Volume 1, Issue 3 (2014) Tropical Plant Research

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

1(3): 0112, 2014
Research article
Phenology, growth and survival of Vatica lanceaefolia Bl.: A

critically endangered tree species in a moist tropical forest of
Northeast, India
Mrigakhi Borah and Ashalata Devi*
Department of Environmental Science, Tezpur University, Tezpur, Sonitpur, Assam, India
*Corresponding Author: ashalatadevi12@gmail.com [Accepted: 28 September 2014]
Abstract: An attempt has been made to unravel the major phenophases, seedling survival and
growth of Vatica lanceaefolia, a critically endangered tree species in two different micro sites of
Hollongapar Gibbon Wildlife Sanctuary, Assam. The study was carried out for a period of 24
months to investigate various phenophases with respect to seasonal variations of the year and, to
understand the growth and survival of the seedlings in two micro sites (gap and understory) in
relation with the prevailing meteorological parameters of the study area. Leaf flushing was
observed twice in a year in the month of December and May, while flowering and fruiting occurs
during pre-monsoon season (April and May). The seedlings showed better survival in gap (66.6%)
compared to the understory (46.6%) and relative growth rates of the seedlings in terms of height
and collar diameter varied significantly across the months and also between the micro
environmental conditions of the two micro sites (P<0.05). Wet monsoon season favoured the
survival and growth of seedlings. Relative humidity (P<0.05), average temperature (P<0.05) and
rainfall (P<0.05) of the study area exhibited positive correlation with the growth of V. lanceaefolia
seedlings in both the micro sites. This is the pioneer study on this species which will be helpful for
developing proper conservation strategies and will serve as a baseline for further research on this
species to improve the status, distribution and multiplication of the species.
Keywords: Phenology - Seedling growth - Micro sites - Temperature - Rainfall - Relative
humidity.
[Cite as: Borah M & Devi A (2014) Phenology, growth and survival of Vatica lanceaefolia Bl.: A critically
endangered tree species in moist tropical forest of Northeast India. Tropical Plant Research 1(3): 112]
INTRODUCTION
Forest ecology and management gives the scientific knowledge of the interrelated patterns, ecosystems
processes, flora and fauna of the forests. General community patterns in leafing, flowering and fruiting for many
species, of which particular forest types are composed, determines the status of the forest (Frankie et al. 1974,
Opler et al. 1980). Vegetative and generative phases of plants show species-specic dependences on
geographical location, weather and environmental conditions. Therefore in todays world species-specific
ecological studies have become crucial to understand the status of a particular species in its habitat with the
changing global climate.
Vatica lanceaefolia is an evergreen tree species which is distributed in moist tropical forests of Bangladesh,
Myanmar and in three states of India namely Arunachal Pradesh, Nagaland and Assam (Shiva & Jantan 1998).
The species is listed in IUCN Red list category as critically endangered (CR) under criteria A1cd, C2a, version
2.3 (IUCN 2014). The plant is an important source of non-timber forest product (NTFP) and is used for
firewood and charcoal making and, the bark yields a clean, white aromatic oleoresin which is used as incense in
Bangladesh, Myanmar and India (Shiva & Jantan 1998).
Plant phenological study has a great significance because it provides knowledge about the plant
growth pattern as well as provides knowledge on the effects of environment and selective pressure on
flowering and fruiting behaviour (Zhang et al. 2006). The reproductive success of an individual plant is
dependent on its ability to pass through several phenological events that occur during its life cycle: germination,
establishment, growth to adulthood, and finally the production and dispersal of viable seed (Harper 1977).
www.tropicalplantresearch.com 1
Received: 07 August 2014 Published online: 31 October 2014
Borah & Devi (2014) 1(3): 0112
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Phenology of vegetative phases is important, as cycles of leaf flush and leaf fall are intimately related to
processes such as growth, plant water status and gas exchanges (Reich 1995). Phenological study is also
essential for seed procurement of plant species. The knowledge on phenology of plants has helped to understand
the influence of phenological events on feeding, movement patterns, and sociality of insects, birds and mammals
(Foster 1982a, Prasad 1983, Coates-Estrada & Estrada 1986). The timing of flowering in plants can serve as an
isolating mechanism in plant speciation (Newstrom et al. 1994). The timing of flowering and fruiting in tropical
trees has been attributed to edaphic, climatic and biotic factors and photoperiod, temperature and soil moisture
have been recognized as the main environmental cues for leafing and flowering (Rathcke & Lacey 1985). In
most tropical forests, variation in rainfall is suggested to be the most significant climatic factor that influences
the phenology of flowering and fruiting (Foster 1974, Hilty 1980, Borchert 1983).
India with a wide range of variation in climate, altitude and physiography exhibits enormous variation in the
life cycle of plants of different regions (Koul & Bhatnagar 2005). Several studies on phenology (Boojh &
Ramakrishnan 1982, Shukla & Ramakrishnan 1982, Ralhan et al. 1985, Bhat 1992, Bajpai et al. 2012, Kaur et
al. 2013) were made in different forest types of India. In recent years phenological studies on some forests of
Assam have been reported by few workers (Nath 2012, Devi & Garkoti 2013, Barman et al. 2014, Devi et al.
2014). However, studies on phenology of tropical moist semi evergreen forest and species specific in particular
of North East India, particularly in Assam have been little worked out.
An understanding of the population status and regeneration behaviour is a pre-requisite for developing
conservation strategies for the threatened species (Upadhaya et al. 2009). Successful regeneration of a species in
nature depends on its ability to withstand disturbance stress that plays a key role in seedling survival and
establishment (Rao et al. 1990). Seedling survivorship relies on many factors, both abiotic and biotic (Karst et
al. 2011). The process of seedling growth and development of forest trees largely depends on gaps/canopy
openings in the forest created due to natural disturbance or seedling establishment barriers such as topography
(Koide et al. 2011), thus influences the regeneration and species composition of the forest (Khumbongmayum et
al. 2005). A canopy gap is defined as an area opened by the removal of canopy trees, in which most of the living
plants were < 5 m tall and < 50 % of the height of surrounding canopy trees (Runkle 1982). Gap dynamics has
been described by many researchers in tropical (Brokaw 1985, Lawton & Putz 1988, Khumbongmayum et al.
2005, Sapkota et al. 2009, Arihafa & Mack 2012) and sub-tropical forests (Barik et al. 1992, Arunachalam &
Arunachalam 2000, Griffiths et al. 2007), and are being considered as a process capable of influencing the
structure of plant communities, enhanced diversity of forest systems, as it expands environmental heterogeneity,
and chances for the growth of tree species (Yamamoto 2000). Many workers reported better growth and survival
of tree seedlings in tropical (Augspurger 1984) and subtropical (Khan & Tripathi 1991, Rao et al. 1997) forest in
areas with more sunlight and there are evidences of fast growth and better survival of dipterocarp seedlings in
gap compared to understory (Tuomela et al. 1996, Kuusipalo et al. 1997, dOliveira & Ribas 2011). These
studies have suggested gap dynamics as an alternative management technique for the degraded and over- logged
Dipterocarp forests. Studies on species-specific seedling growth and survival in northeast India is sparse with
only a few documentations (Bharali et al. 2012, Saikia & Khan 2012a, Saikia & Khan 2012b).
The present study was carried out to understand the major phenological changes and, seedling growth and
survival of V. lanceaefolia in the study area. The study examines the spatial and temporal changes of
phenophases of the plant species and seasonal variation of seedling growth and survival in different micro-sites.
MATERIAL AND METHODS

Study area
The study was conducted for two years (2010-2012) in Hollongapar Gibbon Wildlife Sanctuary (HGWLS),
which is situated in Mariani, Jorhat District of Assam, India. It covers an area of 20.98 km2 and situated at
2640" to 2645" N and 9420" to 9425" E and is located in the south bank of the Great Brahmaputra river
system at an altitudinal gradient of 100120m above msl. The forest type of the sanctuary is Eastern Alluvial
Secondary Semi Evergreen Forest (1/2/2B/2S2) (Champion & Seth 1968) under moist tropical forest of India,
dominated by plants namely, Dipterocarpus macrocarpus, Vatica lanceaefolia and Mesua ferrea. The sanctuary
is divided into five compartments by the forest department. Continuous pressure by the people of fringe area
mainly in the form of cattle grazing, fishing, illegal felling of trees and fuel wood collection have threatened the
flora and fauna of this sanctuary.
Climate and soil type
Borah & Devi (2014) 1(3): 0112
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The climate of HGWLS is seasonal with monsoonic pattern of rainfall having four seasons winter
(December to February), pre-monsoon (March to May), monsoon (June to September) and post-monsoon
(October to November). Rainfall data were collected from India Meteorological Department while relative
humidity and temperatures were recorded with the help of a pocket weather station (Kestrel 4000 NV). Winter is
cool and temperature goes down up to 7 C and maximum temperature was recorded 32.4 C in the monsoon
season. Relative humidity ranged from 4095 % during the study period (Fig. 1).
Figure 1. Meteorological parameters of the study area during the study period (July 2010July 2012).
Different micro-environmental variables such as light intensity and edaphic characteristics of the two micro
sites i.e. gap and understory were determined during the study period 20102012. Light intensity was measured
using digital light meter (Extech EasyViewTM30). A total of 20 soil samples, 10 each from gaps and understory
were collected from the depth of 10 to 15 cm after removing litter accumulation and physicochemical
characteristics of the soil was analysed in the laboratory of Department of Environmental Science, Tezpur
University, Assam. Soil type of the sanctuary is sandy clay loam and the surface soil is largely covered by litter
fall. Light intensity and physicochemical parameters of soil of two micro sites of the study site are given in
Table 1.
Table 1. Physico-chemical parameters of soil and environmental variables recorded in the understory and gap
of HGWLS.
Variables Gap Understory
Light intensity (mol m-2 s-1) 1235.4-2993.03 52.29-122.60
Soil texture Sandy Clay Loam Sandy Clay Loam
Sand (%) 67.47 68.83
Silt (%) 10.04 9.58
Clay (%) 22.49 21.59
Bulk density (g/cm3) 1.1 1.23
Water holding capacity (%) 49.07 47.22
Soil pH 4.9 5.2
Conductivity (mS/cm) 0.2744 0.296
Available N (%) 0.0109 0.031
Available P (ppm) 6.06 6.01
Available K(ppm) 45.88 46.12
Organic Carbon (%) 1.548 2.12
Organic matter (%) 2.6438 3.82
Study species
Vatica lanceaefolia is a middle canopy evergreen tree species under the family Dipterocarpaceae (Fig. 2A).
The species is distributed randomly in all the five compartments of the sanctuary. The density of the species
inside the sanctuary is 227 individuals per hectare (Sarkar & Devi 2014). V. lanceaefolia is largely collected by
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villager inhabitant in the fringe of the wildlife sanctuary due to its good quality firewood. Large individuals
(girth > 40 cm) of the species are also illegally cut down by the people of surrounding villages for commercial
purpose. During the study period, cut stumps of the species were found to be highest in compartment no. 1 and 5
having 4 and 3 individuals ha-1, respectively.
Figure 2. Vatica lanceaefolia Bl.: A, Adult Tree; B, Seedling; C, Flowers; D, Germinated seeds on the forest floor.
Phenological investigations
Preliminary investigations on major phenological events of V. lanceaefolia were carried out for a period of
two consecutive years (April 2010-March 2012) in Hollongapar Gibbon wildlife sanctuary. A total of twenty
five adult plants (having gbh > 40 cm) with uniform canopy coverage were tagged using Aluminium sheets and
plastic thread in five different compartments of the study site having five individuals in each compartment for
phenological investigation. Monthly observations for phenological changes were made for six major
phenological phases viz., leaf abscission or senescence of leaves, leaf flushing, flowering, fruiting, and dropping
of fruit and vegetative growth. The phenological characteristics are reported as per Newstrom et al. (1993 &
1994) and phenophases were represented with the help of a phenogram.
Survival and growth of seedlings
Two micro sites namely understory and gap were selected for the study of seedling survival and growth of V.
lanceaefolia in HGWLS. The area of gap was measured using the equation for the area of an ellipse, after
measuring the longest axis and its perpendicular shorter axis by laying down long meter tapes (Sapkota et al.
2009). A gap of 942.48 m2 in size inside the forest area located at 264040.2N and 942053.4E were selected
for the purpose of the study. Thirty seedlings of uniform size and shape within height range of 9 to10.5 cm
without any physical damage or herbivory attack were selected in each of the understory and gap (Fig. 2B). To
Borah & Devi (2014) 1(3): 0112
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analyse the temporal variation of growth of seedlings, plant height (cm) and collar diameter (cm) of each tagged
seedling were measured and recorded at monthly intervals for a period of two years (July 2010-June 2012). The
Relative Growth Rate (RGR) in terms of height (RGRH) and collar diameter (RGRD) was calculated as per the
formula (Hunt 1982).
RGR (tn-1-tn) = lnS(tn)-lnS(tn-1)/ tn-tn-1
Where, S is the plant size, i.e. height (cm) or collar diameter (cm) and t is the time (months).
Seasonal variation for RGRH and RGRD in gaps and understory seedlings of the study site was analysed by
one-way ANOVA and significance in variation of the RGRH and RGRD between the seedlings in gap and
understory was tested using t-test. The correlation between few meteorological parameters viz. relative
humidity, rainfall and average temperature with the relative growth rates of seedlings in both understory and
gaps were analysed. All of these analyses were performed in SPSS software version 16.0.
RESULTS
Phenological observations
Vatica lanceaefolia did not show any significant difference among the phenological events from year to year
during the two years of consecutive study. It was also observed that the meteorological parameters recorded
were more or less same during the two years observation (Fig. 1). The two years study depicts that, leaf
abscission accompanied by large scale leaf flushing of V. lanceaefolia takes place in the month of December.
Flowering takes place once in a year in the month of April, fruit initiation takes place in May with fruit
maturation in late June and dropping of fruit takes place in the month of July. From August to November the
plant species did not show any major event of phenology and it was considered as vegetative growth (Table 2).
In late July, germination of V. lanceaefolia takes place in the forest floor.
Table 2. Monthly phenophases of Vatica lanceaefolia recorded during the study period.
Study years Apr May Jun Jul Aug Sept Oct Nov Dec Jan Feb Mar
2010-2011
20112012
Abbreviations: 1=Leaf abscission, 2=Leaf flushing, 3=Flowering, 4=Fruiting,

5=Dropping of fruit and 6=Vegetative growth.

Seedling survival in two micro sites
1 00
90
Seedling survival (% )
80
70
60
Gap
Understory
50
0
J ul Nov M ar J ul Nov M ar J ul
Months
Figure 3. Survival of seedlings (%) of Vatica lanceaefolia in understory and gap during the
study period.
At the end of the study period, the seedling survival percentage of V. lanceaefolia in the two micro sites viz.
understory and gaps was recorded 46.6 % (N=14) and 66.6 % (N=20), respectively. The seedling survival was
comparatively high in gap compared to the understory (Fig. 3). In the first year observation, the mortality rate of
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seedlings in gap was 23.33 % while in the following second year observation mortality rate was reduced with
13.04 %. Similarly, seedlings of understory also showed higher mortality rate of 30 % in the first year of
observation compared to 26.32 % in the second year of observation. Seedling mortality was high in the month of
January and February, which experience the cool and dry winter season in both the study years.
Variation in relative growth rates of seedlings between two micro sites
Relative growth rates in terms of height (RGRH) and collar diameter (RGRD) of the seedlings recorded in
both the micro sites during the study period shows higher increment during the month of May and August which
corresponds to pre-monsoon and monsoon season and less during February (winter season) and November
(post-monsoon season). However, RGRH and RGRD of seedlings exhibit different increment between the gaps
and understory with response to seasonal changes. RGRH of understory seedlings shows slightly higher growth
rate during winter and post-monsoon compared to the seedlings in gap (Fig. 4).
Figure 4. Relative Growth Rates of Vatica lanceaefolia seedlings recorded in understory (U) and gap (G): A, Height
(RGRH); B, Collar Diameter (RGRD).
RGRH (t=4.362, df=11, P=0.001) and RGRD (t=5.575, df=11, P=0.000) of seedlings varied between gap
and understory and the difference was highly significant. RGRH and RGRD of seedlings of V. lanceaefolia in
both the micro sites also varied significantly (P=0.000) across the months {RGRH(U), t=5.41, df=23;
RGRH(G), t=4.794, df=23; RGRD(U), t=4.450, df=23; RGRD(G), t=4.552, df=23}.
It was observed that relative growth rate in terms of height (RGRH) in both understory (U) and gap (G)
exhibited positive correlation with rainfall, relative humidity and average temperature of the study area during
the study period (July 2010Jun 2012). Relative growth rates in terms of collar diameter (RGRD) in understory
(U) and gap (G) also showed little correlations with these meteorological parameters (Table 3).
Table 3. Correlations of RGRH and RGRD of seedlings of understory and gaps with meteorological parameters.
Data from July 2010June 2011 Data from July 2011June 2012
RF RH AVT RF RH AVT
RGRH(U) R=0.637* R=0.602* R=0.687** R=0.786** R=0.544* R=0.609*
RGRH(G) R=0.702** R=0.665** R=0.781** R=0.686** R=0.560* R=0.675**
RGRD(U) R=0.548* R=0.548* R=0.482ns R=0.452ns R=0.543* R=0.416ns
RGRD(G) R=0.521* R=0.349ns R=0.197ns R=0.673** R=0.395ns R=0.521*
RF=Rainfall, RH=Relative humidity and AVT =Average temperature

*significant at the 0.05 level
**significant at the 0.01 level
ns
not significant
Borah & Devi (2014) 1(3): 0112
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Absolute height and collar diameter of Vatica lanceaefolia seedlings in gap and understory
At the end of the 12 months observation, the absolute height of the tagged seedlings of V. lanceaefolia
reached 20.70.303 cm and 23.90.259 cm in understory and gap, respectively whereas, collar diameter of the
seedlings reached 1.30.055 cm and 1.60.089 cm, respectively for understory and gap. But the difference in
absolute height of the seedlings in understory and gap was not significant (P>0.05). At the end of the two years
study, the absolute height of the tagged seedlings reached 32.22.25 cm and 35.70.72 cm in understory and
gap, respectively. Correspondingly, collar diameter of the seedlings reached 30.29 cm and 3.60.28 cm,
respectively for understory and gap. Variations in absolute height (t=58.428, df=14, P=0.000) and collar
diameter (t=39.884, df=14, P=0.000) of seedlings of V. lanceaefolia between the two micro sites was highly
significant at the end of two years of observation.
DISCUSSIONS
Phenological observations
From the present study it was revealed that, V. lanceaefolia is an annual flowering plant with brief flowering
(< 1 month) duration (Newstrom et al. 1993 & 1994). Bud initiation in V. lanceaefolia takes place after the first
shower of monsoon or in mid-April and flower fully opens in the month of May. White flowers of the plant
species (Fig. 2C) bear a very mild, pleasant fragrance. Flowering takes place almost at the same time in all the
inflorescences of the plant. Occurrence of peak flowering and fruiting records in the months of April to May
corresponds with the increased temperature during the pre-monsoon season or the summer. Increasing day
length, soil moisture and temperature may have induced flowering during warm pre-monsoon period (Foster
1974, Hilty 1980, Rathcke & Lacey 1985). Regular flowering in some tropical deciduous trees after the spring
equinox during MarchJune has been reported by many workers (Felger et al. 2001, Singh & Kushwaha 2006).
After 25 days of flowering fruit initiation takes place as the monsoon rain starts and earlier studies also
suggested that the seasonal availability of water is the primary determinant of fruiting (Foster 1974, Borchert
1983, Bach 2002). Fruit maturation takes place in June during the monsoon period. The need of high moisture
level for the proper development of fleshy fruits has been reported by Zahner (1968), and it was also showed
experimentally that the shortage of soil moisture reduces the rate of enlargement and final size of fruits. Seeds of
V. lanceaefolia germinates without any dormancy period within 6-10 days of dropping, in late July which was
favoured by the sufficient availability of soil moisture content due to heavy precipitation and prevailing warm
temperature (Fig.2D). This relationship of germination with availability of soil moisture has also been supported
by several earlier studies (Foster 1982b, Shukla & Ramakrishnan 1982, White 1994, Bach 1998). In relation to
temperature, genera Vatica are known to germinate at temperatures above 14C (Yap 1981). In general seedlings
of dipterocarps germinate quickly in warm and moist climate (Tompsett 1986).
Flushing and leaf production completes towards the end of the dry season, before the onset of rains. Leaf fall
occurs when temperature declines and day length becomes short during winter which is also supported by earlier
studies (Shukla & Ramakrishnan 1984, Bhat 2001). There are several reports of maximum leaf-fall during the
driest period of the year in different tropical forest types (Richards 1952, Frankie et al. 1974, Opler et al. 1980,
Liberman & Liberman 1984). During dry season leaf abscission may be attributed to avoid water stress. It was
found that leaf flushing and leaf fall in V. lanceaefolia requires low temperature (2025C) and low relative
humidity (4050 %). In February the plant bears completely new leaves in its branches. This has also been
shown for other seasonal tropical forests (Boaler 1966, Frankie et al. 1974). After maturation of leaves, heavy
insect infestation is observed during the study period (personal observation). Studies have shown that seasonal
peaks and depressions for leaf flush and leaf fall are quite common in tropical rain forests with pronounced dry
period (Kramer & Kozlowski 1960, Fogden 1972). In tropics emergence of leaves peaked either in dry season
(Frankie et al. 1974, Whitmore 1984) or in the wet season (Fogden 1972, Proctor et al. 1983). Leaf flushing
during dry season probably permit renovation of the canopy before the onset of monsoon, so that the plant
becomes able to take full advantage of the rainy season to produce and store nutrients for their growth and
development. A small scale of leaf flushing observed in the month of May during the second year of observation
indicates minor difference in phenological events during the two year of study periods. However, such slight
variation could not interpret any remarkable changes in phenological events of the species and it may be stated
that the two year phenological observation of V. lanceaefolia reveals more or less similar pattern of phenophase
which corresponds with the recorded meteorological parameters.
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During the two years of seedling survival and growth study, seedling mortality was recorded highest both in
understory and gap during the cold dry winter season (December to February) which resulted in sudden decline
in the survival percentage of the seedlings in the month of March in both the study years (Fig. 3). Large number
of mortality of seedlings during the winter season may be due to the detrimental effect of soil moisture stress on
the seedlings which has been stated by many workers (Khan et al. 1986, Kikim 1999, Khumbongmayum et al.
2005). From June, with the onset of monsoon and increase in soil moisture content, survival rate of the seedlings
was stabilized. Increase in survival rate of seedlings during the wet season is reported by various researchers
(Tompsett 1986, Lieberman & Li 1992, Bharali et al. 2012). Lower mortality rate recorded in the second year
observation than the first year observation may be attributed due the establishment of the seedling towards the
preliminary stage of sapling.
From the present study it was found that, RGRH, RGRD and absolute height of seedlings of V. lanceaefolia
is affected by different micro-environmental conditions and the seedling survival was favourable in gap (66.6
%) compared to the understory (46.6 %). Better growth and survival is recorded in a large number of plant
species in gaps compared to understory by many workers (Brokaw 1985, 1987, Welden et al. 1991, Nagamastu
et al. 2002). Higher light intensity and the prevailing micro-environmental conditions in gap may have
influence in the better growth rates of seedlings of V. lanceaefolia compared to the understory (Table 1). The
effect of light (Burton & Mueller-Dombois 1984, Connel et al. 1984) and temperature (Sorenson & Ferrel
1973) in regulating the growth of tree seedlings in tropical forests has been reported earlier by many workers.
Seasonal variability in growth response to light environment is an important parameter to determine the growth
of subtropical tree species (Khumbongmayum et al. 2005). Growth in terms of height and collar diameter of the
seedlings increased in both understory and gap during pre-monsoon and monsoon period probably because of
the rainfall, as it shows significant positive correlation with the rainfall of the study area during the study
period, however, growth rate decreases during cold dry seasons from November to February (post-monsoon and
winter) (Fig. 4). These may be attributed to the high moisture content in soil along with the high surface
temperature. The peak seedling growth during rainy season may be because of the increased availability of
nutrients in soil due to rapid decomposition of litter on the forest floor and because of the higher moisture
content of the soil (Mueller-Dombois et al. 1980, Khumbongmayum et al. 2005). It was observed that, relative
growth rates in terms of height and collar diameter of the seedlings in understory and gap also increases with
the increase in relative humidity during the monsoon period. Growth performance was highest in the months of
April to September with higher relative humidity and least in the months of November to February with lower
relative humidity. Growth reduction in plants due to low relative humidity of the air is reported earlier (Grantz
1990).
Prevailing average temperature of the study area also exhibited positive correlation with the seedling growth
performance. This reveals that, seedling growth of V. lanceaefolia is largely influenced by rainfall, relative
humidity and average temperature of the study area. It can be concluded that, seedling growth of V. lanceaefolia
shows better in gaps and growth rate increases with increase in soil moisture regime, ambient temperature and
rainfall during the summer monsoon season.
CONCLUSIONS
The present study reports the timing of occurrence of different phenological phases of Vatica lanceaefolia,
an annual flowering plant. Senescence of old leaves occurs with the onset of large scale leaf flushing as a
simultaneous process in the cold dry winter months. Flowering and fruiting occurs once in a year in pre-
monsoon season. Dropping of mature fruit takes place in late July and correspondingly germination of seeds
starts on the forest floor. The study also reveals that, seedling growth of V. lanceaefolia is significantly affected
by different micro-environmental conditions in terms of their survival and growth parameters. Significantly
higher growth performance was observed in the seedlings growing in gap area compared to the understory
during the study period in HGWLS. Thus, plantation of V. lanceaefolia seedlings in the gaps or in open areas
will be a fruitful measure to multiply the number of this critically endangered plant species in their habitat.
Germination showed dependency on water availability in the soil as it starts in the rainy season without any
dormancy period. Monthly relative growth rate (RGR) in terms of height and collar diameter indicates
dependency of the species on wet season as growth rates were found higher during the rainy hot months
compared to the cool dry months. The long rainy season from April to September (pre-monsoon and monsoon)
during the study period had a positive impact on the growth and development of seedlings in both the micro
sites, which was associated with the major changes in the phenological events of the plant.
Borah & Devi (2014) 1(3): 0112
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Presently the sanctuary harbours a good density of V. lanceaefolia, but continuous anthropogenic pressure
exerted in terms of illegal cutting and felling for the purpose of firewood has emerged as a serious threat to the
survival of this species. So, possible measures should be undertaken to control the illegal logging of this species
by the fringe village people and proper strategies should be adopted to conserve the plant and multiply its
number in the study area in particular and other similar habitats of the species in northeast India in general. This
investigation on phenological characteristics and survival and growth of seedlings on this species is a pioneer
study and data of the present study may be helpful not only in the formulation of conservation strategies but also
in implementing further research aspects of this species viz. reproductive phenology, chemical analysis, genetic
improvement, etc.
ACKNOWLEDGEMENTS
The authors are thankful to the Principal Chief Conservator of Forests (Wildlife), Basistha, Guwahati,
Assam, for his kind permission to carry out the research work in HGWLS. Sincere thanks to forest officials of
Meleng Beat Office, HGWLS especially, Mr. Daben Borah, for his kind assistance during the entire field work.
We also thank Dr. Manoranjan Nath, Dr. Rajeev Basumatary and Dr. Gitamani Dutta for their valuable
suggestions and help.
REFERENCES
Arihafa A & Mack AL (2012) Treefall gap dynamics in a tropical rainforest in Papua New Guinea. Pacific
Science 67(1): 126.
Arunachalam A & Arunachalam K (2000) Inuence of gap size and soil properties on microbial biomass in a
subtropical humid forest of north-east India. Plant and Soil 223: 185193.
Augspurger CK (1984) Pathogen mortality of tropical seedlings: Experimental studies of the effect of dispersal
distance, seedling density and light conditions. Oecologia 61: 211217.
Bach CS (2002) Phenological patterns in monsoon rainforests in the Northern Territory, Australia. Austral
Ecology 27(5): 477489.
Bach CS (1998) Resource patchiness in space and time: Phenology and reproductive traits of Monsoon
Rainforests at Gunn Point, Northern Territory, Australia, Ph.D. Thesis. Northern Territory University,
Darwin, Australia.
Bajpai O, Kumar A, Mishra AK, Sahu N, Behera SK & Chaudhury LB (2012) Phenological study of two
dominant tree species in tropical moist deciduous forest from the northern India. International Journal of
Botany 8(2): 6672.
Barik SK, Pandey HN, Tripathi RS & Rao P (1992) Microenvironmental variability and species-diversity in tree
fall gaps in a subtropical broadleaved forest. Vegetatio 103: 3140.
Barman D, Nath N & Deka K (2014) Phenology of some medicinal plant species of Goalpara District, Assam
(India). Scholars Academic Journal of Biosciences 2(2): 8184.
Bharali S, Paul A, Khan ML & Singha LB (2012) Survival and Growth of Seedlings of Two Rhododendron
Tree Species along an Altitudinal Gradient in a Temperate Broad Leaved Forest of Arunachal Pradesh,
India. International Journal of Plant Research 2(1): 3946.
Bhat DM & Murali KS (2001) Phenology of understory species of tropical moist forest of Western Ghats region
of Uttara Kannada district in South India. Current Science 81: 799805.
Boaler SB (1966) Ecology of a miombo site, Lupa North Forest Reserve, Tanzania II. Plant communities and
seasonal variation in the vegetation. Journal of Ecology 54: 465479.
Boojh R & Ramakrishnan S (1982) Growth strategy of trees related to successional status II. Leaf dynamics.
Journal of Forest Ecology and Management 4: 375386.
Borchert R (1983) Phenology and control of flowering in tropical trees. Biotropica 15(2): 8189.
Brokaw NVL (1985) Gap phase regeneration in a tropical forest. Ecology 66: 682687.
Brokaw NVL (1987) Gap phase regeneration of three pioneer tree species in a tropical forest. Journal of
Ecology 75: 919.
Burton PJ & Mueller-Dombois D (1984) Response of Metrosidros polymorpha seedlings to experimental
canopy. Ecology 5: 779791.
Champion HG & Seth SK (1968) A revised survey of the forest types of India. The Manager of Publications,
Delhi.
Borah & Devi (2014) 1(3): 0112
.
Coates-Estrada R & Estrada A (1986) Fruiting and frugivores at a strangler fig in the tropical rain forest of Los
Tuxtlas, Mexico. Journal of Tropical Ecology 2: 349357.
Connell J, Tracey JG & Webb JL (1984) Compensatory recruitment, growth and mortality as factors
maintaining rainforest tree diversity. Ecological Monograph 54: 141-164.
dOliveira MVN & Ribas LA (2011) Forest regeneration in articial gaps twelve years after canopy opening in
Acre State Western Amazon. Forest Ecology and Management 261: 1722-1731.
Devi AF & Garkoti SC (2013) Variation in evergreen and deciduous species leaf phenology in Assam, India.
Trees 27: 985997.
Devi AF, Garkoti SC & Borah N (2014) Periodicity of leaf growth and leaf dry mass changes in the evergreen
and deciduous species of Southern Assam, India. Ecological Research 29: 153165.
Felger RS, Johnson MB, Wilson MF (2001) The trees of Sonora. Oxford University Press, Oxford, pp. 391.
Fogden L (1972) The seasonality and population dynamics of equatorial forest birds in Sarawak. Ibis 114:
307342.
Foster RB (1974) Seasonality of fruit production and seed fall in a tropical forest ecosystem in Panama, Ph.D.
Dissertation. Duke University, Durham, North Carolina.
Foster RB (1982b) The seasonal rhythm of fruitfall on Barro Colorado Island. In: Leigh EGJr, Rand AS &
Windsor DM (eds) The Ecology of a Tropical Forest: Seasonal Rhythms and Long-Term
Changes. Smithsonian Institution Press, Washington DC, pp. 151172.
Foster R (1982a) Famine on Barro Colorado Island. In: Leigh GJr, Rand AS & Windsor D (eds) The
ecology of a tropical forest, seasonal rhythms and long-term changes. Smithsonian Institution Press,
Washington, pp. 201212.
Frankie GW, Baker G & Opler (1974) Comparative phenological studies of trees in tropical wet and dry
forests in the low lands of Costa Rica. Journal of Ecology 62: 881919.
Grantz DA (1990) Plant response to atmospheric humidity. Plant, Cell & Environment 13(7): 667679.
Griffiths ME, Lawes MJ & Tsvuura Z (2007) Understory gaps influence regeneration dynamics in subtropical
coastal dune forest. Plant Ecology 189: 227236.
Harper JL (1977) Population biology of plants. Academic Press, London, pp. 892.
Hilty SL (1980) Flowering and fruiting periodicity in a premontane forest in Pacific Colombia. Biotropica
12(4): 292306.
Hunt R (1982) Plant growth curves: The Functional Approach to Plant Growth Analysis. Edward Arnold,
London, 248 p.
IUCN (2014) IUCN Red List of Threatened Species, Version 2014.2. Available from:
http://www.iucnredlist.org. (accessed: 22 Sept. 2014).
Karst J, Hoeksema JD, Jones MD & Turkington R (2011) Parsing the roles of abiotic factors in Douglas-fir
seedling growth. Pedobiologia 54: 273280.
Kaur G, Singh BP & Nagpal AK (2013) Phenology of Some Phanerogams (Trees and Shrubs) of Northwestern
Punjab, India. Journal of Botany 2013: 110.
Khan ML & Tripathi RS (1991) Seedling survival growth of early and late successional tree species as affected
by insect herbivory and pathogen attack in sub-tropical humid forest stands of north east India. Acta
Oecologia 12: 569579.
Khan ML, Rai JPN & Tripathi RS (1986) Regeneration and survival of tree seedlings and sprouts in tropical
deciduous and sub tropical forests of Meghalaya, India. Forest Ecology and Management 14: 293304.
Khumbongmayum AD, Khan ML & Tripathi RS (2005) Survival and growth of seedlings of a few tree species
in the four sacred groves of Manipur, Northeast India. Current Science 88(11): 17811788.
Kikim AD (1999) Vegetation dynamics and regeneration of some trees in sub tropical forests of Manipur, Ph.D.
Thesis. Manipur University, Manipur.
Koide RT & Fernandez C (2011) General principles in the community ecology of ectomycorrhizal fungi. Annals
of Forest Science 68: 4555.
Koul M & Bhatnagar AK (2005) Phenology and climate change. Current Science 89: 243244.
Kramer J & Kozlowski (1960) Physiology of trees. McGraw Hill, New York, pp. 642.
Kuusipalo J, Hadengganan S, Adjers G & Sagala APS (1997) Effect of gap liberation on the performance and
growth of dipterocarp trees in a logged-over rainforest. Forest Ecology and Management 92: 209219.
Lawton RO & Putz FE (1988) Natural disturbance and gap-phase regeneration in a wind-exposed tropical cloud
forest. Ecology 69(3): 764777.
Borah & Devi (2014) 1(3): 0112
.
Lieberman D & Li M (1992) Seedling recruitment patterns in a tropical dry forest in Ghana. Journal of
Vegetation Science 3: 375382.
Lieberman D & Lieberman M (1984) The causes and consequences of synchronous flushing in a dry tropical
forest. Biotropica 16: 193201.
Mueller-Dombois D, Jacobi JD, Cooray RG & Balakrishnan N (1980) Ohia rainforest study: Ecological
investigations of the Ohia dieback problem in Hawaii. Miscellaneous Publication, Hawaii Institute of
Tropical Agriculture and Human Resources, Honolulu, HI, 183 p.
Nagamastu D, Seiwa K & Sakai A (2002) Seedling establishment of deciduous trees in various topographic
positions. Journal of Vegetation Science 13: 35-44.
Nath N (2012) Phenological Study of Some Tree Species of Sri Surya Pahar of Goalpara District, Assam, Indian
Journal of Fundamental and Applied Life Sciences 2 (1): 102104.
Newstrom LE, Frankie GW & Baker HG (1994) A New Classification for Plant Phenology Based on Flowering
Patterns in Lowland Tropical Rain Forest Trees at La Selva, Costa Rica. Biotropica 26(2): 141159.
Newstrom LE, Frankie GW, Baker HG & Colwell RK (1993) Diversity of flowering patterns at La Selva. In:
McDade LA, Bawa KS, Hartshorn GS & Hespenheide HA (eds) La Selva: ecology and natural history of a
lowland tropical rainforest. University of Chicago press, Chicago, Illnois.
Opler PA, Frankie GW & Baker HG (1980) Comparative phenological studies of treelet and shrub species in
tropical wet and dry forests in the lowlands of Costa Rica. Journal of Ecology 68: 16788.
Prasad NLNS (1983) Seasonal changes in the herd structure of Blackbuck. Journal of Bombay Natural History
Society 80: 549554.
Proctor J, Anderson JM, Fogden SCL & Vallack W (1983) Ecological studies in four contrasting lowland
rainforests in Gunung Mulu National Park Sarawak. II. Litterfall, litter standing crop and preliminary
observation on herbivory. Journal of Ecology 71: 261283.
Ralhan , Khanna RK, Singh S & Singh JS (1985) Phenological characteristics of the shrub layer of Kumaun
Himalayan forests. Vegetatio 63: 113119.
Rao P, Barik SK, Pandey HN & Tripathi RS (1990) Community composition and tree population structure in a
subtropical broad-leaved forest along a disturbance gradient. Vegetatio 88: 15112.
Rao P, Barik SK, Pandey HN & Tripathi RS (1997) Tree seed germination and seedling establishment in tree
fall gaps and understory in a subtropical forest of north-east India. Australian Journal of Ecology 22: 136
145.
Rathcke & Lacey (1985) Phenological patterns of terrestrial plants. Annual Review of Ecology, Evolution,
and Systematics 16: 179214.
Reich PB (1995) Phenology of Tropical Forests: Patterns, Causes and Consequences. Canadian Journal of
Botany 73: 14174.
Richards PW (1952) The Tropical Rain Forest: An Ecological Study. Cambridge University Press, London.
Runkle JR (1982) Patterns of disturbance in some old-growth mesic forests of eastern North America. Ecology
63: 15331546.
Saikia P & Khan ML (2012a) Seedling survival and growth of Aquilaria malaccensis in different microclimatic
conditions of northeast India. Journal of Forestry Research 23(4): 569574.
Saikia P & Khan ML (2012b) Phenology, Seed Biology and Seedling Survival and Growth of Aquilaria
malaccensis: a Highly Exploited and Red Listed Tree Species of North East India. The Indian Forester
138(3): 289295.
Sapkota IP, Tigabu M, Oden PC (2009) Species diversity and regeneration of old growth seasonally dry Shorea
robusta forests following gap formation. Journal of Forestry Research 20: 714.
Sarkar M & Devi A (2014) Assessment of diversity, population structure and regeneration status of tree species
in Hollongapar Gibbon Wildlife Sanctuary, Assam, Northeast India. Tropical Plant Research 1(2): 2636.
Shiva MP & Jantan I (1998) Non timber forest products from dipterocarps. In: Appanah S & Turnbull JM (eds)
A Review of Dipterocarps: taxonomy, ecology and silviculture. Center for International Forestry Research,
Bogor, Indonesia, Chapter 10, 223 p.
Shukla R & Ramakrishnan S (1982) Phenology of trees in a sub tropical humid forest in north Eastern India.
Vegetatio 49: 103109.
Shukla R & Ramakrishnan S (1984) Leaf dynamics of tropical trees relation to successional status. New
Phytologist 97: 697706.
Borah & Devi (2014) 1(3): 0112
.
Singh KP & Kushwaha CP (2006) Diversity of Flowering and Fruiting Phenology of Trees in a Tropical
Deciduous Forest in India. Annals of Botany 97(2): 265276.
Sorenson FC & Ferrel WK (1973) Photosynthesis and growth of Douglas-fir seedlings which are grown in
different environments. Canadian Journal of Botany 51: 16891698.
Tompsett PB (1986) The effect of desiccation on the viability of dipterocarp seed. In: Nather J (ed) Seed
problems under stressful conditions. Proceeding of the IUFRO Symposium, Federal Research Institute,
Vienna, pp. 181202.
Tuomela K, Kuusipalo J, Vesa L, Nuryanto K, Sagala APS & Adjers G (1996) Growth of dipterocarp seedlings
in artificial gaps: an experiment in a logged-over rainforest in South Kalimantan, Indonesia. Forest Ecology
and Management 81: 95100.
Upadhaya K, Barik SK, Adhikari D, Baishya R & Lakadong NJ (2009) Regeneration ecology and population
status of a critically endangered and endemic tree species (Ilex khasiana Purk.) in north-eastern India.
Journal Forestry Research 20(3): 223228.
Welden CW, Hewett SW, Hubbell SP & Foster RB (1991) Sapling survival, growth and recruitment:
Relationship to canopy height in a neotropical forest. Ecology 72: 3550.
White LJT (1994) Patterns of fruit-fall phenology in the Lope Reserve, Gabon. Journal of Tropical Ecology
10: 289312.
Whitmore C (1984) Tropical rainforests of the Far East. Clarendon Press, Oxford, 2nd edition.
Yamamoto S (2000) Forest Gap Dynamics and Tree Regeneration. Journal of Forestry Research 5: 223229.
Yap SK (1981) Collection, germination and storage of dipterocarp seeds. Malaysian Forester 44: 281300.
Zahner R (1968) Water deficits and growth of trees. In: Kozlowskl TT (ed) Water Deficits and Plant Growth.
Academic Press, New York.
Zhang G, Song Q & Yang D (2006) Phenology of Ficus racemosa in Xishuangbanna, Southwest China.
Biotropica 38(3): 334341.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
1(3): 13 15, 2014
Research article
Pogonatum perichaetiale subsp. thomsonii (Mitt.) Hyvnen -

An uncommon species from western Himalaya
Vinay Sahu and A. K. Asthana*
Bryology Laboratory, CSIR-National Botanical Research Institute Lucknow- 226 001, India
*Corresponding Author: drakasthana@rediffmail.com [Accepted: 10 October 2014]
Abstract: The present study deals with the investigation of Pogonatum perichaetiale subsp.
thomsonii from Watan Village, Pithoragarh. The important characteristics of this species are plants
simple, leaves stiff, tufted and forming a bud like structure when dry, margin sharply toothed in
upper part of the leaves, costa ends in a sharp awn like point. Leaf base 1/4 to 1/5 of the total leaf
length, lamellae 5 6 cells high, end cells of lamellae thick walled, smooth rectangular. The present
study recognizes Pogonatum perichaetiale subsp. thomsonii a rare species from Uttarakhand
which is a new addition to west Himalayan bryoflora of India.
Keywords: Polytrichaceae - Awn like point - Lamellae - End cells - India.
[Cite as: Sahu V & Asthana AK (2014) Pogonatum perichaetiale subsp. thomsonii (Mitt.) Hyvnen - An
uncommon species from western Himalaya. Tropical Plant Research 1(3): 1315]
INTRODUCTION
Genus Pogonatum belongs to family polytrichaceae. This genus is easily recognized by its thick, rough
textured leaves and hairy calyptra. In India this genus is represented by 18 species (Gangulee 1969, Hyvnen
1989, Asthana & Sahu 2012, Sahu & Asthana 2013). Gangulee (1969) described 4 species within section
Cephalotrichum (C. Muell.) Broth. from eastern India (P. perichaetiale ( Mont.) A. Jaeger, P. thomsonii (Mitt.)
A. Jaeger, P. tortipes (Mitt.) A. Jaeger and P. muticum Broth.). Hyvnen (1989) synonmized P. thomsonii
(Mitt.) Jaeag. and P. tortipes (Mitt.) A. Jaeger under P. perichaetiale subsp. thomsonii and P. muticum into P.
neesii (Mll. Hal.) Dozy. Only two valid species were reported in the section Cephalotrichum from India at
present. P. perichaetiale subsp. thomsonii can easily be distinguished from P. perichaetiale subsp. perichaetiale
with serrated leaf margin and aristate leaves. The key characters of this taxon are: leaves aristate, margin
serrulate at top, forming a bud like structure when dry and end cells of lamellae thick walled, quadrate to short
rectangular. Chopra & Kumar (1981) described 6 species from western Himalaya and adjacent plains (P.
perichaetiale, P. thomsonii, P. himalayanum Mitt., P. microstomum (R. Br. ex Schwgr.) Brid., P. neesii, P.
urnigerum (Hedw.) P. Beauv.), out of which 4 taxa are valid. The present study has revealed Pogonatum
perichaetiale subsp. thomsonii as a new addition to Uttarakhand, west Himalayan bryoflora.

Plant specimens were collected from Watan Village, Pithoragarh district of Uttarakhand, western Himalaya,
India. Plants were air dried and transferred to brown packets. For morphological and anatomical study plant
samples were soaked and washed in tap water and were mounted on glass microslide in 30 % glycerine to
investigate under microscope. Sections were cut free hand with a razor blade. Observations were made under
Olympus compound microscope. The measurements were taken with the help of oculometer. The voucher
specimens were deposited in Bryophyte Herbarium, National Botanical Research Institute, Lucknow (LWG).
TAXONOMIC DESCRIPTION
Pogonatum Palisot de Beauvois in Mag. Enc., 5: 329 (1804).
Plants usually dioicous, stiff, robust, erect, simple. Leaves curled to crispate when dry and erectopatent when
moist. Leaves lanceolate from a sheathing bases, margin not bordered, usually serrate at upper portion and
numerous longitudinal lamellae on ventral surface. Leaf costa percurrent to excurrent. Seta long, capsule erect to
inclined, subcylindrical, stomata absent. Peristome teeth 32, sometimes 16, calyptra hairy, cucullate.
Sahu & Asthana (2014) 1(3): 13 15
.
Figure 1. Pogonatum perichaetiale subsp. thomsonii: A, Plant in dry condition; B, Plant in wet condition; C, Leaves; D,
Apical margin of Leaf; E & F, Cross sections of leaves showing Lamellae; G, Apical cells of leaf; H, median cells of leaf;
I, basal cells of leaf.
P. perichaetiale subsp. thomsonii comes under the section Cephalotrichum. The important characteristics of
this section are that plants are small, stiff, leaves tufted at top. Leaf margin dentate at apex or entire, costa
excurrent in a sharp point or ending at the tip into a long awn like point. Lamellae 4 5cells high (sometimes up
to7), end cells bigger quadrate to rectangular, thick walled, smooth and 16 Peristome teeth each having a
bifurcated axial pillar.
Pogonatum perichaetiale subsp. thomsonii (Mitt.) Hyvnen, in a synopsis of genus Pogonatum (Polytrichaceae,
Musci). Acta Bot. Fennica 138: 1 87 (1989). (Fig. 1).
Polytrichum thomsonii Mitt., J. Linn. Soc. Bot. Suppl. 1:155 (1859).
Pogonatum thomsonii (Mitt.) A. Jaeger. Ber. Thtigk. St. Gallischen Naturwiss. Ges. 1873 74: 257 (1875);
Pogonatum tortipes (Mitt.) A. Jaeger. Ber. Thtigk. St. Gallischen Naturwiss. Ges. 1873 74: 257 (1875);
Pogonatum thomsonii var. tibetanum Chen, Sci. Exped. Qomolongma Reg. 235.14 (1962).
Sahu & Asthana (2014) 1(3): 13 15
.
Plants dark brown, erect, simple, 12 15 mm long. Leaves stiff, tufted and forming a bud like structure when
dry, Lower leaves small. Leaves erectopatent, lanceolate from a wider transparent sheathing base, 4 5 mm long
and 0.96 1.12 mm wide, margin sharply toothed in upper part of the leaves. Leaf costa ends in a sharp awn like
point. Leaf base 1/4 to 1/5 of the total leaf length. In cross section of leaf, lamellae covering almost the entire
ventral leaf surface, lamellae 5 6 cells high, end cells of lamellae thick walled, smooth reddish brown,
rectangular with top cell flat or rounded. Apical cells of leaf 12 16 m long and 8 12 m wide, short quadrate.
Basal cells of leaf 20 40 m long and 12 20 m wide, quadrate to rectangular. Leaf costa 140 160 m wide at
base. Sporophyte not seen.
Specimens examined: INDIA, Western Himalaya, Uttarakhand, Pithoragarh, Near Watan Village, 27.09.1990,
V. Nath 205087A (LWG).
Habitat: ca. 3500 m, on soil.
Distribution: India (Simla, Sikkim), Bhutan, South eastern Tibet, Nepal, China.
Hyvnen (1989) synonymized Pogonatum thomsonii and P. tortipes under P. perichaetiale subsp.
thomsonii. In the case of P. tortipes end cells of lamellae are smooth, thick walled, 4 5 cells high, elongated
rectangular with top cells flat and leaf basal part 1/3 of total leaf length, basal cells rectangular up to 145m
long and 24 m wide while in P. thomsonii end cells of lamellae cup shaped with depressed top, lamellae 5 7
cells high, basal leaf cells up to 60m long and 17 m wide (Gangulee 1969). Characteristic end cells of
lamellae and basal leaf portion might be the reason for making P. perichaetiale subsp. thomsonii as separate
subspecies. In our specimens end cells of lamellae are 5 6 cells high, thick walled, smooth, elongated
rectangular, with top cells flat or rounded and basal portion of leaf 1/4 to 1/5 of the total leaf length. P. tortipes
was collected by Hooker in Japanese Expeditions in 1960 63 from Sikkim and it is known in India from that
collection only. Chopra & Kumar (1981) examined the specimen no. 6202 (BM) of P. thomsonii but in that
specimen date of collection and altitude was not mentioned. Pogonatum perichaetiale subsp. thomsonii is very
rare and it has been collected from Pithoragarh, western Himalaya after 30 years. It is still untraced despite
several collections in the area in past few decades. After Hyvnen treatment of this taxon, the plants have been
identified and described from Pithoragarh region of western Himalaya for the first time.
ACKNOWLEDGEMENTS
Authors are grateful to the Director, National Botanical Research Institute (CSIR), Lucknow for
encouragement and providing the facilities and work has been carried out under In house project OLP-0083.
REFERENCES
Asthana AK & Sahu V (2012) Two mosses new to western Himalayan Bryoflora. Phytotaxonomy 12: 6367.
Chopra RS & Kumar SS (1981) Mosses of the western Himalayas and adjacent plains. Published by The
Chronica Botanica Co., New Delhi, India, 142 p.
Gangulee HC (1969) Mosses of Eastern India and adjacent regions Vol I. Published by Author, printed at Sree
Saraswati Press, Calcutta, India, pp. 94180.
Hyvnen J (1989) A synopsis of genus Pogonatum (Polytrichaceae, Musci). Acta Botanica Fennica 138: 187.
Sahu V & Asthana AK (2013) Genus Pogonatum P. Beauv. in Singalila National Park (Darjeeling), eastern
Himalaya, India. Geophytology 43(2): 117124.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
1(3): 1621, 2014
Research article
Species composition and structure of Sal (Shorea robusta

Gaertn. f.) forests along disturbance gradients of Western
Assam, Northeast India
Debajit Rabha
Department of Ecology & Environmental Science, Assam University, Silchar, Assam, India
Corresponding Author: d10rabha@gmail.com [Accepted: 12 October 2014]
Abstract: The present paper deals with the structure and species composition of undisturbed and
disturbed secondary Sal forests of Goalpara district, Western Assam, Northeast India. Species
richness was recorded very low with only 3 species in undisturbed Sal forests compare to the 18
species in disturbed Sal forests. The density and basal area were recorded high in undisturbed
forests than the disturbed one. Shorea robusta is the single dominant species and constitute the
bulk of the stocks in both forests types. Girth class distribution of density revealed the dominance
of middle girth classes in undisturbed forests whereas in disturbed forests 45 % of the total density
recorded in lowermost girth class. Anthropogenic disturbances influence the forests structure,
functions as well as services in both forests types in the present study.
Keywords: Species diversity - Density - Disturbance index - Sal forests.
[Cite as: Rabha D (2014) Species composition and structure of Sal (Shorea robusta Gaertn. f.) forests along
distribution gradients of Western Assam, Northeast India. Tropical Plant Research 1(3): 1621]
INTRODUCTION
Sal (Shorea robusta Gaertn. f.) is one of the dominant tree species in the tropical moist as well as dry
deciduous forests in India (Champion & Seth 1968) and frequently forms a mono-specific canopy (Rautiainen &
Suoheimo 1997). Sal is well known for its high timber value and government always attempted to manage Sal
forests for commercial timber production in order to increase revenue (Gautam & Devoe 2006). Sal tree grows
gregariously and tends to form dense vegetation in its natural habitat. Natural Sal forests have high resilience
capacity and survive through regeneration (Soni 1961, Qureshi et al. 1968). In India, Sal forests are found to
occur gregariously in the northern and central regions and cover approximately 13.30% of the total forest area of
the country (Upreti & Nayaka 2005). There is almost a continuous belt of Sal stretching along the sub-
Himalayan tract from Punjab to Assam (Pandey & Shukla 2003) in the northern Indian region.
In Assam, Sal is a semi-deciduous species and found in the form of high forest and coppice forest confined
specially to the Western part of Assam (Sarma & Das 2012). Champion & Seth (1968) categorized Assams Sal
forests as Tropical Moist Deciduous Forest further divided into Khasi hill Sal forest (3C/C1 1a (ii)) and
Kamrup Sal forest (3C/C2 2d (iv)). Kamrup Sal forests are more prominent and confined in Western part of
the state.
Disturbances not only influence diversity but also regeneration and dominance of tree species (Lawes et al.
2007). Recurrent anthropogenic disturbances treated as a major threat of natural Sal forests which can change its
structure as well as function (Lalfakawma et al. 2009). Due to the ongoing over-exploitation, deforestation,
encroachment and alteration in land use and land cover the mother Sal forests gradually replace by secondary
regenerated Sal forest of the low lying areas of Assam (Deka et al. 2012). Again regeneration was very poor
where soil moisture is inadequate and which experienced higher degree of disturbances such as fire and different
human activities (Padey & Shukla 2001, Chauhan et al. 2008). Chitale & Behera (2012) stated that moisture is
one of the key factors that influence the distribution to shift the Sal forests towards northern and eastern India
due to changing climate. Ahmed & Medhi (2005) estimated that there was shrinkage of 1050.46 hectares reserve
forests and proposed reserve forest areas of Goalpara District during the period 19812002 due to encroachment
for human habitation, pasture and agricultural uses. These are causing loss of Sal forest trees at a very fast rate,
thereby encouraging the spread of mix forest communities (Sarma & Das 2012). Timely, accurate assessment
Rabha (2014) 1(3): 1621
.
and understanding of the dynamics of plant resources is important for their sustainable management, utilization
and biodiversity conservation (Sarkar & Devi 2014). Comparatively a good number of quantitative studies of
community attributes are available for tropical Sal forest of northeast India (Uma Shankar 2001, Ahmed &
Medhi 2005, Lalfakawma et al. 2009, Deka et al. 2012, Sarma & Das 2012, Dutta & Devi 2013) but in Western
part of Assam which constitute the major partion of Kamrup Sal forest (Champion & Seth 1968) have not
received much attention, except few similar studies (Deka et al. 2012, Sarma & Das 2012). Therefore the
present study deals with the species composition and other community attributes of undisturbed and disturbed
Sal forest of Goalpara District, Western Assam, Northeast India.

The study was conducted in undisturbed and disturbed Secondary Sal forest located in Goalpara District,
Western Assam, Northeast India (Fig. 1). The geographical location of Goalpara District is between latitude 25
53'26 30' N and longitude 90 07'91 05' E. The vegetation was analysed by delimiting a total of five 0.1
Figure 1. Location of the study area in Goalpara District, Western Assam, Northeast India.
hectare quadrats randomly in each undisturbed and disturbed Sal forests. The girth of all the trees ( 10 cm
GBH) within the sampling area were measured at breast height (i.e. 1.37 m above the ground) and identified.
The climate is damp and warm humid and average annual rainfall of last five-year period (20082012) was
2173.02 mm yr-1 (Hydromet Division 2013).
Quantitative analysis of tree vegetation for Density and Basal area were done by following Misra (1968).
The importance value index (IVI) is the sum of relative density, relative frequency and relative dominance.
Shannon-Wiener diversity index (Shannon & Wiener 1963) was calculated from the IVI values using the
formula -
H=
Rabha (2014) 1(3): 1621
.
Where, is the proportion of individuals of species and total number of individuals all the species
( /N).
Concentration of dominance (cd.) was measured by Simpson index (Simpson 1949).
Cd. =
Where, is same as Shannon-Wiener diversity index.
A disturbance index for each forest site was calculated following Kanzaki & Kyoji (1986), Pandey & Shukla
(2001) and Borah et al. (2014). The disturbance index (DI) was calculated as the basal area of cut trees
measured at the ground level expressed as fraction of total basal area of all trees:
Basal area of cut stumps
DI % = 100
Total basal area (cut stumps basal area + Standing tree basal area)
RESULTS
Species richness of pure Sal forests is generally very poor. In the present study only 3 species belonging to
three families was recorded in undisturbed forests whereas 18 species representing 14 families in disturbed Sal
forests (Table 1). Disturbances might be responsible for arrival and establishment of new species in disturbed
Table 1. Cumulative results of the undisturbed and disturbed Sal forest of Western Assam, Northeast India.
Parameter Undisturbed Disturbed
Species number 3 18
Family number 3 14
Density (tree ha-1) 410 306
Basal area (m2 ha-1) 26.40 12.90
Shannon index 0.73 2.05
Simpson index 0.60 0.25
Disturbance index (DI %) 7 51
Sal forests besides its high resilience capacity. The density and basal area of the tree species were significantly
lower in the disturbed forests than the undisturbed forests. The encountered density as well as basal area was
410 tree ha-1 and 26.40 m2 ha-1 in undisturbed and 306 tree ha-1 and 12.90 m2 ha-1 in disturbed forests
respectively (Table 1). Disturbance index indicated the degree of disturbance and found high (51%) in disturbed
forests and less (7%) in undisturbed forests (Table 1). Shorea robusta was found dominant in both undisturbed
and disturbed forests based on IVI score (Table 2). IVI score of each species in both forest types are shown in
Table 2.
In each forest type distribution of density and basal area in different GBH classes was shown in figure 2. In
undisturbed forests maximum density (30%) was recorded in 7090 cm GBH class and overall 78% density in
middle girth classes (50 cm to 130 cm GBH class) evidenced the post mass regeneration of that particular forest.
In disturbed forests maximum density (45%) was recorded in lowermost i.e. 1030 cm girth class and it
drastically decrease in successive girth classes hints its past disturbance history and the resilience capacity.
Density of other species was found high in disturbed Sal forests especially in lower girth class (Table 3).
Diversity index was comparatively more in disturbed forests than undisturbed forests (Table 1).
Figure 2. Girth class distribution of tree species in Sal forest: A, undisturbed; B, disturbed.
Rabha (2014) 1(3): 1621
.
Table 2. IVI score of each species in undisturbed and disturbed Sal forests of Western Assam, Northeast India.
Undisturbed Disturbed
S.No. Species name
R.De R.Fr. R.Do. IVI R.De R.Fr. R.Do. IVI
1 Aegle marmelos (L.) Corr. 1.31 4.44 0.47 6.22 - - - -
2 Alstonia scholaris (L.) R.Br. 1.31 4.44 0.09 5.84 - - - -
3 Artocarpus chama Buch.-Ham. 0.65 2.22 0.14 3.02 - - - -
4 Callicarpa arborea Roxb. 2.61 6.67 7.75 17.03 - - - -
5 Dillenia indica L. 0.65 2.22 0.21 3.08 - - - -
6 Dillenia pentagyna L. 3.92 8.89 2.17 14.98 - - - -
7 Ficus religiosa L. 0.65 2.22 3.73 6.61 - - - -
8 Holarrhena pubescens (Buch.-
0.65 2.22 0.02 2.90 - - - -
Ham.) Wall. ex G.Don
9 Litsea monopetala (Roxb.) Pers. 3.92 8.89 1.26 14.07 - - - -
10 Mallotus ferrugineus (Roxb.)
3.27 8.89 1.09 13.24 - - - -
Muell. Arg
11 Mitragyna rotundifolia (Roxb)
1.96 4.44 0.31 6.72 - - - -
O. Kuntze
12 Shorea robusta Gaertn. 62.09 11.11 71.25 144.45 94.14 41.66 90.22 226.04
13 Schima wallichii (DC) Kuntze 7.19 11.11 8.22 26.52 3.41 33.33 5.53 42.27
14 Spondius pinnata 1.96 4.44 0.31 6.72 - - - -
15 Streblus asper Lour. 1.31 2.22 0.03 3.56 - - - -
16 Sterculia villosa Roxb. 2.61 4.44 0.62 7.68 - - - -
17 Terminalia bellirica (Gatertn.)
2.61 6.67 2.17 11.45 2.43 25.00 4.24 31.68
Roxb.
18 Toona ciliata M. Roem. 1.31 4.44 0.17 5.92 - - - -
Total 100 100 100 300.00 100 100 100 300.00
*R.De.= Relative density; R.Fr.= Relative frequency; R.Do.= Relative dominance; IVI= Importance Value Index.
Table 3. Density of Sal and other species in different girth class in undisturbed and disturbed Sal forest of Western Assam,
Northeast India.
Density (tree ha-1)
GBH Class (cm) Species
Undisturbed Disturbed
Sal 40 72
10-30
Other species 8 66
Sal 8 18
30-50
Other species 2 14
Sal 322 76
50-130
Other species 12 24
Sal 16 30
>130
Other species 2 6
DISCUSSIONS AND CONCLUSION

In present study only 3 species was found in undisturbed Sal forests agreement with the study carried out by
Stainton (1972) in Pure Sal forests of Nepal. More species number (18 species) in disturbed Sal forests might be
due to the anthropogenic disturbances which favour arrival and establishment of new species. In the present
study overall species number is quite low compare to the other studies reported from different part of Northeast
India (Uma Shankar 2001, Lalfakawma et al. 2009, Deka et al. 2012, Sarma & Das 2012, Dutta & Devi 2013).
The density of undisturbed forests was found 410 tree ha-1 and it is comparable to other studies done in different
Sal forests of the country such as 294559 tree ha-1 in Central India (Jha & Singh 1990), 484 tree ha-1 in Eastern
Himalaya, Meghalaya (Uma Shankar 2001), 408 trees ha-1 in Gorakhpur, India (Padey & Shukla 2003), 438 tree
ha-1 in moist Sal forests of West Bengal, India (Kushwaha & Nandy 2012), 422 tree ha-1 in Doboka reserve
forest, Assam, NE India (Dutta & Devi 2013). Comparatively less density in disturbed forests especially >50 cm
GBH class trees (136 tree ha-1 in disturbed against 340 tree ha-1 in undisturbed forests) might be due to the
various anthropogenic disturbances (Table 2). In the present study the basal area was recorded 26.40 m2 ha-1 in
undisturbed and 12.90 m2 ha-1 in disturbed Sal forests. Similar basal area (729 m2 ha-1) was reported from Sal
forest of Central India (Jha & Singh 1990). High density of other species in disturbed forests might be due to the
canopy gaps resulted from the disturbances. Disturbances enabled increased light intensity and ultimately
change the environmental condition make favourable for other lights demanding successional species (such as
Rabha (2014) 1(3): 1621
.
Schima wallichii, Callicarpa arborea etc.) leading towards mix forest communities. Species richness of
disturbed forest is a cumulative outcome of differential responses of species to disturbances (Sagar et al. 2003).
Kushwaha & Nandy (2012) reported that climatic conditions-mainly the rainfall, disturbance regimes and the
management practices influenced the species composition and community structure of Sal forests while in the
present study differences occur mainly because of the disturbance regimes.
Sal is a very important tree species and usually harvested for its timber. The Sal forests of Goalpara district
of Assam were exposed to different intensities of fire and anthropogenic disturbances in the past. Sarma & Das
(2012) stated that Sal forests of Western Assam has been facing great biotic pressures such as illegal felling,
firewood collection, encroachment of peripheral areas leading to pure Sal forests to mix forests which was also
reflected in the present study. Weeds and creeper also greatly influenced the regeneration of Sal forests. The
major threats such as illegal tree felling, firewood collection, encroachment of peripheral areas might be due to
the inadequate conservation strategy, negligence of concerned forest departments. The ongoing disturbances, if
not control then these undisturbed pure Sal forests may degrade and convert to the mix forests in the very near
future.
ACKNOWLEDGEMENTS
Author is thankful to the forest department of Goalpara district for permission and support during the
fieldwork.
REFERENCES
Ahmed M & Medhi D (2005) Encroachment causes shrinkages of forests in Goalpara district, Assam. In: Kumar
A (ed) Environmental Biology S.B. Nangia A.P.H. Publishing Corporation, New Delhi, pp. 167171.
Borah N, Devi AF, Garkoti SC, Das AK & Hore DK (2014) Structural and compositional variations in
undisturbed and disturbed tropical forests of Bhuban hills in south Assam, India. International Journal of
Biodiversity Science, Ecosystem Services and Management 10: 919
Champion HG & Seth SK (1968) A Revised Survey of the Forest Types of India. Govt. of India publications,
New Delhi.
Chauhan PS, Negi JDS, Singh L & Manhas RK (2008) Regeneration of Sal forests of Doon Valley. Annals of
Forestry 16 (2): 178182.
Chitale VS & Behera MD (2012) Can the distribution of Sal (Shorea robusta Gaertn. f.) shift in the northeastern
direction in India due to changing climate? Current Science 102(8): 11261134.
Deka J, Tripathi OP & Khan ML (2012) High dominance of Shorea robusta Gaertn. in alluvial plain Kamrup
Sal forest of Assam, N. E. India. International Journal of Ecosystems 2 (4): 6773.
Dutta G & Devi A (2013) Plant diversity sand community structure in tropical moist deciduous sal (Shorea
robusta Gaertn.) forest of Assam, northeast India. Journal of Environmental and Applied Bioresearch 1 (3):
16.
Gautam KH & Devoe NN (2006) Ecological and anthropogenic niches of Sal (Shorea robusta Gaertn. f.) forest
and prospects for multiple-product forest management- a review. Forestry 79: 81101.
Hydromet Division (2013) Hydromet division, India meteorological department. Available from:
http://www.imd.gov.in/section/hydro/distrainfall/webrain/assam/goalpara.txt. (accessed: 19 Sep. 2014).
Jha CS & Singh JS (1990) Composition and dynamics of dry tropical forest in relation to soil texture. Journal of
Vegetation Science 1: 609614.
Kanzaki M & Kyoji Y (1986) Regeneration in subalpine coniferous forests: mortality and pattern of death of
canopy trees. The Botanical Magazine Tokyo 99: 3752.
Kushwaha SPS & Nandy S (2012) Species diversity and community structure in Sal (Shorea robusta) forests of
two different rainfall regimes in West Bangal, India. Biodiversity and Conservation 21: 12151228.
Lalfakawma, Roy S, Vanlalhriatpuia K & Vanalalhluna PC (2009) Community composition and tree population
structure in undisturbed and disturbed tropical semi-evergreen forest stands of north-east India. Applied
Ecology and Environmental Research 7: 303318.
Lawes MJ, Joubert R, Griffiths ME, Boudreau S & Chapman CA (2007) The effect of the spatial scale of
recruitment on tree diversity in Afromontane forest fragments. Biological Conservation 139: 447456.
Misra R (1968) Ecology Workbook. Oxford & IBH Publication co., New Delhi.
Padey SK & Shukla RP (2001) Regeneration strategy and plant diversity status in degraded Sal forests. Current
Science 81: 95102.
Rabha (2014) 1(3): 1621
.
Padey SK & Shukla RP (2003) Plant diversity in managed Sal (Shorea robusta Gaertn. f.) forest of Gorakhpur,
India: species composition, regeneration and conservation. Biodiversity Conservation 12: 22952319.
Qureshi IM, Shrivastava PBL & Bora NKS (1968) Sal (Shorea robusta) natural regeneration De-Novo effect of
soil working and weeding on the growth and establishment. Indian Forester 94: 591598.
Rautiainen O & Suoheimo J (1997) Natural regeneration potential and early development of Shorea robusta
Gaertn. f. forest after regeneration felling in the Bhabar-Terai Zone in Nepal. Forest Ecology and
Management 92: 243251.
Sagar R, Raghubanshi AS & Singh JS (2003) Tree species composition, dispersion and diversity along a
disturbance gradient in a dry tropical forest region of India. Forest Ecology and Management 186: 6171.
Sarkar M & Devi A (2014) Assessment of diversity, population structure and regeneration status of tree species
in Hollongapar Gibbon Wildlife Sanctuary, Assam, Northeast India. Tropical Plant Research 1(2): 2636.
Sarma SK & Das RK (2012) Community Structure of Sal (Shorea robusta, Gaertn.f) Forests of Western Assam,
India. The Botanica 59-61: 6777.
Shannon CE & Wiener W (1963) The Mathematical Theory of Communication. University of Illinois Press,
Urbona, USA.
Simpson EH (1949) Measurement of diversity. Nature 163: 688.
Soni RC (1961) Recent trends in Sal natural regeneration techniques with particular reference to B3 Sal. Proc.
Silva. Conf., Dehra Dun.
Stainton JDA (1972) Forests of Nepal. John Murray, London.
Uma Shankar (2001) A case of high tree diversity in a Sal (Shorea robusta) dominated lowland forest of Eastern
Himalaya: Floristic composition, regeneration and conservation. Current Science 81: 776786.
Upreti SDK & Nayaka S (2005) Shorea robusta-an excellent host tree for lichen growth in India. Current
Science 89(4): 594595.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
1(3): 2226, 2014
Research article
Comparative evaluation of nutritional, biochemical and

enzymatic properties of the mycelium of two Pleurotus species
Ashutosh Rajoriya1, Anuradha Panda2 and Nibha Gupta1*
1
Plant pathology and Microbiology division, Regional Plant Resource Centre, Bhubaneswar-751015, Odisha
2
MITS School of Biotechnology, Bhubaneswar, Odisha
*Corresponding Author: nguc2003@yahoo.com [Accepted: 20 October 2014]
Abstract: Aim of the present studies focuses on nutritional, antioxidant and extracellular
enzymatic activity of mycelium of Pleurotus sajor-caju and Pleurotus florida. Results shows that
both of the mushroom mycelium possess multiple nutritional, antioxidant components along with
good extracellular enzymatic activities. Methanolic extract of Pleurotus sajor-caju showed higher
phenolic and flavonoid content (1.010.57 mg.gm-1 and 0.140.01 mg.gm-1) than Pleurotus florida
(0.450.05 and 0.110.01 mg.gm-1). A high alkaloid content was exhibited in P. sajor-caju than P.
florida, apart from the antioxidant components. P. sajor-caju showed high protein and
carbohydrate content i.e. 10.551.62 mg.gm-1 and 32.167.16 gm 100gm-1 respectively, as
compared to P. florida which showed less amount of protein and carbohydrate content (8.50015
mg.gm-1 and 8.301.09 gm 100gm-1). Enzymatic screening showed good activity of amylase and
lipase where as Xylanase and protease activity in both the mushroom mycelium was negative.
Overall studies revealed that both the mushroom mycelium are potential source of antioxidants and
extracellular enzymes, especially flavonoids, amylase and lipase.
Keywords: Pleurotus - Mycelium - Nutritional - Antioxidants - Enzymatic.
[Cite as: Rajoriya A, Panda A & Gupta N (2014) Comparative evaluation of nutritional, biochemical and
enzymatic properties of the mycelium of two Pleurotus species. Tropical Plant Research 1(3): 2226]
INTRODUCTION
Wild edible mushroom have been integrated part of the diet, especially among rural, urban dwellers and
tribal people. Some of the common edible mushrooms, which are predominantly consumed in India are
Pleurotus sajor-caju, P. florida, P. platypus, P. djamor, Volvariella volvacea and Calocybe indica (Pan et al.
2008, Ramkumar et al. 2010). Pleurotus species is known as oyster mushrooms, which are widely spread
saprophytic macrofungi and distributed throughout the temperate and tropical forests of the world (Gunde-
Cimerman 1999). Oyster mushrooms are now in second rank among the cultivated mushrooms in the world
(Chang 1991) and are known to have potent antitumor, antimicrobial activities (Zhang et al. 1994, Gerasimenya
et al. 2002). Pleurotus sp are rich in minerals (Ca, P, Fe, K and Na) and vitamin C, B-complex (alarrmak
2007). Apart from the different nutritional and antioxidant components mushroom mycelium possess different
enzymes (Nonaka et al. 1997, Bose et al. 2007, Kadimaliev et al. 1998). Pleurotus is known for the different
cellulolytic and amylolytic enzymes (Sawiska & Kalbarczyk 2011, Jonathan & Adeoyo 2011). Recently
Pleurotus ostreatus and P. sajor-caju is characterized for the protease activity (Choi & Shin 1998, Ravikumar et
al. 2012). In Odisha mainly oyster mushroom Pleurotus sajor-caju and Pleurotus florida are grown
commercially. Both of them are liked by local people on account of unique characteristic of aroma and taste. In
the present work, this was taken into the consideration because reports suggests that fruit body of both of the
species possess good nutritional and antioxidant components along with industrially important enzymes and
since mycelium is the miniature of fruit body, therefore same behaviour was expected from the respective
mushrooms, hence it intended to evaluate the mushrooms nutraceuticals (Nutritional and Pharmaceutical)
potential.
Rajoriya et al. (2014) 1(3): 2226
.
Nutritional analysis
Protein estimation was done by the method given by Bradford (1976). Estimation of carbohydrates was
carried out by following phenol sulphuric acid method (Dubois et al. 1956, Hedge & Hofreiter 1962). Reducing
sugars in the mycelium was done by following dinitrosalicylic acid method (Miller 1972). Non reducing sugar
was calculated by following the formula of Nazarudeen (2010).
Antioxidant analysis
One gm of fresh mycelium sample was disintegrated with 10 ml of methanol. Samples were stirred for 15
minutes for effective extraction and centrifuged at 3000 rpm for 20 minutes. Supernatants were referred as
methanolic extract and kept at 4 C until analysis (Puttaraju et al. 2006). The DPPH activity was estimated in the
methanolic extracts by colorimetric method (Chan et al. 2007). Ascorbic acid equivalent Antioxidant Capacity
(AEAC) was calculated by calibrating the value of above absorbance in standard ascorbic acid curve and
expressed in mg per gram of dried sample. Ferric Reducing Antioxidant Power (FRAP) assay was done by
following the method of Benzie & Strain (1996) and Athavale et al. (2012) and. The total phenolic content in
the mycelium were determined through Folin-phenol method with slight modifications (Singleton & Rossi
1965). The flavonoid content of sample was estimated by using aluminium chloride colorimetric technique and
flavonoid content was expressed in terms of mg quercetin equivalents per gram of extract (Chang et al. 2002).
The concentration of -carotene and lycopene in mushroom mycelium extracts was estimated
spectrophotometrically (Nagata & Yamashita 1992, Barros et al. 2007). Alkaloid content in the mushroom
mycelia was quantified spectrophotometrically (Srividya & Mehrotra 2003). Tannin content was estimated in
the sample by Folin denis reagent tannic acid was served as standard and expressed in mg.gm-1 (Schanderl
1970).
Extracellular enzymatic activity
i) Amylase activity: All the mushroom mycelium was screened for the extracellular amylase activity for
which starch agar media was used. After the appreciable amount of the growth in plates they were
flooded with 1% iodine solution. Clear zone around the mycelial growth was recorded for the starch
hydrolysis activity.
ii) Cellulase activity: The medium containing 0.5% sodium salt of carboxymethylcellulose was used for the
tests. After the mycelial colonization plates were flooded with congo red solution (0.2%) and washed
with 1M NaCl solution followed by the incubation period of 15 minutes.
iii) Lipase activity: Spirit blue agar media was used for the screening of lipase activity. After the requisite
amount of growth of mushroom mycelium a clear zone or precipitate was observed for the positive
organism.
iv) L-Asparaginase activity: For screening of L- Asparaginase activity, medium containing 1% L- asparagine
was used where L-asparagine served as an active ingredient, after the mycelial growth plate was flooded
with Nesslers reagent. Plates showing pink coloration after the addition were recorded as extracellular
L- asparaginase producer.
v) Protease activity: Gelatin agar media was used for the screening of protease producing organism, for
which centre inoculation was done, after the incubation of 10 days plates were flooded with the reagent
containing 15% HgCl2 and 20% HCl.
vi) Xylanase activity: Medium containing xylan was used for the screening of Xylanase activity in
mushroom mycelium. After the appreciable growth in the plate it was flooded with 0.1% Congo red,
incubated for 30 minutes and washed with 1M NaCl subsequently. Plate was observed for the formation
of clear zone for the production of Xylanase enzyme.
RESULTS AND DISCUSSION

In the present studies moderate to high nutritional components and antioxidant activities with varying levels
of phenolics, proteins and alkaloids were recorded in the two species of Pleurotus (Table 1). Relatively higher
amount of the protein content (10.551.62 mg.gm-1 and 8.500.15 mg.gm-1) in both of the species was observed
as compared to the other species of Pleurotus as reported by Jean-Phillip (2005). Carbohydrate content in the
Pleurotus sajor-caju and Pleurotus florida was 32.16 and 8.30 gm 100gm-1, respectively which was less than
the cultivated variety of Pleurotus as reported by Paz et al. (2012) but much more than the reports of Boda et
al. (2012). Pleurotus along with many other types of edible mushrooms have been known as a potent source of
Rajoriya et al. (2014) 1(3): 2226
.
nutrients as well as natural antioxidants. Findings from this research showed that fungal mycelia studied have
antioxidant capacity where FRAP and DPPH free radical scavenging activities assay showed a remarkable
difference between both the species. P. sajor-caju showed higher DPPH scavenging activities than P. florida i.e.
Table 1. Nutritional components and antioxidant activities of Pleurotus spp.

S. No. Parameters P. sajor-caju P. florida
1 Protein (mg/gm) 10.551.62 8.500.15
2 Carbohydrates (gm/100gm) 32.167.16 8.301.09
3 Red. Sugars (mg/gm) 24.371.04 12.911.51
4 Non Red. Sugars(gm/100gm) 29.727.09 7.001.02
5 DPPH scavenging (%) 45.530.01 9.95 1.14
6 AEAC(mg/gm) 0.101.69 0.020.00
7 FRAP (mg AEAC/gm) 0.790.10 0.060.01
8 Phenolics (mg/gm) 1.010.57 0.45 0.05
9 Flavonoids (mg/gm) 0.140.01 0.11 0.01
10 Beta carotene (mg/gm) 0.0380.012 0.0180.005
11 Lycopene (mg/gm) 0.0160.001 0.0070.002
12 Tannins (mg/gm) 5.180.64 4.480.86
13 Alkaloids (mg/gm) 0.490.01 0.470.08
DPPH- 2, 2-Diphenyl-1-picryl hydrazyl
AEAC- Ascorbic acid Equivalent Antioxidant Capacity.
FRAP- Ferric Reducing Antioxidant Power
(45.530.01%) and (9.95 1.14%) along with their corresponding AEAC value which was 0.101.69 mg.gm-1
and 0.020.00 mg.gm-1, respectively. Phenolic and flavonoid content in both of the species confirms the study
of Vamanu (2012) and Jeena et al. (2014) in P. ostreatus. High amount of - carotene (0.0380.012 mg.gm-1)
and lycopene (0.0180.005 mg.gm-1) content was recorded in P. sajor-caju where as comparatively less amount
of the same was recorded in P. florida. Presence of -carotene and lycopene was less as compared to the
investigations of Pal et al. (2010). Alkaloids are responsible for different cytotoxic and antimicrobial properties
(Ozcelik et al. 2011) Present study revealed the high amount of alkaloid was recorded in P. sajor-caju
(0.490.01 mg gm-1) and P. florida (0.470.08 mg.gm-1). Tannins are responsible for different biological
activities such as antioxidant, antimicrobial and antitumor activities (Yoshizawa et al. 1987, Yoshida et al.
1989, Yoshida et al. 2009). Tannin content in P. florida and P. sajor-caju ranged from 4.485.18 mg.gm-1.
Comparatively P. sajorcaju, exhibited better scavenging of free radicals including high levels of protein,
carbohydrate, reducing sugars, phenol along with both FRAP and DPPH scavenging activities than P. florida. In
the present studies of the enzymatic activity of these mushroom mycelium, P. florida showed higher amylase
and lipase activity than P. sajor-caju, both the species were negative for the Xylanase and protease activity
(Table 2).
Table 2. Extracellular enzymatic activity of Pleurotus sajor-caju and P. florida.

Sl. No. Species Amylase Protease Xylanase L-Asparaginase Cellulase Lipase
1 P. sajor-caju + - - - + ++
2 P. florida +++ - - + - +++
Note: (++++): very good activity; (+++): good activity; (++): moderate activity; (+): poor activity; (-): No activity.
CONCLUSION
Wide range of applications from the mushroom fruit bodies and mycelium has attracted much attention in
regard of nutritional components and secondary metabolites. Present analysis showed varying range of bioactive
compounds along with versatile production of extracellular enzymes. The preliminary study on quantification of
antioxidants and screening of the enzymes can be the future prospective for the exploration of these mushroom
mycelium for development of bioactive compounds.
ACKNOWLEDGEMENTS
The financial assistance obtained from Ministry of Environment and Forests, Govt. of India (Project no. 22-
24/2010 CS.I) is gratefully acknowledged by the authors (AR and NG).
Rajoriya et al. (2014) 1(3): 2226
.
REFERENCES
Arnon DI (1949) Copper enzymes in isolated chloroplast, polyphenol oxidase in Beta vulagris. Plant Physiology
24: 115.
Athavale A, Jirankalgikar N, Nariya P & Des S (2012) Evaluation of in-vitro antioxidant activity of
panchagavya: a traditional ayurvedic preparation. International journal of pharmaceutical sciences and
research 3(8): 25432549.
Barros L, Ferreira MJ, Queiros B, Ferreira ICFR & Baptista P (2007) Total phenols, ascorbic acid, B-carotene
and lycopene in portuguese wild edible mushrooms and their antioxidant activities. Food chemistry 103:
413419.
Benzie IFF & Strain JJ (1996) The Ferric Reducing Ability of Plasma (FRAP) as a measure of Antioxidant
Power: The FRAP Assay. Analytical Biochemistry 239: 7076.
Boda RH, Wani AH, Zargar MA, Ganie BA, Wani BA & Ganie SA (2012) Nutritional values and antioxidant
potential of some edible mushrooms of Kashmir valley. Pakistan Journal of Pharmaceutical Sciences 25(3):
623627.
Bose S, Mazumder S & Mukherjee M (2007) Laccase production by the white-rot fungus Termitomyces
clypeatus. Journal of Basic Microbiology 47: 127131.
Bradford MM (1976) A Rapid and sensitive method for the quantification of Microgram quantities of the
protein utilizing the principle of protein dye binding, Analytical Biochemistry 72: 248258.
alarrmak N (2007) The nutrients of exotic mushrooms (Lentinula edodes and Pleurotus sp) and an estimated
approach to the volatile compounds. Food Chemistry 105: 11881194.
Chan EWC, Lim YY & Omar M (2007) Antioxidant and antibacterial activity of leaves of Etlingera Species
(Zingiberaceae) in Peninsular Malaysia. Food Chemistry 104 (4): 15861593.
Chang ST (1991) Cultivated mushrooms. In: Arora DK, Mukerji KG & Marth EH (eds) Hand book of applied
mycology Vol. 3. Marcel Dekker Inc. New York, pp. 221240.
Choi HS & Shin HH (1998) Purification and characterization of cysteine protease from Pleurotus ostreatus.
Bioscience Biotechnology and Biochemistry 62(7): 14161418.
Dubois M, Gilles KA, Hamilton JK, Rebers PA & Smith F (1956) Colorimetric method for determination of
sugars. Analytical Chemistry 28: 350356.
Gerasimenya VP, Efremenkova OV, Kamzolkina OV, Bogush TA, Tolstych IV & Zenkova VA (2002)
Antimicrobial and antitoxical action of edible and medicinal mushroom Pleurotus ostreatus (Jacq. ex Fr.)
Kumm. extracts. International Journal of Medicinal Mushrooms 4: 106.
Gunde-Cimerman N (1999) Medicinal Values of the Genus Pleurotus. International Journal of Medicinal
Mushrooms 1: 6980.
Hedge JE & Hofreiter BT (1962) In: Whistler RL & Be Miller JN (eds) Methods in Carbohydrate Chemistry
Vol. 17. Academic Press, New York, USA, pp. 420.
Jean-Philippe Sharon Rose (2005) Antioxidant properties of some edible fungi in the genus Pleurotus, Masters
Thesis. University of Tennessee, USA.
Jeena GS, Punetha H, Prakash O, Chandra M & Kushwaha KPS (2014) Study of in vitro potential of some
cultivated Pleurotus species (Oyster mushrooms). Indian journal of natural product and resources 5(1): 56
61.
Jonathan SG & Adeoyo OR (2011) Evaluation of ten wild nigerian mushrooms for amylase and cellulase
activities. Mycobiology 39(2): 103108.
Kadimaliev DA, Nadezhina OS, Atykian NA, Revin VV, Parshin AA, Lavrova AI & Dukhovskis PV (2008)
Increased secretion of lignolytic enzymes by the Lentinus tigrinus fungus after addition of butanol and
toluene in submerged cultivation. Prikladnaia Biokhimiia Mikrobiologiia 44 (5): 582588.
Miller GI (1972) Use of dinitrosalicylic acid reagent for determination of reducing sugars. Analytical Chemistry
31: 426.
Nagata M & Yamashita I (1992) Simple method for simultaneous determination of chlorophyll and carotenoids
Tomato fruit. Nippon Shokuhin Kogyo Gakkaish 39(10): 925928.
Nazarudeen A (2010) Nutritional composition of some lesser known fruits use by ethinic communities and local
folks of Kerala. Indian Journal of traditional Knowledge 9(2): 398402.
Nonaka T, Dohmae N, Hashimoto Y & Takio K (1997) Amino acid sequences of metalloendopeptidases
specific for acyl-lysine bonds from Grifola frondosa and Pleurotus ostreatus fruiting bodies. Journal of
Biological chemistry 272: 3003230039.
Rajoriya et al. (2014) 1(3): 2226
.
zelik B, Kartal M & Orhan I (2011) Cytotoxicity, antiviral and antimicrobial activities of alkaloids,
flavonoids, and phenolic acids. Pharmaceutical Biology 49(4): 396402.
Pan Y, Wang K, Huang S, Wang H, Mu X, He C, Ji X, Zhang J & Huang F (2008) Antioxidant activity of
microwave-assisted extract of longan (Dimocarpus longan Lour.) peel. Food Chemistry 106: 12641270.
Pal T, Ganguly S, Tahsin KS & Acharya K (2010) In vitro free radical scavenging activity of wild edible
mushroom, Pleurotus squarrosulus (Mont.) Singer. Indian journal of experimental biology 47: 12101218.
Paz MF, Breyer CA, Longhi RF & Oviedo MSVP. (2012) Determining the basic composition and total phenolic
compounds of Pleurotus sajor-caju cultivated in three different substrates by solid state bioprocess. Journal
of Biotechnology and Biodiversity 3(2): 1114.
Prabu M & Kumuthakalavalli R (2014) Nutritional and phytochemical studies on Pleurotus florida (mont.)
singer and Calocybe indica P&C. World Journal of Pharmaceutical Research 3(3): 49074913.
Puttaraju NG, Venkateshaiah SU, Dharmesh SM, Urs SM & Somasundaram R (2006) Antioxidant activity of
indigenous edible mushrooms. Journal of Agricultural and Food Chemistry 54: 97649772.
Ramkumar L, Ramanathan T, Thirunavukkarasu P & Arivusevam N (2010) Antioxidant and radical scavenging
activity of nine edible mushrooms extract. International Journal of Pharmacology 6(6): 950953.
Ravikumar G, Gomathi D, Kalaiselvi M & Uma C (2012) A protease from the medicinal mushroom Pleurotus
sajor-caju; production, purification and partial characterization. Asian Pacific Journal of Tropical
Biomedicine 2(1): S411S417.
Schanderl SH (1970) In: Method in food analysis. Academic press, New York, London, pp. 709.
Sawiska A & Kalbarczyk J (2011) Evaluation of enzymatic activity of Pleurotus ostreatus regarding stages of
mycelium development. Acta Scientiarum Polonorum: Hortorum Cultus 10 (2): 195202.
Srividya N & Mehrotra S (2003) Spectrophotometric Method for the estimation of Alkaloids Precipitable with
Dragendroffs reagent in plant materials. Journal of AOAC international 86(6): 11241127.
Vamanu E (2012) In vitro Antimicrobial and Antioxidant Activities of Ethanolic Extract of Lyophilized
Mycelium of Pleurotus ostreatus PQMZ91109. Molecules 17: 36533671.
Yoshida T, Hatano T, Ito H & Okuda T (2009) Structure diversity and antimicrobial activities of ellagitannins.
In: Quideau S (ed) Chemistry and Biology of Ellagitannins. World Scientific, Singapore pp. 55-93.
Yoshida T, Mori K, Hatano T, Okumura T, Uehara I, Komagoe K, Fujita Y & Okuda T (1989) Radical-
scavenging effects of tannins and related polyphenols on 1, 1-diphenyl-2-picrylhydrazyl radical. Chemical
and Pharmaceutical Bulletin 37: 19191921.
Yoshizawa S, Horiuchi T, Fujiki H, Yoshida T, Okuda T & Sugimura T (1987) Antitumor promoting activity of
()-Epigallocatechin gallate, the main constituent of Tannin in green tea. Phytotherapy Research 1: 44-47.
Zhang J, Wang G, Li H, Zhuang C, Mizuno T, Ito H, Suzuki C & Okamoto H (1994) Antitumorpolysaccharides
from a Chinese mushroom, yuhuangmo,the fruiting body of Pleurotus citrinopileatus. Bioscience
Biotechnology and Biochemistry 7: 11951201.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
1(3): 2735, 2014
Research article
Study on relationship and selection index in chickpea

M. A. Samad1*, Nibadita Sarker2 and A. C. Deb2
1
Department of Botany, University of Rajshahi, Rajshahi-6205, Bangladesh
2
Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh
*Corresponding Author: samad_ru_bt_bd@yahoo.com [Accepted: 06 November 2014]
Abstract: Correlation, path coefficients and selection index were studied in eight irradiated
chickpea lines for eleven quantitative characters to design the selection strategy towards higher
yield. Genotypic correlation coefficients were higher than the phenotypic correlation coefficients.
Seed weight per plant (SW/P) was positively correlated with days to maximum flower (DMF),
number of primary branches at maximum flower (NPBMF), number of secondary branches at
maximum flower (NSBMF), plant weight after fully dry (PWFD), pod weight per plant (PdW/P)
and number of seeds per plant (NS/P) both at phenotypic and genotypic levels. PdW/P and NS/P
exhibited high significant positive correlation on SW/P. NS/P had the highest positive direct effect
of 1.077 and 1.334 on SW/P at both levels, respectively whereas PdW/P showed positive direct
effect of 0.346 at genotypic levels. Regarding selection NPBMF and root weight after fully dry
showed highest genetic gain among the combinations of selection indices.
Keywords: Correlation - Irradiation - Path Coefficients - Path Diagram - Yield.
[Cite as: Samad MA, Sarker N & Deb AC (2014) Study on relationship and selection index in chickpea.
Tropical Plant Research 1(3): 2735]
INTRODUCTION
Yield is the ultimate goal of a breeding program. Seed yield being most important trait, is governed by many
physiological changes within the plant and influenced by many environmental factors. So, the breeder needs
some index traits to select elite genotype for higher yield. Information on correlation, path-coefficients and
selection index analyses is of much use to plant breeders for selection and breeding genotypes with increased
yield potential. Correlation coefficients in general show associations among independent characteristics and the
degree of linear relation between these characteristics. It is not sufficient to describe this relationship when the
causal association among characteristics is needed (Toker & Cagirgan 2004). Correlation does not provide the
adequate picture of the relationship among traits. In such cases, it is inevitable to study a method which takes
into account the causal relationship between the variables in addition to the degree of such relationship. Path-
coefficient analysis measures the direct influence of one variable upon the other and permits separation of
correlation coefficients into components of direct and indirect effects. The plus point of this analysis is that it
allows the partitioning of correlation coefficient into its components (Dewey & Lu 1959). In addition, selection
index disclose about the primary yield components. The present study was undertaken to find out the association
of different traits and their contribution to define seed yield.

BARI (Bangladesh Agriculture Research Institute) Chola (Cicer arietinum L.) 1, 2, 3, 4, 5, 6, 7 and 8 were
taken from Regional Agriculture Research Station, Ishourdi, Pabna, Bangladesh as materials. All the above
varieties originated from ICRISAT line except BARI Chola 5 which is from local cultivar of Pabna,
Bangladesh. Selected varieties of chickpea were irradiated with irradiation source of Co60 at the Institute of
Food and Radiation Biology, Atomic Energy Research Establishment, Savar, Dhaka, Bangladesh. Layout of the
experimental field and trial of the irradiated varieties as lines was conducted in the research field of the
department of Botany, University of Rajshahi under randomized complete block design with 4 replications in
two consecutive years namely 20072008 (Y1) and 20082009 (Y2). Each replication having 4 blocks and each
block having 8 plots. Each plot contains 3 rows and per row there are 5 hills. In each hill, one plant was
maintained for data. Gap between blocks and that between plots were 5 cm. The same between rows and that
Received: 24 August 2014 Published online: 31 December 2014
Samad et al. (2014) 1(3): 2735
.
between plants were 70 and 25 cm, respectively. The data of eleven quantitative traits viz., days to maximum
flower (DMF), number of primary branches at maximum flower (NPBMF), number of secondary branches at
maximum flower (NSBMF), plant height at maximum flower (PHMF), plant weight after fully dry (PWFD),
root weight after fully dry (RWFD, number of pods per plant (NPd/P), pod weight per plant (PdW/P), number of
seeds per plant (NS/P), seed weight per plant (SW/P) and 1000-seed weight were collected on individual plant
basis following C.G.S system. The data were analyzed by Excel software.
Statistical Analysis
i. Correlation coefficient: The correlation coefficient at phenotypic (rp) and genotypic (rg) levels were
calculated as follows:
rp = (2p12)/ (2P11 2P22)1/2
rg = (2g12)/ (2g11 2g22)1/2
Where, 2P12 and 2g12 express phenotypic and genotypic co-variance of character 1 and 2
whereas, P11 and 2g11 represent phenotypic and genotypic variance of character 1 and 2p22 and 2g22 indicate
2
variance at phenotypic and genotypic levels of character 2.

ii. Path-coefficient: The path-coefficient analysis was carried out using the formula of Wright (1921 &
1923) as illustrated by Dewey & Lu (1959). The path-coefficient analysis was done at both phenotypic and
genotypic levels by solving the simultaneous equations using matrix method. The form of equation is as
follows:
rxy = pxy + rx2 P2y ++ rx3pxy + ..................................rxnPny
Where, rxy = correlation between one components character and yield. Pxy = Path-coefficient
between the same character and yield. rx2, rx3,....rxn= Represent correlation coefficient between that character and
each of the other yield components in turn.
iii. Selection index: The expected genetic advance from straight selection {GA(S)} and from discriminant
function {GA(D)} was calculated as follows:
GA (S) = (Z/P) (gyy)/(tyy)1/2
GA (D) = (Z/P) (b1g1y + b2g2y)1/2
Where, Z/P = the selection differential in standard units, for the present study it was 2.06 at
5% level of selection. gyy and tyy = the genotypic and phenotypic variances of character. b1, b2, . . . . .bn = the
relative weights for character. g1y, g2y . . . . . = the genotypic co-variances of independent character with y. The
expected gain from the discriminant function over straight selection was calculated for all the functions as
shown below:
Expected gain (%) = [{GA (D)/GA(S)}-1] 100
RESULTS
Correlation coefficients
In most of the cases the genotypic correlation coefficients were higher than the phenotypic correlation
coefficients (Table 1). The results indicated that SW/P was positively correlated with DMF, NPBMF, NSBMF,
PWFD, PdW/P and NS/P both at phenotypic and genotypic levels. On the other hand, RWFD, NPd/P and 1000-
SW were negatively correlated with SW/P at phenotypic and genotypic level, while PHMF was correlated with
SW/P at phenotypic and genotypic levels with positive and negative values, respectively. The genotypic
correlation coefficient of SW/P with PdW/P and NS/P were high and greater than the phenotypic correlation
coefficients of those characters and exhibited significant values. The genotypic correlation coefficients of
NPBMF and NSBMF with SW/P were significant at 5% and 1% levels, whereas DMF with SW/P showed
significant value at 5% level. The genotypic correlation coefficients of PHMF and RWFD with SW/P expressed
significance with negative values at 5% level and 1000-SW had negative significant values at 5% and 1% levels.
In this analysis, DMF was positively correlated with NSBMF, PHMF, NPd/P, PdW/P and NS/P both at
phenotypic and genotypic levels. RWFD and 1000-SW correlated with DMF positively at both levels and
NPBMF correlated negatively and positively with DMF at phonotypic and genotypic levels, respectively.The
character, PWFD correlated with DMF positively and negatively at phonotypic and genotypic levels,
respectively. In all the cases genotypic correlation coefficient was higher than the phenotypic correlation
Samad et al. (2014) 1(3): 2735
.
coefficient excluding PWFD, RWFD and 1000-SW. The genotypic correlation coefficients of NPd/P, PDW/P
and NS/P with DMF were significant at 5% level, whereas NPBMF with DMF showed significant values at both
levels. RWFD had negative significant genotypic correlation coefficient value at 5% level.
Table 1. Phenotypic (rp) (upper diagonal) and Genotypic (rg) (lower diagonal) correlation coefficients between yield and yield contributing
traits in chickpea.
Traits DMF NPBMF NSBMF PHMF PWFD RWFD NPd/P PdW/P NS/P 1000-SW SW/P
DMF -0.036 0.149 0.139 0.069 -0.008 0.076 0.048 0.079 -0.055 0.059
NPBMF 0.844** 0.279 0.135 0.064 -0.175 0.294 0.244 0.299 -0.294 0.251
NSBMF 0.191 0.741* 0.228 0.088 -0.065 0.126 0.124 0.132 -0.009 0.121
PHMF 0.246 0.166 -1.314** 0.044 0.027 0.007 -0.008 0.033 -0.033 0.009
PWFD -0.062 0.647 -0.158 0.405 0.100 0.169 0.165 0.159 -0.098 0.141
RWFD -0.989** -0.952** -0.891** 0.220 -0.278 -0.187 -0.082 -0.185 0.222 -0.111
NPd/P 0.812* 1.200** 0.737* -0.448 0.497 -0.855** 0.910** -0.006 0.007 -0.003
PdW/P 0.789* 1.098** 0.585 -0.607 0.756* -0.502 0.964** 0.882** -0.022 0.936**
NS/P 0.766* 1.169** 0.769* -0.295 0.411 -0.939** -0.031 0.943** -0.252 0.939**
1000-SW -0.534 -1.156** -0.832** -0.512 -0.584 0.972** 0.0318 -1.213** -1.218** -0.011
SW/P 0.816* 1.139** 0.925** -0.768* 0.423 -0.754* -0.025 1.005** 0.969** -1.260**
Note: DMF- Days to maximum flower; NPBMF- Number of primary branches at maximum flower; NSBMF- Number of
secondary branches at maximum flower; PHMF- Plant height at maximum flower; PWFD- Plant weight after fully dry; RWFD-
Root weight after fully dry; NPd/P- Number of pods per plant; PdW/P- Pod weight per plant; NS/P- Number of seeds per plant;
1000-SW-Thousand seed weight ; SW/P- Seed weight per plant.
*& ** indicated significant at 5% and 1% levels respectively.
NPBMF showed positively correlation with NSBMF, PHMF, PWFD, NPd/P, PdW/P, NS/P at phonotypic
and genotypic levels. RWFD and 1000-SW expressed negative correlation with NPBMF. The genotypic
correlation coefficient was higher than the phenotypic correlation coefficient in all the cases except RWFD and
1000-SW. The genotypic correlation coefficients of NPd/P, PdW/P and NS/P with NPBMF were significant at
5% and 1% levels. NSBMF exhibited significant rg value at 5% level. It is observed that RWFD and 1000-SW
correlated with NPBMF significantly but in negative direction.NSBMF exhibited that this character correlated
positively both at phenotypic and genotypic levels with NPd/P, PdW/P and NS/P. But this character correlated
negatively with RWFD and 1000-SW at phenotypic and genotypic levels, respectively. In addition, PHMF and
PWFD correlated positively with NSBMF at phenotypic level but negatively at genotypic level. It is noticed that
NSBMF had significant rg values at 5% level with NPd/P and NS/P in positive direction and PHMF, RWFD and
1000-SW showed negative significant rg values. The character, PHMF correlated positively both at phenotypic
and genotypic levels with PWFD and RWFD but negatively with PdW/P and 1000-SW. On the other hand, it
showed positive and negative correlation at phenotypic and genotypic levels with NPd/P and NS/P, respectively.
PWFD correlated positively with NPd/P, PdW/P and NS/P both at phenotypic and genotypic levels and also
indicated that the value of the genotypic correlation coefficient was higher than that of the phenotypic
correlation coefficient. But PWFD positively and negatively correlated at phenotypic and genotypic levels with
RWFD but 1000-SW had negative correlation at both levels. In addition, PdW/P showed significant rg value at
5% level. RWFD had positive correlation both at phenotypic and genotypic levels with 1000-SW but negatively
with NPd/P, PdW/P and NS/P. NPd/P and NS/P had significant rg values in negative direction while, 1000-SW
in positive direction.
NPd/P exhibited positive correlation both at phenotypic and genotypic levels with PdW/P and1000-SW and
NS/P had negative correlation. Besides, PdW/P revealed significant correlation with NPd/P.The character,
PdW/P was positively correlated both at phenotypic and genotypic levels with NS/P and negatively with 1000-
SW. The value of rg and rpof NS/P with PdW/P had significant positive values while, 1000-SW with PdW/P had
negative rg value.NS/P correlated negatively with 1000-SW and the value of phenotypic correlation coefficient
was lower than genotypic correlation coefficient. The genotypic correlation coefficient of NS/P with 1000-SW
was significant at 5% and 1% levels but in negative direction.
Path-coefficient analysis at phenotypic level (Fig. 1)
It was observed from the table 2 that NS/P had the highest positive direct effect (1.077) on seed weight per
plant (SW/P). DMF, NSBMF, PHMF, PWFD and PdW/P showed negative and NPBMF, RWFD, NPd/P and
1000-SW expressed least positive direct effect on seed weight per plant (SW/P). DMF had negative direct effect
of -0.008 and indirect positive effect with NPd/P (0.006) and NS/P (0.085) on SW/P. The positive direct effect
Samad et al. (2014) 1(3): 2735
.
Figure 1. Path diagram of different yield contributing traits at phenotypic level.
of NPBMF on SW/P was 0.012. The character, NPBMF exhibited high indirect positive effect of 0.322 through
NS/P. NSBMF had negative direct effect of -0.014 on SW/P. The high indirect positive effect of this character
via NS/P was 0.142. The character, PHMF showed negative direct effect with a value of -0.018 on seed weight
per plant. But it had indirect positive influence through NPBMF, RWFD, NPd/P, PdW/P and NS/P.
Table 2. Path-coefficient analysis showing direct (in bold) and indirect effects of yield components on yield of chickpea at
phenotypic and genotypic levels.
Traits DMF NPBMF NSBMF PHMF PWFD RWFD NPd/P PdW/P NS/P 1000-SW
DMF P -0.008 -0.0004 -0.002 -0.002 -0.0006 -0.0003 0.006 -0.004 0.085 -0.014
G -0.099 -0.011 -0.029 -0.043 0.008 -0.145 -0.008 0.273 1.023 -0.152
NPBMF P 0.0003 0.012 -0.004 -0.002 -0.0005 -0.008 0.023 -0.018 0.322 -0.074
G -0.084 -0.013 -0.112 -0.029 -0.082 -0.140 -0.012 0.380 1.560 -0.330
NSBMF P -0.001 0.003 -0.014 -0.004 -0.0007 -0.003 0.010 -0.009 0.142 -0.002
G -0.019 -0.009 -0.151 0.231 0.020 -0.131 -0.007 0.203 1.026 -0.237
PHMF P -0.001 0.002 -0.003 -0.018 -0.0004 0.001 0.0005 0.0006 0.035 -0.008
G -0.024 -0.002 0.198 -0.176 -0.052 0.032 0.004 -0.210 -0.393 -0.146
PWFD P -0.0006 0.0008 -0.001 -0.0008 -0.008 0.004 0.013 -0.012 0.171 -0.024
G 0.006 -0.008 0.024 -0.071 -0.127 -0.041 -0.005 0.262 0.550 -0.166
RWFD P 0.00006 -0.002 0.0009 -0.0005 -0.0008 0.043 -0.014 0.006 -0.200 0.056
G 0.098 0.012 0.134 -0.039 0.035 0.147 0.008 -0.174 -1.253 0.277
NPd/P P -0.0006 0.004 -0.001 -0.0001 -0.001 -0.008 0.077 -0.068 -0.006 0.002
G -0.081 -0.015 -0.111 0.079 -0.063 -0.126 -0.010 0.334 -0.041 0.009
PdW/P P -0.0004 0.003 -0.002 0.0001 -0.001 -0.004 0.070 -0.074 0.950 -0.006
G -0.078 -0.014 -0.088 0.107 -0.096 -0.074 -0.009 0.346 1.258 -0.346
NS/P P -0.0006 0.004 -0.002 -0.0006 -0.001 -0.008 -0.0004 -0.066 1.077 -0.063
G -0.076 -0.015 -0.116 0.052 -0.052 -0.138 0.0003 0.327 1.334 -0.347
1000-SW P 0.0005 -0.004 0.0001 0.0006 0.0008 0.010 0.0005 0.002 -0.271 0.250
G 0.053 0.015 0.126 0.090 0.074 0.143 -0.0003 -0.420 -1.625 0.285
Note: DMF- Days to maximum flower; NPBMF- Number of primary branches at maximum flower; NSBMF- Number
of secondary branches at maximum flower; PHMF- Plant height at maximum flower; PWFD- Plant weight after fully
dry; RWFD- Root weight after fully dry; NPd/P- Number of pods per plant; PdW/P- Pod weight per plant; NS/P-
Number of seeds per plant; 1000-SW- Thousand seed weight; SW/P- Seed weight per plant.
PWFD had direct negative effect (-0.008) on SW/P and showed high indirect positive effect (0.171) on
SW/P via NS/P. RWFD had positive direct effect on SW/P and the value was 0.043. RWFD exhibited high
Samad et al. (2014) 1(3): 2735
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indirect positive effect (0.056) on SW/P via 1000-SW. Number of pods per plant (NPd/P) had positive direct
effect (0.077) on SW/P and exhibited high positive indirect effect (0.004) on SW/P through NPBMF. The
character, PdW/P showed the negative direct influence with a value of -0.074 on SW/P and expressed highest
positive indirect effect (0.950) through NS/P. The total effect of NPd/P on SW/P was 0.936 (the second highest
value).NS/P indicated positive direct effect (1.077) on SW/P. NS/P showed indirect positive influence (0.004)
on SW/P via NPBMF. The total effect of NS/P on SW/P was 0.939 (highest value). 1000-SW indicated positive
direct effect (0.250) on SW/P. It showed the highest indirect positive influence (0.010) on SW/P via RWFD.
Although it exhibited indirect positive effect through all the characters except NPBMF and NS/P on SW/P, the
values were least and negligible. The total effect of NS/P on SW/P was -0.011.
Path-coefficient analysis at genotypic level (Fig. 2)
This table 2 exhibited that the highest positive direct effect was expressed by NS/P on SW/P and it was
followed by PdW/P, 1000-SW and RWFD. The characters, DMF, NPBMF, NSBMF, PHMF, PWFD and NPd/P
showed negative direct effect.
DMF had negative direct effect (-0.099) on SW/P. But this character contributed to SW/P through positive
indirect effects of PWFD, PdW/P and NS/P. NPBMF showed low direct negative influence on SW/P which was
-0.013. This character also contributed to SW/P indirectly through NS/P and PdW/P. The total effect of NPBMF
on SW/P was 1.139 (the highest value). NSBMF had direct negative effect with a value of -0.151on SW/P. This
character expressed through positive indirect effect of NS/P, PdW/P, PWFD and PHMF. The maximum indirect
effect was exhibited by NS/P (1.026). PHMF expressed negative direct effect on SW/P with a value of -0.176.
This character showed positive indirect effect on SW/P through NSBMF, RWFD and NPd/P with the values of
0.198, 0.032 and 0.004, respectively.
Figure 2. Path diagram of different yield contributing traits at genotypic level.
PWFD had negative direct effect on SW/P was -0.127 and the positive indirect influence through DMF,
NSBMF, PdW/P and NS/P were 0.006, 0.024, 0.262 and 0.549, respectively. More or less, it contributed to
SW/P in spite of having indirect negative effect through NPBMF, PHMF, RWFD, NPd/P and 1000-SW. RWFD
had positive direct effect on SW/P and the value was 0.147. RWFD exhibited indirect positive effect on SW/P
via most of the characters except PHMF, PdW/P and NS/P. Its total effect was -0.754 due to indirect negative
effect on SW/P via NS/P (-1.253).
NPd/P had negative direct effect of -0.010 on SW/P. The character, PdW/P showed the positve direct effect
of 0.346 on SW/P. It contributed to SW/P greatly through NS/P (1.258) followed by PHMF. It showed negative
indirect effect via DMF, NPBMF, NSBMF, PWFD, RWFD, NPd/P and 1000-SW. The total effect of this
Samad et al. (2014) 1(3): 2735
.
character on SW/P was 1.005.NS/P had the highest positive direct effect (1.334). The character NS/P showed
the positive indirect influence through PHMF, NPd/P and PdW/P and other characters expressed negative
indirect effect on SW/P. The total effect of this character on SW/P was 0.969. The character, 1000-SW indicated
positive direct effect (0.285) on SW/P. It exhibited indirect positive effect through all characters except NPd/P,
PdW/P and NS/P on SW/P. The total effect of NS/P on SW/P was -1.260.
Selection index
The results obtained for different indices, containing SW/P and its components with expected gain in
percentage over straight selection are shown in table 3. The maximum genetic gain of 570.49249% was
exhibited when NPBMF (2) and RWFD (6) were included in the discriminant function. This value was followed
Table 3. Expected grain in percentage in seed weight per plant over straight selection from the use of
various selection indices in chickpea genotypes. Index which showed high value is presented only.
Selection Expected Selection Expected Selection Expected Selection Expected
Index Gain Index Gain Index Gain Index Gain
DMF(1) 53.967 8+11 104.337 4+6+11 180.105 3+4+6+11 138.833
NPBMF(2) 1118.189 1+2+3 128.757 4+8+11 79.100 3+4+8+11 59.964
NSBMF(3) -397.918 1+2+4 123.037 6+8+11 93.099 3+6+8+11 70.155
PHMF(4) 272.021 1+2+6 148.169 1+2+3+4 95.050 4+6+8+11 71.394
PWFD(5) -56.164 1+2+8 61.056 1+2+3+6 112.783 1+2+3+4+5 12.005
RWFD(6) 476.857 1+2+11 145.061 1+2+4+6 108.035 1+2+3+4+6 84.92
NPd/P(7) -105.106 1+3+11 84.752 1+2+4+11 108.905 1+2+3+4+11 89.627
PdW/P(8) 13.305 1+4+6 61.467 1+2+6+11 126.603 1+2+3+6+11 103.632
NS/P(9) -153.254 1+4+11 85.140 1+2+8+11 65.882 1+2+3+8+11 52.615
1000-SW(10) -118.038 1+6+11 100.218 1+3+4+11 65.561 1+2+4+6+11 96.808
SW/P(11) 381.482 2+3+4 261.351 1+3+6+11 76.689 1+2+4+8+11 51.682
1+2 174.667 2+3+6 357.340 1+4+6+11 76.444 1+2+6+8+11 59.445
1+4 61.3769 2+3+8 118.126 2+3+4+6 210.381 1+3+4+6+11 59.625
1+6 69.980 2+3+11 284.938 2+3+4+8 88.167 2+3+4+5+11 52.314
1+11 113.285 2+4+5 61.008 2+3+4+11 190.200 2+3+4+6+8 79.254
2+3 531.598 2+4+6 284.288 2+3+5+6 51.659 2+3+4+6+11 162.82
2+4 375.941 2+4+11 238.974 2+3+5+11 66.902 2+3+4+8+11 85.761
2+5 77.667 2+5+6 71.495 2+3+6+8 104.540 2+3+5+6+11 60.457
2+6 570.492 2+5+11 84.216 2+3+6+11 231.595 2+3+6+8+11 99.261
2+8 162.569 2+6+8 138.978 2+3+8+11 112.169 2+4+5+6+11 59.030
2+11 384.049 2+6+11 298.668 2+4+5+6 56.053 2+4+6+8+11 93.208
3+4 141.311 2+8+11 139.454 2+4+5+11 65.418 3+4+6+8+11 54.910
3+6 175.808 3+4+6 133.333 2+4+6+8 101.879 1+2+3+4+5+6 11.548
3+11 252.579 3+4+11 162.111 2+4+6+11 200.379 1+2+3+4+6+11 80.227
4+6 214.057 3+6+11 202.553 2+4+8+11 104.758 1+2+3+6+8+11 47.666
4+11 217.258 3+8+11 76.636 2+5+6+11 75.493 2+3+4+6+8+11 76.849
6+11 282.698 4+6+8 50.494 2+6+8+11 121.88
Note: DMF- Days to maximum flower; NPBMF- Number of primary branches at maximum flower;
NSBMF- Number of secondary branches at maximum flower; PHMF- Plant height at maximum flower;
PWFD- Plant weight after fully dry; RWFD- Root weight after fully dry; NPd/P- Number of pods per
plant; PdW/P- Pod weight per plant; NS/P- Number of seeds per plant; 1000-SW- Thousand seed weight;
SW/P- Seed weight per plant.
by 531.59816% genetic gain which was obtained when NPBMF (2) and NSBMF (3) were included in the
discriminant function. The next high genetic gain of 384.04907% was obtained when NPBMF (2) and SW/P
(11) were included in the discriminant function and followed by 282.6984% (6+11) and 252.57943% (3+11).
The characters, DMF (1) NPBMF (2), PHMF (4), RWFD (6), PdW/P (8) and SW/P (11) showed positive
expected gain and among them NPBMF (2) exhibited highest genetic gain.
In the discriminant function analysis, when selection indices contained three characters, the maximum
genetic gain was recorded as 357.34037% for 2+3+6, followed by 298.66798% for 2+6+11, 284.93796% for
2+3+11, 284.28844% for 2+4+6, 261.35085% for 2+3+4, 238.97406% for 2+4+11, 202.55362% for 3+6+11
and 162.11135% for 3+4+11. In the same way, when four characters were included in the discriminant function
viz., 2+3+6+11 gave the highest genetic gain of 231.595803 and followed by 210.381174%, 200.379694% and
190.200556% for 2+3+4+6, 2+4+6+11, and 2+3+4+11, respectively. Similarly, when five characters were
Samad et al. (2014) 1(3): 2735
.
included in the discriminant function, the combination like 2+3+4+6+11 exhibited the highest genetic gain of
162.82608% followed by 103.63273% for 1+2+3+6+11. In case of six characters combination of discriminant
function components like 1+2+3+4+6+11 had the highest genetic gain of 80.22788% followed by 76.849099%
for 2+3+4+6+8+11.When the combinations of 7, 8, 9, 10 and 11 quantitative characters were considered the
expected genetic gain was found to be low and negative in most of the cases in the present discriminant function
analysis.
DISCUSSION
In the present study, SW/P was positively correlated with DMF, NPBMF, NSBMF, PWFD, PdW/P, and
NS/P both at phenotypic and genotypic levels (Table 1). Seed yield per plant exhibited positive association with
NPB/P as reported by Thakur & Sirohi (2009) in chickpea followed by Singh et al. (1990) in chickpea and
Yucel et al. (2006) who obtained the positive correlation of PBN, SBN and SN with SY. Saleem et al. (2002) in
chickpea observed the positive correlation of DTF and TWP with SY. Roy et al. (2006) in bush bean got
positive correlation of DTF with SY/P followed by Singh et al. (1987) in chickpea and Singh (1985) in pea. On
the other hand, RWFD, NPd/P and 1000-SW were negatively correlated with SW/P at phenotypic and genotypic
levels. While PHMF positively and negatively correlated with SW/P at phenotypic and genotypic levels,
respectively.
The genotypic correlation coefficient of SW/P with PdW/P and NS/P were high and greater than the
phenotypic correlation coefficient of those traits and exhibited significant value. Thus, it can be inferred that
selection based on any one of two traits either alone or in combination, would result in identifying high yielding
genotypes. The genotypic correlation coefficient of DMF, NPBMF and NSBMF with SW/P were significant.
PHMF, RWFD and 1000-SW with SW/P expressed significant but negative values. Ali et al. (2009) in chickpea
reported the significant association of seeds per plant and primary branches per plant with grain yield per plant
and negative correlation of PH with GY/P. Khan & Qureshi (2001) in chickpea also observed the significant
association of PB/P and SB/P with GY followed by Sharma and Saini (2010) and Khan (1985) in mungbean.
In correlation analysis, DMF was positively correlated with NSBMF, PHMF, NPd/P, PdW/P and NS/P both
at phenotypic and genotypic levels. Saleem et al. (2002) in chickpea observed the positive correlation of NSB,
PH and NPP with DTF followed by Ali et al. (2009) regarding correlation of PH with DTF. NPBMF showed
positive correlation coefficient with NSBMF, PHMF, PWFD, NPd/P, PdW/P and NS/P at phonotypic and
genotypic levels. These results confirmed the findings of Khan & Qureshi (2001). NSBMF exhibited that this
trait was positively correlated both at phenotypic and genotypic levels with NPd/P, PdW/P and NS/P. But this
trait was negatively correlated with RWFD and 1000-SW at both levels, respectively. Qureshi et al. (2001) got
the positive correlation of SB/P with GY and negative correlation of 100-GWT with SB/P reported by Ali et al.
(2009). The trait, PHMF was positively correlated both at phenotypic and genotypic levels with PWFD and
RWFD. PWFD was positively correlated with NPd/P, PdW/P and NS/P both at phenotypic and genotypic levels
and also indicated that the value of the genotypic correlation coefficient was higher than that of the phenotypic
correlation coefficient. RWFD had positive correlation both at phenotypic and genotypic levels with 1000-SW
and NPd/P exhibited positive correlation both at phenotypic and genotypic levels with PdW/P and 1000-SW. In
addition, PdW/P was positively correlated both at phenotypic and genotypic levels with NS/P. NS/P was
positively correlated with 1000-SW and the value of phenotypic correlation coefficient was higher than
genotypic correlation coefficient.
The path coefficient analysis based on SW/P as a dependent variable at phenotypic level revealed that all
traits, except NPBMF, RWFD, NPd/P, NS/P and 1000-SW exhibited negative direct effects (Table 2). Among
positive direct effects NS/P had the highest value of 1.077 on SW/P. It means a slight increase in any one of the
above traits may directly contribute towards SW/P. For NPd/P (0.077), the direct effect was positive followed
by RWFD and 1000-SW while, their association with SW/P were observed to be negative, indicating the
importance of restricted selection model for exploitation of the direct effects noticed.
In path-coefficient, the highest positive direct effect was expressed by NS/P on SW/P and followed by
PdW/P, 1000-SW and RWFD at genotypic level. Compared to the correlation analysis at genotypic level, path-
coefficient of SW/P and its components indicated that NS/P and PdW/P exerted the highest direct influence,
with values of 1.334 and 0.346, respectively. Though RWFD and 1000-SW on SW/P showed positive direct
effects but regarding correlation the relationship was negative. Uddin et al. (1990) in chickpea and Yucel et al.
(2006) reported that SN was the major contributor to SW/P. Ali & Shaikh (1986) in mungbean observed that
Samad et al. (2014) 1(3): 2735
.
100-SW had a positive direct effect on SW/P, though a significant negative correlation was found and followed
by Roy et al. (2006).
In selection study, the maximum genetic gain of 570.49249% was exhibited when NPBMF (2) and RWFD
(6) were included in the discriminant function followed by 531.59816% (2+3). Table 3 also revealed that among
the individual traits namely, DMF (1), NPBMF (2), PHMF (4), RWFD (6), PdW/P (8) and SW/P (11) showed
positive expected gain and among them NPBMF (2) exhibited highest genetic gain, followed by RWFD (6),
SW/P (11) and PHMF (4) and the remaining traits indicated negative genetic gain. The highest genetic gain of
NPBMF (2) as showed individually might be due to environment as it is obvious that influence on individual
trait is more than over multiple traits.
CONCLUSION
During selection emphasis should be given on PdW/P and NS/P as they exhibited high correlation and high
positive direct effect on yield. Besides, NPBMF and RWFD may be considered as primary yield components
because they showed highest genetic gain among the combinations of selection indices and also exhibited
positive genetic correlation with yield and positive direct effect on phenotypic level.
ACKNOWLEDGEMENTS
Authors wish to express their gratitude to Ministry of Science and Technology, Bangladesh for supporting
the funding during this research work. Authors also thankful to Dr. A. N. K. Mamun, Principal Scientific
Officer, Food and Radiation Biology, Bangladesh Atomic Energy Research Establishment, Savar, Dhaka.
Authors are also grateful to late Professor Dr. Abdul Khaleque of the Department of Botany of Rajshahi
University for his technical suggestion and assistance during this work. Lastly, Authors sincere thanks are due
to Dr. Rumman Ara, Dr. Abul Kalam Azad, Dr. Rahimul Alam, Hosne Ara Banu and Dr. Anuradha Roy for
their heartfelt co-operation and inspiration during this work.
REFERENCES
Ali MA, Nawab NN, Abbas A, Zulkiffal M & Sajjad M (2009) Evaluation of selection criteria in Cicer
arietinum L. using correlation coefficients and path analysis. Australian Journal of Crop Science 3(2): 6570.
Ali MS & Shaikh MAQ (1986) Path-coefficient analysis in summer mungbean (Vigna radiata L. Wilczek).
Bangladesh Journal of Agricultural Research 1: 813.
Dewey DR & Lu KH (1959) A correlation and path-coefficient analysis of components of crested wheat grass
seed production. Agronomy Journal 51: 515518.
Khan IA (1985) Correlation and path-coefficients of yield components in mungbean (Phaseolus aureus Roxb.).
Botanical Bulletin-Academia Sinica 26: 1320.
Khan MR & Qureshi AS (2001) Path-coefficient and correlation analysis on the variation induced by gamma
irradiation in M1 generation of chickpea (Cicer arietinum L.). Online Journal of Biological Sciences 1(3):
108110.
Qureshi ST (2001) Genotype-environment interaction for quantitative traits in chickpea (Cicer arietinum),
M.Phil.Thesis. Quaid-i-Azam University, Islamabad, Pakistan.
Roy SK, Karim MA, Islam AKMA, Bari MN, Mian MAK & Tetsushi H (2006) Relationship between yield and
its component characters of bush bean (Phaseolus vulgaris L.). South Pacific Studies 27(1): 1323.
Saleem M, Tahir MHN, Kabir R, Javid M & Shahzad K (2002) Interrelationships and path analysis of yield
attributes in chickpea (Cice rarietinum L.). International Journal of Agriculture and Biology 4(3): 404406.
Sharma LK & Saini DP (2010) Variability and association studies for seed yield and yield components in
chickpea (Cicer arietinum L.). Research Journal of Agricultural Sciences 1(3): 209211.
Singh KB, Bejiga G & Malhotra RS (1990) Associations of some characters with seed yield in chickpea
collections. Euphytica 49: 8388.
Singh KN, Santoshi US, Singh HG & Singh IB (1987) Interrelationships among certain quantitative traits in
pea. Farm Science Journal 2: 102104.
Singh RK (1985) Genotypic and phenotypic variability correlations in pea. Indian Journal of Agricultural
Sciences 55: 147150.
Thakur SK & Sirohi A (2009) Correlation and path-coefficient analysis in chickpea (Cicer arietinum L.) under
different seasons. Legume Research 32 (1): 16.
Toker C & Cagirgan MI (2004) The use of phenotypic correlations and factor analysis in determining characters
for grain yield selection in chickpea (Cicer arietinum L.). Hereditas 140: 226228.
Samad et al. (2014) 1(3): 2735
.
Uddin MJ, Hamid MA, Rahman ARMS & Newaz MA (1990) Variability, correlation and path analysis in
chickpea (Cicer arietinum L.) in Bangladesh. Bangladesh Journal of Plant Breeding and Genetics 3: 5155.
Wright S (1921) Correlation and causation. Journal of Agricultural Research 20: 557585.
Wright S (1923) The theory of path-coefficients a reply to Niles criticism. Genetics 8: 239255.
Yucel DO, Anlarsal AE & Yucel C (2006) Genetic variability, correlation and path analysis of yield and yield
components in chickpea (Cicer arietinum L.). Turkish Journal of Agriculture and Forestry 30: 18388.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
1(3): 3642, 2014
Research article
Enzymatic antioxidant activities in eight wild edible fruits of Odisha

Madhulita Patnaik and Uday Chand Basak*
Seed Bank & Seed Biology Division, Regional Plant Resource Centre,
R & D Institute of Forest and Environment Department, Bhubaneswar-751015, Odisha, India
*Corresponding Author: uc_basak07@yahoo.co.in [Accepted: 11 November 2014]
Abstract: Fruits are considered to be rich in antioxidants but the total antioxidant potential is yet
to be unveiled systematically in case of wild edible fruits. The present paper attempts to focus on
assessing the levels of enzymatic activity of Superoxide dismutase (SOD), Catalase and
Peroxidase enzymes in selected eight wild edible fruit species of Odisha viz. Aegle marmelos,
Calamus guruba, Limonia acidissima, Phyllanthus acidus, Phyllanthus emblica, Syzigium cumini,
Ziziphus mauritiana and Ziziphus oenoplia. As per the results inferred, SOD enzyme activity of
Phyllanthus acidus (2.66 OD-1.min-1.mg-1 protein) was found to be the highest and almost equal
to Ziziphus oenoplia (2.15 OD-1.min-1.mg-1 protein). Catalase enzyme activity was found to be
very high in Calamus guruba (4.2 104 I.E.U.) followed by Phyllanthus acidus (4.08 x 104
I.E.U.), Aegle marmelos (3.77 104 I.E.U.) and Syzigium cumini (3.65 104 I.E.U.). Peroxidase
enzyme activity was highest in Calamus guruba (0.975 OD-1.min-1.gm-1 tissue wt.) and at par with
Aegle marmelos (0.645 OD-1.min-1.gm-1 tissue wt.). From this study, it could be concluded that
wild edible fruits like Aegle marmelos, Calamus guruba, Phyllanthus acidus, Syzigium cumini and
Ziziphus oenoplia can be identified as beneficial fruit species that possess significant free radical
scavenging levels. Exploration and higher intake of such fruits with functional attributes could
impart promising therapeutic potential.
Keywords: Antioxidant - Catalase - Peroxidase - ROS - SOD.
[Cite as: Patnaik M & Basak UC (2014) Enzymatic antioxidant activities in eight wild edible fruits of Odisha.
INTRODUCTION
Antioxidants are compounds that undergo oxidation terminating the chain reaction by reacting with free
radicals and chelating catalytic metals (Patel et al. 2010) and results in neutralization of free radicals and
simultaneous inhibition of oxidation of other vital molecules (Sies 1996). Free radicals are mainly derived from
oxygen (reactive oxygen species/ROS) and nitrogen (reactive nitrogen species/RNS) (He & Hader 2002, Apel &
Hirt 2004), and generated in our body by various endogenous systems, exposure to different physicochemical
conditions (such as exposure to ultraviolet light and toxic chemicals), inflammatory or pathophysiological states
(Kagan et al. 2002, Devasagayam et al. 2004). Deleterious effects of these radicals include lipid peroxidation
(Jacob & Burri 1996) causing damage of the cell membrane of phospholipids, lipoprotein (Devasagayam et al.
2003), proteins oxidation (Stadtman et al. 1992), loss of enzyme activity etc (Halliwell & Gutteridge 1997)
resulting in various diseases such as cancer, alcoholic liver cirrhosis, arteriosclerosis, arthritis,
neurodegenerative disorders etc (Lee et al. 2000, Middleton et al. 2000, Yoshikawa et al. 2000).
Antioxidants play a curative role in chronic ailments such as heart disease, diabetes, hypertension, stroke,
gastritis, Alzheimers disease, AIDS (Pourmorad et al. 2006) and decreasing the risk of cardiovascular and
degenerative diseases by reduction of oxidative stress and counteraction of macromolecular oxidation (Ramana
et al. 2011). Currently available synthetic antioxidants like butylated hydroxy anisole (BHA), butylated hydroxy
toluene (BHT), tertiary butylated hydroquinone and gallic acid esters show low solubility and moderate
antioxidant activity (Barlow 1990) along with negative health impact. Hence, scientists have now focused on
isolation and characterization of natural antioxidants from herbs, spices, seeds, cereals, fruits and vegetables by
extraction, fractionation and purification (Dillard & German 2000, Wang & Linn 2000). Natural antioxidants are
basically of two types i.e., enzymatic and non-enzymatic antioxidants. Antioxidant enzymes include catalase,
superoxide dismutase (SOD), peroxidase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate
Patnaik & Basak (2014) 1(3): 3642
.
dehydrogenase and ascorbate oxidase etc (Bandyopadhyay et al. 1990) while non-enzymatic antioxidants
includes -Tocopherol (vitamin E), Ascorbic acid (vitamin C), carotenoids, flavonoids and related polyphenols,
-lipoic acid, glutathione etc which all together work in synergy to counterbalance oxidative stress (Thiele et al.
2001).
In recent years, there has been growing interest in functional foods, i.e., foods that can provide not only basic
nutritional and energetic requirements, but also an additional physiological benefit (Savikin et al. 2009). Fruits
are considered to be major sources of dietary antioxidant compounds. Fruits possess self defense mechanism for
protection from oxidative stress by the activation of many antioxidant defense enzymes (Jacob 1995) etc. The
consumption of fruits with a high antioxidant composition has been associated with a lowered incidence of
chronic, degenerative diseases (Cox et al. 2000) including cancer, coronary heart disease, inflammation,
arthritis, immune system decline, brain dysfunction, cataracts, altitude sickness (Kumpulainen & Salonen 1999),
digestive, stomachic complications, various biological activities like lipid lowering effect (Vijaya et al. 2009)
etc.
The beneficial effects of fruits are hypothesized to owe, at least in part, to antioxidants (Benzie & Strain
1999). Based on this idea, there has been a strong demand of therapeutic and chemo preventive antioxidant
agents with limited cytotoxicity to enhance the antioxidant capacity of the body and help attenuate the damage
induced by ROS. One most commendable and sort after strategy is to identify fruits with high content of natural
antioxidants that would help increase the bodys immunity to many diseases.
Since there is no concrete report regarding the antioxidant potential of wild edible fruits in Odisha, many of
them remain undocumented, unexploited and understudied except very few of them (Basak et al. 2013).
Emphasizing on the possible beneficiary effects of naturally derived antioxidants and recognition of fruits with
high antioxidant activities, this study was undertaken to examine and compare the total antioxidant enzyme
activity of superoxide dismutase (SOD), catalase and peroxidase enzymes in eight selected wild edible fruits
namely Aegle marmelos, Calamus guruba, Limonia acidissima, Phyllanthus acidus, Phyllanthus emblica,
Syzigium cumini, Ziziphus mauritiana and Ziziphus oenoplia for enrolling better understanding of antioxidant
properties of these wild edible fruits to therapeutic purposes and signifying the need for domestication and its
role for industrial development with a view to popularizing their uses by the wider society.

Plant material
Eight wild edible fruits namely Aegle marmelos, Calamus guruba, Limonia acidissima, Phyllanthus acidus,
Phyllanthus emblica, Syzigium cumini, Ziziphus mauritiana and Ziziphus oenoplia were shortlisted and selected
owing to their immense popularity, widespread consumption and distribution (Table 1).
Sample collection & preparation
Fresh samples (edible ripen stages) were used for enzyme activity analysis. These fruits were collected from
botanical garden of the institute and various forest blocks of Odisha during 201112 and enzymatic analysis of
superoxide dismutase (SOD), Catalase and peroxidase enzymes was performed in the institutional research
laboratory.
Superoxide dismutase (SOD) enzyme assay
The extracts were assayed for SOD activity photochemically, using the assay system consisting of
methionine, riboflavin, and NBT (Beauchamp et al. 1971). The photochemical procedure was chosen as being
independent of other enzymes and proteins and, therefore, more reliable in the case of crude extracts than
enzymic assay systems (McCord et al. 1969). The original assay (Beauchamp et al. 1971) was modified
(Constantine et al. 1977). The reaction mixture was composed of 1.3 M riboflavin, 13 mM methionine, 63 M
NBT, 0.05M sodium carbonate (pH 10.2), and the appropriate volume of extract. The initial rate of the reaction
was determined as increase of absorbance at 560 nm. Under the described conditions, the increase of absorbance
in absence of SOD was 0.100 absorbance unit/5 min and was linear up to 15 min. In the presence of SOD, the
reaction was inhibited and the amount of inhibition was used to quantitate the enzyme. Each extract was assayed
twice and the results varied less than +0.005 absorbance unit/5 min.
Peroxidase enzyme assay
The extracts were assayed for peroxidase enzyme activity using o-dianisidine spectrophotometrically
(Queseda et al. 1992) with slight modifications (Putter 1974, Malik & Singh 1980). The oxidized yellow/orange
colored O-dianisidine is measured at 430nm. 0.2ml enzyme extract and 0.1ml freshly prepared O-dianisidine
Patnaik & Basak (2014) 1(3): 3642
.
solution is added to 3.5 ml phosphate buffer in a clean dry cuvette. Water blank is used in the assay. The assay
mixture is brought to 2830 C and then 0.2 ml of 0.2M H2O2 was added and mixed. The initial absorbance was
read at 430nm and then at every 30seconds intervals up to 3minutes. The assay is repeated with diluted extracts
if rate of increase is very high. Increase in absorbance was plotted against time. The enzyme activity is
expressed in terms of rate of increased absorbance per unit time per mg protein or tissue weight.
Catalase enzyme assay
One unit of catalase activity is defined as that amount of enzyme which breaks down I mol of H 2O2 min-1
under the defined assay conditions (Chance & Maehly, 1955) with slight modifications. Five milliliters of the
assay mixture for the catalase activity comprised: 0.2M of phosphate buffer (pH 6.8), 0.4N of H2O2, and I ml of
the twice diluted enzyme extracted. After incubation at 25C for 1 min, the reaction was stopped by adding 10
ml of 2% (v/v) H2SO4, phosphate buffer and the residual H2O2 was titrated against 0.01N KMnO4 until a faint
purple color persisted for at least 15 sec. A control was run at the same time in which the enzyme activity was
stopped at "zero" time.
Table 1. Experimental wild edible fruits selected for antioxidant enzyme analysis.
S. N. Fruit species Local Name Fruiting season Medicinal uses
1. Aegle Bela MayJune The fruit is an astringent and used for treatment of
marmelos Asthma, anaemia, fractures, healing of wounds, high
blood pressure, diabetes mellitus, intestinal tonic, chronic
constipation, indigestion, hemorrhoids, intermittent fever,
hypochondria, melancholia and for heart palpitation,
diarrhea and dysentery.
2. Calamus Kanta Beta December The fruit is traditionally used as a tranquilizer and a
guruba February general wonder drug. It is a sedative, hypotensive,
muscle relaxant, cures cough, cold and pulmonary
disorders.
3. Limonia Kaitha November Fruits are refrigerant, stomachic, stimulant, astringent,
acidissima January aphrodisiac, diuretic, cardiotonic, tonic to the liver and
lungs; cures cough, hiccup and dysentery; good for
asthma, consumption, tumours, ophthalmia and
leucorrhoea.
4. Phyllanthus Nara Koli JuneOctober Fruits are a good remedy for different types of
acidus ailments like emetic and purgative (hypertension and
respiratory). Its hepatoprotective, anti-bacterial, anti-
diabetic, anti-nociceptive, cathartic, liver tonic, laxative
etc. Cures coughs, asthma, bronchitis, poulticing, soles,
rheumatism, sudorific gonorrhea and skin disorders.
5. Phyllanthus Amla November Fruits are antioxidant, anti-diabetic, hypolipidemic,
emblica January antibacterial, anti-inflammatory, antiulcerogenic,
hepatoprotective, gastroprotective, and possess
chemopreventive properties. Also prevents ulcer and used
for treatment of diarrhea, jaundice etc.
6. Syzigium Jamu Koli JulyAugust Fruits are considered to be anti-diabetic, well known
cumini antioxidant, digestive, anthelmintic and astringent to
bowels. It lowers blood pressure and used for treatment of
sore throat, bronchitis, asthma, dysentery, ulcers, chronic
diarrhea, enteric disorders, treat cold, cough, fever, skin
problems etc.
7. Ziziphus Bara Koli November The fruits are applied on cuts and ulcers; are employed
mauritiana February in pulmonary ailments and fevers; the dried ripe fruit is a
mild laxative, halts nausea, vomiting and abdominal pains
in pregnancy, liver trouble, asthma and fever, checks
diarrhea, burning sensations, cough, wound, skin disease,
ulcers, stomatitis, sexual weakness and general debility.
8. Ziziphus Kantei Koli October Pacifies vitiated pitta, kapha, worms, peptic ulcer,
oenoplia January stomach pain and wounds. Also used for sore throats,
dysentery and inflammation of the uterus.
Patnaik & Basak (2014) 1(3): 3642
.
RESULTS
In present investigation, eight wild edible fruits were selected for their antioxidant enzyme potential which
would prove to be an essential parameter for free radical scavenging activity in human health and nutrition
owing to their widespread consumption and assimilation in many of the forest blocks of Odisha. The research
findings pertaining to the antioxidant enzyme activity of superoxide dismutase, catalase and peroxidase are
shown below in table 2.
Table 2. Antioxidant enzyme activity analysis.
SOD Catalase Peroxidase
S. N. Fruit sample Local Name (OD-1.min-1.mg-1 (I.E.U.) in 1gm (OD-1.min-1.gm-1
protein) fresh wt. tissue tissue wt.)
1. Aegle marmelos Bela 0.6050 3.77 104 0.6450
2. Calamus guruba Kanta Beta 0.9320 4.20 104 0.9750
3. Limonia acidissima Kaitha 0.1800 2.65 104 0.2790
4. Phyllanthus acidus Nara Koli 2.6600 4.08 104 0.0087
5. Phyllanthus emblica Amla 0.0067 1.24 104 0.0129
6. Syzigium cumini Jamu Koli 0.0047 3.67 104 0.0081
7. Ziziphus mauritiana Bara Koli 0.0130 1.8 104 0.2100
8. Ziziphus oenoplia Kantei Koli 2.1500 2.85 104 0.0900
Superoxide dismutase enzyme activity
The SOD enzyme activity varied from 0.0047 to 2.66 OD-1.min-1.mg-1 protein as shown in figure 1. Highest
SOD enzyme activity was observed in Phyllanthus acidus (2.66 OD-1.min-1.mg-1 protein) followed by Ziziphus
oenoplia (2.15 OD-1.min-1.mg-1 protein) and Calamus guruba (0.932 OD-1.min-1.mg-1 protein). The SOD
enzyme activity was found to be lowest in Syzigium cumini (0.0047 OD-1.min-1.mg-1 protein). SOD enzyme
activity was observed in the rest of the four wild edible fruits in the following decreasing trend: Aegle marmelos
> Limonia acidissima > Ziziphus mauritiana > Phyllanthus emblica.
Figure 1. Comparison of SOD enzyme activity in eight wild edible fruits.

Catalase enzyme activity
The catalase enzyme activity varied from 1.24 104 I.E.U. to 4.2 104 I.E.U. per gram of fresh tissue in the
selected eight wild edible fruits as shown in figure 2. Calamus guruba was recorded to be the fruit with highest
catalase enzyme activity (4.2 104 I.E.U. per gram of fresh tissue) and Phyllanthus emblica was observed to
possess the lowest enzymatic activity (1.24 104 I.E.U. per gram of fresh tissue). The enzyme activity was
observed in the decreasing trend among fruits: Phyllanthus acidus > Aegle marmelos > Syzigium cumini >
Ziziphus oenoplia > Limonia acidissima > Ziziphus mauritiana.
Patnaik & Basak (2014) 1(3): 3642
.
Figure 2. Comparison of catalase enzyme activity in eight selected wild edible fruits.
Peroxidase enzyme activity
Highest peroxidase enzyme activity was observed in Calamus guruba with a total enzyme activity of 0.975
OD-1.min-1.gm-1 tissue weight while the lowest enzyme activity was recorded in Syzigium cumini with enzyme
activity observed to be 0.0081 OD-1.min-1.gm-1 tissue wt. For rest of the wild edible fruit species the enzyme
activity value ranged from 0.6450.0087 OD-1.min-1.gm-1 tissue weight (Fig. 3).
Figure 3. Comparison of peroxidase enzyme activity in eight selected wild edible fruits.
DISCUSSION
The present study was undertaken to assess the free radical scavenging capacity of the selected eight wild
edible fruits of Odisha, India. From the results, it was observed that Calamus guruba and Phyllanthus acidus
showed promising activities of catalase and SOD. Similarly, Aegle marmelos and Calamus guruba showed high
peroxidase enzyme activity. From our experiment it was deducted that wild edible fruits like Aegle marmelos,
Calamus guruba, Phyllanthus acidus and Ziziphus oenoplia possessed high enzyme activity levels. Since
antioxidant enzymes altogether work in a network, therefore these fruits, possessing high enzymatic activities of
Patnaik & Basak (2014) 1(3): 3642
.
catalase, peroxidase and SOD can efficiently serve as potential antioxidant additives in human diet thereby
preventing oxidative damage by reactive oxygen species.
The ability of the fruit extracts to quench superoxide radicals from reaction mixture is reflected in the
decrease of the absorbance at = 560 nm. The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity. The observed increase in SOD activity suggests that the
Phyllanthus acidus, Ziziphus oenoplia and Calamus guruba have an efficient protective mechanism in response
to ROS. SOD catalyzes the breakdown of endogenous cytotoxic superoxide radicals to H2O2 which is further
degraded by CAT. Thus, they play a crucial role in maintaining the physiological levels of O2 and H2O2
(Arivazhagan et al. 2000).
CONCLUSION
Our results support the possible use of the eight analyzed fruits possessing good antioxidant enzyme activity
levels, as natural antioxidants to replace the synthetic additives as well as their use in the production of
functional foods with high antioxidant activity that are capable of blocking the action of reactive oxygen species
involved in oxidative stress. Evaluation of these fruit extracts has provided interesting results that might be
beneficial for the pharmacological use of these plants in clinical trials. All these fruits with their free radical
scavenging activity and various extents of antioxidant activity provide a valuable source of nutraceutical
supplements, chemo preventive and therapeutic agents. It is suggested that further research needs to be
conducted for selecting the fruits with high antioxidant levels and clarify the importance and role of these
antioxidants on pathogenesis of various diseases.
ACKNOWLEDGEMENTS
The authors are thankful to the department of Forest & Environment, Govt. of Odisha for supporting this
research project under State Plan Grant.
REFERENCES
Apel K & Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress and signal transduction. Annual
Review of Plant Biology 55: 373399.
Arivazhagan S, Balasenthil S & Nagini S (2000) Garlic and neem leaf extracts enhance hepatic glutathione and
glutathione dependent enzymes during N-methyl-Nitrosoguanidine (MNNG)-induced gastric
carcinogenesis. Phytotherapy Research 14: 291293.
Bandyopadhyay U, Das D & Banerjee RK (1990) Reactive oxygen species: Oxidative damage and
pathogenesis. Current Science 77: 658666.
Barlow SM (1990) Toxicological aspects of antioxidants used as food additives. In: Hudson BJF (ed) Food
Antioxidants. Elsevier, London, pp. 253307.
Basak UC, Mahapatra AK & Mishra S (2013) Assessment of protective antioxidant mechanisms in some ethno-
medicinally important wild edible fruits of Odisha, India. Agrotechnology 2(3): 14.
Beauchamp CO & Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to
acrylamide gels. Analytical Biochemistry 44: 276287.
Benzie IFF & Strain JJ (1999) Ferric reducing/antioxidant power assay: direct measure of total antioxidant
activity of biological fluids and modified version for simultaneous measurement of total antioxidant power
and ascorbic acid concentration. In: Packer L & Orlando FL (eds) Methods in Enzymology, Vol. 299.
Academic Press, pp. 1527.
Chance B & Maehly AC (1955) Assay of catalase and peroxidases. Methods in Enzymology 2: 764775.
Constantine NG & Stanley KR (1977) Superoxide Dismutases: Occurrence in higher plants. Plant Physiology
59: 309314.
Cox BD, Whichelow MJ & Prevost AT (2000) Seasonal consumption of salad vegetables and fresh fruit in
relation to the development of cardiovascular disease and cancer. Public Health Nutrition 3: 1929.
Devasagayam TPA, Boloor KK & Ramsarma T (2003) Methods for estimating lipid peroxidation: Analysis of
merits and demerits (mini review). Indian Journal of Biochemistry and Biophysics 40: 3008.
Devasagayam TPA, Tilak JC, Boloor KK, Sane KS, Ghaskadbi SS & Lele RD (2004) Free Radicals and
Antioxidants in Human Health: Current Status and Future Prospects (Review). Journal of the Association
of Physcians of India 52: 794-804.
Dillard CD & German JB (2000) Phytochemicals: Nutraceuticals and human health. Journal of the Science of
Food and Agriculture 80: 17441756.
Patnaik & Basak (2014) 1(3): 3642
.
Halliwell B & Gutteridge JMC (1997) (eds) Free Radicals in Biology and Medicine. Oxford University Press,
Oxford.
He YY & Hader DP (2002) Reactive oxygen species and UV-B: effect on Cyanobacteria. Photochemical and
Photobiological Sciences 1: 729736.
Jacob RA (1995) The integrated antioxidants system. Nutrition Research 15(5): 755766.
Jacob RA & Burri BJ (1996) Oxidative damage and defense. The American Journal of Clinical Nutrition 63:
985990.
Kagan VE, Kisin ER & Kawai K (2002) Towards mechanism based antioxidant interventions. Annals of the
New York Academy of Sciences 959: 18898.
Kumpulainen JT & Salonen JT (1999) Natural Antioxidants and Anticarcinogens In: Nutrition, Health and
Disease. The Royal Society of Chemistry, UK, pp: 178187.
Lee KG, Mitchelle AE & Shibamoto T (2000) Determination of antioxidant properties of aroma extracts from
various beans. Journal of Agricultural and Food Chemistry 48: 48174820.
Malik CP & Singh MB (1980) In: Plant Enzymology and Histoenzymology. Kalyani Publishers, New Delhi, 53
p.
McCord JM & Fridovich I (1969) Superoxide dismutase: an enzymic function for erythrocuprein
(hemocuprein). The Journal of Biological Chemistry 244: 60496055.
Middleton E, Kandaswamy C & Theoharides TC (2000) The effects of plant flavonoids on mammalian cells:
implications for inflammation, heart disease and cancer. Pharmalogical Reviews 52: 673751.
Patel VR, Patel PR & Kajal SS (2010) Antioxidant activity of some selected Medicinal plants in Western
Region of India. Advances in Biological Research 4(1): 2326.
Putter J (1974) In: Methods of Enzymatic Analysis. Academic Press New York, 685 p.
Quesada MA, Sanchez-Roldan C, Heredia A, Valpuesta V & Bukovac M (1992) Peroxidase isoenzymes in the
pericarp of seeded and seedless Redhaven peach fruit. Journal of Plant Growth Regulation 11: 16.
Ramana G, Reddy CS & Rao CV (2011) In-vitro and in-vivo anti-oxidant activity of Ficus racemosa Linn. fruit
extract and Aegle marmelos root and leaf extracts. Journal of Pharmacy Research 4(7): 20782081.
Savikin K, Zdunic G, Jankovic T, Tasic S, Menkovic N, Stevic T & Dordevic B (2009) Phenolic content and
radical scavenging capacity of berries and related jams from certificated area in Serbia. Plant Foods for
Human Nutrition 64: 212217.
Sies H (1996) (ed) Antioxidants in Disease, Mechanisms and Therapy. Academic Press, New York.
Stadtman ER (1992) Protein oxidation and aging. Science 257: 12201225.
Thiele JJ, Schroeter C, Hsieh SN, Podda M & Packer L (2001) The antioxidant network of the stratum corneum,
Oxidants and Antioxidants in Cutaneous Biology. Current Problems in Dermatology 29: 2642.
Vijaya C, Ramanathan M & Suresh B (2009) Lipid lowering activity of ethanolic extract of leaves of Aegle
marmelos (Linn.) in hyperlipidaemic models of Wistar albino rats. Indian Journal of Experimental Biology
47(3): 182.
Wang SY & Lin HS (2000) Antioxidant activity in fruits and leaves of blackberry, raspberry, and strawberry
varies with cultivar and developmental stage. Journal of the Science of Food and Agriculture 48: 140146.
Yoshikawa T, Toyokuni S, Yamamoto Y & Naito Y (2000) (eds) Free Radicals in Chemistry Biology and
Medicine. OICA International, London.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
1(3): 4348, 2014
Research article
Ecology and phenology of plant communities of Gentianaceae in

Montane Grasslands of Karnataka, Southern India
Ashwini H. S., Avinash K. S., Shravanakumar S. and Y. L. Krishnamurthy*
Department of PG Studies and Research in Applied Botany, Jnana Sahyadri, Kuvempu University,
Shankaraghatta -577451, Karnataka, India
*Corresponding Author: murthy_ylk@yahoo.co.in [Accepted: 14 November 2014]
Abstract: The Gentianaceae family represents 33 species of plants belonging to nine genera with a
sub species reported in India. The studies on diversity, distribution and phenology of this family in
montane grasslands of Karnataka have been under taken. A total of 60 quadrats of 50 m line
transect with alternate 11 m subquadrats were laid for enumeration. A total of 12 species of the
family Gentianaceae were occurred. Where, Swertia lawii is one of the dominant species in the
study area. Important value index (IVI) of S. lawii represents 50.62. Most of these species are
restricted in distribution particular habitat and they are highly seasonal. All these species are in
threatened condition due to anthoprogecinc pressure.
Keywords: Gentianaceae - Montane - Grasslands - Herbs.
[Cite as: Ashwini HS, Avinash KS, Shravanakumar S & Krishnamurthy YL (2014) Ecology and phenology of
plant communities of Gentianaceae in Montane Grasslands of Karnataka, Southern India. Tropical Plant
Research 1(3): 4348]
INTRODUCTION
Karnataka, one of the Southern states of India has 3.83 million hectares of the recorded forest area which is
around 20 per cent of its geographical area. The Western Ghats of Karnataka are one of the 35 global priority
hotspots for conservation and one of the two in Indian subcontinent (Conservation International 2005).
The state is endowed with great diversity of climate, topography and soil. The forest types such as deciduous,
semi evergreen, evergreen, scrub, shola and grasslands commonly called montane grasslands. The montane
grasslands are located on the high plateau. The valleys of hills contain forests with stunted evergreen trees. The
coexistence of two contrasting vegetation types creates a landscape of interest to ecologists (Thomas & Palmer
2007). Where forests are found in the depressions or folds of the mountains and are separated by grasslands.
Tropical montane habitats exhibit high endemism with several species restricted to narrow elevational bands
(Robin & Nandine 2012).
The plant species in the family Gentianaceae are distributed in hilly regions particularly in grassy patches.
The family has nine genera and 33 species and 1 sub species (Shahina 2014). Now the genus Limnanthemum
S.P.Gmel., has been excluded from this family and placed in Menyanthaceae. The plants of Gentianaceae family
distributed in wide habitat ranges starting from coastal regions to high elevation montane grasslands. They are
delicate herbs highly seasonal which are ironically facing threatened conditions due to mining, grazing and other
anthropogenic disturbances. Hence, in this study an attempt had been made to explore plant species belong to
Gentianaceae in montane grasslands of Karnataka.

Diversity and Distribution
The study area has been spread in Chikmaglur, Shimoga and Kodagu districts montane grasslands namely
Kemmannugundi, Bababuden giri, Mullayyana giri, Kodachadri and Pushpagiri (Fig. 1). The area comprised the
edge of the forest and montane grasslands which are having iron rich rocky plateau. These places mainly contain
iron and boxite ores. Field exploration in different grasslands has been carried out from 2011 to 2013. A size of
50 m long transects with five 11 m quadrat alternating with an interval of 10 m is used for survey by following
the method of Bhatt & Utkarsh (1999). From the 60 quadrats all the Gentianaceae plant species were listed and
identified with available taxonomic literature (Gamble 1935, Yoganarasimhan & Razi 1981, Saldanha 1984,
Ashwini et al. (2014) 1(3): 4348
.
Ramaswamy et al. 2001, Inghalhalikar 2007, Gowda 2004, Bhat 2003) and voucher specimens were prepared
and deposited in herbarium of Department of Applied Botany. Kuvempu University, Shankaraghatta, Karnataka.
The habitat characteristics of each area were noted. Meso-habitat details were maintained with respect to
exposure of sunlight, wind, slope, and humidity and their habitat were also noted. Meso habitat levels were
sorted based on ordinal scaling (Jongman et al. 1987, Negi & Gadgil 1997). The pooled quadrat information for
each transect was analysed compositional features including diversity, density, frequency and abundance by
using standard literature (Simpson 1949, Shannon & Weiner 1963, Misra 1968, Brilliant et al. 2012).
Figure 1. Map showing locations of study sites in three districts of Karnataka.

Phenology
Detailed phenological records of the 12 species of Gentianaceae namely phenophases of vegetative growth,
flowering stage, and fruit set stage, seed set, and dispersion were observed from June 2013 to January 2014
because these species occur only during this season of the year. Observations were made at monthly intervals
and during peak periods fortnightly. The data was collected by selecting 30 individuals from each species in all
the study sites. Phenological observations were marked in about 10 per cent of individual under observation and
considered it as initiated and peaked when it occurred in more than 80 per cent of individuals (Jeeshna &
Paulsamy 2011).

Diversity of Gentianaceae members in montane grasslands of Karnataka
A total of 12 species among 4 genera were occurred in 3 districts of Western Ghats of Karnataka (Table 1).
The plant species here require high amount of moisture content in the soil for seed germination. The plant
species thus grow up after the first precipitation in the month of June, slight increase in the temperature in the
month of September they undergo reproductive stage. On the onset of summer they undergo dispersion. Most of
this genus prefers montane grasslands as their natural habitat where the precipitation and wind effect is high
during monsoon. Most of these species prefer high altitude grasslands due to high precipitation and atmospheric
humidity.
Swertia lawii is one of the dominant species in the study area, which showed higher density (3.58) followed
by S. beddomi (2.50), Exacum bicolor (0.90) and S. minor with lower density (0.07). The data showed that S.
Ashwini et al. (2014) 1(3): 4348
.
Table 1. Distribution, habitat type and meso- habitat levels of study sites in montane grasslands of Karnataka.
Meso habitat level*
S.N. Locality name Altitude (in ft) Habitat type Name of species
Sn Wn Sl Hm
Exacum bicolor
Exposed E. sessiles
grasslands, soil E. petiolare
1 Kodachadri 37504000 3 3 3 3
rich areas, along Hoppea fastigiata
with grass Canscora diffusa
Swertia beddomi
Exacum bicolor
Exposed surface,
Canscora diffusa
2 Kemmannugundi 46764872 soil rich areas, 3 3 2 3
Swertia lawii
along with grass
S. beddomi
Canscora pauciflora
Exposed surface, C. decussata
iron rich rocky Swertia lawii
3 Bababuden giri 53005500 3 3 2 2
plateau, exposed S.beddomi
grasslands S. minor
Gentiana quadrifaria
Canscora diffusa
Exposed surface,
C. pauciflora
iron rich rocky
4 Mullayyana giri 60006050 3 3 3 2 C. decussata
plateau, exposed
Swertia lawii
grasslands
S. beddomi
Exposed Canscora diffusa
grasslands, soil C. perfoliata
5 Pushpa giri 4090 3 3 2 2
rich areas, along C. pauciflora
with grass C. decussata
Note: Sn- Exposure to sun, Wn- Exposure to Wind, Sl- Habitat slope, Hm- Humidity.
*
1,2,3 represents low, moderate and high levels of meso-habitat condition ranked based on ordinal scaling.
lawii species are abundant (7.17) and it is followed by Exacum sessile (5.83), S. beddomi (4.29).The frequency
of S. beddomi (0.58) and S. lawii (0.50) showed higher frequency of occurrence whereas S. minor with lower
frequency of occurrence. The equitability is the evenness with which the individuals were spread out among the
species in a community, the value in the study area is 0.79. Important value index (IVI) of S. lawii is 50.62,
which is higher when compared with all other species (Table 2).
Table 2. Species composition with their diversity parameter of the Gentianaceae members of montane
grasslands of Karnataka.
S.N. Species name Density Frequency Abundance IVI
1 Exacum bicolor 0.90 0.33 2.70 19.80
2 Exacum sessile 0.58 0.10 5.83 8.87
3 Exacum petiolare 0.33 0.08 4.00 5.97
4 Hoppea fastigiata 0.33 0.10 3.33 6.53
5 Canscora diffusa 0.67 0.25 2.67 14.77
6 Canscora perfoliata 0.22 0.12 1.86 6.01
7 Canscora pauciflora 0.13 0.10 1.33 4.66
8 Canscora decussata 0.70 0.33 2.10 17.93
9 Swertia lawii 3.58 0.50 7.17 50.62
10 Swertia beddomi 2.50 0.58 4.29 43.32
11 Swertia minor 0.07 0.07 1.00 2.90
12 Gentiana quadrifaria 0.67 0.37 1.82 18.76
Simpsons diversity index is a measure of diversity that takes into account the number of species present, as
well as the relative abundance of each species. As species richness and evenness increase, so does diversity. The
value of Simpsons index ranged between 0 and 1. With this index, 1 represents infinite diversity and 0, no
diversity (Simpson 1949). The Simpsons index of the montane grasslands of Karnataka is calculated as 0.80 and
the Dominance Index (D= 1-Simpson) of the study area is 0.192 which indicated moderate biodiversity in the
area even though these are grasslands. Shannon index (Shannon & Weiner 1963) is a diversity index, taking into
account the number of individuals as well as number of taxa, which is 1.98.
Ashwini et al. (2014) 1(3): 4348
.
Ashwini et al. (2014) 1(3): 4348
.
Phenology
The phenophases observed for the 12 species are shown in table 3.The phenophases of all these species
occurred in different time. Most of these species have responded differently to the environment based on the
elevation. The species Exacum and Swertia sprouted tillers during June. These species produced tillers during
rainfall and longer rainy days influence sprouting. A very slight raise in temperature with little shower
frequently in the month of September lead to flowering of the Exacum bicolor, fruit set starts in October where
atmospheric temperature is going to raise. In the winter months, wind blow is higher in these open grasslands
and also under fully sunny days lead to seed set and dispersion of seeds.
Whereas, other two species of Exacum such as E. sessiles and E. petiolare the photoperiod or flowering
stage started during October subsequently fruiting within a week. Seed set and dispersion of these species occur
until cool month of December. Similarly, all the Swertia species underwent vegetative phase in the month of
June as they require high amount of rainfall. The population of Swertia start blooming in the month of October
where they required high amount of sunlight to initiate the flowering. The mass blooming of these flowers make
these areas as a botanical paradise. The flowers remain blooming for 2 to 3 days. Seed set occurred, and
dispersion occurs until the month of January. Hoppea fastigiata is a small week herb found along the foothills.
The vegetative phase starts at the month of August and flowering and fruiting, and dispersion occur in the month
of October.
Gentiana quadrifaria is a small perennial, herbaceous plant with very attractive blue coloured flowers.
Vegetative phase of these plants was observed during the warm month of May when pre-monsoon showers
starts. Due to scarcity of water and desiccation, flowers of this plant were not found during February to May.
Fruiting and dispersion succeeded immediately after flowering. The members of the genus Canscora showed
their vegetative phase in the rainy month of September where they require small amount of sunlight and little
moisture. In this species, flowering phase was observed in the month of October and it followed by fruit set and
dispersal.
Preference of sunlight
Plant species of Gentianaceae prefer about 80 per cent exposed habitats followed by shade (19 per cent) or
partial shade (1 per cent) and high sun light influence for growth and flowering (Fig 2). The light is a very
necessary factor for blooming, where most of the species are ephemerals and last for few hours to one day. Only
few species like Exacum bicolor, Swertia lawii, S.beddomi and Canscora diffusa have extended their flowering
time to 2 to 4 days, where sufficient sunlight and humidity is available. We observed that the major preferred
habitats are exposed grasslands, which have sufficient amount of sunlight, humidity and wind effect.
Figure 2. Population status of different habitats of species of Gentianaceae in montane grasslands of Karnataka.
CONCLUSION
The flowers of the family Gentianaceae are well known for their attractive, colourful and varied forms. Most
of the species prefer high altitude grasslands with high amount of sun light, wind, and humidity for flower
blooming and seed dispersion and also required high amount of soil moisture content. The rock surface covered
by cryptogamic plants and tall grasses, are the most suitable habitats for species survival and distribution.
However, Exacum petiolare, Canscora diffusa, Swertia lawii, S. beddome and S minor have made adaptation to
survive on the rocky plateau where water and moisture availability is low in blooming season. Gentianaceae
members adopt exposed surfaces for the survival, because most of the species prefers anemophily and seed
dispersal by wind; it requires open exposed high elevation grasslands. Population of these species facing threat
now days because of human interference. Tourists existing activities exploit the very rare endemic plant species
Ashwini et al. (2014) 1(3): 4348
.
by trampling, littering plastic, glass, tin materials all along and invasion of many exotic plant species are the
main threats for these local land blooms. Most of these plant species have high medicinal value. Hence,
conservation of these species in their natural habitat is an urgent need.
ACKNOWLEDGEMENTS
Authors are gratefully acknowledging the financial support by the University Grants Commisition, New
Delhi (File.no.41-388/2012 (SR) for studies. Both authors are grateful to Dr. Gopalakrishna Bhat, Poorna Prajna
College, Udupi, Karnataka who have helped identification of plant species. Thanks are also due to Karnataka
Forest Department, Bengaluru for giving permission for field studies.
REFERENCES
Bhat GK (2003) Flora of Udupi, Manipal press limited. Manipal. Karnataka. 913p.
Bhatt HR & Utkarsh G (1999) Herb species diversity of Western Ghat. International Publication, Mangalore, pp.
6192 International book Distributors. In: Hussain SA & Achar KP (eds) Biodiversity of the Western Ghats
complex of Karnataka. Resource Potential and Sustainable Utilisation. Biodiversity Initiative Trust,
Mangalore, pp. 6593.
Brilliant R, Varghese VM, Paul J & Pradeepkumar AP (2012) Vegetation analysis of montane forest of Western
Ghats with special emphasis on RET species. International Journal of Biodiversity and conversation 4: 652
664.
Conservation International (2005) www.conservation.org (accessed: 23 Sep. 2014).
Gamble JS (1935) Flora of the presidency of Madras Vol 1, 2 & 3. Botanical survey of India, Calcutta.
Gowda B (2004) Vanaspathi Kosha - Plant wealth of Sringeri, Karnataka. Kalpatharu Research Academy
Publication. Bangalore, pp. 224.
Inghalhalikar S (2007) Flowers of Sahyadri. Corolla publications, Pune, India, pp. 209.
Jongman RH, Brakter CJF & Tongeren Van OFR (1987) Data analysiss in community and land scape ecology -
Pudoc, wageningen, The Netherlands.
Jeeshna MV & Paulsamy S (2011) Phenology of Exacum bicolor Roxb., an endangered medicinal herb of
Kannur and Wayanad districts, Kerala, India. Maejo International Journal of Science and Technology 5:
358364.
Misra R (1968) Ecology work book. Oxford and IBH publishing company, Calcutta, pp. 242.
Negi HN & Gadgil M (1997) Species diversity and community ecology of mosses: A case study from Grawhal
Himalaya. International Journal of ecology and environmental sciences 23: 445462.
Ramaswamy SN, Rao MR & Arkal GD (2001) Flora of Shimogha district Karnataka. Directorate of
Prasaranga, University of Mysore, Mysore.
Robin VV & Nandini R (2012) Shola habitats on sky islands: status of research on montane forests and
grasslands in Southern India. Current Science 103: 14271437
Saldanha CJ (1984) Flora of Karnataka, Vol 1 & 2. Oxford and IBH publishing co. Pvt. Ltd., New Delhi.
Shannon CE & Weiner W (1963) The Mathematical theory of communication. University of Illinois Press.
Urhana, 117 p.
Shahina PM (2014) Taxonomic studies on Gentianaceae Juss. in South India, Ph.D. Thesis. Submitted to
University of Calicut, Malappuram, Kerala, India.
Simpson EM (1949) Measurement of diversity. Nature 163: 688.
Thomas SM & Palmer MM (2007) The montane grasslands of the Western Ghats, India: Community ecology
and conservation. Community Ecology 8: 6773.
Yoganarasimhan SN & Razi BA (1981) Flora of Chikkamgaluru district, Karnataka, India. International book
distributors, Dehradun, pp. 407.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
1(3): 4961, 2014
Research article
Analysis of physico-chemical parameters, genotoxicity and

oxidative stress inducing potential of soils of some agricultural
fields under rice cultivation
Mandeep Kaur1, Rajneet Kour Soodan1, Jatinder Kaur Katnoria1, Renu Bhardwaj1,
Yogesh B. Pakade2 and Avinash Kaur Nagpal1*
1
Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar-
143005, Punjab, India
2
Institute of Himalayan Bioresource Technology (IHBT) Palampur-176061, Himachal Pradesh, India
*Corresponding Author: avnagpal@rediffmail.com [Accepted: 01 December 2014]
Abstract: In India, agricultural soil has been deteriorated by various on-going practices involving
application of chemical fertilizers, pesticides and effluents. Presently, Amritsar (Punjab), an
agricultural land, is undergoing rigorous cultivation of wheat and rice crops which consequently
increased the application of chemical pesticides and fertilizers for high yield. These agricultural
practices are not only pulling out the essential nutrients from the soil but also adding up huge
quantity of heavy metals and other dreadful contaminants. Keeping this in view, the present study
was planned to assess the physico-chemical parameters and genotoxic potential of soil of four
agricultural fields of Amritsar, India by employing Allium cepa root chromosomal aberration
assay. The responses of different antioxidative enzymes in A. cepa on exposure of Allium bulbs to
different soil samples were also analyzed. In case of physico-chemical parameters cadmium was
found more (9.7030.0 mg-1.kg-1) than the typical range (36 mg-1.kg-1). The genotoxicity in A.
cepa (treated with agricultural soil samples) revealed induction of different types of chromosomal
aberrations in both modes of treatment (in situ and root dip). Among anti-oxidative enzymes, the
activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) were low and
gluthione-S-transferase (GST) and dehydro-ascorbate reductase (DHAR) were high in treated
bulbs as compared to control A. cepa bulbs. Moreover, the results obtained in this study clearly
show harmful consequences of agricultural soils of Amritsar in terms of mitotic abnormalities as
well as their harmful effect on antioxidant defense system of crop plants cultivated at that
particular field.
Keywords: Agricultural soil - Heavy metals - Anti-oxidative enzymes - Root chromosomal
aberration assay - Allium cepa.
[Cite as: Kaur M, Soodan RK, Katnoria JK, Bhardwaj R, Pakade YB & Nagpal AK (2014) Analysis of
physico-chemical parameters, genotoxicity and oxidative stress inducing potential of soils of some
agricultural fields under rice cultivation. Tropical Plant Research 1(3): 4961]
INTRODUCTION
Soil is found to be one of the key elements which sustain life on earth. It acts as an important part of all
terrestrial systems, providing habitat for micro-organisms, plants, and animals (Deyn & Van der Putten 2005);
and also act as a storage system for several natural resources (Achazi 2002). Soil is mainly composed of
minerals, organic matter having different texture, structure, consistency, colour, chemical, biological and other
features. It forms a loose covering of mineral particles that finely cover the earth's surface (Birkeland 1999).
Soil has important ecological functions in recycling resources and has purification property as well. Soil
supports life through main five processes, biomass productivity, detoxification of pollutants, cycling of C, N, P,
S, and H2O; and also acts as carbon sink (Hansen et al. 2008, Blakeslee 2010, Lal 2004).
Enormous studies have revealed that human beings are accidentally or mistakenly exposed to different kinds
of contaminants in soil, water, air and food by different direct and indirect routes of exposure like inhalation,
Received: 04 September 2014 Published online: 31 December 2014
Kaur et al. (2014) 1(3): 4961
.
ingestion and dermal contact which result in different acute and chronic health problems (Bhatnagar 2001,
Rekha & Prasad 2006). There are about 3 million cases registered worldwide every year for pesticide poisoning
out of which 220,000 are fatal (Bolognesi 2003). Pesticide poisoning results in dreadful ailments like cancer,
chronic kidney diseases, sterility among males and females, endocrine disorders, suppression of the immune
system, neurological and behavioral disorders, especially among children (Agnihotri 1999). The increase in
chromosomal damage and reproductive abnormalities has been reported in agricultural workers (Lander et al.
2000, Bhatanagar 2001). Many other reports confirmed the alarming level of pesticide residues in water, air,
soil, food commodities and even in biological materials like human blood, fat, milk etc (Gupta 2004). The
microbial populations of soil also get affected due to drastic changes in soil pH, alkalinity and organic matter
(Zwietan 2004). Keeping this in mind, the present study was planned to analyse four soil samples collected from
rice cultivated fields of Amritsar, Punjab, India, as per following objectives:
1. Estimation of physico-chemical parameters including heavy metals viz, cadmium, chromium, nickel, zinc,
manganese and lead of the soil samples.
2. Genotoxicity assessment of soil samples using A. cepa root chromosomal aberration assay
3. Analysis of effect on activity of different anti-oxidative enzymes in A. cepa bulbs treated with soil samples.

The present study pertains to analysis of four soil samples collected from rice cultivated fields of Amritsar,
Punjab, India, with respect to their physico-chemical parameters including heavy metals (cadmium, chromium,
nickel, zinc, manganese and lead), genotoxicity by A. cepa root chromosomal aberration assay and potential to
influence activity of anti-oxidative enzymes in A. cepa bulb.
Site description and Collection of soil samples
Soils under rice cultivation from four different sites (agricultural fields) of Amritsar district of Punjab were
collected. Four sites included Site 1 (S1) Vill. Heir, Verka Block, Amritsar; Site 2 (S2) Vill. Akalgarh, Dhapian,
Teh., Baba Bakala, Amritsar; Site 3 (S3) Dera Ramdass, Amritsar and Site 4 (S4) Vill. Chabba, Amritsar (Fig.
1). Random sampling method was adopted for soil collection. Soil was collected from 56 different sites of each
agricultural field by digging soil to depth of 1520 cm (101020 cm3 approximately) and pooled together to
form one representative sample. The samples were brought to laboratory, dried at room temperature for 72 h and
finally ground into fine powder (Cabrera & Rodriguez 1999a). Washed sand was considered as negative control
for further analysis.
Figure 1. The location of the study area and distribution of the sampling sites. (S1- Verka Block; S2- Baba Bakala;
S3- Dera Ramdass; S4- Chabba Village).
Kaur et al. (2014) 1(3): 4961
.
Physico-chemical analysis
For physico-chemical analysis, the soil extract was prepared by suspending soil in distilled water in ratio of
1:5 (w/v), shaken on mechanical shaker for 12 h at room temperature. The physico-chemical properties of soil
samples were determined by following standard protocols given in Trivedi et al. (1985) with slight
modifications. pH was measured by pH meter model pH system 361 make Shimadzu. The parameters like
calcium, magnesium and alkalinity were determined titrimetrically whereas, nitrates and phosphates were
estimated using spectrophotometer (model 2202, make Systronics). The contents of sodium and potassium were
estimated by flame photometer (model CL 26 D, make ELICO).
Heavy metals analysis
Heavy metal analysis in soil samples were carried out in triplicate as given below. 1 g of soil was digested in
glass digestion tube of 250 ml along with 15 ml of nitric acid (HNO3) at 140C and the content was evaporated
till dryness. The dried sample was further treated with 3 ml of perchloric acid (HClO 4) for oxidation from the
sample solution for 30 min at 245C. The content was cooled down after digestion, filtered and final volume
was made up to 50 ml with distilled water. The heavy metals measurement was performed at Institute of
Himalayan Bioresource Technology IHBT Palampur with a Shi-madzu model AA 6300 Atomic Absorption
Spectrophotometer (Tokyo Japan). The radiation source was Hallow cathode lamps (HAMA- MATSU
PHOTONICS K.K. JAPAN) of metal (Chand et al. 2011).
Genotoxic potential
For estimation of genotoxic potential, the soil extract was prepared by suspending soil in distilled water in
ratio of 1:2 (w/v), shaken on mechanical shaker for 12 h at room temperature (Cabrera & Rodriguez 1999b).
The genotoxic potential of soil extracts was estimated by using A. cepa root chromosomal aberration assay.
Fresh and young onions were purchased from local market. The primary roots of uniform sized onion bulbs
were removed with the help of forceps. For In situ treatment, the denuded bulbs were grown directly in small
pots containing soil whereas in root dip treatment, the bulbs were placed on couplinjars filled with distilled
water for 2436 h for rooting. After 2436 h, A. cepa bulbs with freshly emerged roots of size 12 cm were
treated with five concentrations (20, 40, 60, 80 and 100 %) of the soil extract and distilled water (negative
control) for 3 h. After treatment, the bulbs were thoroughly washed, root tips were plucked and fixed in
Farmers fluid (glacial acetic acid and ethanol; 1:3). At least 9 root tips were squashed in aceto-orecin to prepare
slides. The slides were screened under microscope to score different types of aberrations taking approximately
900 dividing cells. The chromosomal aberrations were apportioned into physiological (attributed to spindle
inhibition) and clastogenic (attributed to direct breaking action on chromosomes). Different types of
physiological aberrations included laggards, vagrants, stickiness, delayed anaphases, and c-mitosis while
clastogenic aberrations included chromatin bridges and chromosomal breaks. Some physiological aberrations
such as deviation of chromosome from poles at anaphase, asteroid structure at anaphase, deviation of alignment
of chromosome at metaphase, which could not be included among any of the categories, were counted as
abnormal metaphase and abnormal anaphases under other physiological aberrations.
Anti-oxidative enzymes
The denuded bulbs were placed on different soil samples contained in small pots for 72 h under saturated
conditions. Anti-oxidative enzymes activity was checked by preparing supernatant of 1 gm of treated onion
bulb, homogenized in pestle and mortar in 3 ml of chilled phosphate buffer (0.01 M, pH 7.6) and centrifuged
(7000 rpm at 4C for 15 min). The supernatant was used for estimating total protein content (Lowry et al. 1951)
and activities of different anti-oxidative enzymes spectrophotometrically. The activity of catalase was
determined by method of Aebi (1984); Superoxide dismutase activity was estimated according to the
methodology of Kono (1978); the level of GST was determined according to protocol of Habig et al. (1974); the
enzymatic activity of DHAR was measured by method of Dalton et al. (1986) and APX activity was measured
according to the protocol of Nakano & Asada (1987).
Statistical Analysis
Arithmetic means of activation of antioxidant enzymes: CAT, SOD, GST, APX and DHAR and mean
concentrations of physico-chemical parameters and heavy metals Cd, Cu, Ni, Mn, Pb, and Zn in soil samples
were calculated. The dependence of activity of antioxidant enzymes in A. bulbs and concentrations of physico-
chemical parameters including heavy metals in soils were calculated by correlation matrix (significance level at
p<0.05). Correlation matrix was developed using Microsoft excel 2007.
Kaur et al. (2014) 1(3): 4961
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RESULTS
Table 1. Physico-chemical parameters of soil samples of four different agricultural fields of Amritsar.
Sample codes*
Parameter
S1 S2 S3 S4
Bulk density (g.cc-1) 1.880.006 1.890.010 1.010.003 1.000.003
Water holding capacity (%) 31.930.56 24.961.74 31.081.58 36.171.33
pH 8.200.058 8.200.000 8.250.000 8.140.003
Alkalinity (meq 100g-1) 1.230.033 2.330.203 2.330.033 1.200.000
Calcium (mg.g-1) 90.62.667 80.160.00 80.160.00 53.332.66
Magnesium (mg.g-1) 189.32.66 259.800.0 446.506.6 406.702.66
Nitrates (mg.g-1) 0.0150.00 0.0160.00 0.0090.00 0.0040.00
Phosphates (g.g-1) 0.6140.03 0.650.012 0.740.025 0.760.032
Potassium (mg.g-1) 0.350.005 0.260.004 0.121E-09 0.320.002
Sodium (mg.g-1) 0.130.000 0.390.015 0.100.004 0.070.002
Soil texture
Sand 74.700.13 69.100.21 55.600.38 69.920.05

Silt 0.770.319 0.330.127 0.390.035 0.280.139
(%)
Clay 24.530.23 30.560.34 44.010.34 29.780.34
Cd (3-6)** 30.01.48 25.31.78 22.00.71 9.701.08

Cu (135-270)** 58.10.12 19.20.33 28.40.15 20.80.08
Heavy metals
Ni (ND)**
(mg kg-1)
24.70.50 27.20.14 26.70.43 27.90.74

Zn (300-600)** 96.50.11 98.10.34 90.60.20 61.60.46
Mn (ND)** 345.52.2 282.51.34 422.13.6 318.51.64
Pb (250-500)** 24.80.95 19.60.36 25.00.62 22.10.36
All values are Mean SD. of 3 observations for each parameter

*S1- Soil sample from Vill Heir, Amritsar; S2- Soil sample from Vill, Akalgarh, Dhapian, Teh. Baba Bakala,
Amritsar; S3- Soil sample from Dera Ramdass, Amritsar; S4- Soil sample from Chabba Village, Amritsar; Cd-
Cadmium; Cu- Copper; Ni- Nickel; Zn- Zinc; Mn- Manganese; Pb- Lead
**
Indicates safe limits, India for heavy metals in agricultural soils (mg.kg-1) (Awasithi 2000).
Physico-chemical parameters
The results for different physico-chemical parameters are shown in table 1. Bulk density of all soils studied
was found in the optimal range (11.89 g.cc-1). Water holding capacity (WHC) was found to be low (24.96
36.17 %) in all the sites. pH was found to be slightly alkaline (8.148.25) in all the soil samples. The contents of
calcium (53.3390.6 mg.kg-1), magnesium (189.3446.50 mg.kg-1) and phosphates (0.6140.76 g.kg-1) were
found in permissible/safe limits while nitrates (0.0040.016 mg.kg-1) were found to be less in all samples. The
potassium content was found in the range of 0.120.35 mg.g-1 while sodium ranged from 0.070.39 mg.g-1 in all
soil samples studied. Soil textural composition of different soil samples showed higher concentration of sand
(55.6074.70 %) followed by clay (24.5344.01 %) whereas the concentration of silt (0.280.77 %) was found
to be very low.
Heavy metals
Soil samples collected from different agricultural fields showed varied levels of heavy metals. The content of
manganese (Mn) ranged from 282.5422.1 mg.kg-1. Following manganese, Zinc (Zn) was the second most
abundant metal determined in range of 61.698.1 mg.kg-1. The concentration of Copper (Cu) (19.258.1 mg.kg-
1
), lead (Pb) (19.625 mg.kg-1) and nickel (Ni) (24.727.9 mg.kg-1) were found to be in safe limits required in
agricultural soils while cadmium was found much higher (9.7030 mg.kg-1).
Genotoxic potential
Genotoxicity of all agricultural soil samples was assessed by employing A. cepa root chromosomal
aberration assay. Different types of physiological (stickiness, delayed anaphase, vagrants, laggards etc.) and
clastogenic (chromosomal breaks, bridges etc.) chromosomal aberrations were observed following treatments
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Among all samples, the onion bulbs treated with soil of Site 4 (S4) showed maximum content of proteins
(0.15 mg g-1) whereas bulbs treated with soil of Site 2 (S1) showed minimum content (0.075 mg g -1). Catalase
activity was found low in two sites (S3 and S4) of the four sites while higher in other two sites (S1 and S2). The
activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) were found lower and activity of
gluthione-S-transferase (GST) and dehydro-ascorbate reductase (DHAR) were found to be higher as compared
to control in A. cepa bulbs treated with all agricultural soil samples (Table 2).
Table 2. Activity of different anti-oxidative enzymes in onion bulbs treated with different agricultural soil samples.
Superoxide Glutathione-S- Ascorbate Dehydroascorbate
Catalase (CAT)
Dismutase (SOD) Transferase (GST) Peroxidase (APX) Reductase (DHAR)
Sites (U min-1.mg-1.g-1
(U min-1.mg.g-1 (U min-1.mg-1.g-1 (U min-1.mg-1.g-1 (U min-1.mg-1.g-1
.protein-1) -1 -1 -1
.protein ) .protein ) .protein ) .protein-1)
NC* 0.230.031 109198.84 0.260.055 0.600.175 0.370.062
S1 0.500.024 459.4058.58 0.870.054 0.160.012 0.860.013
S2 0.390.035 493.40104.4 0.470.018 0.110.023 0.590.025
S3 0.150.011 917.60370.6 0.520.010 0.470.185 0.640.022
S4 0.150.009 845.50242.4 0.350.027 0.160.016 0.420.031
Note: NC- Negative control (washed sand); U- Units; S1- Soil sample from Vill Heir, Amritsar; S2- Soil sample from
Vill, Akalgarh, Dhapian, The. Baba Bakala, Amritsar; S3- Soil sample from Dera Ramdass, Amritsar; S4- Soil sample
from Chabba Village, Amritsar.
DISCUSSION
Physico-chemical parameters
Vast application of chemicals like pesticides and chemical fertilizers results in deterioration of soil and crop
quality of a particular field. Bulk density of all soils studied was found in the optimal range (12 g.cc-1) which is
required for better growth of plants (1.02.0 g.cc-1). Increase in soil bulk density due to deforestation and
subsequent cultivation period was earlier reported by many scientists (Mulugeta et al. 2005, Mojiri et al. 2012).
Water holding capacity depicts good physical condition of soil. In present study, water holding capacity (WHC)
was found to be low (24.9636.17 %) in all the sites as compared to suitable range (6080%) obtained in other
soils studied (Castillo & Torstensson 2007). The low WHC in present study can be due to its sandy texture
which results in limited storage of water. The similar results were reported earlier by Longwell et al. (1963) in
Tennessee soils. pH is an important property which can directly affect the solute concentration and absorption in
the soil and also assure maximum availability of essential nutrients to plants required for growth and
development. The pH values for different soil samples observed in the present study ranged from 8.148.25.
These results are in conformity with earlier studies on the soils of Kano Urban agricultural land (Dawaki et al.
2013) and agricultural soil of Vishakapatnam (Srinavas & Kumar 2001). However in some studies, this range
has been considered higher as compared to the ideal range for rice cultivated soils i.e. 5.56.5 (Focht 1979,
Bandara et al. 2005). The contents of calcium, magnesium and phosphates were found under permissible limits
while nitrates were found to be less in all samples as shown in table 1. Decrease in nitrate content in our study
can be attributed to its sandy texture as reported earlier (Gaines & Gaines 1994). Other important factor which
can be responsible for low nitrates in agricultural soils can be successive cultivation as reported by Eyayu et al.
(2009). Also, high nitrate contents in different agricultural soils were reported in previous studies (Rai et al.
2011). The potassium content was found in the range of 0.120.35 mg.g-1 while sodium ranged from 0.070.39
mg.g-1 in all soil samples studied. Almost similar results for potassium and sodium were earlier reported by
Udotong et al. (2008) in soils of wetlands of Eket, Nigeria. Soil textural composition of different soil samples
showed higher concentration of sand followed by clay whereas the concentration of silt was found to be very
low. The same variation in soil texture of different soils has also been reported earlier (Mohapatra et al. 1996).
Similarly, many other authors also reported lower clay content in cultivated lands (Eyayu et al. 2009, Mojiri et
al. 2012) might be due to selective removal of clay from the surface by erosion.
Heavy metals
The total concentrations of the heavy metals in agricultural soils are shown in table 1. These are important
and very essential micronutrients required for healthy plant growth (Delbari & Kulkarni 2011). Both manganese
and zinc were found high but under safe limits (Awashthi 2000). Similar results were reported in agricultural
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soils of Abobo area, Western Ethopia (Yitbarek et al. 2013). The concentration of Copper (Cu), lead (Pb) and
nickel (Ni) were found to be in permissible limits. Cadmium (Cd) was found much higher as compared to its
safe limits required for any agricultural soil as given by Awashthi (2000). As cadmium is highly soluble in soil
and is extremely toxic in its nature so its presence in the soil is totally undesirable and harmful. In present study,
the increase in cadmium can be due to excessive use of phosphate based fertilizers containing high content of
Cd as earlier reported by McLaughlin et al. (1996). Moreover, many scientists in the past studies had witnessed
significant increases in cadmium in fertilized soils as compared to unfertilized ones (Williams & David 1973,
Mann et al. 2002). High cadmium toxicity/stress may sometimes leads to death of plants as reported in seedlings
of bean (Mo & Li 1992). High amounts of heavy metals especially Cd and Pb in the plants adversely affect the
absorption and transport of essential elements, disturb the metabolism, and showed direct impact on growth and
reproduction (Xu & Shi 2000). Metal uptake by plants can be affected by metal concentration in soils, soil pH,
cation exchange capacity, organic matter content, types and varieties of plants, and plant age (Alloway & Davies
1971).
Genotoxic potential
In case of genotoxicity study, different types of physiological (stickiness, delayed anaphase, vagrants,
laggards) and clastogenic (chromosomal breaks, bridges) chromosomal aberrations were observed following
treatments (in-situ and root dip) with different soil samples studied using Allium cepa assay (Fig. 2 & 3). Lah et
al. (2008) also evaluated the genotoxicity of soil from six different sites of agricultural and industrial areas using
Tradescantia MCN assay. In present study, genotoxicity has shown negative correlation with lead and positive
with sodium content in the soil (Table 3). Since, lead is not soluble in water, thus it can be possible that lead is
not fully translocated into plant system. Due to increase in sodium content, lead can be possibly replaced by
sodium (important nutrient) which reached plant easily. Besides, the mobility, solubilty and bioavailability of
lead in soil is largely controlled by complex interactions governed by several biogeochemical factors (Dumat et
al. 2006, Kopittke et al. 2008, Lawal et al. 2010, Vega et al. 2010, Arias et al. 2010, Bi et al. 2010, Liu et al.
2010), which may resist the availability of lead in the plants. So this may be the main reason that present soils
under study do not showed lead toxicity and thus induced moderate genotoxic effect in all soil samples.
Figure 2. Percent aberrant cells in Allium cepa root tips following in situ and root dip treatment.
It was reported that heavy metals show deleterious effects on cell division of plants (Mo & Li 1992). Duan
& Wang (1995) observed in his study that when beans were treated with low doses of Cd, Pb and Zn, the period
of cell division elongates while with increased dose period remain shorter. Genotoxicity caused by heavy metals
in plants affects the synthesis and the duplication of DNA and chromosomes by inducing different chromosomal
aberrations. Pohren et al. (2013) also reported different types of chromosomal abnormalities in barley and A.
cepa under heavy metals stress respectively. In our study cadmium was found higher than the safe limits as
compared to other toxic metals. Cadmium generally has a capability to bind with the nucleotides causing direct
damage to DNA by modifications in base structure and ultimately leads to lesions, DNA strand breaks,
exchanges of sister chromatids, destruction of DNA-proteins crosslinks, effect on activity of different anti-
oxidative enzymes and inhibition of DNA repair enzymes (Badisa et al. 2007, Lin et al. 2007, Markovska et al.
2009, Unyayar et al. 2010). The same results were observed by Zhao & Mo (1997) in his study where he found
Kaur et al. (2014) 1(3): 4961
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that continuous exposure of beans, garlic and A. cepa with Cd, Pb, Hg resulted in different mitotic abnormalities
viz., polyploidy, C-karyokinesis, chromosomal bridges, rings, fragments, chromosomal fusion, micro-nucleated
cells and nuclear decomposition.
Figure 3. Allium cepa root tip cells treated with different soil samples showing spectrum of aberrations: AC,
Normal stages of cell division; DI, Different types of chromosomal aberrations.
A very common deleterious effect of pollutants is to produce high amounts of free radicals and other reduced
oxygen species. In plants, the main sources of ROS (reactive oxygen species) production include pathogens,
heavy metals, herbicides, air pollutants, drought and UV-B rays. ROS has been identified as the superoxide
radical (O2-), hydroxyl radical (-OH), hydroperoxyl radical (HO2*), hydrogen peroxide (H2O2), alkoxy radical
(RO), singlet oxygen (1O2) and excited carbonyl (RO*) which are found to be highly cytotoxic to plants
(Karuppanapandian et al. 2011, Vellosillo et al. 2010). ROS production and removal must be controlled strictly
in order to avoid oxidative stress in plants. High levels of these reactive oxygen species can cause irreplaceable
damage to biomolecules such as lipids, proteins and DNA. Plants possess complex anti-oxidative defence
system which comprised of enzymatic (SOD, CAT, APX, GST, DHAR, MDHAR, detoxifying lipid
peroxidation (LP) products like ascorbate and glutathione) and non-enzymatic (tocopherols, carotenoids and
phenols) components which play very important role in scavenging these ROS. These systems are mostly
located in organelles like chloroplasts, mitochondria and peroxisomes. Enzymatic components can convert the
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Kaur et al. (2014) 1(3): 4961
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potentially harmful superoxide radical and hydrogen peroxide to water and molecular oxygen, thus preventing
cellular damage (Scandalios 2005).
Catalase showed low activity in two sites (S3 and S4) while high in other two sites (S1 and S2) as compared
to control. Heavy metal stress leads to accumulation of H2O2 that can result in inactivation of catalase activity
(Olteanu et al. 2011). Decrease in CAT activity with high content of cadmium was reported by Fornazier et al.
(2002) and Sandalio et al. (2001). Dey et al. (2007) also reported a decline in CAT activity in wheat seedlings
grown in the presence of cadmium chloride and lead nitrate and Allium cultivars under the stress of soil moisture
respectively. Vitoria et al. (2001) determined increase in CAT activity in radish following exposure to high
cadmium concentrations. Increase in CAT activity is adapted possibly to overcome the damage caused to tissue
metabolism by toxic levels of reducing H2O2 (Karuppanapandian et al. 2011). These two reports confirmed that
in present soils studied, cadmium may be the main culprit which leads to this type of CAT nature. Other reports
stated that other than heavy metals, salinity and drought conditions may be the other reasons for decrease in
CAT activity (Boo & Jung 1999, Karuppanapandian & Manoharan 2008, Hojati et al. 2010).
Superoxide dismutase activity was found lower in present study. Similar reduction in activity of SOD was
earlier reported in pine roots due to cadmium toxicity (Schutzendubel et al. 2001). Contradictory to our results,
Sharma et al. (2010) found increase in activity of SOD in plants of family Brassicaceae in response to heavy
metal stress whereas Dixit et al. (2001) reported similar findings in pea roots and leaves. It was reported that
cadmium increases the binding of metal ions to sulphydryl group of enzymes which ultimately increase the
phytotoxicity of metals (Van Assche & Clijsters 1990) and resulted in low activity of SOD (Somashekaraiah et
al. 1992, Guan et al. 2009). In another study, inefficiency of SOD in ROS scavenging has reported in rice crops
under water deficit conditions (Boo & Jung 1999). Therefore in our study cadmium can be one of the factors
that played a major role in inducing low activities of SOD.
The activity of gluthione-S-transferase (GST) was found high as compared to control in the present study.
According to Marrs (1996), GST is involved in detoxification of herbicides, heavy metals and pathogen attack.
Fatima & Ahmad (2005) also determined increase in GST activities in A. cepa under high stress of heavy
metals. During stress conditions, lipid peroxidation products like hydroperoxides, epoxides, organic
hydroperoxides and oxidative products of DNA degradation acts as substrates for GST. All these products get
conjugated to glutathione and ultimately detoxified and get stored in vacuoles of cytosol. High activity of
scavenging these toxic peroxidation products in barley vacuoles via GST were described earlier (Tommasini et
al. 1993). Apart from this, ascorbate peroxidase also mediated this conjugation and detoxification of oxidized
glutathione to unsaturated phenylpropanoids (cinnamic and coumaric acids) in plants (Dean & Devarenne
1997).
All soils studied showed low activity of ascorbate peroxidase (APX). Perioxidase catalyses the oxidation of
phenols, amines and act as biomarkers of sublethal toxicity caused by heavy metals so called as stress enzyme
(Zhang et al. 2007). Low activity of APX with exposure to high content of cadmium in pine roots was reported
earlier (Schutzendubel et al. 2001). According to Schutzendubel & Polle (2002), cadmium induced inhibition
can be associated with high H2O2 accumlation and growth retardation as he reported in popular roots. Fatima &
Ahmad (2005) observed increase in APX activity in A. cepa under high stress of heavy metals. Boo & Jung
(1999) reported low activity of APX in detoxification of ROS in rice under water deficit conditions. Increase in
DHAR levels as compared to control were observed in our study. Increase in DHAR activity was previously
reported in many plants in response to stresses like drought, metal toxicity and chilling effect (Sharma & Dubey
2005 & 2007, Yoshida et al. 2006, Maheshwari & Dubey 2009). Contradictory to our findings, Fatima &
Ahmad (2005) reported no significant change in DHAR activity in A. cepa under high stress of heavy metals.
CONCLUSION
Present study showed clearly that cadmium act as one of the major factor governing genotoxicity in A. cepa
root tip cells by induction of different mitotic abnormalities and showed significant variations in the antioxidant
enzymes of A. cepa. It can be emphasized that the A. cepa test system responds to contaminants that are existing
in the areas of study. A. cepa test system was found to be quick, simple, highly sensitive and capable of
identifying genotoxicity of soil samples. This test system can be used as useful biomarker/ indicator for the
detection of pollutants in the any ecosystems viz., air, water and soil. This information can be used for
generating developmental strategies for soil management and as well as in implementation of risk assessment
procedures in future.
ACKNOWLEDGEMENTS
Kaur et al. (2014) 1(3): 4961
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Thanks are due to University Grants Commission (UGC) for financial assistance. The authors wish to thank
Dr. P.S Ahuja, Director, Institute of Himalayan Bioresource Technology (IHBT) for permission to carry out
heavy metal analysis.
REFERENCES
Achazi RK (2002) Invertebrates in risk assessment development of a test battery and of short term biotests for
ecological risk assessment of soil. Journal of Soils & Sediments 2: 174178.
Aebi H (1984) Catalase in Vitro. In: Colowick SP, Kaplan NO (eds) Methods in Enzymology. Acadamic Press,
Florida. 105, U.S.A., pp. 114121.
Agnihotri NP (1999) Pesticides: safety evaluation and monitoring. AICRP on Pesticide Residues: Division of
Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi, pp. 1173.
Alloway BJ & Davies BE (1971) Trace element content of soils affected by base metal mining in Wales.
Geoderma 5: 197208.
Arias JA, Peralta-Videa JR, Ellzey JT, Ren M, Viveros MN & Gardea-Torresdey JL (2010) Effects of Glomus
deserticola inoculation on Prosopis: enhancing chromium and lead uptake and translocation as confirmed by
X-ray mapping, ICP-OES and TEM techniques. Environmental & Experimental Botany 68: 139148.
Awashthi SK (2000) Prevention of Food Adulteration Act No. 37 of 1954. Central and State Rules as Amended
for 1999; Ashoka Law House: New Delhi, India, pp. 2000.
Badisa VLD, Latinwo LM, Odewumi CO, Ikediobi CO, Badisa RB, Ayuk-Takem LT, Nwoga J & West J
(2007) Mechanism of DNA Damage by Cadmium and Interplay of Antioxidant Enzymes and Agents.
Environmental Toxicology 22: 144151.
Bandara WMJ, Kumaragamage D, Wickramasinghe DB & Weerawarne SBA (2005) A site specific fertilizer
management strategy to increase the rice yield in LCIZ. Journal of Soil Science Society of Sri Lanka 17: 32-
44.
Bhatanagar VK (2001) Pesticides pollution: Trends and Perspectives, ICMR Bulletin 31: 18788.
Bi X, Ren L, Gong M, He Y, Wang L & Ma Z (2010) Transfer of cadmium and lead from soil to mangoes in an
uncontaminated area, Hainan Island, China. Geoderma 155: 115120.
Birkeland PW (1999) Soils and Geomorphology Vol 164 (3rd edition). New York: Oxford University Press,
U.S.A., pp. 772773.
Blakeslee TR (2010) Greening deserts for carbon credits. Available from:
http://www.renewableenergyworld.com/rea/news/article/2010/02/greening-deserts-for-carbon-credits
(accessed: 23 Oct. 2012).
Bolognesi C (2003) Genotoxicity of pesticides: A review of human biomonitoring studies. Mutation Research
543: 251272.
Boo YC & Jung J (1999) Water deficit-induced oxidative stress and antioxidtive defences in rice plants. Journal
of Plant Physiology 155: 255261.
Cabrera GL & Rodriquez DMG (1999a) Genotoxicity of soil from farm land irrigated with wastewater using
three plant bioassays. Mutation Reserach 426: 211214.
Cabrera GL & Rodriquez DMG (1999b) Genotoxicity of leachates from a landfill using three plant bioassays.
Mutation Research 426: 207210.
Castillo MD & Torstensson L (2007) Effect of biobed composition moisture, and temperature on the
degradation of pesticides. Journal of Agriculture and Food Chemistry 55: 57255733.
Chand P, Sharma R, Prasad R, Sud RK & Pakade YB (2011) Determination of essential and toxic metals and its
transversal pattern from soil to Tea Brew. Food and Nutrition Sciences 2: 11601165.
Dalton DA, Russell SA, Hanus FJ, Pascoe GA & Evans HJ (1986) Enzymatic reactions of ascorbate and
glutathione that prevent peroxide damage in soybean root nodules. Proceedings of the National Academy of
Sciences 83: 38113815.
Dawaki UM, Dikko AU, Noma SS & Aliyu U (2013) Heavy Metals and Physicochemical Properties of Soils in
Kano Urban Agricultural Lands. Nigerian Journal of Basic and Applied Sciences 21: 239246.
Dean JV & Devarenne TP (1997) Peroxidase-mediated conjugation of glutathione to unsaturated
phenylpropanoids-evidence against glutathione S-trasferase involvement. Physiologia Plantarum 99: 271
278.
Delbari SA & Kulkarni DK (2011) Seasonal varations in heavy concentrations in agricultural soils in Teheran-
Iran. Bioscience Discovery 2: 333340.
Kaur et al. (2014) 1(3): 4961
.
Dey SK, Dey J, Patra S & Pothal D (2007) Changes in the antioxidative enzyme activities and lipid peroxidation
in wheat seedlings exposed to cadmium and lead stress. Brazilian Journal of Plant Physiology 19: 5360.
Deyn GBD & Van der Putten WH (2005) Linking aboveground and belowground diversity. Trends in Ecology
& Evolution 20: 625633.
Dixit V, Pandey V & Shyam R (2001) Differential oxidative responses to cadmium in roots and leaves of pea
(Pisum sativum L cv. Azad). Journal of Experimental Botany 52: 11011109.
Duan C & Wang H (1995) Studies on the cell gene-toxicity of heavy metals to beans and micro-nuclear
techniques. Acta Botanica Sinica 37: 1424.
Dumat C, Quenea K, Bermond A, Toinen S & Benedetti MF (2006) Study of the trace metal ion influence on
the turnover of soil organic matter in cultivated contaminated soils. Environmental Pollution 142: 521529.
Eyayu M, Heluf G, Tekaliign M & Mohammed A (2009) Effects of land-use change on selected soil properties
in the Tera Gedam Catchment and adjacent agroecosystems, north-west Ethiopia. Ethiopian Journal of
Natural Resources 11: 3562.
Fatima RA & Ahmad M (2005) Certain antioxidant enzymes of Allium cepa as biomarkers for the detection of
toxic heavy metals in wastewaters. Science of the Total Environment 346: 256273.
Focht DD (1979) Microbial kinetics of nitrogen losses in flooded soils. In: Nitrogen and Rice. International Rice
Research Institute, Manila, Philippines. pp. 119135.
Fornazier RF, Ferreira RR, Vitria AP, Molina SMG, Lea PJ & Azevedo RA (2002) Effects of cadmium on
antioxidant enzyme activities in sugar cane. Plant Biology 45: 9197.
Gaines TP & Gaines ST (1994) Soil texture effect on nitrate leaching in soil percolates. Communications in Soil
Science and Plant Analysis 25: 25612570.
Guan Z, Chai T, Zhang Y, Xu J & Wei W (2009) Enhancement of Cd tolerance in transgenic tobacco plants
overexpressing a Cd-induced catalase cDNA. Chemosphere 76: 623630.
Gupta PK (2004) Pesticide exposure-Indian scene. Toxicology 198: 8390.
Habig WH, Pabst MJ & Jakoby WB (1974) Glutathione S-Transferases. The first enzymatic step in mercapturic
acid formation. The Journal of Biological Chemistry 249: 71307139.
Hansen J, Sato M, Kharecha P, Beerling D, Berner R, Masson-Delmotte V, Pagani M, Raymo M, Royer DL and
Zachos JC (2008) Target atmospheric CO2: Where should humanity aim? The Open Atmospheric Science
Journal 2: 217231.
Hojati M, Modarres-Sanavy SAM, Karimi M & Ghanati F (2010) Responses of growth and antioxidant systems
in Carthamus tinctorius L. under water deficit stress. Acta Physiologiae Plantarum 33: 105112.
Karuppanapandian T & Manoharan K (2008) Uptake and translocation of tri- and hexa-valent chromium and
their effects on black gram (Vigna mungo L. Hepper cv. Co4) roots. Journal of Plant Biology 51:192201.
Karuppanapandian T, Wang HW, Prabakaran N, Jeyalakshmi K, Kwon M, Manoharan K & Kim W (2011) 2,4-
dichlorophenoxyacetic acid-induced leaf senescence in mung bean (Vigna radiata L. Wilczek) and
senescence inhibition by co-treatment with silver nanoparticles. Plant Physiology and Biochemistry 49: 168
177.
Kono Y (1978) Generation of superoxide radical during auto oxidation of hydroxylamine and an assay for
superoxide dismutase. Archives of Biochemistry and Biophysics 186: 189195.
Kopittke PM, Asher CJ, Kopittke RA & Menzies NW (2008) Prediction of Pb speciation in concentrated and
dilute nutrient solutions. Environmental Pollution 153: 548554.
Lah B, Vidic T, Glasencnik E, Cepeljnik T, Gorjanc G & Logar RM (2008) Genotoxicity evaluation of water
soil leachates by Ames test, comet assay, and preliminary Tradescantia micronucleus assay. Environmental
Monitoring and Assessment 139: 107118.
Lal R (2004) Soil carbon sequestration impacts on global climate change and food security. Science 304: 1623
1627.
Lander F, Knudsen LE, Gamborg MO, Jarventaus H & Norppa H (2000) Chromosome aberrations in pesticide-
exposed green house workers. Scandinavian Journal of Work, Environment & Health 21: 283288.
Lawal O, Sanni A, Ajayi I & Rabiu O (2010) Equilibrium, thermodynamic and kinetic studies for the
biosorption of aqueous lead (II) ions onto the seed husk of Calophyllum inophyllum. Journal of Hazardous
Materials 177: 829835.
Lin A, Zhang Xu, Chen Mei & CAO Q (2007) Oxidative stress and DNA damages induced by cadmium
accumulation. Journal of Environmental Sciences 19: 596602.
Kaur et al. (2014) 1(3): 4961
.
Liu X, Peng K, Wang A, Lian C & Shen Z (2010) Cadmium accumulation and distribution in populations of
Phytolacca americana L. and the role of transpiration. Chemosphere 78: 11361141.
Longwell TJ, Parks WL & Springer ME (1963) Moisture characteristics of Tennessee Soils. University of
Tennessee Agricultural Experiment Station Bulletins 367, pp. 46. (Available from:
http://trace.tennessee.edu/utk_agbulletin/303).
Lowry OH, Rosebrough NJ, Farr A & Randall RJ (1951) Protein measurement with the Folin phenol reagent.
The Journal of Biological Chemistry 193: 205220.
Maheshwari R & Dubey RS (2009) Nickel-induced oxidative stress and the role of antioxidant defence in rice
seedlings. Plant Growth Regulation 59: 3749.
Mann SS, Rate AW & Gilkes RJ (2002) Cadmium accumulation in agricultural soils in Western Australia.
Water, Air, & Soil Pollution 141: 281297.
Markovska YK, Gorinova NI, Nedkovska MP & Mtteva KM (2009) Cadmium-induced oxidative damage and
antioxidant responses in Brassica juncea plants. Biologia Plantarum 53(1): 151154.
Marrs KA (1996) The functions and regulations of glutathione S-transferases in plants. Annual Review of Plant
Physiology and Plant Molecular Biology 47: 127158.
McLaughlin MJ, Tiller KG, Naidu R & Stevens DP (1996) Review: The behavior and environmental impact of
contaminants in fertilizers. Australian Journal of Soil Research 34: 154.
Mo W & Li M (1992) Effects of Cd on the cell division of root tip in bean seedlings. Bulletin of Botany 9: 30
34.
Mohapatra D, Das B, Sahoo PK & Chakraborthy V (1996) Metal pollution in harbor sediments of Pardip port,
East coast of India. Indian Journal of Environmental Protection 16: 724729.
Mojiri A, Aziz HA & Ramaji A (2012) Potential decline in soil quality attributes as a result of land use change
in a hillslope in Lordegan, Western Iran. African Journal of Agricultural Research 7: 577582.
Mulugeta L, Karltun E & Olsson M (2005) Assessing soil chemical and physical property responses to
deforestation and subsequent cultivation in smallholders farming system in Ethiopia. Agriculture,
Ecosystems & Environment 105: 373386.
Nakano Y & Asada K (1987) Purification of ascorbate peroxidase from spinach chloroplasts: its activation in
ascorbate-depleted medium and reactivation by mono-dehydroascorbate radical. Plant and Cell Physiology
28: 131140.
Olteanu Z, Oprica L, Truta E & Zamfirache MM (2011) Behaviour of antioxidative enzymes and of soluble
protein in wheat seedlings after lead-induced stress. Annals of the ''Alexandru Ioan Cuza'' University Sect.II
a. Genetics and Molecular Biology 01/2011; 12(2): 7585.
Pohren RdeS, da Costa TC & Vargas VMF (2013) Investigation of sensitivity of the Allium cepa test as an alert
system to evaluate the genotoxic potential of soil contaminated by heavy metals. Water Air Soil Pollution
224: 1460.
Rai S, Chopra AK, Pathak C, Sharma DK, Sharma R & Gupta PM (2011) Comparative study of some
physicochemical parameters of soil irrigated with sewage water and canal water of Dehradun city, India.
Archives of Applied Science Research 3: 318325.
Rekha SN & Prasad RN (2006) Pesticide residue in organic and conventional foodrisk analysis. Chemical
Health and Safety 13: 1219.
Sandalio LM, Dalurzo HC, Gomez M, Romero-Puertas MC & del Ro LA (2001) Cadmium- induced changes in
the growth and oxidative metabolism of pea plants. Journal of Experimental Botany 52: 21152126.
Scandalios JG (2005) Oxidative stress: molecular perception and transduction of signals triggering antioxidant
gene defenses. Brazilian Journal Of Medical and Biological Research 38: 9951014.
Schutzendubel A & Polle A (2002) Plant responses to abiotic stresses: heavy-metal induced oxidative stress and
protection by mycorrhization. Journal of Experimental Botany 53: 13511365.
Schutzendubel A, Schwanz P, Teichmann T, Gross K, Langenfeld-Heyser R, Godbold A & Polle A (2001)
Cadmium-induced changes in antioxidative systems, H2O2 content and differentiation in pine (Pinus
silvestris) roots. Plant Physiology 127: 887898.
Sharma I, Pati PK & Bhardwaj R (2010) Regulation of growth and antioxidant enzyme activities by 28-
homobrassinolide in seedlings of Raphanus sativus L. under cadmium stress. Indian Journal of Biochemistry
& Biophysics 47: 172177.
Sharma P & Dubey RS (2005) Lead toxicity in plants. Brazillian Journal of Plant Physiology 17: 3552.
Kaur et al. (2014) 1(3): 4961
.
Sharma P & Dubey RS (2007) Involvement of oxidative stress and role of antioxidative defense system in
growing rice seedlings exposed to toxic concentrations of aluminium. Plant Cell Reports 26: 20272038.
Somashekaraiah BV, Padmaja K & Prasad ARK (1992) Phytotoxicity of cadmium ions on germinating
seedlings of mung bean (Phaseolus vulgaris): involvement of lipid peroxides in chlorophyll degradation.
Physiologia Plantarum 85: 8589.
Srinavas K & Kumar S (2001) Physico-chemical characteristics of agricultural soils of Vishakhapatnam. Indian
Journal of Environmental Protection 21: 822824.
Tommasini R, Martinoia E, Grill E, Dietz KJ & Amrhein N (1993) Transport of oxidised glutathione into barley
vacuoles: evidence for the involvement of glutathione S-conjugate ATPase. Zeitschrift fr kologie und
Naturschutz 48C: 867871.
Trivedi RK, Goel PK & Trisal CL (1985) Aquatic ecosystem. In: Practical Methods in Ecology and
Environmental Sciences. Enviro Media Publications, Karad, India, pp. 57113.
Udotong IM, Joh OUM & Udotong IR, (2008) Microbiological and physicochemical studies of wetland soils in
Eket, Nigeria. World Academy of Science, Engineering and Technology 20: 837842.
Unyayar S, Guzel Deger A, Celik A, Cekic, FO & Cevik, S (2010) Cadmium-induced antioxidant status and
sister-chromatid exchanges in Vicia faba L. Turkish Journal of Biology 34: 413422.
Van Assche F & Clijsters H (1990) Effects of metals on enzyme activity in plants. Plant, Cell & Environment
13: 195206.
Vega F, Andrade M & Covelo E (2010) Influence of soil properties on the sorption and retention of cadmium,
copper and lead, separately and together, by 20 soil horizons: comparison of linear regression and tree
regression analyses. Journal of Hazardious Materials 174: 522533.
Vellosillo T, Vicente J, Kulasekaran S, Hamberg M & Castresana C (2010) Emerging complexity in reactive
oxygen species production and signaling during the response of plants to pathogens. Plant Physiology 154:
444448.
Vitoria AP, Lea PJ & Azevedo RA (2001) Antioxidant enzymes responses to cadmium in radish tissues.
Phytochemistry 57: 701710.
Williams CH & David DJ (1973) The effect of superphosphate on the cadmium content of soils and plants.
Australian Journal of Soil Research 11: 4356.
Xu Q & Shi G (2000) The toxic effects of single Cd and interaction of Cd with Zn on some physiological index
of Oenanthe javanica (Blume) DC. Journal of Nanjing Normal University (Natural Science Edition) 23(4):
97100.
Yitbarek T, Gebrekidan H, Kibret K & Beyene S (2013) Impacts of land use on selected physicochemical
properties of soils of Abobo area, western Ethiopia. Agriculture, Forestry and Fisheries 2: 177183.
Yoshida S, Tamaoki M, Shikano T, Nakajima N, Ogawa D, Ioki M, Aono M, Kubo A, Kamada H, Inoue Y &
Saji H (2006) Cytosolic dehydroascorbate reductase is important for ozone tolerance in Arabidopsis
thaliana. Plant Cell Physiology 47: 304308.
Zhang F, Wang Y, Lou Z & Dong J (2007) Effect of heavy metal stress on antioxidative enzymes and lipid
peroxidation in leaves and roots of two mangrove plant seedlings (Kandelia candel and Bruguiera
gymnorrhiza). Chemosphere 67: 4450.
Zhao B & Mo H (1997) Detoxification of ascorbic acid and molysite on the root growth of garlic under
cadmium pollution. Journal of Wuhan Botanical Research 15: 167172.
Zwietan VL (2004) Impacts of fertilizers on soil biota. In: Lines-Kelly R (ed) Soil Biology in Agriculture.
Proceedings of a workshop on current research into soil biology in agriculture. Department of Primary
Industries. Tamworth, NSW, pp. 7279.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
1(3): 6271, 2014
Research article
Assessment of forest structure and woody plant regeneration on

ridge tops at upper Bhagirathi basin in Garhwal Himalaya
C. M. Sharma, Ashish K. Mishra*, Om Prakash, Suchita Dimri and Pratibha Baluni
Department of Botany, HNB Garhwal University, Srinagar Garhwal-246174, India
*Corresponding Author: ashishmishramlg@gmail.com [Accepted: 05 December 2014]
Abstract: Mechanism to maintain species coexistence in ridge top due to drastic climatic variation
could be assessed by their forest structure and composition studies. Variations in forest
composition, structure and their regeneration status are described here for four different ridge top
sites of the Garhwal Himalaya. A total of 2439 individuals were recorded in the study area which
belonged to 17 tree species in 16 genus and 9 families. The most abundant family was Pinaceae
whereas Ericaceae and Rosaceae were found as co-dominant families. Simpson dominance value
was highest on Sukhee top (0.84), moderate in Harshil (0.76) whereas lowest in Raithal (0.65).
The total stem density was highest (710 stems ha-1) at the Harshil and lowest (552 stems ha-1) at
the Sukhee top, and for total basal cover, the highest value (92.07 m2.ha-1) was recorded in Dharali
whereas the lowest value (75.18 m2.ha-1) was found at Harshil ridge top. Importance Value Index
showed that Abies spectabilis, Betula utilis, Cedrus deodara, Pinus wallichiana, Acer caesium and
Quercus semecarpifolia are the dominant species of the ridge tops. Taxus baccata, Acer
acuminatum, Aesculus indica, Juglans regia, Ilex dipyrena and Sorbus cuspidata were reported
with poor regeneration status. The study seeks to understand the current forest composition of the
ridge top but also may be helpful in predicting their adaptation to the climatic changes.
Keywords: Importance value index - Phyto-sociology - Ridge top - Species richness.
[Cite as: Sharma CM, Mishra AK, Prakash O, Dimri S & Baluni P (2014) Assessment of forest structure and
woody plant regeneration on ridge tops at upper Bhagirathi basin in Garhwal Himalaya. Tropical Plant Research
1(3): 6271]
INTRODUCTION
Numerous studies provide evidence for the ecological responses to recent climate change (Rosenzweig et al.
2007). Climate-induced species range shifts have been reported along altitudinal (Cannone et al. 2007, Pauli et
al. 2007, Holzinger et al. 2008) and latitudinal gradients (Lesica & McCune 2004, Walther et al. 2005, Lemoine
et al. 2007). However, again not all the species seem equally responsive. It is expected that changing climate
may shift the vegetation towards the ridge tops which may change the composition of the forests in near future
(Singh et al. 2012). The ridge tops (having uniform environmental conditions) are the proper places, where the
effects of climate change can be compared and monitored to predict the future migration of species. As high
altitudinal vegetation are directly influenced by global warming (Korner 2003) so these habitats are therefore
considered very sensitive to climatic change (Theurillat & Guisan 2001). Composition of vegetation and its
analysis on ridge tops can effectively predict the influence of climate change on migration of woody species.
The forest vegetation is positively correlated to changes in microenvironments, which are variable on different
slope and in aspects. Plant life at high elevation is mostly governed by abiotic factors like temperature and snow
fall so ridge top vegetation composition greatly affected by environmental factors (Kammer & Mohl 2002).
Regeneration is a key process deciding the floristic composition of the community. It refers to recruitment,
survival and growth of seedlings or sprouts (Lalfakawma 2010). The wealth of forest and the future composition
of the forests depend on the potential regenerative status of tree species within a forest stand in space and time
(Henle et al. 2004). Regeneration depends on the ability of a species to initiate new seedling and sapling;
survival and then the ability of seedling and sapling to grow. Presence of sufficient number of seedlings,
saplings, and young trees in a given population indicate a successful regeneration (Saxena & Singh 1984).
Forest health and viability is observed by population structure like presence of sufficient number of seedlings,
saplings and young trees in a given population (Pokhriyal et al. 2010) and the number of seedling of any species
can be considered as the regeneration potential of that species (Negi & Nautiyal 2005). The density of species
Sharma et al. (2014) 1(3): 6271
.
regeneration is expected to vary spatially due to forest structure and phyto-geographical condition (Ward et al.
2006). A successful regeneration is indicated by. Natural regeneration is a central component for tropical forest
ecosystem dynamics (Getachew et al. 2010) and is essential for preservation and maintenance of biodiversity
(Rahman et al. 2011). Forest phytosociological assessment is very helpful to understand the status of tree
population, regeneration and diversity for conservation purposes (Bajpai et al. 2012, Mishra et al. 2013a). In
forest management, regeneration study show current status and hints the future of any forest (Mishra et al.
2013b). Climate has a large influence on plant recruitment (Adler & Lambers 2008), whereas; regeneration
patterns of species population can address climate change by adaptive evolution or by migrating association to
survive in their favourable climate. It is need of the time to understand evolution and associated migration
potential of different life forms and their adaption to climate change and survival ship (Woodward & Kelly
2008), as forests are greatly affected by changes in climate water availability and temperature (Breckle 2002).
Forest composition reflects population structure and deciding the regeneration potential of tree species.
Although several studies on the structure of plant communities, and productivity on various aspects of temperate
Himalayan forests have been carried out by numerous workers but ridge top forest structure and regeneration
still needs to be evaluated. In Garhwal Himalayan region, fragmentary and scanty descriptions are available on
the impact of Climate Change on vegetation and species migration. However, almost nothing is known about the
response of forest ecosystems to warming temperatures due to lack of base line data from ridge top areas.
Keeping in view the aforesaid facts, the present study was undertaken to assess the woody plant diversity,
species composition and regeneration status on ridge top in Garhwal Himalaya to assess impact of climate
change and species migration evaluation.

Study area
In this study, we had selected four undisturbed ridge tops viz., Raithal, Dharali, Sukhee top and Harshil (>
2500 m above sea level (asl)) in Uttarkashi district of Garhwal Himalaya, Uttarakhand, India (Fig. 1). Detail
Figure 1. Map of the study area (District Uttarkashi, Uttarakhand) with locations of the ridge tops sites being studied marked
as: S1-Raithal top; S2-Dharali top; S3-Sukhee top; S4-Harshil top.
study of ridge tops location and dominant species composition shown in table 1. The ridge top selected had
nearly similar type of edaphic-climatic conditions. The district lies in the upper catchment of two great rivers of
India viz., Ganges (called Bhagirathi towards upstream) and the Yamuna. The study area experienced a
temperate monsoon climate with a mean annual rainfall of 2000 mm and three main distinct seasons in a year
i.e., cool and relatively dry winter (December to March); the warm and dry summer (mid-April to June); and a
warm and wet period (July to mid-September) called as the monsoon or rainy season. Frost is common during
winter season, while the higher elevations experience heavy spells of snowfall, which may persist up to April
May in shady locations. Mean minimum monthly temperature ranged from 7.12C (Jan) to 23.20C (Jul) and
mean maximum monthly temperature ranged from 17.56C (Jan) to 33.35C (Jul) (Suyal et al. 2010). The area
is characterized by undulating topography with gentle slopes on Northern, North eastern and North western
faces and somewhat steep slopes on Southern and South western directions. The soil types found in the region
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are brown and black forest soils and podozolic soils. Soils are generally gravelly and large boulders are common
in the area. Numerous high ridges, deep gorges and precipitous cliffs, rocky crates and narrow valleys are part of
the topography of the region. Geologically, the rocks were complex mixture of mainly sedimentary, low grade
metamorphosed with sequence capped by crystalline nappe (Valdiya 1980).
Table 1. Description of study sites location, distribution and dominant woody plant species in Bhagirathi Basin, Garhwal
Himalaya.
Study site Altitude Latitude Longitude Dominant tree species
(Above Sea Level)
Raithal 2962-3523 m 07834' 55"-07832' 53" 3048'58"-3050'48" Q. semecarpifolia
Dharali 2740-3677 m 07847' 35"-07846' 31" 3101'04"-3103'02" A. spectabilis, C. deodara
Sukhee top 2814-3222 m 07843' 19"-07843'26" 3100'10"-3159'57" P. wallichiana, A. spectabilis
Harshil 2730-3460 m 07844' 11"-07844'03" 3101'31"-3101'18" A. spectabilis , B. utilis
Phytosociological study
Phytosociological parameters were studied by laying out 10 permanent sample plots of .01 ha size for trees
on the ridge top in each studied site. The data regarding regeneration status of sapling and seedling was
collected by laying four quadrat of 55 m and eight quadrats of 11 m respectively. The size and number of
quadrat were standardized using the species area curve (Misra 1968). Voucher specimens of plant species were
collected from studied forests and identified with the help of Flora of Gangotri National Park (Pusalkar & Singh
2012) and herbarium of H.N.B. Garhwal University, Srinagar. Important community parameters such as
frequency, density, abundance, basal area and importance value index (IVI) of all the plant species were worked
out by following Misra (1968) and MullerDombois & Ellenberg (1974). The tree species diversity index (H')
was determined by using Shannon Wiener information function H (H = - pi ln pi; where, pi= ni/N; and ni =
number of each species, N= total number of all species) (Shannon & Wiener 1963). The dominance index was
calculated by Simpsons index (1949) through the formula (Cd = (ni/N)2) (Simpson 1949). Hill equation use
to calculate Hill diversity index N0, N1 & N2 (Hill 1973).
Mean stem density (density/100 m2) of tree, sapling and seedling were considered to calculate regeneration
status. We follow (Uma Shankar 2001) to calculate regeneration status with in different categories of tree life
form stages.
On the basis of phytosociological study overall woody plant species richness was observed to be highest
with 11 species in Sukhee top, whereas, lowest 6 species in Harshil. The mean stand stem density was 613 stem
ha-1 (which ranged between 552710 stem ha-1) and mean value of Total Basal Area (TBA) was 84.37 m2.ha-1
(which ranged from 75.1892.07 m2.ha-1) which is quite similar to Pandey (2001) who reported stem density as
7921111 stem ha-1 and TBC 56126 m2.ha-1 from Garhwal Himalaya whereas Gairola et al. (2011) reported
high tree girth ranging between 84.25 to 35.08 m2.ha-1 and low total stem density (9901470 stem ha-1) from
moist temperate forest in the Garhwal region. A monotonic decline in the number of species with increasing
elevation has often been considered a general pattern (Brown 1988, Stevens 1992). Therefore, limitation of
species in hot spot area show the high altitude effect and almost similar results 17 species richness of study on
ridge site of Garhwal Himalaya (Rawat & Chandra 2014). A total of 2439 individuals were recorded in the study
area which belonged to 17 tree species in 16 genus and 9 families. The most abundant family was Pinaceae with
6 species whereas Ericaceae and Rosaceae were co-dominant families represented by 2 species each. Forests in
the study area were mature high girth values as they were undisturbed. According to Saxena et al. (1978) trees
with higher girth point out the best presentation of a species in a particular environmental setup whereas lower
girth either separate the chance occurrence of the species in the area or showed presence of the biotic
disturbances in the past. Simpson dominance index was highest in Sukhee top (0.84), moderate in Harshil (0.76)
whereas; lowest in Raithal (0.65). Along the altitude, the geographic and climatic conditions change sharply
(Kharkwal et al. 2005). The dominance values in this study are much higher than of 0.31 to 0.42 (Mishra et al.
2000) and 0.11 to 0.93 (Tiwari & Singh 1985) which were reported from different parts of Uttarakhand
Himalaya. Sorensen similarity value was higher in Dharali-Harshil whereas lowest similarity was found in
Raithal-Harshil (Table 2). Raithal forest was dominated by Quercus semecarpifolia (IVI 167.33), Sukhee top
forests dominated by Pinus wallichiana (IVI 86.15) whereas Dharali and Harshil were similarly dominated by
Betula utilis, Abies spectabilis and Pinus wallichiana. Importance value index of most common dominant
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.
species of four forest sites are depicted in figure 2, which shows Abies spectabilis, Betula utilis, Cedrus
deodara, Pinus wallichiana, Acer caesium and Quercus semecarpifolia are the main dominated forest species
Table 2. Phytosociological description of ridge top composition of four different sites in Bhagirathi Basin.
S. N. Studied parameters Raithal Dharali Sukhee top Harshil
1 Studied qua 20 28 24 16
2 Covered Area (ha.) 0.5 0.7 0.6 0.4
3 Species richness 8 8 11 6
4 Density (stem ha-1) 552 634 565 710
5 TBC (m2.ha-1) 84.32 92.07 89.9 75.18
6 Simpson Index 0.65 0.78 0.84 0.76
7 Shannon Index 1.47 1.66 2.07 1.50
8 Hill Index
H0 8 8 11 6
H1 4.35 5.26 7.92 4.48
H2 1.54 1.28 1.19 1.54
9 Jaccard Similarity
Raithal 1.00 0.33 0.18 0.27
Dharali 0.46 0.75
Sukhee Top 0.54
Harshil 1.00
Figure 2. Important Value Index of most common dominant tree species of the ridge tops.
which survived and adopted themselves in ridge top area. High frequent distribution on ridge top was shown by
species viz., Quercus semecarpifolia in Raithal, Pinus wallichiana and Cedrus deodara in Dharali, Acer caesium
in Sukhee top whereas Abies spectabilis and Betula utilis. Details of forest phytosociological attribute
frequency, density (stem ha-1), TBC (m2.h-1) and importance value index of different ridge tops are shown in
table 3.
Ridge top population structure
Population structure and regeneration status of tree species in terms of proportions of seedlings, saplings and
adults varied greatly (Table 4). The tree density and species richness in different diameter classes showed a
reverse J-shaped pattern on the ridge tops, whereas, on Sukhee top forest DBH (Diameter at breast height) class
density pattern was bell shaped (Fig. 3 & 4). Shrestha et al. (2007) and Ghimire & Lekhak (2007) have already
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.
defined such shape size-class distribution in Himalayan region. Abies spectabilis followed by Cedrus deodara
(at almost 3 sites), Betula utilis, Picea smithiana and Quercus semecarpifolia showed satisfactory regeneration
status in almost all sites (Table 4), showing their wide adaptability and tolerance to the changing climatic
conditions. However Acer caesium was reported in non-recruiting phase in site 1 (Raithal) and site 4 (Harshil).
Other species with similar trends were Taxus baccata, Juglans regia, Acer acuminatum, Sorbus cuspidata,
Aesculus indica and Ilex dipyrena.
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.
Figure 3. DBH (Diameter at breast height) class based distribution patterns of forest population attributes: Stem density
and Species richness in studied ridges top.
140 35
R a ith a l
D h a r a li
120 30
S u k h e e to p
H a r s h il
T B H (m h a )
100 25
2 -1
)
-1
80 20
T D (m h a
60 15
40 10
20 5
0 0
0 -2 5
00
-1 2 5
-1 5 0
-1 7 5
-2 0 0
-2 2 5
-2 5 0
-2 7 5
-3 0 0
-3 2 5
2 5 -5
5 0 -7
7 5 -1
100
125
150
175
200
225
250
275
300
D B H C la s s I n t e r v a l
Figure 4. DBH (Diameter at breast height) class based distribution patterns of forest population attributes: Total
diameter and Basal cover in studied ridge tops.
Status of dominant woody species composition, distribution and regeneration

Abies spectabilis, Acer caesium, Betula utilis, Cedrus deodara, Picea smithiana and Pinus wallichiana were
reported as dominant species of the ridge tops in the present study (Fig. 5), which was similar with different
parts of Himalaya (Kunwar & Sharma 2004, Ghimai et al. 2010). IVI of the dominant species at four sites was
quite lower than Kunwar and Sharma (2004) from mid Nepal may be due to climatic effect. Abies spectabilis
had highest value in Harshil (95.9) followed by Dharali (92.7) and lowest in Raithal (38.28). It occupied highest
TBC values in Dharali (39.5 m2.ha-1) (Table 3). In all study area its regeneration pattern found in good
regeneration categories. Acer caesium was dominant and widely distributed in Sukhee top followed by Dharali
whereas in other two sites it had low occurrence. Regeneration of this species was good in Sukhee top whereas
in other site its regeneration was subject of concern. Betula utilis showed satisfactory with wide distribution in
almost all girth classes in study area. Dharali top was dominated by Cedrus deodara and Picea smithiana.
Cedrus deodara was absent in Raithal top whereas at other three sites it was reported with good regeneration.
Picea smithiana showed healthy regeneration in all sites whereas it was totally absent in Raithal. Pinus
Sharma et al. (2014) 1(3): 6271
.
wallichiana was widely distributed in three study sites with good regeneration state. Sustainable regeneration of
Pinus wallichiana already reported from different parts of Himalaya (Kimmins 1987, Shimano 2000).
Figure 5. Distribution of the dominant plant species of the studied ridge tops: A, Height (Ht);
B, Diameter at breast height (DBH).
CONCLUSIONS
High altitude Himalayan forest is facing significant/drastic change owing to changing climatic conditions.
Quantification of the current forest composition and regeneration dynamics of the ridge tops is crucial in order
to assess the role of climate change in predicting effect on future species coexistence and species shift in
Himalayan range. Abies spectabilis, Betula utilis, Cedrus deodara, Pinus wallichiana, Acer caesium and
Quercus semecarpifolia were observed as the dominant species of the ridge tops with good regeneration
potential whereas Taxus baccata, Acer acuminatum, Aesculus indica, Juglans regia, Ilex dipyrena and Sorbus
cuspidata were poorly distributed with poor regeneration status. It can thus be concluded that the species with
poor and non-recruiting regeneration status and rare distribution may be affected by the warming temperatures
on ridge tops whereas dominant species with good regeneration potential may adapt and shift in opinionated
microclimate. In the current scenario the species with poor regeneration status are required to be earmarked so
that proper measures could be adopted for their conservation as they are susceptible to the changing climatic
conditions which may result in their complete extermination and hence lead to change of the future composition
of the ridge tops. This study may be constructive to assess biological impacts of climate change which have
focused on species abundance and distribution in search of the predicted systematic shifts in both upwards and
downwards direction.
Sharma et al. (2014) 1(3): 6271
.
ACKNOWLEDGEMENTS
The authors are thankful to Department of Science and Technology, Government of India, New Delhi, India
for providing financial support (Project No. SERB/SR/SO/PS/14/2010).
REFERENCES
Adler PB & Lambers JHR (2008) The influence of climate and species composition on the population dynamics
of ten prairie forbs. Ecology 89: 30493060.
Bajpai O, Kumar A, Mishra AK, Sahu N, Pandey J, Behera SK & Chaudhary LB (2012) Recongregation of tree
species of Katerniaghat wildlife sanctuary, Uttar Pradesh, India. Journal of Biodiversity and Environmental
Sciences 2(12): 2440.
Breckle JW (2002) Walters Vegetation of the Earth. Springer Verlag, Berlin, 4 (527), pp. 300.
Brown JH (1988) Species diversity. In: Myers AA & Giller PS (eds) Analytical biogeography: An integrated
approach to the study of animal and plant distribution. Chapman and Hall, New York, pp. 5789.
Cannone N, Sgorbati S & Guglielmin M (2007) Unexpected impacts of climate change on alpine vegetation.
Frontiers in Ecology and the Environment 5: 360364.
Gairola S, Sharma CM, Suyal S & Ghildiyal SK (2011) Composition and diversity of five major forest types in
moist temperate climate of the western Himalayas. Forest Study China 13(2): 139153.
Getachew T, Demel T, Masresha F & Erwin B (2010) Regeneration of seven indigenous tree species in a dry
Afromontane forest, Southern Ethiopia. Flora 205: 135143.
Ghimire B, Mainali KP, Lekhak HD, Chaudhary RP & Ghimeray AK (2010) Regeneration of Pinus wallichiana
AB Jackson in a trans-Himalayan dry valley of north-central Nepal. Himalayan Journal of Sciences 6(8):
1926.
Ghimire B & Lekhak HD (2007) Regeneration of Abies spectabilis (D. Don) Mirb. in sub-alpine forest of Upper
Manang, north-central Nepal. In: Chaudhary RP, Aase TH, Vetaas OR, Subedi BP (eds) Local Effects of
Global Changes in the Himalayas: Manang, Nepal. Tribhuvan University, Kathmandu and University of
Bergen, Norway, pp. 139149.
Henle K, Lindenmayer DB, Margules CR, Saunders DA & Wissel C (2004) Species survival in fragmented
landscapes: where are we now? Biodiversity and Conservation 13: 18.
Hill MO (1973) Diversity and evenness: a unifying notation and its consequences. Ecology 54: 427432.
Holzinger, Hulber K, Camenisch M & Grabherr G (2008) Changes in plant species richness over the last century
in the eastern Swiss Alps: Elevational gradient, bedrock effects and migration rates. Plant Ecology 195:
179196.
Kammer PA & Mohl A (2002) Factors controlling species richness in alpine plant communities: an assessment
of the importance of stress and disturbance. Arctic, Antarctic and Alpine Research 34: 398407.
Kammer PM, Christian S & Philippe C (2007) Increasing species richness on mountain summits: upward
migration due to anthropogenic Climate change or recollection? Journal of Vegetation Science 18: 301306.
Kharkwal G, Mehrotra P, Rawat YS & Pangtey YPS (2005) Phytodiversity and growth form in relation to
altitudinal gradient in the Central Himalayan (Kumaun) region of India. Current Science 89(5): 873878.
Kimmins JP (1987) Forest ecology. New York: Macmillan Publishing Company. pp. 531.
Korner C (2003) Alpine plant life. Functional plant ecology of high mountain ecosystems. Springer, Berlin.
Kunwar RM & Sharma SP (2004) Quantitative analysis of tree species in two community forests of Dolpa
district, mid-west Nepal. Himalayan Journal of Sciences 2 (3): 2328.
Lalfakawma (2010) Disturb and perish, conserve and flourish regenerating forests: a review Science vision 10:
37.
Lemoine N, Schaefer HC & Bohning-Gaese K (2007) Species richness of migratory birds is influenced by
global climate change. Global Ecology Biogeography 16: 5564.
Lesica P & McCune B (2004) Decline of arctic-alpine plants at the southern margin of their range following a
decade of climatic warming. Journal of Vegetation Science 15: 679690.
Mishra A, Sharma CM, Sharma SD & Baduni NP (2000) Effect of aspect on the structure of vegetation
community of moist bhabar and tarai Shorea robusta forest in Central Himalaya. Indian Forester 126(6):
634642.
Mishra AK, Behera SK, Singh K, Chaudhary LB, Mishra RM, Singh B (2013a) Influence of abiotic factors on
community structure of understory vegetation in moist deciduous forests of north India. Forest Science and
Practice 15(4): 261273.
Sharma et al. (2014) 1(3): 6271
.
Mishra AK, Bajpai O, Sahu N, Kumar A, Behera SK, Mishra RM & Chaudhary LB (2013b) Study of plant
regeneration potential in tropical moist deciduous forest in northern India. International Journal of
Environment 2(1): 153163.
Misra R (1968) Ecology work book. Oxford and IBH publishing company, Calcutta.
Mueller-Dombois D & Ellenberg H (1974) Aims and methods of vegetation ecology. John Wiley & Sons, New
York.
Negi CS & Nautiyal S (2005) Phytosociological studies of a traditional reserve forest- Thal Ke Dhar,
Pithoragarh, Central Himalayas (India). Indian Forester 131: 519534.
Pande PK (2001) Quantitative vegetation analysis as per aspect and altitude, and regeneration behavior of tree
species in Garhwal Himalayan forest. Annuls of Forest 9(1): 3952.
Pauli H, Gottfried M, Reiter K, Klettner C & Grabherr G (2007) Signals of range expansions and contractions of
vascular plants in the high Alps: observations (1994-2004) at the GLORIA master site Schrankogel, Tyrol,
Austria. Global Change Biology 13: 147156.
Pokhriyal P, Uniyal P, Chauhan DS & Todaria NP (2010) Regeneration status of tree species in forest of Phakot
and Pathri Rao watersheds in Garhwal Himalaya. Current Science 98: 171174.
Pusalkar PK & Singh DK (2012) Flora of Gangotri National Park, Western Himalaya, India. Botanical Survey
of India, Kolkata.
Rahman MH, Khan ASA, Roy B & Fardusi MJ (2011) Assessment of natural regeneration status and diversity
of tree species in the biodiversity conservation areas of Northeastern Bangladesh. Journal of Forestry
Research 22: 551559.
Rawat VS & Chandra J (2014) Vegetational Diversity Analysis across Different Habitats in Garhwal Himalaya.
Journal of Botany 2014: 15.
Rosenzweig C, Casassa CG, Karoly DJ, Imeson A, Liu C, Menzel A, Rawlins S, Root TL, Seguin B &
Tryjanowski P (2007) Assessment of observed changes and responses in natural and managed systems. In:
Parry ML, Canziani OF, Palutikof JP, van der Linden PJ & Hanson CE (eds) Climate Change 2007:
Impacts, adaptation and vulnerability. Contribution of Working Group II to the Fourth Assessment Report
of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge, UK, pp. 79
131.
Saxena AK, Pandey U & Singh JS (1979) On the ecology of Oak forest in Naini Tal hills, Kumaun Himalaya.
In: Singh JS & Gopal B (eds) Glimpses of ecology. International Scientific Publishers, Jaipur, pp. 167180.
Saxena AK & Singh JS (1984) Tree population structure of certain Himalayan forest assodations and
implications concerning their future composition. Vegetation 58: 6169.
Shannon CE & Wiener W (1963) The mathematical theory of communication, 1st Editions. University of Illinois
Press, Urbana, IL.
Shimano K (2000) A power function for forest structure and regeneration pattern of pioneer and climax species
in patch mosaic forests. Plant Ecology 146: 207220.
Shrestha BB, Ghimire B, Lekhak HD & Jha PK (2007) Regeneration of treeline birch (Betula utilis D. Don)
forest in trans-Himalayan dry valley in Central Nepal. Mountain Research and Development 27: 259267.
Simpson EH (1949) Measurement of diversity. Nature 163: 688688.
Singh CP, Panigrahy S, Thapliyal A, Kimothi MM, Soni P & Parihar JS (2012) Monitoring the alpine tree line
shift in parts of Indian Himalayas using remote sensing. Current Science 102: 559562.
Stevens GC (1992) The elevational gradient in altitudinal range: an extension of Rapoports latitudinal rule to
altitude. American Naturalist 140: 893911.
Suyal S, Sharma CM, Gairola S, Ghildiyal SK, Rana CS & Butola DS (2010) Phytodiversity (Angiosperms and
Gymnosperms) in Chaurangikhal Forest of Garhwal Himalaya, Uttrakhand, India. Indian Journal of Science
and Technology 3(3): 267275.
Theurillat JP & Guisan A (2001) Potential impact of climate change on vegetation in the European Alps: a
review. Climate Change 50: 77109.
Tiwari JC & Singh SP (1985) Analysis of woody vegetation in a mixed oak forest of Kumaun Himalaya.
Proceedings' in Indian Natural Science Academy 51(B): 232247.
Uma Shankar (2001) A case of high tree diversity in a Sal (Shorea robusta) - dominated low land forest of
eastern Himalaya: Floristic composition, regeneration and conservation. Current Science 81(7): 776786.
Sharma et al. (2014) 1(3): 6271
.
Valdiya KS (1980) Stratigraphic scheme of the sedimentary units of the Kumaon lesser Himalaya. In: Valdiya
KS & Bhatiya SB (eds) Stratigraphy and correlations of the lesser Himalayan formations. Hindustan
Publication Corporation, Delhi, India, pp. 748.
Walther GR, Berger S & Sykes MT (2005) An ecological footprint of climate change. Proceedings of the
Royal Society: Biological Science 72: 14271432.
Ward JS, Worthley TE, Smallidge PJ & Bennett KP (2006) North-east forest regeneration handbook: A guide
for forest owners, harvesting practitioners, and public officials. USDA Forest Service, Newton Square, PA.
Woodward FI & Kelly CK (2008) Responses of global plant diversity capacity to changes in carbon dioxide
concentration and climate. Ecological Letters 11: 12291237.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
1(3): 7279, 2014
Research article
Expression of chitinase with antifungal activities in

ripening Banana fruit
P. W. H. K. P. Daulagala
Department of Botany, Faculty of Natural Sciences, Kandy Regional Centre, The Open University of
Sri Lanka, Polgolla, Sri Lanka
Author: pdaulagala@yahoo.com [Accepted: 18 December 2014]
Abstract: The banana (Musa acuminata) is a climacteric fruit of a great economic importance. In
this present study, the expression of chitinases and their antifungal activities were investigated.
The ripening stages of banana were defined according to the peel colour index (PCI). In the dye-
labelled assay with carboxymethyl chitin remazol brilliant violet 5R (CM-chitin- RBV 5R),
chitinase activity was detected in both pulp and peel extracts. The activities increased markedly at
the onset of ripening and peaked at PCI 5 stage. On sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) activity gels, two chitinases of apparent molecular masses of 26 kDa
and 31 kDa were detected in both pulp and peel extracts and they showed an accumulation during
the subsequent ripening. This result was in consistent with the pattern of chitinase activity obtained
in dye-labelled assay. Interestingly, both pulp and peel extracts inhibited the growth of the fungus
Botrytis cinerea on plates and caused lysis of hyphal tips. In some instances, with bursting of the
fungal tips, flowing out of the cell contents could be observed.
Keywords: Banana (Musa acuminata L. AAA) - Fruit ripening - Chitinase - Hyphal tip lysis.
[Cite as: Daulagala PWHKP (2014) Expression of chitinase with antifungal activities in ripening Banana fruit.
INTRODUCTION
Fruit ripening is a complex metabolic process. After a phase of active cell division followed by cell
expansion, the growth and development of the fruit decline and the ripening begins. This is accompanied by
some physiological changes including changes in aroma, texture, colour and flavour and some biochemical
changes including the synthesis of phytoalexins (Hammerschmidt 1999, Fraser et al. 2007) and pathogenesis-
related (PR) proteins (Van Loon 1997, Datta & Muthukrishnan 1999). Such changes make the fruits more
attractive to animals and more likely to be attacked by pathogens (Derckel et al. 1998). Since fruit ripening is a
tightly regulated process (Giovannoni 2001), all these changes are very likely brought about by activation or
repression of specific genes.
Systemic acquired resistance (SAR) is a distinct signal transduction pathway that plays an important role in
the ability of plants to defend themselves against pathogens. SAR pathway is induced by most pathogens that
cause necrosis of tissues (Anfoka & Buchenauer 1997). SAR provides protection in uninfected parts of the plant
against pathogens and it is correlated with the expression of pathogenesis-related (PR) proteins (Mauch et al.
1984, Lawrence et al. 1996, Daulagala & Allan 2003). Among the PR proteins, the most intensively studied and
well characterized in plant-pathogen interactions are chitinases and -1, 3-glucanases. Although chitin, the
substrate of the chitinase enzyme does not exist in plant cell walls, chitinases are widely distributed in plants
(Neuhaus 1999, Kitajima et al. 2010). Further, -1, 3-glucanses are another group of PR proteins that attack
glucans cross-linked with chitin in fungal cell walls.
A defensive role of PR proteins in plant systems has been suggested based on the induction of their synthesis
upon pathogen infection, and on their in vitro and in vivo antifungal activities. Expression of chitinase-encoding
genes is thought to be an important biochemical mechanism of plant defense against fungal pathogens as
chitinases can degrade chitin in fungal cell walls and inhibit fungal growth (Mauch et al. 1984, Lawrence et al.
1996). Some plant chitinases have direct antifungal activity, causing rapid lysis of fungal hyphal tips and
germinating spores. This antifungal activity of chitinases is synergistically potentiated by -1, 3-glucanses.
Daulagala (2014) 1(3): 7279
.
Chitinases (EC 3.2.1.14) are hydrolytic enzymes. Basically there are three activity classes of chitinases; -
(1,4)-N-acetylglucosaminidases, exochitinases (chitobiosidases) and endochitinases. -(1,4)-N-
acetylglucosaminidases cleave N-acetylglucosamine (NAG) oligomers of the chitin chain and generate N-
acetylglucosamine monomers. Chitobiosidases cleave diacetylchitobiose units from non-reducing end of the
chitin chain and release disaccharides. Endochitinases randomly cleave glycosidic linkages at internal sites
along the chitin chain and produce low molecular mass oligomers like diacetylchitobioses and chitotrioses
(Guthrie et al. 2005).
Banana (Musa acuminata L. AAA) is a globally important fruit, especially in developing countries and
millions of people subsist on bananas as one of their major energy sources. It is a climacteric fruit, characterized
by a green storage phase, followed by a burst in ethylene release. Consistent with the ethylene production,
numerous biochemical changes occur during ripening of banana, including the synthesis of volatile compounds,
alterations in carbohydrate composition, changes in phenolic compounds and breakdown of chlorophyll in peel
(Seymour 1993). A number of enzymes associated with banana ripening have been identified and characterized.
Some of these enzymes are involved in carbohydrate metabolism and cell wall degradation (Seymour 1993, Da
Mota et al. 2002), whereas others showed antifungal activity (Peumans et al. 2002). Differential screening of
cDNA libraries representing banana pulp at different ripening stages yielded several up-regulated genes
(endochitinase, -1, 3-glucanase and methallothionin) as well as down-regulated genes (starch synthase, class
III chitinase, and jacalin-related lectin (Clendennen & May 1997). In a recent study, it was found that in
naturally ripened banana, the expression of endochitinase gene MaECHI1 gradually increased consistent with
the timing of the ripening process (Liu et al. 2012). This suggested that this gene was up regulated during
ripening which correlated with the previous work performed with this gene expression (Xu et al. 2007).
In this study, the chitinase activity and the isoenzyme expression on gels were assessed during the ripening
process of banana fruits to see how the pattern of expression and the functionality of the enzyme are associated
with the ripening process. Further the antifungal activity of the extracts against the fungus Botrytis cinerea was
also investigated.

Plant material
Unripe banana fruits (Musa acuminata cv. Giant Cavendish) were obtained from a commercial source (Asda
Supermarket) in Aberdeen, UK (imported from Chiquita Brands Inc., Cincinnati, Ohio, USA). Only those fruits
normal and healthy in appearance without physical injuries or disease symptoms were selected. Ripening was
allowed to proceed naturally under normal laboratory conditions. Fingers from different hands were mixed and
randomly taken for the experiments. During ripening, pulp and peel of fruits representing five subsequent
ripening stages were collected. Stages of ripening were scored by a peel colour index on a scale from colour
number 1 to 5 as PCI 1 to PCI 5 (1 = very green, internal tissue hard, 2 = more green than yellow, 3 = more
yellow than green, pulp becoming soft, 4= yellow with green tips and 5 = all yellow, pulp very soft) (Customer
Service Department, Chiquita Brands, Inc., Cincinnati, Ohio, USA). Tissues from five individual fruits were
pooled to obtain a uniform sample for each ripening stage.
Botrytis cinerea culture
Botrytis cinerea used in this study was isolated from Yorkshire Chinese cabbage (YCC) (Brassica
campestris sub sp. pekinensis var. Kasumi) from a field at Triffitt Nurseries, Allerthorpe, Humberside. The
culture was maintained on Potato Dextrose Agar (PDA) plates at 21C. Routine subculture was done by
transferring a plug of medium from a stock plate onto a fresh PDA plate.
Preparation of protein extracts
To extract total protein, frozen tissues at each specific ripening stage were immersed in liquid nitrogen and
ground to homogeneity in a mortar. Powdered tissues were further ground in a mortar with the extraction buffer
(0.1M sodium citrate buffer with additives, pH 5.0) at the level of 3 ml.g-1 fresh weight. The homogenates were
transferred to Eppendorf tubes and cellular debris was pelleted by centrifugation at 11,600 g for 15 min. The
supernatant fractions were stored at -20C until required.
Chitinase assay with Carboxymethyl-chitin Remazol Brilliant Violet 5R
The assay developed by Wirth & Wolf (1990) was used. Reaction mixtures contained the extract (1:100
dilution), 0.1M sodium citrate buffer (pH 5.0) and carboxymethyl-chitin remazol brilliant violet 5R (CM-chitin-
RBV 5R) solution (2 mg.ml-1 in water) (Loewe Biochemical Gmbh Co., Germany). The microcentrifuge tubes
Daulagala (2014) 1(3): 7279
.
containing 800 l of reaction mixture (50 l of extract, 200 l of substrate and 550 l of buffer) were incubated
at 37C for 1 h. The reaction was stopped by adding 200 l of 2 N HCl and then by cooling on ice for 10 min.
The tubes were centrifuged (Chillspin, MSE, UK) at 11,600 g for 5 min. The coloured supernatants were gently
pipetted in to cuvettes and absorbances were photometrically determined at 550 nm.h-1 against a blank. The
blank was the reaction mixture without the enzyme extract (with more extra buffer equal to the volume of the
extract). The enzyme activity was expressed as the change in absorbance at 550 nm.h-1. The assays were done in
triplicate for each sample.
Visualization of chitinase isozyme patterns in activity gels
SDS-PAGE was carried out as described by Trudel & Asselin (1989) using glycol chitin impregnated
separating gels, with the buffer system of Laemmli (1970). For activity gels, 12% separating gels containing
0.1% (w/v) glycol chitin and 4% stacking gels were used. Glycol chitin was synthesized from glycol chitosan
(Sigma) according to Trudel & Asselin (1989). Samples were prepared in non-reducing loading buffer (without
-mercaptoethanol). After electrophoresis at 200V, molecular weight markers were trimmed off from the gel
and stained with Coomassie blue with shaking at room temperature. The remainder of the gel was incubated for
overnight at 30C with reciprocal shaking in 25 mM sodium acetate buffer pH 5.0 (renaturing buffer) containing
1% (v/v) Triton X-100. Following the incubation, gels were stained with freshly prepared 0.01% (w/v)
calcofluor white M2R (Sigma, UK) in 0.5 M Tris-HCl buffer (pH 8.9). After 5 min, the brightener solution was
removed and gels were destained by incubating for 1 h in distilled water at room temperature. Lytic zones,
where chitin had been digested, were detected by inspection of gels in a UV illuminator, where they appeared as
dark bands against the fluorescent background of intact glycol chitin (Trudel & Asselin 1989). The destained
portion of the gel containing molecular markers was used to calibrate the Calcofluor-stained activity gel.
Assays of antifungal activity
The crude protein extracts were tested in vitro for antifungal activity against B. cinerea. The effect was
tested in two methods,
1. Inhibition of growth of B. cinerea on PDA plates:
Two agar plugs (10 mm diameter) bearing Botrytis growth were cut from a growing culture and placed
firmly (face down) in the centre of two potato dextrose agar (PDA) plates. Wells were cut with a 10 mm
diameter cork borer, around the plates approximately 2.5 cm away from the centre. The wells were filled to
capacity (200 l) with filter-sterilised (0.2 m Acrodisc, Gelman Sciences, UK) crude enzyme extracts prepared
from both peel and pulp tissues. Commercially available chitinases from Serratia marcescens at 10 mg.ml-1
concentration and boiled enzyme extract were used as controls. All plates were incubated at 21C and observed
daily for any zones of inhibition.
2. Lysis of hyphal tips of B. cinerea:
The method used by Zhu & Gooday (1992) was used with some alterations. Petri dishes were poured with 10
ml of PDA (to give a thin layer) and the agar was overlaid with cellophane (British Celanese Co., PT300) which
had been boiled in distilled water for 30 min and autoclaved. A spore suspension of B. cinerea was stab
inoculated on to the centre of the medium and the plates were incubated at 21C. After 48 h of incubation,
triangular agar blocks containing growing mycelium were cut (still with the Cellophane) and kept them on clean
glass slides. Using a micropipette, filter sterilized enzyme extracts (20 l drops) obtained from banana pulp (PCI
1) were added directly to the growing colony margins by lifting the cellophane with forceps. The extraction
buffer was used as the control. The enzyme extract and the buffer were used with 1.5% (w/v) sorbitol solution,
osmotically compatible with B. cinerea. The agar blocks were observed under the microscope for any swelling
and/or bursting of hyphal tips and photographed.
RESULTS
Chitinase assay with carboxymethyl-chitin remazol brilliant violet 5R
Chitinase assay using the substrate CM-chitin-RBV 5R is based on the precipitability of a non-degraded,
highly polymerized substrate when acid is added to the reaction mixture. This method is very useful to detect the
presence of endochitinase activity in protein extracts. Chitinase activities of extracts from both banana pulp and
peel increased progressively and markedly, during natural ripening from PCI 1 to PCI 5, reaching the maximum
in ripe fruit (PCI 5). The activities of pulp extracts at all five stages of ripening were slightly higher than that of
the peel extracts and the activities obtained at PCI 5 (all yellow) for both pulp and peel extracts were
approximately twice that of respective PCI 1 (very green) stage (Fig. 1).
Daulagala (2014) 1(3): 7279
.
Figure 1. Chitinase activities of banana fruits with CM-chitin RBV 5R at five different stages of ripening (PCI 1 to PCI 5):
A, Pulp extracts; B, Peel extracts.
Visualization of chitinase isozyme patterns in activity gels
To trace major changes in the chitinase expression pattern, crude extracts from peel and pulp of banana were
analysed by SDS-PAGE activity gels. Total protein extracts were prepared from equal fresh weights of pulp and
peel and equal volumes of both extracts were loaded in wells. The results of the SDS-PAGE analysis clearly
demonstrated dramatic changes in the accumulation of chitinase in the pulp and peel during ripening. As shown
in figure 2, chitinases of apparent molecular masses of 26 and 32 kDa were the predominant proteins recovered
from the extracts. Unlike in the pulp, the 26 kDa chitinase was present in low amounts in peel throughout the
ripening process. In pulp, it showed a strong accumulation during the later stages of ripening. The 32 kDa
chitinase which was relatively in low concentrations in peel at PCI 1 stage showed a gradual increase with the
onset of ripening. But in pulp, the abundance of 32-kDa chitinase progressively increased from PCI 1 to PCI 5.
Figure 2. Pattern of chitinase accumulation in banana pulp and peel extracts during five subsequent stages of ripening PCI 1
to PCI 5. Molecular markers are indicated on the left. Sg chitinase from S. griseus as a positive control.
Assays of antifungal activity
1. Inhibition of growth of B. cinerea on PDA plates
The fungus B. cinerea was screened for its ability to be inhibited by extracts of banana. The fungus was allowed
to grow on PDA medium for 2 days at 21C before adding the extracts to the wells. It was interesting to note
that after overnight incubation of these plates with extracts in wells, there was a detectable inhibition of growth
of B. cinerea. Further additions of extracts and incubation of plates produced crescents of inhibition of growth
on plates (Fig. 3). The crescents of inhibition with pulp extracts as shown in figure 3B were more prominent
than that of peel in figure 3A, and this observation showed a pattern similar to the results obtained in dye assay
and activity gels. Botrytis grew towards the well 6 in plate (A), which contained S. griseus chitinases and this
showed that growth of B. cinerea was not affected by the chitinases of S. griseus. With boiled extract, no growth
inhibition was observed in well 6 in figure 3B.
2. Lysis of hyphal tips of B. cinerea
Examination by light microscopy of the mycelium treated with banana extracts from pulp (PCI 1) showed
swelling and subsequent bursting of hyphal tips of B. cinerea (Fig. 4A). However, no such lysis was observed in
Daulagala (2014) 1(3): 7279
.
the control experiment with the sample extraction buffer (Fig. 4B). Although the chitinase activity in pulp
tissues increased with ripening from PCI 1 to PCI 5, there was a slight difference in time taken for the fungal
tips to burst with each extract. The pulp extract of PCI 1 ripening stage contained relatively low chitinase
activity compared with the other pulp extracts, but within 2-3 min of adding the pulp extracts on to the mycelial
tips, the bursting initiated with all extracts. But in contrast, the extracts from peel took more time than the pulp
to initiate bursting. The number of burst tips was fewer with peel extracts compared to the corresponding pulp
and most of the time there were more swollen tips than burst tips with peel extracts.
Figure 3. Antifungal activity of banana extracts: A, The wells in plates contained peel extracts from well 1 (PCI 1) to well 5
(PCI 5) and well 6 contained chitinases from S. griseus; B, The wells in plates contained pulp extracts from well 1 (PCI 1) to
well 5 (PCI 5) and well 6 contained boiled enzyme extract of PCI 3.
Figure 4. Light micrographs of B. cinerea hyphae: A, after incubation with crude protein extracts (Note the swollen (narrow
arrows) and lysed (wide arrows) hyphal tips with the banana pulp extract); B, after incubation with extraction buffer.
DISCUSSION
Ripening of most fruits generally involves the accumulation of sugars and other nutrients and softening and
breakdown of cellular structure of the tissues. The eventual result of all these events makes the fruits as an
excellent target for pathogens (Robinson et al. 1997). These observations have led several researchers to
hypothesize that different proteins should accumulate during maturation and ripening of fruits (Derckel et al.
1998). It is important to note that the pattern, abundance and the type of proteins reported in a particular study,
during ripening of a certain fruit, may not be the same when comparing with another study performed in a
different laboratory. As an example, in banana, the stages of ripening can be indicated according to the colour
development in peel, from green to yellow. One can divide this whole process into 5, 6 or 7 different stages
depending on how the visual colour development in the peel is determined. The whole process can be divided
into pre climacteric, early climacteric, climacteric and post climacteric, depending on the respiration rate and
ethylene production. Furthermore, fruits can be kept in natural atmosphere to ripen or they can be exposed to
ethylene to induce ripening. These aspects may result in differences of conclusions in different studies.
Banana is a climacteric fruit and the onset of ripening is characterized by a large increase in ethylene
synthesis. It is believed that ethylene regulates the expression of genes involved in ripening. Several genes
including a chitinase gene have been isolated from bananas that were up-and down-regulated during ripening. In
Daulagala (2014) 1(3): 7279
.
the study by Liu et al. (2012), they found that in naturally ripened banana fruit, the expression of endochitinase
gene MaECHI1 gradually increased, consistent with the timing of the ripening process. This result suggested
that this gene was up-regulated during ripening, which correlated with previous work performed with this gene
expression library (Xu et al. 2007). Differential screening of cDNA libraries representing banana pulp at
different ripening stages also yielded a number of up-regulated (endochitinase, -1, 3-glucanase, BanTLP, and
methallothionin) as well as several down-regulated (starch synthase, class III chitinase, and jacalin-related
lectin) genes (Clendennen & May 1997).
In the dye-labelled assay with CM-chitin RBV, chitinase activities were detected in both pulp and peel and
they increased during ripening. On SDS-PAGE activity gels, two chitinases of apparent molecular masses of 26
kDa and 31 kDa were detected in both pulp and peel tissues and they showed an accumulation during the
subsequent ripening. Similarly, Clendennen et al. (1998) identified and characterised an abundant protein of 31
kDa (P31) from pulp of banana. But in contrast to the present study, Clendennen et al. (1998) reported that the
abundance of P31 decreased as ripening proceeded. Furthermore this P31 was partially purified and polyclonal
antiserum was raised against the protein. The P31 antiserum recognized a single 31 kDa polypeptide in banana
pulp that was not present in peel, corm meristem or root tissues. From these results, they indicated that P31 was
fruit-specific and the physiological role of P31 is not for plant protection, but as a storage protein in banana
pulp. Because of the unavailability of any other plant part, such as corm, leaves or roots, it is unknown whether
these two abundant chitinases of 26 and 32 kDa assessed in this present study are fruit-specific or present in
other parts of the plant. Biochemical studies confirmed that several abundant pulp proteins like BanTLP (Barre
et al. 2000), -1,3-glucanase (Peumans et al. 2000), and a class I chitinase that is considered as the major
banana allergens (Sanchez-Monge et al. 1999) are encoded by genes up-regulated during climacteric ripening.
In this present study, glucanase activities of peel and pulp extracts were determined using the same protocol of
the chitinase assay with CM-chitin RBV solution, except that the substrate for glucanase was 4 mg ml-1
carboxymethyl-curdlan remazol brilliant blue (CM-curdlan-RBB) solution. Although there are reports on
banana glucanases in pulp of ripe banana fruits (Peumans et al. 2000), all attempts made towards determining
the glucanase activity with this substrate were unsuccessful. Only the positive control (zymolyase) gave
positive results in the assay (results not shown).
The chitinase activity obtained from banana pulp extracts in the present study was tremendous. Having such
activity, both pulp and peel extracts were tested against the fungus B. cinerea. The fungus B. cinerea is an
important pathogen of stored and transported fruits, vegetables, ornamental crops and nursery stocks and it
occurs geographically wherever the host is present. Interestingly, the growth of fungus on plates was inhibited
with both pulp and peel extracts. In addition, inhibition of germination of conidia was observed in in vitro
experiments (results not shown). Although germination of conidia was inhibited, lysis of tips of germ tubes was
not observed with the extracts. But extracts caused lysis of hyphal tips of Botrytis grown on agar. In some
instances, the fungal tips started to burst within the first minute of appliying the pulp extract to the hyphae and
subsequently the cell contents started to flow out. Lysis did not occur at some hyphal tips at the time of
observation, but some of them were swollen. Similar observations have been obtained by Mauch et al. (1988),
when they treated various fungi including Fusarium solani f. sp. phaseoli (strain W8), Fusarium solani f. sp.
pisi (ATCC 38136) Alternaria solani and Botrytis cinerea with the crude extracts of pea pods containing
chitinase and -1,3-glucanase activities. In fact, the antifungal activity of chitinases has been found in fruits,
such as fig (Li et al. 2005), grape (Fernandez-Caballero et al. 2009), and papaya (Chen et al. 2007), and two
homologous chitinases that inhibit Fusarium oxysporum were isolated from Gold bananas (Ho & Ng 2007). The
work done with banana by Toledo et al. (2012) using two-dimensional fluorescence difference gel
electrophoresis (2D- DIGE) found that one class III chitinase was in down regulated spots detected in pre-
climacteric fruits. In contrast, the two chitinase isoforms were accumulated during ripening and these two up-
regulated isoforms revealed in their study could be important in pathogen resistance or in adaptation to
postharvest conditions. The strong inhibition of the in vitro growth of B. cinerea obtained in this present study
also suggested that the chitinase-rich banana extracts had an antifungal activity. In addition, the lack of growth
inhibitory effect with boiled enzyme extract in well 6 in Figure 3B showed that the inhibition of Botrytis growth
from wells 1-5 in plate (B) was not due to any contaminating ions or any other molecules present in extracts.
CONCLUSIONS
The results presented in this study demonstrated that banana fruits had increasing chitinase activities during
ripening. The chitinases detected were constitutive, and showed antifungal activities under in vitro conditions.
Daulagala (2014) 1(3): 7279
.
Though it was not detected in this study, there are reports showing the accumulation of -1,3 glucanases during
ripening of banana fruits. Therefore induction of these two major PR proteins, which act synergistically against
many fungi, will be a better way to enhance disease resistance against pathogens.
ACKNOWLEDGEMENTS
I would like to dedicate this paper to the late Professor G.W. Gooday, who supervised me and whose
knowledge and enthusiasm will remain an inspiration to me. I am grateful to Dr. Eunice J. Atkins for advising
and guiding me throughout the preparation of this manuscript. Financial assistance by the Commonwealth
Scholarship Commission is gratefully acknowledged.
REFERENCES
Anfoka G & Buchenauer H (1997) Induction of systemic resistance in tomato and tobacco plants against
cucumber mosaic virus. Journal of Plant diseases and Plant protection Protection 104: 506516.
Barre A, Peumans WJ, Menu-Bouaouiche L, Van Damme EJM, May GD, Fernandez Herrera A, Van Leuven F
& Rouge P (2000) Purification and structural analysis of an abundant thaumatin-like protein from ripe
banana fruit. Planta 211: 791799.
Chen Y, Hsu L, Huang I, Tsai T, Lee G & Shaw J ( 2007) Gene cloning and characterization of a novel
recombinant antifungal chitinase from papaya (Carica papaya). Journal of Agricultural and Food Chemistry
55: 714722.
Clendennen SK, Lopez-Gomez R, Gomez-Lim MA, Arntzen CJ & May GD (1998) The abundant 31-
kilodalton banana pulp protein is homologous to class-III acidic chitinases. Phytochemistry 47: 613619.
Clendennen SK & May GD (1997) Differential gene expression in ripening banana fruit. Plant Physiology 115:
463469.
Da Mota RV, Cordenunsi BR, Do Nascimento JR, Purgatto E, Rosseto MR & Lajolo FM (2002) Activity and
expression of banana starch phosphorylases during fruit development and ripening. Planta 216: 325333.
Datta SK & Muthukrishnan S (1999) Pathogenesis-Related Proteins in Plants. CRC Press LLC, Boca Raton,
FL, USA.
Daulagala PWHKP & Allan EJ (2003) L-form bacteria of Pseudomonas syringae pv. phaseolicola induce
chitinases and enhance resistance to Botrytis cinerea infection in Chinese cabbage. Physiological and
Molecular Plant Pathology 62: 253263.
Derckel JP, Audran JC, Haye B, Lambert B & Legendre L (1998) Characterization, induction by wounding and
salicylic acid, and activity against Botrytis cinerea of chitinases and -1,3-glucanases of ripening grape
berries. Physiologia Plantarum 104: 5664.
Fernandez-Caballero C, Romero I, Gon O, Escribano MI, Merodio C & Sanchez- Ballesta MT (2009)
Characterization of an antifungal and cryoprotective class I chitinase from table grape berries (Vitis vinifera
cv. cardinal). Journal of Agricultural and Food Chemistry 57: 88938900.
Fraser PD, Enfissi EMA, Halket JM, Truesdale MR, YU D, Gerrish C & Bramley PM (2007) Manipulation of
phytoene lebels in tomato fruit: Effects on Isoprenoids, Plastids, and Intermdiary Metabolism. Plant Cell 19:
31943211.
Giovannoni J (2001) Molecular regulation of fruit ripening. Annual Review of Plant Physiology and Plant
Molecular Biology 52: 725749.
Guthrie JL, Khalif S & Castle AJ (2005) An improved method for detection and quantification of chitinase
activities. Canadian Journal of Microbiology 51: 491496.
Hammerschmidt R (1999) Phytoalexins: what have we learned after 60 years? Annual Review of
Phytopathology 37: 285306.
Ho VSM & Ng TB (2007) Chitinase-like proteins with antifungal activity from emperor banana fruits. Protein
and Peptide Letters 14: 828831.
Kitajima S, Kamei K, Taketani S, Yamaguchi M, Kawai F, Komatsu A & Inukai Y (2010) Two chitinase-like
proteins abundantly accumulated in latex of mulberry show insecticidal activity. BMC Biochemistry 11: 6
13.
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature 227: 680685.
Daulagala (2014) 1(3): 7279
.
Lawrence CB, Joosten MHAJ & Tuzun S (1996) Differential induction of pathogenesis-related proteins in
tomato by Alternaria solani and the association of a basic chitinase isozyme with resistance. Physiological
and Molecular Plant Pathology 48: 361377.
Li Y, Yang Y, Hsu JSF, Wu D, Wu H & Tzen JTC (2005) Cloning and immunolocalization of an antifungal
chitinase in jelly fig (Ficus awkeotsang) achenes. Phytochemistry 66: 879886.
Liu JH, Zhang J, Xu BY, Zhang JB, Jia CH, Wang JS & Jin ZQ (2012). Expression analysis of banana
MaECHI1 during fruit ripening with different treatments. African Journal of Biotechnology 11: 12951
12957.
Mauch F, Hadwiger LA & Boller T (1984) Ethylene: Symptom, not signal for induction of chitinase and b-1, 3-
glucanases in pea pods by pathogens and elicitors. Plant Physiology 76: 607611.
Mauch F, Mauch-Mani B & Boller T (1988) Antifungal hydrolases in pea tissue. II. Inhibition of fungal growth
by combination of chitinase an beta-1, 3-glucanase. Plant Physiology 88: 936942.
Neuhaus J (1999) Plant chitinases. In: Datta SK, Muthukrishnan S (eds) Pathogenesis-related proteins. CRC
Press, Boca Raton, FL, USA, pp. 77106.
Peumans WJ, Barre A, Derycke V, Rouge P, Zhang W, May GD, Delcour JA, Van Leuven F & Van Damme
EJM (2000) Purification, characterization and structural analysis of an abundant -1, 3-glucanase from
banana fruit. European Journal of Biochemistry 267: 11881195.
Peumans WJ, Proost P, Swennen RL & Van Damme EJM (2002) The abundant class III chitinase homolog in
young developing banana fruits behaves as a transient vegetative storage protein and most probably serves as
an important supply of amino acids for the synthesis of ripening-associated proteins. Plant Physiology 130:
10631072.
Robinson SP, Jacobs AK & Dry IB (1997) A class IV chitinase is highly expressed in grape berries during
ripening. Plant Physiology 114: 771778.
Sanchez-Monge R, Blanco C, Diaz-Perales A, Collada C, Carrillo T, Aragoncillo C & Salcedo G (1999)
Isolation and characterization of major banana allergens: identification as fruit class I chitinases. Clinical
and Experimental Allergy 29: 673680.
Seymour GB (1993) Banana. In: Seymair GB, Taylor JE & Tucker GA (eds) Biochemistry of Fruit Ripening.
Chapman & Hall, London, 83106.
Toledo TT, Nogueira SB, Cordenunsi BR, Gozzo FC, Pilau EJ, Lajolo FM & Nasimento JRO (2012) Proteomic
analysis of banana fruit reveals proteins that are differentially accumulated during ripening. Postharvest
Biology and Technology 70: 5158.
Trudel J & Asselin A (1989) Detection of chitinase activity after polyacrylamide gel electrophoresis. Analytical
Biochemistry 178: 362366.
Van Loon LC (1997) Induced resistance in plants and the role of pathogenesis-related proteins. European
Journal of Plant Pathology 103: 753765.
Wirth SJ & Wolf GA (1990) Dye labeled substrates for the assay and detection of chitinase and lysozyme
activity. Journal of Microbiological Methods 12: 197205.
Xu BY, Su W, Liu JH, Wang JB & Jin ZQ (2007) Differentially expressed cDNAs at the early stage of banana
ripening identified by suppression subtractive hybridization and cDNA microarray. Planta 226: 529539.
Zhu WY & Gooday GW (1992) Effects of nikkomycin and echinocandin on differentiated and undifferentiated
mycelia of Botrytis cinerea and Mucor rouxii. Mycological Research 96: 371377.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
1(3): 8086, 2014
Research article
Diversity, utilization and sacred values of Ethno-medicinal plants

of Kumaun Himalaya
Anita Mehra1*, Omesh Bajpai2 and Hema Joshi1
1
Department of Botany, S.S.J. Campus Almora, Kumaun University, Nainital, Uttarakhand, India
2
Biodiversity, Conservation & Management Group, G.B. Pant Institute of Himalayan Environment &
Development, Kosi-Katarmal, Almora, Uttarakhand, India
*Corresponding Author: anitamehra2690@gmail.com [Accepted: 20 December 2014]
Abstract: Kumaun Himalaya is characterized by a rich cultural and biological diversity as well as
a rich heritage of traditional medicine system. The Himalayan people have a close association with
nature. People used plants for fuel fodder medicine fruits etc. They considered some plants
synonyms of god and worship because of the divine value of plant in Kumaun Himalayas. The
present paper deals with ethno medicinal use and sacred value of some important plans of Kumaun
region. The information was based on oral communication and interview with local people, rural
persons, and vaidyas. During the study it was observed that 58 species of ethno-medicinal plants
belonging to 38 families are being used in the folk-medicine system by the indigenous people of
this region. In which 11 species were trees, 30 were herbs, 14 were shrubs and 02 were climbers.
These identify the plants that need conservation and protection. A total 15 sacred plant recorded
from 13 families which have high sacred value in Himalayan region and used in various ritual.
Keywords: Medicinal plants - Sacred plants - Vaidyas - Kumaun Himalaya.
[Cite as: Mehra A, Bajpai O & Joshi H (2014) Diversity, utilization and sacred values of Ethno-medicinal plants
of Kumaun Himalaya. Tropical Plant Research 1(3): 8086]
INTRODUCTION
The forests of India have been the source of traditional medicines form ancient time. The Charak Samhita, a
document on herbal therapy written about 300 BC, reports on the production of 340 herbal drugs and their
indigenous uses (Vedprakash 1991). The unique diversity of medicinal plants in the region is manifested by the
presence of a number of native (31%), Endemic (15.5%) and Threatened elements (14%) of total Red Data
Book plant species of Indian Himalaya Region (Samant et al. 1998). Documentation on ethno-botanical
knowledge was done by Maikhuri et al. (2000) and Nautiyal et al. (2001). While a wide-ranging review has
described a rich diversity and use of medicinal flora within Uttarakhand (Joshi 2002). The pharmaceutical sector
is using 280 medicinal plant species, out of which 175 are from the Indian Himalayan Region (Dhar et al. 2002).
Various plants and their products which are being used by human day to day need to use in Havan (burning of
herbal ingredients and other religious activities like Katha, Vrat, festivals, Pathpuja, Pitrasharadha ceremony.
The use of alternative medicine is growing because of its moderate costs and increasing faith in herbal
medicine. According to the World Health Organization (WHO), as many as 80% of the worlds people depend
on traditional medicine to meet their primary health care needs (Cotton 1997). Non-sustainable collection
methods and harvesting cause threat and many valuable medicinal herbs are becoming rare due to their
continuous utilization (Swe & Win 2005) International agencies such as the World Wildlife Fund (WWF) and
United Nations Educational, Scientific and Cultural Organization (UNESCO) as part of their people and plants
initiative, are promoting research on ethno botanical knowledge and the integration of peoples perceptions and
practices in resource management at the local level.

The Himalaya extend from west to east in a massive arc for about 2500 km. Covering an astounding area of
612,021 km2, the vast mountain chain passes through the Indian States of Jammu and Kashmir, Himachal
Pradesh, Uttarakhand, Sikkim and the Himalayan kingdoms of Nepal and Bhutan From west to east the
Mehra et.al. (2014) 1(3): 8086
.
Himalayas are divided broadly into three mountainous regions Eastern Himalaya, Central Himalaya and
Western Himalaya. The western Himalaya is one of the well-defined and better known phyto-geographic
regions of the Indian subcontinent in his book sketch of the flora of British India; Hooker (18751897)
recognized the western Himalayan botanical zone extending from Kumaun to Chitral. Kumaun in Uttarakhand
hills comprises the six district of Nainital, Alomra, Bageshwar, Champawat, US Nagar and Pithoragarh. The
Almora district lies between 29 30' N to 30 20' N latitudes and 79 20' E to 80 20' E longitudes. It is located in
the central part of Kumaun region of Uttarakhand (India). The study area covers 3629.66 km2 (Fig. 1).
Figure 1. Map showing the study area.
The area rich in traditional knowledge and vegetation Present study is based on intensive field surveys made
during 2013 the villages were visited for identification of medicinal and sacred plant species. Data on local
name botanical name family medicinal and other use were collected recorded and tabulated. The collected
information was re-examined by consulting important works on medicinal plants and ethno-botany and
identification of plant species was made with the help of available literature (Nair & Mohanan 1998, Samant &
Palni 2000, Samant et al. 2001, Brahmvarchaswa 2003). The status of the ethno-medicinal plants was compared
with Red Data Book (IUCN 1993).

Diversity and utilization pattern
The present study records 58 species of ethno-medicinal plants representing 38 families in which
Solanaceae, Rutaceae, Asteraceae and Fabaceae families were showing largest number of medicinal plant
species in study site (Fig. 2; Table 1). Various parts such as whole plant (25 spp.), fruits (20 spp.), roots (46
spp.), seeds (8 spp.), bark (17 spp.), leaves (11 spp.) and flowers (4 spp.) were used for the treatment of various
ailments like in asthma, bronchitis, constipation, cough, diabetes, fever, intestinal complaints, leprosy, piles,
respiratory disease and stomach ache (Fig. 3A). Qualitative analysis of present study reveals that a total of 58
plants used differently of which 53 % were herbs, 25 % shrubs, 20 trees and 2% climbers (Fig. 3B).
Table 1. List of medicinal plants traditionally used by villagers in the study area.
Sl. Local name Botanical name Family Status Part use Habitat Ethno medicinal use
1 Lahsun Allium sativum L. Alliaceae CU Whole plant Herb Clove with the mustard oil used in joint
pain.
2 Aam Mangifera indica L. Anacardiaceae CU Seed, fruit Tree Powder of seed used in diarrhoea locally
people used milk and mango pulp for
weight gaining.
3 Brahmi Centella asiatica (L.) Urb. Apiaceae CO Leaves Herb Leaves used as a brain tonic.
4 Patti Artemisia parviflora Roxb. Asteraceae CO Leaves Shrub Used in skin diseases, burns cuts, wounds
ex D.Don and Fumes are insect repellents.
Mehra et.al. (2014) 1(3): 8086
.
5 Kalabasa Eupatorium odoratum L. Asteraceae CO Leaves, Stem Herb Extract of plant is used to cure cuts.
6 Gha buti Ageratum conyzoides (L.) L. Asteraceae CO Leaves Herb Extract of leaf used in stop bleeding.
7 Muli Raphanus sativus L. Brassicaceae CU Whole plant Herb The roots and leaves used to cure jaundice,
piles and kidney stone.
8 Sarson Brassica rapa L. Brassicaceae CU Whole plant Herb 2-3drops of seed oil put in ear in sepsis and
also used in toothache.
9 Kilmora Berberis asiatica Roxb. ex. Berberidaceae EN Root Shrub The juice of fresh roots is used for curing
DC. diabetes and jaundice. Extract of root used
as eye drop in eye disease.
10 Bathua Chenopodium album L. Chenopodiaceae CO Leaves Herb Green vegetable used in bladder stone and
anaemia.
11 Harar Terminalia chebula Retz. Combretaceae CO Fruit Tree Locally used in cough and throat disorder.
Cure vomiting and used in formation of
Triphla with Awala and Harar.
12 Bhera Terminalia bellirica Combretaceae CO Fruit Tree Fruit is given 2-3 times a day in hyper
(Gaertn.) Roxb. acidity.
13 Karela Momordica charantia L. Cucurbitaceae CO Fruit Climber Continuous use of fruit juice control
diabetes.
14 Burans Rhododendron arboretum Ericaceae CO Flower Tree Flowers are eaten raw or made into juice to
Cowan. cure stomach diseases but Young flower
and leaves are poisonous.
15 Awala Emblica Officinalis Euphorbiaceae CO Fruit Herb Fresh fruit juice used in eye sight
Gaertn improving and anaemia.
16 Gahat Vigna unguiculata (L.) Fabaceae CU Seed Herb Boiled seed or Dal is used in kidney stone
Walp. and localized abdominal tumour.
17 Methi Trigonella foenum - Fabaceae CU Seed Herb Decoction of seed with honey is beneficial
graecum L. in piles.
18 Akhrot Juglans regia L. Juglandaceae CO Bark, Nut, Tree The bark is boiled in water after filtration it
Leaf is used as mouthwash, very useful in loose
teeth.
19 Tulsi Ocimum tenuiflorum L. Lamiaceae CO Whole plant Herb The leave used with common salt in
toothache leave also used in fever and cold.
20 Piperment Mentha piperita L. Lamiaceae CU Leaf Herb Crushed leaves are used in nausea vomiting
and typhoid.
21 Pudina Mentha longifolia (L.) L. Lamiaceae CU Leaf Herb People used in the acidity, stomach pain,
gastrointestinal disorders, cough, cold and
chronic fever.
22 Stawar Asparagus recemosus Asparagaceae CO Root Shrub Dry root powder with milk help in cure eye
Willd. disease and used as a tonic.
23 Alovvera Aloe barbadensis Mill. Liliaceae CU Leaf Herb Leaves juice used in jaundice, fiver, liver
disease, piles, and skin disease.
24 Bakain Melia azedarach L. Meliaceae Bark Tree The bark is boiled in water. After filtration
R it is used as mouthwash, very useful in
loose teeth.
25 Guduci Tinospora sinensis (Lour.) Menispermaceae R Stem, Climber Stem and leaves juice is used in fever,
Merr. Leaves body heat, burning sensation, diabetic,
urinary problem and anaemia.
26 Peepal Ficus religiosa L. Moraceae CO Tree The juice of its leaves extracted by holding
them near the fire can be used as the ear
drop. Its power bark has been used to heal
the wounds. The roots are chewed to
prevent gum diseases.
27 Timul Ficus auriculata Lour. Moraceae CO Fruit Tree Fruits are eaten raw and cooked as
Vegetable.
28 Kafal Myrica esculenta Buch.- Myricaceae CO Bark, Fruit Tree Bark powder inhale is useful in headache.
Ham. ex D. Don Fruits are edible.
29 Kela Musa balbisiana Colla. Musaceae CU Fruit Herb The fruit is given with milk to cure body
weakness.
30 Til Sesamum Indicum L. Pedaliaceae CU Seed Herb It prevents hair loss and It is used for
massaging patients who suffer from body
pain and joint pain.
31 Devdar Cedrus deodara (Roxb. ex Pinaceae CO Bark, Wood Tree Its fumes are used as a snake repellent.
D.Don) G.Don
32 Pine Pinus roxburghii Sarg. Pinaceae CO Wood Tree Resin use to heal crack. Wood and Resin.
Wood used in snake bite and scorpion sting.
33 Dub Cynodon dactylon (L.) Poaceae CO whole plant Herb The extract of plant used in cure nasal
Pers. bleeding.
Mehra et.al. (2014) 1(3): 8086
.
34 Chalmori Rumex hastatus D.Don Polygonaceae CO Leaves Herb Leaves juice is given in abdominal stomach
pain.
35 Hisalu Rubus ellipticus Sm. Rosaceae CO Fruit Shrub Juice of fruits is administered orally in
cholera.
36 Ghingaru Pyracantha crenulata Rosaceae CO Fruit Shrub The fruit used in anaemia.
(Roxb. ex D.Don) M.Roem.
37 Pya Prunus Cerasoides Buch.- Rosaceae CO Bark, Seed Tree The juice of the bark is applied externally
Ham. ex D.Don to treat backaches. Seed are chewed in case
of kidney stone.
38 Mangishta Rubia cordifolia L. Rubiaceae R Root Herb Paste of root applied externally in
leucoderma.
39 Belpatri Aegle marmelos (L.) Correa Rutaceae CO Fruit Tree Local people used ripe fruit to cure the
digestive disorder.
40 Timur Zanthoxylum armatum DC. Rutaceae T Whole plant Shrub It is used in curing various common
ailments such as toothache, common cold,
cough, and fever, as it is believed to give
warmth to the body.
41 Carry Patta Murraya koenigii (L.) Rutaceae CO Leaves Shrub Paste of leaf applies in skin disease.
Spreng.
42 Lemon Citrus medica L. Rutaceae CU Fruit Tree Juice of fruit is used as refrigerant drink
and allaying thirst. It is beneficial in cough
and throat disorder with ginger. Juice is
also useful in diarrhoea and liver trouble.
43 Ber Ziziphus mauritiana Lam. Rhamnaceae R Whole plant Shrub Powdered leaf is given in pain in joint pain.
Leaves chewed in scorpion sting. Root
made into paste is applied to snake bite.
44 Mamiri, Pilijari Thalictrum foliolosum DC. Ranunculaceae VU Root Herb Root is used in jaundice
45 Reetha Sapindus mukorossi Gaertn Sapindaceae CO Fruit Tree Used to control external parasites, hair and
skin diseases and to expel leach
46 Silfoda Bergenia ligulata Engl. Saxiferagaceae VU Root Herb The plant has been recognized for its role
in dissolving kidney and bladder stone.
47 Kutki Picrorhiza kurroa Royle ex. Scrophulariaceae EN Root Herb Roots used to cure digestive disorders,
Benth. asthma, fever, piles, ring worm, jaundice,
anaemia, heart disease, malarial fever,
worms infestation in children, indigestion.
48 Ekalveer Verbascum thapsus L. Scrophulariaceae CO Leaf Herb Leaf paste is rubbed on chest to relieve
pain due to cold. Leaves are useful in fever.
49 Dhatura Datura stramonium L. Solanaceae CO Seed Shrub The seed, leaves after roasting are applied
locally to relive pain.
50 Makoi Solanum nigrum L. Solanaceae CO Fruit, Herb Fruits are used to treat eye diseases,
Root dysentery and fever. Seeds and roots are
used to treat liver related problems.
51 Mirchi Capsicum annuum L. Solanaceae CU Fruit Herb Paste of fruit is applied on scorpion sting.
52 Ashwgandha Withania somnifera (L.) Solanaceae CO Leaves, Herb To improve memory and weakness in
Dunal Root humans. The leaves are applied to tumours.
The roots are regarded as useful in
rheumatism.
53 Baigain Solanum melongena L. Solanaceae CU Stem Herb In dog bite wounds portion is brunt with
the help of burning woody stem.
54 Swina Urtica parviflora Roxb. Urticaceae CO Leaf Shrub Flogging by leaf in bone fracture.
55 Jtamansi Valeriana wallichii DC. Valerianaceae CR Whole plant Herb The essential oil of the root and rhizome is
having antibacterial property.
56 Van haldi Hedychium spicatum Sm. Zingiberaceae VU Rhizome Herb The powder of root is useful in the
treatment of liver complaints, and it also
used in treating fevers, vomiting, diarrhoea,
inflammation, pains.
57 Haldi Curcuma longa L. Zingiberaceae CU Rhizome Herb The powder of rhizome considered as a
good antiseptic, 1:10 mixture of rhizome
powder with boiled water (filtered) use as
eye drop in eye diseases. Juice of crushed
raw Haldi with milk used in physical
damage.
58 Punarnva Boerhavia diffusa L. Nyctaginaceae CO Root Herb Juice of fresh roots is used as eye drops.
Root juice is used in urinal disorder.
Watery extract of the root is given in
jaundice.
Note: CR, Critically Endangered; EN, Endangered; VU, Vulnerable; R, Rare; CU, Cultivated; CO, Common.
Mehra et.al. (2014) 1(3): 8086
.
Among the important works on Himalayan plants Osmastons (1922) Forest Flora for Kumaun is notable
but, however, more emphasis was laid on their systematic rather than their ethno-medicinal used (Gupta 1960 &
1968, Shah & Joshi 1971, Pangtey 1980). Recently Kumari et al. (2012) explore the diversity and ethno-
medicinal significance of medicinal plant of Almora. The present attempt has been designed to explore the
utilization pattern ethno-medicinal significance and sacred values of the floral habitat of this region.
Figure 2. Ethno- medicinal plant belonging to different families.
Figure 3. Ethno medicinal plants: A, Utilization pattern; B, Life form diversity.
Rarity
Using new IUCN criteria, 10 species of medicinal plans have been categorized in different categories,
Critically Endangered (CR) (Valeriana wallichii); Endangered (EN) (Berberis asiatica, Picrorhiza kurroa);
Vulnerable (VU) (Bergenia ligulata, Hedychium spicatum, Thalictrum foliolosum); Rare (R) (Melia azedarach,
Tinospora sinensis, Rubia cordifolia, Ziziphus mauritiana) (Fig. 4).
Sacred plans and there values
A total 15 sacred plant recorded from 13 families which have high sacred value in Himalayan region these
plant species used in different ceremony. These plant species are regularly used by used by local people. The
uses of each plant are enumerated in table 2. Dhiman (2003) have discussed the sacred plans and their medicinal
important. The religious aspect of plant less explore in western Himalaya region. This religious aspect of plants
is a tool of biodiversity conservation.
Mehra et.al. (2014) 1(3): 8086
.
Figure 4. Number of plant species under different IUCN categories (CR, Critically Endangered; EN, Endangered;
VU, Vulnerable; R, Rare; CU, Cultivated; CO, Common).
Table 2. List of sacred plants and there values.
Local
S.N. Botanical name Family Status Habitat Sacred value
name
1 Aam Mangifera indica L. Anacardiaceae Common Tree Used for decoration of new homes and
Havankund (a sacred place). Wood is used in
worship and Hwans (scarify)
2 Patti Artemisia parviflora Asteraceae Common Shrub Plants are worshipped on Sunday
Roxb. ex. D.Don
3 Sarson Brassica rapa L. Brassicaceae Cultivated Herb Seeds of plants are used for yielding oil and
this oil is considered as pure for lighting lamp
(Deepak).
4 Bhang Cannabis sativa L. Cannabaceae Common Shrub Used in worship of lord Shiva.
5 Tulsi Ocimum tenuiflorum L. Lamiaceae Common Herb This sacred plant worshiped because associated
with Lord Vishnu.
6 Guduci Tinospora sinensis (Lo Menispermaceae Rare Climber Used in Hawan or worship.
ur.) Merr.
7 Timul Ficus auriculata Lour. Moraceae Common Tree Leaves of tree are used in any religious activity
because considered pure for the gods.
8 Kela Musa balbisiana Musaceae Common Herb The whole banana tree is worshipped in Katha
Colla being regarded as representation of Lord Satya
Narayan.
9 Til Sesamum indicum L. Pedaliaceae Common Herb The oil used in all sacred rituals Generally
black color Til is used in religious work like
Havan
10 Dub Cynodon dactylon (L.) Poaceae Common Herb it is used in all rituals of Hindu to please lord
Pers. Ganesh
11 Kush Saccharum Poaceae Common Herb Kush is used during Janeau and Shradha
spontaneum L. ceremony
12 Pya Prunus cerasoides Rosaceae Common Tree The leaves and branches of tree are worshipped
Buch.-Ham. ex D.Don during Katha, Jneu& marriage ceremony.
13 Belpatri Aegle marmelos (L.) Common Tree leaves are used in worship of Lord Shiva
Correa Rutaceae
14 Timur Zanthoxylum armatum Rutaceae Threatened Shrub Branches or sticks of plantsare worshipped in
DC. Jneusanskar considered symbolic of folk god
Narsingh.
15 Haldi Curcuma longa L. Zingiberaceae Culivated Herb Rhizome used in various religious activities.
The rhizome used in dyeing of cloths in festival.
Sacred plans and there values

A total 15 sacred plant recorded from 13 families which have high sacred value in Himalayan region these
plant species used in different ceremony. These plant species are regularly used by used by local people. The
uses of each plant are enumerated in table 2. Dhiman (2003) have discussed the sacred plans and their medicinal
Mehra et.al. (2014) 1(3): 8086
.
important. The religious aspect of plant less explore in western Himalaya region. This religious aspect of plants
is a tool of biodiversity conservation.
Conclusion
Religious beliefs and rituals are closely related to the management of the ecosystems the sacred value of a plant
can conserve this plant. Sacred value is an important tool of biodiversity conservation. Villagers use herbal medicine
because of easy access and no side effect the presents findings help in conservation of these medicinal plants. In
present study a total. Medicinal plant comes in threatened category the frequent use of these plant is a point of
concern since this practice may put extinction of these species. The indigenous knowledge about sacred and
medicinal plant pass orally from generation to generation there is an urgent need of investigation and documentation.
REFERENCES
Brahmverchas (2003) In: Ayurved Ka Pran: Van ausdhi vigyan. Shantikunj, Haridwar.
Cotton CM (1997) Ethnobotany, Principles and Applications. Wiley & Sons, UK.
Dhiman AK (2003) Secred plan and here medicinal use. Daya Publications, New Delhi.
Dhar U, Manjkhola S, Joshi M, Bhatt A, Bisht AK & Joshi M (2002) Current status and future strategy for
development of medicinal plants sector in Uttaranchal, India. Currant Science 83 (8): 956964.
Gupta RK (1960) Some Useful and Medicinal Plants of Nainital in Kumaun Himalaya. Journal of Bombay
Natural History Society 68: 309329.
Gupta RK (1968) Floral Nainitalensis. NavYug Traders, New Delhi.
IUCN (1993) Draft IUCN Red List Categories, Gland Switzerland.
Joshi BD (2002) A brief review on the flora of medicinal importance and prospects of developing a sustainable
network of small scale pharmaceutical industries in Uttaranchal. Himalayan Journal of Environment and
Zoology 16(2): 233.
Kumari P, Joshi GC & Tewari LM (2012) Indigenous uses of threatened Ethno-medicinal plants used to cure
different diseases by Ethnic people of Almora District of Western Himalaya. International Journal of
Ayurvedic & Herbal Medicine 2: 4.
Maikhuri RK, Nautiyal S, Rao KS & Saxena KG (2000) Indigenous knowledge of medicinal plants and wild
edibles among three tribal sub-communities of Central Himalayas, India. Indigenous Knowledge and
Development Monitor 8(2): 713.
Nair CKN & Mohanan N (1998) In: Medicinal plants of India. Nag Publishers, Delhi.
Nautiyal S, Rao KS, Maikhuri RK, Semwal RL & Saxena KG (2001) Traditional knowledge related to
medicinal and aromatic plants in tribal societies in a part of Himalaya. Journal of Medicinal and Aromatic
Plant Sciences 22(4A) & 23(1A): 528541.
Osmaston AE (1927) A Forest Flora of Kumaun. Allahabad.
Pangtey YPS (1980) Some Wild Edible Fruit Plants of Kumaun Hills In: Singh JS, Singh SP & Shastri C (eds)
Science and Rural Development in Mountains. Gyanodaya Publication, Nainital.
Samant SS & Palni LMS (2000) Diversity, distribution and indigenous uses of essential oil yielding medicinal
plants of the Indian Himalayan Region. Journal of Medicinal and Aromatic Plant Sciences 22: 671684.
Samant SS, Dhar U & Palni LMS (1998) Medicinal Plants of Indian Himalaya: Diversity Distribution and
Potential Value. Gyanodaya Prakashan, Nainital.
Samant SS, Dhar U & Palni LMS (2001) Himalayan Medicinal Plants: Potential and Prospects, Gyanodaya
Prakashan, Nainital.
Shah NC & Joshi MC (1971) An Ethnobotanical Study of Kumaun Region of India. Economic Botany 25: 414
422.
Swe T & Win S (2005) Herbal gardens and cultivation of medicinal plants in Myanmar regional consultation
on development of traditional medicine in the South East Asia region. Department of Traditional Medicine,
Ministry of Health, Myanmar, Pyongyang, DPR Korea, 2224 June 2005, World Health Organization
(Regional office for South-East Asia).
Vedprakash (1991) In: Samant SS, Dhar U & Palni LMS (eds) Indian Medicinal Plant: Current Status in
Himalayan Medicinal Plants: Potential and Prospects. Gramodaya Prakashan, Nainital, pp. 4563.
Hooker JD (18751897) The flora of British India. L. Reeve & Co., Covent Garden, UK.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
1(3): 87 88, 2014
Short communication
Growth of Papaya grown in pot culture of different soil

compositions
Swagatika Sahu1, Sujata Dash2 and Nibha Gupta2*
1
Project trainee, TACT, Bhubaneswar, Odisha, India
2
Regional Plant Resource Centre, Bhubaneswar, Odisha, India
*Corresponding Author: nguc2003@yahoo.co.in [Accepted: 09 November 2014]
[Cite as: Sahu S, Dash S & Gupta N (2014) Growth of Papaya grown in pot culture of different soil
compositions. Tropical Plant Research 1(3): 8788]
Papaya is known as anti-proliferative agent and consists of high nutritional value. It is reported that the
application of microbial inoculants individually and/or along with different fertilizers effect the growth and
development of papaya in field conditions (Mamtha et al. 2002, Wei et al. 2006). Hybrid papaya (Scarlet
princess) (Carica papaya L.) is a high yielding variety. In present study, an attempt was made towards effect of
different fertilizer treatment and phosphate solubilizer on growth of hybrid papaya grown in pot cultures.
An experiment was carried out in 9 inch poly bags filled with fumigated sandy loam soil. The pot soil was
added with different fertilizers individually and/or in combination. The treatments used were [1] control, [2]
super phosphate (5 mg.pot-1), [3] Potash (5 mg.pot-1), [4] Shymala-a combination of N P K (5 mg.pot-1), [5]
Multiplex Annapurna (5 g.pot-1), [6] Garden Samrat (5 g.pot-1), [7] Nirmal Bio-power (5 g.pot-1), [8] broth
culture of fungi phosphate solubilising Aspergillus sp. (5 ml.pot-1), [9] Fungi + Super phosphate (5 ml + 5 mg
pot-1), [10] Fungi + Super phosphate + Multiplex Annapurna (5 ml + 5 mg + 5g pot-1), [11] Fungi + Multiplex
Annapurna (5 ml + 5 g pot-1). The plants were watered daily and final observation on various growth parameters
were recorded after 60 days.
Where, 1= control, 2= Super phosphate (5 mg.pot-1), 3= Potash (5 mg.pot-1), 4= Shymala-a

combination of N P K (5 mg.pot-1), 5= Multiplex Annapurna (5 g.pot-1), 6= Garden Samrat (5
g.pot-1), 7= Nirmal Bio-power (5 g.pot-1), 8= Broth culture of fungi phosphate solubilising
Aspergillus sp. (5 ml.pot-1), 9= Aspergillus sp. + super phosphate (5 ml + 5 mg pot-1), 10=
Fungi + Super phosphate+Multiplex Annapurna (5 ml + 5 mg + 5 g pot-1) and 11= Fungi +
Multiplex Annapurna (5 ml + 5 g pot-1).
Figure 1. Height of Papaya plant grown in different treatments.
Results obtained on growth parameters exhibited good performance of hybrid papaya grown in different
treatment as compared to control (Fig. 1). The plants grown under treatment of Annapurna showed good leaf
Sahu et al. (2014) 1(3): 87 88
.
Where, 1= Untreated control, 2= Super phosphate (5 mg.pot-1), 3= Potash (5 mg.pot-1), 4=

Shymala-a combination of N P K (5 mg.pot-1), 5= Multiplex Annapurna (5 g.pot-1), 6=
Garden Samrat (5 g.pot-1), 7= Nirmal Bio-power (5 g.pot-1), 8= Broth culture of fungi
phosphate solubilising Aspergillus sp. (5 ml.pot-1), 9= Aspergillus sp. + super phosphate (5 ml
+ 5 mg pot-1), 10= Fungi + Super phosphate+Multiplex Annapurna (5 ml + 5 mg + 5 g pot-1)
and 11= Fungi + Multiplex Annapurna (5 ml + 5 g pot-1).
Figure 2. Dry biomass and root shoot ratio of papaya plants grown in different treatments.
number, shoot height and root length. In similar way treatment of Garden Samrat exhibited good plant growth in
terms of biomass (Figs. 2 & 3). Inoculation of Aspergillus sp. in pot soil did not show any effect on growth
enhancement of papaya. However, the combination of Aspergillus sp. with super phosphate and Multiplex
Annapurna exhibited the highest dry biomass as compared to other treatments and untreated control. Since,
growth and development of any plants depend upon several edaphic and environmental factors too (Wei et al.
2006), further experimentation on standardization of inoculum density, soil factors and quantification of
fertilizer treatments are required to reach any conclusion.
A B
Figure 3. A & B, Effect of different treatments on growth of papaya.
REFERENCES
Mamatha G, Bagyaraj DJ & Jaganath S (2002) Inoculation of field-established mulberry and papaya with
arbuscular mycorrhizal fungi and a mycorrhiza helper bacterium. Mycorrhiza 12: 313316.
Wei XD, Zou HL, Chu LM, Liao B, Ye C, M & lan CY (2006) Field released transgenic papaya affects
microbial communities and enzyme activities in soil. Plant Soil 285: 347358.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
1(3): 8991, 2014
Short communication
Potential for exploitation of Dendrocalamus stocksii (Munro.)

shoots: New report from Peninsular India
Sowmya Chandramouli1*, Syam Viswanath1 and Udaykumar Nidoni2
1
Tree Improvement and Genetics, Institute of Wood Science and Technology,
Malleswaram, Bangalore - 560003, Karnataka, India
2
Dept. of Agriculture Processing and Food Engineering, University of Agricultural Sciences,
Raichur - 584101, Karnataka, India
*Corresponding Author: sowmya.chandramouli@gmail.com [Accepted: 14 November 2014]
[Cite as: Chandramouli S, Viswanath S & Nidoni U (2014) Potential for exploitation of Dendrocalamus stocksii
(Munro.) shoots: New report from Peninsular India. Tropical Plant Research 1(3): 8991]
Dendrocalamus stocksii (Munro.) is an extremely manageable thorn less, mid-sized bamboo species with
great economic and socio-cultural importance found naturally distributed in Central Western Ghats from
Kasargod (Kerala) to Ratnagiri (Maharashtra) (Fig. 1). This is an extremely manageable species with a great
economic and ecological importance (Singhal & Gangopadhyay 1999). Previous studies have indicated that this
species has problem in seed setting coupled with sporadic flowering behaviour (Beena 2012). Due to solid
nature of culms, it has multifarious uses in agrarian ecosystems along the Central Western Ghats and is
maintained in field bunds/farm boundaries and in homesteads (Viswanath et al. 2013).
Figure 1. A typical Dendrocalamus stocksii clump with loosely spaced culms. (Inset: Emerging shoots)
Chandramouli et al. (2014) 1(3): 8991
.
It is a component of various agricultural implements, used in farm structures and small construction, live
fencing and as navigation tool in country boats etc. In recent times due to the scarcity of cane/rattan this species
is increasingly been seen as a substitute in furniture industry due to its typical anatomical characteristics like the
presence of non- predominant nodes, solid nature of culms and good culm wall thickness. Owing to its
multifarious uses and perceived importance, National Bamboo Mission (NBM) has prioritized this species for
large scale cultivation in Peninsular India. Despite its marked presence in the region, there is a lack of awareness
on the utilization potential of the species as edible shoots. Though this species is endemic to Central Western
Ghats, there are no reports on the edible properties of the shoots. The possibility of exploiting shoots of this
species for edible purposes was explored.
Bamboo shoots are used as vegetable in many south Asian nations. Shoots are reported to be high in
proteins, fibre, essential amino acids, bioactive compounds and minerals, and low in fat, which makes it an
excellent food for direct consumption and in nutraceuticals. The macronutritional composition (ash, protein,
carbohydrates, fat, crude fibre) (AOAC 1998, 2005) and total cyanogen content (Bradbury et al. 1999) of the
species was analyzed in comparison with other commonly consumed species in the region like Bambusa bambos
and Dendrocalamus strictus and the well-known sweet bamboo Dendrocalamus asper which is solely
cultivated for edible purposes. Study revealed that the macronutritional composition of D. stocksii was on par
with the other three species and also the cyanogenic glucosides responsible for the pungent taste and bitterness
in the shoots are found to be low in D. stocksii (Figs. 2 & 3). The percentage of edible portion from D. stocksii
(35%) was also as good as Bambusa bambos (31%), Dendrocalamus strictus (52%) and D. asper (41%).
Figure 2. Macronutrient composition in g/100g of fresh Figure 3. Total cyanogen content in ppm in fresh
bamboo shoots of Bambusa bambos, Dendrocalamus strictus, bamboo shoots of Bambusa bambos, Dendrocalamus
D. asper and D. stocksii. strictus, D. asper and D .stocksii.
India, which is the second largest producer of bamboo shoots after China, the food potential seems grossly
underutilized. This may be primarily due to lack of awareness about the edible characteristics of the shoots.
Consumption of tender shoots is conned mainly to the Northeastern states and few parts of Southern peninsula
like Coorg, South Canara in Karnataka and in Wayanad, Kerala where they are part of the traditional cuisine
during monsoon when the shoots emerge. In other parts, especially surrounding forest areas, shoots of species
like Bambusa bambos and Dendrocalamus strictus which generally occur in the wild are consumed. Restrictions
imposed by Forest department on the harvest of bamboo from the forests of Western Ghats have hampered the
exploitation of bamboo shoots for edible purposes. Although multipurpose species like D. stocksii, B. balcooa,
D. asper, D. brandisii and D. hamiltonii are widely cultivated, its potential as food is poorly recognized and
there is a lack of awareness on sustainable management. Observations on the average number of new shoots
emerging D. stocksii clumps per year at IWST field station indicate that around 1820 new shoots emerge as
compared to 1015 shoots in B. bambos and D. strictus and 810 shoots in D. asper grown under same
conditions which indicate that after scientifically harvesting 2030% of the emerging shoots for edible purposes,
the mature culms can still be exploited for other commercial uses. The emerging shoots could serve as
additional source of nutrition during monsoon. Since the mature culms also have very high utility value,
sustainable harvest of juvenile shoots could serve as an additional source of income for the farmers in Central
Western Ghats and the species could truly be recommended as a multi-purpose bamboo species for large scale
commercial cultivation.
Chandramouli et al. (2014) 1(3): 8991
.
REFERENCES
AOAC-Association of Official Analytical Chemists (1998) Official Methods of Analysis of AOAC International,
16th Edition, 4th Revision. AOAC International, Gaithersburg, MD.
AOAC-Association of Official Analytical Chemists (2005) Official Methods of Analysis, 18th Edition. AOAC,
Arlington. VA.
Bradbury MG, Egan SV & Bradbury JH (1999) Determination of all forms of cyanogens in cassava roots and
cassava products using picrate paper kits. Journal of the Science of food and agriculture 49: 9399.
Beena VB (2012) Reproductive Biology and Biochemical changes associated with flowering of Dendrocalamus
stocksii and Ochlandra travancorica, Ph.D. Thesis. Cochin University of Science and Technology. Kerala,
India.
Singhal RM & Gangopadhyay G (1999) Bamboos in India and database. Publication Division, ICFRE,
Dehradun, India, 147 p.
Viswanath S, Joshi G, Somashekar PV, Rane AD, Sowmya C & Joshi SC (2013) Dendrocalamus stocksii
(Munro.): A potential multipurpose bamboo species for Peninsular India. Institute of Wood Science &
Technology Technical Bulletin No.11. IWST publication, Bangalore.

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ISSN (E): 2349 1183

ISSN (P): 2349 9265

Phenology, growth and survival of Vatica lanceaefolia Bl.: A

MATERIAL AND METHODS

Abbreviations: 1=Leaf abscission, 2=Leaf flushing, 3=Flowering, 4=Fruiting,

Survival and growth of seedlings

RGRH(U) R=0.637* R=0.602* R=0.687** R=0.786** R=0.544* R=0.609*

RGRH(G) R=0.702** R=0.665** R=0.781** R=0.686** R=0.560* R=0.675**

RGRD(U) R=0.548* R=0.548* R=0.482ns R=0.452ns R=0.543* R=0.416ns

RGRD(G) R=0.521* R=0.349ns R=0.197ns R=0.673** R=0.395ns R=0.521*

RF=Rainfall, RH=Relative humidity and AVT =Average temperature

Pogonatum perichaetiale subsp. thomsonii (Mitt.) Hyvnen -

MATERIAL AND METHODS

Species composition and structure of Sal (Shorea robusta

MATERIAL AND METHODS

DISCUSSIONS AND CONCLUSION

Comparative evaluation of nutritional, biochemical and

RESULTS AND DISCUSSION

Table 1. Nutritional components and antioxidant activities of Pleurotus spp.

Table 2. Extracellular enzymatic activity of Pleurotus sajor-caju and P. florida.

Study on relationship and selection index in chickpea

MATERIAL AND METHODS

variance at phenotypic and genotypic levels of character 2.

Figure 1. Path diagram of different yield contributing traits at phenotypic level.

Figure 2. Path diagram of different yield contributing traits at genotypic level.

Enzymatic antioxidant activities in eight wild edible fruits of Odisha

MATERIAL AND METHODS

Figure 1. Comparison of SOD enzyme activity in eight wild edible fruits.

Ecology and phenology of plant communities of Gentianaceae in

MATERIAL AND METHODS

Figure 1. Map showing locations of study sites in three districts of Karnataka.

RESULTS AND DISCUSSION

Analysis of physico-chemical parameters, genotoxicity and

MATERIAL AND METHODS

Sand 74.700.13 69.100.21 55.600.38 69.920.05

Clay 24.530.23 30.560.34 44.010.34 29.780.34

Cd (3-6)** 30.01.48 25.31.78 22.00.71 9.701.08

24.70.50 27.20.14 26.70.43 27.90.74

All values are Mean SD. of 3 observations for each parameter

Assessment of forest structure and woody plant regeneration on

MATERIAL AND METHODS

Status of dominant woody species composition, distribution and regeneration

Expression of chitinase with antifungal activities in

MATERIAL AND METHODS

Diversity, utilization and sacred values of Ethno-medicinal plants

MATERIAL AND METHODS

Figure 1. Map showing the study area.

RESULTS AND DISCUSSION

Figure 2. Ethno- medicinal plant belonging to different families.

Figure 3. Ethno medicinal plants: A, Utilization pattern; B, Life form diversity.

Sacred plans and there values

Growth of Papaya grown in pot culture of different soil

Where, 1= control, 2= Super phosphate (5 mg.pot-1), 3= Potash (5 mg.pot-1), 4= Shymala-a

Where, 1= Untreated control, 2= Super phosphate (5 mg.pot-1), 3= Potash (5 mg.pot-1), 4=

Figure 3. A & B, Effect of different treatments on growth of papaya.

Potential for exploitation of Dendrocalamus stocksii (Munro.)

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RGRH(U) R=0.637* R=0.602* R=0.687 R=0.786 R=0.544* R=0.609*

RGRH(G) R=0.702 R=0.665 R=0.781 R=0.686 R=0.560* R=0.675**