Volume 5, Issue 2 (2018) Tropical Plant Research

ISSN (Online): 2349-1183; ISSN (Print): 2349-9265
TROPICAL PLANT RESEARCH 5(2): 121–128, 2018

The Journal of the Society for Tropical Plant Research DOI: 10.22271/tpr.2018.v5.i2.017
Research article
The effect of pH on the biological control activities of a

Trichoderma sp. against Fusarium sp. isolated from
the commercial onion fields in Sri Lanka
Abeyratne G. D. D.1* and Deshappriya N.2
1
Department of Botany, Faculty of Science, University of Kelaniya, Sri Lanka
2
Department of Botany, Faculty of Applied Sciences, University of Sri Jayawardenepura, Sri Lanka
*Corresponding Author: dinushika.makola@gmail.com [Accepted: 25 May 2018]
Abstract: Trichoderma spp. are effective biocontrol agents of plant pathogenic fungi and possess
varied mechanisms of control. Chemical and physical factors including pH are known to have an
influence on these mechanisms as well as on the growth and sporulation of Trichoderma. Hence,
the effect of pH on the biocontrol mechanisms of a Trichoderma sp. isolated from the soils of
commercial onion fields of Matale district Sri Lanka was evaluated in this study. The inhibition of
growth of a Fusarium sp. isolated from onion seedlings with damping off disease was evaluated
using dual culture assay and volatile metabolite plate assay at pH 4, 5, 6 and 7. Both tests indicated
that growth inhibition of the Fusarium sp. tested was most effective at pH 6. The rate of
sporulation of the Trichoderma sp. in PDA plates with amended pH values (4, 5, 6 and 7) was also
estimated and a significantly high (p≤0.05) rate of sporulation (5×107 spores ml-1) was observed at
pH 6 after 49 days. The effect of pH on the survival of Trichoderma evaluated using pot
experiments showed that the pH values 4.6–5.0 facilitated the highest survival rate of 7.9×105 cfu
ml-1. Effect of Trichoderma sp. on the growth of Fusarium sp. at different pH values was
evaluated under greenhouse conditions and a significantly low level of Fusarium colonies were
isolated from seedlings treated with Trichoderma at pH 4.6. This is the first report of the effect of
pH on the activities of Trichoderma spp. isolated from Sri Lankan soils.
Keywords: Soil-born pathogen - Biocontrol - pH.
[Cite as: Abeyratne GDD & Deshappriya N (2018) The effect of pH on the biological control activities of a
Trichoderma sp. against Fusarium sp. isolated from the commercial onion fields in Sri Lanka. Tropical Plant
Research 5(2): 121–128]
INTRODUCTION
Trichoderma species are ubiquitous soil-borne Ascomycetes with biocontrol capabilities against diverse
phytopathogenic fungi including Sclerotinia cepivorum, Penicillium sp. (Mishra et al. 2014), Fusarium sp.
(Gunaratna & Deshappriya 2013). The antagonistic properties of Trichoderma are based on multiple
mechanisms including competition for nutrients, mycoparasitism and antibiosis (Howell 2003, Gunaratna et al.
2014). A biocontrol agent incorporated into soil has to possess a high level of competence to survive in the soil
and compete specially with soil inhabitant microorganisms. A high rate of proliferation and maintenance of
viability for an extended period of time are characteristics that will contribute towards such competence.
Various external factors such as temperature, pH, heavy metal ions and water availability are reported to have an
influence on the growth, proliferation, and development as well as the biocontrol efficacy of Trichoderma spp.
(Kredics et al. 2003). Furthermore, Agosin et al. (1997) and Roy et al. (2015) confirmed that acidic condition
accelerate sporulation and stabilize the survival of Trichoderma.
Therefore, in the study, the effect of pH on sporulation and survival of Trichoderma sp. was tested under
laboratory and greenhouse condition. The effect of pH on biocontrol activity of Trichoderma on the growth of a
Fusarium sp., a common soil-borne pathogen isolated from commercial onion fields in Sri Lanka was evaluated
through volatile metabolite plate assay, dual culture plate assay, and formulation of restrictive coiling structure.
Once the optimal pH value for the most efficient biocontrol activity as well as for the highest growth rate
www.tropicalplantresearch.com 121
Received: 28 January 2018 Published online: 31 May 2018
https://doi.org/10.22271/tpr.2018.v5.i2.017
Abeyratne & Deshappriya 2018
proliferation and survival of Trichoderma sp. is evaluated, pH of the soil can be adjusted before applying the
inoculum to the field and monitored the soil pH. Although, the evaluations were carried out for Trichoderma sp.
isolated from the soils of commercial onion fields in Sri Lanka, the findings were found to be similar to those of
others from different parts of the world and thus can be applied under different soil conditions (Metcalf 1997,
Coventry et al. 2006).
MATERIAL AND METHODS

Sample collection
Seedlings showing damping off disease symptoms were randomly collected through Z shape from three
commercial onion fields (0.10 km2) in Rangirigama model village in Sigiriya (N07°58′50.9″, E80°43′07.1″)
and Galewela (N07°47′55.9″, E080°33′15.3″) in Matale district in Sri Lanka during May, 2015. Twenty five
seedlings were collected from each field.
Isolation of the pathogenic fungi from diseased seedlings
Selected roots from onion seedlings with symptoms were separated and rinsed with running tap water for 10
minutes. Approximately 1 cm length segments were cut from these and surface sterilized with 1% sodium
hypochlorite (v/v) for 1 minute. Surface sterilized roots were finally rinsed with sterilized distilled water and
dried on a sterilized filter paper. The cut edges of each segment were trimmed using a sterilized scalpel. The
trimmed root segments were aseptically transferred into Petri plates containing Potato Dextrose Agar (PDA)
supplemented with tetracycline (50 mg l-1). Four root segments were placed per each PDA plate. Three
replicates were prepared and incubated at room temperature (32±2ºC). The fungal colonies growing from the
plated segments were separately sub-cultured onto a fresh PDA medium supplemented with tetracycline (50 mg
l-1) and incubated at room temperature (32±2ºC). Pure cultures of the pathogenic fungi were prepared using the
hyphal tip method (Dhingra & Sinclair 1995) and identified by studying the morphological features (Domsch et
al. 1980). Trichoderma sp. used in the study has been isolated from soils of commercial onion fields in
Galewela in Matale district in a previous study (Gunaratna et al. 2015). Cultures were maintained at 4ºC until
use.
Preparation of PDA plates with different pH values
pH of sterilized molten Potato Dextrose Agar (PDA) was adjusted to 4, 5, 6 and 7 by adding NaOH (1 mol
dm ) and HCl (1 mol dm-3) and checked by pH papers. The media was then dispensed into petri dishes and
-3
allowed to solidify.
Sporulation of Trichoderma sp. at different pH values
To determine the rate of sporulation of the Trichoderma sp., at different pH values, a 5 mm diameter
mycelial disc cut from the margin of a 7 day old Trichoderma culture was placed in the center of each PDA
plate with different pH values (pH 4, 5, 6 and 7). There were 3 replicate plates for each pH value. The plates
were incubated at room temperature (32±2ºC) for 49 days. To enumerate the number of spores produced at each
pH value, spore suspensions were prepared from each plate by adding of 10 ml distilled water and dislodging
the spores by using a glass spreader. The spore concentration of each suspension was determined using a
haemocytometer. The results were analyzed using two-way ANOVA to understand the significant difference of
two independent variables (pH and time) on the dependent variable (number of spores).
Effect of pH on the antagonistic activities of the isolated Trichoderma sp.
1. Growth inhibition of Fusarium sp. at different pH levels- Dual Culture assay: PDA plates with the pH values
amended to 4, 5, 6 and 7 were used for the test. Mycelial discs (5 mm diameter) were cut using a sterile cork
borer from the margin of a 7 day old Trichoderma and Fusarium cultures maintained in PDA at the room
temperature (32±2ºC). The discs were placed at the opposite ends of each plate with each pH value at equal
distance from the periphery. In the control plates, the Trichoderma disc was replaced by a 5 mm diameter
disc of sterile PDA.
Inoculated plates were arranged in completely randomized manner in the laboratory and incubated at
room temperature (32±2ºC) for three days. The radial growth of Fusarium and Trichoderma colonies were
measured. Percentage average radial growth inhibition was calculated in relation to growth of the controls
using the formula: I% = (r1-r2) /r1 multiplied by 100, where I is inhibition of radial mycelia growth; r 1 is
radial growth of the Fusarium in the control; r2 is radial growth of Fusarium in the presence of Trichoderma
isolate. There were three replicate plates for each pH value tested. The interface of the two colonies was
observed under the light microscope (10×40) for the presence of structures that may facilitate antagonism
Tropical Plant Research (2018) 5(2): 121–128
(Hajieghrari et al. 2008).

2. Volatile metabolite production at different pH levels: PDA plates with the pH values amended to 4, 5, 6 and 7
were used for the test. To determine the effect of volatile metabolites of Trichoderma sp. at different pH
values on mycelial growth of Fusarium sp., 7 day old cultures grown on PDA were used. Discs (5 mm
diameter) were cut from the edge of each colony using a sterile cork borer. Each disc was placed in the
center of PDA plates with each test pH value separately. The lid of the plate inoculated with Fusarium was
removed and it was replaced by the bottom of the PDA plate inoculated with the Trichoderma disc. The
plates were pasted together with adhesive tape. In the control plate, Trichoderma disc was replaced by a
sterile agar disc. There were three replicate plates for each treatment. The plates were arranged in completely
randomized manner in the laboratory and incubated at room temperature (32±2ºC) for 3 days. The radial
growth of Fusarium colonies was measured. Percentage inhibition of average mycelial growth in relation to
growth of their respective control was calculated using the formula: I% = (r1-r2) /r1 multiplied by 100,
where I is inhibition of radial mycelia growth; r 1 is radial growth of the Fusarium in the control; r2 is radial
growth of Fusarium in the presence of Trichoderma isolate (Hajieghrari et al. 2008).
Survival of the isolated Trichoderma sp. at different pH levels under greenhouse conditions
1. Preparation of soil samples with different pH values: pH of soil samples was amended to result in values of 5,
6 and 7 by adding coir dust, burnt rice husk or dolomite and slaked lime to 1 kg of soil (pH 4.6) from a
commercial onion field in Sigiriya at the rates shown in table 1. The mixture was saturated with water. After
obtaining the required pH value, the soil samples were incubated in the greenhouse for 2 days and pH of the
soil was reconfirmed before use.
2. Preparation of spore suspensions: Ten days old Fusarium and Trichoderma cultures growing on PDA were
used to prepare spore suspensions. Sterilized distilled water (10 ml) was added to each culture and spores
were dislodged using a sterilized spreader. The spore suspensions were collected separately and spore
concentrations were adjusted to 1×105 spores ml-1 using sterilized distilled water. Trichoderma spore
suspension (1 ml, 1×105 spores ml-1) was added to 5 g of each pH adjusted and non-adjusted (pH 4.6) soil
sample in a pot (20 cm3) and incubated at room temperature (32±2ºC). Enumeration of the number of
Trichoderma colonies in the treated soil samples was done by dilution plate method at four day intervals and
the number of colonies at different pH levels were calculated as follows:
Number of fungal colonies in the soil sample = (n/d) × (10 mL/0.1 mL) × 1.00 g
n = number of colonies
d = dilution factor
Effect of the isolated Trichoderma sp. on the growth of Fusarium sp. at different pH levels under greenhouse
conditions
Table 1. Components of the soil samples of each pH value.
pH Material added (g) to 1 kg of soil
5 50 g of coir dust
6 27 g of burnt rice husk
7 50 g of compost + 30 g dolomite + 8 g slaked lime
To obtain pots with the soil of the required pH, 25 g of soil amended to give each pH value i.e. pH 5, 6 and 7
(prepared by adding materials at the rates mentioned in table 1) was added to pots (20 cm3). Pots with 25 g of
pH non-adjusted, natural soil in the commercial onion fields (pH 4.6) served as the controls. Fifty onion seeds
(Landmark Agro-Tech Private Limited) were soaked in tap water for 24 hours and planted in a plastic pot (200
cm3) containing 150 g of soil mixed with 50 g of compost. After 8 days, five onion seedlings of the same size
were removed and transferred to each pot (20 cm3) with pH adjusted soil and the controls and pots placed in a
complete randomized design in the greenhouse. Out of these replicates, 4 pots were treated with 10 ml Fusarium
spore suspension (1× 105 spores ml-1) and other two pots were treated with sterile distilled water. Two days after
planting, 2 pots out of 4 pots treated with the Fusarium spore suspension were randomly selected from each pH
value and 10 onion seedlings were removed from each. The fresh weight of the roots was measured. The roots
were surface sterilized as mentioned above and the root segments were plated on PDA (4 per PDA plate) and
incubated for 3 days at room temperature (32±2ºC). After the microscopic observation and the identification, the
number of colonies of Fusarium sp. on the plates was counted and the number of Fusarium colonies in 1.00 g of
roots of seedlings growing at each pH value was calculated. Five days after planting, 10 ml of Trichoderma sp.
suspension (1×105 spores ml-1) was added to remaining 2 pots of each pH value (i.e. pH 4.6, 5.0, 6.0 and 7.0)
which were inoculated previously with Fusarium sp. Two days after the addition of Trichoderma spore
suspension, seedlings were removed and roots were separated from the seedlings. The weight of the roots was
measured. They were surface sterilized and cultured on PDA plates (4 per PDA plate) and incubated for three
days at room temperature (32±2ºC). The number of colonies of Fusarium sp. on the plates was counted and the
number of Fusarium colonies in 1.00 g of root in each pH value was calculated.
Statistical analysis
Significance difference among the treatments of dual culture plate assay, volatile metabolite test, sporulation
of Trichoderma in laboratory and greenhouse condition were statistically analyzed using ANOVA and Tukey’s
pairwise comparison using MINITAB 16.
RESULTS
Effect of pH on the rate of sporulation of the isolated Trichoderma sp.
The highest rate of sporulation of 5×107 spores ml-1 was shown at pH 6 after a 49 day incubation period (Fig.
1). This was significantly different (p≤0.05) from sporulation rates at pH 4, 5 and 7. The rate of sporulation was
higher at pH 6 even after 7 days. However, there was no significant difference in the sporulation rates at
different pH values until the cultures were 49 days old.
8
Log value of spore concentration of
6
pH
Trichoderma
5 4
4 5
3 6
7
2
0
day 7 day 14 day 21 day49
Time
Figure 1. Sporulation of Trichoderma sp. at different pH.
Effect of pH on the antagonistic activities of the isolated Trichoderma sp.
1. Growth inhibition of Fusarium sp. at different pH levels- Dual culture plate assay and Volatile metabolite
production: The highest percentage inhibition of the colony growth of Fusarium due to the restrictive
coiling structures and volatile metabolites produced by Trichoderma were shown at pH 6 (Tables 2 & 3). No
significant difference (p≤0.05) in percentage inhibition of colony growth of Fusarium was observed at pH 5
and 6.
Table 2. Results obtained from the dual culture plates at different pH values.
pH Dual Culture plate assay Number of Coiling Structures
% Inhibition ± SE
4 34.00±1.00a 5.7±0.33a
5 50.19±1.49b 11.00±0.58b
6 56.79±0.36b 10.00±1.15b
7 37.74±0.99a 9.70±0.88b
Note: Each data value represents the mean of 3 replicates ± standard error. Mean values sharing
common letters are not significantly different by Tukey’s multiple comparison test (p≤0.05).
2. Survival of the isolated Trichoderma sp. in soil samples with different pH values: There was a significant
difference (p≤0.05) between the number of Trichoderma colonies isolated from neutral pH (7) and acidic
[pH 4.6 (natural soil of commercial onion fields), 5, 6] soils tested. The numbers were similar and high at pH
values 4.6 and 5, whereas they were low at pH 6 and 7 throughout the period of evaluation (Table 4).
Table 3. Mean % inhibition of colony growth of Fusarium sp. in the presence of volatile metabolites
produced by Trichoderma sp. at different pH values.
pH % Inhibition ± SE
4 10.59±0.7C
5 23.00±0.9A
6 28.93±2.2A
7 26.69±1.4B
Note: Each data value represents the mean of 3 replicates ± standard error. Mean values sharing
common letters are not significantly different by Tukey’s multiple comparison test (p≤0.05).
Table 4. Survival of Trichoderma sp. in soil samples with different pH values

under greenhouse conditions.
Day Treatment Number of Trichoderma colonies
(pH) (cfu ml-1)
4 4.6 7.98×105
5 7.62×105
6 2.24×105
7 2.34×105
8 4.6 7.82×105
5 7.41×105
6 2.14×105
7 2.24×105
12 4.6 7.79×105
5 7.08×105
6 2.09×105
7 2.14×105
Note: Each data value represents the mean of 5 replicates. (Dilution Factor = 10-3)
Effect of the isolated Trichoderma sp. on the growth of Fusarium sp. at different pH values under greenhouse
conditions
The number of Fusarium colonies isolated from roots of onion seedling grown in soil with different pH
values was considered as an indication of colonization of the roots of the onion seedlings by the pathogen. A
reduction in the number of colonies of Fusarium sp. in Trichoderma sp. treated plants was considered as an
indication of control of pathogen growth. The number of colonies of Fusarium sp. isolated decreased at pH 4.6
and pH 5 after inoculation with Trichoderma sp. However, the number of colonies of Fusarium sp. was constant
at 73–76 colonies g-1 of onion roots throughout the period of evaluation at pH 6. There was an increase of
number of colonies of Fusarium isolated from seedlings grown at pH 7 even after inoculation of Trichoderma
sp. According to Tukey’s pair-wise test, there was a significant difference in the number of colonies of
Fusarium sp. isolated from seedlings grown at pH 5 as compared to those grown in soils with pH 4.6, 6.0 and
7.0 (Table 5). This is in agreement with the results of the tests on the survival of Trichoderma at different pH
levels where the optimal pH was indicated as 5.
Table 5. Mean number of colonies of Fusarium sp. per gram of soil and difference of number of Fusarium colonies as a
percentage, before and after inoculation of Trichoderma sp.
pH A B Difference of the number of Fusarium colonies
as a Percentage
4.6 57.38± 0.31c 29±1.05c 49.46 % (Decrease)
5 42.62±0.87d 27.33±1.45c 35.87 % (Decrease)
6 75±1.06b 74±1.65b 1.33 % (Decrease)
7 150±1.15a 188±1.73a 25.33 % (Increase)
Note: A- Mean number of colonies of Fusarium sp. before inoculation of Trichoderma sp.; B- Mean number of colonies
of Fusarium sp. after inoculation of Trichoderma sp.
Each data value represents the mean of 3 replicates ± standard error. Mean values sharing common letters are
not significantly different by Tukey’s multiple comparison test (p≤0.05).
DISCUSSION
Fungicide application is one of the most common methods used to control fungal pathogens. Biological
control of pathogenic organisms is an eco-friendly, risk-free alternative method in agriculture (Benítez et al.
2004). Erwinia herbicola (Kempf & Wolf 1989), Fusarium equiseti (Horinouch et al. 2010), Trichoderma sp.
(Gveroska & Ziberoski 2011) are some of the efficient biological control agents that use in agriculture. Among
the biological control agents, Trichoderma is a fungal genus of cosmopolitan distribution and high
biotechnological value as performs effective biocontrol mechanisms (Hermosa et al. 2013). Previous studies on
Trichoderma revealed that different pH conditions affect the growth, sporulation, survival and the mycoparasitic
activity of Trichoderma under in-vivo and in-vitro conditions (Bhai et al. 2010, Zehra et al. 2014).
Sporulation is an important characteristic of biocontrol agents as their efficiency and competence of
biocontrol is closely associated with the ability to compete with pathogens in the soil along with their ability to
control plant diseases. Carreras-Villasen et al. (2012) reported that sporulation of Trichoderma is a low pH
dependent process and T. harzianum shows the highest sporulation at pH 5.5. The studies of Ali et al. (2015)
and Zehra et al. (2014) confirmed that the pH 6 was the best for the growth and sporulation of different
Trichoderma spp. under laboratory conditions, which is in agreement with the observations of the present study.
Furthermore, Bandyopadhyay et al. (2003) and Singh et al. (2014) reported that Trichoderma isolates exhibit
optimum growth and sporulation rate at pH values ranging from 2 to 7.
The biocontrol ability shown by Trichoderma sp. can occur by means of several antagonistic mechanisms
such as competition for nutrient, mycoparasitism and antibiotic production. Dual culture technique was used to
observe the mycoprasitic activity of the Trichoderma sp. in this study. The production of enzymes plays a major
role in mycoparasitism. According to the study done by Elad et al. (1980) the extracellular enzymes of
Trichoderma spp. have been able to digest cell wall components of Sclerotium rolfsii. Furthermore, Kredics et
al. (2003) examined the effect of pH on the extracellular enzymes of Trichoderma which responsible for the
mycoparasitic activity under in-vitro conditions. They were able to identify that β-glucosidase was activated
under pH 5 while xylosidase was activated at pH 6. Asran-Amal et al. (2010) identified the pH 6–7 is the
optimal pH range for chitinase activity secreted by Trichoderma sp. Similar studies by Ali et al. (2012) and
Petrisor et al. (2016) indicated that the antagonistic potential of Trichoderma sp. against Rhizoctonia solani and
Pythium ultimum was optimal at pH 4.5–5.5. Moreover, Raza et al. (2013) reported of a similar influence of pH
on volatile antibiotic metabolite production by Trichoderma under in-vitro conditions where pH 6 was optimal
for the production of volatile antifungal compounds. Therefore, these findings support the observations of the
present study.
In the present study, different materials (i.e. coir dust, compost, dolomite, slaked lime) were used to alter the
pH of the soil which caused only an alteration of the pH of the growth medium and did not show a reduction of
growth of Fusarium in the media. The fact that the substances used to change the pH values did not change the
growth of Fusarium was further evidenced by the colony numbers before treatments with Trichoderma being
fairly high (150 at pH 7) and also by observing that there was an increase (25.33%) in the non-effective
treatments with the biological control agent (Trichoderma sp.). Therefore it is clear that the materials did not
influence the growth of Fusarium, but only changed the pH of the growth medium and any change in colony
numbers of Fusarium was caused by the effect of pH on the bio control agent and the mechanisms of control.
The suitability of an acidic pH range for the survival of Trichoderma sp. was also reported by Bhai et al.
(2010) where they observed that the pH range 4.5–5.5 was suitable for the growth, sporulation, and survival of
Trichoderma sp. than alkaline conditions under greenhouse conditions. Longa et al. (2007) observed that growth
of T. atroviride declined in soils with alkaline pH levels. Similarly, Hader et al. (1983) showed that pH 4.5 is
the optimal soil pH for T. koningii and T. harzianum for the control of a Pythium sp. which is causing seed rot of
radish and pea.
CONCLUSION
According to the results of the present study, pH values have an effect on the sporulation, the volatile
metabolite production of the selected Trichoderma sp. and the ability to inhibit mycelial growth of the Fusarium
sp. tested. Moreover, pH 6 is optimal for the sporulation, mycoparasitic activities and volatile metabolite
production of Trichoderma sp.
ACKNOWLEDGEMENTS
The authors wish to thank the Department of Botany, University of Kelaniya, Sri Lanka for the facilities
provided to carry out the research.
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Research article
Analysis of plant species diversity and forest structure in Arero

dry Afromontane forest of Borena zone, South Ethiopia
Wakshum Shiferaw1*, Mulugeta Lemenih2 and Tadesse Wolde Mariam Gole3
1
College of Agricultural Sciences, Natural Resources Management, Arba Minch University, Ethiopia
2
Farm Africa, Addis Ababa, Ethiopia
3
Environmental Coffee Forest Forum (ECFF), Addis Ababa, Ethiopia
*Corresponding Author: waaqsh@yahoo.com [Accepted: 26 May 2018]
Abstract: Accelerated human population growth mostly coupled with poverty has enhanced
negative impacts on forest resources in Ethiopia. This study aims to assess the vegetation structure,
composition, and plant species diversity and regeneration status of Arero dry Afromontane forest.
Concentric circular plots of 1m, 3m and 10m radius were used for recording seedling, sapling and
tree species respectively. As the result, 84 species that belong to 44 families were recorded in the
forest. The Shannon-Wiener diversity indices and evenness of woody species were H’=2.67 and
J’=0.79 respectively. The total density and abundance of woody species were 272 stems ha per
hectare and 427 individuals per all of the sampled plots. The forest is floristically diverse and rich
as compared to many similar forests in Ethiopia. The structural analyses of the population of some
dominant species experience poor regeneration. This implies that current management practices
are not satisfactory to sustain forest conditions.
Keywords: Arero - Afromontane forest - Plant diversity - Forest structural analyses.
[Cite as: Shiferaw W, Lemenih M & Gole TWM (2018) Analysis of plant species diversity and forest structure
in Arero dry Afromontane forest of Borena zone, South Ethiopia. Tropical Plant Research 5(2): 129–140]
INTRODUCTION
Tropical forests are the habitat of numerous species of living things which constitute biodiversity through
webs of life. Forests support various life forms including human beings who dwell in settlements in and around
forests (Mukhopadhyay et al. 2007). According to FAO (2007), 11.9% of Ethiopia’s land area is covered with
forests including closed forest plus woodlands. The same report narrated that between 1990 and 2000, 141,000
ha forest of Ethiopia were lost every year, which equals an average annual deforestation rate of 0.93%. On the
other hand, between 2000 and 2005, the rate of deforestation increased by 1.03% to 10.4% per year i.e.
2,114,000 ha of forest cover lost in 15 years between 1990 and 2005. Gatzweiller et al. (2007) estimates that the
area of closed forest has declined to about 3–4% in the country.
Despite the declining of vegetation cover, Ethiopia hosts rich biological diversity. This can be attributed
from its great geographical diversity with high and rugged mountains, flat-topped plateau and deep gorges,
incised river valley and rolling plains (Teketay 1999, Kassa et al. 2016, Tadesse et al. 2017). The size of the
Ethiopian higher plant flora is estimated to about 6000 species out of which about 12% are probably endemic to
Ethiopia (G/Hiywot 2003). The total number of woody plants is estimated to be 1000 (Biru 2003). However,
unsustainable utilization of these resources has resulted in the declining of the country’s biodiversity at a much
faster rate.
In Ethiopia, accelerated human population growth mostly coupled with poverty has enhanced the negative
human impact on the forest resources. Among the tropical forests, dry forests have been preferred for human
settlement than wetter forest zones, due to different biological and ecological reasons (W/Mariam 1998). In rural
area in Ethiopia where the livelihood of 83% of the population resides and dependent on renewable natural
resources, the pressure on forest resources is high. These causes depletion and deterioration of the forest
resources which have resulted in reducing agricultural productivity and hence the quality of life (Merga 2006).
To improve the conservation of the remaining natural forests in Ethiopia, the remnant forest resources have
Received: 01 October 2017 Published online: 31 May 2018
Shiferaw et al. 2018
been portioned into 58 National Forest Priority Areas (NFPAs) covering an area of 3.6 million ha (FSCDD
1990). These areas comprise natural forests, plantations, and non-forested land. Arero forest is one of these
delineated priority forest areas of Oromia National Regional State in South Ethiopia.
Like most forests of the country, Arero forest is experiencing deforestation and degradation. It is obvious
that these changes cause dynamics in the composition and forest structures. Several studies covering wider
disciplines have been conducted in the areas which have contributed to the improved understanding of the
ecological and socio-economic conditions and for better management of the forest. For instance, among several
studies (Haugen 1992, Coppock 1993, Coppock 1994, Mengistu 1998, Angassa 2002, Angassa & Beyene 2003,
Dale et al. 2005, Adelio et al. 2005, Takele 2006, Worku 2006). However, informations are lacking regarding
forest conditions in Arero forest. Therefore, this study aims to respond for research questions such as: 1) What
are the overview of vegetation structure in the forest? 2) What are the statuses of composition and plant species
diversity? and 3) What are the regeneration status of plant species in the forest?
MATERIALS AND METHODS

Description of the study area
This study was carried out in Oromiya Regional National State, Borena Zone in Arero dry afromontane
forest (Fig. 1). The forest is located 670 km south of Addis Ababa on the left-hand side of the high way running
to Moyale town, 96 km from Yabelo town capital of Borena Zone. The boundary of the forest is approximately
7 km from the district town of Meta Gefersa. The forest is located 38°48′ and 38°55′ East and 4°45′ and 4°48′
north and at an altitude ranging from 1,606 to 1,805 masl. Arero forest has a total area of 29,226.39 ha.
Figure 1. Map of the study area.

The population of Arero district were estimated to be 74,119 out which 11,859 or 16% are categorized as
semi and sedentary farmers, while 62,260 (84%) are pastoralists and mixed farmers. There are about 12,595
households in the district, of which 3,108 households are members of different forest user groups. They are
organized by SOS Sahel Ethiopia. The forest user groups are Borana and Guji people (FSCDD 1990).
Since there is no meteorology station at Arero district, data from the nearest Mega Meteorology Station was
used. Hence, based on 20 (1984–2004) years meteorological data the mean monthly rainfall at the nearby station
was 47.1mm. The mean annual rainfall of the district was 532.2 mm. The rain fall regime in Borena dry lands is
bimodal with two rainy seasons (Fig. 2). The main rainy season, known as the long rainy season is between
March and May with a peek in April, and a short rainy season is between September and November, with a peek
in October. The mean monthly minimum and maximum temperature of Arero district was 16.2°C and 18.3°C,
respectively. The mean annual temperature was 18.9°C. The dominant soil types found at Arero district are
Chromic and Eutric Luvisol, Calcaric and Eutric Fluvisol and Chromic, Eutric and Calcari. Its bottom lands of
the Borana rangeland are predominated by vertisols (OBPED 2000). Arero forest is upland dry evergreen forest
dominated by Juniperus procera Hockst. ex Endl., but also consists of plant species such as Olea europaea L.
subsp. cuspidata (Wall. ex G.Don) Clf., Terminalia brownii Fresen., Croton macrostachyus Del., Carissa edulis
Wahl., Ehretia cymosa Thonn., Acokanthera schimperi (A . DC.) Schweinf., Dodonaea angustifolia L. f.,
Balanites eagyptica (L.) Del., Calpurina aurea (Ait.) Benth., and Acacia tortilis (Frossk.) Hayne (Demesa
2002).
Figure 2. Climatic diagram of Mega, Borena zone, Ethiopia. (Source: Worku 2006)
Data collection
Line transects were laid on the basis of the differences in landscapes such as altitudinal ranges, drainage
patterns, size and heterogeneity of the forest. Then, the distance between two consecutive parallel transect lines
was 500 meters. Along the transect lines, sample plots were laid down at intervals of 300 meters. Concentric
circular plots of 1m, 3m and 10m radius of were established along transect lines (Fig. 3). Then, 50 plots were
distributed randomly on 19 transect lines.
Figure 3. Size of sample plots.

In each plot, the percentage of ground cover was estimated as the percentage in 1m radius circle under the
shade or canopy of trees. Then, local names for the woody seedlings and herbaceous plants and the number of
seedling species were recorded in the field. All of the shrubs, saplings and woody climbers (vines, liana) in 3m
radius circle were also recorded and identified. Trees and climbers were collected in the largest circle, with a
radius of 10 m (Ostrom 2007). The DBH of saplings and trees were measured using caliper. Diameter tape was
used for tree diameters beyond caliper size which measures circumference, then later converted to diameter.
For regenerated seedlings (height less than 1.0 m), only their number were recorded. Individual woody
categorization was made at height less than 1.0 m and DBH less than 2.5 cm for seedlings. The height 1.0–3.0 m
and DBH 2.5–10 cm for saplings, and height greater than 3.0 m and DBH greater than or equals to 10.0cm for
tree species were measured (Ostrom 2007). Height was measured using the hypsometer. Where slope,
topography and/or crown structure made it difficult to use hypsometer, height was estimated visually. For plot
location, compass was used to direct the straight line between transect lines and successive plots. For species
that were difficult to identify in the field, herbarium specimens were collected, pressed, dried and transported to
the national herbarium in the Department of Biology in Addis Ababa University for proper identification.
Data processing and analyses
Species diversity in the forest was estimated using Shannon Wiener Diversity Index and species richness
(Kent & Coker 1992):
∑
Where, H’= Shannon diversity index,
Pi = Proportion of individual found in the ith species.
The Ratio of observed Shannon index to maximum diversity (H max = lns) can be taken as a measure of
evenness (J’) (Kent & Coker 1992).
Equitability (evenness)
∑
Where, X= the Shannon diversity index number

H’= Shannon diversity index,
Pi = Proportion of individual found in the ith species.
The basal area per tree is calculated using the formula (Lamprecht 1989):
Where, d= DBH and Π= 3.14

The Importance Value Index (IVI) indicates the importance of species in the system and calculated with
three components as follows (Kent and Coker 1992),
The Importance Value Index (IVI) of each woody species is:
To analyse the population structure of woody species, all individuals of each species encountered in the plots
were grouped into arbitrary diameter classes and histograms were developed using the diameter classes versus
the number of individuals categorized in each of the classes using Microsoft Excel.
RESULTS
Floristic composition and diversity of plant species
Table 1. Number, percentage and life form of collected species in Arero forest.
S.No. Life form Number Percentage
1 Trees 30 34.48
2 Shrubs 9 10.35
3 Climbers 3 3.45
4 Herbs 45 51.72
In this study, 84 plant species representing 44 families were recorded in the forest including trees, shrubs and
herbaceous plants (Appendix A). From all the species collected 51.72% were herbs, 34.48% trees, 10.35%
shrubs and 3.45% were climbers (Table 1). Of all families, Poaceae constitutes the highest number of species
(11.5%) followed by Fabaceae (10.3%), Acanthaceae (5.8%) and Euphorbiaceae (4.6%). The results showed
that species diversity (H’) and evenness (J’) in the forest were 2.67 and 0.79 respectively (Table 2).
Table 2. Summary of the diversity parameters for woody forest species in Arero forest.
Plant species
Characteristics
Woody Herbaceous
Evenness (J’) 0.79 0.64
Diversity (H’) 2.67 2.44
Importance value of woody species
The results showed that 4777 seedlings, 78 saplings and 194 trees stems per ha were recorded in the forest.
The total density of all woody species was found to be 272 stems per ha in the forest excluding seedlings (Table
3). The density of few woody species, for instance Olea europaea L., subsp. cuspidata (Wall. ex G.Don) Clf.,
Juniperus procera Hockst. ex Endl., Scolopia theifolia Gilg and Acokanthera schimperi (A.DC.) Schweinf were
higher in the forest (Table 3). The results also revealed that Olea europaea L. subsp. cuspidata (Wall. ex
G.Don) Clf., Juniperus procera Hockst. ex Endl., Acokanthera schimperi (A.DC.) Schweinf, Scolopia theifolia
Gilg, Teclea simplicifolia (Engl.) verdoorn, Psydrax schimperiana A. Rich Bridson and Nuxia congesta R.Br. ex
Fresen were the most frequent species in the forest. The dominance of the species also showed that Juniperus
procera Hockst. ex Endl., Olea europaea L. subsp. cuspidata (Wall. ex G.Don) Clf., Psydrax schimperiana A.
Rich Bridson, Acokanthera schimperi (A.DC.) Schweinf. and Scolopia theifolia Gilg species were the five top
dominant species in the forest (Table 3). Considering the individual species, Olea europaea L., subsp. cuspidata
(Wall. ex G.Don) Clf., Juniperus procera Hockst. ex Endl., Scolopia theifolia Gilg, Teclea simplicifolia (Engl.)
verdoorn, and Acokanthera schimperi (A.DC.) Schweinf were the five top abundant species in the forest (Table
3). Like Juniperus procera Hockst. ex Endl., Olea europaea L. subsp. cuspidata (Wall. ex G.Don) Clf.,
Scolopia theifolia Gilg, Acokanthera schimperi (A.DC.) Schweinf and Teclea simplicifolia (Engl.) verdoorn
species had the highest IVI of the total woody species in the forest (Table 3).
Table 3. List of woody species in Arero forest.
Species Ab D/ha F RF BA/ha RDo RD IVI
Acokanthera schimperi (A.DC.) Schweinf 34 22 38 8.60 0.56 2.66 7.96 19.23
Euclea divinorum Hiern 6 4 4 1.08 0.01 0.06 1.41 2.54
Ruttya fruticosa Lindau 26 17 4 1.08 0.40 0.01 6.09 7.17
Olea europaea L., subsp. cuspidata (Wall. ex G.Don) Clf. 79 50 76 19.35 2.80 19.39 18.50 57.25
Commiphora campestris Engl. 2 1 2 0.54 0.01 0.10 0.47 1.11
Pavetta abbyssinica Fresen 3 2 6 1.61 0.03 0.24 0.70 2.55
Nuxia congesta R.Br. ex Fresen 28 18 28 7.53 0.22 1.64 6.56 15.72
Psydrax schimperiana A. Rich Bridson 25 16 32 8.60 0.46 3.42 5.85 17.88
Boscia punctulata Decne. 18 11 2 0.54 0.02 0.12 4.22 4.87
Dodonaea angustifolia L. f. 20 13 6 1.61 0.03 0.23 4.68 6.52
Scolopia theifolia Gilg 38 24 36 9.68 0.22 1.66 8.90 20.24
Papea Cappensis Eckl & Zeyh 2 1 4 1.08 0.05 0.39 0.47 1.93
Juniperus procera Hockst. ex Endl. 61 39 52 13.98 8.75 65.26 14.28 93.52
Haplocoelum foliolosum (Hiern) Bullock 15 10 8 2.15 0.12 0.91 3.51 6.58
Acacia tortolis (Forssk) 3 2 2 0.54 0.03 0.24 0.70 1.48
Acacia seyal DC. 1 1 2 0.54 0.01 0.05 0.23 0.82
Zanthoxylum usambarense (Engl.) Kokwaro 1 1 2 0.54 0.01 0.04 0.23 0.82
Waagaa (OL) 1 1 2 0.54 0.01 0.05 0.23 0.82
Diospyros abyssinica (Hiern) F.white 4 3 6 1.61 0.06 0.43 0.94 2.98
Teclea simplicifolia (Engl.) verdoorn 36 23 36 9.68 0.20 1.49 8.43 19.60
Cissus petiolata Hook.f. 1 1 2 0.54 0.01 0.08 0.23 0.86
Baddaa (OL) 1 1 2 0.54 0.01 0.04 0.23 0.81
Secamone punctulata Decne. 2 1 4 1.08 0.40 0.00 0.47 1.55
Cissampelos pareira L. 4 3 8 2.15 0.80 0.02 0.94 3.10
Combretum collinum Fresen 5 3 6 1.61 0.10 0.76 1.17 3.54
Ormocarpum muricatum Chiov. 3 2 4 1.08 0.05 0.39 0.70 2.17
Ficus vasta Forsek 1 1 2 0.54 0.01 0.11 0.23 0.88
Fonqolcha (OL) 5 3 4 1.08 0.03 0.20 1.17 2.45
Gaaddallaa (OL) 2 1 2 0.54 0.01 0.04 0.47 1.05
Total 427 272 372 100 15.65 100 100 300
Note: Ab= Abundance/50 plots (seedlings +saplings +trees), D/ha= Density per hectare, F= Frequency, RF= Relative Frequency,
BA/ha= Basal Area per hectare (saplings + trees), RDo= Relative Dominance, RD= Relative Density, IVI= Importance Value Index,
OL= Oromo Language.
Population structure of whole woody species

The DBH was classified into ten DBH classes and tree height was also classified into nine classes (Fig. 4).
The results showed the existence of variations in diameter and height classes of the vegetation of species in the
forest. The abundance of stems in general was very high at medium diameter classes. Good abundance of
diameter class was found in 3rd diameter class (Fig. 4).
Figure 4. Percentage distribution of the whole woody species in Arero forest: A, DBH classes; B, height classes.
Diameter and height distribution of woody species
The population structure of six top most abundant species in the forest were separetely presented in (Fig.
5A–F). Based on the analyses of population structures, the study species encountered at the study area were
grouped in to three categories: type I, II ,and III. The results for both diameter class showed that Juniperus
procera Hockst. ex Endl., and Psydrax schimperiana (A. Rich) Bridson were grouped as type II in the forest,
while, Acokanthera schimperi (A.DC.) Schweinf, Olea europaea L., Scolopia theifolia Gilg., subsp. cuspidata
(Wall. ex G.Don) Clf. and Teclea simplicifolia (Engl.) verdoorn were grouped as type III. However there were
no species grouped as type I distribution pattern for diameter.
Figure 5. Distributions of individual structure in different DBH class for six most abundant tree species in Arero forest: A,
Juniperus procera Hockst. ex Endl.; B, Psydrax schimperiana (A. Rich) Bridson; C, Acokanthera schimperi (A.DC.)
Schweinf; D, Olea europaea L.; E, Teclea simplicifolia (Engl.) verdoorn; F, Scolopia theifolia Gilg.
Regeneration status of woody species

Regeneration status of the forest as a whole was examined using the result of diameter and height class
distributions of the vegetation as a whole (Fig. 4). Therefore, the frequency histograms of these species as an
individual vegetation of the forest were none of an inverted “J’’ shape. In comparison to trees, the abundance of
seedlings and saplings of the study species were found to be 110 and 9 respectively per total sampled plots in
the forest.
DISCUSSIONS
The forest is richer in species compared to Menagesha Suba forest which had 82 species (Zewdie 2007) and
Harena forest with 85 plant species (Nigatu 1987). In present study, the number of woody species including
trees, shrubs and climbers were 42, which were comparable with findings made by Mekonnen (2006) which had
39 woody species for Dilfaqar National Park.
Patterns of plant species diversity are used to be noted for ranking conservation activities because they show
the underlying ecological processes that are important for management and conservation (Senbeta 2006). In this
particular study, the result showed the existence of high diversity in the case of whole woody species
encountered in the forest. However, as it was the case in species diversity in the forest for instance had relatively
less value of Shannon diversity index (H’=2.67) as compared to Chilimo forest (H’=2.77) (W/Mariam 1998), on
Peninsula of Zegie (H’=3.72) (Alelign et al. 2007) and (H’=2.87) (Mekonnen 2006) in Dilfaqar Regional Park.
The reason might be due to the dominance of few species, which could be caused by selective logging of some
species (Senbeta 2006). According to Kent & Coker (1992), low Shannon evenness is an indication the
existence of unbalance distribution of the individuals of species encountered in this study area and this probably
due to impact of livestock grazing, human influence, and drought in this forest area. On the other hand, the
Shannon diversity index of the forest was comparable with Shi & Zhu (2009) in southwestern China (H’= 1.82–
3.29) and Sudhakar et al. (2008) in Chandoli National Park, northern Western Ghats, India (H’= 2.0–3.2).
Lower evenness indicates the dominance of a few species. Therefore, the implications of evenness values is that,
when there is a high evenness value in a given forest, the location of conservation sites might not be of such
importance compared to when the evenness value of the forest is low (Senbeta 2006). The evenness index of this
particular forest was relatively less than other findings by (Adekunle 2006) for instance in southwest Nigeria
(J=0.91). However, it was comparable with findings by Shi and Zhu (2009) (J=0.58–0.90) in Southwestern
China. In this particular forest the density of woody species was also comparable with reports made by
Sudhakar et al. (2008) in India which ranged from 112 to 406.8 stems per ha.
Few species like Acokanthera schimperi (A.DC.) Schweinf, Dodonaea angustifolia L.f. and Ruttya fruticosa
Lindau were overtaking in the forest. Most of the seedlings and saplings were frequent in sampled plots which
constituted to these species. The dominance and /or abundance of the species contributed a number of factors
such as disturbance factors, successional stage of the forest or survival strategies of the species. On the other
hand, some plant species may have a wide range of dispersal mechanisms and/or rapid reproduction strategies.
Some studies for instance Senbeta (2006) in the Afromontane rainforests of Southwest and Southeast Ethiopia
and report made by Bekele (2000) in dry Afromontane of Southern Wollo in Ethiopia indicated that in early
successional development many pioneer species may establish and grow together in high density until they
reach the climax stage where many individuals were eliminated due to competition. This might also be due to
the species are able to survive and flourish after disturbance tend to be those that reproduce rapidly and
abundantly and dispersed widely.
The species with high IVI were the most dominant while the lower ones were less in the forest. This might
be due to fire damage, natural selection and livestock grazing or trampling of other species. These showed also
that the species were among the best adapted, dominant and with more or less good population status in the
forest. Woody species with high resistance to anthropogenic disturbance and those with efficient regeneration
capacity have relatively high chance of remaining dominant in the forest. Important value indices in the species
showed similar trends with findings made by Worku (2006) and Addo-Fordjour et al. (2009). Woody species
that constitute the lowest IVI depicted that the species could be prioritized for conservation. Therefore, those
species with least IVI should need conservation measure, as they are important in terms of ecological, social and
economic services they provide.
Some classes in lower and higher diameter classes were represented with few individuals and species.
Species richness decreased at lower and higher diameter classes. In other words, the vegetation structure of the
forest was a disturbed shape distribution of type II category. Population structures of woody plant species in
arbitrarily diameter-height size classes were determined to provide the overall regeneration profile of the
vegetation and species (Worku 2006, Lalfakawma et al. 2009). Information on the population structure of a tree
species indicates the history of the past disturbance to that species and the environment and hence used to
forecast the future trend of the population of that particular species. Population structure is also an extremely
useful tool for indicating management activities and may be the most important for assessing both the potential
of a given resource and the impact of resource extraction (Peters 1996). The pattern of the height class
distributions was also varied from species to species (Fig. 4B). The height class distribution shows the
representation of relatively high individuals in 3rd class, but low individuals in lower and higher classes. In other
words, there were the declines of species abundance in lower and higher diameter classes.
The results for both diameter classes showed that Acokanthera schimperi (A.DC.) Schweinf, Teclea
simplicifolia (Engl.) verdoorn, Scolopia theifolia Gilg, and Psydrax schimperiana A. Rich Bridsonwere grouped
as type II in the forest, while , Juniperus procera Hockst. ex Endl., and Olea europaea L., subsp. cuspidata
(Wall. ex G.Don) Clf. were grouped as type III. However, there were no species grouped as type I distribution
pattern for diameter class and these could also be poor regeneration status of the forest probably due to high
impact of livestock grazing, house construction, fuel wood and moisture stress in the forest. This argument
agrees with findings made by Senbeta et al. (2007) in Afromotane forests of Ethiopia. As numbers of seedlings
are greater than saplings which showed that the woody species had healthy regeneration profiles. However, as
regeneration profiles of most top six woody species were seen their diameter and height classes were not
healthy. The low in number of seedlings and saplings of woody species in the forest might be due to impact of
livestock grazing; selected cutting for house construction particularly true for saplings in the study area
(Lalfakawma et al. 2009). According to G/Hiywot (2003), composition and density of seedlings and saplings
would indicate the status of the regeneration of a given tree species. Accordingly, the regeneration of woody
species was poor in the forest. These could be due to afro mentioned reasons.
CONCLUSIONS
Arero forest is floristically diverse and rich as compared to many similar forests in Ethiopia. However, the
structural analyses of the population of some dominant species experience poor regeneration. This study also
showed that Juniperus procera Hockst. ex Endl., Olea europaea L. subsp. cuspidata (Wall. ex G.Don) Clf.,
Psydrax schimperiana A. Rich Bridson, Acokanthera schimperi (A.DC.) Schweinf, Scolopia theifolia Gilg,
Teclea simplicifolia (Engl.) verdoorn and Nuxia congesta R.Br. ex Fresen species were found to be the dominant
and frequent species in the forest.
This also implies that current management practices are not satisfactory to sustain the forest conditions.
Indeed, it deserves concerted effort by local traditional Gadaa System of Oromo People and SOS Sahel Ethiopia
and other institutions to improve its conservation and sustainable use of forests.
Unless improved management interventions are made the sustainability of contribution to livelihoods from
the forest will be at stake in the future. Furthermore, detail study in the forest such as soil seed banks and
carbon potential of the forest will be conducted to show complete diversity and biomass of the forest for carbon
sequestration for biodiversity conservation and climate change mitigation in the region.
ACKNOWLEDGEMENTS
The Authors expresses their sincere thanks to Volkswagen (VW) foundation through “The Role of
Institutions for Forest Resources and Livelihood Management in East African Forest Landscapes (IFLEA)”
project for financial and Oromiya Agricultural Research Institute (OARI) for logistics support of the study.
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Supporting information
Appendix 1: List of plant species in Arero forest per all sampled plots.
Appendix 1: List of plant species in Arero forest per all sampled plots.
Scientific Vernacular Family Growth
name name name habit
Acokanthera schimperi (A.DC.) Schweinf Qaraaruu Apocynaceae S
Euclea divinorum Hiern Mi’eessa Ebenaceae S
Olea europaea L., subsp. cuspidata (Wall. ex G.Don) Clf Ejersa Oleaceae T
Chionothrix latifolia Rendle Garbicha Amaranthaceae T
Commiphora terebinthina Vollesen Sangaa igguu Burseraceae S
Calpurnia aurea (Ait.) Benth. Ceekata Fabaceae T
Dichrostachys cinerea (L.) weight & Arn Jirmee Mimosaceae T
Acacia brevispica Harms Hamarressa Mimosaceae T
Rhus nathlensis Krauss Daboobessa Anacardiaceae T
Dodonaea angustifolia L. f. Ittacha Sapindaceae T
Jasminum grandiflorum L subsp. floribundum (R. Br. ex Dogimaasa Oleaceae H
Fresen.) P.S.Green
Haplocoelum foliolosum (Hiern) Bullock Cannaa Sapindaceae T
Olea capensis (L) Subsp. marocarpa (C.A. wright verde) Gagamaa Oleaceae T
Unknown Dambisuu Unknown H
Pavetta abbyssinica Fresen Korkorree Rubiaceae T
Cissampelos pareira L. Var. hirsuth (Buck. ex DC ) Forman Gaalee Menispermaceae C
Chrysopogon aucheri (Boiss.) Alaloo Orobanchaceae H
Leptothrium senegalense (Kunth) Clayton Ilmogoor Poaceae H
Unknown Laabbessa Unknown H
Pavonia urens Var glabrescens Icinnii Gramineae H
Fuerstia africana T.C.E.Fr. Hancabbii Lamiaceae H
Ruttya fruticosa Lindau Xuuxoo Acanthaceae S
Stylosanthes fruticosa (Retz.) Alston Egajii Fabaceae H
Aspilia mossambicensis (Oliv.) Wild Hadaa Asteraceae H
Dyschoriste radicans Nees Qilxiphee Acanthaceae H
Solanum spp Hiddii loonii Solanaceae H
Phyllanthus sepialis Muell. Arg. Dhirrii warseesoo Euphorbiaceae H
Hibiscus micranthus L. f. Bungaala Malvaceae H
Scolopia theifolia Gilg Muka diimaa Germniaceae T
Nuxia congesta R.Br. ex Fresen Muka daalacha Logniaceae T
Trianthema salsoloides Oliv. Qanxallaa Aizoaceae H
Acanthus sennii Chiov. Sookoruu Acanthaceae H
Eragrostis cilianensis (All.) Vign. Ex Janchen Saagetuu Poaceae H
Digitaria velutina (Forssk.) P. Beauv Raaphuphaa Poaceae H
Unknown Fonqolcha Unknown T
Caralluma speciosa (N.E. Br.) N.E. Br. Mata buttoo Asclepiadaceae H
Tagetes minuta L. Sunkii Asteraceae H
Teclea simplicifolia (Engl.) verdoorn Hadheessa Rutaceae T
Pavetta gardenifolia A.Rich Gaadallaa Rutaceae T
Boscia punctulata Decne. Dhumaayo Capperidaceae T
Cynodon dactylon (L.) Pers. Sardoo Poaceae H
Papea Cappensis Eckl & Zeyh Biiqqaa Sapindaceae T
Achyranthes aspera L. Darguu hoolaa Amaranthaceae H
Ormocarpum muricatum Chiov. Karraa Fabaceae T
Dombeya aethiopica Gilli Sililaalcha Sterculiaceae H
Juniperus procera Hockst. ex Endl., Hindheessa Cupressaceae T
Cenchrus ciliaris L. Mata guddisa Poaceae H
Heteropogon contortus (L.) Roem. & Schult. Seericha Poaceae H
Acacia tortolis (Forssk) Xaddacha Fabaceae T
Ormocarpum trichocarpum (Taub.) Engl Buttiyyee Fabaceae S
Acacia seyal DC. Waacuu Fabaceae T
Panicum maximum Jacq. Lolloqaa Poaceae H
Chlorophytum spp Miirtuu Unknown H
Indigofera spp Agagaroo harree Fabaceae H
Asparagus aethiopicus L. Sarittii Asparagaceae H
Sida ovata Forsk Qarxaxummee Malvaceae S
Blepharis maderaspatensis (L.) Roth. Darguu Acanthaceae H
Psydrax schimperiana A. Rich Bridson Gaallee Rubiaceae T
Pellaea calomelanos (Sw.) Link. Filaa tarrii Sinopteridaceae H

Zanthoxylum usambarense (Engl.) Kokwaro Gadaa Rutaceae T
Acalypha fruticosa Forssk. Dhirrii Euphorbiaceae S
Crotalaria distantiflora Bak.f. Dacisaa Fabaceae H
Diospyros abbyssinica (Hiern) F.white Lookoo Ebenaceae T
Oldenlandia corymbosa L. Bilixuu Portulacaceae H
Microchloa kunthii Desv. Areeddoo Poaceae H
Gnidia stenophylla Gilg. Aarsaa Thymelaeaceae H
Combretum collinum Fresen Lu’oo Combretaceae T
Ficus vasta Forsek Qilxaa Moraceae T
Steganotaenia araliacea acea Hochst Ex A.Rich Luqaanluqqee Apiaceae H
Eleusine jaegeri Pilg. Coqqorsa Poaceae H
Vigna membranacea A.Rich. Cimphaa Fabaceae H
Erica arborea L. Saatoo Ericaceae S
Commelina diffusa Burm.f. Qaayyoo Commeliaceae H
Ormocarpum mimosoides Butiyyee Loasaceae H
Triumfetta pentandra A.Rich Gurbii Tiliaceae H
Alectra sessiliflora (Vahl) Kuntze Gurroo Scrophulariaceae H
Biophytum spp Handaada Oxalidaceae H
Cissus petiolata Hook.f. Arayyee Vitaceae H
Secamone punctulata Decne. Dikicha Asclepiadaceae C
Tephrosia hildebrandtii Vatke Jiraa gubataa Fabaceae H
Tetradenia riparia (Hochst.In C.Krauss) Midhaan dubraa Lamiaceae H
Clutia lanceolata Forssk. Darguu Euphorbiaceae S
Crabbea velutina S. Moore Baballaa Acanthaceae H
Acacia oerfota (Forssk.) Schweinf. waangaa Fabaceae T
Note: H= Herbs, T= Trees, S= Shrubs, C= Climber.
Research article
Assessment of air pollution impact on micromorphological and

biochemical properties of Pentas lanceolata Forssk.
and Cassia siamea Lam.
Lohith Kumar, Hemanth kumar N. K.* and Shobha Jagannath
Department of Studies in Botany, University of Mysore, Manasagangotri, Mysuru-570 006, Karnataka, India
*Corresponding Author: nkhemanthkumar@yahoo.in [Accepted: 27 May 2018]
Abstract: In the present study an attempt was been made to assess the air pollution effect on
micromorphological and biochemical parameters of Pentas lanceolata and Cassia siamea. There
was a decrease in number of stomata in P. lanceolata of the polluted site compared to control but
in C. siamea numbers of stomata were increased in the polluted area when compared to control.
The number of clogged stomata was less in control area samples when compared to polluted
sample. A number of epidermal cells in C. siamea of polluted and control sites showed a
significant difference. Stomatal index of both species was found to be reduced in polluted site
when compared to control. Leaf surface area in both the plant species decreased from control to
polluted area and leaf colour changes from green to pale yellow/dark in a polluted area of both the
plant species. Chlorophyll a, b and total chlorophyll content in both the plants were found to be
significantly different in control and polluted plants. Ascorbic acid, relative water content, pH and
Air Pollution Tolerance index was found to be significantly different between control and polluted
plants. Based on the present study results two plant species i.e., P. lanceolata and C. siamea are
categorized in to intermediate and sensitive respectively. Thus they can be considered as bio
indicators of air pollution.
Keywords: Air pollution - APTI - Chlorophyll - Ascorbic acid.
[Cite as: Kumar L, Hemanth kumar NK & Jagannath S (2018) Assessment of air pollution impact on
micromorphological and biochemical properties of Pentas lanceolata Forssk. and Cassia siamea Lam. Tropical
Plant Research 5(2): 141–151]
INTRODUCTION
Pollution is one of the most pervasive problems in the world due to a rapid increase in population,
urbanization and industrialization. The flow of pollutants to air, soil and water is increasing with every passing
year and causing irreparable damage to the earth. Environmental pollution consists of five basic types of
pollution viz. air, water, soil, noise and light pollution. Among them, air pollution is by far the most harmful
form of pollution. The substances that pollute the environment are called as pollutants, these pollutants include
gases (sulphur oxides, oxides of nitrogen, carbon monoxide, hydrocarbons, etc.), particulate matter (smoke,
dust, fumes, aerosols), radioactive materials and others (Chauhan et al. 2004).
Over the years there has been a continuous increase in human population, transportation and vehicular traffic
and industries which have resulted in further increase in the concentrations of gaseous and particulate pollutants
(Joshi & Swami 2009). Air pollution can directly affect the plants via leaves or indirectly via soil acidification.
When exposed to airborne pollutants, most plants showed physiological changes before exhibiting visible
damage to leaves (Liu & Ding 2008). Automobiles are responsible for the maximum amount of air pollutants
and the crop plants are very sensitive to gaseous and particulate pollution and these can be used as indicators of
air pollution (Joshi & Swami 2009). Pollutants can cause leaf injury, stomatal damage, premature senescence,
decrease photosynthetic rate, disturb membrane permeability and reduce growth and yield in pollution sensitive
plant species (Tiwari et al. 2006).
Air pollution stress leads to stomatal closure, which reduces carbon dioxide availability to leaves and
inhibits carbon fixation. The net photosynthetic rate is commonly used as an indicator of the impact of increased
Received: 22 May 2017 Published online: 31 May 2018
Kumar et al. 2018
air pollutants on tree growth (Woo et al. 2007). Sulphur dioxide (SO2), Oxides of nitrogen (NOX) and carbon
dioxide (CO2) as well as suspended particulate matter (SPM) when absorbed by the leaves may cause a
reduction in the concentration of photosynthetic pigments viz., chlorophyll and carotenoids, which directly
affected the plant productivity (Joshi & Swami 2009).
Chlorophyll measurement is an important tool to evaluate the effect of air pollutants on plants as it plays an
important role in plant metabolism and the reduction in chlorophyll content corresponds directly to plant growth
(Joshi & Swami 2009). Chlorophyll is an index of productivity of the plant. Certain pollutants are known to
increases the total chlorophyll content, whereas others decreased (Agbair & Esiefarienrhe 2009). Studies on
Jasminum sambac (L.) Aiton with respect to foliar epidermal features brought out by a conspicuous decrease in
the size of epidermal cells and stomata, and increase in epidermal cells, stomatal and trichome frequency in
plants of the polluted population. This modification of epidermal trait could be indicative of environmental
pollution and could, therefore be used as bio-indicator of air pollution (Kulshreshtha et al. 1994a). An index to
identify the tolerance of air pollutants was developed which is known as air pollution tolerance index (APTI) by
Singh & Rao (1983). The usefulness of evaluating APTI for the determination of tolerance as well sensitiveness
of plant species were followed by several workers (Liu & Ding 2008, Dwivedi et al. 2008). Therefore the
present study was undertaken to evaluate the effects of air pollution on micro-morphological and biochemical
parameters of Pentas lanceolata (Forssk.) Deflers and Cassia siamea (Lam.) Irwin & Barneby.
P. lanceolata, commonly known as Egyptian Starcluster, belongs to the family Rubiaceae. It is known for its
wide use as a garden plant where it often accompanies butterfly gardens. Cassia siamea also known as Siamese
cassia, Kassod tree, Cassod tree and Senna siamea, Cassia tree, is a legume in the subfamily Caesalpinioideae.
It is a medium-sized, evergreen tree growing up to 18 m with beautiful yellow flowers. It is often used as a
shade tree in Cocoa, Coffee and Tea plantations.

Collection of plant materials
Leaves of Pentas lanceolata (Forssk.) Deflers and Cassia siamea (Lam.) Irwin & Barneby were collected
from the K.R circle Mysuru where main city bus station is located at this junction and Chikkalli which is 5.47
km away from the Mysuru city and has negligible traffic, which served as polluted and control area respectively
(Fig. 1).
Micromorphological studies
Leaf sample collected from polluted and control sites were processed for micro morphological studies
following the method of Ahmad & Yunus (1985). The mature leaf samples of polluted and control sites were cut
into small bits and taken in separate test tubes. 30% of Nitric acid solution was added to each test tube. Then it
was boiled in a boiling water bath for 5–10 minutes. Then the leaf bits were washed in distilled water and
stained with 2% safranin. Excess stain was removed by washing with distilled water and mounted using glycerin
jelly. The following micro-morphological parameters were studied it includes number of stomata, number of
epidermal cells, number of closed stomata, number of opened stomata, number of clogged stomata, stomatal
index, size of the stomatal aperture, number of trichomes and size of the trichome. Stomatal index is calculated
following the method of Salisbury (1927).
Where,
S= number of stomata.
E= number of epidermal cells.
Biochemical studies
The leaves from both plant species were plucked randomly and brought to the laboratory in polythene bag
and preserved at 4ºC until further analysis. The samples were analyzed for different biochemical parameters like
pH (Sing & Rao 1983), Total chlorophyll content (Arnon 1949), Ascorbic acid (Behrens & Madere 1994) and
Relative Water Content (Sing & Rao 1983) all these parameters were analyzed within 24 hours of their
harvesting.
Chlorophyll estimation
The chlorophyll content of leaves was determined following the method of Arnon (1949). The volume of
extraction was made up to 100 mL by using 80% acetone. The absorbance was read by using spectrophotometer
at 645 nm, and 663 nm. The amount of Chlorophyll a, b and total chlorophyll present in the sample was
calculated using the following the formula,
Where,
A645 = Value of Optical Density at 645 at absorption spectra.
A663 = Value of Optical Density at 663 at absorption spectra.
V = Volume of extract.
W = Weight of leaf material.
Figure 1. Location of Pentas lanceolata: A, Polluted; B, Control and Cassia siamea: C, Polluted; D, Control.
Total ascorbic acid
The total ascorbic acid content was determined following the method of Behrens & Madere (1994). One
gram of fresh leaves were ground with 5 ml of Phosphate buffer (pH 7.4) and made up to 10 ml using Phosphate
buffer, centrifuged at 3000 rpm for 15 minutes. 0.2 mL of supernatant was taken and 1 mL of 5% TCA was
added and mixed well. Solution without sample served as blank, 1 mL of 2% DNPH (2-4 Dinitrophenyl
hydrazine) reagent was added to this mixture and placed in a boiling water bath for 15 min. and cooled to room
temperature. 7 mL of 80% chilled H2SO4 was added and the absorbance was read at 540 nm. The total ascorbic
acid content was determined with a standard curve prepared by using ascorbic acid.
Relative water content (RWC)
Relative water content of leaf sample was determined following the method of the Singh & Rao (1983),
based on the percentage moisture on over dry weight basis by using the formula:
Kumar et al. 2018
Leaf extract pH
One gram of fresh leaves were homogenized in 10 ml of deionized water. The extract was filtered and the p H
of leaf extract was determined by digital pH meter.
Air Pollution Tolerance Index (APTI) determination
Air Pollution Tolerance Index (APTI) was determined following the method of Singh & Rao (1983).
[ ]
Where,
A = Ascorbic acid content (mg g-1)
T = Total chlorophyll mg g-1 fresh weight
P = pH of leaf extract
R = Relative water content of leaf (%).
RESULTS
The present study showed that air pollution causes significant changes in leaf micromorphology of two plant
species Pentas lanceolata (Forssk.) Deflers and Cassia siamea (Lam.) Irwin & Barneby growing at polluted
sites in Mysuru when compared with the same plant species growing at the unpolluted site. Air pollution
directly affects the micromorphological parameters of the leaf (Table 1). Leaf surface area in both the plant
species decreased from control to polluted area and leaf colour changes from green to pale yellow/dark in
polluted area of both the species respectively (Fig. 2). There was a decrease in number of stomata in P.
lanceolata of polluted site (48.8) compared to control (17.4) area but in C. siamea number of stomata were
increased in polluted area (150.1) when compared to control (91.2). In both the species clogged stomata (Fig. 3)
were observed, number of clogged stomata were less in control area samples when compared to polluted
sample. A number of epidermal cells in C. siamea of polluted and control sites showed a significant difference
(405.2) and (201) respectively. But in P. lanceolata no such significant differences were observed. Stomatal
index is an important parameter to determine the physiological changes in the plant due to air pollution.
Stomatal index of both species is reduced in polluted site. Stomatal index of P. lanceolata in control is 23.53, it
has been reduced to 11.82 in the polluted site, similarly in C. siamea (35.07) in control and (27.10) in polluted
site.
Figure 2. Leaf size and colour of Pentas lanceolata: A, Polluted; B, Control and Cassia siamea: C, Polluted; D, Control.
Kumar et al. 2018
Figure 3. Microphotographs of stomata of leaf sample from polluted and control areas of Pentas lanceolata: A, Polluted; B,
Control and Cassia siamea: C, Polluted; D, Control.
Figure 4. Microphotographs of trichomes of Pentas lanceolata: A, Polluted; B, Control and Cassia siamea: C, Polluted; D,
Control.
In leaf sample from polluted area number of trichome increased significantly in P. Lanceolata. In C. siamea
there is no such significant difference among polluted and control site (Fig. 4). Stomatal pore size showed a
significant difference in both the species. In P. lanceolata stomatal pore size in control site, it is 677.6 mm and it
has been reduced to 262 mm in polluted site and similarly, in C. siamea stomatal pore size is reduced in the
polluted (231.16mm) site than control (86.94 mm) site. In P. lanceoloata there was significant increase in the
number of trichomes in polluted site (17.8) compared to control (1.6) site not only in number but also in length
and breadth of trichomes, in P. lanceolata trichome length in polluted site was 45.6 mm and in control site it is
44.2 mm no such differences were observed among polluted and control samples in case of C. siamea.
Biochemical parameters
1. Chlorophyll content: The chlorophyll a, b and total chlorophyll content in both the plants were found to be
significantly different (Fig. 5). In both, the plant species of control and polluted sites do not show a
significant difference in chlorophyll a content but they showed a very significant difference in chlorophyll b
content i.e., in P. lanceolata 0.41 mg g-1. Fresh weight (F.Wt.) in control area and 0.16 mg g-1. F.Wt. in
polluted area. In C. siamea 0.15 mg g-1. F.Wt. in control area and it has been decreased to 0.02 mg g-1. F.Wt.
in polluted area. Total chlorophyll content in both the species was significantly reduced in polluted area
compared to unpolluted area. So it clearly indicated that chlorophyll b and total chlorophyll content in both
the species is adversely affected by the air pollution.
Figure 5. Changes in Chlorophyll content of leaf sample from polluted and control areas. (Mean ± SD followed by the same
letters are not statistically significant between the concentration when subjected to SPSS package version 14.0 according to
Turkey’s mean range test at 5% level. Chl a= Chlorophyll a, Chl b= Chlorophyll b, TCH= Total chlorophyll content)
2. Ascorbic acid content: Ascorbic acid content in both the plant species of control and polluted area were
presented in figure 6. In P. lanceolata reduction in the ascorbic acid content of polluted area (6.56 mg g -1)
sample compared to the unpolluted area (18.33 mg g-1) but in C. siamea an increase of the ascorbic acid
content of polluted area (31.5 mg g-1) when compared to unpolluted (16.43 mg g-1) area.
Figure 6. Changes in Ascorbic acid content of leaf sample from polluted and control areas. (Mean ± SD followed by the
same letters are not statistically significant between the concentration when subjected to SPSS package version 14.0
according to Turkey’s mean range test at 5% level)
Kumar et al. 2018
3. Leaf extract PH: The pH value of both the plants of polluted and control sites are found to be significantly
different (Fig. 7). In P. lanceolata pH value is increased in the polluted sample (6.0) compared to control
sample (5.4) but in C. siamea pH value is decreased in the polluted sample (2.62) compared to control (4.43)
respectively.
Figure 7. Changes in pH content of leaf sample from polluted and control areas. (Mean ± SD followed by the same letters
are not statistically significant between the concentration when subjected to SPSS package version 14.0 according to
Turkey’s mean range test at 5% level)
4. Relative water content: The relative water content in both the plants differed considerably (Fig. 8). Relative
water content is increased in polluted (73.33%) sample of P. lanceolata compare to control (65.00%) site
respectively. And in C. siamea relative water content was increased in control (54.6%) samples and
decreased in polluted (49.9%) samples.
Figure 8. Changes in Relative water content of leaf sample from polluted and control areas. (Mean ± SD followed by the
same letters are not statistically significant between the concentration when subjected to SPSS package version 14.0
according to Turkey’s mean range test at 5% level)
Air pollution tolerance index
The ambient air quality monitoring data at K. R. circle for two months April and May 2016, was obtained
from KSPCB, Mysuru (Table 2 & Fig. 9). The concentration of SO2, NO2, and SPM were within the permissible
limits of CPCB. P. lanceolata showed APTI values of 17.00 and 11.34 in control and polluted area sample
respectively and C. siamea do not show significant difference among the plants of polluted (13.39) and control
(14.02) area.
Figure 9. Changes in APTI values of polluted and control areas. (Mean ± SD followed by the same letters are not
statistically significant between the concentration when subjected to SPSS package version 14.0 according to Turkey’s mean
range test at 5% level)
Table 2. Ambient Air Quality Monitoring at K.R. Circle.

S.No. Month (2016) SO2 (µg m-3) NO2 (µg m-3) SPM (µg m-3)
1 April BDL 22.2 53
2 May BDL 21.1 56
Note: BDL= Below detecting level.
DISCUSSION
The response of leaf character to air pollution will indicate the adverse effect of air pollution, which can be
used as bioindicator. Both Pentas lanceolata (Forssk.) Deflers and Cassia siamea (Lam.) Irwin & Barneby in
the present investigation showed a reduction in leaf size in the polluted area compared to unpolluted area. A
similar reduction in leaf area was also observed in leaves of Newbouldia laevis by Kayode & Otoide (2007) and
Albizia lebbeck (L.) Benth. under the stress of air pollution (Seyyednejad et al. 2009). The stomatal index was
found to be decreased in both the plant species under polluted sites as compared to control site. Vehicular
pollution is known to affect the stomatal index and it has been reported to decrease in some plants
(Bhiravamurthy & Kumar 1983, Pavana et al. 2014, Amulya et al. 2015, Thara et al. 2015). A number of
epidermal cells per unit area has also been observed to be affected by the vehicular pollution and their number
increased in C. siamea in polluted site compared to control site. Salgare & Iyer (1991) have reported the
increase in a number of epidermal cells due to air pollution but in P. lanceolata number of epidermal cells was
decreased in polluted area. A similar study was carried out by Bhiravamurthy & Kumar (1983) have reported a
decrease in the number of epidermal cells per unit area due to air pollution.
Clogging of stomata and length of trichomes are also affected by air pollution. The number of clogged
stomata is more in polluted site compared to control site and length of trichomes decreased in C. siamea in the
polluted area but P. lanceolata trichome length has increased. The decrease in trichome number and length in
polluted population has been observed in Lantana and Calatropis (Kulshreshtha et al. 1994a, b). On the
contrary increase in trichome number has been observed. According to Sharma (1977) trichomes help trap the
particulate matter falling directly on the leaf surface which otherwise block the stomata pores and adversely
affect the process of gaseous exchange. This increased number of trichomes seems to be another adaptation of
the stress of air pollution providing an outline defense.
Reduction in the concentration of chlorophyll content in leaves of the polluted area was observed in both the
plant species. Similar changes in the concentration of pigments were also observed in leaves of six tree species
exposed to air pollution due to vehicle emission by Joshi & Swami (2009). Leaves from the polluted area had
significantly lower chlorophyll content than control (Tripathi & Prajapathi 2008, Stevovic et al. 2010).
In the present investigation increase in ascorbic acid content in P. lanceolata and decrease in ascorbic acid
content in of C. siamea in polluted site gives an idea about variation in the ascorbic acid content of two different
species under air pollution stress and their tolerance to air pollution. Increase in ascorbic concentration with
respect to the control leaves was also reported by Jyothi & Jaya (2010). The leaf pH values increased in P.
lanceolata in the polluted area compared with that of control. The leaf pH values decreased in C. siamea in the
polluted area compared with that of control. Similarly, increased and decreased pH values in plants growing at
the polluted site were observed by Patel & Kousar (2011). The increased relative water content in P. lanceolata
was observed in polluted sites. Similar increased relative water content was observed by Patel & Kousar (2011).
In C. siamea relative water content was decreased in the polluted sample. Relative water content is associated
with protoplasmic permeability in cells, which causes loss of water and dissolved nutrients, resulting in early
senescence of leaves (Agrawal & Tiwari 1997).
Among two plant species investigated for APTI P. lanceolata showed more tolerance to air pollution than C.
siamea. Different plant species shows considerable variation in their susceptibility to air pollution. The plants
with high and low APTI can serve as tolerant and sensitive species respectively. Also, the sensitivity levels of
plants to air pollutants differ from shrubs and trees with identical values, a tree may be sensitive but a shrub may
be tolerant to given pollutant. Therefore, the indices for different plant types should be considered separately.
The result of present study reveals that these two plant species (i.e. Pentas lanceolata and Cassia siamea) are
categorized in to intermediate and sensitive respectively based on their APTI values. Thus, they can be
considered as bio indicators of air pollution.
ACKNOWLEDGEMENTS
The authors are thankful to the Head, Department of Studies in Botany, University of Mysore, for providing
all the necessary requirements.
Kumar et al. 2018
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Research article
Effect of various plant growth regulators on in vitro seed

germination and shoot organogenesis in Tectona grandis L.f.
Jay Prakash Mishra, Deepti Bhadrawale, Upasana Yadav,
Naseer Mohammad and Fatima Shirin*
Genetics and Plant Propagation Division, Tropical Forest Research Institute, PO: RFRC,
Mandla Road, Jabalpur-482021, Madhya Pradesh, India
*Corresponding Author: fatimashirin@yahoo.com [Accepted: 22 June 2018]
Abstract: In the present study efforts were made to enhance in vitro seed germination and achieve
shoot organogenesis through different explants of Tectona grandis, a hardwood timber species
known throughout the world for its high-value wood. Seed germination was significantly affected
by seed inoculation on different strengths of MS medium and different GA 3 concentrations.
Different strengths of MS media combined with different GA3 concentration significantly affect
seed germination in vitro. Maximum seed germination (96%) was obtained on 0.4% GA3
combined with half strength MS medium. The effect of types of explants (leaf, internode,
hypocotyl and root of seedlings), different concentrations of thidiazuron (TDZ) (0.1, 1.0 and 10
μM) and auxins (IBA, IAA and NAA) on callus induction and shoot formation was investigated.
After 30 days of inoculation, it was observed that explant type had a significant effect on callus
formation. Maximum callus was obtained on internode explants (56.17%) which were statistically
on par with callus obtained on leaf explants (54.32%). The effect of TDZ on callus and shoot
formation was also observed. TDZ with higher concentration (1 μM) gave maximum callus
(48.6%). Whereas lower concentration of TDZ (0.1 μM) was effective for shoot formation from
both internode and hypocotyl explants. IAA and NAA were also helpful in shoot formation
whereas IBA retards shoot formation. For shoot organogenesis internode was found to be most
responsive explant type followed by hypocotyls and leaf. Roots were found to be the least
responsive explant.
Keywords: Auxins - Callus - Explant - Gibberellic acid - Shoot organogenesis.
[Cite as: Mishra JP, Bhadrawale D, Yadav U, Mohammad N & Shirin F (2018) Effect of various plant growth
regulators on in vitro seed germination and shoot organogenesis in Tectona grandis L.f. Tropical Plant
Research 5(2): 152–159]
INTRODUCTION
Tectona grandis L.f. (Teak) is distributed naturally in many countries of South-East Asia. It is a paragon
amongst the hardwood timber species. It has been planted in a number of tropical countries because of its
amenability for plantations coupled with desirable wood properties and durability. India is considered to be the
center of diversity for teak and it is widely distributed in central and south India, occupying about 13% of the
total forest area (Tewari 1992). The Indian region is considered to be the center of genetic diversity of teak
(Anonymous 2006). Tectona grandis is found in a variety of habitats and climatic conditions from arid areas
with only 500 mm of rain per year to very moist forests with up to 5000 mm of rain per year. In teak growing
areas, average annual rainfall is usually 1250–1650 mm with a 3–5 months dry season It occurs in natural
forests between 9° to 26° N latitude and 73 o to 104o E longitude, which includes southern and central India,
Myanmar, Laos People’s Democratic Republic and northern Thailand (White 1991).
Teak is generally propagated from seeds. Various methods of vegetative propagation have been used, such
as budding and grafting (Mahmood & Somasundaram 1975), rooting of cuttings (Lahiri 1974) and rooting of
buds cut from stock stumps raised in polypots (Mahmood & Somasundaram 1975). The conventional methods
of vegetative propagation have limitations. They are slow and time-consuming. Teak being a cross-pollinated
Received: 04 January 2018 Published online: 30 June 2018
Mishra et al. 2018
species, progeny raised from seed show wide variation. It also has an irregular seed-bearing habit and the
production of seed tends to be much lower than the requirement (Lee & Rao 1981). The viability of seed is poor
and is affected by the season when the seed is collected and by the storage conditions (Department of Forestry.
New Guinea 1962). The seeds are enclosed in hard coats and show very poor germination rates in spite of
various pretreatments (Joshi & Kelkar 1971, Dabral & Amin 1975, Dabral 1976, Davidson & Fairlamb 1976).
Mathew & Vasudeva (2003) also reported low seed germination in seeds collected from clonal seed orchards.
These characteristics have become a major problem in large-scale planting. In the face of the rising demand for
teak planting stock, the traditional tree improvement and multiplication programmes are not productive enough.
A rapid means of producing a large number of plants for afforestation is offered by micropropagation Gupta et
al. (1980) estimated that though subculture, 3000 viable plants from a single seedling or 500 plants can be
obtained from a single bud of a mature plant in one year.
Genetic improvement of teak by conventional breeding methods is still difficult due to the long reproductive
cycle. It takes more than a decade from the initiation of a genetic improvement program until improved seeds
are available and need another 4–5 decades until the timber from the first rotation of improved planting stock
can be harvested. Therefore, special attention must be given for producing genetically engineered teak to by-
pass the long period required for natural genetic crosses and selection (Widiyanto et al. 2009). However, no
report of the successful genetic transformation of teak has been published to date (Widiyanto et al. 2009). In
attempts to develop the transformation procedure of teak, many researchers have studied the regeneration ability
of teak callus tissues to develop shoots and found that the frequency of adventitious shoot formation from teak
callus was either very low or absent (Widiyanto et al. 2003, Widiyanto et al. 2005). Therefore, in the present
study, two experiments were conducted. Firstly attempts were made to study the effect of GA 3 treatments and
MS medium strengths on seed germination. The second experiment was conducted to investigate the effect of
thidiazuron (TDZ), auxins and explant types on adventitious shoot organogenesis in teak.

Collection and preparation of seeds
Dried teak fruits were collected from phenotypically superior trees of Tectona grandis present in Tropical
Forest Research Institute, Jabalpur. The fruits were opened with a nutcracker. The seeds were thoroughly
washed with distilled water and then immersed and agitated in 0.1 % Cetrimide solution for 20 min on a Rotary
shaker. Thereafter, the seeds were washed with distilled water 3–4 times and treated with 0.1% streptomycin
and 0.2% Bavistin® solution for 10 min and again rinsed with distilled water 3–4 times. Next, in the laminar
airflow, the seeds were treated with 0.1 % mercuric chloride solution for 5 min and rinsed thoroughly three
times with sterile distilled water to completely wash off the compound.
Germination of seed
The surface-sterilized seeds were inoculated individually in semi-solid MS medium. In order to optimize the
germination, the seeds were inoculated on three strengths of MS medium supplemented with four concentrations
of GA3. A two way factorial randomised experiment was conducted to study the effect of different strengths of
MS medium (Full, ½ and ¼ strength of MS salts), different concentrations of GA 3 (0, 0.1 %, 0.2 % and 0.4%)
and their interactions on germination (%) in the seeds of teak. The experiment consisted of 12 treatments and
three replicates. There were 10 explants per treatment. The observations for germination percentage were
recorded 10, 15 and 30 days after seed inoculation.
Callus formation
After germination, the in vitro raised seedlings produced 2–3 nodes in 20–25 days. The different parts of
these seedlings were used for callus and shoot formation. A two way factorial randomised experiment was
designed to study the effect of different strengths of TDZ (0.1, 1 and 10 μM), different auxins (0.1 μM) (IBA,
IAA and NAA) and their interactions on callus formation/organogenesis in the different explants obtained from
the seedling viz., internode, leaves, roots and hypocotyl. The experiment consisted of 9 treatments and three
replicates. There were 10 explants per treatment and data was recorded at an interval of 15 and 30 days.
Culture conditions
After inoculation, the test tubes containing seeds were incubated in dark conditions for 7 days to induce
germination. After germination, the cultures were transferred in light condition under a controlled set of
environmental conditions in the culture room. They were incubated at 25±2C with 16 hr photoperiod provided
by cool white fluorescent tubes of 40 watts (approx. 45 µmol m-2 s-1). The inorganic salts used for the
preparation of culture medium were obtained from SRL Chemicals and Qualigens Pvt. Ltd., India and
phytohormones and B vitamins from Sigma Chemicals Pvt. Ltd., India. The medium contained 3% (w/v)
sucrose as carbon source and 0.8% (w/v) agar (Microexpress Pvt. Ltd., India). The pH of the medium was
adjusted to 5.8 using 0.1 N NaOH or 0.1 N HCl before autoclaving for 15 min at 1.06 kg cm -2 (121°C). Each
seed was cultured in a 2.5 cm × 15.0 cm glass test tube (Borosil India Ltd.) containing 10 ml sterilized semi-
solid medium. for culture initiation and 400 ml culture bottles containing 45 ml semi-solid medium for
organogenesis.
The seed germination rate was expressed as a percentage, which was calculated by using the following
equation: (number of germinated seeds/total number of seeds inoculated per treatment) X 100. All experiments
were conducted in complete randomized design (CRD) and data were analyzed by two-way analysis of variance
using SX statistical package. The significance of the data was ascertained by F-test and the Critical Difference
(C.D.) values at p = 0.05 computed for comparing means of various treatments. The factorial combination was
used to study the interactions of the treatments. Arc sine transformation was used to transform data expressed in
percentage (Gomez & Gomez 1984).
RESULTS
Seed Germination
Effect of GA3 treatment: Germination of seeds started one week after inoculation of seeds. The germination
percentage of seeds after 10, 15 and 30 days of inoculation was significantly affected by different concentrations
of GA3 (Table 1). The seed germination obtained on 0.1%, 0.2% and 0.4% GA3 treatments was 42.59%, 42.58%
and 38.88%, respectively. After 15 days of seed inoculation, germination increased to 57.40% on 0.1% GA3,
49.99% on 0.2% and 49.99% on 0.4% GA3 treatment, respectively. After 30 days of inoculation, maximum seed
germination (96.29%) was obtained on treatment of 0.4% GA3, which was statistically on par with germination
obtained on 0.2% GA3 treatment (92.59%) (Fig. 1A–D). Even after one month, seed germination obtained on
control (treatment without GA3 treatment) was very low (9.25%).
Figure 1. In vitro seed germination in seeds of Tectona grandis: A, Seed inoculation; B, Germination after 10 days; C,
Germination after 15 days; D, Germination after 30 days; E–L, Callus formation in leaf, internodes, hypocotyls and root on
MS medium supplemented with 1 μM TDZ & 0.1 μM IBA (E), 0.1 μM TDZ & 0.1 μM NAA (F), 1 μM TDZ & 0.1 μM IBA
(G), 10 μM TDZ & 0.1 μM IAA (H) after 15 days of inoculation and 10 μM TDZ & 0.1 μM IBA (I), 1 μM TDZ & 0.1 μM
NAA (J), 1 μM TDZ & 0.1 μM IBA (K), 10 μM TDZ & 0.1 μM IAA (L) after 30 days of inoculation.
Mishra et al. 2018
Effect of MS medium strengths: Highly significant effect of MS medium strengths was observed on germination
of seeds after 30 days of inoculation. Maximum germination of seeds (79.16%) was obtained on 1/2 strength
MS medium which was significantly higher than seed germination obtained on any other MS medium strength.
Least germination of seeds (61.10%) was obtained on full strength MS medium.
Table 1. Effect of different strengths of MS medium, GA3 and their interactions on seed germination in Tectona grandis after
10, 15 and 30 days of inoculation. (The data expressed in parenthesis are arc sine transformed)
GA3 MS strength (MS) MS strength (MS) MS strength (MS)
(%) 10 days Mean 15 days Mean 30 days Mean
(G) Full Half Quarter Full Half Quarter Full Half Quarter
0 0 11.10 3.70 0 16.66 11.10 9.25 0 16.66 11.10 9.25
0
(0.04) (0.04) (16.04) (5.37) (0.04) (19.77) (16.04) (11.95) (0.04) (19.77) (16.04) (11.95)
49.99 44.44 33.33 42.59 72.21 55.55 44.44 57.40 72.21 100 77.77 83.33
0.1
(44.98) (41.75) (35.24) (40.66) (58.43) (48.23) (41.75) (49.47) (58.43) (89.94) (62.15) (70.17)
44.44 49.99 33.33 42.58 49.99 61.11 38.88 49.99 83.33 100 94.44 92.59
0.2
(41.73) (44.98) (34.76) (40.49) (44.98) (51.96) (38.49) (45.14) (71.18) (89.96) (81.93) (81.02)
49.99 33.33 33.33 38.88 49.99 49.99 49.99 49.99 88.88 100 100 96.29
0.4
(44.98) (34.76) (34.76) (38.17) (44.98) (45.45) (44.98) (45.14) (73.91) (89.96) (89.96) (84.61)
36.10 31.94 27.77 43.04 45.82 36.10 61.10 79.16 70.83
Mean
(32.93) (30.38) (30.20) (37.11) (41.35) (35.31) (50.89) (72.41) (62.52)
CD(0.05)
Variable
10 days 15 days 30 days
G 8.92 10.96 10.57
MS N.S. N.S. 9.61
G*MS N.S. N.S. N.S
Adventitious shoot organogenesis
The objective of this experiment was to investigate the effect of types of explant (leaf, internode, hypocotyl
and root of seedling), different concentrations of TDZ (0.1, 1.0 and 10 μM) and auxins (IBA, IAA and NAA) in
MS medium on callus induction and shoot formation.
Callus formation
Leaf curling and elongation of leaves started after 7–8 days of inoculation on media. The swelling was
observed on the surface of internodes after 7–8 days of inoculation. Callus induction was initiated on the leaf
pieces, internodes and hypocotyls after 10–12 days of inoculation. Colour of the root explants changed to green
from brown after 7–8 days and the length of roots increased. Callus formation on the root explants started after
15 days of inoculation. The data for callus formation was recorded after 15 and 30 days of inoculation (Fig. 1E–
L).
Effect of explants type and auxins
Table 2. Effect of explant type, auxins and their interactions on callus induction in Tectona grandis after 15 and 30 days
of inoculation. The data expressed in parenthesis are arc sine transformed.
Auxins (0.1 μM)(A)
Explant type
15 days 30 days
(E) Mean Mean
IBA IAA NAA IBA IAA NAA
14.81 0.00 14.81 9.87 48.15 55.55 59.26 54.32
Leaf
(16.95) (0.04) (17.11) (11.36) (45.49) (49.82) (50.71) (48.67)
22.22 25.92 25.92 24.69 59.26 55.55 53.70 56.17
Internode
(23.03) (25.35) (27.09) (25.15) (54.05) (49.98) (50.48) (51.51)
22.22 18.52 9.25 16.66 40.74 44.44 27.77 37.65
Hypocotyl
(24.77) (20.86) (13.37) (19.67) (39.41) (39.98) (28.17) (35.85)
9.25 7.40 0.00 5.55 24.07 9.25 1.85 11.73
Root
(10.53) (9.28) (0.04) (6.61) (22.52) (10.53) (2.70) (11.92)
17.13 12.96 12.50 43.05 41.20 35.65
Mean
(18.82) (13.88) (14.40) (40.37) (37.58) (33.02)
CD(0.05)
Variable
15 days 30 days
E 8.95 15.25
A 7.75 13.21
E*A 15.46 26.42
It was observed that explant type had a significant effect on callus induction after 15 days of inoculation.
Maximum callus was formed on the surface and cut ends of internodes (24.69%) which were statistically on par
with callus formed on hypocotyls (16.66%). Least callus induction was obtained on root explant. After 30 days
of inoculation, it was observed that explant type had a significant effect on callus formation (Table 2).
Maximum callus was obtained on internode explants (56.17%) which were statistically on par with callus
obtained on leaf explant (54.32%). The effect of auxins and interaction between explant type and auxins was
found to be statistically not significant for callus induction after 15 and 30 days of inoculation.
Effect of explant type and thidiazuron (TDZ)
The effect of TDZ on callus formation was also observed to be significant after 30 days of inoculation (Table
3). Maximum callus was obtained on 1 μM TDZ (48.67%) which was statistically on par with callus obtained on
10 μM TDZ (40.74 %).
Table 3. Effect of explant type, thidiazuron (TDZ) and their interactions on callus induction in Tectona grandis after
15 and 30 days of inoculation. The data expressed in parenthesis are arc sine transformed.
TDZ (μM) (T)
Explant type (E) 15 days 30 days
Mean Mean
0.1 1 10 0.1 1 10
5.55 14.81 9.25 9.87 38.89 61.11 62.96 54.32
Leaf
(6.61) (15.52) (11.95) (11.36) (36.58) (53.39) (56.06) (48.67)
37.03 24.07 12.96 24.69 50.00 70.37 48.15 56.17
Internode
(35.50) (24.26) (15.70) (25.15) (48.16) (62.29) (44.07) (51.51)
14.81 24.07 11.11 16.66 14.81 53.70 44.44 37.65
Hypocotyl
(16.95) (27.44) (14.62) (19.67) (18.53) (47.30) (41.74) (35.85)
3.70 9.25 3.70 5.55 18.52 9.25 7.40 11.73
Root
(3.95) (11.95) (3.95) (6.61) (16.10) (10.37) (9.28) (11.92)
15.28 18.05 9.25 30.55 48.61 40.74
Mean
(15.75) (19.79) (11.56) (29.84) (43.34) (37.79)
CD(0.05)
Variable
15 days 30 days
E 8.95 15.25
T 7.75 13.21
E*T 15.46 26.42
Shoot formation
Shoot primordia were visible after 30 days of inoculation. For shoot organogenesis hypocotyl was found to
be most responsive explant type followed by internode. The lowest concentration of TDZ (0.1 μM) was
effective for shoot formation on internode explants of teak (Fig. 2A–B). Shoot formation was obtained on
treatments of IAA and NAA but not on IBA.
Figure 2. Shoot formation after 30 days of inoculation: A–B, Internode explants on MS medium supplemented with 0.1 μM
TDZ & 0.1 μM IAA (A) and 0.1 μM TDZ & 0.1 μM IBA (B); C–D, Hypocotyl explants on MS medium supplemented with
0.1 μM TDZ and 0.1 μM IAA (C) and 0.1 μM TDZ and 0.1 μM NAA (D).
Mishra et al. 2018
With hypocotyl explants also, lower concentrations of TDZ (0.1 and 1 μM) were more helpful for shoot
formation than higher concentration (10 μM) (Table 4) (Fig. 2C–D). Shoot formation was obtained with all
three auxins (IAA, IBA and NAA).
Table 4. Effect of thidiazuron (TDZ), different auxins and their interactions on number of shoots formed on
internode in Tectona grandis after 30 days inoculation.
TDZ (μM) (T)
Auxins
Internode Hypocotyl
(0.1μΜ)(A) Mean Mean
0.1 1 10 0.1 1 10
IBA 0.00 0.00 0.00 0.00 0.26 0.10 0.00 0.12
IAA 0.06 0.00 0.00 0.02 0.20 0.20 0.10 0.16
NAA 0.13 0.00 0.00 0.04 0.06 0.06 0.01 0.04
Mean 0.06 0.00 0.00 0.17 0.12 0.03
CD(0.05)
Variable
Internode Hypocotyl
A 0.08 0.20
T 0.08 0.20
A*T 0.12 0.35
DISCUSSION
The increasing demand of teak for plantation purposes by forest departments as well as private companies
has necessitated research on unconventional methods for the improvement of productivity. The potential
benefits of the use of clonal planting stock in reforestation programs have long been recognized. However, to
achieve the maximum possible genetic gain for teak improvement, both sexual reproduction and vegetative
multiplication must be followed. This can be accomplished through micropropagation using seeds as explants by
germinating them under in vitro conditions.
In vitro propagation has been successfully applied in Tectona grandis and it has become an alternative tool
to overcome some problems occurring in sexual regeneration (Widiyanto et al. 2005). Regenerative organs such
as pre-existing shoots, meristem shoot-tips, nodal segments or seedling organs have been widely used as
explants (Gupta et al. 1980, Mascarenhas et al. 1987, Apavatjrut et al. 1988, Devi et al. 1994). Adventitious
shoot formation from callus tissues has been proposed for the regeneration of genetically engineered tissues of
many species (Siemens & Schieder 1996, Tawfik & Noga 2001, Lee & Pijut 2017).
Gibberellic acid is used in laboratory and greenhouse conditions to trigger germination in seeds that would
otherwise remain dormant. It is a naturally occurring plant growth regulator which may cause a variety of
effects including the stimulation of seed germination in some cases. GA3 occurs naturally in the seeds of many
species and is produced commercially by growing Gibberella fujikuroi (Sawada) Wollenw. fungus cultures in
vats, then extracting and purifying the GA3 (Yamaguchi 2008, Santner et al. 2009). The GA3 treatments
accelerated the process of seed germination and germination started early in treated seeds as compared to
control. It stimulates the synthesis and production of the hydrolases, especially amylase, resulting in the
germination of seeds. Presoaking seeds in GA3 solution will in many cases cause the rapid germination of many
types of highly dormant seeds which would otherwise need cold treatment, after-ripening or ageing, or other
prolonged pretreatments. Similar to our results Ribeiro et al. (2009) reported a positive effect of GA3 on seed
germination in Annona crassiflora Mart. Similarly, in Juglans regia L. also enhanced seed germination was
obtained (Kaur et al. 2006).
Generally, media high in salt and sugar content reduced germination efficacy. Maximum germination was
obtained on the lower strength of MS media (1/2). Least germination of seeds (61.10%) was obtained on full
strength MS medium. These results are in agreement with Ranasinqhe & Berlyn (1996) who reported only 50%
seed germination of teak on full strength MS medium as opposed to 60% on half strength. This effect could
partly be attributed to the role minerals play as osmotica. Because any germination process is preceded by
imbibition, anything that affects this process is likely to affect germination.
Adventitious shoot organogenesis
The induction of callus growth and organogenesis or differentiation of shoot buds directly or indirectly from
explants, is accomplished by the differential application of growth regulators and control of physical conditions.
In teak only a very few reports are there for adventitious organogenesis.
Callus formation
The type of explant used had a significant effect on callus formation (%). Maximum callus was obtained on
internode explant which was statistically on par with hypocotyl explant. But the effect of auxins, TDZ and their
interactions was found to be statistically non-significant for callus formation. Similarly, Kozgar & Shahzad
(2012) also did not achieve any enhanced results on combination treatment of BA, kinetin and TDZ in teak.
Shoot formation
Internode and hypocotyls explants of teak proved to be the most responsive explant type among the four
explants tried. Widiyanto et al. (2005) also reported shoot organogenesis in teak on calli induced from
internodal segments on Woody Plant Medium containing 1.0 μM thidiazuron (TDZ) in combination with 0.01
μM indole butyric acid (IBA).
CONCLUSION
The present investigation concentrated on finding out the most suitable GA3 concentration and MS media
strength for in vitro seed germination of Tectona grandis on one hand and on the other hand to screen out the
most suitable explant type and plant growth regulator type and concentration for shoot organogenesis. Looking
at the results of the present study pre-treatment of teak seeds with 0.4 % GA3 and germination on 1/2 strength
MS medium can be recommended. For shoot organogenesis internode was found to be most responsive explant
type followed by hypocotyls and leaf. Roots were found to be the least responsive explant.
ACKNOWLEDGMENTS
The authors are grateful to Dr. N. Kulkarni, Director, Tropical Forest Research Institute, Jabalpur for
providing necessary encouragements, guidance and facilities provided during the entire period of study.
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multiplication of Tectono grandis (teak) by tissue culture. Plant Science Letters 17: 259–268.
Joshi MD & Kelkar SP (1971) Germination of seed in dry teak (Tectono grandisi (I) Preliminary studies in fruit
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Research article
Environmental impact assessment of University of Ibadan

international conference centre on the adjoining forest
O. Chukwu1*, O. Y. Ogunsanwo2, W. A. Danladi1 and O. M. Oluyinka1
1
Department of Social and Environmental Forestry, University of Ibadan, Nigeria
2
Department of Forest Production and Products, University of Ibadan, Nigeria
*Corresponding Author: onye20042000@yahoo.com [Accepted: 24 June 2018]
Abstract: Trees are seen as obstacles of development in the developing countries. Despite global
recognition of the role of forests in mitigation of climate change, deforestation and forest
degradation continues to increase. However, there is dearth of information on the impact of
situating University of Ibadan International Conference Centre (UIICC) on the University’s
Tectona grandis plantation. Hence, provision of baseline information on the impact of the
Conference Centre on the environment will help management decisions on mitigation of its
negative effects. Environmental impact assessment of UIICC on the adjoining forest was
conducted. The data used for this study was obtained from all living T. grandis (332 trees) found
within seven randomly laid sample plots of 25 m × 25 m. Diameters at several points of tree stems
and height (total and merchantable) were measured. Hence, biomass of each sampled tree was
estimated. Point coordinates were also obtained to map out the boundaries of the various land
cover within the study area. The impact assessment results revealed that about 7066 trees with
merchantable volume of 601.55 m3 and total volume of 1266.58 m3 were removed for building of
UIICC covering about 9.309 ha. About 274.99 t C and 1009.28 t CO2 e carbon and carbon dioxide,
respectively were loss. Furthermore, loss of biodiversity and habitat for animals (birds, reptiles and
rodents) and disruptions of biogeochemical cycles were among the identified negative impacts.
Planting of avenue trees within the conference centre, proper waste disposal and use of renewable
energy were recommended as mitigation measures.
Keywords: Biomass - Deforestation - EIA - GIS - Tectona grandis.
[Cite as: Chukwu O, Ogunsanwo OY, Danladi WA & Oluyinka OM (2018) Environmental impact assessment
of University of Ibadan international conference centre on the adjoining forest. Tropical Plant Research 5(2):
160–166]
INTRODUCTION
The increasing rate of deforestation as a result of urbanization and its diverse effect on man and the
environment has become a phenomenon of concern to the world. Inyang & Esohe (2014) asserted that
deforestation exposes the forest land, mountains, hills and even valleys to erosion, subsequently, floods,
landslides and mudslides, loss of wild life and increasing loss of deserts follow. However, deforestation is a
recurring problem in Nigeria (Inyang & Esohe 2014). Hence, the annual rate of deforestation in Nigeria is about
3.5% which is approximately 350,000 to 400,000 hectares per year (FAO 2005).
The issue of sustainability, environment protection, health and the need to incorporate environment to
management of development has triggered several international treaties and legislations. These gave birth to
environmental impact assessment (EIA) (Anago 2002). EIA according to Munn (1979), refers to the need to
identify and predict the impact of projects, operational procedures, policies, legislative proposals and
programmes on the environment and human’s well-being and/or health and further interpret and communicate
information about the impacts.
In Nigeria, legislations have been put in place such as the Environmental Impact Assessment Decree number
86 of 1992 and Sections 20, 17 (2) and 16 (2) of the 1999 constitution of the Federal Republic of Nigeria
(Anago 2002). Therefore, by the provisions of these laws, all developmental projects must operate within
Received: 17 February 2018 Published online: 30 June 2018
Chukwu et al. 2018
environmental sustainability levels and ensure they do not constitute adversely impact on the ecosystem
integrity.
There is dearth of information on the impact of University of Ibadan International Conference Centre
(UIICC) on the adjoining forest (Teak) which have not been exploited, as such, this study was able to bring out
the various impact and ways forwards towards ameliorating the environment in line with EIA Decree number 86
of 1992 and section 20 and 16 (2) of the 1999 constitution of the Federal Republic of Nigeria. Therefore, the
objective of this study was to evaluate the impact of University of Ibadan International Conference Centre
(UIICC) on the adjoining forest with a view to providing baseline information for ecological management and
mitigation of its negative effects.

Study area
This study was carried out at the University of Ibadan International Conference Centre (UIICC) and the
adjoining forests consisting of; Tectona grandis L.f. plantation, Pinus caribaea Morelet plantation, natural
forest and farm/bare land at its Northeastern fringes. The area located along Oyo road, Ibadan in Akinyele Local
Government Area of Oyo State, which lies between latitudes 7º 45.106' N to 7º 45.834' N and longitudes 3º
90.942' E to 3º 90.508' E (Fig. 1), within the tropical rainforest. With mean altitude of 227 m above sea level
and total land area of 47.21 ha.
Figure 1. Map Showing the study area and different land cover.
University of Ibadan teak plantation was established over a period of three years (1951–1953) and managed
by the Department of Forest Resources Management (now; Department of Forest Production and Products and
Department of Social and Environmental Forestry). Until recent years, some part of the planation was proposed
to be used for construction of University of Ibadan-Five (5) Star Hotel. Later between the years 2009–2012,
through the University of Ibadan Endowment Fund, some part uncompleted buildings of the proposed UI-Five
(5) Star Hotel in the plantation were then converted to the present University of Ibadan International Conference
Centre. The UIICC cuts across large portion of the teak plantation which was extended to the UI-second gate
along Oyo road.
Data collection
Global positioning system (GPS) was used to collect coordinates round the boundaries of the study area.
Coordinates of the boundaries of the natural forest, Tectona grandis plantation, Pinus caribaea, Agricultural
lands and UIICC were collected.
Inventory data used in this study was collected from seven randomly laid sample plots (SPs) of size 25 m ×
25 m (0.0625 ha) managed by the Faculty of Renewable Natural Resources, University of Ibadan. Total
enumerations of Tectona grandis found within each SP were made. The following tree growth variables were
measured; diameter breast height (cm), diameters at the base; middle and top (cm), total height (m) and
merchantable height (m). A total number of three hundred and thirty-two (332) trees were enumerated. Spiegel
relaskop was used to measure total and merchantable heights of individual trees. Diameter over bark of
individual trees at breast height (1.3 m) was measured; the point of the measurement was recorded from the
uphill sides of the trees and on the inside of the lean for leaning trees. Climbers, loose bark and epiphytes were
lifted above the measuring tape during measurement. For trees with deformations at 1.3 m, the measurement
was made at the sound point on the stem above the abnormality.
Data analysis
Point coordinates obtained from the study area were saved in text (tab delimited) file format in Microsoft
Excel spread sheet; then loaded into Quantum Geographic Information System (QGIS) for mapping and
demarcations of land cover types and estimation of areas covered by each land use. The Coordinate Reference
System (CRS) was set to WGS84 in respect to the study region.
The following tree variables were computed:
i. Basal Area (BA) (Chukwu & Osho 2018) [1]
ii. Total volume (Vt) ( ) (Akindele 2005) [2]
iii. Merchantable volume (Vm) ( ) (Akindele 2005) [3]
Where Dbh= diameter at breast height (m), MHT = Merchantable height (m), D b = Diameter at the base
(m); Dm = Diameter at mid-point (m), Dt = Diameter at the top (m).
However, basal area, merchantable volume, total volume and number of trees were computed per hectare.
Hence, the number of tree removed was estimated as;
̅
( ) [4]
Where; = number of tree removed, ̅= average number of tree per plot and UIICC = University of Ibadan
International Conference Centre
The aboveground biomass (AGB) for each tree, the ecological condition of the forest was considered. Hence,
a biomass equation developed by Olayode et al. (2015) for Tectona grandis Linn. f. plantations within the
Southwestern Nigeria Tropical Rainforest, was adopted:
[5]
Where; lnB =natural logarithm of the tree aboveground biomass and Dbh= Diameter at breast height
Equation [5] was used to compute AGB for each tree. The biomass stock (kg ha-1) of each sampling plot was
obtained by dividing the sum of all the individual biomass weights (in kilogrammes) by the area of the sampling
plot (0.062 ha). Hence, the AGB value was converted to tonnes per hectare upon dividing by 1000. Later,
biomass value was converted into carbon stock upon multiplying by the default carbon fraction of 0.47 (IPCC
2006). Tree belowground biomass (BGB) was calculated following Mokany et al. (2006):
( ) [6]
Where; BGB= tree belowground biomass and AGB = tree aboveground biomass
Tree carbon (TC) was then obtained by the addition of AGB and BGB. Hence, its carbon dioxide equivalent
(CO2e) was estimated by multiplying TC by default carbon dioxide fraction of 3.67 (Pearson et al. 2007).
Furthermore, mean plot stand variables was computed and average for each stand variable (per ha) was
multiplied by the area covered by UIICC (9.309 ha) to estimate for the growth variables value for T. grandis
removed.
RESULTS
The results of land cover classification reveals that, maximum area (19.348 ha) is covered by the natural
forests, the international conference center (9.309 ha), farm/bare land (8.322 ha), teak plantation (7.951 ha) and
minimum (2.284 ha) by pine plantation (Fig. 2).
The data used in this study comprise of tree growth variables measured from 7 SPs of Tectona grandis stand
in the University of Ibadan, Nigeria. A total of 332 trees were measured and the summary statistics presented in
table 1. The distribution of Dbh ranged from 21.56 to 68.29 cm, THT ranged from 7.58 to 27.80 m, MHT
Chukwu et al. 2018
ranged from 3.01 to 15.45 m, Vm ranged from 0.0104 to 0.383 m3, Vt ranged from 0.0247 to 0.5996 m3 and BA
ranged from 0.0365 to 0.3663 m2.
Figure 2. Land cover classification of the study area.
Table 1. Summary statistics of tree growth variables.

Descriptive Statistics
Tree Variables
Minimum Maximum Mean ± SE
Db (cm) 24.27 115.42 53.9445 ± 0.8488
Dbh (cm) 21.56 68.29 39.8368 ± 0.4361
Dm (cm) 15.00 57.00 34.4608 ± 0.4360
Dt (cm) 10.00 41.00 22.1084 ± 0.3604
THT (m) 7.58 27.80 18.6170 ± 0.1860
MHT (m) 3.01 15.45 8.9390 ± 0.1373
BA (m2) 0.0365 0.3663 0.1296 ± 0.0028
Vm (m3) 0.0104 0.383 0.0852 ± 0.0029
Vt (m3) 0.0247 0.5996 0.1793 ± 0.0056
Note: SE= standard error, Db= diameter at the base, Dbh= diameter at breast height, Dm= diameter
at middle, Dt= diameter at top, THT= total height, MHT = merchantable height (m), BA=Basal area,
Vm= Merchantable volume and Vt= Total volume. The Total number of tree= 332.
The result of the study shows that the tree density (N) ranges from 624 to 928 tree ha -1 with a mean value of
759 tree ha-1. The basal area (BA) from 82.77 to 121.73 (mean 98.34) m2 ha-1; merchantable volume (Vm) from
52.39 to 85.78 (mean 64.62) m3 ha-1; total volume (Vt) from 109.58 to 161.84 (mean 136.06) m3 ha-1;
aboveground biomass (AGB) from 42.27 to 62.75 (mean 50.89) t ha-1; above ground carbon (AGC) from 19.87
to 29.49 (mean 23.92) t C ha-1; belowground carbon (BGC) from 4.67 to 6.93 (mean 5.62) t C ha -1 and Tree CO2
from 90.04 to 133.68 (mean 108.42) t CO2 e (Table 2).
Table 2. Summary statistics of tree stand variables.
Plot N BA Vm Vt AGB AGC BGC Tree CO2
(Tree ha-1) (m2 ha-1) (m3 ha-1) (m3 ha-1) (t ha-1) (t C ha-1) (t C ha-1) (t CO2 e)
1 672 93.04 57.26 121.27 47.72 22.43 5.27 101.65
2 768 92.36 58.58 133.21 48.36 22.73 5.34 103.02
3 688 94.23 62.31 134.03 48.95 23.00 5.41 104.27
4 928 121.73 77.61 161.84 62.75 29.49 6.93 133.68
5 624 82.77 52.39 109.58 42.27 19.87 4.67 90.04
6 736 86.89 58.42 121.39 45.40 21.34 5.01 96.71
7 896 117.34 85.78 171.09 60.82 28.58 6.72 129.56
Mean 759 98.34 64.62 136.06 50.89 23.92 5.62 108.42
Note: N= Number of tree, BA= basal area, Vm= merchantable volume, Vt= total volume, CO2= Carbon dioxide, AGB=
aboveground biomass, AGC= above ground carbon, BGC= belowground carbon.
Furthermore, about 7064 Tectona grandis trees, with estimated basal area (BA) of 915.4471 m2;
merchantable volume (Vm) of 601.5476 m3; total volume (Vt) of 1266.583 m3; aboveground biomass (AGB)
473.74 t; above ground carbon (AGC) 222.67 t C; belowground carbon (BGC) 52.32 t C and Tree CO2 1009.28
t CO2 e were removed from the study area to build UIICC (Table 3).
Table 3. Estimated quantity of Tectona grandis variables removed of the study locations.
N BA Vm Vt AGB AGC BGC Tree CO2
(Trees) (m2) (m3) (m3) (t) (t C) (t C) (t CO2 e)
7066 915.45 601.55 1266.58 473.74 222.67 52.32 1009.28
Note: N= Number of tree, BA= basal area, Vm= merchantable volume, Vt= total volume, CO2= Carbon dioxide,
AGB= aboveground biomass, AGC= above ground carbon, BGC= belowground carbon.
DISCUSSION
In this study, information on the tree growth variables from the University of Ibadan Teak plantation was
presented in table 1. Effort was directed towards estimating number of trees with corresponding stand variables
for the portion of the plantation used to construct the University of Ibadan International Conference Centre
(UIICC).
The result of the spatial analysis shows that the UIICC perimeter fencing covered about 9.309 ha which is
about 19.7% of the total forest area and 53.9% of the teak plantation. This result revealed that more than half of
the teak plantation was deforested for the construction of the conference centre. However, Mohammed et al.
(2013) averred that trees are seen as obstacles to development and their removal is viewed as the first stage in
development. Adeniyi and Omojola (1999) confirmed that most land development programmes and projects in
Nigeria have evolved without an appreciation of the value of land use and land cover information. Hence, this
has given rise to uncontrolled conversion of forests into other land cover types (Mohammed et al. 2013).
High tree carbon value of 29.54 t C ha-1 with corresponding tree CO2 values of 108.42 t CO2 e was estimated
for the T. grandis in the study area. Similar results were obtained by Olayode et al. (2015) for Teak plantations
in Osho Forest Reserve (29.36 t C ha-1) and Shasha Forest Reserve (24.36 t C ha -1) in Southwestern Nigeria.
Teak plantation was confirmed by studies to store substantial amount of carbon (Khanduri et al. 2008,
Boonyanuphap & Kongmeesup 2016). Olayode et al. (2015) affirmed that the high value obtained from Osho
forest reserve was because the plantation has not been harvested. This further confirms the result of this study as
UI Teak plantation is still within its first rotation.
Negative impacts
This study revealed that about 53.9% (9.309 ha) of the teak plantation was deforested. Wilcox (1995) stated
the forest degradation is usually accompanied by species extinction, reduction in biodiversity and decrease in
primary productivity. The long-term effect of this pressure is usually destruction of the quality and quantity of
rainforests (Mohammed et al. 2013). The bare land within UIICC and fence demarcating the Teak Plantation
has been shown in figure 3.
Figure 3. Bare land within UIICC and fence demarcating the Teak Plantation
Hence, about 7066 teak tree stands were removed from the plantation to construct the UIICC. These large
numbers of tree removed constitute high environmental degradation. Gibbs et al. (2007) upheld that trees
(aboveground tissues) are reservoir for majority of atmospheric carbon sequestered in tropical forests. Malhi
(2010) also stressed the importance of tropical forests as a major influence on global patterns of biodiversity,
ecosystem ecology, productivity and biogeochemical cycles. However, the removal of more than a half of the
teak trees in the study area implies loss of habitat for animals (birds, reptiles and rodents) and great deficit to the
environment (Fig. 4).
Furthermore, about 274.99 tonnes of tree carbon with a corresponding carbon dioxide value of 1009.28 t
CO2 e was loss following the destruction of the plantation. Couple with the exposure of the deforested land to
direct sunlight over years. Hence, this exposure tends to increase in both soil and atmospheric temperature and
decrease in relative humidity of the area leading to the degrading of the land area under investigation. Stern
(2007) revealed that, 18% of total global anthropogenic greenhouse gas emissions are contributed by
deforestation and forest degradation.
Suggested Mitigations for Negative Impacts
According to FAO (2006), global carbon retention resulting from reduced deforestation, increased forest
regrowth and more agroforestry could make up for about 15% of carbon emissions from fossil fuels over the
Chukwu et al. 2018
next 50 years. Therefore, planting of avenue trees within the premises of the conference centre is necessary. It is
suggested that renewable source of energy (Solar system) should be adopted and environmental friendly means
for waste disposal (bio-digester) for operations in the conference center.
Figure 4. Some buildings and Hall Complex within UIICC.
CONCLUSION
This study has provided baseline information on the environmental impact of the University of Ibadan
International conference centre on the adjoining forest and the status of the Tectona grandis stand in the study
area. Nevertheless, the assessment revealed high level of deforestation and forest degradation, coupled with
large amount of carbon loss to the environment as the result on situating the UIICC within the Teak forest.
However, Government law enforcement agencies should ensure that environmental impact assessments are
conducted before any developmental project is executed as prescribed by law, also ensuring strict compliance
with the EIA recommendations during and after the project.
ACKNOWLEDGEMENTS
Authors appreciation goes to the Forest Biometrics and Remote Sensing Unit, Department of Social and
Environmental Forestry, University of Ibadan, Nigeria for technical and material support toward the success of
this research work.
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Research article
Inventory of trees in the urban landscape: A case study in Andhra

University, Visakhapatnam, Andhra Pradesh
J. Prakasa Rao
Department of Botany, Andhra University, Visakhapatnam-530 003, Andhra Pradesh, India
*Corresponding Author: jprakasarao@gmail.com [Accepted: 25 June 2018]
Abstract: Documentation of existing flora of the urban environment is important to determine

existing resources and to set target for future improvement. An understanding of the flora in
regional level must play an important role in elucidating the larger patterns of distribution of
biodiversity. Floristic inventory of trees of Andhra University campus in Visakhapatnam city was
carried out during 2010 to 2016. This study resulted in record of 175 tree species pertains to 132
genera under 53 families. The data presented will be a valuable source of information for
management of tree resources in the Andhra University campus.
Keywords: Medicinal plants - Trees - Urban landscape - Visakhapatnam.
[Cite as: Rao JP (2018) Inventory of trees in the urban landscape: A case study in Andhra University,
Visakhapatnam, Andhra Pradesh. Tropical Plant Research 5(2): 167–179]
INTRODUCTION
Trees are the largest and perennial growth forms. A tree is defined as a woody plant that attains diameter of
10 cm or more at the breast height (130 cm above ground) (Reddy et al. 2007). Trees provide basic needs of
human beings in the form of air, food, timber, paper, fuel wood, medicine etc. (Sandhyarani et al. 2007, Bajpai
et al. 2015, Truyen et al. 2015, Borah et al. 2016, Dutta et al. 2016, Sarkar & Devi 2017). They are also
providing other basic products and drugs (Reddy et al. 2009, Mehra et al. 2014). Trees act as noise filters, air
purifiers; sequester carbon and pollutant traps (Pherson et al. 1997, Beckett et al. 2000, Chave et al. 2005,
Vivek P & Parthasarathy 2015). Trees of the concretized urban environment provide food and residence to many
of birds and animals (Fernandez-Zuricic 2000). Urban greening and urban forests are particularly important to
healthy cities in developing countries (Thaiutsa et al. 2008). Urban greenery that includes streets with trees,
parks, educational institutions play vital role in conservation of tree cover, environment as well. It can decrease
the urban island heat effect (Chow & Roth 2006). Results of tree inventory and assessment of urban
environment can be a useful for excellent urban planning and conservation of important tree species (Cy 2006).
Understanding of the distribution of tree species plays an important role in illuminate the larger patterns of
distribution of biodiversity. Trees are useful for analysis of species-area and species-individual relationships
because they are easy to locate precisely and to count. Tropical trees are especially interesting subjects because
of there is rich in species diversity (Condit et al. 1996). Documentation of existing green cover of the urban
environment is important to determine existing resources and to set target for future improvements (Miller
1996). Due to escalating urbanization, green space and urban trees become increasingly important in developing
countries (Cy 2006). Number of threatened species also existed in urban areas. Urbanization is one of the major
threats to the natural vegetation (Sodhi et al. 2010). Present study focused on exploration on tree diversity and
their distributional status in the Andhra University Campus urban landscape.

Study area
Andhra University was established in 1926 located between 17º 35' to 17º 40' N, 83º 20' to 83º 25' E, with an
elevation of 60 m in Visakhapatnam City with an area of 200 hectares (Fig. 1, 2). Campus has tropical humid
climate with an average annual temperature between 18ºC and 45ºC and an average rain fall of 1000–1200 mm.
Received: 26 February 2018 Published online: 30 June 2018
Rao 2018
Data collection
During the period of 2010–2016 author was observed tree diversity and their distribution in Andhra
University Campus. All taxa were identified up to species level with the help of floras and literature (Gamble &
Fischer 1915–1935, Venkateswaralu et al. 1972, Pullaiah, 1997, Pullaiah & Chennaiah 1997, Pullaiah &
Moulali 1997, Bose et al. 1998, Pullaiah & Sandhyarani 1999, Rao et al. 1999, Reddy & Reddy 2008, Subbarao
& Kumari 2008, Rao et al. 2010, Rao 2011, Rao et al. 2015, Rao 2016). Data such as botanical name, family,
distributional status and occurrence were provided, local (Telugu) names and common names also provided as
far as possible. All the plant species were arranged according to alphabetical order of their scientific names.
Nomenclature as far as possible has been made up-to date (www.plantlist.org) (Table 1). Some of the plant
photographs were provided for easy identification (Figs. 6–8).
Figure 1. Map of the Study area.
Figure 2. A, Route map of Andhra University Campus; B, Satellite (Google Earth) map of University North Campus; C,
Satellite (Google Earth) map of University South Campus.
RESULTS
A total of 175 tree species pertaining to132 genera under 53 families were recorded from the Andhra
University Campus (Table 1, Fig. 3). Among the 175 trees, 5 (2.85%) species i.e. Araucaria columnaris (Forst.)
Hk., Thuja orentalis L., Cycas circinalis L., Cycas revoluta Thunb. and Cycas sphaerica Roxb. are
gymnosperms and remaining 170 (97.14%) species are belonging to angiosperms. Among the angisperms 160
(91.42%) species are dicots and 10 (5.71%) species are monocots.
Ficus is the dominant genus with 6 (3.42%) species fallowed by 3 genera Euphorbia, Hibiscus and Citrus
each with 4 (2.28%) species, 6 genera Tabebuia, Terminalia, Cycas, Diospyros, Albizia and Ixora are with
3(1.71%) species 16 genera Annona, Alstonia, Plumeria, Bauhinia, Caesalpinia, Senna, Anogeissus, Cordia,
Phyllanthus, Dalbergia, Lagerstroemia, Ochna, Mussaenda, Murraya, Manilkara and Sterculia are with 2
(1.14%) species and remaining 106 genera are with single (0.57%) species were reported.
Among the 53 families Rubiaceae is the dominant family with 14 (8%) species fallowed by Caesalpiniaceae
12 (6.85%) species, 2 (3.77%) families Euphorbiaceae and Mimosaceae are with 11 (6.28%) species, Arecaceae
with 10 (5.71%) species, Moraceae with 9 (5.14%) species, 2 (3.77%) families Bignoniaceae and Fabaceae are
with 8 (4.57%) species, 2 (3.77%) families Apocynaceae and Rutaceae are with 7 (4%) species, 3 (5.66%)
families Combretaceae, Malvaceae and Myrtaceae are with 5 (2.85%) species, 3 (5.66%) families
Anacardiaceae, Lythraceae and Sapotaceae are with 4 (2.28%) species, 4 (7.54%) families Annonaceae,
Cycadaceae, Ebenaceae and Sterculiaceae are with 3 (1.71%) species, 6 (3.42%) families Bombacaceae,
Cordiaceae, Meliaceae, Ochnaceae, Sapindaceae and Verbenaceae are with 2 (1.14%) species and remaining 27
(50.94%) families are with single (0.57%) species were reported.
200
175
150
132
100
50 53
0
Species Genera Family
Figure 3. Details of species richness, genus and families of the study area.
Table 1. Trees inventory and their details in Andhra University campus. [D.St.- Distributional Status, Occ.- Occurrence; C- Common, O-
Occasional, R- Rare, NAT- Natural, CULT- Cultivated, AVN- Avenue, ORN- Ornamental, INT- Introduced in to the Botanical Garden]
S.No. Name of the plant Family Local (Telugu) Name Common Name D.St. Occ.
1 Acacia auriculiformis Benth. Mimosaceae Australia tumma Australian Oke C INT
2 Adenanthera pavonina L. Mimosaceae Bandi gurivinda Peacock flower fence R NAT
3 Aegle marmelos (L.) Correa Rutaceae Maaredu Bengal Quince R INT
4 Ailanthus excelsa Roxb. Simaroubaceae Peddamanu Indian Tree of Heaven R AVN
5 Alangium salvifolium (L. f.) Wangerin Alangiaceae Uduga Sage-leaved alangium R NAT
6 Albizia chinensis (Osbeck) Merr. Mimosaceae Chinduga Chinese albizia R NAT
7 Albizia lebbeck (L.) Willd. Mimosaceae Dirisena, Siresha puspam East Indian Walnut O NAT
8 Albizia saman (Jacq.) Merr. Mimosaceae Nidraganneru Rain Tree O NAT
9 Alstonia scholaris (L.) R.Br. Apocynaceae Edakula pala Devil tree, white O AVN
cheesewood
10 Alstonia venenata R.Br. Apocynaceae Chinna edakula pala Poison Devil Tree R INT
11 Anacardium occidentale L. Anacardiaceae Zeedimamidi Cashe Nut C CULT
12 Annona reticulata L. Annonaceae Rama phalam Bull's heart O CULT
13 Annona squamosa L. Annonaceae Seeta phalam Custard apple O CULT
14 Anogeissus acuminata (Roxb. ex DC.) Combretaceae Pasi chettu Button tree R INT
Guill. & Seneg.
15 Anogeissus latifolia (Roxb. ex DC.) Combretaceae Sirimanu Axlewood R NAT
Wall.
16 Araucaria columnaris (Forst.) Hk. Araucariaceae Christmas tree Monkey puzzle tree O ORN
17 Areca catechu L. Arecaceae Pokachekka Areca palm R ORN
Rao 2018
18 Artocarpus heterophyllus Lam. Moraceae Panasa Honey Jack tree O CULT

19 Azadirachta indica A. Juss. Meliaceae Vepa Neem R NAT
20 Bauhinia purpurea L. Caesalpiniaceae Devakanchanam Pink bauhinia C ORN
21 Bauhinia tomentosa L. Caesalpiniaceae yellow bauhinia R ORN
22 Bixa orellana L. Bixaceae Mitayirangu Lipstick tree R CULT
23 Bombax ceiba L. Bombacaceae Erra buruga Red silk cotton R NAT
24 Borassus flabellifer L. Arecaceae Tati chettu Palmyra Palm O NAT
25 Brownea coccinia Jacq. Caesalpiniaceae Rose of Venezuela R ORN
26 Butea monosperma (Lam.) Taub. Fabaceae Moduga Flame of the forest R ORN
27 Caesalpinia coriaria (Jacq.) Willd. Caesalpiniaceae Divi Divi American Sumach O NAT
28 Caesalpinia pulcherrima (L.) Caesalpiniaceae Chinna turayi Peacock Flower C ORN
29 Callistemon lanceolatus (Sm.) Sweet Myrtaceae Bottle brush Bottle brush R CULT
30 Calophyllum inophyllum L. Clusiaceae Ponna chettu Alexandrian laurel R ORN
31 Carica papaya L. Caricaceae Boppayi Papaya O CULT
32 Caryota urens L. Arecaceae Jeeluga Fish tail palm O ORN
33 Cascabela thevitia (L.) Lippold Apocynaceae Pachha ganneru Yellow oleander O ORN
34 Cassia fistula L. Caesalpiniaceae Reela Golden shower C ORN
35 Cassine glauca (Rottb.) O. Kuntze Celastraceae Nirija Ceylon Tea R NAT
36 Casuarina equisetifolia Forst. & Forst f. Casuarinaceae Sarugudu Sae oke R ORN
37 Ceiba pentandra (L.) Gaertn. Bombacaceae Pachha buruga White silk cotton R NAT
38 Citrus aurantifolia (Christm.) Rutaceae Nimma Limon R CULT
Swingle
39 Citrus aurantium L. Rutaceae Narinja Orange R CULT
40 Citrus limon (L.) Burm. Rutaceae Dabba Lemon R CULT
41 Citrus sinensis (L.) Osbeck Rutaceae Bathai Sweet orang R CULT
42 Cocos nucifera L. Arecaceae Kobbari, tenkaya Coconut plam O CULT
43 Cordia dichotoma Forest. Cordiaceae Nakkeri Earth cake tree O NAT
44 Cordia sebestena L. Cordiaceae Aloe Wood tree O ORN
45 Couroupita guinensis Aubl. Lecythidaceae Naga lingam Cannon ball tree R ORN
46 Crateva magna (Lour.) DC. Capparaceae Sacred garlic pear R NAT
47 Cycas circinalis L. Cycadaceae Fern palm R ORN
48 Cycas revoluta Thunb. Cycadaceae Madana kamashi Japanese sago palm R ORN
49 Cycas sphaerica Roxb. Cycadaceae Kodhada chettu R ORN
50 Dalbergia latifolia Roxb. Fabaceae Rose wood Rose wood R INT
51 Dalbergia sissoo Roxb. Fabaceae Sosso Sisso R INT
52 Delonix regia (Boj. ex Hook.) Rafin. Caesalpiniaceae Tureyi Gulmohar O AVN
53 Dichrostachys cinerea (L.) Wighth & Mimosaceae Veluturu Sickle Bush R NAT
Arn.
54 Diospyros chloroxylon Roxb. Ebenaceae Illenda O NAT
55 Diospyros ferrea (Willd.) Bakh. Ebenaceae Pisinika Black ebony O NAT
56 Diospyros malabarica (Desr.) Kostel. Ebenaceae Neeti tumiki Malabar ebony R INT
57 Dypsis lutescens (H.Wendl.) Beentje Arecaceae Bamboo palm R ORN
& J.Dransf.
58 Elaesis guuneensis Jacq. Arecaceae Palm oil chettu Oil palm R INT
59 Enterolobium timbouva Mart. Mimosaceae Earpod tree R AVN
60 Erioglossum rubiginosum Bl. Sapindaceae Rusty sapindus R INT
61 Erythrina variegata L. Fabaceae Badita Indian Coral tree R NAT
62 Erythroxylum monogynum Roxb. Erythroxylaceae Devadari Bastard Sandal, Red R NAT
cedar
63 Eucalyptus globulus Labill. Myrtaceae Jamail Southern blue-gum C CULT
64 Euphorbia antiqiorum L. Euphorbiaceae Jemudu Antique Spurge R ORN
65 Euphorbia caducifolia Haines. Euphorbiaceae Katte jemudu Leafless Milk Hedge R ORN
66 Euphorbia pulcherrima Willd. ex Euphorbiaceae Christmas Star O ORN
Klotzsch
67 Euphorbia tirucalli L. Euphorbiaceae Katimandu Pencil cactus R ORN
68 Ficus amplissima Sm. Moraceae Jeechettu Bat tree O NAT
69 Ficus benghalensis L. Moraceae Marri Indian Fig tree O NAT
70 Ficus benjamina L. Moraceae Weeping fig C ORN
71 Ficus elastica Roxb. Moraceae Rabbaru chettu Indian Rubber R ORN
72 Ficus hispida L. f. Moraceae Bodda marri Wild Fig O NAT
73 Ficus racemosa L. Moraceae Cluster fig tree C NAT
74 Ficus religiosa L. Moraceae Raavi Wisdom tree C NAT
75 Flacourtia indica (Burm.f.) Merr. Flacourtiaceae Kanavegu chettu Indian plum R NAT
76 Flueggea virosa (Roxb. ex Willd.) Royle Euphorbiaceae Bushweed C NAT
77 Gardenia latifolia Ait. Rubiaceae Bikki Papra R ORN
78 Gliricidia sepium (Jacq.) Kunth ex Walp. Fabaceae Madri Quickstick C ORN
79 Grevillea robusta A. Cunn. ex R.Br. Proteaceae Silver oke Silver Oke R INT
80 Guaiacum officinale L. Zygophyllaceae Guaiacwood R ORN
81 Guazuma ulmifolia Lam. Sterculiaceae Bastard Ceder R INT
82 Haldinia cordifolia (Roxb.) Ridsd. Rubiaceae Kamba Haldu R INT
83 Hamelia patens Jacq. Rubiaceae Hamelia C ORN
84 Hibiscus mutabilis L. Malvaceae Muddamandara Cotton rosemallow R ORN
85 Hibiscus rosa-sinensis L. Malvaceae Mandara China rose C ORN
86 Hibiscus schizopetalus (Dyer) Hook. f. Malvaceae Coral hibiscus O ORN
87 Hibiscus tiliaceus L. Malvaceae Etagogu Coast cotton tree R ORN
88 Hymenodictyon orixense (Roxb.) Rubiaceae Dudippa Bridal Couch, R INT
Mabb. bandaaru-chettu
89 Ixora coccinia L. Rubiaceae Rama banum flame of the woods C ORN
90 Ixora finlaysoniana Wall. ex G.Don Rubiaceae Tellaguttupulu Fragrant Ixora, White O ORN
Siamese Ixora
91 Ixora pavetta Andrews Rubiaceae Korivi Torch tree O NAT
92 Jacaranda mimosifolia D. Bignoniaceae Jacaranda O AVN
93 Jacquinia ruscifolia Jacq. Theophrastaceae Jaqinia R ORN
94 Jatropha curcas L. Euphorbiaceae Adavi amudam, Pedda Barbados nut R INT
nepalam
95 Kigelia africana (Lam.) Benth. Bignoniaceae Enugu kaaya Susage tree O AVN
96 Lagerstroemia indica L. Lythraceae Chinna gogu Bonnet flower R ORN
97 Lagerstroemia speciosa (L.) Pers. Lythraceae Varagogu Queen's pride R ORN
98 Lannea coromandelica (Houtt.) Merr. Anacardiaceae Gumpina Indian ash tree O NAT
99 Lawsonia inermis L. Lythraceae Gorinta Henna plant R ORN
100 Leucaena leucocephala (Lam.) de Wit Mimosaceae Chandra chettu Subabul C NAT
101 Licuala grandis Wndl. Arecaceae Ruffled Fan Palm O ORN
102 Litsea deccanensis Gamble Lauraceae Naaramamidi Deccan Tallow Laurel R INT
103 Madhuca indica J. Gmelin Sapotaceae Ippa Indian Butter tree R NAT
104 Magnolia champaca (L.) Baill. ex Magnoliaceae Chettu champanga Champak O ORN
Pierre
105 Mangifera indica L. Anacardiaceae Mamidi Mango C NAT
106 Manihot glaziovii Müll. Arg. Euphorbiaceae Rabbaru chettu Ceara rubber tree R INT
107 Manilkara hexandra (Roxb.) Dubard Sapotaceae Pala Ceylon Iron wood R NAT
108 Manilkara zapota (L.) P. Royen Sapotaceae Sapota Sapodilla R CULT
109 Melaleuca bracteata F. Muell. Myrtaceae Black tea-tree, River O ORN
tea-tree
110 Memecylon edule Roxb. Melastomaceae Alli Kaayam, Delek R ORN
bangas
111 Millingtonia hortensis L. f. Bignoniaceae Kada malle Cork tree R AVN
112 Mimusops elengi L. Sapotaceae Pogadu Spanish cherry C AVN
113 Mitragyna parviflora (Roxb.) Korth. Rubiaceae Neer kadamba Water cadamba R NAT
114 Morinda coreia Buch.-Ham. Rubiaceae Togaru Tohari wood O NAT
115 Moringa oleifera Lam. Moringaceae Munaga Drum Stick R CULT
116 Muntingia calabura L. Elaeocarpaceae BP paluu Chinese Cherry O AVN
117 Murraya koenigii (L.) Spreng. Rutaceae Karivepaku Curry leaf R NAT
118 Murraya paniculata (L.) Jack Rutaceae Puvelaga Orange Jasmine R ORN
119 Mussaenda erythrophylla Schum. & Rubiaceae Red Flag Bush O ORN
Thonn.
120 Mussaenda frondosa L. Rubiaceae Dhobi tree O ORN
121 Neolamarckia cadamba (Roxb.) Rubiaceae Kadamba Kadam R AVN
Bosser
122 Nerium oleander L. Apocynaceae Erra ganneru Oleander C ORN
123 Nyctanthes arbor-tristis L. Oleaceae Parijatam Night-flowering R ORN
Jasmine
124 Ochna obtusata DC. Ochnaceae Ramdhan Champa, R NAT
Mickey-mouse
125 Ochna squarrosa L. Ochnaceae Bird's-eye bush O ORN
126 Parkia biglandulosa Wight & Arn. Mimosaceae Tennis ball Badminton Ball Tree R AVN
Rao 2018
127 Peltophorum pterocarpum (DC.) Caesalpiniaceae Coffee pod Rusty Shield Bearer C AVN
Barker ex Heyne
128 Phoenix sylvestris (L.) Roxb. Arecaceae Eeta Wield date palm C NAT
129 Phyllanthus acidus (L.) Skeels Euphorbiaceae Racha Usiri Star gooseberry O CULT
130 Phyllanthus emblica L. Euphorbiaceae Usiri Indian Goose Berry O CULT
131 Pisonia grandis R.Br. Nyctaginaceae Aravapappu kura Lettuce tree C ORN
132 Pithecellobium dulce (Roxb.) Benth. Mimosaceae Chema chinta Manilla tamarind O NAT
133 Plumeria alba L. Apocynaceae Nuru vahalu Temple tree O ORN
134 Plumeria rubra L. Apocynaceae Deva ganneru Temple tree O ORN
135 Polyalthia longifolia (Sonner) Thw. Annonaceae Naramamidi, Asoka Ashoka C ORN
136 Pongamia pinnata (L.) Pierre Fabaceae Ganuga/Kamu Indian Beech tree C AVN
137 Posoqueria latifolia Aubl. Rubiaceae Monkey apple R ORN
138 Premna mollissima Roth Verbenaceae Nelli Dusky leaved fire R NAT
brand
139 Prichardia pacifica Seem. & Wendl. Arecaceae Fiji Fan Palm O ORN
140 Prosopis chilensis (Molina) Stuntz Mimosaceae Tella tumma, Kanche Chilean mesquite C NAT
tumma
141 Psidium guajava L. Myrtaceae Jama Guava R CULT
142 Pterocarpus santalinus L. f. Fabaceae Erra chandanam Red sandal C INT
143 Punica granatum L. Lythraceae Danimma Pomegranate R CULT
144 Putranjiva roxburghii Wall. Euphorbiaceae Puttaranjivi Lucky Bean Tree O INT
145 Ravenala madagascariensis Sonn. Streliziaceae Travellar palm Travellar palm R ORN
146 Ricinus communis L. Euphorbiaceae Amudam Caster C NAT
147 Roystonea regia (H.B. & K.) O.F. Arecaceae Bojja chettu Royal palm O ORN
Cook
148 Santalum album L. Santalaceae Chandanam, Gandam Sandal wood C INT
149 Sapindus emarginatus Vahl. Sapindaceae Konkudu, Ritta kaya Soap nut C NAT
150 Saraca asoca (Roxb.) de Wilde Caesalpiniaceae Ashoka chettu Ashoka Tree R INT
151 Semecarpus anacardium L. Anacardiaceae Nallazeedi Marking nut R INT
152 Senna siamea (Lam.) H.S. Irwin & Caesalpiniaceae Chetuu tangedu Kassod tree C AVN
Barneby
153 Senna spectabilis (DC.) H.S. Irwin & Caesalpiniaceae Yellow cassia O ORN
Barneby
154 Sesbania sesban (L.) Fabaceae Avisa Egyptian riverhemp R INT
155 Shorea robusta Gaertn. f. Dipterocarpaceae Guggilam Sal R INT
156 Spathodea campanulata P. Beauv. Bignoniaceae Tulip African tulip O AVN
157 Sterculia foetida L. Sterculiaceae Seema badam Jangle Badam O INT
158 Sterculia urens Roxb. Sterculiaceae Kovela, Tapasi Gum karaya R INT
159 Streblus asper Lour. Moraceae Barnika Sand Paper Tree, R NAT
Toothbrush tree
160 Switenia mahagoni (L.) Jacq. Meliaceae Mahagani mahogany R AVN
161 Syzygium cumini (L.) Skeels Myrtaceae Neredu Indian Cherry C AVN
162 Tabebuia argentea (But. & K. Schum) Bignoniaceae Silver Trumpet Tree O AVN
Britt.
163 Tabebuia aurea (Silva Manso) Benth. Bignoniaceae Caribbean trumpet O AVN
& Hook.f. ex S.Moore tree
164 Tabebuia rosea (Bertol.) DC. Bignoniaceae Rosy trumpet tree O AVN
165 Tamarindus indica L. Caesalpiniaceae Chinta Tamarind C NAT
166 Tecoma stans (L.) Kunth Bignoniaceae Swarna ganneru Yellow trumpet flower C ORN
167 Tectona grandis L. f. Verbenaceae Teku Teak C CULT
168 Terminalia arjuna (Roxb. DC.) Combretaceae Tella maddi Arjun tree O AVN
Wight. & Arn.
169 Terminalia bellirica (Gaertn.) Roxb. Combretaceae Tanikaya Beleric R AVN
170 Terminalia catappa L. Combretaceae Badam Indian Almond C AVN
171 Thespesia populnea (L.) Soland. ex Malvaceae Ganga raavi India tulip tree O AVN
Correa
172 Thuja orentalis L. Cupressaceae Tree of life C ORN
173 Wendlandia heynei (Roem. & Schult.) Rubiaceae Tellapucu Tilki R INT
Santapau & Merchant
174 Wrightia tinctoria R.Br. Apocynaceae Ankudu Ivory wood C NAT
175 Ziziphus mauritiana Lam. Rhmnaceae Regu Chinese date O NAT
As per number of individuals and distributional status, all trees are classified into 3 categories namely
common, occasional and rare. Among the 175 tree species in the University Campus, 36 (20.57%) species are in
common, 53 (30.28%) species are in occasional and 86 (49.14%) species are in rare (Fig. 4).
As per occurrence of the trees in the University Campus all trees are classified as natural (NAT), cultivated
(CULT), avenue (AVN), ornamental (ORN) and introduced in to the Botanical Garden (INT). The highest
numbers of trees 59 (33.71%) species are growing as ornamental trees, followed by natural 46 (26.28%) species,
introduced 26 (14.85%) species, avenue 24 (13.71%) species and 20 (11.42%) species are cultivated species
(Fig. 5).
Common
21%
Occassional
30%
Rare
49%
Figure 4. Distributional status of Trees in the University Campus.
70
59
60
50 46
40
30 24 26
20
20
10
0
NAT ORN AVN INT CULT
Figure 5. Details of occurrence of the trees in the University Campus. [NAT- Natural, CULT- Cultivated, AVN- Avenue,
ORN- Ornamental, INT- Introduced in to the Botanical Garden]
Cycas circinalis L., Dalbergia latifolia Roxb., Pterocarpus santalinus L.f., Saraca asoca (Roxb.) de Wilde,
Aegle marmelos (L.) Correa, Sterculia urens Roxb. and Ochna obtusata DC. trees are red listed medicinal plants
in Andhra Pradesh (Reddy & Reddy 2008, Rao & Rao 2014). While, Cycas sphaerica and Pterocarpus
santalinus are endemic tress endemic to Eastern Ghats and endemic to Andhra Pradesh respectively (Rao et al.
2014).
Due to the effect of massive cyclone Hud hud with wind speeds 175 km/h on 12 th October 2014
(https://en.wikipedia.org/wiki/Cyclone_Hudhud) several trees were uprooted in the University Campus.
Colvillea racemosa Bojer, Commiphora caudata (Wight & Arn.) Engl., Strychnos nux-vomica L., Limonia
acidissima Groff, and Capparis grandis L. f. trees were disappeared from the Campus and Borassus flabellifer
L., Peltophorum pterocarpum (DC.) Barker ex Heyne trees were decreased in their number of individuals.
Syzygium cumini (L.) Skeels, Mangifera indica L., Tamarindus indica L., Pterocarpus santalinus L. f. have lost
their canopy but now recovered. After cyclone, University authorities planted several trees in the Campus,
among them Spondias pinnata (L. f.) Kurz and Acrocarpus fraxinifolius Arn., are newly introduced species and
now they are growing well.
Rao 2018
Figure 6. A, Ailanthus excelsa Roxb.; B, Anogeissus acuminata (Roxb. ex DC.) Guill. & Seneg.; C, Artocarpus
heterophyllus Lam.; D, Bombax ceiba L.; E, Butea monosperma (Lam.) Taub.; F, Cassia fistula L.; G, Ceiba pentandra (L.)
Gaertn.; H, Couroupita guinensis Aubl.
Figure 7. A, Cycas circinalis L.; B, Diospyros chloroxylon Roxb.; C, Erioglossum rubiginosum Bl.; D, Ficus religiosa L.;
E, Hymenodictyon orixense (Roxb.) Mabb.; F, Mitragyna parviflora (Roxb.) Korth.; G, Pterocarpus santalinus L. f.; H,
Santalum album L.
Rao 2018
Figure 8. A, Sapindus emarginatus Vahl.; B, Saraca asoca (Roxb.) de Wilde; C, Shorea robusta Gaertn. f.; D, Sterculia
foetida L.; E, Sterculia urens Roxb.; F, Terminalia arjuna (Roxb. DC.) Wight. & Arn.; G, Terminalia bellirica (Gaertn.)
Roxb.; H, Wrightia tinctoria R.Br.
DISCUSSION
A total of 73 trees species reported from Pondicherry University Campus (Parthasarathy 2010); 109 tree
species reported from Banaras Hindu University campus (Singh 2015); 39 trees reported from Adikavi Nannaya
University Campus (Rao 2016); 448 species reported from Ramachandrapuram mandala included 97 tree
species (Kumar et al. 2015a). The present research reports 175 tree species from Andhra University campus
revealed that the campus has rich plant diversity. University flora resembles to the nearest forest Kambalakonda
Wildlife Sanctuary in the Visakhapatnam city in their wild species and comparatively University has rich tree
diversity than the sanctuary with 175 tree species and 73 tree species respectively (Naidu et al. 2012) because
the campus has a good number of cultivated, avenue, ornamental and introduced tree species.
Present study also supports that Ficus is the predominant genus in India with other floristic studies (Gadgil et
al. 1996, Sandhyarani et al. 2007, Rasingam & Parathasarathy 2009, Kumar et al. 2015b). Presence of the
Rubiaceae, Caesalpiniaceae, Euphorbiaceae, Mimosaceae, Arecaceae, Moraceae, Bignoniaceae, Fabaceae,
Apocynaceae and Rutaceae have made the University Campus seems to be a forest ecosystem (Gadgil et al.
1996, Kudavul & Parthasarathy 1999, Chittibabu & Parthasarathy 2000, Sandhyarani et al. 2007, Pitchairamu
et al. 2008, Selvamony et al. 2008, Reddy et al. 2009, Sahu et al. 2010).
CONCLUSIONS
It is healthy sign occurrence of 175 tree species in University Campus but it is necessary to enhance the flora
of with more number of native species, emphasizing more on endemic, red list and medicinal plants. This
activity will amplify ex-situ conservation of these species and also spark awareness among students on
biodiversity conservation. I also suggest in-vitro propagation of red list species like Pterocarpus santalinus L.f.
and Saraca asoca (Roxb.) de Wilde), Aegle marmelos (L.) Correa and Sterculia urens Roxb. which are already
existing in the University Campus. The present strongly supports that, the introduction of native trees into the
urban landscape.
ACKNOWLEDGEMENTS
Author is thankful to University authorities’ and also grateful to Botany Department staff members and his
friends for their great encouragement during the studies.
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Research article
Exploring the potential of some phytodyes as histological stains

in wood anatomy
A.J. Akinloye* and H.C. Illoh
Botany Department, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria
*Corresponding Author: akinloye_johnson@yahoo.com [Accepted: 07 July 2018]
Abstract: The potential of some phytodyes were examined to obtain cheap, non- toxic and eco-
friendly stains for use in wood anatomy. Phytodyes from Enantia cholorantha, Harungana
madagascariensis, Hisbiscus sabdariffa, Sarcocephalus latifolius, Sphenocentrum jollyanum, and
Sorghum bicolor were used to stain wood sections and macerates. All the extracts had good
affinity for wood fibre and other lignified tissues except Hibiscus sabdariffa. The results of the
absorption spectrum of these dyes revealed a range of wavelength of absorption between 300.00
and 900.00 nm. These wavelengths fall within the visible region of the electromagnetic spectrum
confirming the presence of colour imparting chromophores in the dyes. Each of the phytodye has
minimum of two peaks, indicating that each dye had a minimum of two colour imparting
chromophores. All the dyes were acidic with pH range of 2.80 to 5.90. The histochemical reactions
of all the phytodyes were similar in that they all imparted their colours indiscriminately on all cells
but fibre and lignified tissues took up the dyes more deeply. They were specific when used with
Alican blue which have affinity for thin walled cells. Therefore, this study revealed that these dyes
could be used solitarily or in combination with artificial dyes for wood histological staining.
Keywords: Phytodye - Non-toxic - Wood anatomy - Macerate - Vessel element - Fibre.
[Cite as: Akinloye AJ & Illoh HC (2018) Exploring the potential of some phytodyes as histological stains in
wood anatomy. Tropical Plant Research 5(2): 180–192]
INTRODUCTION
The use of colourants dates back thousands of years in all societies around the globe. Even before people
began to spin yarn and weave cloth, they applied coloured earth, plant saps and juices directly to their skin; this
was the first type of cosmetic (Maria 2009). In Nigeria, and among Yorubas, dyes from the heartwood of
Pterocarpus osun Craib had been in use since time immemorial, as cosmetics, for painting human body during
war and as medicine for treating skin diseases (Akinloye et al. 2010). The indigo colour from the young leaf of
Lonchocarpus cyanescens (Schum & Thorn) Benth is used among Yoruba tribe of Nigeria to make natural
textile material (adire), to paint houses floors and walls, as well as to colour young chicken so as to protect them
from predators (such as hawk etc). Many other plant dyes were known among Yorubas and used for various
purposes (Akinloye et al. 2010). Among the Hausas tribe in Nigeria, dyes from the leaf of Lawsonia inermis L.,
leaf sheath of Sorghum bicolor Linn and many other herbal dyes have been in use as cosmetics, medicine,
curing of animal skin (leather), differentiating of animal and as household dyes. The use of vegetable dyes in
India dated back to the Indua period between the 2 th and the 4nd millennium BC. The historical record of indigo
is patchy but references were records made by Marco Polo who saw indigo at present day Quilon in the state of
Kerla in 1298 (Maria 2009). Since the use of natural Phytodyes does not cause health hazard nor environmental
pollution, it is of immense importance to explore these sources of dye for use in plant histology. In Nigeria,
there are few workers that have examined the potentials of Phytodyes for use in histopathology (Eom et al.
2001, Avwioro et al. 2005a, b), in plant histology (Akinloye et al. 2010).
The extraction of novel natural dyes from plants is in its infancy in Nigeria. Nigeria can play a vital role in
this field for the development of natural herbal dyes on account of its varied climate and rich flora forest.
Therefore, the present study investigated the potentials of six Phytodyes for use in Plant histology.
The following plants have been carefully selected for the extraction of Phytodyes. The selection was based
Received: 18 February 2018 Published online: 31 July 2018
Akinloye & Illoh 2018
on availability and the presence of dye in part of the plants as indicated below,
1. Enantia chlorantha Oliv (stem bark)
2. Harrungana madagascariensis Lam. ex Poir (stem bark)
3. Hibiscus sabdariffa L. (flower calyx)
4. Sarcocephalus latifolius (S.M) Bruce (root)
5. Sphenocentrum jollyaum Piers (root)
6. Sorghum bicolor L. (leaf sheath)

Collection of Plant Materials for Pyhtodyes Extracts
The six plants used were collected in various locations (Table 1).
Table 1. Collection sites and voucher number of the plant species used for phytodyes.
Voucher
Species Collection Site GPS Location Collector
number
Enantia chloratha PSP Akure Forest Reserve, + 7′ 79′ 0′ N IFE 17408 Akinloye A. J.
Akure, Ondo State, Nigeria + 5′ 1′ 60′ E
Harugana madagascariensis Along Road 7, Opposite + 7′ 31′ 10.68′ N IFE 17409 Akinloye A. J.
International School, Senior + 4′ 32′ 12.35′ E
Staff Quarters, Obafemi
Awolowo University, Ile-
Ife, Osun State, Nigeria
Hibiscus sabdariffa Offa, along the way to Ipee, + 8′ 9′ 11′ N IFE 17410 Akinloye A. J.
Kwara State, Nigeria. + 4′ 39′ 5.61′ E
Sarcocephalus latifolius General Hospital Road, + 6′ 43′ 5.53′ N IFE 17242 Akinloye A. J.
Uromi, Edo State, Nigeria; + 6′ 19′ 9.29′ N
and
Along Ife-Ibadan Express + 7′ 22′ 21.47′ N
way, Ikire, Osun State, + 4′ 10′ 20.66′ E
Nigeria.
Sphenocentrum jollyanum Botanical Garden, Obafemi + 7′ 31′ 20.47′ N IFE 17411 Akinloye A. J.
Awolowo University, Ile- + 4′ 31′ 21.79′ E
Ife, Osun State, Nigeria
Sorghum bicolor Ipee, via Offa, Kwara State, + 8′ 9′ 13.73′ N IFE 17412 Akinloye A. J.
Nigeria. + 4′ 40′ 5.62′E
Extraction of the Dyes
Dyes were extracted from different parts of the selected six-plant species using Sohxlet extractor with
ethanol as solvent according to (Akinloye et al. 2010). The vegetative part of each plant species used for dye
extraction was collected and dried to reduce the moisture content and then pulverized. One-kilogram (1 kg)
powder of plant material from each species was used. The extraction was continued until all the dyes were
extracted from the sample.
Spectrophotometery
Each of the extracted dyes was concentrated to obtain the solid dye. The 1 mg of each sample was dissolved
in 10 mL of absolute ethanol while 10 mL of absolute ethanol served as blank. Each of the dye samples was run
on UV/ visible spectrophotometers, which automatically subtracted the effect of the blank and plotted the graph
of absorbance against wavelength to determine the number of colour imparting chromophore in each dye
(Popoola et. al. 1994, Akinloye et al. 2010).
Determination of pH of the Phytodyes
The pH of each extracted dye was taken using digital Mettler Toledo pH meter (Akinloye et al. 2010)
Staining
Wood sections and the macerates were stained in each of the extracted dyes for 5 minutes, rinsed in water,
dehydrated and differentiated in series of grades of ethanol (50%, 70%, 80%, 90% and 100%). The stained
sections and macerates were mounted in DPX mountant on clean slides and appropriately labelled. Another set
of wood sections were counter stained with Alcian blue (Akinloye et al. 2010)
Microscopy
Microscopical observations of the prepared slides of the wood sections and macerates were made at 10x, 40x
and 100x objective using LEICA DM500 binocular light microscope. The action of the herbal dyes on the wood
sections and macerates were recorded.
Photomicrography
Photomicrographs of the slides showing effects of the Phytodyes on the wood tissues were taken using
Accu-scope trinocular microscope (ACCU-scope 33001 LED Trinocular microscope with 3.2 MP CMOS digital
camera).
RESULTS
Spectrophotometery
All the extracted dyes and conventional dyes have multiple wavelengths with corresponding absorbance and
multiple peaks. The recorded wavelengths of the dye extracts and the conventional stains fall within visible
region of the electromagnetic spectrum and this shows the presence of colour imparting chromophore in the dye
extracts (Figs. 1–9; Table 2).
No. P/V Wavelength (nm) Abs
1 Peak 762.00 0.042
2 Peak 670.00 0.145
3 Peak 603.00 0.263
4 Peak 330.00 0.324
Figure 1. Electronic absorption on Alcian blue.

1 Peak 810.00 0.008
2 Peak 532.00 1.467
3 Peak 343.00 0.009
Figure 2. Electronic absorption on Safranin O.
Electronic absorption spectrum of conventional stains-Alcian blue and Safranin O are as shown in (Figs. 1 &
2). All the phytodyes used have minimum of two peaks. Hibiscus sabdariffa (Fig. 5) and Sarcocephalus
latifolius (Fig. 6) had two peaks; Harungana madagascariensis (Fig. 4) and Sorghum bicolor (Fig. 7) had three
peaks; Enantia chlorantha (Fig. 3) had four peaks: and Sphenocentrum jollyanum (Fig. 8) had five peaks. The
number of peaks corresponds to the number of chromophores in each phytodyes. The quality or sharpness of the
colour of the extract depends on the narrowness or broadness of the peak. Narrow peak tends to have sharp and
1 Peak 778.00 0.005
2 Peak 729.00 0.006
3 Peak 423.00 0.188
4 Peak 339.00 0.890
Figure 3. Electronic absorption on Enantia chlorantha.

1 Peak 764.00 0.022
2 Peak 387.00 1.028
3 Peak 330.00 0.973
Figure 4. Electronic absorption on Harungana madagascariensis.

1 Peak 764.00 0.022
2 Peak 387.00 1.028
3 Peak 330.00 0.973
Figure 5. Electronic absorption on Hibiscus sabdariffa.

1 Peak 448.00 0.166
2 Peak 330.00 0.338
Figure 6. Electronic absorption on Sarcocephalus latifolus.

1 Peak 739.00 0.039
2 Peak 465.00 0.390
3 Peak 330.00 0.849
Figure 7. Electronic absorption on Sorghum bicolor.

1 Peak 768.00 0.006
2 Peak 709.00 0.007
3 Peak 653.00 0.010
4 Peak 405.00 0.082
5 Peak 335.00 0.323
Figure 8. Electronic absorption on Sphenocentrum jollyanum.

VIOLET - Sphenocentrum jollyanum

LIGHT GREEN - Enantia chlorantha
BLACK - Hibiscus sabdariffa
YELLOW - Harungana madagascariensis
DEEP GREEN - Sarcocephalus latifolius
BROWN - Sorghum bicolor
RED - Safranin O
BLUE - Alcian blue
Figure 9. Electronic absorption on all the dyes used.

bright colouration while the broad peak tends to have dull colouration. Each of the Phytodye had one or more
narrow peaks so this made their colouration to be bright. The combination of all the electronic absorption
spectra of all the dyes used is as shown in figure 9.
Table 2. Electronic absorption spectra of the crude dye extracts and two exotic dyes used.
Dye Source Wavelength Absorbance
Enartia chlorantha 778.00 -0.005
729.00 -0.006
423.00 0.188
339.00 0.890
Harungana madagascariensis 764.00 0.022
387.00 1.028
330.00 0.972
Hibiscus sabdariffa 367.00 0.094
330.00 0.146
Sarcocephalus latifolius 448.00 0.166
330.00 0.338
Sorghum bicolor 739.00 0.039
465.00 0.390
330.00 0.849
Sphenocentrum jollyanum 768.00 0.006
709.00 0.007
653.00 0.010
405.00 0.082
335.00 0.323
Alcian blue 762.00 0.042
670.00 0.145
603.00 0.263
330.00 0.324
Safranin O 810.00 -0.008
532.00 1.467
343.00 -0.009
The action of Phytodyes on wood tissues
1. Enartia chlorantha: Dyes from Enantia chlorantha crystalized easily with brillant orange colour and
indiscriminately imparted its colour on all cells but fibres and other thick-walled cells took the stain more
deeply. It is a good stain for wood anatomy (Figs. 10 to 13).
Figure 10. Single staining wood transverse sections T.S. with Phytodyes (X400): A, Diospyros dendo Welw. ex Hiern T.S.
wood stained with Sorghum bicolor; B, D. dendo T.S. wood stained with Enantia chlorantha; C, D. dendo T.S. wood stained
with Hisbiscus sabdariffa; D, D. dendo T.S wood stained with Harungana madagascariensis; E, D. dendo T.S. wood stained
with Sphenocentrum jollyanum; F, D. dendo T.S. wood stained with Sarcocephalus latifolius. [SV= Solitary vessel, PM=
Pore multiple, F= Fibre]
Figure 11. Single staining wood tangential transverse sections TLS with Phytodyes (X400): A, Diospyros physocalycina
Gürke TLS wood stained with Sorghum bicolor; B, D. physocalycina TLS wood stained with Sphenocentrum jollyanum; C,
D. physocalycina TLS wood stained with Sarcocephalus latifolius; D, D. physocalycina TLS wood stained with Hisbiscus
sabdariffa; E, D. physocalycina TLS wood stained with Harungana madagascariensis; F, D. physocalycina TLS wood with
Enantia chlorantha. [F= Fibre, R= Ray, VE= Vessel element]
Figure 12. Staining wood radial longitudinal sections (RLS) of Diospyros iturensis (Gürke) Letouzey & F. White with
Phytodyes (X400): A, Stained with Enantia chlorantha; B, Stained with Harungana madagascariensis; C, Stained with
Hisbiscus sabdariffa; D, Stained with Sorghum bicolor; E, Stained with Sphenocentrum jollyanum; F, Stained with
Sarcocephalus latifolius. [F= Fibre, R= Ray]
Figure 13. Wood macerates of Diospyros piscatoria Gürke (400x): A, Stained with Hibiscus sabdariffa showing anatomical
features of fibre and vessel element with two tails; B, Stained with Harungana madagascariensis showing anatomical
features of vessel element with no tail; C, Stained with Enantia chlorantha showing anatomical features of fibre and vessel
element with one tail; D, Stained with Sphenocentrum jollyanum showing anatomical features of fibre and vessel element
with two tails; E, Stained with Sorghum bicolor showing anatomical features of fibre and vessel element with one tail; F,
Stained with Sarcocephalus latifolius showing anatomical vessel element with one tail. [F= Fibre, VE= Vessel Elements]
Figure 14. Counter staining extracted Phytodyes with Alcian blue (400x): A, Diospyros barteri Hiern T.S. wood stained
with dye extract from Enantia chlorantha and counter stained with Alcian blue; B, D. barteri T.S. wood stained with dye
extract from Harungana madagascariensis and counter stained with Alcian blue; C, D. barteri T.S. wood stained with dye
extract from Hibiscus sabdariffa and counter stained with Alcian blue; D, D. barteri T.S. wood stained with dye extract from
Sarcocephalus latifolius and counter stained with Alcian blue; E, D. barteri T.S. wood stained with dye extract from
Sphenocentrum jolltanum and counter stained with Alcina blue; F, D. barteri T.S. wood stained with dye extract from
Sorghum bicolor and counter stained with Alcian blue. [FE= Fibre took up yellow colour of Enantia chlorantha, FHA=
Fibre took up yellow colour of Harungana madagascariensis, FH= Fibre took up yellow colour of Hibiscus sabdariffa, FA=
Fibre took up yellow colour of Sarcocephalus latifolius, FSP= Fibre took up yellow colour of Sphenocentrum jollyanum,
FSO= Fibre took up wine colour of Sorghum bicolor, PA= Parenchyma cell took up blue colour of Alcian blue]
2. Harungana madagascariensis: Dyes from Harungana madagascariensis crystalized easily and imparted its
yellow colour on cells but fibres and other thick wall cells took the stain more deeply. It is a good stain for
wood anatomy (Figs. 10 to 13).
3. Hibiscus sabdariffa: Dye from Hibiscus sabdariffa form slurry and does not crystalized but rather formed
slurry and is highly soluble in water. It turned black in contact with water so it washed off easily when the
sections were rinsed in water; the dye retained by the cells appeared faintly yellow. It is also good for wood
anatomy but the other Phytodyes are better (Figs. 10 to 13).
Figure 15. Counter staining extracted Phytodyes with Alcian blue (400x): A, Diospyros suaveolens Gürke T.L.S. wood
stained with dye extract from Enantia chlorantha and counter stained with Alcian blue; B, D. suaveolens T.L.S. wood
stained with dye extract from Harungana madagascariensis and counter stained with Alcian blue; C, D. suaveolens T.L.S.
wood stained with dye extract from Hibiscus sabdariffa and counter stained with Alcian blue; D, D. suaveolens T.L.S. wood
stained with dye extract from Sarcocephalus latifolius and counter stained with Alcian blue; E, D. suaveolens T.L.S. wood
stained with dye extract from Sphenocentrum jollyanum and counter stained with Alcian blue; F, D. suaveolens T.L.S. wood
stained with dye extract from Sorghum bicolor and counter stained with Alcian blue. [FE= Fibre took up yellow colour of
Enantia chlorantha, FHA= Fibre took up yellow colour of Harungana madagascariensis, FH= Fibre took up yellow colour
of Hibiscus sabdariffa, FA= Fibre took up yellow colour of Sarcocephalus latifolius, FSP= Fibre took up yellow colour of
Sphenocentrum jollyanum, FSO= Fibre took up wine colour of Sorghum bicolor, PA= Parenchyma cell took up blue colour
of Alcian blue]
Figure 16. Counter staining Safranin O with Alcian blue (X400): A, Diospyros suaveolens Gürke T.S. wood stained with
Safranin O and Alcian blue; B, D. suaveolens T.S. wood stained with Safranin O and Alcian blue; C, D. suaveolens T.L.S.
wood stained with Safranin O and Alcian blue; D, D. suaveolens T.L.S. wood stained with Safranin O and Alcian blue; E, D.
suaveolens T.L.S. wood stained with Safranin O and Alcian blue; F, D. suaveolens T.L.S. wood stained with Safranin O and
Alcian blue. [FSO= Fibre took the yellow colour of Safranin O stain, PRA= Parenchyma and Ray cells took the blue colour
of Alcian blue stain]
4. Sarcocephalus latifolius: Dye from Sarcocephalus latifolius crystalized easily and imparted its brilliant
yellow colour on all cells but fibre and thick-walled cells took the stain more deeply. It is a good stain for
wood anatomy (Figs. 10 to 13).
5. Sphenocentrum jollyanum: Dye from Sphenocentrum jollyanum crystalized easily and imparted its brilliant
yellow colour on all cells, however, fibre and other thick-walled cells took the stain more deeply. It is a good
stain for wood anatomy (Figs. 10 to 13).
6. Sorghum bicolour: Dye from Sorghum bicolour crystalized easily and imparted its brilliant wine colour on
all cells but fibre and other thick-walled cells took the stain more deeply. It is a good stain for wood anatomy
(Figs. 10 to 13).
Double staining Diospyros species wood sections with the extracted Phytodyes and Alcian blue
Counter staining any of the Phytodyes with Alcian blue produced brilliant contrast (Figs. 14 & 15). All the
six Phytodyes were highly selective because they have affinity for fibre and other lignified cells especially when
they were used together with stains that have affinity for thin wall cells (e.g. parenchyma cells) such as Alcian
blue (Figs. 14 & 15). The results of counter staining of any of the Phytodyes with Alcian blue (Figs. 14 & 15) is
similar to that of Safranin O and Alcian blue (Fig. 16).
pH of the Phytodyes
The pH of all the extracted Phytodyes fall within the acidity region ranging from 2.80 to 5.90.
Table 3. pH of the phytodye extracts and exotic dyes.
Phytodye extract of pH
1 Enantia chloratha 5.4
2 Harugana madagascariensis 5
3 Hibiscus sabdariffa 2.8
4 Sarcocephalus latifolius 5.2
5 Sphenocentrum jollyanum 5.1
6 Sorghum bicolor 5.9
Exotic Dye pH
7 Alcian blue 3.25
8 Safranin O 7.61
DISCUSSION
All the six phytodye extracts distinctively imparted their colours on all cells and tissues but more deeply on
fibre and other lignified or thick wall tissues except for Hibiscus sabdariffa whose colour was faintly retained by
these tissues when rinsed in water because it is highly soluble in water and turned black when in contact with
water. The action of these Phytodyes on plant wood tissues shows that they can be used as good stains in wood
anatomy (Figs. 10 & 15).
The results of the absorption spectra of the dye extract from the six Phytodyes revealed a range of
wavelength of absorption between 300.00 nm and 900.00 nm which fall within the visible region of the
electromagnetic spectrum confirming the presence of colour imparting chromophores in the dye extracts (Table
2, Fig. 2–9). Popoola et al. (1994) also discovered colour imparting chromophores in Zingiber officinale L.
which belongs to the family Zingeberaceae. Akinloye et al. (2010) also mentioned colour imparting
chromophores in the dyes from Bixa orellana L., Curcuma domestica Valeton and Pterocarpus osun Craib. The
peak of a colour is determined by the predominant wavelength of absorption in it. Each of the Phytodye extracts
has minimum of two peaks, indicating that each dye had a minimum of two colour imparting chromophores
(Figs. 2–9). Popoola et al. (1994) made similar observation of a single peak on Zingiber officinale.
The quality of the colour of the extract is a product of the sharpness of its absorption spectrum which in the
case of Enantia chlorantha, Harungana madagascariensis and Sphenocentrum jollyanum were represented by
more or less narrow range of intensity profile (Figs 3, 4 & 8). This observation indicates the brightness of these
dyes (Figs. 10 & 15) and support the observation of Popoola et al (1994) in their study of dye in Zingiber
officinale. However, the broad absorption bands of Hibiscus sabdariffa, Sarcocephalus latifolius and Sorghum
bicolor (Figs 5–7) do not agree with their sharpness or brightness because these three dyes imparted their
colours brightly on tissues. Hibiscus sabdariffa imparted its bright colour on tissues better than when tissues
were rinsed in water. Combining each of these Phytodyes with Alcian blue make them to behave strictly
specific for fibre and vessel members just as it was observed by Akinloye et al. (2010) (Figs. 14 & 15).
Consequently, this study has shown that Phytodyes used could be successfully employed in plant histological
studies just as noted by Gaur & Chandel (1998) in Crocus sativus L. and Lawsonia inermis L. in which they
successfully utilized Phytodyes for differentiating inactive living and dead nematodes in plant tissues during
bioassays and other investigations.
The results of the pH revealed that all the dye extracts are acidic (Table 3). Hibiscus sabdariffa dye extract
was the most acidic with pH 2.8. Sorghum bicolor with the pH of 5.9 was the least acidic of all the dye extracts.
The high acidity of Hibiscus sabdariffa is probably responsible for its high solubility in water. The
histochemical reactions of all the Phytodyes were similar and the same with that of Safranin O in that they all
imparted their colours indiscriminately on all cells except fibre and other thick walled cells and lignified tissues
which took up the dyes more deeply (Figs. 10 & 13). They were specific when used with Alcian blue or any
stain that has affinity for thin walled cells such as parenchyma cells (Figs. 14 & 15). Phloroglucinol is a known
stain for lignin and it has pH of 4.97 which is in the acidic region. Alcian blue is a known stain for thin walled
cells such as parenchyma cells and it has pH of 3.25 which is acidic. Therefore, acidity or and alkanility may not
be the best parameter to determine the affinity of these Phytodyes for tissues or cells.
CONCLUSION
Dyes from the selected plants are good replacement for the exotic imported stains being used in wood
anatomy. This will reduce our over dependence on imported exotic stains and thus save our country the more
needed foreign exchange.
Further research work can also be done on application of the Phytodye extracts on other plant parts and their
utilization in other areas of Biology such as Microbiology, Zoology and Biochemistry. Isolation and
identification of prominent chromophores that are responsible for the colouration in the Phytodyes will be of
immence scientific and economic interest. Further work can be carried out to check if these dyes can be used to
detect the presence or absence of ergastic substances such as protein, fat and oil, starch granules and cellulose in
plant tissues.
ACKNOWLEDGEMENTS
Authors are thankful to the Botany Department, Obafemi Awolowo University for providing necessary
facilities. Special thank also goes to Prof J. O. Faluyi of Botany Department, Obafemi Awolowo University for
his support during the course of this work.
REFERENCES
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histological stain for collagen fibres African Journal of Biotechnology 4(5): 460–462.
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the dye in Ginger Rhizome (Zingiber officinale L.) Pakistan Journal of Science Industrial Research 37(5):
217–220.
Research article
A modified and improved protocol development for in vitro clonal

propagation of Santalum album L. from internodal explants
Parul Bhargava*, N. Ravindra and Gyan Singh
State Forest Research Institute, Kanpur, Uttar Pradesh, India
*Corresponding Author: shuklaparul7@gmail.com [Accepted: 12 July 2018]
Abstract: Indian sandalwood (Santalum album) is a medium-sized evergreen semi root parasitic
tree. It is a commercially important species for its heartwood essential oil, extensively used in
medicines, cosmetics, perfume and incense industry. Sandalwood harvesting usually involves
removal of the entire tree resulting in critical loss of the genetic diversity. Commercial plantation
of the species is not widely done due to nonavailability of good quality planting material in
sufficient quantity. Production of sandalwood plantlets through vegetative propagation in regular
nurseries is also difficult and time-consuming. Hence, there is a need to develop such planting
material/plantlets through tissue culture technique, which provide high yielding clones of
candidate plus trees of sandalwood. In the present study, an efficient plant regeneration protocol
for in vitro propagation of Santalum album has been developed. Plant propagation using nodal
explants was achieved on Murashige and Skoog’s (MS) medium. MS solution contains a mixture
of macroelement like NH4, KNO3, Mg, Ca, KH2 and microelements like Zn, H3BO4, MnSo4, Cu and
Co, sucrose as a source of carbohydrate and vitamins. Effect of plant growth regulators (PGR) like
6-Benzylaminopurine (BAP) and Kinetin (Kn) on shoot induction and multiplication and Indole-3
Butyric acid (IBA) and Naphthalene Acetic acid (NAA) on rooting was studied. Highest shoot
multiplication with longest shootlets (2.90 cm) was achieved on MS medium containing 0.5 mg L-
1
BAP and 5.0 ml L-1 IBA after 30 days of culture. New shoots were repeatedly harvested up to
three subculture passages on the fresh medium of same concentration at 4-week intervals.
Microshoots treated with 50.0 ml L-1 IBA for 48 h produced roots on growth regulator free, half-
strength MS medium followed by one-week incubation in the dark. Hence, this protocol is a
simple, rapid and highly reproducible to obtain more number of quality plants of Santalum album
within a short period.
Keywords: Micropropagation - In vitro establishment - Culture medium - Plant growth regulators
- Shoot multiplication.
[Cite as: Bhargava P, Ravindra N & Singh G (2018) A modified and improved protocol development for in vitro
clonal propagation of Santalum album L. from internodal explants. Tropical Plant Research 5(2): 193–199]
INTRODUCTION
India has a rich diversity of forest tree species that have been well known for their utility as well as aesthetic
value. Our indigenous species like teak, rosewood, and sandalwood are amongst the most valuable timbers in
the word. These trees once abundant in the forests throughout the country have been depleted in recent times to
meet the increasing demand for wood plantation (Murlidharan 1997).
Sandalwood or Chandan (Santalum album L.) belongs to the Santalaceae family, a medium-sized evergreen
semi root parasitic tree, highly valued for its fragrant heartwood, which contains sandal oil that is used in
perfumes, cosmetics, medicine, and also in agarbathi (incense sticks) industries (Srinivasan et al. 1992).
Santalum album has the highest oil content (about 6%) among the species of the genus Santalum. The
sandalwood and oil demand (80–90%) in the international market has been fulfilled by Indian sandalwood for
decades. However, the production of sandalwood declined from 4000 tons in 1950 to 2000 tons in 1990 and to
about 1000 tons in 1999 (Ananthapadmanabha 2000). The acute shortage in supply and the demand imbalance
resulted in the closure of several sandalwood based industries in India and other Asian countries.
Received: 12 February 2018 Published online: 31 July 2018
Bhargava et al. 2018
Santalum album is recalcitrant to in vivo and in vitro propagation, for which only limited success has been,
achieved so far (Sanjaya et al. 2003). Natural regeneration and artificial propagation occur mainly by seeds. On
the other hand, vegetative propagation is accomplished by grafting, air layering, and with root suckers, but the
production of clones is insufficient and time-consuming (Srimati et al. 1995).
Previous reports on in vitro propagation have focused on adventitious bud regeneration from in vitro grown
seedling explants such as hypocotyl (Bapat & Rao 1978, Rao & Bapat 1992), endosperm (Sita et al. 1979),
zygotic embryo (Rai & McComb 2002), and somatic embryogenesis through a callus phase (Sita et al. 1980).
Although small numbers of plants were regenerated, the methods are still a long way from being optimized for a
large number of plantlet productions. Indeed, the bottleneck is in vitro rooting, which limits the widespread
application of micro propagation techniques in sandalwood. There are very few efforts found for in vitro clonal
propagation of candidate plus tree (CPT) of S. album through axillary shoot proliferation. Sufficient references
on this aspect are also not available. There is, therefore, a need to develop clonal techniques to produce disease-
resistant and high oil-yielding clones of CPTs of Santalum album. The present research study was designed to
fill the gap for true to type large scale in vitro plantlet production of Santalum album.
MATERIAL AND METHODS

Plant material
The plant materials were collected from healthy, disease-free mature trees of Santalum album, growing in
the nursery of State Forest Research Institute (SFRI), Kanpur (Fig. 1). Nodal explants containing axillary buds
were mainly selected for in vitro micro propagation of Santalum album L.
Figure 1. Santalum album L. at State Forest Research Institute (SFRI), Kanpur, Uttar Pradesh, India.
Surface sterilization
The long shoots obtained from the mother plant after removing the leaves were gently scrubbed with a
cotton swab dipped in 70% alcohol. Cut and trimmed nodal segments containing axillary buds were gently
scrubbed all along its length especially around the node bearing the bud (Fig. 2). Explants were sterilized using
0.1% of Bavistin and 0.1% paradigm antibiotic solution for removing bacterial contamination and in Mercuric
chloride solution at 0.1% concentration for fungal contamination of each for 5–10 minutes. Surface sterilized
explants were thoroughly washed 3–4 times with sterile distilled water to wash off the sterilizing agent. The
surface sterilized explants were trimmed at both the ends with the help of scalpel blade and inoculated on
Murashige and Skoog’s (MS) medium without hormones (Control). 3–4 explants were inoculated in each
culture flask. Finally, the cultures were kept in the culture room under controlled conditions and observations
recorded regularly.
Figure 2. Santalum album L.: A, Surface sterilized nodal explants; B, Surface sterilized trimmed explants; C, Proliferated
explants.
Effect of plant growth regulator on shoot development and multiplication
To find out the best auxin and cytokinin effect and their optimum concentration for multiple shoot
production and subsequent growth, various concentrations of Indole butyric acid (IBA) and 6-
benzylaminopurine (BAP) were used in Murashige and Skoog’s (MS) medium (Murashige & Skoog 1962).
Nodal shoot segments isolated from in vitro grown shoots on MS medium without hormones (Control) were
subcultured on MS medium supplemented with 0.5 mg l-1 BAP and 5 mg l-1 IBA. After four weeks, proliferated
shoots higher than 2.0 cm of original explants were repeatedly subcultured up to five times on Murashige and
Skoog’s medium containing the mixture of IBA and BAP of same concentration at 4-week intervals. Data were
recorded for each subculture cycle.
In vitro root induction in S. album
In vitro raised shoots of 3.0–4.0 cm in length were used for in vitro root induction. Micro shoots were pulse
treated (0–72 h) with 50 mg l-1 Indole-3-butyric acid (IBA) and subsequently transferred to half-strength
Murashige and Skoog’s medium without hormones, with 30.0 gm 1-1, sucrose, and 7.0 gm l-1 agar-agar.
Cultures were incubated in the dark for one week and subsequently shifted to light at 28±1 °C with a 16-h
photoperiod provided by cool white fluorescent lamps (Philips, India). The percentage of root induction, number
of roots, and root length were recorded at the end of the eighth week.
Pre-hardening and transfer of plantlets to the containers
In vitro rooted shoots were carefully removed from the culture medium and transferred into 400-ml glass
bottles containing the autoclaved soilrite mixture. The transplanted plantlets were maintained at 28±1 °C
temperature and 16-h photoperiod. The plantlets were sprayed with one-quarter Murashige and Skoog’s liquid
medium at 15-day intervals to boost the growth of plantlets at pre-hardening. Once adjusted to these initial
shocks these plantlets are ready to move out of the laboratory conditions for further stages of growth at
acclimatization in germination house.
Data collection and statistical analysis
Each treatment consisted of 5 replications, and each experiment was repeated twice, to confirm the results.
The data were analyzed by one-way analysis of variance (ANOVA).
RESULTS
Shoot initiation
The nodal shoot segments exhibited maximum bud break and the highest number of shoots when cultured on
full strength Murashige and Skoog’s medium without hormones (Table 1). The nodal shoot segments cultured
during November–January yielded maximum bud break (95%) per explants. Poor shoot development was
observed when explants were cultured in May–July on optimum shoot induction medium (Fig. 3).
Table 1. Effect of different concentration of growth hormones in MS medium on bud break of Santalum album L.
Nutrient medium Number of explants inoculated Bud break/explants
MS media without hormones (Control) 100 100
MS + 1ml BAP 100 85
MS + 1.5 ml BAP 100 85
MS + 1ml Kn 100 70
MS + 1.5 ml Kn 100 60
MS + 1ml BAP + 1ml Kn 100 50
MS + 1.5 ml BAP + 1.5 ml + Kn 100 50
Note: Data recorded after 30 days of culture
Figure 3. Effect of collection time on the survival of internodeal explants of Santalum album L.
Shoot multiplication
The combined use of BAP (0.5 mg l-1) and IBA (5 mg l-1) significantly enhanced the shoot number per shoot
segment (Table 2; Fig. 4A). Subculturing within 4 weeks was essential to maintain healthy shoot growth;
moreover, a repeated subculture of the original explant (nodal shoot segment) every 4 weeks on the fresh
medium of a mixture of BAP and IBA of same concentration produced a crop of shoots (Fig. 4B). Enhanced rate
of shoot multiplication was observed up to the fifth subculture, and the gradual decline was recorded after the
fifth subculture.
Table 2. Effect of different concentration of growth hormones in Murashige and Skoog’s medium on shoot
multiplication and shoot length of Santalum album L.
Nutrient medium Mean Number of shoots/explants Mean shoot length (cm)
MS + 0.1 ml BAP+ 1ml IBA 1.67 1.40
MS + 0.2 ml BAP+ 2ml IBA 1.75 1.40
MS + 0.3 ml BAP+ 3ml IBA 1.80 1.56
MS + 0.4 ml BAP + 4ml IBA 2.00 2.00
MS + 0.5 ml BAP + 5ml IBA 4.00 2.90
MS + 0.1 ml IBA + 1ml BAP 1.20 1.35
MS + 0.2 ml IBA + 2ml BAP 1.20 1.40
MS + 0.3 ml IBA + 3ml BAP 2.00 1.50
MS + 0.4 ml IBA + 4ml BAP 2.04 1.75
MS + 0.5ml IBA + 5ml BAP 2.04 1.85
Note: Data recorded after 30 days of culture.
Figure 4. Santalum album L.: A, Development of multiple shoots from nodal shoot; B, Shoot multiplication and elongation
during subculture.
Figure 5. Santalum album L.: A, In vitro rooting of pulse treated shoots after 8 weeks; B, In vitro rooted microshoots; C,
Elongation of in vitro root and shoot; D, In vitro microshoots in soilrite.
In vitro rooting
The duration of pulse treatment had a significant effect on rooting capacity; the best results were obtained
with micro shoots treated with 50 ml l-1 IBA for 48h and subsequently cultured on half-strength Murashige and
Skoog’s medium (Table 3; Fig. 5). However, shoots exposed to prolonged pulse treatment leads to necrosis, and
no root induction was observed in control. Cultures were incubated in the dark for one week and subsequently
shifted to light at 28±1 °C with a 16-h photoperiod provided by cool white fluorescent lamps. Out of the
different auxins (IBA, NAA, and IAA) tested, the micro shoots treated with 50 ml l-1 IBA showed maximum
rooting (50%), and no root induction was noted for IAA and in control without pulse treatment.
Table 3. Effect of duration of pulse treatment of Indole butyric acid (50 ml l-1) on in vitro root induction in
Santalum album L.
Pulse treatment (h) Root induction % Root length (cm)
Control (water) 00.00 00.00
1 00.00 00.00
5 04.16 0.15
10 08.33 0.62
24 33.34 0.53
48 50 3.32
72 08.16 0.45
Note: Data recorded after 30 days of culture.
DISCUSSION
The use of preexisting buds for propagation reduces the possibility of variation among the progeny and
therefore can be safely applied for rapid propagation of field-grown CPTs of sandalwood. We optimized shoot
multiplication and novel rooting techniques for mass multiplication of the species without the interference of
callus. This method is quite common for the propagation of Fragaria indica (Indre & Dhar 2000), Acacia
mearnsii (Marguerite et al. 2001), Tectona grandis (Mendoza et al. 2007) and Eucalyptus hybrid (Brondani et
al. 2011).
The combined use of cytokinin and auxin was emphasized in micropropagation of Ficus carica (Kumar et
al. 1998) and Syzygium travancorium (Ajith et al. 1999). The season of explant collection also influenced shoot
development from individual explants; this fact may be due to long flowering and seed setting habit of trees.
These observations are in concordance with Sharma et al. (2003) in Crataeva adansoni, where shoot
initiation is highly influenced by season. The application of IBA by a pulse treatment was used in adventitious
rooting of Maytenus emarginata (Rathore et al. 1992) and Tectona grandis (Siril & Tewari 1999).
The results obtained in this study are very encouraging and the standardized protocol developed in the course
of study will also provide a great opportunity to raise high quality in vitro clonal plantlets of Santalum album.
The present investigation will also take another step forward in demonstrating the application of root-trainer
technology and compost as a major potting medium ingredient for quality plant production; finally, it will help
in the steady establishment of in vitro raised plantlets in field conditions. Because Santalum album is a slow-
growing species, it is too early to evaluate the superiority of in vitro raised plants.
CONCLUSION
This study provides an efficient in vitro propagation method which could be commercially feasible for
Santalum album, by providing a protocol for producing genetically uniform plants from selected genotypes.
Explants collected in November to January showed maximum survival and explants inoculated on Murashige
and Skoog’s media without hormones provided the maximum number of bud break or proliferation.
The concentrations of 0.5 mg l-1 BAP combined with 5mg/l IBA on full MS medium produced mass
multiplication and longest shoots. The effect of pulse treatment of 50 mg l-1 IBA on in vitro multiplied shoots
for 48 hours followed by transferred in half strength MS media provided a maximum percentage of root
induction and root length.
ACKNOWLEDGMENTS
We wish to thank B.K. Singh, PCCF, Research and Training, and M.P. Singh APCCF / Director, Forest
Research Institute, U.P. Kanpur for providing necessary facilities and valuable suggestions during the research
study. Ashok Kumar, technical assistant for helping in entire research work and CAMPA, for providing funds.
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seed production areas, plus trees and clonal seed orchards for sandal (Santalum album L.). In: Srimati RA,
Venkateshan KR & Kulkarni HD (eds) Recent advances in research and management of sandal (Santalum
album L.) in India. New Delhi, pp. 281–299.
Srinivasan VV, Sivaramakrishnan K, Rangaswamy CR, Ananthapadmanabha HS & Shankara Narayana KH
(1992) Sandal (Santalum album L.). Institute of Wood Science and Technology, Bangalore, India, pp. 1–60.
Research article
Evaluation of phytochemicals in the leaf extract of

Clitoria ternatea Willd. through GC-MS analysis
Anupsingh Vijaysingh Thakur1, Sonu Ambwani1*, Tanuj Kumar Ambwani2,
A. H. Ahmad3 and Dharmendra Singh Rawat4
1
Department of Molecular Biology and Genetic Engineering, CBSH, G. B. Pant University of
Agriculture and Technology, Pantnagar 263145, India
2
Department of Veterinary Physiology and Biochemistry, CVAS, G. B. Pant University of
Agriculture and Technology, Pantnagar 263145, India
3
Department of Veterinary Pharmacology and Toxicology, CVAS, G. B. Pant University of Agriculture and
Technology, Pantnagar 263145, India
4
Department of Biological Sciences, CBSH, G. B. Pant University of Agriculture and Technology,
Pantnagar 263145, India
*Corresponding Author: sonuambwani@yahoo.co.in [Accepted: 18 July 2018]
Abstract: Clitoria ternatea is a perennial herb of India which is reported to possess several
therapeutic properties. It is also found in China, Philippines and Madagascar. It is a vigorous,
persistent, herbaceous perennial legume. Most of the plant parts are reported to possess
therapeutic properties. In the traditional system of medicine, Clitoria ternatea has been utilized
for treatment of worm infestation, infertility, skin problems, tonsillitis, cough, asthma
traditionally etc. In the present study, fifty percent hydromethanolic extract of leaf of Clitoria
ternatea (CTE) was prepared and subjected to various biochemical qualitative tests and GC-MS
analysis to detect the presence of various phytoconstituents in CTE. Biochemical tests confirmed
the presence of various phytochemicals viz., saponins, resins, tannins, flavonoids, alkaloids,
glycosides, etc. GC-MS analysis revealed the occurrence of thirty compounds in CTE. The main
phyto-composition of Clitoria ternatea is predicted to be Butyl-2-methyl-propylphthalate
(20.11%), Butyl-2-methylpropylphthalate (10.39%), Butylocty-lphthalate (11.29%),
Diisononylphthalate (3.54%) etc., whereas, Butyl-2-ethyl-hexyl-phthalate was major phyto-
constituents with 30.19% of total constituents. Thus it could be inferred that the therapeutic
potential of CTE is because of different phytochemicals present in the extract prepared.
Keywords: Clitoria ternatea - GC-MS analysis - Phytochemicals.
[Cite as: Thakur AV, Ambwani S, Ambwani TK, Ahmad AH & Rawat DS (2018) Evaluation of phytochemicals
in the leaf extract of Clitoria ternatea Willd. through GC-MS analysis. Tropical Plant Research 5(2): 200–206]
INTRODUCTION
Clitoria ternatea Willd. commonly known as butterfly-pea, blue-pea and cordofan-pea belong to
the Fabaceae family. It is a perennial herb found in India, China, Philippines and Madagascar. It is widely found
in the humid, low land tropics, occurring naturally as well as in cultivated form (Devi et al. 2003, Gupta et al.
2010). Varieties (white-flower and blue flower) of C. ternatea are found in India, China, Madagascar and
Philippines. It is popularly called as “Shankpushpi” in India as the flowers of this plant resemble a conch shell
(Kulkarni et al. 1988).
Clitoria ternatea is widely used as a nervine tonic since ancient time and is believed to promote memory and
intelligence (Kulkarni et al. 1988). In Ayurvedic system of medicine, it has been used as a memory enhancer,
nootropic, antistress, antidepressant, anticonvulsant, tranquilizing and sedative agent (Jain et al. 2003,
Mukherjee et al. 2008). Several studies have been carried out to explore the medicinal properties likes
anthelmintic, anti-hyperglycemic, anti-inflammatory, anti-diarrheal, antioxidant, hepatoprotective,
Immunomodulatory, anti-histaminic; cholinergic activity of C. ternatea (Devi et al. 2003, Chauhan et al. 2012).
Received: 06 March 2018 Published online: 31 July 2018
Thakur et al. 2018
Leaves contain sitosterol, kaempferol-3-monoglucoside, kaempferol-3-rutinoside, kaempferol-3-

neohesperiodoside, kaempferol-3-O-rhamnosyl-(1,6)-glucoside, kaempferol-3-O-rhamnosyl-(1,6)-galactoside
and kaempferol-3-O-rhamlnosyl-(1,2)-Ochalmnosyl-(1,2)-O-[rhamnosyl-(1,6)]-glucoside. Lactones aparajitin
and clitorin from leaves were also reported. The leaves also contain an essential oil, colouring matter and
mucilage (Tiwari & Gupta 1959, Rao et al. 2009, Shekhawat & Vijayvergia, 2010, Sarumathy et al. 2011).
Keeping in view the mentioned facts, the present study was planned to explore phytochemicals in the fifty
percent hydro-methanolic leaf extract of C. ternatea (CTE) through biochemical and GC-MS analyses.

Plant material
The authentic plant material i.e., leaves of C. ternatea were obtained from the Medicinal Plant Research and
Development Centre (MRDC), GBPUA&T, Pantnagar, Uttrakhand, India.
Preparation of Extract of Clitoria ternatea (CTE)
Leaves were washed properly, shade dried and ground into a fine powder and stored in sterile containers in a
cool dry place till further use. Extraction was carried out by using solvents with different polarities. The
hydromethanolic extract was prepared as described by Ukwuani et al. (2012). 50 gm of the powder was allowed
to soak in 500 ml 50% methanol (v/v) for 48 hours under continuous agitation in a shaking incubator. The
mixture was first filtered through muslin cloth, then through Whatmann filter paper No 1. The filtrate was then
kept in the rotatory evaporator (45ºC). Finally, the extract was obtained by drying the filtrate under hot
circulating air at 40ºC followed by lyophilization. The percent yield was calculated by dividing quantity of the
plant extract obtained from dry leaves powder by 50. The prepared extract was kept at -20ºC in air tight
container till further use.
Phytochemical Analyses of CTE
Qualitative phytochemical tests for the identification of carbohydrates, resins, tannins, saponins, flavonoids,
alkaloids, steroids, phenols and glycosides were carried out for 50% hydromethanolic extract of C. ternatia
leaves (CTE) as per the methods described by Trease & Evans (1983), Harborne (1998), Sazada et al. (2009)
and Thakur et al. (2018a, b).
Characterization of CTE by GC-MS analysis
The samples were analyzed at the commercial facility of GC-MS analysis available at Advanced
Instrumentation Research Facility (AIRF), Jawaharlal Nehru University, New Delhi, with the following
parameters as described earlier by Thakur et al. (2018a, b).
A. Sample preparation: 200 mg of the medicinal plant extract was dissolved in 2 ml of methanol and then
filtered through a syringe filter (0.22µ). A finally prepared sample of each extract was loaded in GC-MS
column.
B. GC-MS analysis: GC MS analysis was carried out by splitless injection of 1µl of the sample onto Shimadzu QP2010
GC-MS assembly fitted with a column, coupled with a mass detector. Following parameters were used during
analysis of an extract of medicinal plants. Column Oven Temperature was set at 100.0°C, the pressure was
175.1 kPa with total Flow of 16.3 ml/min, column flow was 1.21 ml min-1, linear velocity was 28.9 cm sec-1
and purge flow was 3.0 ml/min. Mass detector was set with start time 6.00 min and end time 40.49min. The
spectrum of the unknown component was compared with the spectrum of the known components stored in
the NIST and WELLY library as well as TOX Library. Various phytochemicals in the plant extract with the
name of the compound along with its molecular weight and structure were determined.
RESULTS
Percent Yield of CTE
Total of 6.23 g of the hydromethanolic extract was prepared from 50 g of leaves of Clitoria ternatea with
percent yield of 12.46%.
Phytochemical analyses of CTE
As per the biochemical tests conducted, CTE showed the presence of all the tested phytochemicals, viz.
carbohydrates, tannins, saponins, flavonoids, alkaloids, steroids, phenols and glycosides (Table 1).
GC-MS analysis of CTE
The major phyto-composition of C. ternatea is predicted by comparison with Tox library and was found to
be Butyl-2-methyl-propylphthalate (20.11%), Butyl-2-methylpropylphthalate (10.39%), Butylocty-lphthalate
(11.29%), Diisononylphthalate (3.54%) etc., whereas, Butyl-2-ethyl-hexyl-phthalate was major phyto-

constituents with 30.19% of total constituents. Upon comparison with NIST and WELLY library, the major
phyto-constituent of C. ternatea was predicted to be 1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester
with 20.11 % among total phyto-constituents (Fig. 1; Table 2, 3).
Table 1. Phytochemicals present in the leaf extract of Clitoria ternatea Willd..
S.No. Phytoconstituents CTE
1. Protein +
2. Carbohydrates +
3. Resins +
4. Tannins +
5. Saponins +
6. Flavonoids +
7. Alkaloids +
8. Steroids +
9. Phenols +
10. Glycosides +
Figure 1. Chromatogram showing peaks for phytoconstituents in the leaf extract of Clitoria ternatea Willd.
Thakur et al. 2018
Table 2. Phyto-constituents present in the leaf extract of Clitoria ternatea after determining the retention time peaks with TOX
(PERFUMERY and DRUG) Library. [CAS- Chemical Abstracts Service]
PEAK R TIME AREA AREA% NAME Formula CAS No Mol. Wt.
1 10.508 31452 00.21 Lauric acid ME C13H26O2 111-82-0 214
2 11.676 3031299 20.11 Butyl-2-methylpropylphthalate C16H22O4 17851-53-5 278
3 11.913 248936 01.65 Pentadecanoic acid ME C16H32O2 7132-64-1 256
4 12.025 1566771 10.39 Butyl-2-methylpropylphthalate C16H22O4 17851-53-5 278
5 12.151 145092 00.96 Decyloctylphthalate C26H42O4 119-07-3 418
6 12.396 4551903 30.19 Butyl-2-ethylhexylphthalate C20H30O4 85-69-8 334
7 12.529 1701712 11.29 Butyloctylphthalate C20H30O4 84-78-6 334
8 12.668 364432 02.42 Diisooctylphthalate C24H38O4 27554-26-3 390
9 12.85 38739 00.26 Amfetamineintermediate C9H9NO2 705-60-2 163
10 12.942 178606 01.18 Decyltetradecylphthalate C32H54O4 0-00-0 502
11 12.992 44006 00.29 Isopropylbenzene C9H12 98-82-8 120
12 13.143 166239 01.10 Diethylphthalate C12H14O4 84-66-2 222
13 13.233 65562 00.43 3-methylhexane C7H16 589-34-4 100
14 13.289 534008 03.54 Diisononylphthalate C26H42O4 28553-12-0 418
16 13.486 185302 01.23 Lignoceric acid ME C25H50O2 2442-49-1 382
17 13.596 657030 04.36 Pcc C12H20N2 3867-15-0 192
18 13.811 247004 01.64 Cyclotetradecane C14H28 295-17-0 196
19 13.968 491009 03.26 Decyltetradecylphthalate C32H54O4 0-00-0 502
20 14.058 200979 01.33 Decyldodecylphthalate C30H50O4 0-00-0 474
21 14.301 67108 00.45 2-methylpentane C6H14 107-83-5 86
22 14.738 177895 01.18 Diethylphthalate C12H14O4 84-66-2 222
23 14.858 56097 00.37 Decyldodecylphthalate C30H50O4 -- 474
15077249 100.00
Table 3. Phyto-constituents present in the leaf extract of Clitoria ternatea after determining the retention time peaks with NYST and
WELLEY Library. [CAS- Chemical Abstracts Service]
PEAK R TIME AREA AREA% Name Formula CAS No Mol. Wt.
1 10.508 31452 0.21 Dodecanoic Acid, Methyl Ester C13H26O2 111-82-0 214
2 11.676 3031299 20.11 1,2-Benzenedicarboxylic Acid, Bis (2- C16H22O4 84-69-5 278
Methylpropyl) Ester
3 11.913 248936 1.65 Octadecanoic Acid, Methyl Ester C19H38O2 112-61-8 298
4 12.025 1566771 10.39 1,2-Benzenedicarboxylic Acid, Dibutyl Ester C16H22O4 84-74-2 278
5 12.151 145092 0.96 1,2-Benzenedicarboxylic Acid, Bis(2- C16H22O4 84-69-5 278
Methylpropyl) Ester
6 12.396 4551903 30.19 1,2-Benzenedicarboxylic Acid, Butyl 2- C20H30O4 85-69-8 334
Ethylhexyl Ester12652777
7 12.529 1701712 11.29 1,2-Benzenedicarboxylic Acid, Butyl Decyl Ester C22H34O4 89-19-0 362
8 12.668 364432 2.42 2-([(2-Ethylhexyl) Oxy] Carbonyl) Benzoic Acid C16H22O4 4376-20-9 278
9 12.85 38739 0.26 1-Penten-4-Yn-3-Ol, 1-Chloro-3-Ethyl C7H9ClO 113-18-8 144
10 12.942 178606 1.18 Di-Isopentylphthalate C18H26O4 0-00-0 306
11 12.992 44006 0.29 2-Propanone, 1,1-Dichloro- C3H4Cl2O 513-88-2 126
12 13.143 166239 1.1 Phthalic Acid, 4-Cyanophenyl Nonyl Ester C24H27NO4 0-00-0 393
13 13.233 65562 0.43 Phthalic Acid, Bis(7-Methyloctyl) Ester C26H42O4 20548-62-3 418
14 13.289 534008 3.54 1,2-Benzenedicarboxylic Acid, Mono (2- C16H22O4 4376-20-9 278
Ethylhexyl) Ester
15 13.381 235288 1.56 1,2-Benzenedicarboxylic Acid, Diundecyl Ester C30H50O4 3648-20-2 474
16 13.486 185302 1.23 Hexanoic Acid, 2-Methyl126532777 C7H14O2 4536-23-6 130
17 13.596 657030 4.36 1,2-Benzenedicarboxylic Acid, Dioctyl Ester C24H38O4 117-84-0 390
18 13.811 247004 1.64 9-Octadecenoic Acid (Z) C18H34O2 112-80-1 282
19 13.968 491009 3.26 1,2-Benzenedicarboxylic Acid, Dipentyl Ester C18H26O4 131-18-0 306
20 14.058 200979 1.33 Di-Hexylphthalate C20H30O4 334
21 14.301 67108 0.45 Phthalic Acid, 4-Cyanophenyl Nonyl Ester C24H27NO4 3087
22 14.738 177895 1.18 1,2-Benzenedicarboxylic Acid, Butyl Octyl Ester C20H30O4 84-78-6 334
23 14.858 56097 0.37 1,2-Benzenedicarboxylic Acid, Diundecyl Ester C30H50O4 3648-20-2 474
24 18.156 90780 0.6 Di-N-Octyl Phthalate C24H38O4 117-84-0 2832
15077249 100
DISCUSSION
Clitoria ternatea L. is a perennial twining herb with several medicinal properties. Various plant parts have
different phytochemicals that are responsible for various pharmacological activities. The fatty acid content of
Clitoria ternatea seeds includes palmitic, stearic, oleic, linoleic, and linolenic acids as well as a water soluble
mucilage, delphinidin 3, 3’, 5’-triglucoside useful as a food dye, beta-sitosterol (Sinha 1960, Debnath &
Chakravarti 1975, Joshi et al. 1981, Macedo & Xavier-Filho 1992, Husain & Devi 1998). Phytochemistry helps
in standardizing the herbal preparations so as to get the optimal concentrations of known active constituents and
in preserving their activities. Shekhawat & Vijayvergia (2010) studied the presence of metabolites in various
plant parts of C. ternatea. Rai (2010) reported the presence of tannins, flavonoids and steroids in the ethanolic
extract of C. ternatea that are known to be the reason for the antioxidative potential of the plants. The salient
phytoconstituents present in C. ternatea are pentacyclictriterpenoids such as taraxerol and taraxerone.
Phytochemical screening of the roots showed the presence of ternatins, alkaloids, flavonoids, saponins, tannins,
carbohydrates, proteins, resins, starch, taraxerol and taraxerone (Trease & Evans 1983). Leaves of C. ternatea
are reported to contain beta-sitosterol, 3-rutinoside, 3-neohisperidoside, 3 monoglucoside, 3- o- rhamnosyl
Glycoside, kaempferol- 3- o-rhamnosyland essential oils. The flower contains delphinidin-3, 5-diglucoside,
delphinidin-3ß- glucoside, and malvidin- 3ß - glucoside, kaemphferol, p-coumaricacid. Rootcontains ß-
carotene, stigmast- 4- ene- 3, 6, diene, taraxerol & teraxerone, starch, tannins & resins (Tiwari & Gupta 1959).
The present study also reports the presence of carbohydrates, tannins, saponins, flavonoids, alkaloids, steroids,
phenols and glycosides in CTE.
For many chronic and degenerative diseases, oxidative stress is considered one of the leading cause
(Vadlapudi & Naid 2010). Petals of C. ternatea have been reported to exhibit potent anti-oxidant activity
(Kankonen et al. 1999, Shan et al. 2005, Hinneburg et al. 2006). Aqueous extracts of petals showed stronger
anti-oxidant activity in comparison to ethanolic extracts (Kamkaen & Wilkinson 2009). Aqueous leaf extracts of
C. ternatea was subjected to various enzymatic and non-enzymatic antioxidantive analyses to explore their
antioxidant potential. In vitro antioxidant capacity was also determined using different assays such as Ferric
reducing power assay (FRAP), Reducing activity assay, diphenypicrylhydrazyl (DPPH) assay and Hydroxyl
radical scavenging activity. C. ternatea has shown significant antioxidative properties which were found to be
comparable with standard antioxidants used in the study (Rao et al. 2009). Several workers reported its
medicinal value such as anti-imflammatory (Devi et al. 2003), anti-oxidant (Sarumathy et al. 2011),
immunomodulatory, hypoprotective (Daisy et al. 2004, Solanki & Jain 2011) etc.
Sarumathy et al. (2011) prepared an ethanolic extract of the aerial part of C. ternatea and subjected it to GC-
MS analysis. Seven compounds wereidentified in this plant by GC-MS viz., n-hexadecanoic acid (48.77), 1-
butanol, 3-methyl-acetate (30.27), propane, 1,1,3-triethoxy-( 3.92), Z, Z, Z-1, 4, 6, 9-nonadecatetraene (4.60),
undecanoic acid (2.80), 3-trifluoroacetoxy pentadecane (3.59) and 4-ethyl - 5-octyl- 2, 2- bis(trifluoromethyl) -
cis 1, 3 - dioxalone - (6.05) through coupled GC-mass spectroscopy. In the present study, a complex mixture of
24 different compounds was detected through GC-MS analysis in CTE. Butyl-2-methyl-propylphthalate
(20.11%), Butyl-2-methylpropylphthalate (10.39%), Butylocty-lphthalate (11.29%), Diisononylphthalate
(3.54%) and Butyl-2-ethyl-hexyl-phthalate (30.19%) were found to be the major compounds in CTE of total
constituents. Thus it could be inferred from the present study that the presence of various phytochemicals as
revealed through biochemical and GC-MS analyses may be responsible for antioxidative and medicinal potential
of C. ternatea leaf extract. However, the advanced analysis is required to further harness the medicinal potential
of C. ternatea.
ACKNOWLEDGMENTS
Authors are thankful to the Director, MRDC, G.B.P.U.A. &T., Pantnagar, for providing the plant material.
The facilities provided by Director Experiment Station; Dean, Veterinary & Animal Sciences, GBPUA&T,
Pantnagar, to carry out the present study, are duly acknowledged. Master’s thesis grant provided to A.V. Thakur
by DBT, New Delhi, India is duly acknowledged.
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Research article
Morphological, biochemical and DNA barcoding characteristics

for some Lantana L. cultivars growing in Egypt
M. S. A. Soliman1*, Z. A. Shedeed1 and M. M. Soliman2
1
Botany and Microbiology Department, Faculty of Science, Helwan University, Cairo, Egypt
2
Botanical Gardens Department, Horticulture Research Institute, Agricultural Research Center, Giza, Egypt
*Corresponding Author: prof.msoliman@yahoo.com [Accepted: 25 July 2018]
Abstract: Lantana cultivars are aromatic shrubs or herbs cultivated for ornamental purposes for its
different flower colors in addition to its use as hedges in Egypt. The similarity of the
morphological characteristics between Lantana camara and L. depressa varieties lead to carry out
the present study where five varieties cultivated in Helwan University Garden, Egypt of red, dark
purple, pale yellow, dark yellow and mixed colors flowers varieties has been done on the bases of
morphological, chemical study to determine total antioxidant compounds, total phenolics and
flavonoids, total anthocyanin content, total carotenoids content, total proteins, and nucleic acids
contents for the flowers of each cultivar, in addition to the molecular levels on the bases of DNA
barcoding level where the Internal Transcribed Spacer (ITS) discriminate and confirm the
identification of four Lantana ornamental plant varieties at the variety level.
Keywords: Ornamentals - Antioxidant - Phenolics - Anthocyanins - Carotenoids - Nucleic acids.
[Cite as: MSA Soliman, ZA Shedeed and MM Soliman (2018) Morphological, biochemical and DNA barcoding
characteristics for some Lantana L. cultivars growing in Egypt. Tropical Plant Research 5(2): 207–216]
INTRODUCTION
Lantana species are found in different tropical and subtropical environments. Most of Lantana plants are
either Spanish flag (species of section Lantana and their hybrids, including Lantana camara L., L. depressa
Small, L. hirsuta Martens & Galeotti, L. horrida Kunth, L. splendens Medik., L. x strigocamara R.W.Sanders,
etc.), or trailing lantana [L. montevidensis (Spreng.) Briq.] Baldwin (1995). Numerous cultivars of the Spanish
flag exist; including 'Irene', 'Christine' and 'Dallas Red' (all tall-growing cultivars) and several recently
introduced shorter ones. The shorter cultivars may floret more regularly than the taller cultivars for example,
Lantana montevidensis gives blue or white flowers all year round.
Lantana cultivars cultivated for ornamental purpose where leaves have medicinal value; used for snake bites
and treatment of tetanus disease, in addition to its use as hedges in Egypt as well as in Kenya, Tanzania and
Uganda (Soliman & Amer 2002). Although lantana cultivars are generally being somewhat toxic, usually
rejected by herbivores, they may still become infested with pests.
The edibility of Lantana berries is contested. Lantana berries are edible when ripe (Boulos 2002, Bell et al.
2007, Borsch & Quandt 2009), though like many fruits are mildly poisonous if eaten while still green. Some
scientists indicated from their experimental investigations that both undeveloped and developed Lantana fruits
are actually fatal, while others claimed that the developed ones are not fatal (CBOL Plant Working Group
2009). Extracts of Lantana camara may be used for protection of cabbage against the aphide (Calonje et al.
2009). The essential oils of L. camara and L. montevidensis are used as a source of plant-derived natural
products with resistance-modifying antibacterial activity (Erlânio et al. 2012). Also, Lantana can used to
produce furniture which is resistant to sun, rain, and termite damage as an alternative to highly priced cane and
bamboo in Soliga, Korava and Palliyar in southern Karnataka, India (Chase et al. 2005).
Lantana cultivars are aromatic shrubs 0.4–2.0 m or herbs; stems carbrid-hairy or prickly; leaves opposite, 2–
8 × 2–5 cm, ovate, toothed, acute, crenate-serrate, the base subcordate; petiole 0.8–1.8 cm; glandular-punctate;
flowers in axillary heads 2.5–4.0 cm diameter; peduncle 1.5– 6.0 cm; bracts 4–8 × 1–2 mm; sessile, in axillary
pedunculate heads or spike-like inflorescences; flowers bracteates; calyx 3–4 mm, membranous, truncate or
Received: 11 March 2018 Published online: 31 July 2018
Soliman et al. 2018
sinuate-dentate; corolla 0.8–1.2 cm, red, yellow, purple, orange or whitish; corolla- tube densely
puberulentoutside; limb obscurely 2–8 mm, 4–5 lobed; stamens 4, didynamous, included; ovary 2-locular, each
locule with 1 ovule; style short; stigma thick, oblique; fruit a fleshy drupe separating into two 1-locular pyrenes.
The fruit of L. camara is a berry-like drupe which turns from green to dark purple when mature. Both
vegetative (asexual) and seed reproduction occur. The spreading of Lantana camara seeds which are produced
by the individual plant (nearly 12.000 fruit per plant) was facilitated by fowls and other creatures.
Lanatana L. species found mainly in tropical America, few tropical and South Africa (Sharma 1981, Boulos
2002). In Egypt, Lanatana L. has been classified as L. camara and L. viburnoides (Forssk.) Vahl by Boulos
(2002), both have the same morphological characters except that the stems of L. camara covered with recurved
prickles and inflorescence not elongated after anthesis.
Owing to wide discriminating breeding throughout the Centuries (17 th and 18th) to use as a decorative plant,
there are now diverse L. camara cultivars. Lantana L. sub-classification depending on morphological character
may be not successful. Accordingly, depending on the secondary products in plants, such as flavonoids and
anthocyanin pigment, are abundant and are suitable as biochemistry indicator in chemotaxonomy (Harborne
1967). Additionally, the phenolic components of plants can be valuable in sub-classifiaction of plants.
Polyphenols have been used in the chemotaxonomy of various crops like Sorghum bicolor L. (Dicko et al.
2002); Galanthus caucasicus (Baker) Grossh., Magnolia obovata Thunb., Cocculus laurifolius DC., Veratrum
lobelianum Bernh. (Tsakadze et al. 2005) and Theobroma cacao L. (Niemenak et al. 2006). Moreover,
anthocyanins was used in chemotaxonomy to place Crocus species and cultivars into seven chemotypes
(Nørbæk et al. 2002).
Carotenoids are yellow, orange, and red pigments that are widely distributed in nature, it play an important
role in photosynthesis and furnishing flowers and fruits with distinct colors. While the carotenoid composition
of flower petals varies from species to species. List of carotenoid composition in flower petals and discussion
for the possible causes of flowers qualitative diversity has been reported by Ohmiya (2011). In addition, the
qualitative composition and localization of carotenoids in leaflets of ten species of the
genus Ceratozamia (Cycads) was used to differentiate between them )Cardini & Morassi 2005).
Recently, DNA barcoding is a molecular tool that uses a short locus from a standardized genome position to
provide fast and accurate species identification since their first application by (Porter & Collins 1991, Kress &
Erickson 2007). ITS1 and ITS2 are widely used for phylogeny reconstruction, (Baldwin et al. 1995, Liston et al.
1996) and it is suitable for evolutionary studies at the species or generic level, Poczai & Hyvonen (2010). This
technique is helpful in taxonomic, ecological, and evolutionary as stated by Lahaye et al. (2008) and Ragupathy
et al. (2009). Although, the most important characteristic features of a DNA barcode are its universality,
specificity on variation and easiness on employment. Several different loci, and combinations of there have been
suggested as suitable barcodes for land plants (Pennisi 2007, Ford et al. 2009). For instance, the nuclear internal
transcribed spacer (ITS) region and the plastid intragenic spacer trnH-psbA have been proposed for flowering
plants (Kress et al. 2005).
Internal Transcribed Spacer (ITS) is one of the most used polymorphic regions is, a space of non-coding
RNA situated between structural ribosomal RNAs on a common precursor transcript. ITS spacer is known to be
partitioned into ITS1 and ITS2 separated by 5.8S ribosomal cistron, in which the RNA poly-cistronic precursor
transcript will be in this order 18S rRNA, ITS1, 5.8S rRNA, ITS2, 26S (Wheeler & Honeycutt 1988). As a part
of the transcriptional unit of rDNA, the ITS spacers 1 and 2 are therefore present in all organisms (Calonje et al.
2009). The aim of this work is to discriminate and confirm the identification for four of five Lantana ornamental
plants at the molecular level, using ITS regions in addition to some chemical characteristics of the five plant
varieties. The present work aimed to link different Lantana species by their chemical analysis for Lantana
flower-corolla- may which show certain lines between these different species. Then confirm their morphological
identification through reference sequences in database and GeneBank.

Samples collection
Five samples were collected from Helwan Univ. campus Garden, Cairo, Egypt; have been identified
according to the inflorescence color petals; (1) Multicolor, (2) Pale yellow, (3) Deep purple, (4) Ded and (5)
Deep yellow flowers. About fifty flowers for each specimens for determining and characters description.
Total phenolics and flavonoids content
Extraction was carried out according to Stanković (2011). Then the extract was used for the determination of
their content.
Total phenolic compounds content was determined by the method of Savitree et al. (2004). 0.5 ml of each
extract of samples was mixed with 2.5 ml of Folin-Ciocalteu reagent (10 folds diluted) and 2.0 ml of 7.5%
sodium carbonate, then reading the absorbance at 760 nm after incubation for 30 minutes at room temperature.
The total phenol content of the samples was calculated as mg g-1 d. wt. using the standard curve of gallic acid.
Total flavonoids content was estimated according to Zhishen et al. (1999) method, flowers extract (1.0 ml)
was mixed with 0.30 ml NaNO2 solution (10%) and the total volume completed to 5.3 ml with dis. water. 0.30
ml AlCl3 solution (10%) and 2.0 ml 1% NaOH solution were added after incubation period. The absorbance was
measured at 510 nm versus the blank. The total flavonoids content of the samples was calculated as mg/g -1d.wt
using the standard curve of quercetin (0–12 mg ml-1).
Nucleic acids content
Nucleic acids content was carried out by Marmur (1961) method for extraction and Dische & Schwartz
(1973) method for DNA determination. The plant extract (1 ml) and 2 ml of diphenylamine solution were
incubated for 10 minutes at 100°C. The absorption was measured at 595 nm and DNA was calculated as mg g -1
F. Wt. by using DNA calibration curve. RNA was determined using the method of Ashwell (1957). The flower
corolla extract (3 ml), 3 ml of FeCl3 reagent and 0.3 ml orcinol solution were incubated in a boiling water bath
for 40 minutes. The absorbance was measured at 670 nm and RNA expressed as mg g-1 F.Wt. by using RNA
calibration curve.
Total soluble proteins content
Total soluble proteins content was determined according to Lowry et al. (1951) method. A sample of the
extract was mixed with 1 ml freshly prepared solution of 2% sodium carbonate in 4% sodium hydroxide and
0.5% copper sulphate in 1% sodium tartrate. Folin reagent (0.1 ml) was added after 10 minutes of incubation
and the optical density of the mixture was measured after 30 minutes at 700 nm. Total protein content was
calculated as mg g-1 d.wt. using calibration curve of albumin.
Total Anthocyanin Content
Total Anthocyanin Content was determined according to Hodges & Nozzolillo (1996) method. Fresh flowers
petals were crushed in (2 ml) acidified methanol in a mortar. Centrifuge and incubate sample in a water bath at
50°C for one hour. The absorbance was measured at 540 nm and 600 nm. The amount of total anthocyanin
contents was calculated in the original sample at 520 nm and 600 nm, expressed as mg l −1.
TLC separation of Anthocyanin
TLC separation of Anthocyanin was carried out according to Simona et al. (2008) method. Pigments were
extracted from the flower petals of different color flowers by using 5 ml ethanol to homogenize 1 g of the petals
tissue. Then the homogenate was heated for few minutes, and 100 µl of each extract was applied on the silica
gel plates of 20 × 20 cm, Merck. The used running solvent mixture was contained: 1-butanol: acetic acid: water
(4:1:5, v/v/v). The Rf value for each band was then calculated.
Total Carotenoids Content
Total carotenoids Content was estimated following Metzener et al. (1965) method amended by Lichtenthaler
(1987). Fresh leaves was homogenized with 85% acetone and centrifuged. Then the supernatant was measured
at 3 wave lengths 452 nm, 645 nm, 664 nm. The total carotenoids content was expressed as µg g -1 fresh weight
of tissue. The concentration of carotenoids was calculated according to the following equations:
Chlorophyll a = 10.3 E664 - 0.918 E645
Chlorophyll b = 19.7 E645 - 3.87 E664
Carotenoids = 4.3 E452 (0.0265 Chl.a + 0.426 Chl.b)
Molecular DNA extraction
Molecular DNA extraction of Floral plant petal samples for the five Lantana varieties; Multicolor, pale
yellow, dark purple, red and dark yellow was carried out using SIGMA® Plant High Molecular DNA extraction
KIT®, Plant tissue was disrupted by grinding in liquid nitrogen and DNA was released with detergent and
chaotropic agents.
After 10 minute precipitation and centrifugation by filtration column, cell debris with polysaccharides and
proteins were removed. Then, microcentrifuge spin colums used for DNA purification.
DNA quality was tested using agarose gel electrophoresis, visualized by pre-added RedSafe® (5 µl per 100
ml) under UV light and quantified using Eppendorf® Spectrophotometer X100 device, about 50 μg of DNA
Soliman et al. 2018
were obtained from 2 g ground powder of dry plant material. PCR and sequencing Two primers, ITS1 (5’-TCC
GTA GGT GAA CCT GCG G-3’) and ITS4 (5’-TCC TCC GCT TATTGA TAT GC-3’) were used to amplify
the internal transcribed spacer (ITS) according to White et al. (1990). PCRs of 50 ul reaction mixture (1X Flexi
buffer, 50ng DNA template, 2.5 m MMgCl2, 10 µM dNTPs, 0.4uM of each primer, and 1U Promega© Green
Go Taq™ enzyme) were performed, standard PCR profile with 55ºC annealing temperature was used to amplify
ITS. Results were tested on 1.5% agarose gel electrophoresis and visualized by pre-added 1x RedSafe® using a
UV light. When successful, amplified fragments were cleaned and concentrated using Thermo Gene JET PCR
Purification Kit #K0702. Cleaned fragments were sequenced by private service (Macrogen, Netherlands).
Sequence chromatograms were compiled using Bioedit V3 to assemble the sequences. All sequences were
manually aligned, while gaps inserted to preserve nucleotide homology. Ambiguous regions were deleted from
the analyses. All Haplotype sequences were submitted into the GeneBank database
(http://www.ncbi.nlm.nih.gov; accessions KR998497– KP998498 for ITS).
To identify the evaluative position and study phylogenetic relationships of the four varieties, the aligned
sequences were analyzed by maximum likelihood (ML) analysis implemented in MEGA6 (Tamura et al. 2013).
Tree inference options were set to Nearest Neighbor Interchange. Gaps/missing data were treated as partial
deletions with site coverage cut off 95%. A bootstrap analysis with 1000 replicates was carried out in order to
study the clade support values. The available ITS sequencing by BLASTn tool (NCBI) generate the trees in all
methods. Maximum Composite Likelihood model was followed in our analysis. The rate variation among sites
was modeled with a gamma distribution (Shape parameter = 0.48). The consensus tree was obtained after
bootstrap analysis, with 1,000 replications, with values above 50% was reported. The analysis involved 22 and
26 nucleotide sequences for ITS.
RESULTS
Morphological identification: Based on the morphological aspect, the main morphological characters have
been represented in (Table 1; Fig. 1). The phenolics and flavonoids content: The present results indicated that
the red flowers contain the premier content of phenolics and flavonoids followed by the deep yellow petals.
Further, deep purple had the lowest content of phenolics and flavonoids (Table 2; Fig. 2). The high phenolics
and flavonoids content of red flowers make a distinction of them from other species.
Table 1. Morphological characters for Lantana taxa studied of different inflorescences colors.
Character Multi Colour Pale Yellow Deep Yellow Deep Purple Red
Habit Shrub Herb Herb Herb Shrub
Width (cm) 0.5 0.35 0.4 0.3 0.5
Stem
Surface Prickly Smooth Smooth smooth Rough

Shape Square Square Square Square Square
Habitate Erect stem Weak stem Weak stem Weak stem Erect stem
Blade Max width (cm) 4.5 1.6 2.5 1.8 3.6
Leaves
Blade Max long (cm) 6.0 3.0 3.3 3.0 7.0

Petiole length (cm) 1.5 0.5 0.8 0.3 1.3
Decussate Decussate Decussate Decussate Decussate
Textile Rough Smooth Rough Smooth Rough
Blade Large Small Small Small Large
position Opposite Opposite Opposite Opposite Opposite
Shape Ovate Lancelate to ovate Lancelate Ovate Ovate
Margin Serrate Serrate Serrate Crenate Coronate
Apex Acuminate Acute Acute Acute Acuminate, acute
Color Dark green Pale green Pale green Pale green Pale green
Number of flowers/ Infl. 36 26 30 20 36

Inflorescence
Fruit Berry Berry Berry Berry Berry

Peduncle Corium Corium Long raches Corium Corium
Peduncle length (cm) 4.8 3.8 2.8 6.2 3.6
Figure 1. The collected samples of different colors: A, Multicolor; B, Pale yellow; C, Deep purple; D, Red; E, Dark yellow
samples respectively.
Table 2. Biochemical analysis for different colors inflorescences of Lantana taxa studied.
Biochemical character Multi Colour Pale Yellow Deep Yellow Deep Purple Red
Anthocyanin content (mg g-1 f.wt.) 16.7 ± 0.5 14.0 ± 0.06 16.1 ± 0.07 27.4 ± 1.0 37.0 ± 0.06
Carotenoids content (µg g-1 f. wt.) 3.58 ±0.07 11.93±0.05 13.33±0.15 1.03 ±0.05 2.03 ± 0.02
Total phenolic compounds 93.25 ±4.5 77.97±0.83 102.8 ± 3.6 46.01 ±0.62 126.7 ± 3.0
Total flavonoids 4.14 ±0.16 4.90 ±0.23 6.32 ±0.62 1.54 ± 0.24 7.88 ± 0.29
DNA content (mg g-1 f.wt.) 19.8 ±0.26 11.7 ±0.52 14.6 ±0.25 15.7 ± 0.95 20.1 ± 0.14
RNA content (mg g-1 f.wt.) 23.6 ± ]0.10 40.5 ± 1.0 39.5 ± 0.17 36.8 ± 0.0 34.3 ± 0.17
Total Protein (mg g-1 d.wt.) 83.37 ± 2.1 176.0 ± 1.8 204.1 ± 1.1 154.1 ± 4.7 145.8 ± 2.7
Figure 2. TLC of anthocyanin pigment in 1. Multicolor, 2. Pale yellow, 4. Red 5 deep yellow colors of lantana corolla
flowers (showing characteristic arrowed band in red colored lantana flower).
The anthocyanin and total carotenoids content: The present study showed that, the petals of red flowers
showed the highest content of anthocyanin. The anthocyanin content of red flowers was more than 2-fold of
other flowers (pale yellow, deep yellow and mixed colors). In contrast, the red flowers showed the lowest
content of the carotenoids from the other species and the yellow corolla shown the highest carotenoids content
(Table 2; Fig. 3).
Soliman et al. 2018
Figure 3. rbcL based maximum likelihood for collected samples of different Lantana specimens distinguished, Sample 1, 2, 4 and 5 representing Multicolor,
pale yellow, red and dark yellow respectively.
The TLC separation of anthocyanin: The present study showed in the TLC chromatogram of red Lantana
petals, the presence of 3(a, b and d) spots. One of them appeared only in the red Lantana (b band, Rf = 16). In
the case of pale yellow, deep yellow and multicolor petals of Lantana, the chromatogram showed 3 spots (a, c
and d).
The nucleic acids content: The nucleic acid extract showed dissimilar values of RNA content in the order of
pale yellow > deep yellow > red > mixed colors (Table 2; Fig. 4). These results showed a relationship between
the pale yellow and the deep yellow flowers of various Lantana varieties. The results revealed the maximum
DNA value for the red flower (20.1) which powerfully related to mixed color flowers (19.8). The DNA content
values showed the following order dark yellow > pale yellow> deep purple.
Figure 4. DNA Barcoding graph for Lantana samples 1. multicolor, 2. Pale yellow, 3. Deep purple, 4.red and 5 deep yellow.
The total protein content: The protein content values in table (2) were ordered in the following deep yellow>
pale yellow> red> multi-colors flowers. The results indicated that the yellow corolla contained the maximum
content of proteins. The red corolla protein content value fallen in-between the yellow and the multi-colors ones.
Molecular Identification and DNA Barcoding: Using both regions (ITS), the phylogenetic analysis was
performed using four off five samples together with GeneBank accessions (sample 1 ‘multicolor’, sample 2
‘pale yellow, sample 3 ‘deep purple’, sample 4 ‘red’ and sample 5 dark yellow flowers respectively). The trees
was rooted between these samples that revealed twomain clads, the first clade possessed three samples
(multicolor, pale yellow and dark yellow varieties) belong to Lantana depressa, while the second clade of ITS
phylogenetic analysis, showed that, sample 4 ‘red variety’ does belong to the same species, and not specifically
to Lantana depressanor to Lantana camara.
DISCUSSION
Species of the genus Lantana, belonging to the family Verbenaceae. They have been of great interest for
their ornamental, phytochemical, biological and pharmacological studies. The different varieties of Lantana
have been reported to contain different types and levels of phytochemicals.
The maximum content of total phenolic, flavonoid compounds and anthocyanin of the red flower petals
provides significant evidence for the distinction of this species from others. The flavonoid pathway has a
pleiotropic role in plants, and one must consider that a single deviation in the pathway may have multiple
phenotypic effects, Mary & Michael (2000). So there is an obstacle to link the phenotype to molecular changes.
Soliman et al. 2018
Now, the genes of the flavonoid pathway are known, so it is essential to associate a phenotype to the gene
family member responsible for flavonoid biosynthesis pathway. The unique pathway of the flavonoid
biosynthesis in this species made it dissimilar from others.
Also, the highest content of DNA and anthocyanin pigment in the red flower petals confirmed the previous
revealing. The water-soluble flavonoid pigments represent a class of phenylpropanoid secondary plant
metabolites. The most important classes with regard to flower color are anthocyanins, flavonols and flavones.
Flavonols and flavones as accessory pigments turned into anthocyanins giving the flowers their distinct colors
(Goto 1987) and possess antioxidant properties (Stintzing et al. 2002, Einbond et al. 2004).
In the present work, the TLC analysis showed special band in the pigment extract of red flower with Rf 16
reflects the presence of cyaniding 3 glucoside,as a component of anthocyanin, Harborne (1967). While the
malvidin 3 glucoside, Harborne (1967) with its band Rf 26 was observed in pale yellow, deep yellow and multi-
color flowers and did not appear in red flower extract. The band with 50 Rf was observed in all extracts. This
established another report of the correlation between the different species.
The biochemistry analysis of different colored flowers shows that; Flower color varied according to the
different chemical composition of scent where In Lantana, the red flower variety exhibited significant
differences between other varieties, while the multicolor and deep yellow flower varieties showed a certain
association. The red flower variety was strongly associated to higher pheolics, flavonoids, anthocyanin pigment
and DNA content than the other studied varieties (with other flower colors) were strongly associated with
terpenoid compounds. This reflected the conserved biosynthetic pathways and the genetics of each variety
which may have a critical role in determining specific associations between floral color and floral constituents.
The molecular identification and DNA Barcoding results show the first clade, possessed three samples 1, 3
and 5 (multi-color, pale yellow and deep yellow varieties) belong to Lantana depressa, while the second clade
of ITS phylogenetic analysis, showed sample 4 of red variety does belong to the same species, and not
specifically to Lantana depressa nor Lantana camara. This result was confirmed with the biochemistry analysis
(of the present study) of the varieties of Lantana where red flower variety has been separated from other
varieties (camara which included multicolor, pale yellow and deep yellow colored flowers) by their biochemical
structure. In spite of (Kress et al. 2005, Chase et al. 2005) conclusion that the nuclear ITS region found to have
a better resolution toward species and its varieties identification, The present result might be due to the
insufficient similar sequences of these species in the GeneBank database. Even though the ITS was more
efficient, it cannot be relied on as a single DNA barcoding region due to its variation within a single species
(e.g. Lantana camara) or due to the presence of paralogs, orthologs and pseudogenes of ITS sequence in a
single genome as suggested by (Feliner & Rosello 2007, Soltis et al. 2008), or due to the growing of Lantana
camara wild among Lantana depressa populations, Hammer (2014).
CONCLUSION
The present study provides preliminary assessment data that will be useful for wider application of DNA
barcoding for ornamental plants. With the current development of primers, ITS will be very useful in barcoding
for the ornamental plant species, where it has a better resolution toward species and varieties identification than
the morphological. DNA barcoding together with the biochemical analysis provided a beneficial tool to sub-
classify of different varieties of Lantana L.
ACKNOWLEDGMENTS
The authors are grateful to Prof. Dr. Mohamed Saied Rashed, Genetics Dept., Fac. of Agriculture, Ain
Shams University for the support to carry out the ITS technique in his lab.
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Research article
Accumulation potential of Indian mustard (Brassica juncea var.

arawali) and fenugreek (Trigonella foenum-graecum L.)
planted on lead and nickel contaminated soil
Leela Kaur
Department of Environmental Science, Maharaja Ganga Singh University, Bikaner, Rajasthan, India
*Corresponding Author: leela.kaur@gmail.com [Accepted: 26 July 2018]
Abstract: The aim of the present study was to examine the accumulation potential of Indian
mustard (Brassica juncea var. arawali) and fenugreek (Trigonella foenum-graecum) plants in lead
and nickel contaminated soil. Plants were exposed to higher concentrations of Pb and Ni (800 mg
l-1). EDTA and SA were amended at 2.4 mM concentrations. Plants were harvested and oven-
dried. The accumulation of Pb and Ni in plants was determined and their effects on plant growth
were evaluated for both species. Seed germination rate of T. foenum-graecum plants was higher as
compared to B. juncea arawali plants. In conclusion, the accumulation capacity of fenugreek was
found more than Indian mustard in Pb and Ni contaminated soil.
Keywords: Accumulation - Lead - Nickel - Indian mustard - Fenugreek.
[Cite as: Kaur L (2018) Accumulation potential of Indian mustard (Brassica juncea var. arawali) and fenugreek
(Trigonella foenum-graecum L.) planted on lead and nickel contaminated soil. Tropical Plant Research 5(2):
217–223]
INTRODUCTION
The contamination of soil by toxic heavy metals is a global environmental problem. These heavy metals can
go into soil by natural sources and anthropogenic sources (Ali et al. 2013). Anthropogenic sources of heavy
metal from agriculture, mining, smelting, electroplating, and other industrial activities have resulted in the
deposition of undesirable concentration of metal such as As, Cd, Cr, Cu, Ni, Pb and Zn in soil (Ensley 2000). A
number of techniques such as soil excavation, extraction, immobilization and phytoremediation have been used
to remove the toxic metals from soil. All these methods have advantages and limitations. Phytoremediation
process has been extensively used to remove heavy metals from contaminated soil using plants. Phytoextraction
is one of the types of phytoremediation where contaminated soil is treated by using high biomass containing
crops and natural hyperaccumulating plants. Metal hyperaccumulators are generally slow growing plant species
having low biomass and metal specific too. To enhance the uptake and accumulation of metals in above-ground
parts of plant, chelating agents were introduced. Ethlenediaminetetraacetic acid (EDTA) helps in enhancement
of metal solubility and translocation from roots to shoots. EDTA-assisted phytoextraction has been done by
many studies (Saifullah et al. 2009, Barrutia et al. 2010, Zaier et al. 2010, Lee & Sung 2014). EDTA has
drawback of its persistence in environment due to which another chelating agents have been used such as the
use of organic acids or more degradable aminopolycarboxylic acids. Salicylic acid (SA) has also been used as
chelating agents for phytoextraction of heavy metals (Meng et al. 2009, Duman et al. 2010, Khan et al. 2015,
Elhassan et al. 2016).
Recent phytoextraction research is focused on identification of metal hyperaccumulator plants and selection
of suitable chelants for heavy metal binding (Anwar et al. 2016). The known metal hyper accumulators are
species of Thalspi, Brassica and Alyssum (Sarma 2011). Many studies have been done to determine heavy
metals phytoextraction potential of plants by investigating bioconcentration factor and translocation factor
(Yang et al. 2016, Zhou et al. 2016, Hosman et al. 2017, Lee et al. 2017, Tabasi et al. 2018, Tawatchai et al.
2018).
Lead and nickel metals were chosen for the present study as their concentrations are increasing in soil due to
industrial development and both are considered carcinogenic (Beyersmann & Hartwig 2008). Lead is one of the
Received: 07 January 2018 Published online: 31 July 2018
Kaur 2018
toxic heavy metal which is harmful to human health. Its retention time in soil is about 150 to 5000 years (Shaw
1990). While, Ni is an essential element but its excess concentration have detrimental effects.
Indian mustard and Fenugreek were taken for the present work as both are food crops and their growth is
affected by heavy metal contaminated soil. However, Indian mustard is a known metal hyperaccumulator
(Mourato et al. 2015) and fenugreek have been explored slightly for remediation potential.
The objective of this study was to evaluate the performance of two plants Indian mustard (Brassica juncea
var. arawali) and fenugreek (Trigonella foenum-graecum L.) in phytoremediation of lead and nickel with and
without chelants (EDTA and SA). The present work will further help in investigating a comparison of
phytoremediation capacity of both plants.

Experimental site
A pot experiment was carried out under field conditions to examine the accumulation potential of Indian
mustard and fenugreek in lead and nickel contaminated soil. Experiments were done at the Micromodel
experimental site of the Indian Institute of Technology, Delhi. It is located at 77.09º E longitude and 20.45º N
latitude, and 28 m altitude above sea level. The mean maximum and minimum temperature during the study
period were 18–43 ºC and 3–15 ºC, respectively. Different physical and chemical soil characteristics are
summarised in table 1.
Table 1. Various soil characteristics.
Parameter Unit Value
Texture - Sandy loam
Clay % 16.30
Silt % 14.23
Sandy % 69.47
Electrical Conductivity mS cm-1 0.28
pH - 7.5
Cation Exchange Capacity Cmol kg-1 18.4
Organic Carbon % 0.72
Available N kg ha-1 272
Available P kg ha-1 9.0
Available K kg ha-1 200.7
Total Pb mg kg-1 0.02
Total Ni mg kg-1 4.0
Pot experiment
The seeds of Brassica juncea var. arawali and Trigonella foenum-graecum L. were obtained from the
National seeds Corporation Ltd., Beej Bhawan, Pusa, New Delhi. About 20 seeds were sown in 11×11 cm pots
containing unsterilized field soil, farm yard manure (organic carbon 12.2 %, total N 0.55 %, total P 0.75 %, total
K 2.30 % and pH 7.2) and sand in a 2:2:1 ratio in October 2008. In chemical treatment, Pb, Ni, EDTA and SA
were added as per the designed treatment. The designed treatment for mustard and fenugreek plants was as
follows: (T1) Pb, (T2) Ni, (T3) Pb + Ni, (T4) Pb + Ni + 2.4 mM EDTA, (T5) Pb + Ni + 2.4 mM SA, and (T0)
Control or untreated plants. The concentrations of lead and nickel (supplied as Pb(NO3)2 and Ni(NO3)2) were
800 mg l-1 in all treatments. Dipotassium salt of EDTA was supplied in the treatment. Plants were watered daily
using tap water to maintain optimal growth conditions. Tap water was analysed for lead and nickel
concentration which were found negligible. Seed germination started after the seventh day of sowing and plants
were thinned to 3 plants per pot after 30 days of sowing. Samples of plants were taken one day before and after
chelant treatment and monthly after the treatment. At the end of the experiment the plants were harvested and
then washed accurately, the aerial part was divided from the roots and the two parts were analysed separately to
determine the metal content.
Soil and plant analysis
The organic C, N, P and K were estimated by the methods of Walkley and Black, Micro-kjeldahl, Olsen and
Flame photometer, respectively, in soil and farm yard manure as descried by Rowell (1994). Cation exchange
capacity was carried out using the BaCl2 method (Hendershot & Duquette 1986). Metals concentration in soil
was determined using the aqua regia extraction method (ISO 12914 2012). Plant samples were washed with tap
water and dried at 70ºC for 48 hours. The dried material was digested with aqua regia. All determinations were
performed in triplicate. Metal concentrations in solutions were analysed by ICP-OES (Varian Vista-MPX CCD
Simultaneous ICP-OES, Varian Australia Pty. Ltd) with a Ni detection limit of 0.007 mg l-1 and a Pb detection
limit of 0.01 mg l-1.
Translocation factor
Translocation factor (TF) was calculated to evaluate the potential of B. juncea var. arawali and T. foenum-
graecum L. for phytoextraction. This ratio is an indication of the ability of the plant to translocate metals from
the roots to the aerial parts of the plant (Marchiol et al. 2004). It is represented by the ratio:
Bioaccumulation Factor (BAF)

Bioaccumulation Factor can be employed to quantify toxic element accumulation efficiency in plants by
comparing the concentration in plant and soil (Baker 1981, Ma et al. 2001).
The experiment was conducted as a factorial randomized block design with each treatment replicated thrice.
Statistical analysis of the data was done following analysis of variance (ANOVA); when the ANOVA was
significant the means were separated using least significant difference at P≤ 0.05 level of significance.
RESULTS AND DISCUSSION

Plant growth
Effect of treatments on seed germination and dry weight of plants is represented in table 2. Application of
SA with heavy metal (Pb2+ and Ni2+) treatment helped in reducing the inhibitory effects of these metals on seed
germination along with increased dry weight. Our results are supported by Mishra & Choudhuri (1997) and
Elhassan et al. (2016). Elhassan et al. (2016) also found that salicylic acid enhanced lead uptake as well as all
growth parameters of maize (Zea mays L.).
Table 2. Effect of treatments on seed germination and dry weight in Brassica juncea var. arawali and Trigonella
foenum-graecum L.
Seed germination (%) Dry weight (g)
Treatment B. juncea var. T. foenum-graecum B. juncea var. T. foenum-graecum
arawali arawali
T0 100±0.8 100±0.5 6.2±0.70 3.8±0.31
T1 80±1.3 100±0.6 3.42±0.28 2.7±0.16
T2 85±1.8 95±1.4 2.28±0.30 1.7±0.48
T3 65±2.2 90±1.9 1.3±0.06 1.2±0.19
T4 75±1.5 85±1.2 1.5±0.42 1.4±0.09
T5 90±2.8 95±1.6 2.2±0.60 2.3±0.25
Note: Values represents mean ± SD (n=3).
Rate of seed germination was higher in fenugreek plants as compared to mustard plants in all treatments. A
potential decrease in total plant dry weight was observed in plants treated with combined Pb + Ni. Dry weight
was higher in SA treated plants as compared to Pb + Ni treatment (T3). Alyemeni et al. (2014) also found a
significant increase of dry mass of Cicer arietinum plants with the application of 10-5 M salicylic acid.
Metal concentrations in plant roots and shoots
Accumulation of Pb and Ni in root and shoot part of mustard and fenugreek plants are shown in table 3 and
4. Lead bioaccumulation in roots and shoots of plants was significantly different. Pb and Ni concentration in
root organs of the plants was higher than that in shoot organs in all treatments. Pb concentration increased in
both organs of the plant with EDTA treatment, but it decreased with SA treatment (except in the shoot part of
fenugreek). Applying EDTA increased Pb concentration in roots of mustard plants significantly (P < 0.05) from
370 mg kg-1 in T3 up to 1010 mg kg-1 in the case of application of EDTA soil at T4. Zaier et al. (2010) found
that EDTA addition increases the ability of Brassica napus to accumulate heavy metals (Zn, Mn and Pb) from
sludge-amended soil. Lee & Sung (2014) also investigated the effect of EDTA on phytoremediation of soils
contaminated with Cd, Cu, Pb, Zn, and Ni by Brassica juncea and found that EDTA was most effective for
heavy metal uptake, but had significant effects on plant biomass. Our findings are corroborated with Gill et al.
(2015) and Kanwar et al. (2015) who studied Cr stress on Brassica species and suggested that inhibited growth
of these plants is directly interrelated with its accumulation.
Kaur 2018
Table 3. Accumulation of Pb and Ni in shoots and roots of Brassica juncea var. arawali.
Shoot concentration (mg kg-1 DW) Root concentration (mg kg-1 DW)
Treatment Pb Ni Pb Ni
T0 43±12 79±11 96±16 123±41
T1 280±201 65±15 502±182 119±33
T2 36±9 91±36 83±15 187±121
T3 370±176 217±134 525±246 303±202
T4 1010±425 52±27 16395±989 2876±541
T5 517±228 69±28 3828±782 610±348
Table 4. Accumulation of Pb and Ni in shoots and roots of Trigonella foenum-graecum L.

Shoot concentration (mg kg-1 DW) Root concentration (mg kg-1 DW)
Treatment
Pb Ni Pb Ni
T0 23±8 47±11 68±22 124±25
T1 429±218 58±13 684±250 106±20
T2 31±12 216±135 53±17 634±241
T3 17624±985 5322±576 43140±969 15291±586
T4 401±252 170±112 2888±755 1187±481
T5 512±214 230±98 1653±682 871±364
Bioaccumulation factor and Translocation factor
2
1.8 Pb
1.6 Ni
Bioaccumulation factor
1.4
1.2
1
0.8
0.6
0.4
0.2
0
T0 T1 T2 T3 T4 T5
Treatments
Figure 1. Bioaccumulation factor of Brassica juncea var. arawali treated with lead and nickel.
25
Pb
Bioaccumulation factor
Ni
20
15
10
0
T0 T1 T2 T3 T4 T5
Treatments
Figure 2. Bioaccumulation factor of Trigonella foenum-graecum L. treated with lead and nickel.
Figure 1 and 2 represents the bioaccumulation factor of B. juncea var. arawali and T. foenum-graecum
treated with lead and nickel respectively. Bioaccumulation factor is the measure of the capability of plants to
transport the metals to its aerial organs from the medium. Bioaccumulation factor mainly depends on
environmental efficacy, metals particularity, species-specific characteristics and disposal route. If the value of
bioaccumulation factor is greater than 1.0, it indicates that plants are able to extract metals and these plants can
be selected for phytoremediation of the contaminated soil (Zhao et al. 2003, Luoma & Rainbow 2005, Kamari et
al. 2012). Bioaccumulation of Pb and Ni were highest in fenugreek plants treated with T3 (22.03±1.23 and
6.65±0.72 respectively). The results show that Pb and Ni bioaccumulation by T. foenum-graecum was greater
than B. juncea var. arawali.
Figure 3. Translocation factor of Brassica juncea var. arawali treated with lead and nickel.
Figure 4. Translocation factor of Trigonella foenum-graecum L. treated with lead and nickel.
Translocation factor (TF) is an effective measure for determining the metal phytoextraction from soils
(Kamari et al. 2012). There are three categories of plants based on their translocation factor namely accumulator
(TF>1), excluder (TF<<1), and indicator (TF near 1) (Baker 1981, Ghosh & Singh 2005). Translocation factor
of B. juncea var. arawali and T. foenum-graecum treated with lead and nickel are shown in figure 3 and 4
respectively. Our result revealed less than 1 value of translocation factor which signifies that Pb and Ni could
not be effectively translocated from the roots to the shoots. Overall, fenugreek showed better Pb-translocation as
compared to mustard. On the contrary, mustard was superior in Ni-translocation as compared to fenugreek.
However, translocation factor was less than 1.0 for both plant species. This study revealed that both the plant
species have high accumulation of Pb and Ni in roots and low translocation in shoots, nevertheless they could be
used for phytoremediation of Pb and Ni contaminated soils.
CONCLUSION
Brassica juncea var. arawali and Trigonella foenum-graecum L. showed reduced seed germination and plant
growth under Pb and Ni contaminated soil. Furthermore, both plant species were able to accumulate Pb and Ni
in root and shoot parts. However, B. juncea var. arawali and T. foenum-graecum may be used as an alternative
for phytoremediation of Pb and Ni contaminated soils.
Kaur 2018
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Research article
Nicandra Adans.: A new generic addition for the

flora of Odisha, India
Sidhanta S. Bisoi1*, Anil K. Biswal2 and Mahendra K. Satapathy1
1
Department of Botany, Regional Institute of Education (NCERT), Bhubaneswar-751022, Orissa, India
2
Department of Botany, North Orissa University, Baripada-757003, Orissa, India
*Corresponding Author: sidhantasekhar91@gmail.com [Accepted: 20 August 2018]
Abstract: Nicandra physaloides, finds its extended phyto-geographical range from Deccan
peninsula to part of Eastern Ghats in Odisha, India. The detailed description, correct nomenclature,
ecological notes and distribution along with photographs of the taxon has been provided.
Keywords: Solanaceae - Nicandra physalodes - New generic record - Odisha.
[Cite as: Bisoi SS, Biswal AK & Satapathy MK (2018) Nicandra Adans.: A new generic addition for the flora
of Odisha, India. Tropical Plant Research 5(2): 224–226]
INTRODUCTION
The southern part of the state of Odisha forms a part of Deccan peninsula. The pioneer workers namely,
Haines (1924) and Mooney (1950) have not mentioned any collection from undivided Koraput and Ganjam
districts. A comprehensive flora of Odisha was published by Saxena & Brahmam (1996) after the reorganisation
of modern Odisha in 1936. Recently, additions to the flora of Odisha from Koraput have been made by Mishra
et al. (1999). Moreover, floristic account of pteridophytes of Koraput has been made by Das et al. (1989) and
Panigrahi (1998). Gamble (1892) was to comment that Odisha is the meeting ground for the Himalayan and
south Indian elements (Subbarao & Kumari 2006). The flora of Odisha offers an ideal database for the
phytogeographical analysis of Indian sub-continent (Panda & Das 1997). Gamble (1915–36) cited five species
from Jeypore hills, Koraput in his treaties “The Flora of Madras” based on the collection of R. H. Beddome. In
connection with the project entitled “Exploration of local plant biodiversity used as fun and games by childrens
in tribal and rural pockets of Odisha” funded by NCERT, New Delhi, the authors made an extensive survey in
the Koraput district. The genus Nicandra Adans. is reported from Kharaguda (Semiliguda), Koraput as an
extended distribution of Deccan peninsula. After critical analysis and careful scrutiny of works by Hooker
(1885), Saxena & Brahmam (1996), Dassanayake & Fosberg (1987) and Singh et al. (2001) the identity and
distribution of the taxon has been confirmed. The detailed description, correct nomenclature, ecological notes
and distribution along with photographs of the taxon has been provided. The herbarium specimen has been
deposited in the herbarium, P.G. Department of Botany, North Orissa University, Baripada.
Figure 1. Map showing the collection site in Odisha, India.

Received: 11 March 2018 Published online: 31 August 2018
Bisoi et al. 2018
Figure 2. Herbarium specimen of Nicandra physalodes (L.) Gaertn.
Figure 3. Nicandra physalodes (L.) Gaertn.: A, Plant in natural habitat; B, Flower twig and unripe fruit; C, Ripe fruits.
Nicandra physalodes (L.) Gaertn. Fruct. Sem. Pl. 2(2): 237. t. 131, fig. 2.1791.
Hook, f., Fl. Brit. India 4: 240.1885.
Atropa physaloides L., Sp. Pl. 181.1753.
Annual, erect under shrubs, upto 1.5–2.0 m high; branches spreading. Stems fistular, grooved, glabrous. Leaves
alternate, ovate, ovate-lanceolate or rhomboidal, 5–20 × 2–16 cm, cuneate, margin coarsely sinuate or sinuate-
dentate, apices acute or acuminate, upper surface sparsly pilose, lower surface glabrescent. Flowers in axillary
solitary cymes; pedicels 2.5–3 cm, calyx lobes-5, up to 3 cm, prominently veined, broadly ovate, deeply cordate
at base, apices acute to acuminate, accrescent; corolla 3–4 cm, bluish to purple, white centred, campanulate,
basal half enclosed by sepals. Stamens-5, filaments up to6 mm long, anthers yellow. Carpels-3, ovary superior,
obvoid, style 3–4 mm long, pilose below, stigma yellow, ovules many. Berries globose, 1–2 cm in diam.,
smooth, enclosed by accrescent chartaceous, calyx lobes, yellow on ripe. Seeds discoid, pitted, reddish brown.
Illustration: Dassan. et Fosb. Rev. Handb. Fl. Ceylon, 400. fig. 10.1987
Flowering & Fruiting: November–January.
Specimen Examined: INDIA, Odisha, Kharaguda, Similiguda, Koraput, 15.12.2017, S.S. Bisoi 1568 (NOU).
Ecology: Annual weed, probably sown by birds. Occasional in wasteland, grows well in fertile soil
Distribution: Native of Peru. India (Kashmir to Sikkim, Tamil Nadu, Maharashtra, Karnataka, Kerala). As
informed by local inhabitants, the plant is observed in the locality since more than 20 years. The population of
the taxon is not spreading as invasive species.
ACKNOWLEDGEMENT
The authors are thankful to the Principal, Regional Institute of Education (NCERT), Bhubaneswar for
providing the library and laboratory facilities for this study.
REFERENCES
Das PK, Mishra MK & Panigrahi G (1989) Fern Flora of Koraput District, Orissa. Plant Science Research
11(1): 7–44.
Dassanayake & Forsberg FR (1987) A Revised Hand Book to the Flora of Ceylon, Vol-6. Oxford & IBH
Publishing Co., pp. 399–400.
Gamble JS (1892) The Ferns of Panchmarhi and those of Mahendragiri. Indian Forester 18: 55–57.
Gamble JS (1915–36) Flora of the Presidency of Madras, London. Reprint Edition 1967, Kollkata.
Haines HH (1924) The Botany of Bihar and Orissa, Vol. 3. Adlard & Sons and West Newman Ltd., London pp.
1235–1280.
Hooker JD (1885) The flora of British India, Vol. 4. L. Reeve & Co., London.
Mishra MK, Dash SS & Das PK (1999) Addition to Flora of Koraput. Rheedia 9(2): 163–172.
Mooney HF (1950) Supplement to the Botany of Bihar and Orissa. Catholic Press, Ranchi.
Panigrahi G (1998) Pteridophytic Flora of Orissa. Plant Science Research 20(1&2): 1–45.
Panda PC & Das P (1997) Addition to Bibliography of Systematic Botany in Orissa. Journal of Economic &
Taxonomic Botany 21(1): 129–142.
Saxena HO & Brahmam M (1996) The Flora of Orissa, Vol. 4. Orissa Forest Development Corporation,
Bhubaneswar. pp. 2536–2654.
Singh NP, Mudgal V, Khanna KK & Srivastava SC (2001) Flora of Bihar. Botanical Survey of India, Calcutta.
Subbarao GV & Kumari GR (2006) Phytogeography of Eastern Ghats of India: A Historical Glimpse. ENVIS-
SDNP Newsletter Special Issue: 1–6.
Research article
Effect of salinity on osmolytes and relative water content of

selected rice genotypes
Mohammed Arif Sadik Polash1, Md. Arif Sakil2, Md. Tahjib-Ul-Arif2
and Md. Alamgir Hossain1*
1
Department of Crop Botany, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
2
Department of Biochemistry and molecular biology, Bangladesh Agricultural University,
Mymensingh-2202, Bangladesh
*Corresponding Author: alamgir.cbot@bau.edu.bd [Accepted: 22 August 2018]
Abstract: Reduction in relative water content (RWC) and photosynthesis (dry matter) is a
common effect of salinity stress. In the present study, 7 days old rice plants were exposed to 0
(control) and 100 mM NaCl salinity for 7 days to determine the osmolytes accumulation and
relative water content (RWC) in leaves. We observed that shoot dry matter, relative water content
and K+ content decreased significantly with the increasing of salinity. In contrast, Na+ and proline
content excessively increased in the leaves of salinity stressed plant. The results revealed that both
the organic (proline) and inorganic (K+) osmolytes accumulation may be responsible for stress
alleviation by retaining water in cell.
Keywords: Salinity - Relative water content - Osmolytes - Osmoregulation.
[Cite as: Polash MAS, Sakil MA, Tahjib-Ul-Arif M & Hossain MA (2018) Effect of salinity on osmolytes and
relative water content of selected rice genotypes. Tropical Plant Research 5(2): 227–232]
INTRODUCTION
Salinity is one of the most brutal environmental stresses that adversely influenced the capability of plant to
uptake water, and this quickly causes reduction in growth rate, along with a suite of metabolic changes (Munns
2002, Vaidyanathan et al. 2003). It seriously limits agricultural productivity with significant crop loss through
worldwide (Munns & Tester 2008). Now more than 800 million hectares of land throughout the world are salt
affected which is 6% of the world’s total land area (FAO 2008) and this area is increasing day by day because of
global warming with consequent rise in sea level and increase in tidal surges, particularly in coastal areas in the
globe (Wassmann et al. 2004). In Bangladesh, about 1.5 million hectares out of 2.85 million hectares of coastal
and off-shores land is affected by different degrees of salinity (Murshed 2008).
Salinity exerts its detrimental effect on rice into two phases. Firstly, high concentration of salt in the root
zone makes it harder for root to uptake water (osmotic stress). This is caused due to abscisic acid (ABA)
mediated root signal that limits the availability of water to plant cell, which leads to reduce relative water
content, slower plant growth and finally reduces dry mater accumulation (Fricke et al. 2004, Davies et al. 2005,
Zhang et al. 2006). Secondly, high concentration of salt within the plant commences ionic toxicity
(hyperosmotic stress) that leads to cell death, which is mitigated by either biosynthesis of organic osmolytes by
plant itself or sequestrated role of inorganic osmolyte (K+). Under various environmental stresses, plant cells
have experienced biosynthesis of some organic osmolytes such as sucrose (Hu et al. 2000), glycinebetain (Chen
& Murata 2002), mannitol (Abebe et al. 2003), trehalose (Garg 2002) and proline (Matysik et al. 2002, Misra &
Gupta 2005) that contribute in osmogerulation as well as maintaining cell turgor. Among these compatible
osmolytes, proline is one of the most frequently reported organic osmolyte which involved in osmoregulation in
rice (Matysik et al. 2002). A large number of data indicate a positive correlation between proline accumulation
and adaptation to salt stress in rice (Blum & Ebercon 1976, Hanson et al. 1977, Chandler & Thorpe 1987) whilst
some recent studies reveal that, inorganic osmolyte exhibits a greater role than organic osmolytes in relation to
osmoregulation and maintaining cell turgor in monocot plants (Shabala 2003, Rahman et al. 2007) because
proline biosynthesis is not always rapid in salt stressed plants (Hanson et al. 1979). Plants that contain high
Polash et al. 2018
K+/Na+ ratio can tolerate more salinity over those that contain low K+/Na+ ratio even though they accumulate
proline in several folds higher which is revealed in this experiment. Na+ and Cl- which are cytotoxic to cell
sequestrated in the vacuole of the cell in relation to K+ accumulation in the cytoplasm to balance osmotic
pressure of the ions in the vacuole (Munns 2002) while proline biosynthesis is not always rapid and occurred
when plant espoused to excessive salinity which might damage them fatally. So the present research was
designed to measure the magnitude of RWC (%), osmolytes accumulation and their physiological role in stress
mitigation under salt stress in selected rice genotypes.

Study material
Seeds of seven rice (Oryza sativa L.) genotypes were collected from Department of Genetics and Plant
Breeding, Bangladesh Agricultural University (BAU) and Plant Breeding Division of Bangladesh Institute of
Nuclear Agriculture (BINA). The experimental design was completely randomized design (CRD) with three
replications.
Detail of experimentation
The experiment was conducted in the Plant Physiology laboratory, Department of Crop Botany, Bangladesh
Agricultural University, Mymensingh from December 2016 to January 2017 To investigate the survivability of
seedlings surface sterilized (rinsed with NaOCl) 100 seeds of each rice genotype were placed on water-soaked
filter paper (Whatman filter paper-1) in Petridis with three replication at 25oC temperature with 70% relative
humidity and 400 µmol m-2 s-1 light intensity. Day/night ratio was maintained at 14/10 hours. After 3 days of
germination the seedlings were treated with 0 (control) and 100 mM NaCl salinity. On the 7th day after
Salinization the seedling survivability was recorded. Further, to evaluate the effect of salinity on the seedling
stage, seeds of each rice genotypes were surface sterilized with 10% sodium hypochlorite (NaOCl) for 10
minutes. The surface sterilized seeds were then placed on germinating pot for germination containing normal
water. After germination healthy and vigorous seedlings with the uniform shoot and root were selected and
transferred to perforated cork sheets, each of whole contained 6 seedlings. The sheets were floated on a nutrient
solution (Hoagland solution) in 32 liters plastic trays where the seedlings were grown hydroponically at 25oC
temperature with 70% relative humidity and a light intensity of about 400 µmol m-2 s-1. Day/night ratio was
maintained at 14/10 hours. The seedlings were cultured for 14 days at pH 6.5–7.0 in the growth chamber under
the control environment. At 15th days of normal growth, salinity was imposed with two concentration of salt (0
and 100 mM of NaCl) for 7 days. On the 7th day after Salinization plants were harvested and necessary data
were recorded.
Relative water content
A pre-dawn leaflet sample was taken from 3 plants of each replication in each treatment on 7th day after
salinization and its fresh weight was recorded immediately. The leaf sample was then incubated in deionised
water for 4 hours as described by Sairam et al. (2002) and turgid weight of the leaf sample was recorded. The
leaf sample was then filled into a brown paper bag and oven dried at 70oC temperature for 48 hours and the dry
weight of the sample was taken. The relative water content (RWC) was estimated as follows:
Growth measurement (Shoot dry matter)

For the measurement of shoot dry weight 6 seedlings of each replication of control and salinity treatments
were randomly selected and gently uprooted. The seedlings were separated into roots and shoots. Then the
shoots were dried in an aerated oven at 70oC temperature for 72 hours and shoot dry weights were recorded
expressed as % dry mater (DM).
Proline determination
Proline was determined according to the method of Bates et al., (1973). Briefly, fresh leaf sample (500 mg)
was homogenized in 5 ml of 3% sulphosalisylic acid by the means of a mortar and pestle and then centrifuged at
18000 g for 10 minutes to remove cell debris. Then the supernatant (2 ml) was taken in a test tube glacial acetic
acid (2 ml) and ninhydrin reagent (2 ml) was added. The reaction mixture boiled in a water bath for an hr. Then
the test tube was cooled in ice and toluene (6 ml) was added into the mixture and mixed thoroughly. After that
upper toluene phase was separated into a glass cuvette and free proline was estimated from proline standard (0–
50 µg ml-1) treated in an identical manner.
Na+ and K+ content

About 200 mg dry and ground leaves of each replication in each treatment were placed in a digestion tube
and 2.5 ml of digestion mixture (H2SO4 + HClO4) was added in each tube. After mixing the tube was allowed to
place in a heating block and heated for 5 minutes at 100oC temperature, then for 30 minutes at 180oC
temperature. After cooling 1 ml aliquots of 30% H2O2 were added and the content of digestion tube was
thoroughly mixed. Then the test tube was again heated for 2 hours at 330oC temperature (just below the boiling
point of the digestion mixture). The cooled, clear, digested mixture was diluted by deionised water and filtered.
Then the filtered was volume up to 100 ml and taken for analysis of Na+ and K+ concentration by using flame
photometer.
RESULTS
Seedling survivability
Seedling survivability significantly (P<0.05) decreased with the increasing salinity (Fig. 1). At 100 mM salt
concentration Pokkali (Salt resistant trait) exhibited 10% decrease in seedling survivability while BRRIdhan 29
(salt sensitive trait) exhibited 78.4% decrease over corresponding control. Ashfal, Benapol, Jamainaru, Gunshi
and Mohini showed 15, 23, 20, 36.5, 56.6% decrease in seedling survivability respectively over corresponding
control.
Figure 1. Seedling survivability of some selected rice genotypes under salt stress. [The vertical bars represent the ±SE]
Relative water content
Relative water content significantly (P<0.05) decreased with the increasing salinity (Fig. 2). About 6.6%
RWC declined in pokkali at 100 mM salt concentration compared to corresponding control. The other varieties
Ashfal, Benapol, Jamainaru, Gunshi, Mohini and BRRIdhan 29 with a treatment of 100 mM salinity exhibited a
decrease in relative water content at 8.1, 11.5, 12.1, 14.7, 18.3 and 21.7% respectively over corresponding
control.
Figure 2. Relative water content (%) of some selected rice genotypes under salt stress. [The vertical bars represent the ±SE]
Growth measurement (Shoot dry matter)
Shoot dry weight significantly (P<0.05) decreased with the increasing salinity. Here, pokkali showed
maximum dry matter production (1.89 g) at control which gradually decreased with the increase of salinity and
at 100 mM salt concentration it showed minimum (1.35 g) dry matter production with 28.57% reduction. The
other varieties Ashfal, Benapol, Jamainaru, Gunshi and Mohini with a treatment of 100 mM salinity exhibited a
decrease in shoot dry matter at 30, 30, 39.07, 40, 46.09 and 54.76 % respectively over corresponding control.
Polash et al. 2018
Proline accumulation
Proline accumulation is an exceptional physiological attitude in salinity stressed plant. Table 1 showed a
positive correlation between salinity and proline accumulation. With increasing, salinity proline accumulation
increased in all rice genotype. The rice genotypes pokkali, Ashfal, Benapol, Jamainaru, Gunshi, Mohini and
BRRIdhan 29 at 100 mM salinity showed an increase in proline accumulation about 200%, 209%, 211%, 188%,
283%, 400% and 500% respectively over corresponding control.
Table 1. Effect of salt stress (100 mM) Proline accumulation (µg g-1 FW) of selected rice genotypes.
Leaf proline content (µg g-1 FW)
Genotype
Control Salt stress
ab
Pokkali 10 20 b
a
Ashfal 11 23 a
b
Benapole 9 19 bc
b
Jamainaru 9 17 c
c
Gunshi 6 17 c
c
Mohini 5 20 b
cd
BRRIdhan 29 4 20 b
Note: Values marked with the same letter within the columns do not differ significantly @ 5% level of probability.
Na and K+ content
+
Na+ content showed increasing trend while K+ content showed a decreasing trend in saline stressed plant
(Table 2). At 100 mM NaCl stress Pokkali showed 2.91% Na+ while salt sensitive variety BRRIdhan 29 showed
3.90% Na+. Others such as, Ashfal, Benapol, Jamainaru, Gunshi, Mohini showed 2.39, 2.49, 2.17, 3.06 and
3.41% Na+ respectively. In case of K+ content Pokkali showed 2.20% K+ while salt sensitive variety BRRIdhan
29 showed 1.48% K+. Others such as, Ashfal, Benapol, Jamainaru, Gunshi, Mohini showed 2.67, 2.47, 2.68,
2.28 and 1.63% K+ respectively. Ashfal showed the height K+/Na+ ratio (1.117%) followed by Jamainaru
(1.101%). The lowest K+/Na+ ratio was found in BRRIdhan 29 (0.379%).
DISCUSSION
In the resent study, salinity reduces the seedling survivability (Fig. 1) because the high concentration of salt
in the root zone makes it harder for root to uptake water (Munns & Tester 2008). Water is essential for all
metabolic processes; lack of water may collapse all the metabolic processes of salt treated seedlings and finally
influenced the seedling survivability. Again accumulation of the toxic ions in cellular level may be thought
responsible for decreasing seedlings survivability.
Salinity reduces the relative water content (Fig. 2). When plants are subjected to salinity, firstly they face an
osmotic challenge that reduces water uptake by roots. Besides ABA mediated stomatal closure affects
transpiration pull that lead low/no water uptake by roots, entail low relative water content in the cell (Blatt &
Armstrong 1993).
Dry matter production also hampered during salinity (Fig. 3). Salinity disturbs the normal electron flow for
carbon reduction in the Calvin cycle by impairing water uptake, which acts as an electron donor. Again ABA
mediated stomatal closure limits CO2 fixation, resulting in decreasing carbon reduction by Calvin cycle (Lawlor
& Cornic 2002) that leads to low photosynthesis. As it is proved that dry matter production is associated with
photosynthesis, low photosynthesis rate causes low dry matter production.
Figure 3. Shoot dry matter (g) of some selected rice genotypes under salt stress. [The vertical bars represent the ±SE]
Proline accumulation is a common metabolic process to be involved in stress tolerance mechanism (Lutts et
al. 1999, Sivakumar et al. 2002). In the present study, a significant increase in proline accumulation was
recorded in rice leaf after 7 days of exposure to NaCl salinity (Table 1). The result also revealed that the
magnitude of proline accumulation was positively associated with the concentration of NaCl in the culture
solution. These results are likely to with the findings of some earlier study (Chen & Murata 2002, Abebe et al.
2003). The mechanism that evolved in proline accumulation is, salinity stress causes ABA mediated stomatal
closure that limits the fixation of CO2, resulting in decreasing carbon reduction by Calvin cycle (Lawlor &
Cornic 2002) makes NADP+ non available for electrons acceptance during photosynthesis. In this
circumstances, Photosynthetic reducing power NADPH2 donate election to glutamate for biosynthesis of proline
and regenerate NADP+ for further election acceptance (Wang et al. 2007).
Table 2. Effect of salt stress on Na+ and K+ content (%) of selected rice genotypes.
Genotype Na+ and K+ content (%) at leaf blade
Na+ K+ K+/ Na+
e c
Pokkali 2.19 2.20 1.004b
d a
Ashfal 2.39 2.67 1.117a
d b
Benapole 2.49 2.47 0.991b
e a
Jamainaru 2.17 2.68 1.101a
c c
Gunshi 3.06 2.28 0.745c
b d
Mohini 3.41 1.63 0.478d
a e
BRRIdhan 29 3.90 1.48 0.379e
Note: Values marked with the same letter within the columns do not differ significantly @ 5% level of probability.
Recently some studies revealed that, inorganic osmolyte exhibit a greater role than organic osmolytes in
relation to osmoregulation and maintaining cell turgor in rice which is revealed in this experiment in table 2.
According to Hanson et al. (1977) and Moftah & Miche (1987), Proline biosynthesis is not always rapid in salt-
stressed plants, the maximum accumulation of proline might have occurred when plant espoused to excessive
salinity which might damage them fatally. In these circumstances, K+ an inorganic osmoticum other than proline
might be involved in retaining leaf water content as well as cell turgidity by maintaining ionic balance (Shabala
2003). The evolved mechanism behind this science is Na+ and Cl- are sequestrated in the vacuole of the cell in
relation to K+ accumulation in the cytoplasm and organelles to balance osmotic pressure of the ions in the
vacuole (Munns 2002). Again K+ (known as water buoy) plays an important role in the opening of stomata by
regulating guard cells with its water holding capacity. If the K+ concentration is high in the guard cell it keeps
the stomata open for carbon fixation thus help in plant growth and productivity (Murata et al. 1994, Shabala
2003). So plant that accumulates more proline over control but content less k+/Na+ ratio are less tolerance to
stress and vice-versa (Table 2).
CONCLUSION
From this study, it might be concluded that, salinity affects the seedling survivability and also exhibits the
reduction in relative water content and dry matter accumulation. Photosynthetic impairment induced by salinity
was mitigated by proline biosynthesis whereas its function in maintaining ionic balance and cell turgidity was
not so strong over k+.
ACKNOWLEDGEMENTS
The authors are thankful to the Head, Department of Crop Botany; Director, Dr. Mohammad Hussain
Central Labotary, Bangladesh Agricultural University. For providing all utensil required in the experiment.
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Garg AK, Kim JK, Owens TG, Ranwala AP, Choi YD, Kochian LV & Wu RJ (2002) Trehalose accumulation
in rice plants confers high tolerance levels to different abiotic stresses. Proceedings of the National Academy
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Research article
Comparative study on structural composition and community

association of Nambor Wildlife Sanctuary and its South-
Westward extended Bornewria forest, Assam, India
Kuntala N. Barua*, Girish Gogoi and Protul Hazarika
Ecology and Biodiversity Division, Rain Forest Research Institute, Post Box No. 136,
Sotai, Jorhat-785001, Assam, India
*Corresponding Author: baruahkn@icfre.org [Accepted: 27 August 2018]
Abstract: Knowledge of species composition and diversity are of utmost importance, not only to
understand the structure of a forest community but also for planning and implementation of
conservation strategy of the community. An extensive field study was undertaken to ascertain the
structural composition, species diversity and community association of two forest sites i.e.
Nambor Wildlife Sanctuary (NWLS) and its South-Westward extended Bornewria forest of
Assam, India. The forests represent Tropical moist semi-evergreen and moist mixed deciduous
type. The entire area was embraced with a fragmented block of Gondowana formation. A total of
261 plant species were observed from the two forests sites in floristic assessment. Out of which
247 species were recorded from NWLS and in Bornewria forest 136 were enumerated. The
overexploitation and shifting cultivation adversely affected the total forested area and species
composition of Bornewria forest. Phytosociological studies showed that Vatica lanceaefolia
(15.47) followed by Magnolia hodgsonii (10.97), Castanopsis hystrix (10.02) and Mesua ferrea
(9.56) were dominated in NWLS. However, in case of Bornewria forest, Hydnocarpus kurzii
expressed its dominance with highest IVI values (15.98), followed by Dysoxylum excelsum
(13.52), Mesua ferrea (12.37) and Stereospermum tetragonum (11.87). Plant species diversity was
quantitatively higher in NWLS in comparison to Bornewria forest because of ecological
destabilization and disturbance in their natural abode. Study on regeneration status of NWLS
revealed that 67.42% trees were naturally regenerated. Mesua ferrea and Vatica lanceifolia were
the most ecologically successful species with IVI of 7.66 and 5.27 in the seedling stage. In
Bornewria forest site 42 regenerating tree individuals were recorded. The maximum quantity of
seedlings of Hydnocarpus kurzii was noticed in the forest which showed mass regeneration status
of the species. Both the forest desires to curb the anthropogenic disturbance, so that protect the
integrity of the forest.
Keywords: Phytosociological studies - Plant inventory - Regeneration - Species diversity.
[Cite as: Barua KN, Gogoi G & Hazarika P (2018) Comparative study on structural composition and community
association of Nambor Wildlife Sanctuary and its South-Westward extended Bornewria forest, Assam, India.
Tropical Plant Research 5(2): 233–242]
INTRODUCTION
Floristic composition of vegetation and species diversity reflect the gene pool and adaptation potential of the
community (Odum 1971). Knowledge of species composition and diversity of tree species is of great
importance not only to understand the structure of a forest community but also for planning and implementation
of conservation strategy of the community (Bajpai et al. 2012, Malik 2014, Malik & Bhatt 2015, Masens et al.
2017). It is a prerequisite for the foresters to understand the structural attributes of the forest for better
silviculture and management practices. In forest management, regeneration study not only reflects the current
status but also gives an idea about the possible changes in forest composition in the future (Mishra et al. 2013,
Sharma et al. 2014, Hanief et al. 2016, Malik & Bhatt 2016). Survival and growth of seedlings/saplings
Received: 11 April 2018 Published online: 31 August 2018
Barua et al. 2018
determine the successful regeneration of the species which is one of the most important steps toward achieving
long-term sustainability of forests (Saikia & Khan 2013, Malik 2014, Malik & Bhatt 2016).
However, the structure and nature of the plant community are determined by the species composition and
their ecological amplitude. The quantitative characters are very important in the analysis of comparative
community structure of different forest stands. Study of community structure in the natural forests of various
climatic zones of the country have been done by different authors in the recent past (Nath et al. 2000, Pande et
al. 2001, Pandey & Shukla 2003, Galav et al. 2005, Naithani et al. 2006, Khatri et al. 2009, Sarkar & Devi
2014, Naidu & Kumar 2016, Bajpai et al. 2017). The species diversity and community structure of Nambor
Wildlife Sanctuary and Bornewria forest of Assam have not been explored well and a very little information is
available so far (Prasad 2001). Therefore, the present study was carried out on structural composition, diversity
and community association of the two forest sites.

Study site
Two natural forest stands Nambor Wildlife Sanctuary (NWLS) (primary) and Bornewria forest (secondary)
geographically located at latitude of 26.36° N; longitude of 93.86° E and latitude of 26.24° N; longitude of
93.82° E respectively were selected for the present study in Karbi Anglong Autonomous District Council of
Assam (Fig. 1). The forests are classified as Moist Semi-Evergreen Forest mixed with East Himalayan Moist
Mixed Deciduous Forest (Champion & Seth 1968). The entire area is of great importance in paleohistorical
point of view as it comprises a fragmented block of Gondowana formation. The total forested area of NWLS is
37.0 km2. Bornewria is a South-west ward extended part of original Nambor reserve forest now converted to a
secondary denuded one. Over a period, Bornewria forest has been adversely affected by shifting cultivation
(Jhum), an age-old ecologically hazardous traditional cultivation practice. With the rapid shortening of the
fallow cycle, the farmers shift towards the forested land and forest has been deforested gradually. Invasion of
secondary species such as Hydnocarpus kurzii (King) Warb., Dysoxylum excelsum Blume, Stereospermum
tetragonum DC. etc. changes the original forest scenario.
Figure 1. Map of Nambor Wildlife Sanctuary & Bornewria forest in Assam, India.
The climate is the typically humid sub-tropical type and receiving nearly 1200 mm total average annual
rainfall. The soil comprises old alluvial and red laterite type. The texture is sandy loam, soil pH ranges from 4.8
to 6.31 and soil reaction is acidic. The highest relative humidity was observed to be 90.4% (in the month of
August). The average maximum temperature varies from 28.65–31.24 °C and average minimum temperature
from 14.67–19.38 °C.
Methodology
Phytosociological studies of the two selected forest stands were carried out by randomly laying quadrats of
10m × 10m, 5m × 5m and 1m × 1m for trees, shrubs/saplings and herbs/seedlings replicated with 40, 80 and 160
numbers of quadrats respectively for each site. The species were categorized as a tree (>3 m height), shrub
(height above 0.5 to 3 m) and herb (less than 0.5 m) height (Khan 1961). The size and the number of sampling
quadrats were determined following Mishra (1968) and Kershaw (1973). Species diversity (H') was calculated
by using Shannon and Wiener formula (Shannon & Wiener 1963). The concentration of dominance values was
assessed by Simpson’s index (Simpson 1949). The similarity coefficient for common and rare species was
calculated by following Sorenson and Jaccard’s coefficient (Magurran 1988). Motyka’s index (%) was
calculated by following formula ISmo =2MW/MA+MB X 100 that expresses the similarity percentage for each
pair of vegetation types
Sorensen Co-efficient Ss = 2a/(2a+b+c)
Jaccard’s Co-efficient Sj = a/(a+b+c)
Where a = Number of species occurring in both sample.
b = Species occurring only in sample B
c = Species occurring only in sample A.
Identification of the plant species was done with the help of floras (Kanjilal et al. 1934–40, Hooker 1872–
97) and by consulting herbaria of Botanical Survey of India, Eastern Regional Centre, Shillong and Central
National Herbarium, Howrah. The nomenclature of each plant species was confirmed with the help of databases
like 'The Plant List' and 'Tropicos'. The abundance to frequency ratio (A/F) of different species was computed to
define the distribution pattern of the species (Whitford 1949). This ratio indicates regular (<0.025), random
(0.025 to 0.050) and contiguous (>0.050) distribution (Cottam & Curtis 1956).
RESULTS AND DISCUSSION

Plant Inventory
A total of 261 plant species were found distributed in both the forests during floristic assessment. Out of
which Nambor Wildlife Sanctuary (NWLS) was comprised of 247 species belong to 75 families and 179 genera.
On the other hand, Bornewria forest was comprised of 136 species belong to 54 families and 110 genera. The
breakup of the families, genera, species, dicotyledonous, monocotyledon, ferns and Gymnosperms are shown in
table 1. A total of 30 fern and fern allies were recorded from Nambor WLS whereas, only 10 fern and fern allies
were recorded in the Bornewria forest. Fern and fern allies like Adiantum caudatum L., Angiopteris sp.,
Lygodium flexuosum (L.) Sw., Polypodiodes amoena (Wall. ex Mett.) Ching, Pteris cretica L.,
Sphaerostephanos unitus (L.) Holttum and Stenochlaena palustris (Burm. f.) Bedd. were found in both the
habitats. Gnetum gnemon L., a highly evolved gymnosperm species distributed in the lower strata of both the
forests and established as an important component of the vegetation that reflects the old land history of this
region.
Table 1. Plant inventory of Nambor Wildlife Sanctuary and Bornewria forest of Assam.
Dicots Monocots Ferns Gymnosperms
Numbers % Numbers % Numbers % Numbers % Total
Nambor WLS
Families 49 65 9 12 16 21 1 1.33 75
Genera 120 67.04 34 18.99 24 13.41 1 0.56 179
Species 165 66.80 51 20.65 30 12.14 1 0.40 247
Bornewria Forest
Families 40 74.07 8 14.81 5 9.26 1 1.85 54
Genera 78 71.56 21 19.26 10 9.17 1 0.91 110
Species 100 73.53 25 18.38 10 7.35 1 0.73 136
Artocarpus chama Buch.-Ham., Mesua ferrea L., Morus macroura Miq., Phoebe goalparensis Hutch.,
Aglaia spectabilis (Miq.) S.S.Jain & S.Bennet etc. were the predominant species in moist semi-evergreen forest.
Other trees like Haldina cordifolia (Roxb.) Ridsdale, Lagerstroemia speciosa (L.) Pers., Albizia procera
Barua et al. 2018
(Roxb.) Benth., Bombax ceiba L., Schima wallichii Choisy, Stereospermum tetragonum etc. along with vascular
climbers and epiphytes were common plant species of the moist mixed deciduous forest.
The upper canopy was predominated by deciduous tree species whose leafless period is short viz. Alstonia
scholaris (L.) R. Br., Artocarpus chama, Mallotus nudiflorus (L.) Kulju & Welzen, Morus macroura,
Stereospermum tetragonum, Tetrameles nudiflora R. Br., Zanthoxylum budrunga (Roxb.) DC. etc. Whereas,
middle and lower canopy were of more or less evergreen character. In the middle canopy, trees like Elaeocarpus
sikkimensis Mast., E. tectorius (Lour.) Poir., Canarium resiniferum Bruce ex King, Castanopsis hystrix Hook. f.
& Thomson ex A. DC., Machilus gamblei King ex Hook. f., Vatica lanceifolia (Roxb.) Blume, Mesua ferrea
with evergreen characteristic were found predominant in the primary forest.
The lower canopy vegetation is mainly influenced by the size of seedling population and survivability. The
Density of trees in a forest is largely depending upon the response of the tree seedlings to the prevailing
microenvironment. The present observation noted that the seed germination and survival percentage of
Hydnocarpus kurzii, Vatica lanceifolia, Mesua ferrea, Litsea laeta (Wall. ex Nees) Hook.f. etc. were maximum
and found well established in the forest floor. Better establishment of the tree seedlings was recorded in the
primary forest strand near the periphery rather than the core areas that may be due to the lack of threshold light
intensity available to the seedlings (Whitmore 1975, Abbott 1984, Primack et al. 1985).
Orchids are susceptible and selective for their habitats. The humid forest of NWLS has cradled the rich
heritage of both terrestrial and epiphytic orchids. A total of 23 orchid species were recorded and Acampe
papillosa (Lindl.) Lindl., Bulbophyllum spp. Calanthe sylvatica (Thouars) Lindl., Dendrobium acinaciforme
Roxb., D. lituiflorum Lindl., D. anceps Sw., D. lindleyi Steud., Eria lasiopetala (Willd.) Ormerod, Gastrochilus
calceolaris (Buch.-Ham. ex Sm.) D.Don, G. inconspicuus (Hook.f.) Kuntze, Oberonia mucronata (D.Don)
Ormerod & Seidenf., Zeuxine gracilis (Breda) Blume were the dominant orchids in both the site. An endangered
spectacular orchid Anoectochilus brevilabris Lindl. (Jewel orchid) also reported their occurrence in the dense
damp forest floor of the primary forest, but now severely influenced by biotic interference. In Bornewria forest
(Secondary forest) found only 12 tropical orchids. The ecological destabilization due to various anthropogenic
factors a number of orchid species have been on the verge of disappearance.
A total of 35 species of climbers and scandent shrubs were recorded in the primary natural forest which are
the curious botanical wealth of this forest. Whereas, only 20 species of climbers and scandent shrubs were found
in the secondary forest. Abrus precatorius L., Aristolochia saccata Wall., Beaumontia grandiflora Wall.,
Dioscorea spp., Dischidia major (Vahl) Merr., Dischidia major (Vahl) Merr., Licuala peltata Roxb. ex Buch.-
Ham., Piper spp., Pothos spp., Smithia grandis Baker, Tacca integrifolia Ker Gawl. etc. were the species of
climbers and scandent shrubs found in the primary forest that twisted or straggled the trees which bear the
characteristic of the moist evergreen forest. Some common climbers and plants of straggling habit found
dominated in the secondary forest were Dalhousea bracteata Grah., Mikania micrantha Kunth., Mimosa
rubicaulis Lam., M. himalayana Gamble, Piper griffithii C. DC., Pothos scandens L., Thunbergia grandiflora
(Roxb. ex Rottl.) Roxb., etc.
Vegetation Analysis
Phytosociological studies in the primary natural forest showed that Vatica lanceaefolia (Roxb.) Blume
(15.47) was the predominant tree species followed by Magnolia hodgsonii (Hook.f. & Thomson) H.Keng
(10.97), Castanopsis hystrix (10.02) and Mesua ferrea (9.56). However, in case of secondary forest
Hydnocarpus kurzii expressed its dominancy with highest IVI values (15.98), which was followed by
Dysoxylum excelsum (13.52), Mesua ferrea (12.37) and Stereospermum tetragonum (11.87) (Table 2).
Gnetum gnemon (IVI 22.64) was found dominant in the shrub layer of primary forest followed by
Phlogacanthus curviflorus (Wall.) Nees (IVI 17.38), which is highly medicinal. Maximum number of saplings
of Hydnocarpus kurzii (IVI 15.14) followed by Litsea laeta (IVI 13.01) and Mesua ferrea (11.85) etc. were
found in the secondary forest. The floor of the primary forest was found covered by the herbaceous species like
Cheilocostus speciosus (J.Koenig) C.D.Specht, Oplismenus compositus (L.) P.Beauv., Curculigo orchioides
Gaertn., Alpinia nigra (Gaertn.) Burtt, Selaginella biformis A. Braun ex Kuhn, Setaria palmifolia (J.Koenig)
Stapf, Setaria pumila (Poir.) Roem. & Schult., Cyperus pilosus Vahl, etc. On the other hand, in the secondary
forest the edible fern species Diplazium esculentum (Retz.) Sw. (IVI 24.25) was found dominated in ground
strata followed by Colocasia esculenta (L.) Schott (IVI 19.69) and Cheilocostus speciosus (IVI 15.94) (Table 2).
As reported by Knight (1975) the value of Diversity Index varies from 5.06–5.40 for tropical forest. The
present study showed that Diversity index (H') of the primary forest were 5.7, 4.727, 4.1 for trees,
shrubs/saplings and herbs/seedlings respectively, which was found within the range. On the other hand, the
Table 2. Importance value index (IVI) of Nambor Wildlife Sanctuary and Bornewria forest. [Tree- >3 m, Shrub- 0.5–2 m,
Herb- <0.5 m hight]
Nambor WLS Bornewria forest
SN Name of species
Tree Shrub Herb Tree Shrub Herb
1 Abacopteris lakhimpurensis Ching 2.81 5.61
2 Abroma augusta (L.) L.f. 0.69 2.21
3 Acanthus leucostachyus Wall. ex Nees 5.22
4 Achyranthes aspera L. 5.19
5 Actinodaphne obovata (Nees.) Blume 2.12 2.5 5.5
6 Aesculus assamica Griff. 1.35 1.45
7 Aglaia cucullata (Roxb.) Pellegr. 2.91 1.61 1.09 1.33
8 Alangium chinense (Lour.) Harms 1.59 0.71 3.18
9 Albizia procera (Roxb.) Benth. 2.62
10 Alpinia nigra (Gaertn.) Burtt 12.47 3.67 3.15
11 Alstonia scholaris (L.) R. Br. 0.77 1.27 3 5.95 3.84
12 Amischotolype gracilis (Ridl.) I.M. Turner 10
13 Antidesma bunius (L.) Spreng. 1.09
14 Antidesma montanum Blume 3.07 3.42
15 Artocarpus chama Buch.-Ham 2.24 1.61 3.42 11.15 1.91
16 Artocarpus lacucha Buch.-Ham. 2.18 0.99 1.91
17 Baccaurea ramiflora Lour. 3.83 4.55 5.33 5.64
18 Balakata baccata (Roxb.) Esser 3.22 3.15 7.08
19 Bauhinia malabarica Roxb. 6.99 2.04 3.93 1.14 4.12
20 Benkara fasciculata (Roxb.) Ridsdale 2.6
21 Bischofia javanica Blume 3.66 1.23 1.64 2.27
22 Blumea lacera (Burm.f.) DC. 3.36
23 Boehmeria glomerulifera Miq. 1.95
24 Boehmeria nivea (L.) Gaudich. 2.39
25 Bombax ceiba L. 3.63 7.79
26 Bridelia retusa (L.) A.Juss. 1.49 2.71
27 Bridelia stipularis (L.) Blume 1.51
28 Butea monosperma (Lam.) Taub. 1.32
29 Calanthe sylvatica (Thouars) Lindl. 5
30 Callicarpa arborea Roxb. 4.01 1.51 3.12
31 Callicarpa longifolia Lamk. 5.4 2.79
32 Canarium resiniferum Bruce ex King 3.5 3.96
33 Carex indica L. 5.66
34 Castanopsis indica (Roxb. ex Lindl.) A.DC. 2.36
35 Castanopsis armata (Roxb.) Spach. 5.47 3.75 1.62 8.65 2.49
36 Castanopsis hystrix Hook. f. & Thomson ex A. 10 5.12 0.78 5.94 1.89
DC.
37 Castanopsis tribuloides (SM.) A.DC. 1.56
38 Catunaregam spinosa (Thunb.) Tirveng. 1.39 1.95
39 Chloranthus elatior R. Br. 0.78
40 Chromolaena odorata (L.) R.M.King & H.Rob. 6.75 5.17
41 Chrysophyllum roxburghii G.Don 1.7
42 Chrysopogon zizanioides (L.) Roberty 2.62
43 Chukrasia tabularis A.Juss. 3.24 2.1 2.44 2.91 3.91
44 Cinnamomum glanduliferum (Wall) Meissn. 0.87 5.66
45 Cinnamomum glaucescens (Nees) Hand-Mazz. 4.69 2.55 2.12 4.53
46 Cinnamomum bejolghota (Buch.-Ham.) Sweet 3.94 4.26 1.69 3.26 4.64
47 Clerodendrum indicum (L.) Kuntze 0.89 5.23
48 Clerodendrum laevifolium Blume 3.07
49 Clerodendrum glandulosum Lindl. 3.64
50 Clerodendrum infortunatum L. 2 6.51
51 Coffea benghalensis B.Heyne ex Schult. 5.7 1.78 3.81
52 Colocasia esculenta (L.) Schott 4.39 19.7
53 Combretum decandrum Jacq. 1.17 1.97
54 Cordia dichotoma G. Forst. 0.72 2.85 4.36
55 Cordia myxa L. 2.85
56 Cheilocostus speciosus (J.Koenig) C.D.Specht 16.67 15.9
57 Croton joufra Roxb. 3.88 1.31
Barua et al. 2018
58 Croton caudatus Geiseler 5.61 3.1

59 Croton persimilis Mull. Arg. 1.58 1.69 3.47 4.38
60 Croton tiglium L. 0.74 2.46
61 Curculigo orchioides Gaertn. 13.39 2.38
62 Curcuma zerumbet Roxb. 5 3.27
63 Cyperus pilosus Vahl. 3.32 2.81
64 Dalbergia stipulacea Roxb. 0.92
65 Dalhousiea bracteata (Roxb.) Benth. 3.96 1.97
66 Delima sarmentosa L. 3.25
67 Dicliptera chinensis (L.) Juss. 1.24
68 Dillenia indica L. 5.29 2.69 2.68 6.46 6.38 1.34
69 Dillenia pentagyna Roxb. 4.3 1.48 1.33 5.34
70 Diplazium esculentum (Retz.) Sw. 24.3
71 Drimycarpus racemosus (Roxb.) Hook f. ex 3.81
Marc.
72 Duabanga grandiflora (Roxb.ex DC.) Wall. 2.76 1.22 3.38
73 Dysoxylum excelsum Blume 7.93 3.35 0.78 13.52 6.43
74 Elaeocarpus sphaericus (Gaertn.) K. Schum 1.99 1.09 0.83
75 Elaeocarpus rugosus Roxb. ex G.Don 2.66
76 Elaeocarpus sikkimensis Mast. 1.9
77 Elaeocarpus tectorius (Lour.) Poir. 5.32 2.88 0.71 3.36 0.89
78 Erythrina stricta Roxb. 0.78 2.06
79 Eurya japonica Thunb. 2.63 2.81 1.94
80 Eurya acuminata DC. 3.09 7.87 5.55
81 Evodia meliifolia (Hance ex Walp.) Benth. 3.31 1.03 1.94
82 Ficus hispida L.f. 3.54 3.4 3.05
83 Ficus racemosa L. 0.97 2.05
84 Ficus religiosa L. 1.09 1.41 1.23
85 Ficus sarmentosa Buch-Ham. ex Sm. 2.49 1.03 3.12 1.18
86 Ficus nervosa B.Heyne ex Roth 3.75 6.52
87 Fimbristylis bisumbellata (Forsk.) Bubani 2.75
88 Floscopa scandens Lour. 3.56
89 Flueggea virosa (Roxb. ex Willd.) Royle 1.78 3 3.27 2.84 1.33
90 Garcinia cowa Roxb. ex Choisy 1.87 1.85 1.91 1.16
91 Garcinia xanthochymus Hook.f. ex T.Anderson 3.77 2.4
92 Girardinia palmata (Forssk.) Gaudich. 1.43
93 Globba multiflora Wall. ex Baker 7.72
94 Glochidion ellipticum Wight 1.54
95 Glycosmis pentaphylla (Retz.) DC. 3.23 10.6 2
96 Gmelina arborea Roxb. 1.61 1.77
97 Gnetum gnemon L. 22.64 8.45 12.6 3.11
98 Gomphostemma parviflorum Wall. ex Benth. 0.96
99 Grewia multiflora Juss. 1.95 1.18 4.6 3.07
100 Haldina cordifolia (Roxb.) Ridsdale 4.91
101 Hedychium spicatum Sm. 6.18 1.03
102 Heteropogon contortus (L.) P.Beauv. ex Roem. 1.07
& Schult.
103 Hydnocarpus kurzii (King) Warb. 3.95 2.03 1.11 15.98 15.1 29.6
104 Justicia procumbens L. 4.38
105 Knoxia sumatrensis (Retz.) DC. 5.96 5.19
106 Kydia calycina Roxb. 4.81 1.55 4.77 1.14
107 Lagerstroemia speciosa (L.) Pers. 5.33 1.43 2.08 3.67
108 Lantana camara L. 4.28
109 Leea asiatica (L.) Ridsdale 7.91 1.14
110 Leea indica (Burm. f.) Merr. 5.5 5.67
111 Lepidagathis incurva Buch Ham. ex D. Don. 1.37
112 Litsea monopetala (Roxb.) Pers. 2.1 4.96 2.74 3.65 5.05
113 Litsea glutinosa (Lour) Robin. 3.21 5 1.21
114 Litsea laeta (Wall. ex Nees) Hook.f. 3.7 3.28 0.78 6.01 13 6.56
115 Litsea salicifolia (Roxb. ex Nees) Hook. F. 4.35
116 Litsea lancifolia (Roxb. ex Nees) Fern.-Vill. 3.49 5.02 2.43 4.18 4.97 2.5
117 Litsea nitida (Roxb.) Hook. f. 0.68 5.02 7.41 5.89 1.15
118 Macaranga peltata (Roxb.) Muel. Arg. 8.11 2.95 2.46

119 Machilus gamblei King ex Hook. f. 4.86 0.78
120 Magnolia griffithii Hook. f. & Thomson 2.6 3 3.07
121 Magnolia hodgsonii (Hook. f. & Thomson) H. 11 5.5 2.54 10
Keng
122 Magnolia insignis Wall. 1.15 2.72 3.79 2.06 2.86
123 Magnolia mannii (King) Figlar 3.93 6.97
124 Magnolia montana (Blume) Figlar 2.95
125 Magnolia pterocarpa Roxb. 1.67 1.91
126 Magnolia champaca (L.) Baill. ex Pierre 1.21
127 Mallotus nudiflorus (L.) Kulju & Welzen 1.63 2.42 3.48
128 Mallotus philippensis (Lamk.) Mull. Arg 3.56 5 3.29 3.08 1.06
129 Mangifera indica L. 1.4 1.66 2.18 1.07
130 Mangifera sylvatica Roxb. 1.83
131 Mansonia dipikae Purkayastha 4.19 5.58 1.65 2.97 7.28 7.59
132 Melastoma malabathricum L. 2.37
133 Merremia umbellata (L.) Hallier f. 3.33
134 Mesua ferrea L. 9.56 6.21 7.66 12.37 11.9 6.54
135 Mikania micrantha Kunth. 8.89
136 Mimosa pudica L. 2.07
137 Mimosa himalayana Gamble 3.19 3.09
138 Morus macroura Miq. 2.84 1.76 3.82 1.61
139 Mussaenda roxburghii Hook. f. 3.59 1.71 1.96
140 Nelsonia canescens (Lam.) Spreng. 6.38
141 Neolamarckia cadamba (Roxb.) Bosser 4.32 1.73 2.85 1.33
142 Ocotea lancifolia (Schott) Mez 2.92 6.17 2.82 2.59 2.05 2.89
143 Oplismenus burmanni (Retz.) P.Beauv. 3.24
144 Oplismenus compositus (L.) P. Beauv. 15.61 12.3
145 Oroxylum indicum (L.) Kurz 1.33 2.67 2.59
146 Oxalis corniculata L. 1.07
147 Oxyceros longiflorus (Lam.) T.Yamaz. 6.49
148 Oxyspora paniculata (D. Don) DC. 8.28
149 Panicum repens L. 5.94
150 Phlogacanthus curviflorus (Wall.) Nees 17.38 7.03
151 Phoebe goalparensis Hutch. 2.45
152 Phyllanthus fraternus G.L.Webster 0.68 2.56
153 Piper griffithii C. DC. 2.12 13
154 Polygonum barbatum L. 5.87 4.77
155 Pothos scandens L. 2.83
156 Premna latifolia Roxb. 3.16 4.15 1.02
157 Psychotria calocarpa Kurz 1.92
158 Pteridium equilinun (L.) Kuhn. 2.43 4.43
159 Pteris cretica L. 1.8 3.36
160 Quercus semiserrata Roxb. 1.05
161 Rhamnus napalensis (Wall.) M.A. Lawson 2.5 4.51
162 Schima wallichii Choisy 5.06
163 Schoenoplectiella juncoides (Roxb.) Lye 2.27
164 Selaginella biformis A.Braun ex Kuhn 7.34
165 Setaria palmifolia (J.Koenig) Stapf 5.78 7.26
166 Setaria pumila (Poir.) Roem. & Schult. 3.79 12.9
167 Sida cordifolia L. 2.84
168 Smilax zeylanica L. 2.11
169 Smithia grandis Baker 2.54
170 Spermacoce articularis L.f. 3.03
171 Sphaerostephanos unitus (L.) Holttum 6.47
172 Stephania japonica (Thunb.) Miers 4.87
173 Sterculia villosa Roxb. 4.57 5.07 1.94
174 Stereospermum tetragonum DC. 4.75 3.1 11.87 2.14 4.4
175 Strobilanthes adnatus C.B.Clarke 3.75
176 Syzygium fruticosum DC. 3.6
177 Tabernaemontana divaricata (L.) R.Br. ex 2.35 2.39 2.87 1.44
Roem. & Schult.
Barua et al. 2018
178 Tarennoidea wallichii (Hook.f.) Tirveng. & 0.75 0.5 3.53 3.98
Sastre
179 Terminalia myriocarpa Van Heurck & 1.7 1.22
Mull.Arg.
180 Terminalia bellirica (Gaertn.) Roxb. 4.33 1.18
181 Terminalia chebula Retz. 3.27 1.1 2.41 3.15 1.33 1.15
182 Tetrameles nudiflora R.Br. 5.65 3.96 3.12 1.06
183 Thunbergia grandiflora (Roxb. ex Rottl.) Roxb. 1.77 4.2
184 Toona ciliata M. Roem. 3.09 2.19 2.09 0.96 1.33
185 Triadica cochinchinensis Lour. 8.64 3.27 3.75 5.47 6.73 1.4
186 Urena lobata L. 3.95
187 Vatica lanceaefolia (Roxb.) Blume 15.5 6.08 5.27 1.85 5.15
188 Vitex peduncularis Wall. ex Schaue 1.6 2.08
189 Wrightia coccinea Roxb. ex Hornem. 1.26
190 Zanthoxylum budrunga (Roxb.) DC. 1.83 2.29
191 Ziziphus jujuba Mill. 1.38 1.35 0.92
diversity index of secondary forest were found 4.760 4.491 and 4.016 respectively. The values of plant species
diversity of primary natural forests were always higher in comparison to the secondary forest which indicates
high species richness considering the population of individual species. Concentrations of Dominance (CD) of
the primary forest were found 0.027, 0.042 and 0.046 for trees, shrubs/saplings and herbs/seedlings respectively
whereas, 0.031, 0.0369 and 0.062 for the secondary forest (Table 3). Species diversity and concentration of
dominance are generally inversely related.
Table 3. Diversity Index (H’) and Concentration of dominance (CD) of different canopy height in
Nambor Wildlife Sanctuary and Bornewria forest. [Tree- >3 m, Shrub- 0.5–2 m, Herb- <0.5 m hight]
Nambor WLS Bornewria forest
Indices
Tree Shrub Herb Tree Shrub Herb
H' 5.736 4.727 4.1 4.760 4.491 4.016
CD 0.027 0.042 0.046 0.031 0.0369 0.062
Similarity coefficients of both the Nambor and Bornewria Forest were calculated and compared (Table 4).
The Sorenson’s similarity co-efficient (0.3772) was found higher than Jaccard’s co-efficient (0.2324). Motyka’s
similarity index was found 60.57% between the forest sites (Table 4). The contagious trend of the distribution
pattern of plant species was found in both the forests, which has also been reported by Kershaw (1973) and
Greig-Smith (1957).
Table 4. Similarity co-efficient of studied natural forest.
SN Similarity Indices Nambor WLS Bornewria Forest
1 Sorenson,s co-efficient (Ss) 0.3772
2 Jaccard’s co-efficient (Sj) 0.2324
3 Motyka’s index 60.57
Regeneration performance of a tree species is characterized by its population constitution in different life
phases i.e. tree, sapling and seedling (Pokhriyal et al. 2010) as well as depends upon the existence of a sufficient
number of seedlings and saplings. In NWLS out of 89 reported tree species, 60 species were found regenerated
naturally i.e. regeneration percentage 67.42%. Mesua ferrea and Vatica lanceaefolia were found the most
ecologically successful species with IVI of 7.66 and 5.27 respectively in the seedling stage. Whereas, in
Bornewria forest out of 62 tree species, 42 species were found regenerated naturally i.e. regeneration percentage
67.74%. So, the regeneration percentage in both the forests was found almost similar. A maximum number of
seedlings of Hydnocarpus kurzii (IVI 29.62) was found in the Bornewria forest which showed mass
regeneration status of the species. Seedling diversity was found poor in the Bornewria forest. According to
Mishra et al. (2008) higher numbers of saplings and trees in comparison to seedlings, point out the ability of the
forest to recruit more seedlings.
CONCLUSION
The assessment of structural composition and plant species diversity of two forest sites have indicated that,
Nambor Wildlife Sanctuary (NWLS) (Primary forest) was found richer than Bornewria forest (Secondary forest)
in plant diversity. Numbers of plant species were declined in the Bornewria forest because of ecological
destabilization and disturbance in their natural abode.
ACKNOWLEDGMENTS
The authors would like to express deep sense of gratitude and sincere thanks to Director, Rain Forest
Research Institute, Jorhat whose advice and encouragement were vital. The authors also grateful to the State
Forest Department of Assam, Divisional Forest Officer Golaghat and the staff for permits to work in the Forest
as well as their support and co-operation.
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Research article
Lichen diversity in coal mining affected areas of Makum

coalfield, Magherita, Assam
Sandeep Yadav1*, Ajay Kumar2, Hans Raj1 and H. R. Bora1
1
Forest Research Centre - Bamboo and Rattan, Aizawl, Mizoram, India
2
Rain Forest Research Institute, Jorhat, Assam, India
*Corresponding Author: sandeep080987@gmail.com [Accepted: 27 August 2018]
Abstract: The coal mining industry in Assam was started by the British in 1884. Coal mining
activities are considered hazardous to the sustenance of biodiversity of the region, which is under
Eastern Himalayan province, the richest bio-geographical province of the Himalayan zone and also
falls in one of the mega biodiversity hotspots of the world. Makum Coalfield of North Eastern
Coalfields in Margherita of Tinsukia district of Assam was surveyed and lichen diversity of
different sites was studied. Sites with active mining had very low diversity of lichens. Reclamation
sites have an intermediate number of lichen species; rocks were under the process of lichenization.
Reserve forests near the mining sites had maximum lichen diversity; understorey plants were
abundant with follicolous lichens.
Keywords: Coal mining - Biodiversity - Lichenization - Follicolous.
[Cite as: Yadav S, Kumar A, Raj H & Bora HR (2018) Lichen diversity in coal mining affected areas of Makum
coalfield, Magherita, Assam. Tropical Plant Research 5(2): 243–249]
INTRODUCTION
Any sort of developmental activity in forests usually initiate a series of changes in the status quo and disrupt
the natural forest dynamics. Mining tends to make a notable impact on the environment, the impacts varying in
severity depending on whether the mine is working or abandoned, the mining methods used, and the geological
conditions (Bell et al. 2001). Natural plant communities get disturbed and the habitats become impoverished due
to mining, presenting a very rigorous condition for plant growth. The unscientific mining of minerals poses a
serious threat to the environment, resulting in the reduction of forest cover, erosion of soil in a greater scale,
pollution of air, water and land and reduction in biodiversity (UNESCO 1985). The problems of waste rock
damps become devastating to the landscape around mining areas (Goretti 1998).
Coal mining by both opencast and underground method affects the environment of the area (Dhar 1993). In
the process of mining, huge amounts of water are discharged to the surface to facilitate the mining operation.
The discharged water often contains a high load of Total Suspended Solids (TSS), Total Suspended Solids
(TDS), hardness and heavy metals, which contaminate the surface and groundwater (Tiwary & Dhar 1994).
Sometimes it is acidic in nature and pollutes the water regime (Tiwary et al. 1997).
Coal contains a significant amount of ferrous sulphate in the form of pyrites. The exposure of pyrite to
atmospheric oxygen during mining oxidizes it to produce ferrous sulphate and sulphuric acid. The sulphuric acid
thus formed, lowers the pH of the soil and water in the terrestrial and aquatic environments, respectively, which
affects the organisms inhabiting those environments. Chemicals released from the coal mines, overburden and
tailings also contain a high concentration of metals such as Cu, Cd, Fe, Hg and Zn, which also affect the
organisms adversely. Large-scale denudation of forest cover, scarcity of water, pollution of air, water and soil
and degradation of agricultural lands are some of the conspicuous environmental implications of coal mining
(Swer & Singh 2004).
Just as canaries provide warnings of toxic gases to coal miners, so can the investigation of lichen
communities provide information on potential deterioration of ecosystems stressed by air pollutants (Nash
2008). Lichens have sensitivity, both physiological and ecological, to pollutants and therefore have been
employed almost exclusively to monitor the extent or spread of air pollution particularly SO 2 (Gries et al. 1997).
Received: 23 February 2018 Published online: 31 August 2018
Yadav et al. 2018
Due to lack of cuticle, lichen absorbs both gases and dissolved substances through their surface. Reduction in air
pollution has been reported to result in recovery of lichen abundance (Showman 1990, Kirschbaum et al. 1996).
Murphy et al. (1999) used lichen abundance to indicate whether a rural, coal burning, electrical generating
station in the northern USA has measurable impacts on the surrounding forest. Charak et al. (2009) studied
lichens growing around the Moghla Coal mines, Kalakote, Jammu & Kashmir and revealed that pollutants
released by the open coal mining activities not only effected qualitative distribution but also have an effect on
the quantitative parameters.
The coal mining industry in Assam is more than a century old, started by British in 1884 when huge deposits
of bituminous coal were discovered in this part of the country. Few collieries are operational and huge loads of
coal are recovered every day, however, the impact of coal mining on plant vegetation has not been studied till
date. This study intends to assess the lichen diversity of coal mining sites in Margherita, Assam and draw some
useful inferences.

Study area
The study was confined to the Makum Coalfield of North Eastern Coalfields in Margherita (Fig. 1) and its 10
km buffer area in Tinsukia district of Assam. The area of the buffer zone is approximately 484.8 km 2. The
extent of the study area is from 95° 36′ 12″ E to 96° 01′ 55″ E and 27° 12′ 20″ N to 27° 26′ 27″ N. As many as
16 reserve/proposed reserve forests in four forest ranges fall in this buffer zone. Broadly, the area comes under
Assam Valley Tropical Wet Evergreen Forests (Dipterocarpus- Mesua formation) with interspersed Semi-
Evergreen Forests and scattered Bamboo Brakes (Champion & Seth 1968). Moderately Dense Forests (forests
with the crown cover 40–70%) cover the maximum area followed by Open Forests (crown cover 10–40%) and
Very Dense Forests (crown cover >70%), covering 16,256.1 ha, 7043.8 ha and 2070.4 ha, respectively. Bio-
geographically, the study site is situated in the Eastern Himalayan province, the richest bio-geographical
province of the Himalayan zone and also falls in one of the mega biodiversity hotspots of the world (Myers et
al. 2000).
Figure 1. Location map of the study sites.

The Makum Coalfield covers forest areas amongst hilly terrains within Digboi forest division, Assam (Fig.
2). Coal mining activities of North Eastern Coalfields, Coal India Limited (CIL) are at present confined to
Makum Coalfields with four coal mines under operation, covering an area of 2688.16 hectares. Tirap, Ledo and
Tikak collieries are open cast coal mines, while the Tipong colliery is underground (Anonymous 2008, 2009).
Among the land uses, Agriculture with an area of 9470.2 ha is the dominant land use followed by Scrub Land
(2994.1 ha), Tea Gardens (2940.5 ha), Open Land (2400), major Settlement (1705.0 ha), Sandy Area (1574.4
ha) Agriculture-Crop (903.2 ha), Mine Area (568.2 ha) and major water body (555.9 ha). The present study area
falls in watersheds of River Tirap and Burhi-Dihing flowing across the area.
Figure 2. A, Active Coal mining at location 5 of Tikak colliery; B, Reclamation site at location 4 of Tikak colliery; C,
Boulders in the initial stage of Lichenization at location 4 of Tikak colliery; D, Tree at reclamation site are covered with
algae Trentopholia sp.; E, Reserve forest at Tipong Charlie at Location 1, 2, and 3; F, Salaki Hills at Location 6; G, Tea and
turmeric garden at Salaki hills; H, Tinkupaani Reserve forest at location 7, 8, 9, and 10; I, Rich abundance of corticolous
lichens at Dehing-Patakai at location 11 and 12.
The Study area falls under the humid zone, characterized by high precipitation. High humidity and heavy
rainfall are significant features of evergreen forests in this region. The relative humidity is generally high during
most part of the year, touching about 90 percent during monsoon. Heavy downpour takes place during the
monsoon months of May, June, July & August. The average rainfall recorded from 2010–11 to 2016–17 in this
region shows that highest rainfall occurs during the month of July (428.86 mm) whereas the lowest average
rainfall was recorded during November (17.11 mm).
Data collection
Around 150 lichen specimens were collected from rocks, soil, twigs and barks of different trees from 12
different sites in Makum Coalfield. The collected specimens were investigated morphologically (by Leica EZ4
HD Stereo microscope), anatomically (by Leica DM 750 compound microscope) and chemically (by Thin Layer
Chromatography). The specimens were identified following the publications of Coppins & James (1984),
Awasthi (1991, 2000 & 2007) and Harris (1995). The colour tests were performed with the usual reagents, i.e. K
(5% potassium hydroxide), C (Aqueous solution of calcium hypochlorite) and P (paraphenylenediamine).
Lichen substances were identified with thin layer chromatography (TLC) in solvent system A (toluene: dioxane:
acetic acid; 180:60:8 ml) using the technique of Walker & James (1980).
Yadav et al. 2018
RESULTS
The identification of the specimens collected from study sites (Table 1) reveals the occurrence of 31 lichen
species belonging to 26 genera and 21 families (Table 2; Fig. 3–4). In all of the sites studied, lichens exhibited a
high abundance of low diversified communities. Sites with good forest cover such as location 7 and 10 of
Tinkupaani reserve forest harboured a high number of lichens including species such as Coccocarpia palmicola
and Cladonia coniocrea.
Table 1. Study sites of Makum coalfield.
S.N. Sampling Site Location (Code) Altitude (m) GPS
1. Tipong Charlie no. 8 Location 1 (L1) 175 N 27° 17' 42.9''
E 095° 51' 17.2''
2. Location 2 (L2) 376 N 27° 17' 21.7''
E 095° 50' 47.4''
3. Location 3 (L3) 476 N 27° 17' 02.4''
E 095° 50' 32.6''
4. Tikak Colliery Location 4 (L4) 483 N 27° 16' 02.8''
(Reclamation site) E 095° 43' 08.2''
5. Location 5 (L5) 530 N 27° 16' 15.6''
(Active mining) E 095° 43' 30.7''
6. Salaki Hills (proposed extension site) Location 6 (L6) 300 N 27° 18' 04.4''
E 095° 47' 53.0''
7. Tinkupaani Reserve Forest Location 7 (L7) 186 N 27° 22' 10.8''
E 095° 57' 48.0''
8. Location 8 (L8) 183 N 27° 22' 28.6''
E 095° 57' 52.6''
9. Location 9 (L9) 200 N 27° 21' 55.7''
E 095° 58' 00.2''
10. Location 10 (L10) 180 N 27° 21' 20.5''
E 095° 58' 01.2''
11. Dehing – Patakai Location 11 (L11) 141 N 27° 21' 23.2''
E 095° 31' 46.0''
12. Location 12 (L12) 114 N 27° 21' 05.6''
E 095° 30' 34.4''
13. Saraingpung Range Location 13 (L13) 106 N 27° 20' 41.2''
E 095° 28' 28.4''
Understorey plants in Tipong and Tinkupaani reserve forests were colonized by follicolous lichens such as
Mazosia phyllosema, Strigula antillarum, Strigula smaragdula, Calopadia fusca etc. Sites nearby active mining
such as Location 5 of Tikak Colliery showed a minimum number of genera. Sites near reclamation land such as
Location 4 of Tikak Colliery exhibited less number of lichens indicating disturbed condition; however, many of
the rocks in this area were in the initial process of lichenization. Trees at Saleki Hills (which included the
proposed coal mining extension sites) had already been felled down by local tribal and replaced with tea and
turmeric gardens, and therefore, exhibit a low diversity of lichen communities. The complete list of lichens
found in the study area is given in table 2.
Table 2. List of lichens studied in Makum coalfield.
S.N. Botanical name Family Growth Substratum Abundance
form
1. Bacidia incongruens (Stirt.) Zahlbr. Ramalinaceae Crustose Bark Rare
2. Buellia alboatra (Hoffm.) Th. Fr. Caliciaceae Crustose Bark Rare
3. Calopadia fusca (Mull. Arg.) Vezda Ectolechiaceae Crustose Leaves Common
4. Caloplaca bassiae (Willd. Ex Ach.) Teloschistaceae Crustose Bark Rare
Zahlbr.
5. Chiodecton leptosporum Mull. Arg Roccellaceae Crustose Bark Common
6. Chrysothrix chlorina (Ach.) laundon Chrysothricaceae Leprose Bark Rare
8. Cladonia coniocraea (Florke) Spreng Cladoniaceae Fruticose Soil and rocks Rare
9. Coccocarpia palmicola (Spreng.) Coccocarpiaceae Foliose Bark Rare
Arvidss& D.J. Galloway
10. Collema pulcellum Ach. var. Collemataceae Foliose Bark Rare
subnigrescens (Mull. Arg.) Degel.
11. Cryptothecia striata G. Thor Arthoniaceae Crustose Bark and rocks Common
12. Dirinaria aegialita (Afzel.) B.J. Moore Caliciaceae Foliose Bark and rocks Common
13. Glyphis duriuscula Stirton Graphidaceae Crustose Bark Common

14. Graphis duplicata Ach Graphidaceae Crustose Bark Common
15. Graphis scripta (L.) Ach Graphidaceae Crustose Bark Common
16. Haematomma puniceum (Sw.) A. Haematommataceae Crustose Bark Common
Massal.
17. Heterodermia diademata (Taylor) D.D. Physciaceae Foliose Bark and rock Rare
Awasthi
18. Lecanora indica Zahlbr. Lecanoraceae Crustose Bark Common
19. Leptogium azureum Lecanoraceae Crustose Bark Common
20. Mazosia phyllosema (Nyl.) Zahlbr. Roccellaceae Crustose Leaves Common
21. Parmotrema crinitoides J. C. Wei Parmeliaceae Foliose Bark and rock Common
22. Pertusaria quassiae (Fee) Nyl. Pertusariaceae Crustose Bark Common
23. Phaeographina caesioradians Graphidaceae Crustose Bark Common
(Leighton) Redinger
24. Phaeographis platycarpa Müll. Arg. Graphidaceae Crustose Bark Common
25. Pseudopyrenula pupula (Ach.) Mull. Trypetheliaceae Crustose Bark Common
Arg
26. Strigula antillarum (Fee) Mull. Arg. Strigulaceae Crustose Leaves Common
27. Strigula elegans (Fee) Mull. Arg. Strigulaceae Crustose Leaves Common
28. Strigula smaragdula Fr. Strigulaceae Crustose Leaves Common
29. Tricharia vainioi R. Sant. Gomphillaceae Crustose Leaves Common
30. Trichothelium annulatum (Karst) R. Trichotheliaceae Crustose Leaves Common
Sant.
31. Trypethelium eluteriae Spreng Trypetheliaceae Crustose Bark Rare
Figure 3. A, Trentopholia sp., a terrestrial alga, was of common occurrence in the reclamation sites; B, Leprose thallus of
Chrysothrix chlorine; C, Cladonia coniocraea growing on rock at Tipong Charlie reserve forest; D, Corticolous thallus of
Bacidia incongruens; E, Corticolous thallus of Chiodecton leptosporum; F, Corticolous thallus of Dirinaria aegialita; G,
Corticolous thallus of Glyphis duriuscula; H, Corticolous thallus of Graphis duplicate; I, Corticolous thallus of Pertusaria
quassiae.
Yadav et al. 2018
Figure 4. A, Foliose thallus of Parmotrema crinitoides; B, Corticolous thallus of Phaeographina caesioradians; C,

Corticolous thallus of Phaeographis platycarpa; D, Corticolous thallus of Pseudopyrenula pupula; E, Follicolous thallus of
Strigulaelegans; F, Follicolous thallus of Trichothelium annulatum; G, Corticolous thallus of Collema pulcellum; H,
Corticolous thallus of Letroutia transgressa; I, Corticolous thallus of Haemotomma puniecium
CONCLUSION
Biodiversity may be considered as a non-renewable resource that needs to be managed for its conservation
and sustainable utilization. As far as biodiversity is considered, Makum Coalfield, Assam is situated within a
vivacious habitat that is famous for quite rich flora and fauna. However, biotic interference in this fertile region
over the years has resulted in forest degradation, fragmentation and deforestation and evolution of secondary
forests in place of the primary virgin forest. The direct impacts of mining disturbances to land surfaces are
usually significant, with the likelihood of destruction of biodiversity within natural ecosystems through the
removal of natural soil, plants and animals.
In the open cast mining sites, phorophytes are uprooted and therefore, lichen communities are adversely
affected. Lichen diversity in sites with active or past mining is found to be considerably low, because of the
prevailing harsh and disturbed environmental conditions. On the other hand, sites with good forest reserve cover
have a high abundance of lichen communities. It had been noticed that in reclamation sites, while the vegetation
had successfully been established, lichens were only restricted to the initial process of colonization of rocks
(pioneer stage of Lithosere). The bark of trees of reclamation sites was devoid of lichens and was inhabited by
terrestrial algae, Trentepholia sp. This indicates that coal mining activities are more detrimental for lichens as
compared to other vegetation. A conglomeration of both nitrophylic and oligotrophic species at the sites of
active mining and reclamation sites reveals that the land use is the guiding factor in community dynamics.
However, the presence of terricolous and cyanolichens such as Coccocarpia palmicola and Cladonia coniocrea
in the reserve areas near to the collieries points towards the low atmospheric pollution output and stable
substrate indicating a lichen growth supporting habitat.
ACKNOWLEDGEMENTS
The authors are sincerely grateful to Dr. S. C. Gairola, IFS, Director General, Indian Council of Forestry
Research and Education (ICFRE), Dehradun for his kind patronage. We thank Dr. R. S. C. Jayaraj, IFS,
Director, Rain Forest Research Institute (RFRI), Jorhat, Assam for his continuous support and guidance.
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Research article
Assessment of genetic diversity in natural populations of

Calamus guruba Buch.-Ham. ex Mart.
using ISSR marker
Rajendra Kumar Meena1*, Hans Raj2, Pratibha Sharma2, Sandeep Yadav2,
Rama Kant3 and Maneesh Singh Bhandari3
1
Division of Genetics & Tree Propagation, Forest Research Institute, Dehradun-248195, Uttarakhand, India
2
Advanced Research Centre for Bamboo and Rattan, Aizawl, Mizoram, India
3
Genetics & Tree Propagation Division, Forest Research Institute, Dehradun-248195, Uttarakhand, India
*Corresponding Author: rajnrcpb@gmail.com [Accepted: 27 August 2018]
Abstract: Rattan palms are one of the important nontimber forest produce which forms an integral
part of the tribal population and contribute significantly in the rural economy. The demand of the
rattan resources is substantial but most of the raw material is being collected from natural stands;
and therefore, there is tremendous pressure on natural populations. Calamus guruba is one of the
important rattan species of India and extensively harvested for a wide range of end products. In the
present study, three populations of C. guruba naturally growing in Mizoram were characterized for
their genetic diversity and population structure using Inter-Simple Sequence Repeat (ISSR)
markers. A total of 197 bands generated in PCR amplification of 67 individuals with 10 ISSR
primers. Among all the primers UBC 808 displayed maximum 25, while UBC 807 and UBC 827
showed the lowest 13 numbers of bands. Relatively high genetic diversity (PPB = 98.98 %, h =
0.271, I = 0.341) was recorded for the species with a moderate level of genetic differentiation
(PhiPT = 0.282). Analysis of molecular variance revealed that the genetic differentiation among
the populations is the significant and large proportion of the genetic variation (72%) resides among
the individuals within a population, whereas only 28 % were found among the populations.
UPGMA tree, PCoA plot and STRUCTURE analysis revealed that two ancestral groups captured
the entire genetic variability. The baseline study on C. guruba is of paramount importance in
devising programs for conservation and improvement.
Keywords: Calamus guruba - Genetic diversity - Genetic differentiation - ISSR - Rattan.
[Cite as: Meena RK, Raj H, Sharma P, Yadav S, Kant R & Bhandari MS (2018) Assessment of genetic diversity
in natural populations of Calamus guruba Buch.-Ham. ex Mart. using ISSR marker. Tropical Plant Research
5(2): 250–259]
INTRODUCTION
Rattans are climbing spiny palms known for their lightness, strength, durability and elasticity. About 650
species of rattans have been reported in the world under 22 genera and around 400 of them belong to the genus
Calamus (Govaerts et al. 2014). Calamus genera comprising most of the commercial rattan species; is
predominantly distributed in Asia from the Indian subcontinent, China, Malaysia, Indonesia, Fiji to tropical and
subtropical parts of eastern Australia. India is a home of about 60 species belonging to 4 genera and mainly
distributed in three hotspots viz., Peninsular India (Western Ghats), North East India and Andaman and Nicobar
Islands (Ravikanth et al. 2001, Umashaanker et al. 2004). Throughout their natural range, rattan species are
found in a wide variety of forest and soil types from alluvial plains to moist hill forest up to 2000 m elevation
(Renuka 2002).
Rattans are harvested by local communities either for subsistence or commercial utilization and contribute
significantly to the rural economy (Ravikanth et al. 2001). Many small and large scale industries of furniture,
cottage, handicraft, basketry and sports equipment are solely dependent on rattan for their raw material. With the
increasing preference of cane products; the demand for raw material has been significantly increased locally as
Meena et al. 2018
well as globally, which results in heavy extraction from the natural forest (Senthilkumar et al. 2014).
Continuous and unscientific extraction of rattans along with changes in land use patterns like shifting cultivation
in North East India has reportedly threatened several economically important rattan species (Thomas et al. 1998,
Singh et al. 2004, Raj et al. 2014). A significant change has been recorded in the distribution of rattan palms
over the years that might be attributed to changes in land use pattern, shrinkage of the natural forest cover,
selective exploitation of stems for the furniture and handicrafts, poor natural regeneration etc. (Renuka 2002).
Natural variation is the key requirement for selection of superior genotypes and adaptation towards climate
change; hence, conservation of available genetic resources needs to be accorded the highest priority (Rao & Rao
2000). Genetic variability exists in the base populations bestow them the ability to survive in long-term by
accommodating new selection pressure brought by environmental changes and serves as a resource for tree
breeding and improvement programs (Frankham 2008, Porth & El-Kassaby 2014). Every genetic improvement
programme is based on the assumption that the species has enough genetic variability for the traits of interest
that could be exploited during domestication and selection (Renuka et al. 1998). Despite of the growing
importance and pressure over natural stands of rattans, no serious effort was made to characterize the genetic
variability existed among the natural populations. Knowledgebase for the genetic diversity, population structure
and spatial patterns of genetic variation are highly useful in devising sound conservation plan of the species. As
per the India State of Forest Report, the total forest cover of North East India has been declined significantly
(FSI 2017). Therefore, the concern over the state of genetic resources is paramount, particularly for the species
endemic to the region. Despite of the wide distribution of rattans, most species are endemic. In India, three
geographical regions have specific rattan flora with minimum overlapping distribution pattern (Renuka 2002).
However, unlike the attention received by rattans of the Western Ghats in peninsular India, only limited efforts
were made in addressing the conservation of rattans of North East India (Lyngdoh et al. 2005). Since rattans are
distributed in small pockets and knowledge base for their genetic structure and diversity is only poorly
developed, in situ conservation sites for conservation are yet to be established (Rawat & Ginwal 2009).
Therefore there is an instant need to generate baseline data of natural populations for scientific management and
conservation of this valuable depleting resource for future, along with the cultivation of commercially important
species.
Calamus guruba Buch.-Ham. ex Mart. (Family: Arecaceae) locally referred to as „Thilte‟ in Mizoram, is one
of the most important dioecious species of rattan found in Southeast Asia viz. Malaysia, Indonesia, Borneo and
the Philippines. In the Indian subcontinent, it occurs in the natural forest areas of Odisha, Chhattisgarh, coastal
districts of Andhra Pradesh and lower Western Ghats in the states of Tamil Nadu and Kerala (Sinha et al. 2017).
However, the species also reported to exist in dense tropical and lower subtropical forests of North East India
(http://indiabiodiversity.org/species/show/258795). C. guruba is extensively harvested by forest-dwelling
communities for making a wide range of furniture products and handicraft articles Due to its advantages of the
high-quality flexible canes for furniture manufacturing and the tender shoots as food items; it is one of the most
economically important rattan species of India (Mahzuz et al. 2013). Despite of its enormous usages, the
regeneration potential of wild populations of C. guruba has been declining gradually due to several biotic and
abiotic factors (Sinha et al. 2017). Some populations of Odisha have been genetically characterized using
Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) markers (Sinha &
Panda 2016). In the present study, three populations of C. guruba were explored in Mizoram and characterized
for their genetic diversity and population structure.

Plant Materials
Leaf samples of 67 genotypes representing 3 populations were collected along with geospatial parameters
using GARMIN 650 model of Global Positioning System (GPS) during 2015–17. Two populations (CG01 and
CG02) were located in Zongaw Reserve Forest, an un-notified Community Reserve Forest of Mizoram while
another population was present in Bungthlang forest of Mizoram. The map of the sampled populations in the
distribution range of C. guruba was prepared using the software Arc-GIS ver 9.2 (Fig. 1). Two sampled
genotypes were at least 100 m apart at each site. Fresh leaves were preserved during long field tours by
desiccating with silica gel and desiccated samples were brought to the laboratory of Genetics and Tree
Propagation Division, Forest Research Institute, Dehradun for further genotyping work.
Figure 1. Distribution map of sampled populations of Calamus guruba Buch.-Ham. ex Mart.in Mizoram.
Genomic DNA Extraction
Genomic DNA was extracted using standard protocols of Doyle & Doyle (1987) with minor modifications.
Frozen tissues were grounded in liquid nitrogen and incubated at 60ºC for 60 minutes in pre-heated CTAB
extraction buffer followed by mixing with chloroform-isoamyl alcohol (24:1). The supernatant was pipetted out
after centrifugation and precipitated with equal volume of chilled isopropanol overnight. The precipitated DNA
was first washed with a solution containing 96% ethanol and 0.3 M Sodium acetate followed by washing with
Meena et al. 2018
70% ethanol. After vacuum drying, DNA pellet was re-suspended in 100 µl of 10 mMTris-EDTA buffer (pH
8.0). Qualitative and quantitative analysis of genomic DNA was carried out using 0.8% agarose gel and
Biophotometer (Eppendorf), respectively and diluted for final working concentration of 30 ng µl-1.
PCR amplification with ISSR primers
Ten ISSR primers (Table 1) showing positive amplification in C. guruba were selected in the initial
screening and the annealing temperature was optimized through gradient PCR for clear and distinct banding
pattern. Each sample was subjected to the PCR amplification with the 20 μl PCR reaction mixture containing 60
ng of template DNA, 2 μl of 10x PCR buffer, 1.75 mM MgCl 2, 0.2 mM dNTPs, 0.2 μM primer, 0.6 units of Taq
DNA polymerase and 12.98 μl nuclease free water. The cycling conditions included an initial denaturation at
94°C for 5 min; then 40 cycles of 94°C for 30 Sec, 54–57.5°C for 30 Sec, and 72°C for 1 min; and a final
extension at 72°C for 10 min. The PCR products were electrophoretically separated in 2% agarose gel buffered
with 1x TBE. A 100 bp DNA ladder was used as a size marker. After staining with ethidium bromide (0.5 μg
ml-1), the DNA fragments were visualized and captured using a gel documentation system.
Table 1. Details of ISSR primers used for genotyping.
Optimized annealing
Primers Sequence (5' - 3' )
temperature (°C)
PCP-1 GACGACGACGACGAC 55.8
PCP-2 AGGAGGAGGAGGAGGAGG 55.0
PCP-3 GTGCGTGCGTGCGTGC 54.6
UBC 807 AGA GAG AGA GAG AGA GT 57.5
UBC 808 AGAGAGAGAGAGAGAGC 57.5
UBC 826 ACA CAC ACA CAC ACA CC 54.5
UBC 827 ACA CAC ACA CAC ACA CG 54.5
UBC 841 GAG AGA GAG AGA GAG AYC 55.4
UBC 888 BDB CAC ACA CAC ACA CA 56.2
UBC 890 VHV GTG TGT GTG TGT GT 54.5
Data Analysis
Each band was considered as a locus with two alternative alleles and scored manually as 1 (presence) and 0
(absence) in a binary matrix. Input files in different formats were prepared as per the requirement of different
software. The parameters like observed number of alleles (Na), effective number of alleles (Ne), percentage of
polymorphic bands (PPB), allele frequency, number of private bands (PB), Nei's gene diversity (h) and
Shannon's Information index (I) were calculated to describe the statistics of genic variation for all the loci at
population and species level using software POPGENE ver 1.32 (Yeh et al. 1999) and GenAlex ver 6.5 (Peakall
& Smouse 2012). Total genetic diversity (HT) and proportional variance resided within the populations (HS)
were also calculated in subdivided populations through POPGENE. Analysis of molecular variance (AMOVA)
was performed using software GenAlEx, which allow the partitioning of genetic variation among the
populations. The genetic differentiation between populations was determined using phiPT value (an analogue of
FST) that allows within-population variance to be suppressed and simply calculate population differentiation
based on the genotypic variance in binary data. For interpretation, another measure of genetic differentiation
(GST) was also calculated through POPGENE. The amount of gene flow (Nm) among populations is a measure
of the effective number of migrants per population per generation. It was estimated indirectly from the GST
values at each locus and from the average values over all loci by applying McDermott & McDonald‟s (1993)
formula [Nm = 0.5(1-GST)/GST] for the studied populations using software POPGENE.
Genetic relationship among the genotypes was studied by constructing a dendrogram based on pair-wise
genetic distances among the individual genotypes calculated through Jaccard similarity coefficient using
Unweighted Pair-Group Method with Arithmetic averages (UPGMA) in NTSYSpc ver 2.02 (Rohlf 1998). To
check the consistency of the clusters obtained in UPGMA dendrogram, Principal coordinate analysis (PCoA)
was performed using GenALEx.
The Bayesian model-based clustering method was used to elucidate the genetic structure of the populations
using STRUCTURE software ver 2.2 (Pritchard et al. 2000). Ancestry model with admixture under the
assumption of correlated allele frequencies was used to determine the posterior probability. Simulations were
run 10 times for each value of K (1 to 4) with 50,000 Markov Chain Monte Carlo (MCMC) sampling runs after
a burn-in period of 50,000 iterations.
RESULTS
Gene diversity and genetic differentiation
PCR amplification with 10 ISSR primers generated 197 bands in 67 individuals representing 3 different
populations of C. guruba. Figure 2 presents the polymorphic banding pattern generated by ISSR primer PCP1 in
populations of C. guruba. Among all the primers UBC 808 displayed maximum 25, while UBC 807 and UBC
827 showed the lowest 13 numbers of bands. High genetic variability was revealed by the percentage of
polymorphic bands (PPB) recorded at species (98.98 %) and population level (63.45%). The average percentage
of polymorphic bands (PPB) among the populations was ranged from 52.28% (CG02) to 70.56% (CG03). Mean
genetic diversity was estimated by calculating Shannon‟s Information indices (I) which were recorded as 0.271
for population and 0.341 for species. Among studied populations, the highest degree of variability was recorded
for the population of Bungthlang forest, CG03 (h = 0.191 and I = 0.293) and lowest for a population of Zongaw
Reserve Forest, CG02 (h = 0.162 and I = 0.246) (Table 2). As per the Nei's analysis of gene diversity in
subdivided populations, total genetic diversity (HT) of the species was recorded as 0.215, out of which 81.65%
(0.176) was found to reside within the population (HS).
Figure 2. Band profile generated by ISSR primer PCP1 in 3 populations of Calamus guruba Buch.-Ham. ex Mart. Lane M
represents 100bp DNA ladder; Lane 1, 2,3……n represent “n” samples of the respective populations.
Table 2. Gene diversityover loci for each sampled population of Calamus guruba Buch.-Ham. ex Mart.
Population ID No. of Percentage of No. of Nei's gene Shannon's
Polymorp Polymorphic Private diversity (h) Information Index
hic Bands Bands (PPB) Bands (I)
(PB) Mean (SD) Mean (SD)
CG01 133 67.51 25 0.175 (0.180) 0.273 (0.256)
CG02 103 52.28 7 0.162 (0.194) 0.246 (0.277)
CG03 139 70.56 34 0.191 (0.194) 0.293 (0.271)
Mean of 125 63.45 - 0.176 (0.014) 0.271 (0.024)
Populations
Overall for 195 98.98 - 0.216 (0.178) 0.341 (0.239)
Species
Analysis of molecular variance (AMOVA) showed 72% of the variation existed within the populations and
28% among the populations (Table 3). Variance estimates were based on 999 permutations. Accordingly, the
difference between the individuals within the populations was statistically significant with P value <0.001. High
values obtained for the estimated measures of genetic differentiation (GST = 0.184 and PhiPT = 0.282) indicates
Meena et al. 2018
the moderate level of genetic differentiation among the populations, that was further supported by the presence
of a large number of private bands (bands unique to a single population). A total of 66 private bands were
detected, of which 34 (51.52%) were present in the population of Bungthlang forest (CG03) while rest of the 32
(48.48%) were present in the other two populations from Zongaw Reserve Forest (CG01 and CG02). Nei's
measures of genetic distance were also estimated among the populations which ranges from 0.034 (CG01 and
CG02) to 0.103 (CG01and CG03) (Table 4).
Table 3. Analysis of molecular variance (AMOVA) for populations of Calamus guruba Buch.-Ham. ex Mart..
Source of variation DF SS MS Est. Var. % GD P (rand
var. >= data)
Among populations 2 379.30 189.65 7.63 28 PhiPT= 0.282 0.001
Within populations 64 1240.40 19.38 19.38 72
Total 66 1619.70 27.01 100
Note: DF, Degree of freedom; SS, Sum of Square, MS, Mean Sum of Square; Est. Var.,Estimated variance; %
var.,Percentage of variation; GD, Genetic differentiation; P(rand >= data), Probability for PhiPT is based on standard
permutation across the full data set.
Table 4. Pair-wise Nei's genetic distance and genetic identity among populations.
CG01 CG02 CG03
CG01 0.000 0.9667 0.902
CG02 0.0339 0.000 0.9149
CG03 0.1031 0.089 0.000
Note: Nei's genetic identity (above diagonal) and genetic distance (below diagonal).
Genetic structure and relationship among the populations
The UPGMA tree revealed that genotypes of both the populations of Zongaw Reserve Forest (CG01 and
CG02) were found to be genetically similar and clustered together which later separated into different sub-
cluster of respective populations (Fig. 3). Genotypes of the population of Bungthlang forest (CG03) constitute
another major cluster which was genetically dissimilar from others. Moreover, some genotypes were not
included in any major cluster and remained as an outlier. Clustering pattern resulted from UPGMA dendrogram
was also supported by principal coordinate analysis (PCoA). The PCoA plot showed that the first principal
coordinate accounts for 19.49 % of the total variation and separates population of Bungthlang forest (CG03)
from other two populations of Zongaw Reserve Forest (CG01 and CG02). Second principal coordinate
accounted for only 8.07 % of the variation which couldn‟t able to separate any of these populations (Fig. 4).
Figure 3. Dendrogram based on genetic distances between individual genotypes obtained from ISSR markers using
UPGMA algorithm. Individual genotypes were labelled as decimal points of each population number like CG1.1 to CG1.22
represent samples of population CG01.
Figure 4. Spatial distribution of the genotypes of Calamus guruba Buch.-Ham. ex Mart. in the 2-dimensional plot of
Principal Coordinates Analysis (PCoA) based on genetic distances.
Bayesian clustering method was used to elucidate the genetic structure of the sampled populations. A
posterior probability [Estimated Ln Probability of Data, LnP(D)] was calculated for each K value. The K value
provides the maximum likelihood, called LnP(D) in STRUCTURE, is generally considered as the optimal
number of subdivisions but the distribution of LnP(D) values create a plateau in the plot (Fig. 5A). Thus,
another ad hoc quantity (∆K) was used to overcome the difficulty for interpreting the real K values using the
graphical method suggested by Evanno et al. (2005). The highest value of ∆K with a clear peak was obtained for
K=2 and hence it was considered that two ancestral groups captured the entire variability among the sampled
genotypes (Fig. 5B). As per the inferred ancestries (Q matrix) of the population determined for K=2, all the
genotypes of 3 populations were clearly defined by 2 clusters with proportional membership coefficient of more
than 0.9. Clustering pattern was in accordance to the UPGMA dendrogram i.e. two populations of Zongaw
Reserve Forest (CG01 and CG02) were defined in a single cluster while another population of Bungthlang forest
(CG03) was defined in a separate cluster. FST values for clusters 1 and 2 were recorded as 0.365 and 0.253,
respectively. The estimated membership coefficients of the analyzed individuals in each group were represented
as a bar plot (Fig. 5C). Each individual of the sample is represented by coloured bars separated by vertical line
divided into K coloured segments with the length of each segment being proportional to the estimated
membership in each of the K inferred groups. On, X-axis of bar plot, the numbers 1–67 represents individual
genotypes of the 3 corresponding populations as depicted in the brackets.
Figure 5. Graphic representation of the estimated probability of data for each K value and Bar plot for estimated individual
Q-matrix at optimum K: A, Plot of Bayesian posterior probability of data [LnP(D)] with increasing K; B, Magnitude of ΔK
as a function of K; C, Bar plot for estimated individual Q-matrix at K=2 for the genotypes of Calamus guruba Buch.-Ham.
ex Mart.
Meena et al. 2018
DISCUSSION
Three natural populations of Calamus guruba, a commercially important rattan palm of North East India
were characterized for the genetic diversity and population structure. High level of genetic diversity has been
recorded for the species (PPB = 98.98 %, h = 0.216, I = 0.341) with moderate level of genetic differentiation
(GST = 0.184 and PhiPT = 0.282) as compare to other rattan species viz., Calamus palustris var. malaccensis
Becc. (h = 0.153) in Peninsular Malaysia (Sitisalwana et al. 1998), C. flagellum Griff. ex Mart. (h = 0.201) in
Arunachal Pradesh (Lyngdoh et al. 2005), C. thwaitesii Becc. (h = 0.272–0.333) in central Western Ghats
(Ramesha et al. 2007) and C. vattayila Renuka (h = 0.150) in Kerala parts of Western Ghats (Priya et al. 2016).
Low genetic diversity with very high genetic differentiation (I = 0.146 and GST = 0.726) was reported in the
Indonesian populations of Daemonorops draco due to geographical isolation and apomixtic behaviour of the
species (Asra et al. 2014).
Generally, species with a wide geographical range tends to maintain a high level of genetic diversity than
that of geographically localized species (Hamrick & Godt 1989). Similarly, outcrossing species tend to be
genetically diverse in respect to self-pollinated species (Hamrick & Godt 1996). Based on this assumption, a
high level of genetic diversity was expected in rattan palms because of their wide range of distribution as well as
outcrossing behaviour due to dioecious nature of flowering (Renuka et al. 1998). In the present investigation,
genetic differentiation (GST = 0.184) of populations were found to be comparable to the mean value of other out-
crossing species (GST = 0.23); but relatively lower than the mean value of the widely distributed species (GST =
0.33) (Nybom & Bartish 2000). Although, the Rattans are widely distributed in India; but most of the species
are endemic to specific geographical regions (Renuka 2002). Lower genetic differentiation recorded in C.
guruba may be attributed to its restricted distribution.
Clustering pattern showed two ancestral groups comprising entire variability and most of the genotypes were
clustered in accordance of their geographical distribution i.e. genotypes belong to a particular geographical area
were clustered together. Therefore it can infer that both the populations of Zongaw Reserve Forest are actually
single major population and share common ancestral gene pool but quite different from another population of
Bungthlang forest. Presence of a large number of private bands and high value of differentiation measures
depicted in natural populations indicates that considerable genetic changes adopted by populations in response
to habitat fragmentation, genetic drift, and/or barriers to gene flow. The GST value of 0.184 recorded in C.
guruba will generally be considered as moderate differentiation and indicates that the structuring between
subpopulations is fairly good (De Vicente 2004).
The present investigation revealed that sufficient genetic diversity exists in the sampled populations of the C.
guruba but the populations of both regions are significantly different from each other comprising various unique
alleles. Therefore, conservation measures are required to protect the rattan biodiversity in its natural habitat by
preserving rare alleles. The basic but utterly useful information generated in our study would be highly
beneficial in devising conservation, management and genetic improvement programme for the depleting rattan
genetic resources.
ACKNOWLEDGEMENTS
The authors are grateful to the Indian Council of Forestry Research and Education for financial support.
Directors of Rain Forest Research Institute, Jorhat and Forest Research Institute, Dehradun are highly obliged
for providing field and laboratory facilities during project execution. We are also thankful to forest departments
of Mizoram for their permission and support during sample collection from forests.
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Research article
Effect of oil spillage on abundance and diversity of soil

mesofauna in Bodo city, Niger Delta, Nigeria
S. A. Adeduntan and O. O. Owokotomo*
Department of Forestry and Wood Technology, Federal University of Technology Akure, Nigeria
*Corresponding Author: owokotomoolayinka@gmail.com [Accepted: 28 August 2018]
Abstract: The abundance and species diversity of mesofauna were surveyed in the unpolluted site,
remediated site and crude oil polluted sites to examine the effect of crude oil on the abundance and
diversity of mesofauna. The samples were collected on a line transect. Soil samples were collected
and taken to the laboratory to isolate and identify mesofauna found in the soil samples. The pH,
moisture content and moisture content of the soil samples were also obtained, where the
unpolluted site has the highest pH followed by remediated site while the crude oil polluted site
recorded the least pH. During the research work 37 individuals of mesofauna were encountered
(distributed in 15 species) in the entire studied sites. Mesofauna abundance was highest in the
unpolluted site (31 individuals) followed by the remediated site (4) while crude oil polluted site
recorded the least abundance (2 individuals). Highest species diversity of mesofauna was also
recorded in the unpolluted site (11). The results further explained that the effect of crude oil is
significant (P≥0.05) on the abundance of mesofauna). The relationship between pH and mesofauna
abundance and diversity showed that there were no significant differences (P<0.05). It was further
observed that the pH of the unpolluted site with the mean value of 6.79 is higher than those of
remediated (6.33) and crude oil polluted site (6.12). This work showed that abundance and species
diversity of mesofauna in the study habitats were significantly different. Soil moisture content of
the polluted site (32.29%) is higher than those of remediated (20.17%) and unpolluted site
(20.32%). Soil temperature in the remediated site is the highest value (31.03°C) followed by the
unpolluted site (29.14°C) while the crude oil affected site recorded the least temperature value
(23.11°C).
Keywords: Hydrocarbon - Pollution - Contamination - Ecosystem.
[Cite as: Adeduntan SA & Owokotomo OO (2018) Effect of oil spillage on abundance and diversity of soil
mesofauna in Bodo city, Niger Delta, Nigeria. Tropical Plant Research 5(2): 260–266]
INTRODUCTION
Crude oil, a complex mixture of hydrocarbon, liquid in their natural state is classified into aliphatic, alicyclic
and aromatic compounds (Colombe et al. 2005). Most of these components are known to be toxic in nature to
different biomass (Walker & Colwell 2000), and this has raised considerable concern on the subject of crude oil
pollution especially on agricultural land. Though oil spillage is a widespread phenomenon, it is comparatively
more frequent in the developing countries than in the technologically developed nations. According to Chindah
& Braide (2000), over 6000 spills had been recorded in the 40 years of oil exploitation in Nigeria, with an
average of 150 spills per annum. In the period 1976–1996, 647 incidents occurred resulting in the spillage of
2,369,407.04 barrels of crude oil. With only 549,060.38 barrels recovered, 1,820,410.50 barrels of oil were lost
to the ecosystem. Oil spillage has been known to exhibit various deleterious effects on both plants and
microorganisms. Crude oil spillage on soil generally retard plant growth to a large extent (Lundberg et al. 2003,
Ekpo & Nwaankpa 2005), reduces aeration by blocking air spaces between soil particles hence create an
anaerobic condition (Okiwelu 2011). Oil spillage is known to be a major environmental problem in Nigeria,
most especially in the Niger Delta. It is reported that oil spillage has caused a constant threat to farmlands, crop
plants and forest tree species by being deadly to plant roots through their interaction with soil fauna hence, the
roots die due to a lack of oxygen (Ogri 2001, Agbogidi 2003). It destroys soil fertility, causes alterations in soil
Received: 02 April 2018 Published online: 31 August 2018
Adeduntan & Owokotomo et al. 2018
physicochemical and microbiological properties, thereby having detrimental effects on the terrestrial and aquatic
ecosystems. The initial reaction of the microorganisms as it gets in contact with oil in the soil is a reduction of
activity due to reduced air availability. This has been noted to arise from leaving the resistant and adaptive
microbial strains to proliferate (di Leonardo 2007). This ability, however, depends on a number of factors which
include temperature, the viscosity of the oil, and coarseness of the soil and the level of oil in the environment.
Soil mesofauna exert strong regulatory control over the soil food web and have substantial effects on important
soil characteristics, including the distribution of soil particles, the soils water-holding capacity and water
infiltration rate, the availability of organic compounds and mineralization, immobilization, the availability of N
and other nutrients, the transport of compounds. Species that feed on decaying plant material open drainage and
aeration channels in the soil by removing roots. Faecal material of soil mesofauna remains in channels which
can be broken down by smaller animals. The aims and objectives of this study were to examine the effect of
crude oil on the diversity and abundance of mesofauna and to determine the relationship between pH,
temperature, moisture content and diversity and abundance of mesofauna in Bodo city, Niger Delta.

Study area
The study area is located in Rivers state on the coast of the Gulf of Guinea, east of the city of Port Harcourt
(Fig. 1). It extends across the local government's areas (Khana, Ghokana, Eleme and Tae). This study was
carried out at crude oil spill site, remediated site and an unpolluted site i.e. Bodo city located within Latitude 4°
37′ North of the equator and longitude 7° 16′ east of the meridian.
Figure 1. Gokana Local Government Map showing Bodo City Niger Delta, Rivers State, Nigeria
Data collection
In Bodo community, samples were collected on oil spill polluted site, Remediated site and unpolluted site.
Each soil sample unit was carefully collected along a line transect, in each of the site, soil samples were
collected and bulked for subsequent laboratory analysis. Each soil sample unit was carefully pushed into a
polythene bag which was securely tied at the mouth to prevent desiccation and spilling of soil before reaching
the laboratory. The soils samples were immediately brought to the laboratory where the mesofauna present were
isolated and identified using the floatation method.
On each sample, pH of the soil was determined in water (a soil/water ratio of 2:1) using a pH meter with
glass electrode from each of the six sampling points in each plot. Soil samples were also collected from each
sampling point for determination of pH and mesofauna identification. All the data collected were subjected to
analysis of variance test (ANOVA) at 5% level of probability to test for significance among the treatments.
Mesofauna isolation and identification
Extraction of soil mesofauna was done using Berlese funnel extraction technique (Berlese 1905). Samples
were removed from bag and placed in Berlese extractor. The dirt were placed in an extractor, clouds were
broken up so that the arthropods can emigrate properly from a sample of even consistency. Earthworms were
removed to a separate specimen vial (worm skin mucus is terribly sticky and if the worms were not removed all
of the little critters would get stuck on the worm skin). The clods were broken apart without squashing the soil.
2. 25 watt light bulb was turn on; extraction was carried out for 48 h. A jar was placed containing a small
amount of antifreeze under the Berlese funnel. Antifreeze has the advantage that it does not evaporate. The
extracted samples (critters, dirt, and alcohol or glycol) were placed into one (or more as needed) labeled small
vials. Few drops of cooking oil were added to top of vial, enough to form a thin meniscus. Cap was replaced; the
mixture was also agitated to force the oil into micro drops throughout the solution. The oil was slowly allowed
to (10 min) rise to the top carrying nearly all the arthropods with it. Critters were pipetted from the oil layer into
a Petri plate; the excess oil and glycol from the Petri plate were removed. The critters were sorted into piles of
similar species. Identification was then made. Species were counted and data was put into spreadsheet.
Soil pH determination
The soil pH was determined with the aid of glass electrode pH meter in the soil solution 0.01 mol L -1
chlorine. 10 grams of sieved soil samples was weighed into a container and 20 ml of distil water was added and
stirred together. After stirring the samples the samples were left for one hour after which the samples were
measured with pH meter and their corresponding values were carefully recorded.
RESULTS
Table 1. Effect of oil spillage on the abundance of soil mesofauna in Bodo City, Niger Delta, Nigeria.
S.N. Species Unpolluted soil (A) Remediated soil (B) Polluted soil (C) Total
1 Octolasion lacteum 2 0 0 2
2 Porcellio scaber 4 0 0 4
3 Allolobophora trapezoides 5 0 0 5
4 Aporrectade trapezoide 2 0 0 2
5 Ancistrotermis cavithorax 3 0 0 3
6 Allolobophora calignosa 4 0 0 4
7 Eudrilus eugeniae 1 0 0 1
8 Tucbergia granulata 4 0 0 4
9 Colpoda steinii 3 1 0 4
10 Centipede 1 0 0 1
11 Dorylus fimbriatus 2 1 0 3
12 Oniscus asellus 0 1 0 1
13 Folsomia candida 0 0 1 1
14 Isopod 0 0 1 1
15 Spider 0 1 0 1
Total 31 4 2 37
Thirty-seven (37) individuals of mesofauna were encountered in all the study areas (Table 1). The
Unpolluted site accounts for the highest abundance of mesofauna thirty-one (31) individuals in all, followed by
the remediated site with four (4) individuals while the crude oil polluted site has the least abundance of two (2)
individuals. The result also shows that Allolobophora trapezoids was found to have the highest number of
individual mesofauna five (5) while Eudrilus eugeniae, Oniscus asellus, Folsomia candida, Centipede, Isopod
and Spider were seen to have the least number of individuals (1 each) in their respective habitats. The result of
the effect of oil spillage on mesofauna diversity is also presented in table 2. Fifteen (15) species were
encountered in all the study area, unpolluted area has the highest diversity of 11 species followed by the
remediated area four (4), while the least diversity was found in the crude oil polluted soil. Most of the species
encountered in the study area are, Allolobophora caliginosa, Tucbergia granulate, Dorylus fimbriata,
Allolobophora trapezoids, Aporrectodae trapezoids, Colpoda stenii, Octolasion lacteum, Porcellio scaber,
Folsmia candida and Isopod were found only in the crude oil polluted soil while species like, Oniscus asellus
and spider were found in the remediated soil.
Table 2. Effect of oil spillage on the diversity of soil mesofauna in Bodo City, Niger Delta, Nigeria.
S.N. Species Unpolluted soil (A) Remediated soil (B) Polluted soil (C)
1 Octolasion lacteum * - -
2 Porcellio scaber * - -
3 Allolobophora trapezoides * - -
4 Aporrectade trapezoide * - -
5 Ancistrotermis cavithorax * - -
6 Allolobophora calignosa * - -
7 Eudrilus eugeniae * - -
8 Tucbergia granulata * - -
9 Colpoda steinii * * -
10 Centipede * - -
11 Dorylus fimbriatus * * -
12 Oniscus asellus - * -
13 Folsomia candida - - *
14 Isopod - - *
15 Spider - * -
Total 11 4 2
Note: * Represents the presence of mesofauna; - Represents the absence of mesofauna.
The effect of crude oil has been shown significant (P≥0.05) on the abundance of mesofauna in ANOVA
(Table 3). Since F-cal is greater F-tab, then there is a significant difference at 0.05 levels.
Table 3. Effect of the crude oil on the abundance of mesofauna in Bodo city, Niger Delta, Nigeria.
Source of Variation SS df MS F P-value F crit
Treatment 2.396085 2 1.198043 8.448049 0.017996 5.143253
Error 0.850878 6 0.141813
Total 3.246963 8
There is significant (p≤0.05) difference in the mesofauna abundance of the three habitats based on the
observed mean difference (Table 4). It also shows that there is significant (p≤0.05) difference between
unpolluted soil, remediated soil and crude oil affected soils.
Table 4. Effect of crude oil on mesofauna abundance in Bodo city, Niger Delta, Nigeria.
Treatment Means(103)
Unpolluted soil (A) 1.2307b
Remediated soil (B) 0.3662a
Crude oil polluted soil (C) 0.0001a
Note: Mean with the same alphabets are not significantly different at p≥0.05
The relationship between pH and mesofauna abundance (Y= -281.532X + 45.8131) is not significant
(P≥0.05) (Table 5). The rate at which pH affects the abundance of mesofauna in the habitats is very high (R=
0.9712). The result further shows that a change in pH will cause as high as 94.41% change in mesofauna
abundance.
Table 5. Regression relationships of physicochemical parameters on the abundance and distribution of soil mesofauna in
Bodo City, Niger Delta, Nigeria.
Relationships Equation R R2 P-value Remarks
Relationship between pH and Y = -281.532X + 45.8131 0.9712 0.9441 0.1519 NS
Mesofauna abundance
Relationship between pH and Y = 13.7048X - 82.2423 0.9962 0.9924 0.0555 NS
Mesofauna diversity
Relationship between MC and Y = -1.2652X + 43.0247 0.5431 0.2949 0.6345 NS
Mesofauna abundance
Relationship between MC and Y = -0.4510X + 16.6079 0.6635 0.4403 0.5381 NS
Mesofauna diversity
Relationship between TEMP and Y = 0.5531X - 9.6896 0.4843 0.2345 0.6781 NS
Mesofauna diversity
Relationship between TEMP and Y = 1.3575X - 25.3504 0.3467 0.1202 0.7745 NS
Mesofauna abundance
Note: MC, Moisture content; TEMP, Temperature; pH, Hydrogen potential.
The regression equation showing the relationship between pH and mesofauna diversity (Y= 13.7048X -
82.2423) is not significant (P≥0.05). The rate at which soil pH affects the mesofauna diversity is very high
(R=0.9962). This further shows that a change in pH will cause about 99.24% change in mesofauna diversity.
The regression equation showing the relationship between moisture content and mesofauna abundance (Y= -
1.2652X + 43.0247) is not significant (P≥0.05). The rate at which soil pH affects the mesofauna diversity is
moderately high (R=0.5431). This further shows that a change in moisture content will cause about 29.49%
change in mesofauna abundance. The regression equation showing the relationship between moisture content
and mesofauna diversity (Y= -0.4510X + 16.6079) is not significant (P≥0.05). The rate at which soil moisture
content affects the mesofauna diversity is high (R= 0.6635). This further shows that a change in moisture
content will cause about 44.03% change in mesofauna diversity.

The regression equation showing the relationship between temperature and mesofauna abundance (Y=
1.3575X - 25.3504) is not significant (P≥0.05). The rate at which soil temperature affects bacteria diversity is
low (R= 0.3467). This further shows that a change in temperature will cause about 12.02% change in bacteria
abundance. The regression equation showing the relationship between temperature and mesofauna diversity (Y=
0.5531X - 9.6896) is not significant (P≥0.05). The relationship between temperature and mesofauna diversity is
weak (correlation coefficient R=0.4843). This means that a change in temperature will lead to a small change in
mesofauna diversity.
DISCUSSION
The result showed that unpolluted soil has the highest abundance of mesofauna (31) individuals followed by
the remediated soil (4), while crude oil polluted soil has the least abundance of 2 individuals in the study area.
The low number of soil mesofauna recorded in the polluted site could be because the spills might have killed off
many of the mesofauna by adversely affecting respiration. The oil might have adversely affected their food
source, leading to reduced reproductive rate. Table 2 shows that the highest species diversity of mesofauna was
also high in unpolluted soil (11) followed by remediated soil with 4 species while the least diversity is recorded
in the crude oil polluted soil (2). This result implies that more species of mesofauna tends to live in unpolluted
soil. This conforms to the observation of Rapport (1983) who reported that mesofauna lives where there is a
minimum disturbance of the ecosystem. Which therefore means mesofauna is one of the indicator species of the
unpolluted habitat.
Crude oil has significant effect on the abundance of mesofauna because there is a significant difference
between the unpolluted, remediated and oil polluted soils, this may be due to varying factors that might favor
their existence such as the soil pH, temperature or their microenvironment conditions, such as, humidity and air
composition which may either be sufficient or insufficient around and within the mesofauna immediate habitat.
There is significant (p≤0.05) difference in the mesofauna abundance of the three habitats based on the
observed mean difference. It also shows that there is significant (p≤0.05) difference between unpolluted soil,
remediated soil and crude oil affected soils. The unpolluted soil has the highest mean value which makes it the
best soil habitat. This is however similar to the reports of (Evans 1999), which stated that fauna is not
necessarily more in the natural forest than reserves or plantations.
The summary of regression analysis shows that there is no significant (p ≥ 0.05) interaction between soil pH
and mesofauna abundance and diversity in the three habitats this may be due to the fact that the three habitats
are subjected to the same climatic condition, since they are all within the same ecological zone (tropical
rainforest). It was further observed that the pH of the unpolluted soil is higher than others, which indicates that
the soils of unpolluted soils are more fertile. This suggests that soil pH might play a significant role in
influencing the soil fertility; hence, the diversity and abundance of soil mesofauna are also affected. The result
of this finding is similar to Badejo & Ola-Adams (2000) who reported that the soil pH of the Natural forest is
higher than its surrounding plantations and cultivated land.
The unpolluted soils has the highest pH followed by the remediated soils while the crude oil polluted soils
recorded the least pH, i.e. the pH values of the polluted samples obtained in this study were lower than those of
unpolluted samples. These results are similar to the findings of Amadi et al. (1993) and Chukwuma et al.
(2010). It is also contrary to the findings of Andrade et al. (2004) and Ayotamuno et al. (2004) who observed an
increase in the pH of soils polluted with crude oil.
The polluted soil has the highest moisture content followed by the unpolluted soil while the remediated soil
has the least moisture content. These values are in agreement with the findings of Bossert & Bartha (1984) in
which they concluded that moisture contents ranging from 20% to 80% are generally optimum for hydrocarbon
degradation. The remediated soil has the highest temperature followed by the unpolluted soil while the polluted
soil recorded the least temperature.
CONCLUSION
It can be concluded that oil spill adversely affected both abundance and diversity of soil mesofauna. The test
results obtained from the soil analysis of the oil-spilled impacted sites (Bodo community) compared to the result
of the unpolluted site shows that the total abundance of soil mesofauna from both the oil spilled sites and
remediated sites have provided evidence of severe hydrocarbon contamination of the sites. These conditions
imply reduction in soil fauna composition which is important in decomposition and mineralization, thereby
affecting agricultural productivity.
RECOMMENDATIONS
Having successfully analyzed the soil samples in the Bodo community, it has shown that the crude oil has
affected the diversity and abundance of soil mesofauna. Therefore, in order to minimize the rate of spills in this
community, the following recommendations are suggested.
 Adequate security personnel should be provided to guard oil installation and such security arrangement
should involve people from the host communities to work-in collaboration with government security
forces to improve monitoring of oil facilities to avoid vandalization.
 The constant seminar, training workshop, public enlightenment campaign should be organized for host
communities and other stakeholders in the oil industry to educate them on the negative impact of oil
spillage on soil mesofauna.
ACKNOWLEDGEMENTS
We wish to appreciate the dwellers of the study areas (Bodo community) for giving us an audience and
opportunity to carry out our research on oil spillage in Bodo Niger Delta, Nigeria.
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ISSN (Online): 2349-1183; ISSN (Print): 2349-9265

TROPICAL PLANT RESEARCH 5(2): 121–128, 2018

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