to ensure their
success. On-farm demonstrations and field days are keys to the understanding and appreciation of agro forestry
practices by landowners. Education and training in agro forestry are needed both for natural resource
professionals and college students.
In addition to the traditional model for the transfer of technology from researcher to extension agent to
practitioner, landowners should have greater involvement in all phases of this process. With the assistance of
research and extension personnel, local groups of landowners may analyse their own needs for agro forestry
development, conduct on-farm experiments under real-life conditions, and then choose the practices most
appropriate for their individual properties. Rather than accusing landowners of causing environmental
degradation, they should be approached from a "win-win" perspective. Emphasis should be placed on
participatory decision-making including landowner advisory groups. Research and information development
Mishra & Agarwal (2015) 2(3): 215223
.
should focus on agro forestry practices that afford economic opportunities, increase production efficiency, and
provide cost-effective and pro-active solutions to conservation problems.
CONCLUSION AND FUTURE PROSPECTIVE

The relevance and application of information technology to Agro forestry practices in these days is of
tremendous importance. There are still more areas where IT would be applicable in agroforestry which are yet to
be discovered, but in the immediate future. Virtually, all other human endeavours have come to know that the
benefits of IT is far outstripped its disadvantages. It is therefore suggested that IT should be a tool that all
professions should embraced. Successful application of agroforestry systems depends upon pulling together
diverse sources of information, in a manner that responds to users needs and resources. Computer-based DSTs
that accommodate these tasks can greatly facilitate the decision-making process that seeks to simultaneously
balance environmental and production goals that meet landowner and societal needs. We must go beyond
providing tools that only address the ecological and economic aspects of sustainability and provide those that
also enhance the cultural sustainability of agroforestry systems; that is, it must elicit sustained human attention
over time or else the benefits may be compromised as land ownership changes, as development pressure
increases
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(3): 224229, 2015
Research article
Variability and germination divergence in seed traits of

Stereospermum chelonoides DC.
Anita Tomar
Centre for Social Forestry and Eco-rehabilitation, 3/1, Lajpat Rai Road, New Katra, Allahabad, U.P, India
*Corresponding Author: anitatomar@icfre.org [Accepted: 24 October 2015]
Abstract: The investigation was carried out in two different seed sources viz. Uttarakhand and
Uttar Pradesh of Stereospermum chelonoides. The aim of the study was to determine variability
and germination divergence in seed traits of Stereospermum chelonoides collected from two states.
A variation was observed in germination percent, mean daily germination, peak value, germination
energy and germination value and seed growth parameters (capsule/seed length, capsule/seed
width and seed weight) of two states. The seeds from Uttarakhand found better as per selected
parameters in comparison to the seeds from Uttar Pradesh.
Keywords: Seed sources - Peak value - Mean daily germination - Germination value.
[Cite as: Tomar A (2015) Variability and germination divergence in seed traits of Stereospermum chelonoides
DC. Tropical Plant Research 2(3): 224229]
INTRODUCTION
Stereospermum chelonoides, DC. is a large sized tree, deciduous, branches and usually 9 to 10 m tall and
distributed in sub Himalayan tract, central parts of India. It is commonly called as "Patla and "Padri" and
belongs to the "Bignoniacea" family (Troup 1986, Masoumeh & Deokule 2013).The decoction of the root is
antipyretic and it is useful in asthma, cough and excessive thirst. The bark and all parts contain a napthaquinone
and lepachol (Sandermann & Dietrichs 1957, Joshi et al. 1977). Flowers are used in bleeding disease, sore throat
and diarrhoea; fruits are useful in blood diseases. The root-bark is an ingredient of Dashmoola (Tomar et al.
2013) and Chywanprash (Yashoda et al. 2004). It is regarded as cooling, astringent cardio tonic, bitter, diuretic
and generally used in combination with other medicine; the ashes of this plant are used in the preparation of
alkaline water and caustic pastes. Fruits are useful in hic cough and blood diseases (Negi 2000).
Seeds of different species and of the same species from different provenances behave differently in their
germination response. Similarly a species may be found in a wide variety of climatic regions, but the
germination behaviour may differ according to provenance. Germinability is a measure of the ability of
population of seeds to germinate or the maximum percentage of seeds that will germinate under favourable
conditions. (Bewley & Black 1978). Variation in seed germination is due to a complex of environmental and
genetic factors during seed formation and subsequent handling of treatments (Wang et al. 1982).
Destructive harvesting practices have seriously reduced seed production and caused gradual erosion of its
natural populations. The species is mainly propagated through seeds and collecting them becomes a laborious
process as their pericarps are winged. Another difficulty it faces is poor germination rate and thus propagation
through seeds in the wild is limited (Baul 2006). Hence, steps have to be taken to conserve this tree of great
economic value therefore, its planting and conservation is recommended for future conservation. Keeping this in
view the present study was conducted to study the variability and germination divergence in seed traits of
Stereospermum chelonoides.

A reconnaissance field survey was carried in five sites (Lakhimpur Kheri, Faizabad, Chitrakoot, Allahabad
and Mirzapur) bearing Stereospermum chelonoides trees in the state of Uttar Pradesh for undertaking the present
study (Fig. 1). The Uttarakhand collection was done from only one site which falls in Dehradun. The latitudinal
and longitudinal ranges of all the six sites have been given in table 1.
Received: 05 August 2015 Published online: 31 October 2015
Tomar (2015) 2(3): 224229
.
Fig. 1: studied sites of Stereospermum chelonoides.

Mature capsules were collected during 20122013 from all the six sites from minimum eight to ten selected
plants of each seed sources and packed in marked polythene bags. For Uttar Pradesh, a composite sample of
seed was drawn by mixing the seed collected from different sites for seed studies. Capsule and seeds were
randomly drawn from the pool in order to determine their size and shape. For each individual seed, three
principal dimensions: length, width, and weight were measured.
Table 1. Geographic information of the studied sites of Stereospermum chelonoides forests.
State Forest Divisions Altitude (m) Latitude Longitude
Uttar Pradesh Lakhimpur Kheri 174.0 28 28 29.94 N 80 41 56.32 E
Faizabad 113.0 26 47 00.00 N 82 12 00.00 E
Chitrakoot 92.1 25 14 00.00 N 81 28 00.00 E
Allahabad 102.6 25 15 53.90 N 81 37 18.20 E
Mirzapur 167.0 24 49 16.88 N 82 18 57.71 E
Uttarakhand Dehradun 640.0 30 19 48.00 N 78 03 36.00 E
Germination test were conducted in 10 cm diameter petri dishes lined with Whatman filter papers. Distilled
water was added whenever moisture loss was detected. There were 4 treatments in this experiment including the
control. The experiment was undertaken in completely randomized block design with four replication in each
treatment and twenty five seeds per replication. Results were expressed as germination percentage which was
the percentage of live seeds that had germinated at the end of test. The seeds were inspected every day and were
considered to be germinated when the radicle penetrated the seed coat and reached about 1mm in length
(Teketay 1996). The data of seed germination was recorded and quantified as per ISTA (1976). The parameters
studied were germination percent (%), germination value (GV) calculated as per Czabator (1962) procedure,
mean daily germination (MDG) according to Bonner (1983), germination energy and germination value (Grouse
& Zimmer 1958).
RESULT AND DISCUSSION

Seed traits, namely seed length, width, weight, and germination parameters vary significantly among both
the state seed sources. The capsule and seed characteristics of Stereospermum chelonoides from Uttarakhand
state have been described in table 2. The highest coefficient of variation (CV) of 38.19% was observed in the
capsule length as the capsule length varies from 14.40 to 49.20 cm with mean value 33.91 cm. The number of
seeds per kg varied from 25,64140,000 as this depends on size of the capsules. Lowest coefficient of variation
Tomar (2015) 2(3): 224229
.
was observed in seed length with and without wings (6.606.09%). However seed width shared a variation
10.12%.
Table 2. Variation in Capsules, seed traits of Stereospermum chelonoides of Uttarakhand state.
No. of seeds No. of seeds Capsule Character Seed Character
/capsule /kilogram Length (cm) Width Weight (gm) Length with Length without Width
(mm) wings (cm) wings (cm) (mm)
Mean SD 49.71 16.60 330805320.06 33.91 12.95 12.213.42 32.437.09 3.090.20 1.830.11 4.420.45
Range 2265 25,64140,000 14.4049.20 7.3216.24 2442 2.93.5 1.72.0 3.885.03
C.V. 33.39 16.08 38.19 28.00 21.9 6.60 6.09 10.12
Note: S.D. = Standard deviation; C.V. = Coefficient of variation. (n = 25 x 4)
The characteristics of Stereospermum chelonoides capsule and seed from Uttar Pradesh have been provided
in table 3. The highest coefficient of variation (CV) of 31.9 % was observed in no. of seeds /capsule. This is due
to the fact that the actual values varied from a minimum of 21 seed in one capsule to a maximum of 56 seeds per
capsules.
Capsule length varies from 34.350.0 cm with mean value 43.64 cm. The lowest co-efficient of variation
observed in number of seeds per kg (0.2) as it varied from 2587026000. The variation in seed size may be due
to both internal (maternal, hereditary) and external (environmental) conditions operating at the time of seed
development (Harper et al. 1970) and advantageous for wide range of adaptability. Seed size has been found to
regulate germination and subsequent seedling growth in many species (Baldmin 1942, Langdon 1958, Williams
1967, Kandya 1978, Devagiri 1997, Singh 1998). Both seed sources of S. suaveolens varied significantly in
respect of capsules and seed traits.
Comparatively wider variations were observed in case of capsule characters, number of seeds per capsule
and seed weight. Such genetic variations have been reported in Acacia catechu (Ramachandra, 1996), Acacia
nilotica (Bagchi & Dobriyal, 1990), Dalbergia sissoo (Gera et al. 2000).
Table 3. Variation in Capsules, seed traits of Stereospermum chelonoides of Uttar Pradesh state.
No. of seeds No. of seeds Capsule Character Seed Character
/capsule /kilogram Length (cm) Width Weight (gm) Length with Length without Width
(mm) wings (cm) wings (cm) (mm)
Mean SD 42.4313.55 25906 43.51 43.645.50 18.60 0.93 55.5713.21 3.34 0.60 1.1 10.12 10.070.85
Range 2156 2587026000 34.350.0 17.1119.75 41.683.1 2.13.9 0.981.30 8.2010.75
C.V. 31.9 0.2 12.6 5.00 23.8 17.9 11.0 8.4
Note: S.D. = Standard deviation; C.V. = Coefficient of variation. (n = 25 x 4)
The commencement of germination in Uttarakhand started eight day onwards after sowing and continued up
to 15 days. The seed germination varied significantly (ANOVA; p < 0.01) during the study period. The peak
germination (11.0%) was observed on 10th day and the total germination under laboratory conditions recorded
was 90.0%. (Table 4). Seeds sown achieved 2.43 peak value and 2.33 as mean daily germination, 45.0
germination energy and 5.66 as germination value (Fig. 2).
In Uttar Pradesh the peak germination was observed on 12th day and total germination under laboratory
conditions recorded was 65% (Table 4). Seeds sown achieved 1.73 as mean daily germination, 1.75 peak value,
42.5 germination energy and 3.02 as germination value (Fig. 2).
Table 4. Variation in Uttarakhand and Uttar Pradesh Germination.

Seed germination % Germination
States 8th 9th 10th 11th 12th 13th 14th 15th Period
Total % Energy Value
day day day day day day day day (days)
Uttarakhand 3 3 11 7 5 3 3 1 90 1.41 815 45.0 5.66
Uttar Pradesh 4 4 4 4 5 1 0 4 65 0.58 815 42.5 3.02
Note: The values refer to mean Standard deviation, (n = 25 x 4).
Germination energy is a measure of speed of germination and is assumed to given an idea of the vigour of
seed and seedlings which it produces (Willan 1985). Germination value, an index combining speed and
completeness of germination was influenced by seed size and weight (Baldwin 1942, Czabator 1962, Dunlop &
Barnett 1984). Differences observed for germination percent, germination value and germination energy could
Tomar (2015) 2(3): 224229
.
be genetic in nature because environmental deviations are negligible for experimental conditions and seeds of
both states were stored in similar conditions. This is supported by the reports of Gera et al. (2000) and
Vakshasya et al. (1992) for Dalbergia sissoo and Arya et al. (1995) for Prosopis cineraria. Since the seeds were
germinated under similar condition, variations among the seed sources may be attributed to genetic differences.
Such variations in nursery performances have reported in Acacia albida (Sniezko & Stewart 1989), Acer rubrum
(Townsend 1977) and Prosopis cineraria (Hooda & Bahadur 1996).
Figure 2. Germination values of Stereospermum chelonoides under laboratory conditions.

Variation in germination of seed sources has been reported in Acacia mangium (Salazar 1989), Pinus brutia
(Isik 1986), Betula ermanii (Shembreg & Protemkin 1987), Pinus greggi (Dvorak et al. 1996) Acacia catechu
(Ramachandra 1996) and Pinus roxburghii (Roy et al. 2004). In general pod, seed and germination traits are
supposed to be inherited characters influenced by age, growth, micro and macro habitats of the parent tree (Isik
1986). Larger seed germinate faster and more completed than smaller one probably due to more endosperm
nutrient pool (Kandya 1978). Aldhous (1972) opined that only those seeds which germinate rapidly and
vigorously under favourable conditions, are likely to be capable of producing vigorous seedlings in field
conditions which is of immediate interest, whereas, week or delayed germination is often fatal. Isik (1986)
stated that populations with high germination rate are more vigorous in terminal and root growth. Khalil (1986)
also recommended the detection of fast growing provenances based on germination traits.
CONCLUSION
It emerged from the present study that a large variability exists in the Stereospermum chelonoides growing
naturally in Uttar Pradesh and Uttarakhand particularly for number of seeds/capsule, capsule character and seed
character. The variability of different characters could be utilized for selection of genotypes suitable for the
plantation and utilization. In this study, Uttarakhand seed source had shown better germination as compared to
Uttar Pradesh. However, more comprehensive survey of Stereospermum chelonoides habitat areas of
Uttarakhand is required to select some promising forms of Stereospermum chelonoides.
This study helps to identify the better seed source of S. chelonoides having better yield therefore, the best
seed source selected may improve the poor sites for agroforestry systems and energy plantations in the
wastelands.
ACKNOWLEDGEMENTS
This work was financially supported by Indian Council of Forestry Research and Education (ICFRE),
Dehradun, India.
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2(3): 230239, 2015
Research article
Diversity and carbon stock assessment of trees and lianas in

tropical dry evergreen forest on the Coromandel Coast of India
P. Vivek1 and N. Parthasarathy1*
1
Department of Ecology and Environmental Sciences, Pondicherry University, Puducherry - 605014, India
*Corresponding Author: parthapu@yahoo.com [Accepted: 25 October 2015]
Abstract: The diversity and carbon stock of all woody plants were investigated in ten tropical dry
evergreen forest (TDEF) sites on the Coromandel Coast of India. All trees 10 cm girth at breast
height and all lianas 1 cm diameter, measured at 1.3 m from the rooting point were enumerated.
A total of 81 tree species (26.36.7 species ha-1) and 52 liana species (23.45.7 species ha-1) that
belonged respectively to 34 and 28 families were inventoried from the ten study sites. The
abundance of woody plants in the ten study sites totaled 18705 individuals (9466 trees and 9239
lianas) and the average tree density was 946.6298.9 stems ha-1 and liana density was 923.9403.3
stems ha-1. Trees contributed 61 % and 51 % respectively to the total woody species richness and
abundance. The basal area of trees in the ten study sites ranged from 8.23 m2 ha-1 to 29.48 m2 ha-1
and that of lianas ranged from 0.2 m2 ha-1 to 1.76 m2 ha-1. The aboveground biomass (AGB) of
trees totaled 3025.8 Mg and ranged from 96.9 Mg ha-1 to 576.4 Mg ha-1 across the ten sites. The
liana aboveground biomass ranged from 2.24 Mg ha-1 to 42.13 Mg ha-1 and totaled 153.76 Mg in
the ten sites. The woody plants in the present study sites stocked 1978.24 Mg Carbon and it ranged
from 62.2 Mg C ha-1 to 365.4 Mg C ha-1. Trees accounted for a maximum share of 95 % and lianas
contributed just 5 % to the total woody-plant carbon stock in the study sites. The extent of woody
species diversity and estimated carbon stock of the TDEF sites, underlines the need for biological
conservation of this unique forest type which are fast vanishing due to anthropogenic pressure.
Keywords: Biomass - Allometric equation - Anthropogenic pressure - Wood specific density -
Carbon flux.
[Cite as: Vivek P & Parthasarathy N (2015) Diversity and carbon stock assessment of trees and lianas in
tropical dry evergreen forest on the Coromandel Coast of India. Tropical Plant Research 2(3): 230239]
INTRODUCTION
Forests are one of the major pools of carbon, where plants fix atmospheric carbon into the biological system.
Indeed it is the tropical forest ecosystems that have the potential to hold and sequester large amounts of carbon
than the other forest biomes (Metz et al. 2001). Tropical forests comprise about 40 % of the total terrestrial
carbon stock (Dixon et al. 1994), but uncertainty prevails in their quantitative contribution to the global carbon
cycle (Chave et al. 2005). This uncertainty is largely due to the lack of standard methods for converting field
measurements into biomass estimates (Hall 2012, Liu et al. 2014). The woody biomass of trees and lianas, their
standing crop of litter including the soil organic matter together comprise the key carbon pools in tropical forest
ecosystems (Gibbs et al. 2007). The carbon stocked as aboveground and belowground biomass in woody plants
is impacted directly by human-mediated disturbances. Deforestation currently accounts for about 18 % of the
global carbon emissions (IPCC 2007). Several other factors including selective logging, forest fragmentation
and shifting cultivation are expected to play a major role in altering forest biomass (Houghton 2005). Under the
present scenario of global climate change and increasing deforestation rates, it has become crucial to quantify
the carbon stocks and fluxes particularly in the tropics. Currently, data presented on forest biomass is available
from wide array of sources (e.g. quantitative inventories, output of ecological models and through satellite
products). However, it is the direct assessment of biomass through destructive sampling is likely to give better
estimates on forest carbon stocks. It could also be approached by applying generalized allometric equations
(Brown 1997, Chave et al. 2005) using variables such as diameter, height and wood specific density. Allometric
Vivek & Parthasarathy (2015) 2(3): 230239
.
equations applied for forest inventory data, relate these inventory data to measurements made from destructive
sampling by statistical means, and available for most forest types (Brown 1997, Chave et al. 2005).
Tropical dry evergreen forest (TDEF) is a unique and geographically restricted forest type distributed as
numerous patches along the Coromandel Coast of peninsular India. All the TDEF sites are protected as sacred
forests owing to the religious and traditional believes of the local people. However, the recent developments and
erosion in the belief system, questioned the concept of sacred forests and as a result, some TDEF sites are now
subjected to unprecedented levels of anthropogenic pressure. Meher-Homji (1974) estimated that just 45 % of
the original TDEFs remain. Hence, it is of prime importance to estimate the diversity and carbon stocking
potential of TDEF ecosystem for conservation with the aid of scientific data. Although, there have been many
studies that reported the estimation of carbon stocks of trees in tropical forests, only few studies have included
the liana life-form. Hence, the present study is aimed to investigate woody species (trees and lianas) diversity
and their carbon stocks in TDEF ecosystem.

Study area
Figure 1. Map showing ten tropical dry evergreen forest sites distributed in Cuddalore, Pudukottai and Nagapattinam
districts on the Coromandel Coast of India.
The present study was conducted in ten tropical dry evergreen forest (TDEF) sites located in Cuddalore (11
44 57.88 N latitude and 79 44 50.99 E longitude), Nagapattinam (10 45 56.28 N latitude and 79 50
31.68 E longitude) and Pudukottai (10 22 46.63 N latitude and 78 49 18.35 E longitude) districts of Tamil
Nadu, India (Fig. 1). The tropical dry evergreen forest that occurs as patches along the Coromandel Coast of
peninsular India is characterized by two to three-layered, tree-dominated forests with short stature and sparse
.
ground flora (Parthasarathy et al. 2008, Vivek & Parthasarathy 2015). The rainfall here is tropical dissymmetric
type with most rains received during the north-east monsoon and a little, inconsistent rainfall during the south-
west monsoon. The mean annual rainfall is 1184 mm, 1346 mm and 919 mm in Cuddalore, Nagapattinam and
Pudukottai respectively. The length of dry season is 68 months annually. The mean annual maximum and
minimum temperature are 36.9C and 20.8C in Cuddalore, 34.9C and 22.3C in Nagapattinam and 36.1C and
21.6C in Pudukottai. Soil type varies from hard lateritic, red ferralitic and alluvium to coastal sandy. All the
study sites are community-managed, except the sites Point Calimere 1 and 2, which are a part of Point Calimere
Wildlife Sanctuary (RAMSAR site) and is probably the largest existing TDEF site in India. The community-
managed sites are protected by the local people as sacred groves (sacred forests) dedicated to Gods, based on the
traditional belief system. However, the concentration of human settlements near these study sites makes them
more vulnerable to anthropogenic pressure.
Field inventory
Field work was carried out between April 2013May 2014 in ten 1-ha study plots. Each one-hectare study
plot was further divided into one-hundred 10 10 m sub-grids to facilitate the inventory. During inventory, all
trees 10 cm girth at breast height (gbh) were measured at 1.3 m from the ground level and all lianas 1 cm
diameter were measured at 1.3 m from the rooting point. All the inventoried tree and liana species were
recognized to species-level using regional floras (Gamble & Fischer 19151935, Matthew 1991) and confirmed
with the specimens lodged in the herbarium of Department of Ecology and Environmental Sciences,
Pondicherry University. Diversity indices such as Shannon, Fishers alpha and Simpson index were computed
following Magurran (2004).
Allometric equation and biomass estimation
We used the forest inventory data (dbh values) to estimate the aboveground (AGB) and belowground biomass
(BGB). The aboveground biomass of trees was estimated following the allometric equation of Chave et al.
(2005) using two variables, the diameter and wood specific density (WSD):
AGB est = exp (-1.499 + 2.148 ln (D) + 0.207(ln (D))2 - 0.0281(ln (D))3)
where D is the diameter and is the wood specific density of tree species.
The wood specific density of each tree species was taken from available literature (Mani & Parthasarathy
2007) and also from global wood density database. We used the generalized allometric equation (Pearson 2005)
for few species for which WSD value was not available, using diameter as the only variable.
For lianas, the allometric equation of Schnitzer et al. (2006) was used:
AGB = exp [ 1.484 + 2.657 ln (D)]
where D is the diameter.
The belowground biomass of trees and lianas was calculated by multiplying the aboveground biomass value
with 0.26 (Cairns et al. 1997, IPCC 2003). The carbon stock was estimated to be 50 % of the total biomass
(AGB + BGB) (IPCC 2005).

Woody species diversity
Woody species inventory yielded a total of 133 species including 81 tree species and 52 liana species from ten
study sites (Table 1) and it ranged from a minimum of 29 species ha-1 at site MK to the maximum of 64 species
ha-1 at site PC 1. Trees in the study sites comprised 60 % of the total woody species richness and lianas the rest.
A total of 18705 woody individuals were enumerated from the study sites, of which trees and lianas shared 51 %
and 49 % of the total abundance respectively. Site VV harbored maximum density of trees and lianas (3351
individuals ha-1) and it was minimum at site MK (1194 individuals ha-1). Site PC 1 with greater species richness
had the highest Fishers value and site MK, the lowest (Table 1). Shannon and Simpson index values were
higher for sites PR and SN respectively. Memecylon umbellatum (2318 individuals), Glycosmis mauritiana (740
individuals) and Albizia amara (700 individuals) were the top three abundant tree species forming 42 % of the
total tree species abundance. Among lianas, Strychnos lenticellata (1920 individuals), Combretum albidum (987
individuals) and Reissantia indica (747 individuals) were the predominant species which together accounted for
40 % of the total liana abundance.
.
Table 1. Consolidated details of woody plant (trees and lianas) diversity in ten tropical dry evergreen forest (TDEF) sites
distributed 1-ha each at: Karukkai (KA), Kothattai (KT), Maanadikuppam (MK), Point Calimere 1(PC1), Point Calimere 2 (PC2),
Purangani (PR), Silattur (SL), Sunayakkadu (SN), Suran Viduthi (SV) and Vanniyan Viduthi (VV) on the Coromandel Coast of
India.
Study site Total
Variable
KA KT MK PC1 PC2 PR SL SN SV VV for 10 ha
TWSR (number of spp.) 42 45 29 65 58 42 47 55 64 50 133
Trees 20 25 18 37 27 20 22 27 37 30 81
Lianas 22 20 11 28 31 22 25 28 27 20 52
TWSD (individuals ha-1) 1786 1213 1194 1462 1523 1867 2424 1542 2343 3351 18705
Trees 845 661 786 790 803 948 1211 841 888 1693 9466
Lianas 941 552 408 672 720 919 1213 701 1455 1658 9239
Note: TWSR- Total woody species richness; TWSD- Total woody species density.
Forest biomass
The total woody species basal area in the ten study sites was 176.75 m2 and it varied from a maximum of 30.16
m2 ha-1 at site PC 1 to a minimum of 8.43 m2 ha-1 at site MK (Table 2). Trees in the study sites comprised 96 %
of the total woody species basal area. The total biomass of the woody species recorded in study sites was 3956.4
Mg with the maximum contribution from trees (95 %). The mean woody species carbon stock in the ten study
sites was 197.881.8. Whereas, Tiwari & Singh (1987) estimated 68.5122.5 Mg ha-1 biomass carbon in
Table 2. Basal area (BA), aboveground biomass (AGB), belowground biomass (BGB), total biomass (TB) and carbon stock (CS) of
all woody plants followed by trees and lianas in ten TDEF sites on the Coromandel Coast of India: Karukkai (KA), Kothattai (KT),
Maanadikuppam (MK), Point Calimere 1(PC1), Point Calimere 2 (PC2), Purangani (PR), Silattur (SL), Sunayakkadu (SN), Suran
Viduthi (SV) and Vanniyan Viduthi (VV).
Study site Total
Variable
KA KT MK PC1 PC2 PR SL SN SV VV . for 10 ha
BA (m2 ha-1) 13.99 16.15 8.43 30.16 20.97 11.29 15.13. 19.02. 20.25 21.35. 176.74
Trees 13.38 15.92 8.23 29.48 20.53 10.64 14.36. 18.07. 19.19 19.59. 169.39
Lianas 0.61 0.23 0.2 0.68 0.44 0.65 0.77. 0.95. 1.06 1.76. 7.35
AGB (Mg ha-1) 271.35 300.31 98.84 580.02 315.67 138.07 270.26 391.5. 390.26 383.83. 3140.11
Trees 262.2 296.8 96.6 569.6 307.4 128.1 256.9 353.2. 373.9 341.7. 2986.4
Lianas 9.15 3.51 2.24 10.42 8.27 9.97 13.36 38.30. 16.36 42.13. 153.71
BGB (Mg ha-1) 70.551 78.080 25.698 150.80 82.074 35.898 70.267 101.79 101.46 99.795. 816.41
Trees 68.172 77.168 25.116 148.09 79.924 33.306 66.794 91.832 97.214 88.84. 776.45
Lianas 2.379 0.9126 0.5824 2.7092 2.1502 2.5922 3.4736 9.958 4.2536 10.95. 39.96
TB (Mg ha-1) 341.90 378.390 124.53 730.82 397.74 173.96 340.52 493.29 491.72 483.62. 3956.49
Trees 330.37 373.96 121.71 717.69 387.32 161.40 323.69 445.03 471.11 430.54. 3762.82
Lianas 11.529 4.4226 2.8224 13.129 10.420 12.562 16.833 48.258 20.613 53.083. 193.67
CS (Mg ha-1) 170.95 189.19 62.2692 365.41 198.87 86.984 170.26 246.64 245.86 241.81. 1978.24
Trees 165.18 186.98 60.858 358.84 193.66 80.703 161.84 222.51 235.55 215.27. 1881.39
Lianas 5.7645 2.2113 1.4112 6.5646 5.2101 6.2811 8.4168 24.129 10.306 26.541. 96.8356
Himalayan region of Uttar Pradesh. Ravindranath et al. (1997) reported the average of 63 % Mg C ha-1 from the
values for few forest types studied using harvest method. Thus, the estimated carbon on per hectare basis in the
present study is much higher than the values reported in the previous studies in India. The increased interests in
estimating the biomass and carbon stocks resulted in the evolution of new methods that confounded
comparisons across the different studies. For example, Mani & Parthasarathy (2007) obtained two contradictory
results on aboveground biomass using two different allometric equations for the same dataset. It is of paramount
importance to obtain more accurate estimates on carbon stocks for tropical forests to understand the role of
tropical ecosystems in the global carbon cycle (Brown et al. 1989, Kale et al. 2004, Kuller et al. 2001). The
.
choice of equation is therefore much important when comparing the biomass estimates in regional scale. Among
all the study sites, the relatively undisturbed site PC 1 stocked maximum carbon (365.41 Mg C ha-1). Tree
species such as Manilkara hexandra with 481 individuals stocked maximum carbon (399.4 Mg C), followed by
Drypetes sepiaria (192.20 Mg C) and Albizia amara (165.29 Mg C) (Table 3). The predominant tree species
Memecylon umbellatum and Glycosmis mauritiana contributed at least four-fold lower value (92.12 Mg C and
58.64 Mg C respectively) than that of Manilkara hexandra, possibly due to their major representation in smaller
girth classes. Ventilago madraspatana, an unarmed scrambler was the highest contributor of carbon stock
among the 52 liana species, followed by Acacia caesia (16.72 Mg C) and Derris scandens (11.95 Mg C) (Table
3). Although lianas continue to increase in biomass in tropical forests (Schnitzer & Bongers 2011), they have
not been figured in most forest biomass assessment studies. It is estimated that lianas can add up to 30% of the
total aboveground biomass in tropical forests with dense liana population (Schnitzer & Bongers 2011).
However, in the present study sites, lianas with almost equal abundance as that of the trees, comprised just 5 %
of the total forest biomass. Yet, they may play a major role in reducing the whole forest carbon stock and
sequestration potential by competing aggressively with trees for aboveground and belowground resource
(Schnitzer & Bongers 2011). Lianas usually capitalize and grow well on the disturbed environments and reduces
tree growth and increases the tree mortality rates. This may not be a good sign as lianas do not compensate for
the tree biomass that they displace (van der Heijden & Phillips 2009, Schnitzer & Bongers 2011).
Table 3. Species abundance (Ab), aboveground biomass (AGB), belowground biomass (BGB), total biomass (TB) and total
carbon stock (TCS) of all the 81 tree species and 52 liana species enumerated from 10-ha area distributed 1-ha in each of ten
tropical dry evergreen forest sites
Ab AGB BGB TB TSC
S.No. Woody species
(10-ha) ) (kg) (kg) (kg) (kg)
Tree species
1 Acacia leucophloea (Roxb.) Willd. 4 228.6 59.4 288.0 144.0
2 Alangium salvifolium (L.f.) Wangerin 6 1416.6 368.3 1784.9 892.5
3 Albizia amara (Roxb.) Boivin 700 262366.6 68215.3 330581.9 165290.9
4 Allophyllus serratus (Roxb.) Kurz 7 65.4 17.0 82.3 41.2
5 Anacardium occidantale L. 9 35973.4 9353.1 45326.4 22663.2
6 Atalantia monopylla (L.) Correa 363 44060.3 11455.7 55516.0 27758.0
7 Azadirachta indica A. Juss. 87 73078.3 19000.4 92078.6 46039.3
8 Bauhinia racemosa Lam. 1 213.5 55.5 269.0 134.5
9 Benkara malabarica (Lam.) Tirven. 14 339.6 88.3 427.9 213.9
10 Borassus flabellifer L. 51 64176.7 16685.9 80862.6 40431.3
11 Breynia vitis-idaea (Burm. f.) Fischer 3 7.9 2.1 9.9 5.0
12 Cadaba trifoliata (Roxb.) Wight & Arn. 143 2439.5 634.3 3073.8 1536.9
13 Canthium coromandelicum (Burm.f.) Alston 18 17.1 4.4 21.5 10.8
14 Canthium dicoccum (Gaertn.) Teijsm.& Binn 230 28752.3 7475.6 36227.9 18114.0
15 Carmona retusa (Vahl) Masm 6 22.8 5.9 28.7 14.3
16 Cassia auriculata L. 1 3.8 1.0 4.7 2.4
17 Cassia fistula L. 146 26408.0 6866.1 33274.1 16637.1
18 Cassia roxburghi DC. 8 2631.8 684.3 3316.1 1658.1
19 Cassine glauca (Rottb.) Kuntze 17 710.2 184.7 894.9 447.5
20 Catunaregam spinosa (Thunb.) Tirven 13 43.8 11.4 55.2 27.6
21 Chionanthus zeylanica L. 48 9929.4 2581.6 12511.0 6255.5
22 Chloroxylon sweitenia DC. 398 61783.0 16063.6 77846.6 38923.3
23 Clausena dendata (Willd.) Roemer 248 1627.7 423.2 2050.8 1025.4
24 Commiphora berryi (Arn) Engler 2 329.0 85.5 414.6 207.3
25 Commiphora caudata (Wight & Arn.) Engl. 26 7268.7 1889.9 9158.6 4579.3
26 Cordia obliqua Willd. 7 666.1 173.2 839.2 419.6
27 Dalbergia coromandeliana Prain 2 234.9 61.1 296.0 148.0
.
28 Dalbergia paniculata Roxb. 46 81653.5 21229.9 102883.5 51441.7
29 Dichrostachys cinerea (L.) Wight & Arn. 4 54.5 14.2 68.7 34.3
30 Diospyros ebenum J. Koenig ex Retz. 149 28684.8 7458.0 36142.8 18071.4
31 Diospyros ferrea (Willd.) Bakh. var. buxifolia 103 22402.1 5824.6 28226.7 14113.4
(Rottb.) Bakh.
32 Diospyros montana Roxb. 85 6368.3 1655.8 8024.1 4012.0
33 Dodonea angustifolia L. f. 47 1837.2 477.7 2314.8 1157.4
34 Drypetes sepiaria (Wight & Arn.) Pax & Hoffm. 390 305091.4 79323.8 384415.2 192207.6
35 Ehretia pubescens Benth. 2 5.0 1.3 6.3 3.1
36 Ehretia wightiana Wall. ex G.Don 2 973.4 253.1 1226.5 613.3
37 Eugenia bracteata (Willd.) Roxb. ex DC. 4 41.5 10.8 52.3 26.2
38 Euphorbia antiquorum L. 176 - - - -
39 Ficus amplissima Sm. 1 4238.4 1102.0 5340.4 2670.2
40 Ficus benghalensis L. 17 172077.9 44740.3 216818.1 108409.1
41 Ficus microcarpa L.f. 7 8950.6 2327.1 11277.7 5638.8
42 Ficus religiosa L. 1 84.4 21.9 106.3 53.1
43 Garcinia spicata (Wight & Arn.) J. D. Hook. 161 88635.4 23045.2 111680.5 55840.3
44 Gardenia resinifera Roth 17 3441.7 894.8 4336.5 2168.2
45 Glycosmis mauritiana (Lam.) Yuich. Tanaka 940 9308.1 2420.1 11728.2 5864.1
46 Gmelina asiatica L. 33 2951.0 767.3 3718.2 1859.1
47 Ixora pavetta T.Anderson 36 10299.4 2677.8 12977.2 6488.6
48 Jatropha gossipyfolia L. 1 1.8 0.5 2.3 1.2
49 Lannea coromandelica (Houtt.) Merr. 32 3094.8 804.6 3899.4 1949.7
50 Lepisanthes tetraphlla (Vahl.) Radlk. 313 153606.5 39937.7 193544.2 96772.1
51 Mallotus phillipensis (Lam.) Muell.-Arg. 20 2693.5 700.3 3393.8 1696.9
52 Manilkara hexandra (Roxb.) Dubard 481 634041.8 164850.9 798892.7 399446.3
53 Maytenus emarginata 43 599.5 155.9 755.3 377.7
54 Memecylon umbellatum Burm.f. 2318 146235.1 38021.1 184256.2 92128.1
55 Morinda coreia Buch.-Ham. 14 1162.7 302.3 1465.1 732.5
56 Muntingia calabura L. 4 417.0 108.4 525.4 262.7
57 Ochna obtusata DC. 33 4957.3 1288.9 6246.2 3123.1
58 Pamburus missionis (Wight) Swingle 4 13.8 3.6 17.4 8.7
59 Phyllanthus polyphyllus Willd. 2 87.9 22.9 110.8 55.4
60 Pleiospermium alatum (Wall. ex Wight. & Arn.) 48 12944.2 3365.5 16309.7 8154.8
Swingle
61 Polyalthia korintii (Dunal) Thw. 10 904.4 235.1 1139.6 569.8
62 Polyalthia longifolia (Sonn.) Thw. 4 137.8 35.8 173.7 86.8
63 Pongamia pinnata (L.) Pierre 65 86160.0 22401.6 108561.6 54280.8
64 Premna serratifolia L. 14 7548.2 1962.5 9510.7 4755.4
65 Prosopis juliflora (Sw.) DC. 131 119681.9 31117.3 150799.1 75399.6
66 Pterospermum canescens Roxb. 86 105674.5 27475.4 133149.9 66575.0
67 Pterospermum xylocarpum (Gaertn.) Sant. & 8 13920.2 3619.2 17539.4 8769.7
Wagh.
68 Salvadora persica L. var. wightiana 15 6820.1 1773.2 8593.4 4296.7
(Thwaites) Verdc
69 Sapindus emarginatus Vahl 3 21.0 5.5 26.5 13.2
70 Sapium insigne (Royle) Trimen 16 140.1 36.4 176.5 88.2
71 Securenega leucopyrus (Willd.) Muell.-Arg. 3 12.5 3.2 15.7 7.9
72 Strebulus asper Lour. 5 60.7 15.8 76.5 38.2
.
73 Strychnos nux-vomica L. 2 126.0 32.8 158.8 79.4
74 Syzygium cumini (L.) Skeels 51 211847.6 55080.4 266928.0 133464.0
75 Tamarindus indica L. 4 62402.1 16224.5 78626.6 39313.3
76 Tarenna asiatica (L) kuntz ex Schumann. 510 4990.5 1297.5 6288.1 3144.0
77 Tricalysia sphaerocarpa (Dalz.) Gamble 372 81809.3 21270.4 103079.7 51539.8
78 Vitex altssima L.f. 10 7734.7 2011.0 9745.8 4872.9
79 Walsura trifolia (A. Juss.) Harms 6 683.1 177.6 860.7 430.4
80 Wrightia tinctoria (Roxb.) R. Br. 4 75.5 19.6 95.2 47.6
81 Zizyphus mauritiana Lam. 13 603.0 156.8 759.8 379.9
Liana species
82 Abrus precatorius L. 9 4.4 1.1 5.6 2.8
83 Acacia caesia (L.) Willd. 196 16722.9 4348.0 21070.9 10535.5
84 Adenia wightiana (Wall.exWight & Arn.) Eng. 1 0.3 0.1 0.4 0.2
85 Aristolochia indica L. 5 1.7 0.4 2.1 1.1
86 Asparagus racemosus Willd. 82 70.2 18.3 88.5 44.2
87 Azima tetracantha Lam. 25 357.6 93.0 450.6 225.3
88 Canavalia virosa (Roxb.) Wight & Arn. 7 4.7 1.2 6.0 3.0
89 Cansjera rheedii Gmel. 74 1150.1 299.0 1449.2 724.6
90 Capparis brevispina DC. 141 1971.9 512.7 2484.5 1242.3
91 Capparis divaricata Lam. 1 0.3 0.1 0.4 0.2
92 Capparis rotundifolia Rottl. 28 104.4 27.1 131.5 65.7
93 Capparis sepiaria L. 4 26.8 7.0 33.7 16.9
94 Capparis zeylanica L. 58 1463.9 380.6 1844.5 922.3
95 Carissa spinarum L. 575 2224.3 578.3 2802.6 1401.3
96 Cissus quadrangularis L. 254 409.3 106.4 515.7 257.8
97 Cissus vitiginea L. 293 4972.1 1292.7 6264.8 3132.4
98 Clerodendrum inerme (L.) Gaertn. 13 60.7 15.8 76.4 38.2
99 Coccinia grandis (L.) Voigt 182 960.0 249.6 1209.6 604.8
100 Combretum albidum G.Don 987 10042.2 2611.0 12653.1 6326.6
101 Derris ovalifolia (Wight & Arn.) Benth. 192 9014.1 2343.7 11357.8 5678.9
102 Derris scandens (Roxb.) Benth. 343 11951.2 3107.3 15058.5 7529.3
103 Dioscorea oppositifolia L. 1 0.2 0.1 0.3 0.1
104 Gloriosa superba L. 4 3.0 0.8 3.7 1.9
105 Grewia rhamnifolia Heyne ex Roth 445 11772.3 3060.8 14833.1 7416.6
106 Gymnema sylvestre (Retz.) R.Br. ex Schultes 180 4053.4 1053.9 5107.3 2553.7
107 Hugonia mystax L. 488 6329.6 1645.7 7975.3 3987.6
108 Ichnocarpus frutescens (L.) R.Br. 162 158.9 41.3 200.2 100.1
109 Ipomoea staphylina Roemer & Schultes 5 36.4 9.5 45.9 23.0
110 Jasminum angustifolium (L.) Willd. 309 1123.2 292.0 1415.2 707.6
111 Jasminum sessiliflorum Vahl 61 34.8 9.1 43.9 21.9
112 Olax scandens Roxb. 18 138.4 36.0 174.4 87.2
113 Pachygone ovata (Poir) Miers ex Hook. 56 391.1 101.7 492.7 246.4
114 Plecospermum spinosum Trecul. 30 1061.2 275.9 1337.1 668.6
115 Premna corymbosa (Burm.f.) Rottl. & Willd. 137 459.5 119.5 578.9 289.5
116 Pterolobium hexapetalum (Roth.) Sant.&Wag. 74 208.3 54.2 262.5 131.2
117 Pyrenacantha volubilis Wight 191 209.5 54.5 264.0 132.0
118 Reissantia indica (Willd.) Halle 747 10709.3 2784.4 13493.7 6746.8
119 Rivea hypocrateriformis (Desr.) Choisy. 39 922.1 239.7 1161.8 580.9
.
120 Salachia chinensis L. 8 66.0 17.1 83.1 41.6
121 Sarcostemma acidum (Roxb.) Voigt 57 103.0 26.8 129.8 64.9
122 Scutia myrtina (Burm. f.) Kurz 110 2892.3 752.0 3644.3 1822.1
123 Secamone emetica (Retz.) R. Br. 186 505.1 131.3 636.4 318.2
124 Strychnos lenticellata (Dennst.) Hill 1920 7554.2 1964.1 9518.2 4759.1
125 Symphorema involucratum Roxb. 62 6260.5 1627.7 7888.2 3944.1
126 Tiliacora acuminata (Lam.) Hook.f. & Thoms. 16 23.2 6.0 29.2 14.6
127 Tinospora cordifolia (Willd.) Hook.f.&Thoms. 87 87.0 22.6 109.6 54.8
128 Toddalia asiatica (L.) Lam. 40 301.9 78.5 380.4 190.2
129 Trichosanthes tricuspidata Lour. 2 5.5 1.4 6.9 3.5
130 Tylophora indica (Burm. f.) Merr. 7 4.4 1.1 5.5 2.8
131 Ventilago madraspatana Gaertn. 87 26031.1 6768.1 32799.2 16399.6
132 Wattakaka volubilis (L.f) T. Cooke 102 1048.4 272.6 1321.0 660.5
133 Zizyphus oenoplia (L.) Mill. 136 9759.3 2537.4 12296.7 6148.4
Size-class distribution and carbon stock

Overall, in the ten study sites, 62 % of trees and 70 % of lianas fell within the lowest dbh class (Fig. 2 & 3)
and this observed pattern could have resulted from the greater recruitment and mortality rates in the lowest dbh
class (Vivek & Parthasarathy 2015). The highest dbh class comprised just 7 % of the total tree abundance, yet,
managed to represent 75 % of the total tree carbon stock, suggesting the role of large trees in maintaining the
carbon pools in TDEF ecosystem. Similarly in lianas, the highest dbh class represented by 3 % of the total liana
abundance across the study sites, accounted for 67 % of the total carbon stocked by lianas in the study sites. In
general, the carbon stock of the trees and lianas increased with increasing size-class irrespective of the number
of individuals.
Figure 2. Girth-class wise distribution of tree abundance and their corresponding carbon stocks in tropical dry evergreen
forests on the Coromandel Coast of India.
CONCLUSION
This study provides valuable data on biomass carbon of woody plants, thereby emphasizes the role of TDEF
ecosystem in maintaining carbon pool of the local forest environment and will be helpful in framing
conservation strategies and action plans. The present study also indicates the role of trees, particularly the large
trees in maintaining the carbon stock, but in recent years, the TDEFs on the Coromandel Coast are experiencing
immense pressure that result in reduced tree counts (Baithalu et al. 2012), but the lianas on the other hand
increased drastically (Khadanga 2015). Therefore, we recommend long-term monitoring studies to estimate
.
carbon stocks and dynamics in TDEF ecosystem under the current scenario of climate change and anthropogenic
disturbance.
Figure 3. Diameter-class wise distribution of liana abundance and their corresponding carbon stocks in tropical dry
evergreen forests on the Coromandel Coast of India.
ACKNOWLEDGEMENTS
We thank the Ministry of Environment and Forests for funding this study through a project (No.22/16/2011-
(SG)-RE).
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(3): 240245, 2015
Research article
Vitamin C content of commonly eaten green leafy vegetables in

fresh and under different storage conditions
Umaramani Mathiventhan1* and Sivakanesan Ramiah2
1
Senior Lecturer, Department of Botany, Faculty of Science, Eastern University, Sri Lanka
2
Senior Professor, Department of Biochemistry, Faculty of Medicine, University of Peradeniya, Sri Lanka
*Corresponding Author: umaramanip@yahoo.com [Accepted: 27 October 2015]
Abstract: This study was an attempt to determine the consumption of popular green leafy
vegetables (GLVs) available in Batticaloa district, Sri Lanka and to determine vitamin C content of
fresh and stored (under room temperature 302C and at 4C for 4 days) GLVs. Vitamin C content
was estimated in aqueous extracts using dichloroindophenol titrimetric method. Thirty one species
of GLVs were consumed commonly by the subjects with an average consumption of 59%. Vitamin
C content of fresh GLVs ranged from 5.25 mg/100 g for Centella asiatica to 433.13 mg/100 g wet
weight for Drgea volubilis. Drgea volubilis, which had the highest amount of vitamin C, is
consumed by 80% of the consumers followed by Delonix elata, which is consumed by 52% of the
consumers interviewed. Murraya koenigii, which is used by almost all the consumers interviewed,
is a poor source of vitamin C. Similarly Centella asiatica, which was claimed to be consumed by
90% of the consumers, too was a poor source of vitamin C. The decline in vitamin C content of all
GLVs, ranging from 18% for Aerva lanata to 100% for Moringa oleifera, was higher and
significant (p = 0.000 at 95% confident interval) when stored at room temperature for 4 days than
stored at 4C (ranging from 2.5% for Sauropus androgynus to 70% for Alternanthera sessilis)
except Pisonia grandis. Both Drega volubilis and Delonix elata showed 22.3 and 6.1% increase
in vitamin C content respectively when stored at 4C.
Keywords: Vitamin C - Green leafy vegetables - Drgea volubilis.
[Cite as: Umaramani M & Sivakanesan R (2015) Vitamin C content of commonly eaten green leafy vegetables
in fresh and under different storage conditions. Tropical Plant Research 2(3): 240245]
INTRODUCTION
Green leafy vegetables (GLVs) are rich sources of many nutrients and form a major category of vegetable
group that are rich in health promoting phytochemicals. Their high antioxidant contents have attracted attention
of several investigators (Boxin et al. 2002, Ismail et al. 2004, Gupta et al. 2005).
More than 90% of vitamin C in human diet is supplied by fruits and vegetables (Latif & El-Aal 2007).
Vitamin C is required for the prevention of scurvy and has many biological activities in the human body
(Antonelli 2002). Unfortunately, this vitamin has low thermo stability and high water solubility (Anna 2007). In
view of this, refrigerated storage recommended for increased shelf life of fresh vegetables (Vina & Chaves
2006) would be helpful in retention of their vitamin C content. Latif & El-Aal (2007) reported that simple
packaging of fresh green leafy vegetables in polythene was very effective in reducing weight and moisture
losses during cold storage.
Inadequate number of studies, shortage of primary data and many information gaps exist regarding the
consumption pattern of GLVs as well as the vitamin C content of the GLVs under the existing practice of
storage. Therefore this study is an attempt to find the answers for the above concerns.

Determination of percentage of consumption
Ten popular market places in Batticaloa district namely Arayampathy, Batticaloa, Chenkalady, Eravur,
Kaluwanchikudy, Kallar, Kattankudy, Kiran, Oddamavadi and Valaichenai were selected for the study. During
Umaramani & Sivakanesan (2015) 2(3): 240245
.
the preliminary study it was found that around 20,000 people regularly used these markets. Interviews were
conducted for a period of 10 months and 5 consumers (subjects) were interviewed, on a random basis, during
each visit to the selected market. A total of one thousand subjects were interviewed at the end of the survey (5
consumers 2 times per month 10 places 10 months). Types of GLVs and percentage consumption of each
GLV based on the interview was calculated
Preparation of GLVs, storage and packing
The GLVs collected from field were cleaned and shoots separated. The shoots were washed thoroughly
under running tap-water for 5 minutes to remove soil particles and dirt. Then the washed shoots were placed in a
plastic container with paper towel and allowed to drain for 5 minutes. The cleaned, washed, GLVs were left at
room temperature to dry, and thereafter each GLV was wrapped with paper and then placed in polyethylene
bags. They were stored both at room temperature (30 2C) and 4C (refrigerator) for 4 days.
Estimation of vitamin C (Ascorbic acid)
All chemicals used for the study were of analytical grade and distilled water was used for the preparation of
reagents. Samples of GLVs (10 g of each) were accurately weighed and ground using a mortar and pestle in 20
ml of metaphosphoric acid-acetic acid solution. The mixture was strained through a muslin cloth and the extract
was made up to 100 ml with the metaphosphoric-acetic acid solution. The extracts were prepared using 3
different samples of each GLV and all analyses were carried out in triplicates.
Ascorbic acid content was estimated by the 2, 6-dichloroindophenol (DCP) titrimetic method (Nielsen 2010).
Metaphosphoric acid-acetic acid solution (5 ml) was pipetted separately into three 50 ml Erlenmeyer flasks
followed by 2 ml of the sample extract. The samples were titrated separately with the indophenol dye solution
until a light rose pink colour persisted for 5 seconds. The 2, 6-dichlorophenolindophenol dye was standardized
against standard ascorbic acid. The results were expressed as mg ascorbic acid/100 g wet weight (WW).

Consumption of GLVs
Thirty one species of GLVs were consumed commonly as food by the subjects, which ranged from 15% for
Lactuca sativa to 98% for Murraya koenigii and the average consumption was 59.35%. Among the GLVs, 16
species were consumed for more than 50 % and 15 species were consumed 50% or less by the subjects
interviewed (Table 1). GLVs consumed more than 50% by the subjects were selected to determine the vitamin C
content. The consumption of 16 selected GLVs ranged from 52 to 98% with an average of 79.1% (Table 1).
Table 1. Overall consumption of commonly consumed leafy vegetables for food purposes.
Vernacular names Consumption
No Name of GLVs
(T-Tamil, S- Sinhala, E-English) (%)
1 Aerva lanata* Polpala (s) /Thaenkaipukerai (T) /Hongone 81
2 Allmania nodiflora Kumatiya (S),Kumatti (T) 44
3 Alternanthera sessilis* Mukunuwenna/Ponnangani/Alligator weed 97
4 Amaranthus caudatus* Rana-Tampala/Kerai/Pendant amaranth 94
5 Amaranthus spinosus Mudkerai(T), Spiny amaranth (E) 42
6 Amaranthus viridis* Kura/Kuppaikerai/Green amaranth 76
7 Argyreia pomacea* Manpanchan (T) 60
8 Asteracantha longifolia Neer Mulli(T), Hydrophylla (E) 22
9 Basella alba Nivithi kola (S),Pasali (T), Indian spinach (E) 60
10 Borreria hispida Nathaisuri (T) 38
11 Canthium parviflorum Kara (S),Kaarai (T), Wild jasmine (E) 28
12 Cardiospermum halicacabum* Penela-wel (S)/Mudakottan (T)/ Wintetcherry 82
13 Centella asiatica* Gotukola (S)/Vallarai (T), Indian penny ward (E) 88
14 Cucurbita maxima Kumbala (S),Poosani (T), Pumpkin (E) 50
15 Delonix elata* Vatham-Nairaini (T),Yelow Gul-Mohur (E) 52
16 Drega volubilis* Anguna kola (S) Kurinja (T)/Sneez ward (E) 80
.
17 Gymnema sylvestre Bin nuga (S),Sirukurinja (T), Gymnema (E) 37
18 Ipomoea aquatica Kangkong (S),Vallal (T), Water spinach (E) 49
19 Lactuca sativa Salada (S), Salathu (T), Lettuse (E) 15
20 Mollugo oppositifolia* Hinipala (S)/Thirai (T)/Itch flower (E) 84
21 Moringa oleifera* Murunga (S)/Murungai (T)/ Drum stick (E) 95
22 Murraya koenigii* Karapincha (S)/Karuveppilai (T)/Curry leaf (E) 98
23 Passiflora edulis Kodimathulai (T) 46
24 Pisonia grandis* Wathabanga (S) Ledchakaddai (T), Lettuse tree (E) 67
25 Premna obtusifolia Sihin-Midi (S), Kananthi (T) 39
26 Premna serratifolia Midi (S,) Earumai mullai (T) 24
27 Premna latifolia Maha midi (S), Pasumullai (T) 40
28 Rivex ornate Musutai (T), Midnapore creeper (E) 41
29 Sauropus androgynus* Japanbatukola (S)/ Thavasi murungai (T) 54
30 Sesbania grandiflora* Katuru murunga (S)/ Ahaththi (T) 76
31 Solanam trilobatum* Wel-tibbatu (S), Thuthuvilai(T), Heliotrope (E) 81
Average 59.35
Note: Selected plant species used for determining vitamin C content.
Vitamin C (Ascorbic acid) content

The sixteen GLVs showed varying ascorbic acid content in fresh state and under two temperature storage
conditions for 4 days (Table 2). The vitamin C content of fresh GLVs ranged from 5.25 mg/100 g for Centella
asiatica to 416.2 mg/100 g for Drega volubilis on wet weight basis (W/W). Gupta & Prakash (2009) reported
the ascorbic acid content of C. asiatica was 15.18 mg/100 g (W/W) using the same method as in the present
study. However, the vitamin C content of Centella asiatica in the present study is much lower than that reported
by Gupta & Prakash (2009).
Table 2. Vitamin C contents of fresh and stored (for 4 days) GLVs.

Vitamin C content (mg/100g)
No GLVs
Fresh Room temp. 302 C Refrigeration 4 C
1 Aerva lanata 53.675.10a 43.753.21c 49.294.50b
a c
2 Alternanthera sessilis 36.173.42 6.711.91 10.791.58b
a c
3 Amaranthus caudatus 51.634.15 5.832.55 28.885.41b
a c
4 Amaranthus viridis 35.584.37 6.422.32 24.504.73b
a c
5 Argyreia pomacea 71.86 0.290.88 3.211.16c
a c
6 Cardiospermum helicacabum 85.1711.07 27.713.96 44.635.08c
a b
7 Centella asiatica 5.251.86 2.631.31 4.382.27a,b
a b
8 Delonix elata 183.1713.43 7713.32 194.8316.54a
b c
9 Drega volubilis 416.238.6 37129.14 505.467.08a
a c
10 Mollugo oppositifolia 39.3810.08 7.292.19 27.1310.66b
a c
11 Moringa oleifera 135.335.10 0.00 123.084.03b
a b
12 Murraya koenigii 22.755.57 16.045.16 23.926.49a
a b
13 Pisonia grandis 17.503.71 11.082.55 12.543.15b
a b
14 Sauropus androgynus 66.796.58 47.715.79 65.045.53a
a c
15 Sesbania grandiflora 134.7514.07 42.887.54 83.139.64b
a b
16 Solanam trilobatum 10.52.94 3.52.27 6.422.32b
Note: Values are means of 3 replicates Standard deviation; Statistically significance at 5% level
(Superscripts of letters a, b, c denotes statistically significant, superscripts of same letter denotes statistically
not significant).
The vitamin C content of 14 common vegetables in Nigeria estimated by the Nielsen (2010) procedure was
found to range from 400 to 692 mg/100 g dry weight and Amaranthus caudatus was reported to contain 400
mg/100 g on dry weight (Akindahunsi & Salawu 2005). Amaranthus caudatus was the only GLV investigated
by Akindahunsi & Salawu (2005) and in the present study and a comparison could not be made because of the
different procedures used. Oboh (2005) reported the vitamin C content of Amaranthus cruentus and Solanum
.
macrocarpon estimated by the Nielsen (2010) method as 70.00.5 mg/100 g and 43.570.6 mg/100 g
respectively. The Amaranthus varieties used in the current study had lower vitamin C content than reported by
Oboh (2005) and the difference could be due to different varieties studied. Similarly the vitamin C content of
Solanam trilobatum used in the present study too was lower than the levels reported by Oboh (2005) for
Solanum macrocarpon.
In general, ascorbic acid content of GLVs declined during 4 days of storage and the differences observed in
vitamin C content among selected GLVs in fresh and under two storage conditions are significant (p < 0.05)
(Table 2). Drega volubilis showed the highest content of ascorbic acid (416.238.6 mg/100 g, when fresh) in all
three conditions followed by Delonix elata, Sesbania grandiflora, Moringa oleifera and Sauropus androgynus.
Three species showed lower ascorbic acid content in all three conditions, such as Argyreia pomacea, Centella
asiatica and Solanam trilobatum (Table 2). Vitamin C content of fresh GLVs such as Centella asiatica, Delonix
elata, Murraya koenigii and Sauropus androgynus showed no notable changes when stored in a refrigerator
(Table 2).
The decline in vitamin C content in all the GLVs, ranging from 18% for Aerva lanata to 100% for Moringa
oleifera, was higher and significant when stored at room temperature for 4 days than when stored at 4C
(ranging from 2.5% for Sauropus androgynus to 70% for Alternanthera sessilis) except for Pisonia grandis
(Table 3). Both Drega volubilis and Delonix elata showed 22.3 and 6.1% increase in vitamin C content
respectively when stored at 4C (Table 3).
Table 3: Loss of vitamin C of GLVs stored for 4 days at different temperatures.

Loss during storage (%)
No GLVs Room temp. Refrigeration
302 0C 4 0C
a
1 Aerva lanata 18 8.04b
a
2 Alternanthera sessilis 81.6 70.06b
a
3 Amaranthus caudatus 88.93 44.4b
a
4 Amaranthus viridis 82.3 31.51b
a
5 Argyreia pomacea 96.30 53.70b
a
6 Cardiospermum helicacabum 67.41 47.43b
a
7 Centella asiatica 50.00 14.81b
a
8 Delonix elata 58.23 -6.31b
a
9 Drega volubilis 10.75 -22.29b
a
10 Mollugo oppositifolia 81.48 32.93b
a
11 Moringa oleifera 100.00 9.03b
a
12 Murraya koenigii 30.13 -4.88b
a
13 Pisonia grandis 36.27 28.27a
a
14 Sauropus androgynus 37.69 2.47b
a
15 Sesbania grandiflora 68.37 38.31b
a
16 Solanam trilobatum 67.96 38.52b
Note: Values are means of 3 replicates Standard deviation; Statistically significance
at 5% level (Superscripts of letters a, b, c denotes statistically significant, superscripts
of same letter denotes statistically not significant).
The vitamin C content of Drega volubilis increased significantly by 22.3% when stored in a refrigerator for 4
days compared to the fresh state (Tables 2 & 3). This should be further investigate any biochemical support to
this increasing. Interestingly, Latif & El-Aal (2007) also found that the vitamin C content of fresh GLVs
Raphanus sativus and Anthum graveolans increased during first 4 days of storage at 4C. The same observation
was made by Latif & El-Aal (2007) for Allium kurrat stored at 4C for 4 days. M. oleifera showed complete loss
of vitamin C at room temperature when stored for 4 days (Table 2 and 3). Moringa oleifera turned yellow and
some decayed when stored under room temperature for 4 days. Pisonia grandis showed no notable changes in
vitamin C when stored under room temperature and in a refrigerator.
More than 50% of vitamin C was lost in 10 species when stored at room temperature (302C) such as
Alternanthera sessilis, Amaranthus caudatus, Amarantus viridis, Argyeria pomacea, Cardiospermum
halicacabum, Delonix elata, Mollugo oppositifolia, Moringa oleifera, Sesbania grandiflora and Solanam
trilobatum. Green leafy vegetables such as Alternanthera sessilis, Cardiospermum halicacabum, Delonix elata,
.
Moringa oleifera and Sesbania grandiflora turned yellow when stored at room temperature. Alternanthera
sessilis and Argyeria pomacea lost more than 50% of vitamin C when stored at 4C (Table 3).
The highest percentage vitamin C loss when stored at room temperature was observed for Moringa oleifera
(100%) whereas highest percentage of vitamin C retained when stored at 4C was 98% for Sauropus
androgynus. Seven GLVs namely Aerva lanata, Centella asiatica, Delonix elata, Drega volubilis, Moringa
oleifera, Murraya koenigii and Sauropus androgynus showed more than 80% of vitamin C retention (Table 3).
Latif & El-Aal (2007) also reported that 80% of total vitamin C is retained when stored for 8 days, at 4C1C
packed in polyethylene bags. The rate of loss of vitamin C is low at cold temperature in the current study which
is in agreement with the observation of Latif & El-Aal (2007) and Favell (1998) under similar packing
conditions using polyethylene bags. The variation in decline in vitamin C content in GLVs appears to be due to
species difference, pre and post-harvest conditions, initial vitamin C concentration, auto-oxidation, storage and
enzymatic degradation (Latif & El-Aal 2007, Rivera et al. 2006, Howard 1999). Vitamin C loss continues
during post-harvest handling, processing, cooking and storage (Moreira et al. 2006).
Drega volubilis, which had the highest amount of vitamin C is consumed by 80% of the subjects interviewed
(Table 1). Murraya Koenigii, which is used by almost all the subjects interviewed, is a poor source of vitamin C.
Similarly Centella asiatica, which was claimed to be consumed by 90% of the subjects, too was a poor source of
vitamin C. Furthermore, packaging and storing the vegetables at 4C, which prevents wilting and retain the
freshness of GLVs, preserves the vitamin C content compared to packaging and storing at room temperature.
These observations are valuable in carrying out nutrition awareness among the public. Delonix elata, which was
next to Drega volubilis in ascorbic acid content, was stated to be consumed by 52% of the subjects.
CONCLUSION
Thirty one species of GLVs were commonly consumed by the subjects. The average consumption was about
59% and ranged from 28 to 98%. GLVs consumed for medicinal purpose, ranged from 1.7% for Amaranthus
caudatus to 48.9% for Cardiospermum halicacabum and the average consumption was 20.11%. GLVs
consumed more than 50% by the subjects interviewed, were selected to determine the vitamin C content and
such GLVs also showed a high consumption for their medicinal properties.
Vitamin C content of fresh GLVs ranged from 5.25 mg/100 g wet weight for Centella asiatica to 416.2
mg/100 g wet weight for Drega volubilis. Decline in vitamin C content was observed in GLVs stored at room
temperature. GLVs wrapped with paper and then in polyethylene bags and stored at cool temperature retained
more than 80% of vitamin C. Vitamin C content of Drega volubilis increased by 22% when stored in a
refrigerator for 4 days. This needs further investigation.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(3): 246252, 2015
Research article
Physiological response of broccoli exposed to RuO2 nanoparticle

Imtiyaz Hussain1, Ajey Singh1, Himani Singh1, S. C. Singh2 and N. B. Singh1*
1
Plant Physiology Laboratory, Department of Botany, University of Allahabad, Uttar Pradesh, India
2
Department of Physics, University of Allahabad, Uttar Pradesh, India
*Corresponding Author: singhnb166@gmail.com [Accepted: 28 November 2015]
Abstract: Nanotechnology no doubt is a boom for science but exposure of nanoparticles (NPs) in
the environment is a new concern. Application of NPs in agriculture to increase crop yield is still
debated. In the present study 1040 g.ml-1 concentrations of ruthenium oxide (RuO2) NPs were
exposed to Broccoli (Brassica oleracea var. italica) seedlings in hydroponic culture. RuO2 NPs are
synthesized via Laser ablation method and hence are contamination free. UV-visible spectroscopy
and field emission scanning electron microscope (FE-SEM) is used for the characterization of NPs.
Carotenoids, protein and sugar content decreased with increase in concentrations of RuO2 NPs.
Total chlorophyll content increased to maximum with highest content of Chlorophyll a & b at 10
g.ml-1 of RuO2 NPs, but no stimulatory effect was recorded at highest doses. Lipid peroxidation,
was unaffected by exposure to 020 g.ml-1 NPs, but at 40 g.ml-1 malondialdehyde (MDA)
formation and SOD activity increased by two fold. It is concluded that RuO2 NPs significantly
inhibited the seedlings growth of broccoli by impairing the metabolism of the test plants.
Keywords: RuO2 NPs - Brassica oleracea var. italica - FESEM - Hydroponic culture -
Malondialdehyde.
[Cite as: Hussain I, Singh A, Singh H, Singh SC & Singh NB (2015) Physiological response of broccoli
exposed to RuO2 nanoparticle. Tropical Plant Research 2(3): 246252]
INTRODUCTION
Nanoparticles (NPs) in between 1100 nm size act as bridge between bulk material and atomic or molecular
structure (Kaushik et al. 2010). They possess remarkable and interesting properties due to small size, large
surface area, free dangling bonds, high reactivity other than bulk material of same composition (Daniel &
Astruc 2004). The use of nanomaterials in industries such as medicine (Panatarotto et al. 2003) and agriculture
(Singh et al. 2014, Shekhawat et al. 2014) is increasing rapidly which leads to its exposure in the environment.
Application of NPs in agriculture to increase crop yield is still debated. It is now widely recognized that
sufficient amount of NPs exists in the soil which affects living systems. A broad and mechanistic understanding
of the risks is posed by NPs in the environment, including bioaccumulation through the food chain, thus it is
necessary to adequately protect human and environment.
Ruthenium (Ru) a rare earth element is known to possess useful catalytic properties and have imperative role
in nuclear reactors which become source of Ru in the environment. Cowser & Parker (1958) reported Ru as
radioactive waste in soil. Incorporation of Ru in by plants is already verified (Selders 1950, Klechkwosky 1956,
Goss & Romey1959). Menzel & Brown (1959) found that ruthenium at 0.0018 and 0.18 mgml-1 in hydroponic
solution had no effect on the metabolism of Trifolium pratense. Very limited studies have been done to explore
the response of RuO2 NPs on plants and the toxicity data regarding Ru and its oxides are also scarce. Ru NPs
synthesized using Gloriosa superb L. leaf extract shows antibacterial activity against gram positive & negative
bacterial strains (Kasi et al. 2014).
The essential processes leading to plant adaptation to any stress or toxicity include regulation of water loss
through stomata, metabolic adjustment, toxic ion homeostasis, and osmotic adjustment has been studied
(Hasegawa et al. 2000, Munns & Tester 2008). Imlay & Linn (1998) reported that the reactive oxygen species
(ROS) are responsible for various stress-induced damages to tissues of an organism. Consequently the role of
antioxidant enzymes viz. superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) are
responsible for the quenching of ROS becomes very important (Bowler et al.1992). Thus the present study
Received: 17 August 2015 Published online: 31 December 2015
Hussain et al. (2015) 2(3): 246252
.
aimed to evaluate the potential effects of RuO2 NPs on living systems especially on physiological metabolism of
broccoli plant.

Synthesis of RuO2 NPs
Procedure for the synthesis of NPs was adopted from Singh et al. (2010). In the experiment, 2g of ruthenium
oxide bulk powder was dispersed in 20 ml of double distilled water (DDW). Fundamental (1064 nm) output
from pulsed Nd:YAG laser operating at 40 mJ pulse-1 energy, 10 ns pulse width and 10 Hz rep rate was
bombarded at the centre of the solution column using 25 cm focal length quartz lens for half an hour with
continuous magnetic stirring . In order to avoid laser induced sedimentation or aggregation, ablation process was
carried out for 30 minutes. The weight of the source was measured before and after the ablation process to
estimate concentration of RuO2 NPs. This process was repeated several times to get colloidal solution of RuO2
NPs as stock solution.
Characterization of RuO2 NPs
Characterization of synthesized RuO2 NPs was carried out by UV-visible spectroscopy and field emission
scanning electron microscope (FE-SEM). Lambda 35 Perkin Elmer spectrophotometer was used to record of
UV-visible absorption spectra of colloidal solution of RuO2 NPs. Scanning electron microscopic analysis was
done by JEOL JXA-8230. Very few amount of precipitated powder of NPs was coated on the copper grid and
allowed to magnify the grid to record morphological characters and size of NPs.
Hydroponic culture
The experiment was conducted in the month of February, 2015 in the glass house in the Department of
Botany, University of Allahabad, Allahabad (24o47 and 50o 47N latitude; 81o 91 and 82o 21E longitude; 78
m above sea level). The different concentrations viz. 0, 10, 20, 30 and 40 g.ml-1 of RuO2 NPs considered for C,
R1, R2, R3, R4 treatments respectively were obtained by dilution of stock solution with appropriate amount of
double distilled water (DDW). The nursery of the test plants was raised in nursery beds (11m). Twenty one
days old seedlings were uprooted and washed with tap water followed by DDW until soil was totally removed
from the roots. The properly washed 10 seedlings were transferred in transparent plastic boxes (23179 cm)
filled with 2 litre of half strength Hoagland solution (Hoagland & Arnon 1950). Aeration of medium was done
with the help of bubblers for 12 h daily. The plants were allowed to establish for a week. After a week the
Hoagland solution was replaced with fresh Hoagland solution and the respective concentration of NPs twice at
the interval of seven days were also added. Box containing only Hoagland solution was taken as control. The
boxes were covered with black paper to avoid the algal growth in the nutrient medium. The biochemical
parameters were recorded after 14 days of treatment.
Pigment, Protein and Sugar content
The photosynthetic pigments viz. chlorophyll a, chlorophyll b and carotenoids from the first fully expanded
leaves (10 mg) were extracted with 80% acetone and quantified following Lichtenthaler (1987). Optical density
of supernatant was measured with UV-visible spectrophotometer Lambda 35 PerkinElmer at 663, 646 and 470
nm. The amount of pigments was calculated as:
Chlorophyll a (g.ml-1) = 12.21 x A663 - 2.81 x A645
Chlorophyll b (g.ml-1) = 20.13 x A645 - 5.03 x A663
Carotenoid (g.ml-1) = [1000 x A470 - 2.27(Chl. a) 81.4(Chl. b)] / 227
Where, A is the observed OD
Quantitative analysis of protein was done following Lowry et al. (1951). The absorbance was measured at
650 nm. The amount of protein was calculated with reference to standard curve of bovine serum albumin.
The quantification of total soluble sugars was done following Hedge & Hofreiter (1962). Fresh leaf tissue
(0.05 mg) was homogenized in 5 mL of 95% ethanol. After centrifugation, 1 mL of supernatant was mixed with
4 ml anthrone reagent and heated in boiling water bath for 10 min. After cooling, the absorbance was recorded
at 620 nm. The amount of sugar was determined using the standard curve prepared from glucose.
Lipid peroxidation and SOD activity
The lipid peroxidation (LP) in leaves was measured by determining the malondialdehyde (MDA) content
according to Heath & Packer (1968). The plant material (200mg) was homogenized in 5 ml of 0.1% w/v
Hussain et al. (2015) 2(3): 246252
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trichloroacetic acid and centrifuged at 10,000g for 10 min. One mL of supernatant was mixed with 4 mlof 0.5%
thiobarbituric acid made in 20% trichloroacetic acid. The mixture was then heated at 95 C for 30 min followed
by cooling and centrifugation. The absorbance of supernatant was measured at 532 nm and corrected by
subtracting the non-specific absorbance at 600 nm. The MDA concentration was calculated using the extinction
coefficient of 155 mM-1 and expressed as n mol g-1 FW.
The activity of SOD (EC 1.15.1.1) was estimated by the nitrobluetetrazolium (NBT) photochemical assay
following Beyer & Fridovich (1987). The reaction mixture (4 ml) consisted of 20 mM methionine, 1.3M
riboflavin, 0.15 mM ethylene diamine-tetra acetic acid (EDTA), 0.12 mM NBT, and 0.5ml supernatant. The test
tubes were exposed to fluorescent lamp for 30 min and identical unilluminated assay mixture served as blank.
One unit of enzyme was measured as the amount of enzyme which caused 50% inhibition of NBT reduction.
Statistical Analysis
Treatments were arranged in a randomized block design with three replications. Data were statistically
analyzed using analysis of variance (ANOVA) by using SPSS (Ver.10; SPSS Inc., Chicago, IL, USA. The
treatment means were analyzed by Duncans multiple range test (DMRT) at p < 0.05.

UV-visible absorption spectra of bulk as well as NPs produced after 5, 10 and 30 minutes of laser
irradiations are shown in figure 1. It is evident that with the increase of time of laser irradiation, band edge
absorption shifts towards the shorter wavelength side, which indicates laser induced reduction in the size of
particles. Colloidal solution of NPs produced after 30 minutes of laser irradiation is highly constant in colloidal
form for several months and is used for the experiment. The morphological studies of synthesized NPs have
been carried out by Field Emission Scanning Electron Microscopy magnifications (FE-SEM). It is observed,
NPs constructing nanostructures were in the range of 30113 nm with spherical shape in the under 50,000 X
(Fig. 2). The FE-SEM images of NPs were assembled on to the surface due to the interactions such as hydrogen
bond and electrostatic interactions which is supported by the SEM images of Kathiravan et al. (2015).
Figure 1. UV-Visible spectra of ruthenium oxide NPs at time in terval of 0, 5, 10 and 30 minutes.
Ru exhibited adverse effect on both physiological and biochemical parameters of crop plants. Amounts of
chlorophyll a, chlorophyll b, carotenoids, protein and MDA content and SOD activity were considered as
indicators for the estimation of effects of different concentrations of ruthenium oxide NPs on the health of the
plant.
Hussain et al. (2015) 2(3): 246252
.
Figure 2. FE-SEM image of ruthenium oxide NPs taken at 50,000 X, showing NPs in the range of 30-113 nm.
The present study confirms that the increase in RuO2 NPs concentrations on broccoli seedlings appeared to
have negative impacts, primarily which is evident from a reduced sugar and protein content and the increase in
MDA formation and SOD activity by 2 fold at highest concentration. A decrease in sugar and protein under
unfavourable conditions allows the conservation of energy, thereby launching the appropriate defence response
and also reducing the risk of damage. RuO2NP did not significantly influence the chlorophyll content of plant
seedlings but in all treatments carotenoid content decreased (Table 1). The total chlorophyll content improved
under lower concentrations of RuO2 NPs. The seedlings treated with 10g.ml-1 of RuO2 NPs exhibited maximum
Table 1. Effects of Ruthenium Oxide NPs on pigment content of broccoli seedlings.
Chlorophyll a Chlorophyll b Total Chlorophyll Carotenoids
Treatments
(mg.g-1 FW) (mg.g-1 FW) (mg.g-1 FW) (mg.g-1 FW)
C 2.260.088b 0.560.032c 2.830.120c 0.520.017a
a a a
R1 2.930.088 0.850.034 3.780.070 0.440.008b
R2 2.720.109a 0.680.037b 3.410.140b 0.330.017c
c d c
R3 1.910.075 0.450.033 2.360.041 0.250.014d
R4 2.160.094bc 0.400.006d 2.560.100c 0.200.005e
Note: C= 0 g.ml ; R1= 10 g.ml ; R2= 20 g.ml ; R3= 30 g.ml ; R4= 40 g.ml-1.
-1 -1 -1 -1
Mean SE values followed by same letters within each column are not significantly different at
0.05 (ANOVA and Duncans multiple range test) n=3.
content of chlorophyll a and b, while no effect was recorded at the highest dose of NPs. This might be due to
increase in efficiency of photosystems by Ru NPs and the role of Ru in photosystem is in agreement with the
work of Xiaojun et a.l (2004). They found that Ruthenium tris-bipyridine complex act as photosensitizer, that
plays the role of the p680 chlorophyll in psII. The protein content of leaves decreased significantly, reaching the
minimum value in the plant treated with 30g.ml-1 of RuO2 NPs (Table 2). The decrease of sugar was higher
under low treatments as compared to that of treatments with increased concentration. RuO2 at 20 g.ml-1
concentration caused maximum decrease in sugar content over control (Table 2). Our results on biochemical
parameters are in agreement with the findings of Khuzihko et al. (2011). According to their report RuO2 in
Hussain et al. (2015) 2(3): 246252
.
lower concentration enhanced the photocatalytic activity by taking part in oxygen evolution reaction (OER)
while in excess amount exhibited adverse effect on photocatalytic activity. Moreover, RuO2 also acts as a water
oxidation catalyst (Trasatti & Buzzanca 1971).
Table 2. Effects of Ruthenium Oxide NPs on protein and sugar content, lipid peroxidation and SOD
activity of broccoli seedlings.
Protein Sugar lipid peroxidation Superoxide dismutase
Treatments
(mg.g-1 FW) (mg.g-1 FW) (n mol.g-1 FW) (EU.g-1 FW)
C 26.300.502a 86.230.648a 11.800.357c 46.050.138d
R1 24.390.265b 77.391.243b 12.980.193c 43.270.177e
R2 21.220.451c 53.551.442d 12.390.582c 48.760.280c
R3 19.780.232d 67.341.368c 21.560.238b 85.340.490b
R4 20.140.098d 77.320.779b 27.620.285a 99.791.015a
Note: C= 0 g.ml-1; R1= 10 g.ml-1; R2= 20 g.ml-1; R3= 30g.ml-1; R4= 40 g.ml-1.
Mean SE values followed by same letters within each column are not significantly different
at 0.05 (ANOVA and Duncans multiple range test) n=3.
In our study, H2O2 in connection with other signal molecules may contribute to the control of plant growth
and development at specific checkpoints of the cell cycle (Xiong et al. 2002). Malondialdehyde (MDA)
production, a measure of lipid peroxidation, was unaffected by exposure to 020 g.ml-1 NPs, but at 40 g.ml-1
MDA formation was increased by more than two fold (Table 2). Increase in H2O2 was observed only at higher
concentrations conditions which are in agreement with the significant increase in H2O2 observed in cultivated
tomato (Mittova et al. 2002) and pea plants (Hernandez et al. 2001) under stresses. The seedlings treated with
the lower concentrations R1 and R2treatment of RuO2 NPs exhibited minimum lipid peroxidation while the
highest dose of RuO2 NPs exhibited maximum 128% of lipid peroxidation.
SOD activity significantly enhanced under higher concentrations of RuO2 NPs (Table 2).Because of the
significant damage likely resulting from ROS production and associated toxicity of NPs exposure, the SOD
content of plant leaves was determined. Quantification of SOD activity confirms the results that at the10 g.ml-
1
exposure level had no impact but exposure at 40 g.ml-1 NPs resulted in significantly greater SOD activity.
SOD activity directly modulates the amount of ROS same from what was reported by Gmez et al. (2004)
which found an increase in all SOD isoenzymes of pea chloroplasts under stress. Deficiency of micronutrients
such as Mn and Zn also affects SOD activities in plants (Yu & Rengel 1999) but we provide appropriate
hogland solution as reported earlier (Singh et al. 2014). Thus in our results the activity of SOD under stress
depends on the toxicity level.
CONCLUSION
We have successfully demonstrated the toxic effect of RuO2NPs on broccoli plant. Purity of laser produced
RuO2 NPs with chemical contamination free surfaces helps in the real investigation of NPs effects on the
biological systems. The NPs at 40 g.ml-1 resulted in drastic increase of antioxidant enzyme (SOD) and MDA
content and decrease in carotenoid, protein and sugar content in hydroponic culture. The decrease in sugar
content and protein content is due to diverting of maximum energy towards plants defence mechanism. Based
on findings we conclude that higher doses (>20 g.ml-1) of RuO2 NPs are toxic. Further study is required to
explore the minimum dose of RuO2 NPs which have least impact on biological system that ensures safe
environment release.
ACKNOWLEDGEMENTS
The authors are thankful to the CSIR and UGC, New Delhi, India for providing financial assistance to
Imtiyaz Hussain and IIT Kanpur for providing SEM facility.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(3): 253256, 2015
Research article
Three new records of dicotyledonous plants from Bangladesh

Md. Salah Uddin*, Mohammad Sajid Ali Howlader and Shaikh Bokhtear Uddin
Initiative for Biodiversity Research and Development (IBIRD), Dhaka, Bangladesh
Ecological Genetics Research Unit, Department of Biosciences, University of Helsinki, Helsinki, Finland
Department of Botany, University of Chittagong, Chittagong, Bangladesh
*Corresponding Author: sohailmsu@gmail.com [Accepted: 02 December 2015]
Abstract: Three dicotyledon plant species belong to three different families of angiosperm include
Symphorema involucratum from Verbenaceae, Miliusa balansae from Annonaceae and
Spermacoce ocymoides from Rubiaceae have been recorded for the first time from Bangladesh.
They are collected from Chittagong and Chittagong hill tracts. Each species is presented with
updated nomenclature, taxonomic description, ecology, geographical distribution, places of
occurrence in Bangladesh and field photographs of these three new records are also provided.
Keywords: Bangladesh - Dicotyledon - Angiosperm - New records - Plants.
[Cite as: Uddin MS, Howlader MSA & Uddin SB (2015) Three new records of dicotyledonous plants from
Bangladesh. Tropical Plant Research 2(3): 253256]
INTRODUCTION
Dicotyledons plants are the most successful and dominant plant group (Heywood 1993) having seeds with
two cotyledons and an exogenous manner of growth (Cronquist 1981, Bewley & Black 1994). It is one of the
classes of the great subdivision of flowering plants, the angiosperms (Wagner et al. 1999, McKenna et al. 2009).
About 4,00,000 species of angiospermic plants have so far been recorded of which more than 2,50,000 are
dicotyledons and the remaining are monocotyledons (Leitch & Leitch 2008). Bangladesh is endowed with about
5,000 species of flowering plants (Pasha & Uddin 2013, Rashid et al. 2014) of which more than two third are
dicotyledonous (Khan 19721987). Dicot plants dominate the forests, village groves and woodlands of
Bangladesh (Khan & Banu 1969, Khan & Afza 1968, Khan 19721987).
Here, in this paper, three dicot plant species from three different families includes Verbenaceae, Annonaceae
and Rubiaceae are reported for the first time from Chittagong and Chittagong Hill Tracts for Bangladesh, placed
in Indo-Burma biodiversity hotspot.

The plant specimens of three families include Verbenaceae, Annonaceae and Rubiaceae have been collected
from different areas of Bangladesh, mainly the forest of Chittagong and Chittagong Hill Tracts (Fig. 1) through
repeated field trips.
The collected specimens were preserved at Chittagong University Herbarium (CUH) and examined by using
Microscope. Unnamed specimens were identified and described by consulting relevant floristic literature of
Roxburgh (1805), Gamble (19211935), Matthew (1991), Nair et al. (1983), Henry et al. (1987, 1989), Pasha &
Uddin (2013).
The photographs of fertile specimens in natural habitat were taken during field trips. A taxonomic
enumeration with these three newly recorded from three families are prepared. In the ennumeration, each
species is cited with current nomenclature, taxonomic description, ecology, geographical distribution,
occurrence in Bangladesh.
RESULTS
Taxonomic enumeration
Family: Verbenaceae J. St.-Hil. 1805
Uddin et al. (2015) 2(3): 253256
.
Figure 1. Distribution of three species in Bangladesh.

Genus: Symphorema Roxburgh, 1805
Symphorema involucratum Roxburgh, 1805 (Fig. 2A)
A climbing shrub, with cylindrical branchlets which are hairy when young. Leaf blade is nearly elliptic to
ovate, densely velvety on the underside, and somewhat smooth above. Leaf base is rounded to slightly heart-
shaped, margin nearly entire to toothed. Flowers are borne in beautiful clusters carried on long, velvety stalks.
The bracts just below the flowers are oblong, enlarged in fruit. Sepal cup is tube-like, velvety outside. Flowers
are white, with 6-8 narrowly oblong petals. Fruit is nearly round.
Flowering: MarchApril.
Ecology: Forests or scrub in valleys.
Geographical distribution: India, Myanmar, Sri Lanka and Thailand.
Occurrence in Bangladesh: It was collected from Recha jhiri (2210'56.4"N 9211'18.0"E), Meghla, Sadar
upazila, Bandarban district, Bangladesh.
Family: Annonaceae Juss., 1789

Genus: Miliusa Leschenault ex Alph. de Candolle, 1832
Miliusa balansae Finet & Gagnepain, 1906 (Fig. 2B)
A shrub about 25 m tall, branchlets slightly pubescent. Leaves petiolate, leaf blade elliptic, elliptic-oblong,
or oblong, membranous, glabrous or sparsely puberulent on midvein and secondary veins but glabrescent,
secondary veins 1012 on each side of midvein, base cuneate to rounded and oblique, apex acuminate to shortly
acuminate. Inflorescences axillary, flowers solitary. Pedicel filiform, pendulous, glabrous.Sepals ovate, slightly
pubescent. Petals red; outer petals slightly longer than sepals; inner petals ovate, apex reflexed. Anthers ovoid to
obovoid.Carpels oblong to lens-shaped, slightly pubescent; ovules 2 or 3 per carpel; stigmas terete,
puberulent.Fruiting peduncle slender; monocarps globose.Seeds 13 per monocarp.
Flowering: AprilJuly; fruiting: JulyDecember.
Ecology: Forests or scrub in vallyes.
Geographical distribution: China and Vietnam.
Occurrence in Bangladesh: The plant species was collected from Dulu jhiri (2211'01.7"N 9211'24.0"E),
Meghla, Sadar upazila, Bandarban district, Bangladesh
Uddin et al. (2015) 2(3): 253256
.
Family: Rubiaceae Juss., 1789
Genus: Spermacoce L., 1753
Spermacoce ocymoides Burm. f., 1768 (Fig. 2. C & D)
An annual herb, stem erect rarely prostrate, hairy. Leaves are oppositely arranged. It should be relatively
easy to differentiate from other species with its broadly elliptic (sometimes lanceolate) leaves which have
depressed venation, either hairless or with scattered hairs on veins below and margin. Flower clusters appear at
the end of branches or in leaf axils. Sepals are narrowly triangular, length variable on individual flowers.
Flowers are white, petals much longer than the tube. Stamens protrude out, but are much shorter than the petals.
Style does not exceed the stamens.
Flowering: AprilAugust.
Ecology: Plain land along roads, hill slopes and forest periphery.
Geographical distribution: Tropical Africa, Mauritius, India, Myanmar, Java, Peninsular Malaysia and
Philippines.
Occurrence in Bangladesh: This species was collected from two regions; one is Chittagong University Kata
Pahar road side (2228'15.4"N 9147'23.5"E), Hathazari, Chittagong, Bangladesh and another collection place is
Toitong High School road side (2153'00.2"N 9158'22.5"E), Pekua, Coxs Bazar, Bangladesh.
Figure 2. A, Symphorema involucratum; B, Miliusa balansae; C & D, Spermacoce ocymoides.
DISCUSSION AND CONCLUSION

We have reported three species from three highly diverged families comprising several numbers of species.
The family Verbenaceae consists of about 100 genera and 2600 species mostly pantropical, a few are limited to
temperate regions. In Bangladesh, this family is represented by 19 genera and 68 species (Ahmed et al. 2009).
Uddin et al. (2015) 2(3): 253256
.
Annonaceae family consists of about 130 genera and 2300 species are very largely confined to tropical regions,
15 genera and 42 species in Bangladesh (Ahmed et al. 2008). Rubiaceae includes about 450 genera and 6500
species occur in tropical and subtropical regions, in Bangladesh it has 56 genera and 170 species (Ahmed et al.
2009).
Herein we report three species which were available in other neighboring countries of Bangladesh. Our
findings may increase the possibility of finding more species which were reported from neighboring countries.
ACKNOWLEDGEMENTS
The authors expressed their deepest sense of gratitude and sincere thanks to Dr. Mostafa Kamal Pasha,
Department of Botany, University of Chittagong for his kind co-operation for article preparation.
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Ahmed ZU, Hassan MA, Begum ZNT, Khondoker M, Kabir SMH, Ahmad M & Ahmed ATA (2009)
Encyclopedia of Flora and Fauna of Bangladesh. Vol. 10. Angiosperms: Dicotyledons (Ranunculaceae-
Zygophyllaceae). Asiatic Society of Bangladesh, Dhaka, Bangladesh, 580 p.
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Cronquist A (1981) An integrated system of classification of flowering plants. Columbia University Press, New
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Heywood VH (1993) Flowering plants of the world (No. Ed. 2). BT Batsford Ltd.
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(Monocotyledons). Journal of Asiatic Society, Pakistan 14(2): 217224.
Khan MS (1972-1987) Flora of Bangladesh. Fasc. 139. Bangladesh National Herbarium, Dhaka, Bangladesh.
Leitch A R & Leitch IJ (2008) Genomic plasticity and the diversity of polyploid plants. Science 320(5875):
481483.
Matthew KM (1991) An Excursion Flora of Central Tamil Nadu, India. Oxford and IBH Publishing co., New
Delhi, India, 647 p.
McKenna DD, Sequeira AS, Marvaldi AE & Farrell BD (2009) Temporal lags and overlap in the diversification
of weevils and flowering plants. Proceedings of the National Academy of Sciences 106(17): 70837088.
Nair NC & Henry AN (1983) Flora of Tamil Nadu, India. Series I: Analysis. Vol. 1. Botanical Survey of India,
Coimbatore, India, 184 p.
Pasha MK & Uddin SB (2013) Dictionary of Plant Names of Bangladesh. Janokalyan Prokashani, Chittagong,
Bangladesh, 434 p.
Rashid, MHU, Rashid M E & Rahman MA (2014) Inventory of threatened plants of Bangladesh and their
conservation management. International Journal of Environment 3(1): 141167.
Roxburgh W (1805) Plants of the Coast of Coromandel. Vol. 2. W. Bulmer and Co., London, pp. 46.
Wagner WL, Herbst DR & Sohmer SH (1999) Manual of the Flowering Plants of Hawai'i, Vols. 1 and 2 (No.
Edn 2). University of Hawai'i and Bishop Museum Press, Honolulu Hawaii, USA.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(3): 257263, 2015
Research article
Genotypic variations in the inhibitory potentials of four combined

botanicals on mycelia growth of Macrophomina phaseolina of
cowpea [Vigna unguiculata (L) Walp.]
A. O. Akanmu1, O. J. Olawuyi1, O. B. Bello2*, O. A. Akinbode3,
T. Aroge1, B. Oyewole1 and A. C. Odebode1
1
Department of Botany, P.O. Box 128, University of Ibadan, Ibadan, Nigeria
2
Department of Biological Sciences, Fountain University, Osogbo, Nigeria
3
Plant Pathology Unit, Institute of Agriculture Research and Training, Apata, Ibadan, Nigeria
*Corresponding Author: obbello2002@yahoo.com [Accepted: 07 December 2015]
Abstract: The ethanol extracts of Ficus asperifolia, Mormordica charantia, Anacardium

occidentals and Psidium guajava were evaluated sole and in treatment combinations at 25, 50 and
75mg ml-1 concentration levels against the mycelial growth of Macrophomina phaseolina of
Cowpea. The pathogen was cultured on plates containing botanicals amended Potato Dextrose
Agar (PDA) in three replicates while only ethanol treated PDA tested plates served the control
experiment. The radial growths were recorded at 4th, 6th and 8th day after inoculation. Data
obtained were analysed using the SAS software program version 9.2. The extract of Mormordica
charantia was the most effective in the botanical treatments alone. The most significant inhibition
of Macrophomina phaseolina were observed from the combined treatments of Ficus asperifolia,
Mormordica charantia and Anacardium occidentals (3.11 cm), followed by Mormordica
charantia and Psidium guajava (3.29 cm), then combination of four extracts; Ficus asperifolia,
Mormordica charantia, Anacardium occidentals and Psidium guajava (3.53 cm), then
Mormordica charantia and Anacardium occidentals (3.84 cm). Other treatments, either alone or in
combination produced significant result compared to the control experiment (6.94 cm). However,
the efficacy of botanicals increased with concentration and also significantly correlated with time
and reduction in mycelia extension of the pathogen. More so, variability in the antifungicidal
potentials of the botanicals on Macrophomina phaseolina ranges from 15.93% to 34.06%
according to Eigen proportions. The treatment combinations of; Ficus asperifolia, Mormordica
charantia and Anacardium occidentals at 75mg ml-1 concentration level produced the most
inhibitory effect against Macrophomina phaseolina in vitro. However, the untreated plates did not
show inhibitory effect on the mycelial growth of the pathogen. Therefore, combined treatments of
botanicals could be a potential source in the practise of plant disease control.
Keywords: Macrophomina phaseolina - Mycelial growth - Correlation - Eigen proportion.
[Cite as: Akanmu AO, Olawuyi OJ, Bello OB, Akinbode OA, Aroge T, Oyewole B & Odebode AC (2015)
Genotypic variations in the inhibitory potentials of four combined botanicals on mycelia growth of
Macrophomina phaseolina of cowpea [Vigna unguiculata (L) Walp.]. Tropical Plant Research 2(3): 257263]
INTRODUCTION
Macrophomina phaseolina (Tassi) Goid. belongs to the family Botryosphaeriaceae. It is a highly polyphagus
necrotrophic fungal pathogen that infects more than 500 plant hosts (Wyllie 1988). The plants invaded by this
pathogen are; major food crops, pulse, fiber and oil crops (Su et al. 2001, Mayek-Prez et al. 2001, Dinakaran &
Mohammed 2001, Aly et al. 2007, Khan 2007). Macrophomina phaseolina have a wide geographical
distribution, and is especially found in tropical and subtropical countries with arid to semi-arid climates in;
Africa, Asia, Europe, and North and South America (Wrather et al. 2001). Diseases caused by Macrophomina
phaseolina include ashy seedling blight, seedling damping-off, charcoal rot, stem rot, and root rot leads to
significant yield loss (Emechebe & Lagoke 2002, Wellington et al. 2011). Incidences of these diseases are
Akanmu et al. (2015) 2(3): 257263
.
favoured at low soil moisture (Sandhu et al. 1999, Islam et al. 2012) and high temperatures (3035C). The
comparatively high capacity of Cowpea to withstand drought stress, poor soil conditions and high temperatures
made the crop very valuable especially in arid and Sub-Saharan regions of West Africa where cowpea is a crop
of major economic importance for resource poor farmers (Sanginga et al. 2003).
The persistence of sclerotia of Macrophomina phaseolina in the soil and plant debris (Short et al. 1980)
coupled with its wide host range has resulted in difficulty of the disease control especially on Cowpea where the
pathogen constitutes a major yield-suppressing factor even when under drought stress (Wyllie 1993). The
ineffective chemical control approach against Mormordica phaseolina necessitates a biological control measure
(Mark & Norman 2007). However, disease caused by fungi pathogens and their control with botanicals has been
found to be environmental friendly and effective against the targeted pathogen (Odebode 2006, Akanmu et al.
2013a, Abiala et al. 2013). This study investigates the efficacy of the interactive treatments of the extracts of
Ficus asperifolia, Mormordica charantia, Anacardium occidentals and Psidium guajava alone and in treatment
combinations against the pathogenic Macrophomina phaseolina of Cowpea.

Source of pathogen
The pathogenic strain of Macrophomina phaseolina isolated from Cowpea was obtained from the plant
pathology laboratory, International Institute of Tropical Agriculture (IITA) Ibadan, Oyo state, Nigeria.
Source of plant extracts
Fresh leaves of Ficus asperifolia, Mormordica charantia, Anacardium occidentales and Psidium guajava
were obtained from Botanical garden and authenticated at the herbarium laboratory, both of the Department of
Botany, University of Ibadan, Ibadan, Nigeria.
Preparation of plant extracts
The fresh leaves were washed in clean water to be freed from possible dirt and debris. The cleaned leaves
were soaked in 5% Sodium hypochlorite solution for 10 minutes and rinsed in three exchanges of distilled water
to neutralize the effect of Sodium hypochlorite. The leaves were then air dried for two week weeks after which
they were blended separately into powdery form. The ethanolic extraction was carried out by weighing 2.5 g,
5.0 g and 7.5 g of each powdered extracts separately into 100ml of 75% ethanol, to achieve 25 mg ml -1, 50 mg
ml-1 and 75 mg ml-1 concentration levels respectively.
In vitro control of Macrophomina phaseolina with plant extracts
The Macrophomina phaseolina culture obtained was asceptically subcultured on Potato Dextrose Agar
(PDA) and incubated for 7 days. The biocontrol experiment using plant extracts was carried out by adding 1ml
of the extracts to 9 ml of PDA poured on each plate. This was observed on each concentration level of the four
plant extracts used in this study. For the interactive treatment which involves the combination of two extracts,
0.5 ml of each extracts were added to 9ml of PDA poured per plate. The interaction of three extracts were
prepared by adding 0.33 ml of each extract to 9 ml of PDA while for the different four extracts used, 0.25 ml of
each were added to 9 ml of PDA. The extracts amended molten PDA was initially swirled gently for
homogenization before the plates were poured and allowed to solidify. Using a 5 mm cork borer, the mycelia
growth from the advancing edge of the 7 day old pure culture of Macrophomina phaseolina were picked and
inoculated at the centre of extracts treated PDA plates. The control experiment consisted of the treatments of 1
ml 75% ethanol with 9 ml of PDA. The plates were then incubated for 8 days, during which data on the radial
growth of the pathogen in each plate were taken at the 4th day, 6th day and 8th day of the experiment.
Percentage inhibition of mycelial growth by the leaf extracts was calculated using the formula:
Where: %MGI = % Inhibition of mycelial growth

DC = diameter of control
DT = diameter of test
Data analysis
Data obtained from the radial growths were subjected to the Analysis of Variance (ANOVA) using System
Akanmu et al. (2015) 2(3): 257263
.
Analysis Software package (SAS) 9.1 2009 version, while means were separated by Duncan Multiple Range
Test (DMRT).
RESULTS
Highly significant (p<0.0) result was obtained on the radial growths of Macrophomina phaseolina by
activities of the plant extracts, with respect to period (day) of the study, also, in the interactions involving;
concentration x day, extracts x concentration, extracts x day, extracts x replicates, extracts x concentration x
day, also, extracts x replicate x concentration. While significant (p<0.05) effects were produced in the
interactions of concentration x day and extract x replicates x day (Table 1).
Table 1. Interactive effect of extract, replicate, concentration and day of data collection on the
mycelia growth of Macrophomina phaseolina.
Source df Radial Growth
Concentration 2 1.46ns
Day 2 1624.83**
Extracts 15 33.01**
Replicates 3 0.13ns
Concentration x Day 4 1.32*
Extracts x Concentration 30 8.10**
Replicates x Concentration 6 0.19ns
Extracts x Day 30 3.61**
Replicates x Day 6 0.03ns
Extracts x Replicates 45 1.33**
Extracts x Concentration x Day 60 1.23**
Replicate x Concentration x Day 12 0.51ns
Extract x Replicate x Concentration 90 1.18**
Extract x Replicate x Day 90 0.82*
Error 180 101.21
Corrected total 575 4527.88
Note: ** = Highly significant (p<0.01), * = Significant (p<0.05), ns = not significant.
Considering the sole treatment of the four botanicals tested, Mormordica charantia (4.34 cm) had the
highest mean inhibitory effect of 4.34 cm on the mycelial growths of Macrophomina phasolina, while F.
asperifolia (5.69 cm), Anacardium occidentals (5.59 cm) and Psidium guajava (5.55 cm) which showed slight
inhibitory effect on the pathogen, were not significantly (p>0.05) different from the control (5.69 cm). A more
significant (p<0.05) control of the pathogen was achieved by the effects of the combined treatments of the four
extracts, with the extracts of Mormordica charantia and Psidium guajava (3.29 cm) as well as Ficus asperifolia,
Mormordica charantia and Anacardium occidentales (3.11 cm) on Macrophomina phaseolina (Table 2).
Table 2. Inhibitory effect of interaction of extracts on the mycelia growth of Macrophomina phaseolina.
Extracts Radial growth (cm)
Anacardium occidentals 5.59b
Psidium guajava 5.55b
Ficus asperifolia and Mormordica charantia 4.79c
Ficus asperifolia and Anacardium occidentals 4.61dc
Ficus asperifolia and Psidium guajava 4.63dc
Mormordica charantia and Anacardium occidentals 3.84gh
Mormordica charantia and Psidium guajava 3.29ij
Anacardium occidentals and Psidium guajava 4.76c
Ficus asperifolia, Mormordica charantia and Anacardium occidentals 3.11j
Ficus asperifolia, Mormordica charantia and Psidium guajava 4.36de
Mormordica charantia, Anacardium occidentals and Psidium guajava 4.16efg
Ficus asperifolia, Anacardium occidentals and Psidium guajava 3.95fg
Ficus asperifolia, Mormordica charantia, Anacardium occidentals and Psidium guajava 3.53hi
Control 6.94a
Note: The significant difference (p<0.05) is indicated by different letters along each column.
The growth inhibition at 75 mg ml-1 concentration produced significant (p<0.05) effect, followed by 25 mg
ml-1, while the concentration of 50 mg ml-1 had the least mycelia inhibition. The radial growth of the pathogen
Akanmu et al. (2015) 2(3): 257263
.
increases with time in which the most significant growth was recorded on the 8th day of data collection (7.32
cm), followed by the 6th day (4.63 cm) while the least was recorded on the fourth day (Table 3).
Table 3. Mean performance of botanical concentration, days of observation and replicated treatments
on the mycelia growth of Macrophomina phaseolina.
Observed characters Variables Radial growth (cm)
Extract concentration (mg ml-1) 25 4.57a
50 4.49ab
75 4.40b
Days 4 1.51c
6 4.63b
8 7.32a
Replicates 1 4.50a
2 4.51a
3 4.48a
4 4.45a
Note: The significant difference (p<0.05) is indicated by different letters along each column.
The day of observation is positive and significantly (p<0.05) correlated with concentration of the extract and
mycelia growth with r=0.15 and 0.85. There was negative association between the extracts and the mycelia
growth with r=0.22 while non-significant and negative association occurs between the radial growth and
replicate as well as concentration, no association exists between extracts and days of observation, concentration
and the replicates (Table 4).
Table 4. Effect of Correlation of the extracts; extract concentration, days of observation and
replicates on the radial mycelia growth of Macrophomina phaseolina.
Correlation Extracts Replicates Concentration Day
Replicates 0.00ns
Concentration 0.00ns 0.00ns
ns
Day 0.00 0.00ns 0.15*
Radial growth -0.22* -0.01ns -0.01ns 0.85**
Note: ** = Highly significant (p<0.01), * = Significant (p<0.05), ns = not significant.
The contribution of principal component analysis (PCA) in tables 5 showed variations in the Eigen values
and proportion of the mycelial growth. Prin 1 accounted for the highest variation with the highest Eigen vector
for extract, concentration and day of experimental observation at proportion of 34.06%. The first and fourth
PCA showed positive and more relatedness for the extracts treatments. The extract concentration of the second
PCA had the highest Eigen value which accounted for 25.01% of total variation while the third PCA had the
highest vector for the replicates (Table 5).
Table 5. Contribution of principal component analysis (PCA) to the variation in the radial growth
of Macrophomina phaseolina.
Radial growth Prin1 Prin2 Prin3 Prin4
Extracts 0.706 -0.067 -0.011 0.705
Replicates 0.022 0.389 0.920 0.003
Concentration 0.035 0.919 -0.390 0.047
Day 0.707 0.010 0.002 -0.707
Eigen values 1.360 1.000 1.000 0.637
Proportion (%) 34.06 25.01 25.00 15.930

Research has demonstrated that biological control is a potentially feasible alternative to the use of pesticides
(Jacobsen et al. 2004, Adandonon et al. 2006, Sobowale et al. 2008, Olawuyi et al. 2011, Akanmu et al. 2012,
2013a, Olawuyi et al. 2013). The combined use of biocontrol agents in the control of some important pathogens
affecting crops of economic importance is a new trend in plant disease control experiments. The integrated
approach of biocontrol research adopted in this research using botanicals of; Ficus asperifolia, Mormordica
charantia, Anacardium occidentals and Psidium guajava at different concentrations levels in sole and in
combinations towards the control of the mycelial growth of Macrophomina phaseolina of cowpea is in line with
the observations earlier made (Odebode 2006, Adandonon et al. 2006).
Akanmu et al. (2015) 2(3): 257263
.
Macrophomina phaseolina had been reported as the cause of charcoal rot disease which is a major biotic
factor that limits cowpea productivity worldwide (Ma et al. 2010, Zveibil et al. 2012, Arshad et al. 2012). This
was demonstrated in the reaction of cowpea varieties to infection of Macrophomina phaseolina obtained from
six different leguminous plants in Nigeria, as was earlier investigated by Amusa et al. (2007). The possible
control of this pathogen using botanicals as investigated in this research showed the efficacy of all the extracts
tested when compared to the results obtained in the control experiment. The effectiveness of the botanicals
against Macrophomina phaseolina could be attributed to their antifungal properties as was also discovered by
Arshad et al. (2012) who reported the efficacy of the antifungal properties of different parts of Sorghum
halepense Pers. in the control of Macrophomina phaseolina of cowpea.
The efficacy of the extract of Mormordica charantia which showed the most inhibitoriest potential on the
growth of Macrophomina phaseolina had earlier been reported in the treatment of human and plant diseases
(Virdi et al. 2003, Burger et al. 2010, Jonathan et al. 2012). However, the most significant mycelial inhibition of
Macrophomina phaseolina which was recorded in the combination of the extracts of; Ficus asperifolia,
Mormordica charantia and Anacardium occidentals, compared to the control experiment conformed to the
findings of Akhter et al. (2006) who observed 91% to 100% inhibition of conidial germination of Bipolaris
sorokiniana by plant extracts amended with cow dung and cow urine. The efficacy of combined treatment of
plant extracts was also affirmed in the combined treatments of botanicals and antibiotics against some emerging
drug-resistance microorganisms investigated by Rakholiva & Chanda (2012).
The significant result in interactions of the biological agents with the pathogen is in accordance with
findings of Sobowale et al. (2009), Akanmu et al. (2013b), Olawuyi et al. (2011, 2013). More so, the efficacy of
botanicals increases with concentration while it was also significantly correlated with time and the reduction in
the mycelia extension of the pathogen as similar reported by Akinbode (2010).
The significant correlation of the days of observation with extracts concentration and mycelia growth is an
indication of the positive contribution of the extract and their concentration in inhibition of Macrophomina
phaseolina as similarly observed by Akanmu et al. (2013a) and Olawuyi et al. (2014). The contribution of the
principal component analysis in this study is an indication of variability in the inhibitory potential of the
botanical extracts on the mycelia growth of the pathogen in accordance with the findings of Olowe et al. (2013).
The mycelial growths of Macrophomina phaseolina showed variability which ranges from 15.93% to 34.06%
due to the interactive treatments of botanicals at varying concentration over a period of time according to Eigen
proportion. The variations in the extract performances could be attributed to differences in their anti-fungicidal
properties, as similarly confirmed by Burger et al. (2010), Olawuyi et al. (2010) and Fapounda et al. (2011).
This suggests that there are variations in bioactive of antifungal compounds with varying characteristics and
potentials in their modes of action. However, the effectiveness of botanicals in field condition could
subsequently affirm the findings of this study to prove further their potentials in plant disease control.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(3): 264270, 2015
Research article
Comparative evaluation of salicylic acid and EDTA chelant induced

phytoremediation of lead and nickel using Lemna minor L.
Leela Kaur1*, Kasturi Gadgil2 and Satyawati Sharma3
1
Department of Environmental Science, Maharaja Ganga Singh University, Bikaner, Rajasthan, India
2
ABES Engineering College, Ghaziabad, Uttar Pradesh, India
3
Centre for Rural Development & Technology, Indian Institute of Technology (IIT) Delhi, New Delhi, India
*Corresponding Author: leela.kaur@gmail.com [Accepted: 08 December 2015]
Abstract: The objective of the present study was to study the influence of natural organic agent
SA (salicylic acid) and synthetic organic agent EDTA (ethylenediaminetetraacetic acid) on metal
uptake by Lemna minor L. in Pb and Ni contaminated water. L. minor was treated with Pb and Ni,
each at concentration of 10 mg.l-1. EDTA and SA were added at 2.4 mM concentration. Samples
were collected at an interval of 7 days for four weeks i.e. 7, 14, 21 and 28 day. The nickel
accumulation capacity of L. minor for combined Pb+Ni treatment was lower than individual Ni
treatment accumulation capacity. Salicylic acid significantly enhanced the uptake of Pb and Ni in
single Pb and Ni treatments. However, addition of EDTA could not induce Pb and Ni
accumulation. Based on results it could be said that removal of metals depends on the type and
concentration of chelants and concentration of pollutants in water.
Keywords: Chelant - Duckweed - Heavy metals - Phytoremediation.
[Cite as: Kaur L, Gadgil K & Sharma S (2015) Comparative evaluation of salicylic acid and EDTA chelant
induced phytoremediation of lead and nickel using Lemna minor L. Tropical Plant Research 2(3): 264270]
INTRODUCTION
Heavy metals are persistent pollutants and they are of major concern in the environment due to their toxicity
and harm to plant and animal life. They are released either by natural or anthropogenic sources. Lead and nickel
heavy metals are detected in the waste streams emerging from mining operations, tanneries, electronic
industries, electroplating, batteries and petrochemical industries as well as textile mill products (Johnson 1998).
Lead is an extremely toxic metal and is having many important industrial applications. The permissible limit
of Pb in drinking water is 0.005 mg.l-1 (Patterson et al. 1998). Pb effects kidney, nervous disorder and mental
deficiency (Panchandaikar & Das 1994, Axtell et al. 2003). Pb is the most abundant, globally distributed, best-
recognised dangerous among heavy metals that has been longest use since ancient times (Volesky & Holan
1995, Salt et al. 1998). Pb has been used as an anti-knocking gasoline additive since 1920 and remains an
important component in the manufacturing of the several commercial items such as storage batteries, cable
coverings, casting, sheet lead, pipes and ammunition. Pb has been tested and found to be carcinogenic,
mutagenic, and teratogenic. It affects the reproductive, nervous, muscular and haemopoeitic systems (Volesky &
Holan 1995). Ni causes dermatitis and bronchial problems (Gardea-Torresday et al. 1996).
Metal contamination in water can be treated using a variety of technologies that incorporate chemical,
physical and biological processes. Conventional remediation technologies such as chemical precipitation, ionic
exchange, filtration, electrochemical treatment, reverse osmosis, evaporative recovery and solvent extraction
(Rahmani & Sternberg 1999) are generally too expensive and are not able to remove heavy metals completely
and its byproducts (toxic sludge generation) again need disposal. Phytoremediation has emerged as a cost-
effective and environmental friendly sustainable technique in the last decades (Debusk et al. 1996). It is the use
of plants for pollutant removal from contaminated water or soil (Vujevic et al. 2000). Several studies indicate
that aquatic plants have large potential for the removal of organic and inorganic pollutants from wastewater.
Aquatic plants such as Eichhornia crassipes, Elode Canadensis, Myriophyllum spicatum, Potamogeton
pectinatus, Wolfia globosa, Lemna, Vellisnaria, Hydrilla verticilleta and Typha latifolia have been extensively
Received: 03 September 2015 Published online: 31 December 2015
Kaur et al. (2015) 2(3): 264270
.
used in phytoremediation research. Floating macrophytes (macro algae, duckweed, and water hyacinth) provide
advantages over emergent aquatic plants as they are much easier to harvest (Begonia et al. 2002).
Lemnoideae are limnic vascular plants that belong to the Araceae family and comprise the Landoltia spp.,
Lemna spirodela, Wolfia spp. and Wolffiella spp. They are commonly found in fresh water and brackish
ecosystems in temperate climates and serve as an important food source for various water birds and fish.
Additionally they provide habitats for invertebrates. Lamnaceae plants are easy to culture and handle, have a
high growth rate and are highly sensitive to different pollutants. Lemna minor L. is frequently used in
ecotoxicological research as a representative of higher aquatic plants. It can serve as a hyperaccumulator,
keeping the metal from continuous reintroduction into the ecosystem (Turgut et al. 2004). Lemna minor can be
used in phytotoxicity tests of contaminants, including heavy metals, phenolics and herbicides (Wu et al. 2004).
Studies have revealed that EDTA forms a metal-complex that enhances the mobility of the metal through the
plant (Raskin 1992, Dat et al. 1998, Janda et al. 2000). Salicylic acid, which may act as a component of the
signal transduction system important in defense mechanisms against pathogen attack (Lu et al. 2004), may also
provide protection against certain abiotic stresses e.g. heat stress in mustard seedlings (Saygideger & Dogan
2004) or chilling damage in maize (Janda et al. 1999).
The objective of this research was to investigate the ability of chelants (EDTA and SA) to enhance the
phytoremediation of Pb and Ni in contaminated water.

Duckweed plants were picked up from a stream of water from the main Indian Institute of Technology Delhi
campus. They were identified as Lemna minor L. and were cultured in a water tank in micro model IIT Delhi
(the experimental site). Stock solutions were prepared by using lead nitrate and nickel nitrate salt. Plastic
containers of ten litre capacity were filled with water. 6.0 g initial fresh weight of L. minor was used and treated
with Pb and Ni, each at concentrations of 10 mg.l-1. EDTA and SA were amended at 2.4 mM concentration as
C10H14K2N2O8.2H2O (MERCK) and C6H4OH-COOH (Qualigens). A black line was drawn on the containers so
a six litre water level could be kept constant. Every 1-2 days, the plants were checked and tap water was added
to each container so the six litre water level line remained constant. Samples were collected in an interval of 7,
14, 21 and 28 days.
The biomass weight was taken by drying duckweed plants on filter paper for 10 minutes (fresh weight).
Plants were dried and their dried weights were noted in the lab notebook. Relative growth of control and treated
plants were calculated as follows (Lu et al. 2004):
( )
( )
The dried plant samples were heated in a muffle furnace at 500C for 6 hours. The ash of each sample was
dissolved in 5 ml of 20% HCl to dissolve the residue. Samples were heated on a hot plate to boiling. Required
amount of HCl (20%) was added to avoid sample drying. The resulting solutions were filtered and diluted to 50
ml with deionized water in volumetric flasks. The Pb and Ni content of these plant samples and water samples
were determined using flame atomic absorption spectrophotometer (Electronics Corporation of India Limited
AAS4129) with the following settings: for Pb - wavelength 217 nm, lamp current 5 mA, slit 1 nm, fuel
acetylene and oxidant air and for Ni - wavelength 232 nm, lamp current 5 mA, slit 0.2 nm, fuel acetylene and
oxidant air.

Relative growth
Effect of chelants (EDTA and SA) on relative growth of Lemna minor L. treated to Pb and Ni is shown in
figure 1. Relative growth of L. minor increased with duration of time. In Pb experiment sets, the highest relative
growth was 2.540.13 in Pb+SA combination after 28 days. Furthermore, the lowest relative growth was
1.130.05 in individual Pb treatment after 7 days. However, in Ni experiment sets, the maximum value of
relative growth 1.990.19 in Ni+SA combination after 28 days and the minimum relative growth was observed
in Ni+EDTA combination after 7 days (1.070.025). Nevertheless, the highest and the lowest relative growth in
Pb+Ni experiment sets were measured in Pb+Ni+SA combination after 28 days (2.480.18) and in Pb+Ni
combination after 7 days (1.170.08) respectively.
Kaur et al. (2015) 2(3): 264270
.
Figure 1. Effect of chelants on relative growth of Lemna minor L. treated with Pb and Ni concentrations (10+10 mg.l-1).
Saygideger & Dogan (2004) observed that single and combined Cd and Pb with and without EDTA
concentrations affected growth in L. minor and Ceratophyllum demersum L. after 7 days exposure. Both
macrophytes were adversely affected in 50 mg.l-1 Pb plus 0.5 mg.l-1 Cd combination more than other tested
concentrations. Liu et al. (2004) reported that when Sedum alfredii Hance plants were treated with 200 M
Pb+200 M EDTA+1-100 M IAA (Indole-3-acetic acid) for 12 days, EDTA and IAA had no effects on shoot
biomass.
Metal accumulation
Effect of chelants on Pb and Ni accumulation by Lemna minor L. is shown in figure 23. Pb and Ni
accumulation increased with time in all sets. The highest and the lowest Pb accumulation in Pb experiment sets
were determined as 2.560.12 mg kg-1 for Pb+SA combination after 28 days and 1.100.10 mg kg-1 for
individual Pb for 7 days respectively. Similarly, in Ni experiment sets, the highest Ni accumulation (910101
mg kg-1) was found in the same treatment and same time as in case of Pb. However, the lowest Ni accumulation
(20536 mg kg-1) was obtained in Ni+EDTA after 7 days.
Figure 2. Effect of chelants on accumulation in Lemna minor L.: A, Pb; B, Ni.
Figure 3. Effect of chelants on accumulation in Lemna minor L. by Pb+Ni treatments: A, Pb; B, Ni.
Kaur et al. (2015) 2(3): 264270
.
Effect of EDTA and SA on Pb and Ni accumulation in combined Pb+Ni treatment by L. minor is shown in
figure 3 respectively. The highest and the lowest Pb accumulation were in Pb+Ni+EDTA treated plants and
Pb+Ni treated plants respectively. The highest and the lowest Ni accumulation were in Pb+Ni+SA treated plants
and Pb+Ni+EDTA treated plants respectively. Pb and Ni accumulation increased with time. It shows that in
combined Pb+Ni treatment, Pb and Ni accumulation in L. minor was found to be the utmost in Pb+Ni+SA
treatment. However, addition of EDTA could not induce Pb and Ni accumulation much. Salicylic acid enhanced
the uptake of Pb and Ni in single Pb and Ni treatments. Nickel accumulation capacity of L. minor for combined
Pb+Ni treatment was lower than individual Ni treatment accumulation capacity.
The Pb concentrations in the leaves and roots of Typha orientalis plant increased with increasing of Pb level
in the nutrient solution of 0500 mg.l-1 (Li et al. 2008). Results from this study demonstrated that the plants had
the highest accumulation and translocation under the condition of 500 mg.l-1 Pb+0.5 m moll-1 EDTA. Rahman et
al. (2008) reported that the uptake of inorganic arsenic species into the aquatic plant Spirodela polyrhiza L.
increased by EDTA when plants were exposed to different arsenic species at 6 M and 50 M EDTA for two
weeks.
Metal remained in the residual solution
SA and EDTA absorbed/extracted greater Pb/Ni than control. The best results of Pb and Ni removal were
obtained in SA treatments which may be due to the greater solubility of SA (0.2 g/100 ml at 20C) in water than
EDTA (0.05 g/100 ml at 20C) and metal-SA complexes may have been more available to plants than metal-
EDTA complexes.
In combined Pb+Ni treatments, Pb removal by EDTA was more than SA and Ni removal was best in SA.
This may be due to the competition between Pb and Ni to bind with chelants EDTA and SA. EDTA has strong
complexing capacity with Pb but in Pb treatment, uptake got reduced may be due to reduction in ion activity in
water. SA acts as defender and the protective function of SA includes absorption and distribution of elements.
SA is implicated in the high degree of cellular tolerance towards Ni in the genus Thalspi (Freeman et al. 2005).
SA may help in metal uptake by chelating Pb/Ni in the solution and then release Pb/Ni metal in the plant
system. The differences in uptake between the essential and non-essential metals and the effects of chelants on
their uptake can be explained by two parallel pathways: a selective symplastic pathway and a nonselective
apoplastic pathway (Tandy et al. 2006). Selective uptake of Ni is very efficient and uptake of Pb is very low
under these conditions. Apoplastic (passive) uptake of metal complexes is a function of the complex
concentration in the surrounding solution. This also suggests that higher chelate concentrations may lead to
increased uptake of essential metals. There were no literature on EDTA and SA effect in removal of Pb and Ni
from water. Based on presented results it could be said that removal of metals depends on the type and
concentration of chelants in water.
Ni uptake was inhibited by EDTA. On the other hand, the addition of SA in the medium increased the uptake
substantially. Our results are corroborated with Shi & Zhu (2008). They reported that addition of SA increased
Mn concentration in cucumber plants under excess Mn condition. Similarly, content of Cd in barley treated with
SA was higher than Cd alone treatment (Metwally et al. 2003).
Figure 4. Concentration of remained in the residual solution after treatments: A, Pb; B, Ni.
Concentrations of Pb and Ni (mg.l-1) remained in the water samples after treatments are represented in
figures 45. Pb and Ni content in water were decreased with the passage of time. The lowest value of Pb
Kaur et al. (2015) 2(3): 264270
.
(1.120.12 mg.l-1) remained in the solution after 28 days was in treatment Pb+Ni+EDTA (97% removal) while
the highest value (2.580.34) in the same treatment was after 7 days. Removal potential of Ni was highest in
Pb+Ni+SA treatment for 28 days (99.5%).
Figure 5. Concentration of remained in the residual solution in Pb+Ni treatments: A, Pb; B, Ni.
Removal potential of L. minor with chelant in combined Pb+Ni treatment was better as compared to
individual Pb and Ni treatments (Fig. 67). Akin et al. (1994) observed that removal of lead was decreased by
increasing the concentration of EDTA in Eichhornia crassipes (water hyacinth). Kruatrachue et al. (2002)
studied the combined effects of Pb and humic acid on Pb uptake by L. minor. Humic acid did not significantly
decrease Pb uptake at 50 and 100 mg.l-1 Pb treatment. In the 200 mg.l-1 treatment, the lowest Pb contents were
observed in the presence of 160 mg.l-1 humic acid. Therefore, a high concentration of humic acid could
significantly decrease the Pb uptake. Miretzky et al. (2006) investigated the mechanism of simultaneous metal
removal of Cd, Ni, Cu, Zn and Pb by Spirodela intermedia, L. minor and Pistia stratiotes. L. minor biomass
presented the highest mean removal percentage and P. stratiotes the lowest for all metals tested. Pb and Cd were
more efficiently removed by the three of them. No significant differences were observed in the metal exchange
amounts while using multi-metal or individual metal solutions.
Figure 6. Removal potential of Lemna minor L. after different days: A, Pb; B, Ni.
Figure 7. Removal potential of Lemna minor L. in Pb+Ni treatments with or without chelants: A, Pb; B, Ni.
Kaur et al. (2015) 2(3): 264270
.
CONCLUSION
The results showed that growth of Lemna minor helped in the accumulation of Pb and Ni. SA significantly
increased more Pb and Ni accumulation in L. minor as compared to EDTA when added as chelants in water.
This suggests that L. minor can be used for cleaning water polluted by heavy metals with the help of chelants.
ACKNOWLEDGEMENTS
This research work was financially supported by the University Grants Commission (U.G.C.), New Delhi,
India.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(3): 271275, 2015
Review article
A comprehensive review of effects of water stress and tolerance in

wheat (Triticuma estivum L.)
Muhammad Bilal1*, Irfan Iqbal1, Rashid Mehmood Rana1, Shoaib Ur Rehman2, Qurat-
ul-ain Haidery3, Farah Ahmad3, Ammara Ijaz3 and Hafiz Muhammad Imran Umar4
1
Department of Plant Breeding and Genetics, PMAS Arid Agriculture University, Rawalpindi, Pakistan
2
Chinese Academy of Agriculture Sciences, Beijing, China
3
Department of Biochemistry, PMAS Arid Agriculture University, Rawalpindi, Pakistan
4
Department of Plant Breeding and Genetics, Bahauddin Zakariya University, Multan, Pakistan
*Corresponding Author: m_bilal788@hotmail.com [Accepted: 09 December 2015]
Abstract: Wheat is regarded as one of the most important worldwide cereal crop and utilized as
staple food commodity. Its productivity restricted by several biotic and abiotic stresses but among
all of these,drought is most devastating stress which immediately affects the morphological and
physiological attributes of wheat crop and lead to severe reduction in overall production.
Keywords: Wheat - Staple - Biotic - Abiotic - Drought.
[Cite as: Bilal M, Iqbal I, Rana RM, Shoaib Ur Rehman, Haidery Q-ul-A, Ahmad F, Ijaz A & Umar HMI
(2015) A comprehensive review of effects of water stress and tolerance in wheat (Triticuma estivum L.).
INTRODUCTION
Wheat (Triticum aestivum L.) is regarded as one of most vital cereal crop of world and mainly grow in rain
fed regions in which drought occur which cause serious yield reduction (Rana et al. 2013). Drought stress is a
globally widespread and ever growing environmental phenomenon encountered by wheat crop and long duration
of water deficit lead to severe reduction in overall production (Nezhad ahmadi et al. 2013). Drought stress can
be determined by three factors viz., intensity, time of incidence and duration. Under drought stress conditions
changeable nature of these factors make it complicated for plant breeder to decide which plant trait should be
improved first to improve plant production (Mujtaba & Alam 2002).
Recent research advances associated with crop responses to numerous biotic and abiotic stresses especially
water deficit stress is gaining significant emphasis, as global environment fluctuations situation prognosticates
water deficit conditions (Ullah et al. 2010). Better critical knowledge about drought stress tolerance related to
physiological and morphological characters helps in the screening of germplasm to evaluate genotypes against
drought. One of the superior goals of plant breeders is to make wheat genotypes suitable to drought stress
condition which ensures higher grain yield. Several efforts have been address to enhance the per acre
productivity of wheat crop under water deficit situation by improving the attributes damaged by drought stress.
Numerous previous reports exposed many morphological and physiological attributes correlated with drought in
cultivated wheat varieties. Reviewed article on the drought subject has discussed separately the studies on
morphological basis and studies on physiological basis. The comprehensive overview has been explained below.
1. Morphological Attributes
Wheat yield under drought stress suffer serious moisture deficit throughout its growth period from seedling
to full maturity (Bilal et al. 2015). Under drought condition decreasing pattern was experienced in
morphologically yield contributing characters like plant height (PH), grains per spike, spikes per plant, 1000-
grain weight (TGW) in wheat (Kilic & Yabasanlar 2010). Blum & Pnuel (1990) reported that yield and yield
contributing traits of wheat crop were drastically decreased under least annual precipitation. Drought stress lead
to reduction in number of fertile tillers per plant, grains per spike and 1000-grain weight (TGW) which
ultimately cause noticeably low grain productivity. Relationship between plant height (PH), leaf area and wheat
Bilal et al. (2015) 2(3): 271275
.
grain yield has been noticed at booting and anthesis phase which cause improvement in grain yield under water
deficit condition (Gupta et al. 2001). The decreasing graph in grain number was linked with reduced leaf area
and lower photosynthesis as outcome of drought stress (Fischer et al. 1980). Under drought stress condition
screening of wheat genotypes to evaluate these yield contributing characters are suggested in hybridization
programs for water deficit tolerance. Various research scientists have reported considerably positive relationship
for effective tillers plant1 in wheat which depicted negative relationship with 1000-grains weight (Ali et al.
2008). Considerable positive and worth mentioning association has been observed for grains per spike with total
grain production in diverse bread and durum wheat genotypes, and grains per spike was observed 5068 in
irrigated condition while under stress 3250 grains per spike were found (Jatoi et al. 2011). Thousand grains
weight (TGW) is essential yield enhancingtrait and has been given due consideration during varietal evaluating
procedure. Kilic & Yagbasanlar (2010) investigation depicted that under drought conditions grain filling period
and spikelets per spike were associated with high grain production. Therefore it is suggested that these
morphological attributes should be selected for screening diverse wheat genotypes under drought in successful
breeding schemes. The adverse and unfavorable outcomes of water deficit stress on wheat sensitive stages of
crop growth such as reproductive, booting and grain filling stagecan be minimized by preventing stress at these
stages to develop genotypes showing drought tolerant (Saini & Westgate 1999).
2. Physiological Attributes
Different types of plant physiological responses have been reported by various Plant physiologists in their
findings under drought stress situation. Zaharieva et al. (2001) reported that in globally drought affected areas
physiological mechanism is very handy approach in evaluating and screening the extraordinary genotypes
having drought resistant mechanism. Comprehensive information of physiological mechanisms permits plant
researcher to develop promising genotypes that would be utilized efficiently, continue his growth and
production under water deficit stage (Ashraf & Khan 1993).
Cell-membrane stability (CMS) is of vital important selection criteria of drought tolerant genotypes
(Tripathy et al. 2000). It has been reported that under water stress cell membrane integrity and stability confers
drought resistance (Bewley 1979). The water stress activates the reactive oxygen species which ultimately
decreases membrane stability caused by lipid peroxidation (Menconi et al. 1995). Although many reports
depicted lower lipid peroxidation and higher cell membrane stability (CMS) in drought tolerant wheat and maize
genotypes (Pastori & Trippi 1992). It has been reported by Sairam & Saxena (2000) that higher level of
accumulation of H2O2 under water stress leads to production of hydroxyl radicals, which cause lipid
peroxidation and consequently cell membrane rupture. Damage caused by water deficit stress to cell membrane
is negatively associated with increased activities of superoxide dismutase (SOD) and catalase (CAT) in drought
susceptible and tolerant genotypes (Dhindsa & Matowe 1981). Under drought stress assembly of lower levels of
H2O2 lead to lower damage of cell membrane in wheat drought tolerant genotypes.
Cell membrane stability (CMS) under drought stress depicts the ability of plant tissues to prevent
electrolytes leakage by keeping the cell membrane in safe mood (Sullivan 1971). Estimation of Cell membrane
stability (CMS) via in vitro includes dehydration of leaf tissues by means of polyethylene glycol (PEG) and then
assessment of electrolyte leakage from leaves. Leakage of various solutes, such as organic acids, amino acids,
saccharides, phenolic compounds and hormones from revealed cell membrane stability (CMS) after subject to
dehydration through polyethylene glycol has been reported (Leopold et al. 1981). CMS Values have immense
significance in hybridization programs because these Values predict the drought tolerant varieties (Dhanda et al.
2004). Genotypes having lower CMS value are vulnerable to water deficit condition while the genotypes
showing higher CMS values depicts drought tolerant behaviour. The genotypes having less than 50% values are
tremendously susceptible to drought while genotypes with 7180% values are considered to grow with full
potential under water deficit. Farshadfar et al. (2011) noticed in investigation that under water deficit conditions
cell membrane stability (CMS) depicted positively considerable relationship with tillers per plant, grain yield,
but negative association 100 kernel weights (TGW).
Higher leaves chlorophyll contents is significantly correlated with photosynthesis and regarded as
encouraging selection trait in crop productivity (Teng et al. 2004). It has been reported in many studies that
under drought stress Photosynthesis exhibit direct relationship with wheat grain production because less stomata
opening frequency and low amount of CO2 fixation lead to reduction in photosynthetic amount (Mafakheri et al.
Bilal et al. (2015) 2(3): 271275
.
2010). Severe water deficit stress restricts the photosynthesis by damaging the chlorophyll components (CC)
and changing the photosynthetic machinery (Iturbe-Ormaetxe et al. 1998). Decreased photosynthetic amount
under water deficit condition is an outcome of Inhibition of RuBisCO (ribulose-1, 5-bisphosphate
carboxylase/oxygenase) enzyme activity and development of ATP (Dulai et al. 2006). Many researchers
revealed in their investigations that photosynthesis of higher plants is extremely susceptible to drought stress
and Lower amount of chlorophyll cause chlorosis and reduction in crop growth (Khosh & Ando1995). Higher
concentration of chlorophyll is essential for plants because it depicts the low quantity of photo-inhibition of the
photosynthetic which prevents the carbohydrates losses and eventually enhances growth (Farquhar et al. 1989).
Relative water content (RWC) of leaves has been reported as direct indicator of plant water contents under
water deficit conditions (Lugojan & Ciulca 2011). Drought stress lead to reduction of water status during crop
growth, soil water potential and plant osmotic potential for water and nutrient uptake which ultimately reduce
leaf turgor pressure which results in upset of plant metabolic activities. Momentous pattern of divergence can be
observed in Relative water content (RWC) among diverse genotypes during various plant growth stages. The
highest Relative water content (RWC) might be calculated at crop vegetative stage (Almeselmani et al. 2011).
Under water stress condition decrease in water status and osmotic potential in plants is the ultimate outcome of
lower relative water content. Osmoregulation mechanism plays a phenomenal role in preserving turgor pressure
which helps in soil water absorption and continue plant metabolic activities for its survival.
Proline is well known to occur extensively in higher crop plants and accumulates in higher concentration in
response to different abiotic environmental stresses specially drought stress (Kavi-Kishore et al. 2005).
Accumulation of higher proline concentration in crop plant under water deficit condition is highly associated
with and drought tolerance genotypes depicts its concentration is much higher than drought sensitive genotypes.
It has been found by many scientists that in saline stress soil proline are mainly accumulated in leaves of many
higher halophytic plant (Briens & Larher 1982) but plants grown under drought stress showed much higher
concentration of proline in leaves, shoots, in desiccating pollen and in root apical regions Lansac et al. 1996).
Accumulation of higher concentration of proline permits plants to keep less amount of water potential which
cause accumulation of osmolytes in osmoregulation process which enables the plant to take up water to perform
growth and metabolic activities (Kumar et al. 2003).
Under water deficit condition proline perform many functions like act as osmolyte contribute s in the
maintenance of membrane and protein, scavenging free radicals. Moreover after the severe damage of stresses
proline contents provide adequate reducing agents that assist in mitochondrial oxidative phosphorylation and
production of adenosine triphosphate (ATP) for revival from damages of various stresses (Hare et al. 1998).The
primary site of proline contents accumulation in response to drought stress in crop plant is cytosol (Ketchum et
al. 1991).
CONCLUSION
Wheat (Triticum aestivum L.) being a most vital cereal crop has always been of area of interest to plant
breeders. Since several years numerous efforts have been made to boost up its productivity under various
conditions especially under drought stress condition. This review depicted that drought stress caused extensive
decline in all the studied attributes performance. So there is need to explore several helpful attributes and to
minimize the harmful effect of water stress on wheat crop productivity through development of genotype having
drought tolerant and better performance.
ACKNOWLEDGEMENTS
The authors would like to express their special thanks and grateful to acknowledge the Rashid Mehmmod
Rana for their assistance, guidance, and all kind of support for the successful completion of this review article.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(3): 276281, 2015
Research article
Priming of Abelmoschus esculentus (L.) Moench (okra) seeds with

liquid phosphobacterium: An approach to mitigate drought stress
P. Pravisya and K. M. Jayaram*
Division of Plant Physiology and Biochemistry, Department of Botany,
University of Calicut, Calicut - 673635, Kerala, India
*Corresponding Author: jayaramkm@yahoo.co.in [Accepted: 11 December 2015]
Abstract: The present investigation aimed to evaluate the effect of priming of Abelmoschus
esculentus (okra) seeds with liquid phosphobaterium (LPB) on water stress. The
phosphobacterium is known for its use as inoculants to increase the uptake of soil phosphorus as
well as crop yield. The availability of rain fall is getting decreased every year causing severe
drought and that may adversely affect the agricultural crops in the state of Kerala. So the aim of
the study is to provide a helping hand to the farming community to fight against drought stress. In
the present study the seeds of okra cv. Arka anamika were subjected to priming treatment with 5%
and 10% liquid phosphobaterium, and the parameters like biomass, relative water content,
chlorophyll content, total protein and yield were studied. Priming with liquid phosphobaterium
showed considerable variation in both the physiological and biochemical parameters. Among the
concentrations of liquid phosphobaterium tested seeds primed with 10% liquid phosphobaterium
were found to effective in mitigating the effect of water stress, stimulating early flowering and also
increase in yield.
Keywords: Drought - Re-irrigation - Biomass - Chlorophyll - Protein.
[Cite as: Pravisya P & Jayaram KM (2015) Priming of Abelmoschus esculentus (L.) Moench (okra) seeds with
liquid phosphobacterium: An approach to mitigate drought stress. Tropical Plant Research 2(3): 276281]
INTRODUCTION
Agricultural crop productions have been determined by the availability of soil water and which in turn
related to global climate changes (Cias et al. 2005). Drought is one of the major causes in the field of agriculture
all over the world. Depending upon the period of exposure to drought (water stress) and growth stage of plants,
water scarcity experienced plants responded differently. Drought stress leads to the commendable variation in
the morphological, anatomical, physiological and biochemical parameters of plants which is finally reflected on
yield potential (Kramer 1969, Shintu & Jayaram 2015). Drought, irrespective of the length of exposure of the
plant and severity, adversely affected photosynthesis and other metabolic activities of plants and ultimately the
growth productivity of such plants.
Phosphobacteria (Phosphate solublizing bacteria- PSB) is one of the most useful plant soil microorganisms,
widely used as bio-fertilizer. PSB plays an important role in enhancement of plant growth by improving texture
of soil by adding organic matter to the soil, solublizing the insoluble phosphorous in soil (Bhattacharya & Jain
2000, Ravikumar et al. 2010). Inorder to compensate phosphorus deficiency in soil phosphate fertilizer are
being used widely. However the increased use of chemical fertilizers, cause soil contamination. In such
condition PSB efficiently take part in the utilization of unavailable native phosphates (Lagreid et al. 1999). The
studies conducted by various authors revealed that priming or pre-sowing treatment of seeds with various
chemicals or even with water can enable the plants to improve the health and such plant may become resist
water stress (Chivasa et al. 2000, Harris et al. 2004, Shintu & Jayaram 2015). During drought stress these
microorganisms help to accumulate large amount of compatible solutes and accelerate the production of
antioxidant enzymes in plants and reduce the adverse effect of drought (Mayak et al. 2004). Considering all
these facts the authors made an attempt to study the effect of priming of Abelmoschus esculentus (L.) Moench
Pravisya & Jayaram (2015) 2(3): 276281
.
(okra) seeds with liquid phosphobacterium (LPB), a cheapest method that adapt to overcome the adverse effect
of water stress.

For the present study, seeds of Abelmoschus esculentus (L.) Moench cv. Arka anamika (okra) were procured
from the Regional Agricultural Research Station, Mele Pattambi, Palakkad District, Kerala State. Healthy seeds
were manually selected from the seed lot and were divided to 3 sets. First set was not inoculated with Liquid
phosphobacterium (LPB) and considered as control, the second and third sets were inoculated with different
concentrations of LPB such as 5% and 10% respectively. The pre weighed seeds were surface sterilized with
teepol and 0.1% mercuric chloride solutions and were kept in respective concentrations of LPB solutions for six
hours with continuous shaking. Thereafter the seeds were air dried until the weight became equal to the initial
weight. All the seeds were sown in garden pots filled with potting mixture. The experimental setup was
maintained in the open field of the Department of Botany, University of Calicut (355C temperature). After 17
days of vegetative growth the control and LPB treated plants were again divided in to two sets, one set kept as
irrigated regularly and the other set as non-irrigated for 3 days. After 3 days of water stress the plants were re-
irrigated as in the other case and was continued until taking yield.
The following parameters were studied by using standard procedures: fresh and dry weight of plants
(biomass), relative water content (RWC) (Bars & Weatherly1962), total chlorophyll (Arnon 1949),total protein
(Lowry et al.1951) and yield parameters like fruit length, fruit fresh and dry weight, number of fruits per plant
and seed per pod. All the data were collected as detailed below: on the previous day of commencement of
water stress treatment (0thday), 1stday (24hrs after water stress), 2ndday (48 hrs after water stress), 3rdday (72hrs
after water stress), 24 and 48 hrs after re-irrigation (1stand 2ndday of recovery respectively).
Analysis of variance was performed using SPSS software 16. Means were compared using the Duncan test
at 5% probability level. The data is an average of three independent experiments each with three replicates
(n=9). The data represent MeanStandard Error (SE).
RESULTS
Biomass
The plants showed significant reduction in biomass under water stressed condition and which was prominent
in untreated (control) water stressed plants than LPB treated plants subjected to drought stress (Fig. 1). Plants
treated with 10% LPB exhibited lesser biomass reduction compared to other treatment and control. During re-
irrigation LPB treated plants showed fastest recovery, compared to untreated plants given drought.
25 7
C Cs 5C 5s 10C 10s A C Cs 5C 5s 10C 10s a
B
ab 6 a c b
Fresh weight of plant (g)
20 ab b
Dry weight of plant (g)
a a c d
ab b c e
ab cd d 5 a b d f
c ef a b e
15 c d ef b c
c d e 4 aa e d f
bba a e f d ef f cd
f
cc cd e
bb f
3 cc ef
10
2
5
1
0 0
0th day 1st day 2 nd day 3rd day 1st 2nd 0th day 1st day 2 nd day 3rd day 1st 2nd
stress stress stress recovery recovery stress stress stress recovery recovery
Days Days
Figure 1. Effect of liquid phosphobacterium on Abelmoschus esculentus (L.) Moench (Okra): A, Fresh weight; B, Dry
weight. (C- Control; Cs- Control with drought stress; 5C- 5% LPB treated plants; 5Cs- 5% LPB treated plants with
drought stress; 10C- 10% LPB treated plants; 10Cs- 10% LPB treated plants with drought stress)
Relative water content (RWC)
RWC was observed high in all the irrigated set of plants and among the plants subjected to water stress
treatment, LPB treated plants exhibited high rate of RWC of which highest rate was observed in 10% LPB
.
treated plants (Fig. 2). The same pattern of increase in RWC was noticed during re-irrigation also.
100 C Cs 5C 5s 10C 10s
90 a a a a a a a b
c c b b b e c d c b b c b c d e d c f
Relative water content (%)
80 f f d d e
e f
f e
70
60
50
40
30
20
10
0
0th day 1st day stress 2 nd day stress 3rd day stress 1st recovery 2nd recovery
Days
Figure 2. Effect of liquid phosphobacterium on relative water content of Abelmoschus esculentus (L.) Moench (Okra). (C-
Control; Cs- Control with drought stress; 5C- 5% LPB treated plants; 5Cs- 5% LPB treated plants with drought stress;
10C- 10% LPB treated plants; 10Cs- 10% LPB treated plants with drought stress)
Total Chlorophyll
The total chlorophyll content of untreated water stressed plants was found decreased more significantly
throughout the period of water stress treatment compared to LPB treated water stressed plants (Fig. 3A). Among
the LPB treated plants highest rate of chlorophyll content was observed in 10% LPB treated plants. During re-
irrigation LPB treated plants showed fastest recovery, compared to untreated plants.
Total protein
The okra plants treated with LPB (5% and 10%) exhibited higher rate of accumulation of protein content
compared to control plants (Fig. 3B). But the plants exposed to drought stress the decrease of protein content
was greater in untreated water stressed plants compared to LPB treated plants. The recovery of protein content
was found faster in LPB treated water stressed plants compared to untreated water stressed plants.
C Cs 5C 5s 10C 10s A C Cs 5C 5s 10C 10s B

1600 a a a ab 100
aa ab b b a
b 90 a bc
1400 bb c c c c cf a b d
Total chlorophyll (g/gfw)
cc e d e d e d e d ef 80 a b
Protein content (mg/fw)
1200 f f b
f f 70 bba a b a c
e
c
1000 60 d c e d f
d e c
800 50 cc e e d
600 40 f
f
30 f
400 f
20
200 10
0 0
0th day 1st day 2 nd day 3rd day 1st 2nd 0th day 1st day 2 nd day 3rd day 1st 2nd
stress stress stress recovery recovery stress stress stress recovery recovery
Days Days
Figure 3. Effect of liquid phosphobacteriumon Abelmoschus esculentus (L.) Moench (Okra): A, Chlorophyll; B, Protein. (C-
Control; Cs- Control with drought stress; 5C- 5% LPB treated plants; 5Cs- 5% LPB treated plants with drought stress;
10C- 10% LPB treated plants; 10Cs- 10% LPB treated plants with drought stress)
Yield
Drought stress exhibited a reduction of yield in okra, which was measured by using parameters like length of
fruit, fresh weight of fruit, dry weight of fruit, number of fruit per plant and number of seed per fruit but the
reduction was more prominent in untreated plants than LPB treated plants (Fig. 4). The plants treated with 10%
LPB showed significant increase in yield parameters when compared to 5% LPB treated plants and control
plants.
.
C Cs C Cs 5C C Cs 5C
5C 5s A 5s 10C 10s
B C
100 5s 10C 10s
10C 10s 20 a a
30 b
c
80 c b
25 a 15
b a 60
c c d b d
20 d 10 e c e
e f d 40 f
15 d a b
e
10
5 20 d e c d
5 0 0
0 Fresh weightof Dry weightof No.of fruit per No.of seed per
Length of fruit (cm) fruit (g) fruit (g) plant fruit
Figure 4. Effect of liquid phosphobacterium on yield parameters of Abelmoschus esculentus (L.) Moench (Okra): A, Length
of fruit; B, Fresh and dry weight of fruit; C, Number of fruit per plant and seed per fruit. (C- Control; Cs- Control with
drought stress; 5C- 5% LPB treated plants; 5Cs- 5% LPB treated plants with drought stress; 10C- 10% LPB treated plants;
10Cs- 10% LPB treated plants with drought stress)

The present study showed that the LPB inoculated plants exhibited an increased rate of fresh and dry weight
of which 10% LPB inoculation exhibited higher rate of fresh and dry weight (Fig. 1 AB). The studies
conducted by Singh & Singh (2010) also revealed higher dry matter content in PSB treated fenugreek plants,
which reflected the tolerance of the plants due to bacterial inoculums. Similar pattern of dry matter increase was
observed in finger millet treated with agrochemicals like CaCl2 (Maitra et al. 1998), rice plants treated with
Rhizobacterium (Raj et al. 2012) and black gram treated with bio-fertilizers (Selvakumar et al. 2012). All these
results are in tune with the results obtained in the present study, which may lead to the conclusion that priming
has an important role in nutrient uptake and growth, that may resulted in the high rate of biomass in plants
treated with LPB.
Priming of okra seeds with LPB showed an increase in RWC which was very prominent in 10% LPB treated
plants (Fig. 2). Studies conducted by Yordanov et al. (2003) observed that mild drought helped plants to
regulate water loss and uptake, allowing maintenance of their leaf water content within the limits. According to
Velentovic et al. (2006) RWC in the leaves of maize plants at low water potential decreased significantly
compared to control. Similarly the RWC in drought affected leaves of okra was significantly lower than the
continuously irrigated control plants. These are in confirmation with the results obtained in fenugreek (Singh &
Singh 2010) and tomato (Shintu & Jayaram 2015) and the authors found that the plants inoculated with PSB
exhibited highest level of RWC as compared to non-inoculated plants under controlled condition. All these
observations revealed that the RWC in leaves of drought affected plants were significantly reduced as a result of
relative water uptake and storage by the plants. So it can be presumed that comparatively high RWC in the
leaves of LPB treated plants may be due to the higher activity of water uptake and restoration by these plants.
Thus LPB can be considered as a bio-priming agent in order to tolerate drought stress.
Total chlorophyll content of LPB treated plants was found decreased but the decrease was not lesser than
untreated plants (Fig. 3A). Drought stress produced changes in the total chlorophyll content (Farooq et al.
2009). The results obtained in the present study showed a high rate of chlorophyll pigment in plants raised from
10% LPB treated seeds. Estill et al. (1991) and Ashraf et al. (1994) observed same pattern of changes in
chlorophyll content in stressed alfalfa and wheat respectively. Similarly fenugreek plants treated with PSB also
showed identical results (Singh & Singh 2010). Moreover the studies conducted by Rupa (2007) also revealed
that, under moisture stress conditions there will be degradation in pigment composition which ultimately
induced a decrease in the chlorophyll content. In the present study total chlorophyll content of okra leaves
decreased with increased moisture stress, but it was found increased during the recovery period. Higher
persistence of chlorophyll content in plants under stress due to LPB may be attributed to decreased chlorophyll
degradation and increased chlorophyll synthesis, as suggested by Jayakumar & Thangaraj (1998). So in the
present study also higher level of photosynthetic pigment was obtained in LPB treated drought affected plants
that may be due to non-degradation of chlorophyll. The LPBs regulatory effect on phosphate solubilisation may
be beneficial to the non-degradation of chlorophyll pigments and that may be the reason for getting high
chlorophyll content in the LPB treated drought affected plants.
.
The leaves of LPB treated okra plants exhibited an increase in total protein content which was more
prominent in 10% LPB treatment (Fig. 3B). Similar results were obtained in fenugreek plants treated with
phosphobacterium (Singh & Singh 2010). According to those authors the non-inoculated plants exhibited a
lesser amount of protein than inoculated plants. Selvakumar et al. (2012) opined that double inoculation of
Rhizobium with Phosphobacteria yielded more protein content than single and non-inoculated in black gram.
Rhizobium increased protein content in sunflower by increasing nitrogen uptake (Shehata & EL-Khawas 2003).
Radin (1984) suggested that high phosphorous caused stomatal opening, and facilitate plants to accumulate
more protein in inoculated plants compared to non-inoculated plants. Accumulation of ABA caused by
deficiency of phosphorus is directly proportional to the degree of water stress and thus resulted in stomatal
closure and low photosynthetic rate (Singh & Singh 2010). So in the present study it can be presumed that LPB
may cause to enhance the uptake of insoluble soil phosphorous and thus resulted in increased stomatal opening
and ultimately enhanced the accumulation of protein in okra plants.
In addition to the beneficial effect on growth of plants, bio-priming is also known to increase the yield by its
significant effect during drought on yield parameters of crop under study (Casanovas et al. 2003). In the present
study, maximum yield was obtained in 10% LPB treated and water stressed okra plants which were followed by
its control plants (Fig. 4). These results revealed that the LPB has an important role in increasing the yield and
also to reduce the adverse effect on drought stress. According to Prabhakar & Saraf (1991) the interaction effect
of limited irrigation regimes and phosphorous fertilizers was significant on sorghum grain yield. Chauhan et al.
(1995) observed that inoculation of Azospirillum as a bio-fertilizer markedly increased pod number and seed
yield of Brassica napus plants over the non-inoculated plants. Zodape (2001) suggested that, the increase in
yield with bio-fertilizer application was due to micro-element and plant growth regulator contained in the
fertilizer. So it can be presumed that the increase in yield of LPB treated plants exposed to water stress may be
due to the positive effect of priming of okra seeds with LPB. Priming with LPB also enhanced the potential of
the plant to drought stress by influencing on various physiological as well as biochemical parameters studied. So
it can be concluded that LPB may beneficially affected plants to improve water status of okra plants that may
help them to tolerate water deficit condition to a certain extend and to give high productivity. So, liquid
phosphobacterium can be recommended to the farming community as a priming agent to their vegetable crops,
in order to fight against mild drought stress.
ACKNOWLEDGEMENTS
The first author (PP) gratefully acknowledged to the Govt. of Kerala for providing the financial support
and both the authors are thankful to the Head, Department of Botany, University of Calicut, for providing
laboratory facilities in order to complete the work.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(3): 282287, 2015
Research article
Diversity of invasive alien species in Pantnagar flora

Jyotsna Rastogi*, D. S. Rawat and Satish Chandra
Department of Biological Sciences, College of Basic Sciences and Humanities
G. B. Pant University of Agriculture and Technology, Pantnagar - 263145, Uttarakhand, India
*Corresponding Author: jyotsnarastogi2015@gmail.com [Accepted: 12 December 2015]
Abstract: Biological diversity faces many threats throughout the world and one of the major
threats is caused by invasion of alien species. The present study proves presence of 94 invasive
alien species in flora of Pantnagar, Uttarakhand, India. These 94 invasive alien species (IAS)
belong to 72 genera, under 33 families 85 species are dicotyledons while 9 species are
monocotyledons. On the basis of their nativity maximum IAS have their sourced region as
American continents (74), followed by Africa (8), Europe (5), Mediterranean (3) and Asia & Australia
(2). The taxonomic analysis of IAS reveals dominance of Asteraceae with 18 spp. followed by
Fabaceae, Amaranthaceae, Convolvulaceae, Malvaceae, Solanaceae, Poaceae etc. Among these, 78 IAS
are herbs followed by shrubs (8), grasses (4), sedges (2), trees (1), and climber (1). Such a large number
of invasive alien species in small area of Pantnagar, indicate miserable condition of natural vegetation.
Keywords: Invasive alien species - Diversity - Biological invasion - Nativity - Pantnagar.
[Cite as: Rastogi J, Rawat DS & Chandra S (2015) Diversity of invasive alien species in Pantnagar flora.
INTRODUCTION
The plants that has been introduced by humans intentionally or accidentally from one region to another are
referred as exotic, introduced, foreign, non-indigenous or non-native (Reddy et al. 2008). Invasive alien species
(IAS) are those that occur outside their natural range, spread rapidly and cause harm to other species,
communities and entire ecosystem. Invasive alien species are one of the major causes of biodiversity loss in the
world and impose high cost to agriculture, forestry, and aquatic ecosystem. The introduction of invasive alien
species in the new habitat outside of their natural home range carries significant risk. Plant invasion is defined as
the whole process from the arrival of a new species into a community, its establishment and maintenance in that
community, to its further spread into neighbouring communities (Prieur-Richard & Lavorel 2000). Biological
invasions are posing a great threat to biodiversity, which is already threatened by habitat destruction due to
human population growth. Biological invasions have been recognized as one of the most serious global process
impacting the structure, composition and function of natural and semi natural ecosystems (Mooney & Hobbs 2000,
Vitousek et al. 1997).
The common characteristics of invasive species include rapid reproduction and growth, high dispersal
ability, ability to adapt physiologically to new conditions, the ability to survive on various food types and in a
wide range of environmental conditions and ability of association with humans. Many invasive alien species grow
faster than native plants and reproduce quickly and thus replace indigenous plants and completely alter the
composition of flora of the area they have colonized. The presence of invasive species in an area changes the soil
structure, its profile, composition, nutrient content of soil, moisture availability etc. It has been reported that
agriculture and grazing land, as well as protected areas, are threatened by rapidly growing species of plants that were
introduced during colonialism as garden plants and wind breakers (Hall 2003).
The present study is conducted in the land area covered by G. B. Pant University of Agriculture and Technology
Pantnagar in Udham Singh Nagar, district of Uttarakhand, India. The University campus at Pantnagar is spread in an
area of 12,661acre (51.24 K m2) between the latitudes N 28 59' 36" 29 02' 34" and longitude E 79 28' 33" 79
31' 12" with an altitude range of 213 to 238 m above the sea level. Geographically Pantnagar is situated in the Terai
belt near the outer hills of Kumaon Himalaya. The soil is quite rich in nutrients and soil pH is around 6.85. In
Rastogi et al. (2015) 2(3): 282287
.
Pantnagar area the landscape is completely devoid of natural vegetation and the land is mainly used for agricultural
activities. Such an environment is congenial for invasion by invasive alien species and their presence in the area is
sporadically reported earlier (Gaur & Rawat 2013, Joshi & Rawat 2011, Nisha et al. 2015). However, a complete
account of invasive alien species of angiosperms is not yet available and therefore, attempted in this work.

Invasive alien species (IAS) were searched and collected from the different localities of study area
(Pantnagar). Plant specimen with flower and fruits were collected from different gardens, parks, and research
centres of the university regularly for further study. These localities were visited in different seasons to find out
the exact flowering and fruiting time of IAS. Collected specimens contain information on locality, date, and
other important information as suggested by Jain & Rao (1976). Plant specimen were identified with the help of
different floristic work like Duthie (190329), Bailey (1949), Maheshwari (1963), Raizada (1976), Babu (1977),
Sharma & Pandey (1984), Graf (1992), Gaur (1999), Khuroo et al. (2006), Negi & Hajra (2007), Reddy (2008),
Chandra Sekar (2012), Mehra et al. (2014), Bajpai et al. (2015) and volumes of Flora of India by BSI (Sharma
et al.1993, Sharma & Balakrishnan 1993, Sharma & Sanjappa 1993, Hajra et al. 1995a,b, Hajra et al. 1997,
Singh et al. 2000, Balakrishnan et al. 2012). Collected specimens were pressed and dried according to the
standard method suggested by Jain & Rao (1976) and submitted in the herbarium of G. B. Pant University of
Agriculture and Technology in the department of Biological Sciences, CBSH.

All the invasive alien species collected from Pantnagar area are enumerated in table 1. Each botanical name is
followed by family name, source region (nativity), growth forms and wild/cultivated status.
Table 1. Invasive alien species of Pantnagar flora.
Growth Cultivated
S. No. Name of Species Family Nativity
form or Wild
1. Acacia farnesiana (L.) Willd. Mimosaceae Australia Tree Wild
2. Acanthospermum hispidum DC. Asteraceae Brazil Herb Wild
3. Acmellaradicans (Jacq.) R.K. Jansen. Asteraceae Trop. America Herb Wild
4. Ageratum conyzoides L. Asteraceae Trop. America Herb Wild
5. Ageratum houstonianum Mill. Asteraceae Trop. America Herb Wild
6. Alternanthera paronychioides St. Hill. Amaranthaceae Trop. America Herb Wild
7. Alternanthera philoxeroides (Mart.) Griseb. Amaranthaceae Trop. America Herb Wild
8. Alternanthera pungens Kunth Amaranthaceae Trop. America Herb Wild
9. Alternathera sessilis (L.) DC. Amaranthaceae Trop. America Herb Wild
10. Amaranthus spinosus L. Amaranthaceae Trop. America Herb Wild
11. Anagallis arvensis L. Primulaceae Europe Herb Wild
12. Antigonon leptopus Hook. & Arn. Polygonaceae Trop. America Climber Cultivated
13. Argemone mexicana L. Papaveraceae South America Herb Wild
14. Argemone ochroleuca Sweet Papaveraceae South America Herb Wild
15. Asclepias curassavica L. Asclepiadaceae Trop. America Herb Cultivated
16. Bidens pilosa L. Asteraceae Trop. America Herb Wild
17. Blainvillea acmella (L. f) Philipson Asteraceae Trop. America Herb Wild
18. Blumea lacera (Burm. f.) DC. Asteraceae Trop. America Herb Wild
19. Calotropis gigantea (L.) R.Br. Asclepiadaceae Trop. Africa Shrub Wild
20. Calotropis procera (Ait.) R.Br. Asclepiadaceae Trop. Africa Shrub Wild
21. Cannabis sativa L. Cannabaceae Central Asia Herb Wild
22. Cassia alata L. Caesalpiniaceae South America Shrub Wild
23. Cassia occidentalis L. Caesalpiniaceae South America Herb Wild
24. Cassia tora L. Caesalpiniaceae South America Herb Wild
25. Celosia argentea L. Amaranthaceae Trop. Africa Herb Wild
26. Chenopodium album L. Chenopodiaceae Europe Herb Wild
27. Chenopodium ambrosioides L. Chenopodiaceae Trop. America Herb Wild
28. Cleome viscosa L. Capparaceae Trop. America Herb Wild
29. Convolvulus arvensis L. Convolvulaceae Europe Herb Wild
30. Conyza canadensis (L.) Cronquist Asteraceae South America Herb Wild
31. Corchorus aestuans L. Tiliaceae Trop. America Herb Wild
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32. Corchorus olitorius L. Tiliaceae Trop. Africa Herb Wild
33. Croton bonplandianum Baill. Euphorbiaceae South America Herb Wild
34. Cuscuta reflexa Roxb. Cuscutaceae Mediterranean Herb Wild
35. Cyperus difformis L. Cyperaceae Trop. America Sedges Wild
36. Cyperus iria L. Cyperaceae Trop. America Sedges Wild
37. Datura metel L. Solanaceae Trop. America Shrub Wild
38. Datura stramonium L. Solanaceae Trop. America Shrub Wild
39. Echinochloa colona (L.) Link. Poaceaea South America Grass Wild
40. Echinochloa crusgalli (L.) P. Beauv. Poaceaea South America Grass Wild
41. Eclipta prostrata (L.) L. Asteraceae Trop. America Herb Wild
42. Eichhornia crassipes (C.Martius) Solms Pontederiaceae Trop. America Herb Wild
43. Emilia sonchifolia (L.) DC. Asteraceae Trop. America Herb Wild
44. Euphorbia cythophora Murray Euphorbiaceae Trop. America Herb Wild
45. Euphorbia heterophylla L. Euphorbiaceae Trop. America Herb Wild
46. Euphorbia hirta L. Euphorbiaceae Trop. America Herb Wild
47. Evolvulus nummularius (L.) L. Convolvulaceae Trop. America Herb Wild
48. Gnaphalium pensylvanicum Willd. Asteraceae Trop. America Herb Wild
49. Gomphrena celosioides Mart. Amaranthaceae South America Herb Wild
50. Grangea maderaspatana (L.) Poir. Asteraceae Trop. America Herb Wild
51. Hyptis suaveolens (L.) Poit. Lamiaceae Trop. America Herb Wild
52. Impatiens balsamina L. Balsaminaceae Trop. America Herb Cultivated
53. Imperata cylindrica (L.) Raeusch. Poaceae Trop. America Grass Wild
54. Ipomoea eriocarpa R.Br. Convolvulaceae Trop. Africa Herb Wild
55. Ipomoea fistulosa Mart. ex Choisy Convolvulaceae Trop. Africa Herb Wild
56. Ipomoea hederifolia L. Convolvulaceae Trop. America Herb Wild
57. Ipomoea pes-tigridis L. Convolvulaceae Trop. E. Africa Herb Wild
58. Ipomoea quamoclit L. Convolvulaceae Trop. America Herb Wild
59. Lantana camara L. Verbenaceae Trop. America Herb Wild
60. Leucaena latisiliqua (L.) Gilli. Mimosaceae Trop. America Herb Wild
61. Ludwigia perennis L. Onagraceae Trop. America Herb Wild
62. Malvastrum coromandelianum (L.) Garcke Malvaceae Trop. America Herb Wild
63. Mecardonia procumbens (Mill.) Small Plantaginaceae Trop. America Herb Wild
64. Melilotus albus Medik. ex Desr. Fabaceae Europe Herb Wild
65. Melochia corchorifolia L. Sterculiaceae Trop. America Herb Wild
66. Mimosa pudica L. Mimosaceae Brazil Herb Wild
67. Mirabilis jalapa L. Nyctaginaceae Peru Herb Wild
68. Nicotiana plumbaginifolia Viv. Solanaceae Trop. America Herb Wild
69. Ocimum americanum L. Lamiaceae Trop. America Herb Cultivated
70. Opuntia vulgaris Miller Cactaceae South America Shrub Wild
71. Oxalis corniculata L. Oxalidaceae Europe Herb Wild
72. Parthenium hysterophorus L. Asteraceae North America Herb Wild
73. Peperomia pellucida (L.) Kunth Piperaceae South America Herb Wild
74. Physalis angulata L. Solanaceae Trop. America Herb Wild
75. Physalis minima L. Solanaceae Trop. America Herb Wild
76. Pistia stratiotes L. Araceae Trop. America Herb Wild
77. Portulaca oleracea L. Portulaceae South America Herb Wild
78. Portulaca quadrifida L. Portulaceae Trop. America Herb Wild
79. Prosopis juliflora (Sw.) DC. Mimosaceae Mexico Shrub Wild
80. Rorippa dubia (Pers.) Hara Brassicaceae South America Herb Wild
81. Saccharum spontaneum L. Poaceae Trop. W. Asia Grass Wild
82. Scoparia dulcis L. Plantiganeace Trop. America Herb Wild
83. Sida acuta Burm. f. Malvaceae Trop. America Herb Wild
84. Solanum nigrum L. Solanaceae Trop. America Herb Wild
85. Sonchus asper (L.) Hill Asteraceae Mediterranean Herb Wild
86. Sonchus oleraceus L. Asteraceae Mediterranean Herb Wild
87. Torenia fournieri Linden ex Fourn. Linderniaceae Australia Herb Wild
88. Tribulus terrestris L. Zygophyllaceae Trop. America Herb Wild
89. Tridax procumbens L. Asteraceae C. America Herb Wild
90. Triumfetta rhomboidea Jacq. Tiliaceae Trop. America Herb Wild
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.
91. Typha angustifolia L. Typhaceae Trop. America Herb Wild
92. Urena lobata L. Malvaceae Trop. Africa Shrub Wild
93. Xanthium indicum Koenig Asteraceae Trop. America Herb Wild
94. Youngia japonica (L.) DC. Asteraceae South America Herb Wild
In the present study 94 invasive alien species under 72 genera, belonging to 33 families have been recorded
in Pantnagar area. In IAS flora of Pantnagar dicotyledons are represented by 85 species under 65 genera, and 28
families, whereas monocoyledons are represented by 9 species under 7 genera and 5 families (Table 2). On the
basis of their source regions IAS can be broadly categorized into six major groups viz., American, African,
European, Asian, Mediterranean, Australian. Almost 78% (74 spp.) IAS were introduced from the American
continent followed by Afican continent 8.5% (8 spp.), Europe 5.3% (5 spp.), Mediterranean 3.1% (3 spp.), Asian
and Australian by 2.1% (2 spp). These results are in tune of Reddy (2008), Singh et al. (2010), Chandra Sekar et
al. (2012) where Tropical American elements are recorded as the dominant part of IAS flora. Pantnagar is a hot
and moist subtropical habitat where, tropical American plants have found climatic conditions similar to their
native habitats and thus flourish well (Fig. 1).
Table 2. Families, Genera and species of IAS diversity in Pantnagar area.
Families Genera Species
Number % Number % Number %
Dicots 28 84.84 65 90.28 85 90.42
Monocots 5 15.15 7 9.72 9 9.57
Total 33 100.00 72 100 94 100
80
70
Number of Species
60
50
40
30
20
10
0
American African European Mediterranean Asian Australian
Source regions
Figure 1. Source regions of Invasive Alien flora of Pantnagar.
Family Asteraceae dominates the invasive alien flora with 18 species in 16 genera, followed by Fabaceae (8
spp., 6 genera), Amaranthaceae (7 spp., 4 genera), Convovlvulaceae (7 spp., 3 genera), Malvaceae (7 spp., 6 genera),
Solanaceae (6 spp., 4 genera), Poaceae (4 spp., 3 genera), Euphorbiaceae (4 spp., 2 genera) constituting the eight
dominant families of IAS (Fig. 2). In other regions IAS flora also have the dominance of family Asteraceae
(Singh et al. 2010, Wagh & Jain 2015). The IAS flora of Uttarakhand and India also recorded the highest
member of species from family Asteraceae (Reddy 2008). The dominance of family Asteraceae may be
attributed to its prolific seed production and efficient seed dispersal mechanism. Habit wise analysis showed that
the herbs (78 species) were dominant, followed by shrubs (8 species), grasses (4 species), Sedges (2 species)
trees (1 species), and climbers (1 species) (Fig. 3). Herbs, shrubs and trees may have equal chances of dispersal
to non-native lands of these species but since herbs have shortest life cycle their further proliferation in non-
native land is easier. This seems to be the reason behind large percentage of herbs in IAS flora in Pantnagar and
elsewhere. The genera with the highest number of invasive alien species in Pantnagar region are Ipomoea (5
species), Alternanthera (4 species) and Cassia (3 species). These genera are also recorded having many species
in Reddy (2008), Chandra Sekar et al. (2012). In the recent years IAS have gained considerable notoriety as
being major threats to native species and ecosystem. These IAS proliferates easily because they find no natural
enemies in their new habitat and produce large numbers of propagules. Presence of IAS in any area be it
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.
country, state, district or local, indicates the disturbance in the vegetation of that area. More disturbed is the
vegetation more are the chances of occurrence of invasive alien species in the area.
Euphorbiaceae
Poaceae
Families
Solanaceae
Malvaceae
Convolvulaceae
Amaranthaceae
Fabaceae
Asteraceae
0 5 10 15 20
Number of Species
Figure 2. Eight dominant families of Invasive Alien flora in Pantnagar.
Figure 3. Life forms of IAS in Pantnagar region

Pantnagar is a small land area devoid of natural stands of vegetation and suffer from continuous agricultural
operations and human activities, which seems to be the reasons behind the presence of large number of IAS
flora here. The challenge now is to find ways to manage the invasive aliens that are firmly entrenched in the
area. Though prevention is suggested as the most effective and feasible method for controlling the Invasive
species it cannot be applied after invasion. Rather, now steps should be taken to ensure no further spread of
these weedy species through planting materials, seeds carried from the university to adjacent areas.
ACKNOWLEDGEMENTS
Authors are thankful to Head of Department Biological Sciences, CBSH and incharge of MRDC Pantnagar,
G. B. Pant University of Agriculture and Technology, Pantnagar for providing us constant support during this
entire research.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(3): 288291, 2015
Research article
Phytoconstituents composition and in vitro antibacterial activity

of a blue green alga Anabaena variabilis Ktz. ex Born. et Flah.
Nilu Halder*
Department of Botany, Raja Peary Mohan College, Uttarpara - 712258, W. Bengal, India
*Corresponding Author: niluhalder1@gmail.com [Accepted: 15 December 2015]
Abstract: Cyanobacterial species produce various types of secondary metabolites which are used
in drug development as medicinal importance, dye and pigmentation and, food additives.The
preliminary screening of phytoconstituents analyses in various solvent extracts (benzene,
chloroform, acetone and methanol) of a microscopic blue green alga, Anabaena variabilis Ktz. ex
Born. et Flah., collected from a pond at Diara in Hooghly district, West Bengal, India was done
following standard methods and the results exhibited the presence of alkaloid, terpenoid, phenol,
flavonoid, flavonol and phycocyanin phytoconstituents in those solvent extracts. The antibacterial
activity of the said alga was detected using eight pathogenic bacterial strains out of which three are
Gram positive (Bacillus subtilis, Micrococcus luteus and Staphylococcus aureus) and five are
Gram negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae, Shigella
flexneri and Vibrio cholerae) by agar well diffusion method with minor modifications. Maximum
inhibition zones were observed in acetone and benzene extracts against the same Gram positive
bacteria Bacillus subtilis. This study also indicated that acetone and benzene crude extracts were
best active solvents against most of the studied bacterial strains followed by methanol and
chloroform crude extracts. So, the present work suggested that this alga possessed several
bioactive phytoconstituents which showed antibacterial potentiality on the tested pathogenic
bacteria.
Keywords: Anabaena variabilis - Phytoconstituents screening - Antibacterial activity.
[Cite as: Halder N (2015) Phytoconstituents composition and in vitro antibacterial activity of a blue green alga
Anabaena variabilis Ktz. ex Born. et Flah. Tropical Plant Research 2(3): 288291]
INTRODUCTION
Algae are one of the most important richest sources of novel bioactive compounds which may be used in the
pharmaceutical industries for the development of pharmaceuticals. Cyanobacteria are well known incredible,
primitive and prokaryotic algal group which produce various types of phytoconstituents with biological
activities.
The search of cyanobacterial species with antimicrobial activities has gained much momentum in the recent
times due to growing worldwide concerns about increases in the emergence of antibiotic resistance among the
pathogenic bacteria (Chauhan et al. 2010). Cyanobacteria have been recognized as a good source of antibiotic
with antimicrobial activities during the last two decades (Bhattacharyya et al. 2013). They have potentiality to
produce an elaborate array of secondary metabolites with unusual structures and potent bioactivities. They also
produce industrially important secondary compounds like antibiotic, algicide, cytotoxic, immune suppressive
and enzyme inhibiting agents (Shaieb et al. 2014). The cyanobacterial algal group produces different
biologically active compounds which may be used in drug development and some of the active components
have potentially to inhibit anticancer, antimicrobial, antiviral, anti-inflammatory effects (Seal et al. 2014, 2015).
The aims of the present study were preliminary screening of phytoconstituents and to investigate antibacterial
potentiality of four organic solvent extracts of this alga against human pathogenic bacteria.

Selected species
Halder (2015) 2(3): 288291
.
Anabaena variabilis Ktz. ex Born. et Flah. is a blue-green alga which belongs to the family Nostocaceae
under the order Nostocales of class Cyanophyceae and it is grown in different types of aquatic bodies. The
trichomes contain heterocystous stuctures and the alga acts a bio-fertilizer due to having the capability to fix
atmospheric nitrogen in the soil.
Collection of algal sample
Algal material had been collected in plastic packets and sterilized glass containers from a pond of Diara (N
22 79' E 88 28') of Hooghly district (N 20 30'23 1' E 87 30'80 30'), West Bengal. Detailed study was
made by examining the specimen under Olympus microscope (Model-CH20i). Identification of taxon was
accomplished with the help of authentic literatures (Geitler 1932, Desikachary 1959).
Preparation of algal extracts
For extraction, the selected algal material, collected from pond was washed under running tap water to
remove adhering soil particles, epiphytes and associated debris, if any, and dried up at room temperature.
Benzene, chloroform, acetone and methanol were used for preparing the different solvent extracts. Extracts were
prepared by grinding the algal material in a mortar and pestle at 201 C. In a soxhlet extractor at 5055 C,
extracts were concentrated under reduced pressure in a rotary evaporator and kept deep frozen until tests. Each
time before extracting the powdered drug (marc) was dried up in a hot air oven. The concentrated extracts were
obtained with each solvent were weighed. However, when the pH was out of range it was adjusted to 7.0 before
assay of antibacterial activity.
Preliminary qualitative phytoconstituent tests
All the extracts were subjected to preliminary phytoconstituents screening as described by Trease & Evans
(1989), Harborne (1998) and Silveira et al. (2007).
1. Used Gram positive bacteria
The tested bacterial strains were Bacillus subtilis MTCC441, Micrococcus luteus MTCC1538,
Staphylococcus aureus MTCC3160. These strains were maintained on nutrient agar slant at 4C and sub-
cultured for 24 h before use.
2. Gram negative bacteria
The tested bacterial strains were Escherichia coli MTCC 443, Pseudomonas aeruginosa MTCC2581,
Shigella dysenteriae (clinically isolated), Shigella flexneri MTCC1457, Vibrio cholera MTCC3904. The
maintenance procedure of these bacteria was same as Gram positive bacteria.
Antibacterial assay
The antibacterial activity test of the above said alga was done using agar well diffusion method of Perez et
al. (1990). 0.1 ml of diluted inoculum (105CFU ml-1) of the bacterial strain was swabbed on the nutrient agar
plates. Wells of 5.0 mm size diameter were made into the agar plates with the help of sterilized cork borer (5.0
mm). Using a micropipette, each of 100 l of the algal extract was added to the wells made in plates. The plates
were inoculated aerobically in an upright position at 372 C for 2448 h. Antibacterial activity was evaluated
by measuring the zone of inhibitions (mm) against the bacterial strains. The tests were performed in triplicates
with controls.
Statistical analysis
The results were presented as mean values standard errors (SE). The standard errors were calculated using
statistical software package (SPSS v. 13, Inc.USA).

Qualitative screening of phytoconstituents of organic solvent extracts (benzene, chloroform, acetone and
methanol) of Anabaena variabilis Ktz. ex Born. et Flah. showed the conformity of different types of bioactive
compounds (Table 1). The result of the phytoconstituents screening revealed that this alga contained alkaloid,
tannin, terpenoid, phenol, flavonoid, flavonol and phycocyanin compounds.
In table 2, the result of antibacterial activity of the said algal species against three Gram positive and five
Gram negative bacterial strains was shown. The extracts of this alga confirmed antibacterial activities against
only six tested pathogenic bacteria out of eight bacterial strains. Acetone and benzene extracts exhibited better
antibacterial activities than other two organic solvent extracts. Regarding antibacterial activities, Bacillus
Halder (2015) 2(3): 288291
.
subtilis and Escherichia coli were more susceptible whereas, Staphylococcus aureus and Pseudomonas
aeruginosa showed intermediate results. Micrococcus luteus and Vibrio cholera were comparatively less
susceptible against all the four solvent extracts. Both acetone and benzene extracts executed the higher
inhibition zones of 18.1 mm and 16.5 mm against the same bacteria Bacillus subtilis. Comparatively, somewhat
lower antibacterial activities were recorded by methanol and chloroform extracts of Anabaena variabilis. The
aqueous extract showed poor activities against the tested pathogenic bacteria (Table 2).
Table 1. Presence of phytoconstituents of Anabaena variabilis Ktz. ex Born. et Flah. in four solvents.
Phycocyaninn
Terpenoid
Flavonoid
Glycoside
Flavonol
Alkaloid
Saponin
Tannin
Steroid
Phenol
Benzene extract + - - - - + + + + +
Chloroform extract + - - - - + + + + +
Acetone extract + - - - - + + + + +
Methanol extract + + + - - + + + + +
Note: + = Presence or positive reactions; = Absence or negative reactions.
Table 2. Antibacterial activity of different solvent extracts of Anabaena variabilis Ktz. ex Born. et Flah.
Zone of inhibition (mm) as (Mean SE ) in different bacteria
Solvents
Bs Ml Sa Ec Sd Pa Vc Sf
Benzene 16.50.07 100.03 150.05 16.10.06 - 140.05 110.03 -
Chloroform 14.40.06 8.60.02 120.04 140.05 - 120.04 100.03 -
Acetone 18.10.08 120.04 16.10.06 15.50.07 14.20.06 13.80.04
Methanol 14.60.05 9.00.02 13.20.04 14.80.04 - 130.04 8.00.03 -
Water 9.00.04 - 7.00.03 8.00.03 - 7.00.02 - -
Note: Ec= Escherichia coli, Sf= Shigellaflexneri, Pa= Pseudomonas aeruginosa, Sd= Shigelladysenteriae, Vc=
Vibrio cholerae, Bs= Bacillus subtilis, Ml= Micrococcus luteus, Sa= Staphylococcus aureus; -= Not detected.
Chauhan et al. (2011) carried out in vitro antibacterial evaluation of Anabaena sp. against several clinically
significant pathogenic bacteria and HPTLC analysis of its crude extracts indicated that different solvents
possessed significant antibacterial effects on both Gram positive and Gram negative bacteria. In the current
scenario, similar observation was noticed.
Ethyl acetate solvent among three solvents (chloroform, ethyl acetate and n-butanol) was proved as a most
effective organic solvent for the extraction of the antibacterial compounds in five species of Anabaena namely
Anabaena solitaria, Anabaena variabilis, Anabaena cylindrical, Anabaena spiroides and Anabaena circinalis
(Abdel-Raouf & Ibraheem 2008). Among various solvent extracts (acetone, methanol, ethanol. diethyl ether,
chloroform and hexane) methanol extract of Anabaena circinalis appeared to be the most effective solvent by
showing maximum antibacterial activity against the selected bacterial pathogens viz. Escherichia coli,
Salmonella typhi, Proteus vulgaris, Streptococcus pyogens, Pseudomonas solanocearum (Sivakami et al. 2013).
In this study, it was noticed that acetone and benzene extracts were more effective over the other two solvent
extracts for extraction of antibacterial compounds.
Malathi et al. (2014) while working on the screening of three cyanobacterial strains observed significant
antibacterial activities of Anabaena variabilis against the bacteria Bacillus subtilis (ATCC-11774) and
Pseudomonas aeruginosa (ATCC-15442) in chloroform and methanol crude extracts respectively. In the present
work, in vitro antibacterial activities of this alga showed the similar result. Maximum size of inhibition zone
(9.670.57) of Anabaena BT2 was observed in methanol extract against Pseudomonas sb1 (Yadav et al. 2012)
but in this study, higher inhibition zone (18.1 mm) was observed in acetone extract against a Gram positive
bacteria Bacillus subtilis.
CONCLUSION
It is quite evident from the discussion that the above said alga possessed several bioactive compounds of
pharmaceutical interests and the formation of inhibition zones (mm.) depended on various factors like types of
Halder (2015) 2(3): 288291
.
algal species, solvents used and the kind of tested pathogenic bacterial stains. Therefore, this study could be
used for future research and to produce antibacterial drugs of Cyanobacterial origin.
ACKNOWLEDGEMENTS
The author is indebted to Dr. S. N. Sinha, University of Kalyani, Nadia, West Bengal for his guidance and
supports. The author is also grateful to Dr. T. Seal and K. Chaudhuri, Plant Chemistry Department, BSI,
Howrah for their kind co-operations and suggestions.
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