Volume 6, Issue 1 (2019) Tropical Plant Research

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com ISSN (Online): 2349-1183

ISSN (Print) : 2349-9265
Tropical Plant Research

Volume 6 Issue I 30 April, 2019
https://doi.org/10.22271/tpr.2019.v6.i1
Published by AkiNik Publications

For Society for Tropical Plant Research
at New Delhi, India
ISSN (Online): 2349-1183; ISSN (Print): 2349-9265
TROPICAL PLANT RESEARCH 6(1): 01–07, 2019
The Journal of the Society for Tropical Plant Research DOI: 10.22271/tpr.2019.v6.i1.001
Research article
Phenotypic plasticity of Centella asiatica (L.) Urb. growing in

different habitats of Nepal
Anjana Devkota* and Pramod Kumar Jha
Central Department of Botany, Tribhuvan University, Kathmandu, Nepal
*Corresponding Author: devkotaa@gmail.com [Accepted: 05 February 2019]
Abstract: The plant growing in range of environmental conditions exhibits phenotypic plasticity
that reflects the ability of the plant to allow its establishment in different areas. Centella asiatica,
an important medicinal plant, is widely growing in tropical and subtropical belt of Nepal. We
measured phenotypic characters (density, petiole length, stolon length, SLA, leaf number per
ramet, plant biomass, flower number) and soil attributes (soil pH, soil nitrogen (N), soil organic
carbon (OC), soil organic matter (OM) of 21 C. asiatica populations in three habitats (open
grassland, open agricultural land, shady grassland) of Nepal. Ramet density (105 plants m-2) and
biomass yield (52.5 g m-2) was found highest in partially shaded grassland with soil having 5.83
pH, 0.20% N, 4.26% OC and 7.38% OM. Leaves had 391 cm2 g-1 SLA, 4.13 cm long petiole and
1.76% N. The three sites differed significantly (p<0.001) in petiole length, SLA, leaf N, soil N,
soil OC and soil OM contents. Thus land uses had a significant effect on ramet density and leaf
characters of C. asiatica. Phenotypic plasticity in leaf petiole length and number of flowers per
inflorescence was observed, which appeared to be governed by light availability and height of
associated species. In terms of yield partially shaded grassland was the most suitable natural
habitat of C. asiatica. Evaluation of growth traits and yield in a different habitat help to find the
suitable condition for growth of the plant in nature. This information is helpful in planning
cultivation of C. asiatica.
Keywords: Phenotypic plasticity - Growth traits - Habitats - Yield.
[Cite as: Devkota A & Jha PK (2019) Phenotypic plasticity of Centella asiatica (L.) Urb. growing in different
habitats of Nepal. Tropical Plant Research 6(1): 01–07]
INTRODUCTION
The plants occurring in varying environmental conditions (i.e. soil characteristics and/or light intensity)
exhibit phenotypic plasticity in the form of various important ecological traits which reflects the adaptiveness of
the plant in habitat (Tyler et al. 2007, Zhu et al. 2007, Zhao et al. 2010). Centella asiatica (L.) Urb., an
important medicinal plant, belongs to family Apiaceae is widely distributed in tropical and subtropical region of
Nepal. The plant is indigenous to the warmer regions of both hemispheres and found in different parts of the
world including Africa, Australia, Cambodia, Central America, China, Indonesia, the Lao People’s Democratic
Republic, Madagascar, the Pacific Islands, South America, Thailand, Southern United States of America, and
Viet Nam (Annonymous 1953, Iwu 1993). The plant flourishes in damp, moist and shady habitats and grows by
producing stolons that are characterized by long internodes and nodes, on which are borne reniform-cordate
leaves and sessile flowers in simple umbels. The plant has multiple uses in different parts of the world as
vegetable in Asian and in some western countries (Peiris & Kays 1996), as medicine for treatment of leprosy
and psoriasis, wounds and burns, and for insanity in traditional Indian and Chinese systems of medicine (Bose
1932, Kan 1986), as memory enhancing tonics, mental and stress-related disorders (Sharan & Khare 1991,
Moharana & Moharana 1994), and as cosmetic industry for the preparation of hair oils, tonics and shampoos.
Though Centella asiatica grows widely in a different habitat, evaluation of best habitat condition suitable for
growth of the plant is not studied in detail. In view of this 21 different population of C. asiatica were identified
representing different habitat (open grassland, open agricultural land, shady grassland) of Nepal. Evaluation of
www.tropicalplantresearch.com 1
Received: 19 June 2018 Published online: 28 February 2019
https://doi.org/10.22271/tpr.2019.v6.i1.001
Devkota & Jha 2019
growth traits and yield in a different habitat help to find the suitable condition for the growth of the plant in
nature. This information is helpful in planning cultivation of C. asiatica.
MATERIALS AND METHODS

The Species
Centella asiatica (L.) Urb. a small creeping perennial herb belongs to family Apiaceae has following
morphological characters: slender stem, creeping stolons, green to reddish green in color, interconnecting one
plant to another with kidney-shaped leaves emerging alternately in clusters at the petiole. The runners lie along
the ground and leaves with their scalloped edges rise above on long reddish petioles. The rootstock consists of
rhizome growing vertically down (DPR 1986).
Field sampling and analysis of plant and soil
Preliminary information on the range of ecological habitats for C. asiatica growth was obtained by
reviewing literature (Malla et al. 1997, Press et al. 2000). Potential sites were then visited for field sampling
between 2006 and 2008. Twenty-one different populations of C. asiatica were identified representing different
habitats (open grassland, open agricultural land, partially shaded grassland) and soil characteristics from Nepal.
Detail plant and soil chemical analysis were carried out from the selected habitats. In each population, 2–5 plots
of 10 m × 10 m were subjectively chosen to represent the patches with relatively high density of C. asiatica. In
each plot, 10 quadrats (1 m × 1 m) were sampled randomly. Altogether 54 plots (10 m × 10 m size) have been
categorized into three different habitats considering 18 plots per habitat. Total number of individuals of C.
asiatica was recorded from sampling plots. Because of the rosette habit, each adult with a rosette of leaves and a
root system was considered an individual. Ramet density was calculated in number per square meter (plants m-
2
). To determine aboveground biomass, all individuals of C. asiatica were collected from a single most densely
populated quadrat of the large (10 m × 10 m) plot. Biomass was also estimated in 54 quadrats.
From each population ninety leaf samples of C. asiatica were collected (three from a single ramet) randomly
from each subplot. Collected green leaf samples were shade dried and analysed for total nitrogen content.
Species specific traits: During field visit, individual plants of C. asiatica to study life history traits also
sampled. Healthy and undamaged 40–50 individuals of C. asiatica were completely uprooted and out of which
thirty healthy plants were selected to determine ramet diameter, number of leaves per ramet, number of primary
branches, stolon length, petiole length, number of flowers borne by individual plant.
Ninety mature leaves, three from each plant per population were measured for petiole length (PL) and
specific leaf area (SLA). Petiole length, length and width of leaves were measured in fresh leaves. Then these
leaves were oven dried (60ºC, 48 h) and mass of each leaf was weighed in electric balance (0.001 g). Length and
width of leaves were measured and multiplied by conversion factor following Zobel et al. (1987) for the
determination of leaf area. SLA was calculated by dividing leaf area with per unit dry mass. The number of
leaves (NLN) from each node was also scored. The total number of flowers per mature rosette was also scored.
Dry mass of individual plant per replication was obtained after harvest. Leaf nitrogen (N) content was
determined twenty samples per population as permodified micro Kjeldahl method following the procedure
described by Horneck & Miller (1998).
Soil: About 200 g soil samples from the rooting depth (5–10 cm) of the plant was collected and analysed for
soil pH, organic carbon (OC), organic matter (OM) and total nitrogen (N) content in air-dried soil samples after
passed through fine sieve (mesh size 0.5 mm). A total of thirty soil samples were analysed following the method
described by Gupta (2000).
Numerical Analysis
The significance of the difference in measured attributes among the three habitats was tested by one way
analysis of variance (ANOVA). These three habitats were also compared by multiple range tests (Duncan
Homogeneity test). The multiple range tests allow comparison of the pairs of sites for each attribute. Statistical
Package for Social Science (SPSS version 11.5, 2002) was used for all statistical analysis.
RESULTS
Morphology, distribution and abundance of Centella asiatica
Centella asiatica was found throughout Nepal (eastern, central and western) from <85 to 2300 m asl in a
wide range of habitats from open fallow land, to roadside to shady grassland (Table 1). Population distribution
of C. asiatica in open agricultural fallow land was quite sparse (Table 2), whereas the population distribution
was quite wide in open to shady grassland with sparse density of other associated species.
Tropical Plant Research (2019) 6(1): 01–07
Table 1. Collection sites and habitats of Centella asiatica.

S.N. District Locality Elevation Eco- Habitat Land use
(m asl) region
1. Ilam Phidim 1250 E Open Grassland Grazing by cattle
2. Jhapa Bhadrapur 85 E Agricultural fallow land Fallow land,
grazing of cattle
3 Sunsari Inaruwa 96 E Grassland Cattle grazing
was common
4. Dhankuta Hille 1800 E Grassland Cattle grazing
was common
5. Makwanpur Daman 2300 C Open Grassland Grazing by cattle
6. Makwanpur Hetauda 650 C Grassland Grazing by cattle
7. Chitwan Gauriganj 250 C Open Grassland, Fallow land,
Agricultural Land grazing by cattle
8. Gorkha Palungtar 600 C Open Grassland, Agricultural Grazing by cattle
Fallow land, Shady place
9. Lamjung Way to 740 C Shady grassland Cattle grazing
Besisahar was prohibited
10. Lalitpur Godavari 1550 C Open Grassland Grazing by cattle
11 Kathmandu Kirtipur 1350 C Open and shady Grassland, Grazing by cattle
Agricultural fallow land
12 Kathmandu Matatirtha 1400 C Shady Grassland, Open Fallow land,
Agricultural fallow land grazing by cattle
13 Kaski Pokhara 850 C Open grass land grazing by cattle
14 Kaski Dhampus 1800 C Shady grassland, Agricultural
fallow land
15 Pyuthan Ampchaur 1250 W Open Grassland, Shady Fallow land,
grassland grazing by cattle
16 Dang Lamahi 150 W Open grassland Fallow land,
grazing by cattle
17 Surkhet Birendranagar 650 W Shady grassland Cattle grazing
was prohibited
18 Banke Kohalpur 155 W Open fallow land Fallow land,
grazing by cattle
19 Bardiya Magaragadi 150 W Shady grassland Cattle grazing
was prohibited
20 Kailali Dhangadi 130 W Open grassland Grazing by cattle
21 Kanchanpur Mahendranagar 150 W Open grassland Grazing by cattle
Table 2. Density and morphological characters of Centella asiatica in different habitats. For each parameter significant
difference between mean among different sites are indicated by different letters (Duncan homogeneity test, α = 0.05). F and P
values were obtained by one way analysis of variance (ANOVA).
Attributes Partially shaded Open Open N Mean F value P value
grassland grassland agricultural land
Density (plants m-2) 105.24c ±2.09 82.14b ±1.43 32.12a ±1.57 630 73.16±1.69 22.025 < 0.001
Petiole length (cm) 5.34c ±0.25 3.38b ±0.4 2.16a ±0.89 1890 4.13 ±1.02 47.95 < 0.001
Leaf length (cm) 1.9c ±0.53 1.63b ±0.96 1.57b ±0.34 1890 1.68±0.71 40.21 < 0.001
Leaf width (cm) 3.03b ±0.90 2.59a ±0.80 2.62a ±0.60 1890 2.74±0.77 4.48 <0.001
Stolon length (cm) 6.08b ±1.29 5.78 a ±0.98 5.25a ±1.08 630 5.82±1.19 2.59 0.076
SLA (cm2 g-1) 492b ±41 322a ±26 279a ±29 1890 391±23 8.74 < 0.001
Leaf no/ramet 3.6a ±0.89 3.67a ±1.08 3.74a ±0.82 630 3.69±0.96 0.154 0.857
2 c b
Plant biomass (g m ) 52.5 ±0.89 38.23 ±1.20 20.12 a ±0.28 630 36.95 ±0.79 18.50 < 0.001
Flower no/ramet 3.14 a ±1.14 6.78b ±2.53 10.56c ±2.36 630 6.61±2.01 33.35 < 0.001
a b
Seeds/ ramet 0.24 ±0.03 2.36 ±1.21 9.65c ±0.62 630 4.8±0.3 28.14 < 0.001
Population density and above ground biomass yield varied with habitats (Table 2). Mean values of plant
density and biomass across the habitats were 73 plants m-2 and 37 g m-2, respectively. Highest density was
shared by relatively undisturbed shaded grassland. Partially shaded grassland had the highest ramet density (105
plants m-2) and biomass (52.5 g m-2) yield. Open agricultural land had the least density (32 plants m-2) and
biomass (20 g m-2) yield. Density and biomass production of C. asiatica increased relatively with increasing soil
N and OC (Table 2 & 3).
There was a significant difference in growth (petiole length, specific leaf area, leaf mass and stolon length)
and reproductive traits (flowers/plant, seeds/plant) of Centella asiatica in different habitats. Petiole length
Devkota & Jha 2019
ranged from 2.16 cm at open agricultural land to 5.34 cm at partially shaded grassland (Table 2). There was a
significant difference (p = <0.001) in leaf length and leaf width of individual leaf among the habitat. Specific
leaf area ranged from 279 to 492 cm2 g-1 (mean 391 cm2 g-1, n = 1890). The average number of leaves per ramet
was not affected by habitats. The number of flowers per ramet was highest (10.56) in open agricultural fallow
land. The number of flowers per inflorescence in open agricultural land was three times and two times higher
than in partially shaded grassland and open grassland, respectively.
Table 3. Leaf nitrogen (N) content and soil nutrients (nitrogen and organic carbon) in Centella asiatica habitats.
For each parameter significant difference between mean among the sites are indicated by different letters (Duncan
homogeneity test, α = 0.05). F and P values were obtained by one way analysis of variance (ANOVA).
Attributes Partial shaded Open Open N Mean F value P value
grassland grassland agricultural land
Leaf N (%) 1.95c ±0.74 1.65b ±0.63 1.52a ±0.73 630 1.76±0.72 23.55 < 0.001
b a
Soil pH 5.83 ±0.94 5.57 ±1.02 5.69a ±0.7 630 5.63±0.7 8.62 < 0.001
Soil N (%) 0.20b ±0.07 0.18 b ±0.10 0.16a ±0.07 630 0.18±0.08 14.97 < 0.001
Soil OM (%) 7.38b ±1.21 5.14 b ±1.86 4.41a ±2.21 630 5.64 ±1.76 19.89 0.032
b b
Soil OC (%) 4.26 ±1.21 2.97 ±1.26 2.55a ±1.3 630 3.16±1.26 20.49 0.041
CN ratio 21.3 a ±2.65 16.5a ±2.34 15.93a ±4.32 630 17.91±3.10 0.95 0.386
Nutrient content of soil and plant parts
Leaf N content ranged from 1.52 to 1.95% (average 1.76%). Leaf N content differed significantly (p <0.001)
along C. asiatica habitats. Soil pH ranged from 5.57(open agricultural land) to 5.83 (partially shaded grassland).
There was a significant difference (p<0.001) in soil pH among the habitats. Soil N content ranged from 0.16 to
0.20 % (average 0.182 %) and there was significant difference (p <0.001) among three habitats (Table 3). Soil
organic carbon (OC) ranged from 2.55 % at open agricultural land to 4.26 % at shady land with average 3.26 %
for all sites. Mean soil organic matter (OM) was 5.64 %. There was a significant difference in soil OM and OC
among the sites (Table 3). C/N ratio ranged from 15.93 at open agricultural land to 21.3 at shady grassland.
There was no significant difference in C/N ratio among habitats.
DISCUSSION
Distribution of Centella asiatica
Centella asiatica has wide distribution in terms of elevation (< 95m to about 1900 m asl), habitat types (open
agricultural land, grassland to shady fallow land) and physiography (eastern to western Nepal, Terai plain to
midhills of Mahabharat range in Nepal (Table 1). It was also reported from wetland system of the Terai region
of Nepal (Burlakoti & Karmacharya 2004).
This species has been reported from the shady marshy ground and roadside ditches of Sikkim and Bhutan
from altitudinal range 400 m to 1500 m asl (Grierson & Long 1999). In open agricultural land C. asiatica
distributed sparsely, while in grassland it grows vigorously. Abundant distribution of the species in the swampy
areas of India, the Islamic Republic of Iran, Pakistan and Sri Lanka up to an altitude of approximately 700m
have been reported (Tyler et al. 1988, Farnsworth & Bunyapraphatsara 1992, Iwu 1993).
It appears that deep shade within tall tress was not a suitable microhabitat for C. asiatica. In partially shaded
grassland; density of C. asiatica was higher than in nearby open grassland and open agricultural land (Table 3).
Thus the distribution of this plant in nature is appeared to be determined by light availability.
Abundance
Population density and above ground biomass varied with habitats (Table 2). Mean values of density and
biomass across the habitats were 73 plants m-2 and 37 g m-2, respectively. Highest density was found in
relatively undisturbed moist partially shaded grassland. Partially shaded grassland had the highestramet density
(105 plants m-2) and biomass (53 g m-2) yield. The highest ramet density and biomass production in partially
shaded grassland could possibly due to high nutrient content (Table 2). Lowest ramet density of C. asiatica in
agricultural land could be due to periodic disturbance during agricultural practices. Given the highest plant
density and biomass production in partially shaded grassland, this habitat can be considered as the most suitable
habitats for the growth of this plant under natural conditions. Comparable data on population and abundance of
this species is not available from Nepal and elsewhere (Devkota & Jha 2008); but experimental shading has
shown that plant biomass yield of C. asiatica was the highest under partial shading condition (Mathur et al.
2000).
Variation in growth characters
Variation in growth characters and reproductive outputs among the individuals of Centella asiatica growing
in different habitats (open agricultural land, open grass land and partially shaded grassland) (Table 2) represents
growth responses to resource availability. The variation in petiole length was adaptive response to light.
Plasticity of petiole helps to place the lamina in areas of high light (de Kroon & Hutchings 1995). Herbaceous
dicot plants growing in open habitat often respond to shading by elongating internodes and petiole length
(Huber 1996), and improves access to light (Falster & Westoby 2003). Long petiole raises the leaf lamina and
enables the plant to receive adequate light when density and height of associated species are high. This is a
common strategy of light-demanding herbaceous species. Shortest petiole length of C. asiatica at agricultural
land was due to less density of associated species and more open area as it received a sufficient amount of light.
There is no need to develop long petiole for the plant. Plasticity in lamina and petiole form occurs both between
and within plantsin response to contrasting exposure to light (Niklas 1999, Niinemets & Fleck 2002). Due to
open land both in agricultural as well as grassland, plant easily get sufficient light so no need of the extension of
cell of petiole as well as stolon. This explains the short petiole length in the leaf of C. asiatica in open habitat.
There was a significant difference (p>0.001) in SLA among three habitats. The variation in SLA might be
due to different light intensity (Hughes & Cockshull 1972, Heuvelink & Marcelis 1996) or may due to the
variation of leaf nutrient. The highest SLA value (492 cm2 g-1) of C. asiatica in shady grassland may be due to
the reduction in available light to the leaves when the plant density was high. A positive effect of plant
densityon SLA has been found in other crops, e.g. potato (Vos 1995), tomato (Heuvelink & Marcelis 1996) and
Impatiens capensis (Maliakal et al. 1999). Plants were grown in high light generally have thick leaves with low
SLA (Bjorkman 1981). Average SLA of Centella lies near the median range (14 to 150 g m-2) of global data set
for 2548 species compiled by Wright et al. (2004).
There was a significant difference in the number of flower per ramet among the habitats (Table 2). There
appears trade-off between densities of ramets and number of flower per inflorescence. The number of flower per
inflorescence was highest at open agricultural land where ramet density was the lowest. Failure of the large
proportion of ramets to bear flower at shady grassland may be due to density dependent factors such as
competition for resources (e.g. nitrogen), space as well as the light factor. The lowest number of flower per
inflorescence in shady grassland might also be due to the light factor. Dense growth of associated species could
also be less favourable for the production of flowers. The plants of shady grassland tended to invest less in
sexual fecundity and more in traits ensuring vegetative offshoots. Differential patterns of ramet recruitment and
growth in different habitats also underlie variation in population growth rates in other perennial herbs (Tictin &
Nantel 2004).
Nutrient status
i. Leaf nitrogen content: Land use history and ramet density of Centella asiatica had significant influence
(p<0.001) on the leaf N content. Leaf N content increased with increasing ramet density at these different
habitats. The open agricultural land with lowest leaf N content had lowest ramet density, and relatively low
soil N content, whereas opposite were the cases at shady grassland. Shaded leaves have a higher N
concentration than exposed leaves (Lusk 2002). Plant density varied significantly with soil nitrogen
(p>0.05).It appears that high ramet density and high soil N content may be responsible for high leaf N
content at shady grassland. Though leaf N appeared to be dependent of soil N (correlations, p > 0.03), the
leaf N content of C. asiatica was 8–9 times higher than soil N content. Average leaf N content of Centella
asiatica (1.76%) lies within the range (0.2–6.4 %) of global data set for 2548 species compiled by Wright et
al. (2004). It was also comparable to other tropical herbs in Nepalese grasslands (0.9–2.3 %, Jha 2003). Leaf
N content from 1.5–2.0 % (15–20 mg g-1) has been considered as adequate for growth of most plants
(Chapin & Cleve 1989). Leaf N content of C. asiatica lies within this range.
ii. Soil nutrient: Different habitats had a significant impact on soil N and soil organic carbon (OC) (Table 3).
High soil OC in shady grassland could be due to the addition of organic fertilizer by litter decomposition.
Increase in total nitrogen is due to the increase in organic matter content of the soil (Hatton &Smart 1984).
Low OC in soils of open agricultural land could be the result of continuous utilization of resources of soil by
crop. Likewise, low OC in soils of grazing land may be due to the removal of above ground biomass by
livestock, which removed plant biomass and dilutes the soil OC. Soil OC of C. asiatica habitat lies within
average values (1.34–3.35%) reported for the soil of tropical zone of eastern Nepal (Jha 2003).
Average soil N content of the study sites was comparable to the global average of soil N content which is
2 gk g-1 (0.2%) (Larcher 1995), while the value was close to soil N content of warmer climates (tropical and
subtropical) where soil N is generally >0.1 (Banarjee et al. 1989, Paudel & Sah 2003, Jha 2003). Average
C/N ratio of the soil of C. asiatica habitat is higher than the upper limit of C/N ratio for fertile soil with
Devkota & Jha 2019
stable organic matter (10–12, Biswas & Mukherjee 1999). That was due to the low amount of total nitrogen
in the soil.
CONCLUSION
Centella asiatica exhibits significant differences in certain morphological characters like (ramet density,
petiole lengths, number of flower per inflorescence and stolon length) and species-specific traits (leaf N content)
in relation to habitat conditions (light intensity and soil N, OC, OM) but had no effect on leaf number per ramet.
Long petiole with less number of flower at partially shaded grassland and short petiole with more number of
flower per inflorescence at open agricultural land of C. asiatica indicate light demanding nature of the plant. In
terms of yield partially shaded grassland was the most suitable natural habitat of C. asiatica.
ACKNOWLEDGEMENT
We are thankful to Dr Bharat Babu Shrestha for helping in statistical analysis. Partial financial support for
this research from University Grants Commission, Nepal, is thankfully acknowledged.
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Research article
Effects of chitosan on vegetative organs growth and peroxidases

activities in cassava (Manihot esculenta Crantz) cultivars YACE,
9620A, TMS4(2)1425 and TMS30572
Kouakou Dappah KRA1*, Seu Jonathan GOGBEU2, Kafana SORO3, Kouassi
Joseph KOUAKOU4, Koffi II Nazaire KOUASSI5 and Denezon Odette DOGBO1
1
Laboratory of Biology and Improvement of Plant Production, University Nangui Abrogoua, Côte d'Ivoire
2
Laboratory of Plant Physiology and Pathology, University Jean Lorougnon Guédé, Côte d’Ivoire
3
Laboratory of Floristic Biodiversity, Ecology Research Center, University Nangui Abrogoua, Côte d'Ivoire
4
Laboratory of Biology and Improvement of Plant Production, University Nangui Abrogoua, Côte d'Ivoire
5
Laboratory of Plant Physiology, university Félix Houphouet-Boigny, Côte d'Ivoire
*Corresponding Author: dappahk@yahoo.fr [Accepted: 08 February 2019]
Abstract: In this work, the influence of chitosan on vegetative organs growth and peroxidases
activities in cassava (Manihot esculenta, Euphorbiaceae) plants grown in hydroponic was studied.
The experiment was carried out under a greenhouse lit by natural light at the Ecology Research
Center (Côte d’Ivoire). Six concentrations of chitosan (0, 25, 50, 75, 100 and 125 mg L-1) added to
the culture media were tested on four cassava cultivars (Yace, 9620A, TMS4(2)1425 and
TMS30572). These media are renewed every 15 days and growth of the plants lasted two months.
The results showed that chitosan stimulated the growth of 1.1 to 1.4 according to cultivars and
organs. Peroxidases activities were 1.5 to 2.6 times higher in leaves and roots in presence of 75 or
100 mg L-1 of chitosan depending on the cultivars. This activity is multiplied by 2.3 to 2.6
respectively in leaves and roots of Yacé, 2.6 and 1.5 of TMS30572, 1.9 and 2.2 of TMS4(2)1425.
In 9620A, this factor was less than 2. These results show that chitosan could be a biostimulator in
cassava.
Keywords: Peroxidases activities - Chitosan concentration - Growth - Cassava cultivar -
Vegetative organs.
[Cite as: Kra KD, Gogbeu SJ, Soro K, Kouakou KJ, Kouassi KN & Dogbo DO (2019) Effects of chitosan on
vegetative organs growth and peroxidases activities in cassava (Manihot esculenta Crantz) cultivars YACE,
9620A, TMS4(2)1425 and TMS30572. Tropical Plant Research 6(1): 08–14]
INTRODUCTION
Chitosan is a natural copolymers of N-acetyl-D-glucosamine and D-glucosamine in variable proportion
(Katiyar et al. 2014). It is rare in nature but could be found in cell walls of certain fungi such as zygomycetes
(Tayel et al. 2010). In addition to these organisms, chitosan is obtained after deacetylation of chitin, which is the
second most abundant natural biopolymer in nature after cellulose (Shahidi et al. 1999). This is explained by
fact that chitin is one of the protective cuticle constituents of certain invertebrates teguments, such as insects,
molluscs and crustaceans as well as cell walls of fungi (Rinaudo 2006, Tayel et al. 2010).
Chitosan is a biodegradable and non-polluting polymer for the environment. It has used in many fields,
including agriculture (Dzung 2005, Dzung 2007). Several authors have reported that this molecule influences
positively plants agronomic parameters. Indeed, chitosan stimulates germination (Xue et al. 2002), growth
(Mondal et al. 2012, Piyavadee 2013, Mondal et al. 2016) and plants production (Salachna & Zawadzińska
2014, Salama et al. 2015). It has been used successfully in plants protection against abiotic (Lizarraga-Pauli et
al. 2011, Jabeen & Ahmad 2013, Pongprayoon et al. 2013) and biotic stress (Coqueiro & Piero 2011, Falcón-
Rodriguez et al. 2014).
These different actions of chitosan on plants would be done by metabolism modification. Indeed, works
Received: 18 July 2018 Published online: 28 February 2019
carried out by some authors have shown that this compound stimulates synthesis of several enzymes including
hydrolases, polyphenol oxidases, catalases and peroxidases (Falcón-Rodríguez et al. 2009, Ricardo et al. 2013,
Ben-Shalom et al. 2013).
The Objective of this work was to evaluate chitosan effects on the vegetative organs growth and peroxidases
activities in cassava cultivars (Manihot esculenta Crantz) Yacé, 9620A, TMS4(2)1425 and TM30572 cultivated
in hydroponic.
MATERIAL AND METHODS

Plant material
Stems of cassava cultivars Yace, 9620A, TMS4(2)1425 and TM30572 were obtained at National Center for
Agronomic Research (CNRA) and multiplied on an experimental plot. This plot is located at Azaguié, about 45
km from Abidjan. Among these cultivars, Yace is a local cultivar, 9620A is a cultivar improved by CNRA
whereas TMS4(2)1425 and TM30572 were cultivars proved from International Institute of Tropical Agriculture
(IITA).
Obtaining plants and treatments
Stems of these different cultivars were sterilized with sodium hypochlorite 3.6% (w/v) and alcohol 70%
(v/v). After three rinses with sterile distilled water, stems were cut into cuttings at least 20 cm long and placed in
liquid nutrient medium according to Gogbeu et al. (2012) method. In this medium, different amounts of chitosan
(SIGMA) were added so as to obtain final concentrations of 0; 25; 50; 75; 100 and 125 mg L-1. Seedlings from
cuttings were kept in culture for 2 months under a greenhouse illuminated by natural light. All treatments of a
cultivar were randomly arranged. Each treatment was replicated three times.
Determination of agronomic parameters
Agronomic parameters measured on these 2-month-old seedlings were leaf surface, length of shoot and root.
Measurements were taken on all plants of each treatment. Length of a plant was determined from the insertion
point of shoot on cutting to seedling apex. Length of root was determined from its point of insertion on the
cutting to root apex. These measurements were made using a tape measure. Leaf surface was determined by the
method described by Gogbeu et al. (2015). It consisted in spreading sheet on paper of known dimensions. Initial
mass (M) and surface (S) of paper were determined. Cassava leaf is then spread on this paper and outline of it
was delimited with a pencil. The bounded surface was cut with a blade and sheet of paper was weighed again
and final mass (M’) was obtained. Leaf surface (LS) was determined according to the following formula:
( )
Average leaf surface (ALS) for each treatment was determined according to the following formula:
, with N = total number of leaves.
Estimation of peroxidases activities
After determining agronomic parameters, leaves and roots of these seedlings were used for determination of
peroxidases activities. Leaves taken are those of ranks 3, 4, 5 and 6 denoted f3, f4, f5 and f6, numbered from
apex cauline to base. One (1) g of limb or root was crushed in 5 mL of sodium phosphate buffer (0.2 M, pH
6.8), 0.1 g of polyvinylpyrrolidone and 50 µL of Triton X-100 (1%, v/v). The ground material was centrifuged
at 8000 rpm for 10 min at 4°C. Resulting supernatant was incubated with 3% (w/v) Dowex 50 (SIGMA) for 30
min at 4°C under shaking. The mixture was centrifuged as before. This last supernatant constituted enzymatic
extract used for peroxidases assay. Dosage of peroxidases was done by Criquet et al. (2001). Reaction medium
is composed of 0.1 ml of enzymatic extract, 1 ml of H 2O2 (0.01 M) and 1 ml of guaiacol (0.01 M). Control
consists of 0.1 mL of enzymatic extract and extraction buffer solution. The volume of each medium was
supplemented to 3 mL with extraction buffer solution. Tubes containing reaction media were stirred rapidly and
then incubated for 5 min in dark at 25ºC. At the end of incubation time, tubes were homogenized and
absorbance was determined spectrophotometrically at 470 nm. Enzymatic activity was expressed as absorbance
per minute and per milligram of protein (ΔDO min-1 mg-1prot.).
Determination of proteins
Extracts used for enzyme assay are those used for determination of proteins. The protein assay was
performed according to Bradford (1976) colorimetric method. Amount of protein was determined using a
standard curve made from different concentrations (0 to 100 μg mL-1) of Serum Albumin Bovine solution.
Kra et al. 2019
Statistical analyzes
All experiments conducted in this work were repeated three times. Analysis of variance (ANOVA) was done
at 5% threshold with STATISTICA software 7.1. When p ≤ 0.05, the difference is said to be significant.
Homogeneous groups were determined by test of Duncan.
RESULTS
Agronomic parameters
The analysis in table 1 showed that agronomic parameters evaluated were variously influenced by chitosan
according to cultivars. In most cultivars, 25 and 50 mg L -1 of chitosan did not affect organs growth except
TMS4(2)1425 and 9620A leaves whose presented slight stimulation. For the latter, statistical treatment
performed indicated a significant difference at 5% level. Concentrations 75 and 100 mg L -1 of chitosan induced
growth of all evaluated organs. However, highest stimulation was achieved by 100 mg L -1 treatment except
TM30572 leaves, which slightly increased in presence of 75 mg L-1 of chitosan. Organ growth was inhibited
with an increase in chitosan concentration to 125 mg L-1. Statistical analysis showed a significant difference
apart from Yace leaves.
Table1. Effect of chitosan concentration on growth of stems and roots, and leaf surface of cassava aged 2 months.
Cassava
Chitosan
Concent.
(mg L-1)
Yace 9620A TMS4(2)1425 TMS30572

Stem Root Leaf Stem Root Leaf Stem Root Leaf Stem Root Leaf
(cm) (cm) (cm2) (cm) (cm) (cm2) (cm) (cm) (cm2) (cm) (cm) (cm2)
0 14.22 9.15 39.62 13.52 11.05 36.72 13.7 12.02 38.67 13.97 9.07 40.12
±0.27b ±0.13c ±0.65a ±0.72c ±0.37b ±0.64e ±0.50c ±0.53b ±0.64e ±0.31c ±0.39d ±0.60bc
25 13.85 9.27 39.67 13.6 10.95 37.02 13.27 11.75 39.05 14.5 9.9 39.07
±0.10b ±0.19c ±0.70a ±0.58c ±022b ±0.65de ±0.4c ±0.52b ±0.82de ±0.30c ±0.15c ±0.41c
50 14.35 9.55 40.02 13.65 11.47 37.8 15.2 12.55 40.3 14.57 9.97 39.8
±0.56b ±0.30c ±0.34a ±0.54c ±0.34b ±0.58d ±0.34b ±0.34ab ±0.98d ±0.28c ±0.33c ±0.54c
75 14.87 11.07 41.22 16.6 12.65 40.35 15.82 13.22 42.62 15.55 11.05 42.8
±0.76b ±0.15b ±0.61a ±0.50b ±0.29a ±1.46b ±0.38ab ±0.23a ±1.31b ±0.31b ±0.67b ±1.87a
100 16.72 13.05 40.82 18.02 13.05 42.7 16.65 13.3 44.5 16.8 12.32 40.87
±0.93a ±0.27a ±0.97a ±0.83a ±0.27a ±2.12a ±0.31a ±0.219a ±1.94a ±0.36a ±0.58a ±0.57b
125 13.17 11.17 39.32 13.92 11.45 39.5 15 12.05 41.8 13.82 9 39.32
±0.39b ±0.76b ±0.69a ±0.27c ±0.23b ±1.27c ±0.33b ±0.31b ±1.30
c
±0.48c ±0.37d ±0.40c
Note: In each column, the averages followed by the same alphabetical letter are not statistically different at the 5% threshold (Duncan test).
Peroxidases activities in cassava leaves
Analysis of table 2 showed a variation of peroxidases activities in different leaves of cassava [Yace, 9620A,
TMS4(2)1425 and TMS30572], treated with chitosan. In general, these activities increased with leaf age and
chitosan concentration in all cultivars. In control leaves, peroxidases activities ranged from 0.12 to 0.23 ΔDO
min-1 mg-1prot. for f3 and from 0.35 to 0.41 ΔDO min-1 mg-1prot. for f4 according to cultivars. In leaves 5 and 6,
enzyme activity oscillated from 0.25 to 0.32 ΔDO min-1 mg-1prot. apart from 9620A, for which peroxidases
activities have increased to 0.45 ΔDO min-1 mg-1prot.
In treated leaves, peroxidases activities increased significantly with concentrations in 4 th and 5th leaves where
it was maximum at 75 or 100 mg L-1 of chitosan according to cultivars (Table 2). It went from 0.31 to 0.87 ΔDO
min-1 mg-1 prot., overall, 175 to 260% stimulation at optimal concentration. In leaf 6, increase in activity was
188 to 244% depending on cultivars for same concentrations. Statistical analysis revealed a significant
difference at only 5% between leaf levels and concentrations. Lowest enzymatic activity was detected in leaves
of rows 3 regardless of concentration used.
Peroxidases activities in roots of cassava
Peroxidases activities evaluated in plants roots of different cassava cultivars presented in table 3 shows that
chitosan stimulated activities of these enzymes. Their activities have increased with the concentration of
chitosan and reached optimum at 75 or 100 mg L-1 of chitosan in treatment medium. In Yace cultivar, there was
a marked increase in peroxidase activity from 0.67 to 1.45 ΔDO min-1 mg-1 prot. 50 to 75 mg L-1, respectively.
This latter concentration was most inducing of peroxidases activities (1.45 ΔDO min -1 mg-1 prot). From this
maximum value, peroxidases activities decreased to 1.37 ΔDO min-1 mg-1prot. for 125 mg L-1 of chitosan.
For cultivar TMS30572, the increase in peroxidases activities was gradual until it reached its optimum (0.93
ΔDO min-1 mg-1 prot) at 100 mg L-1 chitosan. Statistical treatment did not show a significant difference at 5%
threshold for treatments of 100 and 125 mg L-1 of chitosan. Peroxidases activities were maximal (1.29 ΔDO
min-1 mg-1 prot) in 9620A for 100 mg L-1 of chitosan. However, it decreased slightly to 125 mg L-1, namely 1.25
ΔDO min-1 mg-1 prot. As for cultivar TMS4(2)1425, the increase in peroxidases activities were consistently 0 to
75 mg L-1 of chitosan. From 75 mg L-1, an increase in activity was observed from 0.78 to its optimum at 1.33
ΔDO min-1 mg-1 prot. for concentration of 100 mg L-1 of chitosan. The activity of enzyme decreased slightly
(1.27 ΔDO min-1 mg-1 prot.) with increasing chitosan concentration.
Table 2. Effect of different concentrations of chitosan on peroxidases activities (ΔDO min -1 mg-1 prot.) in cassava leaves.
Concentrations of chitosan (mg L-1)
Casava
0 25 50 75 100 125
F3 0.24±0.02ab 0.25±0.01a 0.26±0.01a 0.27±0.01a 0.27±0.05a 0.27±0.01a
F4 0.37±0.02d 0.40±0.01d 0.45±0.01c 0.62±0.08b 0.87±0.02a 0.83±0.01a
Yace F5 0.30±0.01c 0.35±0.01d 0.40±0.01c 0.56±0.02b 0.76±0.01a 0.72±0.02a
F6 0.25±0.01d 0.30±0.02c 0.35±0.01b 0.47±0.01a 0.41±0.01a 0.43±0.01a
F3 0.18±0.01d 0.20±0.01d 0.20±0.04d 0.28±0.01c 0.36±0.02b 0.54±0.02a
F4 0.35±0.04c 0.37±0.01c 0.53±0.01b 0.63±0.01a 0.67±0.01a 0.66±0.01a
9620A F5 0.45±0.02d 0.46±0.01d 0.58±0.01c 0.71±0.02b 0.79±0.02a 0.72±0.02b
F6 0.30±0.02de 0.35±0.01d 0.40±0.01c 0.56±0.01b 0.61±0.01a 0.55±0.01b
F3 0.20±0.02c 0.26±0.01ab 0.28±0.01ab 0.33±0.01a 0.28±0.01ab 0.29±0.01a
F4 0.41±0.01d 0.43±0.01d 0.58±0.02c 0.72±0.02b 0.80±0.01a 0.76±0.02b
TMS4(2)1425 F5 0.32±0.02d 0.36±0.02d 0.54±0.01c 0.64±0.01b 0.70±0.02a 0.64±0.02b
F6 0.29±0.01c 0.29±0.01c 0.40±0.01b 0.62±0.01a 0.61±0.01a 0.59±0.01a
F3 0.12±0.10c 0.19±0.01b 0.21±0.02b 0.23±0.02ab 0.28±0.01a 0.28±0.01a
F4 0.37±0.02d 0.38±0.08d 0.50±0.02c 0.65±0.01b 0.75±0.01a 0.71±0.03a
TMS30572
F5 0.30±0.00d 0.31±0.02d 0.56±0.01c 0.79±0.01a 0.75±0.01ab 0.74±0.01ab
F6 0.25±0.02c 0.28±0.01c 0.39±0.01b 0.61±0.01a 0.59±0.01a 0.57±0.02a
Note: F3, Leaves 3; F4, Leaves 4; F5, Leaves 5; F6, Leaves 6.
Number in line marked with the same letters do not differ significantly at P≤ 0.05 (Duncan test).
From the analysis of this table 3, in TMS30572, TMS4(2)1472 and 9620A cultivars, concentration of 100
mg L-1 of chitosan was most stimulating of peroxidases activities. In cultivar Yace, concentration of 75 mg L-1 of
chitosan was more inducing to activity of enzyme. This activity was much higher than that evaluated in the other
three cultivars.
Table 3. Effect of chitosan concentrations on peroxidases activities (ΔDO min -1 mg-1prot.) of cassava plant roots.
Chitosan (mg L-1)
Cultivars
0 25 50 75 100 125
Yacé 0.55 ±0.01a 0.64±0.02b 0.67±0.01b 1.45±0.01d 1.41±0.05d 1.37±0.03c
9620A 0.73±0.04a 0.77±0.02a 0.97±0.01b 1.20±0.01c 1.29±0.01d 1.25±0.01c
TMS4(2)1425 0.57±0.01a 0.63±0.02b 0.74±0.01c 0.78±0.02c 1.30±0.03d 1.30±0.02d
TMS30572 0.61±0.01a 0.65±0.01a 0.72±0.02b 0.82±0.01c 0.93±0.03d 0.92±0.01d
Note: Number in line marked with the same letters do not differ significantly at P≤ 0.05 (Duncan test)
DISCUSSION
Results of this study indicate that chitosan promoted growth of stem, leaf and root organs. Elongation of
stems and roots, and increase of leaf surface would result from cells division and/or their extension. Indeed,
organs growth is done by cells mitoses of meristematic zones which, after cell turgor increase of dimensions.
Whole of merèse and auxèse phenomena is at the origin of organs growth of plants. Work done by some authors
(Dixon et al. 1994, Thain et al. 1995, Amborabe et al. 2004) have shown that chitosan acts on depolarization of
plasma membrane that is manifested by the influx of Ca 2+ and H+, and efflux of Cl- and K+. Activation of ion
channels causes changes in intra and extracellular media. A decrease of calcium in pectocellulosic walls
increases their extensibility; which leads to cell growth or elongation. Growth induction of various plants by
chitosan has been reported by several authors (Asghari-Zakaria et al. 2009, Algam et al. 2010, Saad 2011,
Mondal et al. 2012, Mondal et al. 2013, Ramkissoon et al. 2016). As obtained in this study, their work showed
that 75 mg L-1 and 100 mg L-1 chitosan were the most growth-inducing factors in spinach, okra, beans,
chickpeas and chickens. On the other hand, shoots and roots growth of green anise (Pimpinella anisum L.) was
stimulated after seed treatment by 50, 100 and 200 mg L-1 of chitosan (Batool 2016). Regardless of inducing
concentration, growth obtained is also due in part to activation of nitrogen metabolism (Ke et al. 2001). This
mineral is essential for the synthesis of molecules necessary for the vital functions of plant cells such as
proteins, nucleic acids and chlorophylls.
Kra et al. 2019
Peroxidases activities were detected in leaves and roots of all plants examined. Presence of these enzymes in
various organs demonstrates that they exercise physiological functions therein. Indeed, bibliographical synthesis
carried out on these enzymes by Delannoy et al. (2004), indicated that they are involved in several metabolic
pathways, among others, regulation of certain hormones, synthesis of lignins and defense of plants against
abiotic and biotic stress. Low peroxidases activities in young leaves, increased in mature leaves and then
decreased in older leaves. This variation is related to cell differentiation and more or less important synthesis of
auxin and lignin. After germination of plants in chitosan solution, peroxidases activities were increased and
multiplied by 1.7, 1.9, 2.3 and 2.6 at maximum activity respectively in 9620A, TMS4(2)1425, Yace and
TMS30572 leaves. High peroxidases activities in leaves of rows 4 and 5 could be interpreted by their high
metabolic activity. Indeed, in these leaves, chitosan activates photosynthesis (Mondal et al. 2012), which
produced hydrogen peroxide. However, this compound is a substrate for peroxidases. In addition, work by some
authors has reported that chitosan stimulates key enzymes in the biosynthetic pathway of phenolic compounds
such as phenylalanine ammonia-lyase and tyrosine ammonia-lyase (Khan et al. 2003) and those involved in
plant defense such as peroxidases (Coqueiro & Piero 2011). In total, the presence of hydrogen peroxide and
precursors of lignin biosynthesis would have promoted activation of peroxidases.
Peroxidases activities evaluated in roots of controls was generally higher than that detected in leaves. This
increase was 1.5 to 2.6 depending on cultivars. This result could be interpreted by the fact that germination was
done in an aqueous medium which caused more marked water stress in the roots; these being in direct contact
with the water. In the presence of chitosan, the enzyme activity was similar in the leaves and roots of the same
cultivar except in TMS30572 for which peroxidase activity was 1.7 times stronger in the leaves. Water stress
and chitosan concomitantly have stimulated the activity of peroxidases. These results confirmed those obtained
by Lizarraga-Pauli et al. (2011) and Pongprayoon et al. (2013), on the induction of plant resistance by chitosan
against abiotic stress. In addition, these results are in the same direction as those of El-Hassni et al. (2004) and
Falcón-Rodríguez et al. (2014) whose have obtained the activation of peroxidases after application of this
product. However, this peroxidase activity was a function of the concentration used but could also depend on the
degree of acetylation of chitosan (Coqueiro & Piero 2011).
CONCLUSION
This study showed that chitosan promoted growth of all organs studied and stimulated peroxidases activities
in cassava. Depending on the cultivar, its action was maximum at 75 or 100 mg L -1.
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Short communication
Range extension of Actinodaphne bourneae Gamble (Lauraceae):

An overlooked endemic tree of Western Ghats, India
S. Selvakumar1, R. Kottaimuthu2* and K. Suresh1
1
Department of Botany, Saraswathi Narayanan College, Madurai-625 022, Tamil Nadu, India
2
Department of Botany, Alagappa University, Karaikudi-630 003, Tamil Nadu, India
*Corresponding Author: kottaimuthu@yahoo.co.in [Accepted: 11 February 2019]
[Cite as: Selvakumar S, Kottaimuthu R & Suresh K (2019) Range extension of Actinodaphne bourneae Gamble
(Lauraceae): An overlooked endemic tree of Western Ghats, India. Tropical Plant Research 6(1): 15–17]
INTRODUCTION
The genus Actinodaphne Nees is a member of the family Lauraceae, comprising 100 species worldwide, and
is predominantly found in Southeast Asia and Malaysia (Van der Werff, 2001, Li et al. 2006). The preferred
habitats of these plants are semi-evergreen, evergreen, and shola forests (Robi & Udayan 2016). Actinodaphne
bourneae was first described by Gamble in 1925, based on a specimen collected by A.G. and Lady Bourne in 26
June 1897 from Kodaikanal shola, Pulney Hills. Since then it has not been recollected from the type locality or
any other hill ranges in Western Ghats (Nayar & Sastry 1990). Recently, Matthew (1999) has recollected this
endemic tree from Palni hills, the one and only locality known for India (Chithra & Nair 1999, Singh et al.
2015).
During ethnobotanical inventory on High wavy mountains, the first author has collected some interesting
laurel specimens from the upper Manalar shola forests. Critical studies with protologue, type specimen and
relevant literature it is identified as Actinodaphne bourneae Gamble, an endemic and endangered ray laurel of
Tamil Nadu (Chithra & Nair 1999). The perusal of literature analysis it is found that this endemic tree was
hitherto not reported from Megamalai wildlife sanctuary (Ravikumar 1993; Ravikumar & Lakshmanan 1999,
Ravichandran & Karuppusamy 2016). Thus the present report on extended distribution of this narrow endemic
species is an immense scientific importance from a conservation point of view. Hence it is described in detail
along with relevant notes for the better understanding of the taxa.
SYSTEMATIC ENUMERATION
Actinodaphne bourneae Gamble in Bull. Misc. Inform. Kew 1925: 128. 1925 & Gamble, Fl. Pres. Madras
2: 1231. 1925; V. Chandras. in A. N. Henry et al., Fl. Tamil Nadu 2: 206. 1987; K.M. Matthew, Ill. Fl. Palni
hills t. 605. 1996 & Fl. Palni hills 2: 1065. 1999. (Fig. 1)
Trees, 10–15 m tall; branchlets densely fulvous tomentose; terminal buds perulate; scales imbricate, elliptic-
oblong, 1.0–1.7 cm long, rufous tomentose. Leaves verticillate, estipulate, in whorls of 4–6; petioles 1.5–3.0 cm
long, flat above, rounded beneath; lamina elliptic, 8.0–19 × 1.5–6.0 cm, base cuneate, apex acuminate, margin
entire, densely fulvous-tomentose, glaucous beneath, thinly coriaceous; lateral veins 8–10 pairs, prominent
beneath, fulvous-tomentose on both sides, margin entire. Umbels axillary or lateral, 4–6 flowered, sessile. Male
flowers: greenish-yellow, rufous-tomentose; pedicel 5 mm long, fulvous sericeous; tepals 6 in two whorls,
ovate, 4 mm long, apex acute, sub equal, margin ciliate, fulvous sericeous outside, sparsely villous or glabrous
inside, membranous, 3–5 nerved; stamens 9 in three whorls, outer whorl 3, 4-locular; anthers 1 mm long, ovoid,
yellow; filament 2.5–3.0 mm long, sparsely silky villous at the base, eglandular; middle whorl almost same as
the outer whorl; inner whorl 3, extrorse with 2 glands at base of the filament; glands sessile, sometimes sterile
pistil present. Female flowers: ca. 10 mm long, densely fulvous tomentose, campanulate; tepals 6, ovate, 3–4
mm long, margin ciliate, fulvous tomentose outside, glabrous inside staminodes 9, arranged in three whorls.
Ovary obovoid, 1.0–1.5 mm long, sparsely villous; style ca. 2.5 mm long, sparsely villous; stigma irregularly
lobed, yellow, plumose. Berries globose, 6–8 mm across, greenish, red when ripe.
Flowering & Fruiting: April–November.
Received: 06 October 2018 Published online: 28 February 2019
Selvakumar et al. 2019
Figure 1. Type specimen of Actinodaphne bourneae Gamble [©The Board of Trustees of the Royal Botanic Gardens, Kew.]
Distribution: India (Tamil Nadu). Endemic. It is very rare and sparsely distributed in shola forests of Palni Hills
and Megamalai (present report).
Specimens examined: South India, Pulney Hills, Kodaikanal shola, 26.06.1897, A.G. and Lady Bourne 517
(K!); Theni district, High wavy mountains, Upper Manalar, 1650 m, 11.11.2015, S. Selvakumar 571; same
place, 21.04.2016, S. Selvakumar 815 (Saraswathi Narayanan College Herbarium!).
Ecology and Association: It is growing in shola forest at an altitudinal range 1800–2400 m. The population was
very sparse in its natural habitat and it was found approximately an area of 30 sq. m. Totally 12 mature
individuals, 28 saplings and 35 seedlings were encountered in the study area. Impatiens cordata, Ilex wightiana,
Nothopegia heyneana, Nothopegia vajravelui, Crotalaria beddomeana, Crotalaria walkeri, Photinia integrifolia
var. sublanceolata, Syzygium sriganesanii, Ophiorrhiza brunonis and Psychotria flavida are the commonly
associated plants.
ACKNOWLEDGEMENTS
Authors are grateful to retired Dr. G.V.S. Murthy, ‘Scientist G’, Botanical Survey of India for granting
permission to consult the library and to Royal Botanic Gardens, Kew for making their herbarium images
available online. The author (KS) is thankful to the Tamil Nadu Forest Department for necessary permission.
REFERENCES
Chithra V & Nair VJ (1999) Tamil Nadu. In: Mudgal V & Hajra PK (eds) Floristic Diversity and Conservation
Strategies in India. Vol. 3. Botanical Survey of India, Kolkata.
Gamble JS (1925) New Lauracaeae of Southern India. Bulletin of Miscellaneous Information (Royal Botanic
Gardens, Kew) 1925: 126–132.
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nrDNA ITS and ETS sequences. Acta Phytotaxonomica Sinica 44(3): 272–285.
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District, Tamil Nadu, India. Rheedea 9: 55–75.
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152–140.
Research article
Enhanced rhizome induction and fast regeneration protocol in

liquid culture of Dendrocalamus longispathus Kurz:
A single step culture
Papori Phukan Borpuzari1* and Narendra Singh Bisht2
1
Rain Forest Research Institute, Jorhat-785001, Assam, India
2
Forest Research Institute, Dehradun-248006, Uttarakhand, India
*Corresponding Author: paporis@icfre.org [Accepted: 15 February 2019]
Abstract: The present experimental study was aimed for in-vitro regeneration through nodal
culture of Dendrocalamus longispathus an important bamboo species of north-eastern region.
Single auxillary buds were cultured in different concentration of BAP and Kn incorporated media
for bud breaking and shoot regeneration. Effect of collection period and type of explants is a major
impact on bud breaking. Single step plantlet regeneration has been achieved in the liquid basal
medium Murashigs and Skoogs (MS) with BAP 1.0 + Kn 1.0 shows best regeneration of 6 to 8
numbers of shoots within 3 weeks of culture. Both inoculated intact node and cut node cultures
produced shoots and rhizomes during subcultures. Increased incubation period up to 11 weeks
with serial sub culture produced simultaneous roots and rhizomes in the cultured media containing
BAP 1.0 + Kn 1.0. Culture response of 90% healthy rooted plantlets has been established outside
the lab condition. The whole experiment completed within 12 weeks of culture incubation. Good
growth of established in-vitro plantlets in field of FRCBR, Aizwal is observed after one year.
Keywords: Tissue culture - Bamboo - Auxillary bud - Intact node - Cut node.
[Cite as: Borpuzari PP & Bisht NS (2019) Enhanced rhizome induction and fast regeneration protocol in liquid
culture of Dendrocalamus longispathus Kurz: a single step culture. Tropical Plant Research 6(1): 18–23]
INTRODUCTION
Bamboos are the tallest and largest member of the grass family having several commercial applications
which are widely distributed in India and abundantly occur in northeast region. Demand for bamboo is rising in
all over the world and are considered as one of the most economically important plants for their utility in
handicraft industry, construction, paper making, fishery, human consumption etc. (Scurlock et al. 2000).
Dendrocalamus longispathus (Kurz) is a long-sheath bamboo grows up to 20 m tall and locally known as
'rawnal' in Mizoram (Fig. 1). Native place of the species are Bangladesh, Myanmar and Thiland and widely
distributed across the South and Southeast Asia, particularly in India (northeastern state of Assam, Manipur,
Meghalaya, Mizoram, Tripura and Nepal). The conventional method of propagation of bamboo through seed
possesses several problems like long flowering cycle up to 120 years, poor seed set, as well as low seed viability
etc. In vitro rhizomes are produce shoots and roots giving rise to complete plantlets act as seeds and help in the
early establishment of plants in the field and culm production for both the commercial production and
germplasm conservation (Kapoor & Rao 2006). Hence, considering the importance of rhizome in bamboo many
researchers like Shirgurkar et al. (1996) observed in vitro rhizome formation and micropropagation in
Dendrocalamus strictus (Roxb) Nees. In vitro rhizome formation was also reported in Dendrocalmus hamiltonii
Nees & Am. ex Munro on prolonged sub-culturing of plantlets raised through somatic embryogenesis (Godbole
et al. 2002). Rhizome formation was also induced in Bambusa bambos (L.) Voss by Kapoor & Rao (2006). In
the present study we report an easy method of micropropagation and in vitro rhizome induction of D.
longispathus in a single step on cut and uncut node cultures in the liquid basal medium of Murashigs & Skoogs
(1962).
MATEIALS AND METHODS

The experimental study was conducted with the nodal segment of lateral branches collected from the
Received: 30 September 2018 Published online: 28 February 2019
bamboo nursery of Advanced Research Centre for Bamboo and Rattan, Aizwal, Mizoram, during the month of
January to June. The average temperature of the site is 15 to 30°C, RH 84 to 91 %, rainfall 182 mm and lies
between 23° 30' N to 92° 43' E. Nodal segments with axillary buds covering with leaf sheaths were carefully
removed and placed under slow running tap water to wash out dust particles and prepared in larger size explants
for bud breaking and initiation of culture (Fig. 2). Collected explants are washed in 2% Teepol solution and
shake for 15to 20 min in 500 ml Erlenmeyer conical flask very carefully to prevent from rupturing of the tender,
soft auxilary bud. The teepol solution is removed by washing with several times in tap water followed by rinse
in distilled water. Surface sterilization is done with 0.1% v/v mercuric chloride solution for 7 to 8 min and
washed in sterile distilled water until to remove the traces. BA has been found to be superior over other
cytokinins for shoot regeneration in a number of earlier reports in different bamboo species. These sterilized
nodal segments were cultured in three different basal liquid media viz Murashiges and Skoogs (MS), ½ strength
of MS and SH (Schenk & Hildebrandt 1972) with 30 gm l-1 sucrose for bud breaking and shoot regeneration
with BAP five different concentration (1.0, 2.0, 3.0, 4.0 and 5.0 mg l-1) alone and in combination with BAP
1.0 mg l-1 and Kn (1.0, 2.0 and 3.0 mg l-1). pH of the medium adjusted to 5.6 to 5.8 prior to autoclaving at 121°C
in 15 psi for 15 min and the cultures are maintained at 25±2°C under continuous illumination of 3000 lux
provided by daylight cool white fluorescent tubes maintaining 16-hr photoperiod. Proliferated 3–5 axillary
shoots with intact node were subculture in the same medium. Multiplied shoots were supported by sterile filter
paper bridges and shoots were transferred regularly at the interval of 7 days into fresh medium. After shoot
initiation, culture incubated for 3 weeks and divided into two parts i.e. pre cultured uncut node left intact with
differentiated shoots and pre- cultured cut node with differentiated shoots. Culture continued in the same
medium and multiplied shoots were counted after 30 days to evaluate the multiplication rate and rhizome
production in each culture medium. Control of browning for the species is the best methodology as to adopt for
frequent sub culturing after 7 days of culture so as to maintain healthy cultures. Longer sub-culture durations
usually lead to longer and pale shoots which gradually turn brown to black instead of enhancing the
multiplication rate further (Mudoi & Borthakur 2009, Singh et al. 2012). All the experiments were repeated
twice with five replicates each. Later, regenerated plants were divided into several parts and planted in the poly
bags for field plantation.
Figure 1. Mature Dendrocalamus longispathus in natural habitat.
Figure 2. A, Aseptic explants before inoculation; B, Bud breaking and shoot initiation.
Borpuzari & Bisht 2019
RESULTS
Figure 3. Rhizome induction and plant regeneration of Dendrocalamus longispathus (Kurz): A, Shoot multiplication; B,
Rooting of regenerated shoots; C, Number rhizome buds and rooting; D–G, Rooted rhizome of intact node explants; H–I,
Rooted rhizomes of cut node explants; J, Deflasked plantlets after 11weeks of culture in the shoot regeneration media; K,
Hardening under shaded area; L, Plants after field establishment.
Results of the study reveals single step plantlet regeneration in the liquid basal medium of MS along with
BAP 1.0 + Kn 1.0 with optimum 6 to 8 numbers of shoots within 3 weeks of culture in the pre-cultured node left
intact. Both the explants produced rhizomes along with healthy shoots. Shoots initiated from the axillary buds of
the explants, with a maximum number of shoots after a period of 30 days. Increased incubation period up to 11
weeks with serial subculture produced simultaneous roots and rhizomes in the same medium from pre-cultured
cut node with differentiated shoots. Both the explants response for root initiation but differs in regeneration time
and percentage of rooting and produced rhizomes along with the regenerated shoots. All regenerated shoots
were nicely green and healthy in appearance and small roots were visible from the basal part of the regenerated
shoots after 14 days of culture on the shoot multiplication medium without changing any hormones. Cultures
were produced 2 to 4 numbers of rhizomes from the base of the regenerated shoots and later developed into
shoots. After 60 days of incubation, the shoots at the nodes were grown to 2.5 to 5.0 cm in length with roots.
Rooted shoots were isolated and maintained in the same medium until fully developed plant lets about to thirty
days. To minimize the level of contamination we have success in simple initial steps of surface sterilization. For
acclimatization, the rooted plants were transferred to paper cups containing sterilized soil and covered with a
plastic film to retain moisture. The plastic cover was gradually opened day by day during the acclimatization
period 8 days. These plants were transplanted to garden pots, containing a mixture of sand and soil in 1: 1 and
watered regularly. After 15 days acclimatization plantlets were established. 90% of the culture possesses healthy
and plantlet establishment outside the laboratory has been completed within 12 weeks.
DISCUSSION
Considering the basal media for optimum result in the present investigation, similarly in several bamboo
species MS is the most widely used medium for bud breaking as reported in Dendrocalamus hamiltonii (Sood et
al. 1994, Agnihotri et al. 2009); D. giganteus Munro (Ramanayake & Yakandawala 1997); D. asper (Schult)
Backer (Arya et al. 2008); Bambusa vulgaris Schrad (Rout & Das 1997); B. edulis Carrière, Bambusa
odashimae Hutus. ex D. Z. Li & Stapleton (Lin & Chang 1998); B. balcooa Roxb (Das & Pal 2005, Negi &
Saxena 2011) etc. Again, Singh et al. (2011, 2012), reported that MS was found to be as better in his study when
compared with MS, B5 (Gamborg et al. 1968) and NN (Nitsch & Nitsch 1969). Simultaneously the size of the
explants plays an important role in the regeneration of shoots, which was supported by Anand et al. 2013 in
Bambusa bambos that larger explants are more suitable than smaller one to initiate the culture within a short
time because of its high endogenous hormonal effect. Some researchers also reported that nodal bud sprouting
for shoot formation is generally depends on their physiology of the explants tissue, collection of time and year
and culture (Saxena & Dhawan 1994, Ramanayake et al.1995, Ramanayake & Yakandawala 1997, Singh et al.
2011, 2012). In the case of present study, stems collected after February showed a low frequency of bud-break
and gradually decrease to July. It was observed that the nodal segments from the upper portion of stem showed
higher percentage of bud break. According to the report of Saxena & Bhojwani (1993) bud break frequency in
D. longispathus was strongly influenced by the juvenility stem showed the higher percentage of bud break.
According to the report of Saxena & Bhojwani (1993) bud break frequency in D. longispathus was strongly
influenced by the juvenility of lateral shoots, the position of axillary bud on the branch and the season in which
cultures were initiated. Similarly, our result of bud breaking was a response from (January to April) and gave
the best response in terms of decreased contamination. Early shoot initiation and increased the percent of bud
breaking with the higher number of shoots in D. asper is reported by Singh et al. (2011) while, Singh et al.
(2012) reported that the early summer i.e. April–June was best for explants collection period and the
establishment of D. hamiltonii with low rate of contamination. Incorporation of BAP into the medium improved
the axillary bud proliferation was reported by Nadgir et al.(1984), Dekkers & Rao (1989), Hirimburegama &
Gamage (1995) and Arya et al. (2006); while, Kn alone was found to be less effective by Ramanayake &
Yakandawala (1997), Arya et al. (2006) and Singh et al. (2011). Synergistic effect of the two cytokinins BA and
Kn was reported best for shoot multiplication in D. giganteus reported by Arya et al. (2006) and B. bambusa,
glaucescens (Wolld.) Merr., Bambusa multiplex (Lour.) Raeusch. ex Schult. by Shirin & Rana (2007).
The response in the liquid medium of bamboos, higher rates of shoot multiplication and improved growth
was observed by several workers (Saxena & Bhojwani 1993, Sood et al. 2002, Das & Pal 2005, Arya et al.
2006, Shirin & Rana 2007, Ogita et al. 2008). Alternatively, use of the liquid medium is more economical as
compared to solid. Several authors reported on good growth and shoot multiplication rate in liquid medium than
agar gelled such as in Dendrocalamus hamiltonii, Bambusa tulda Roxb. by Saxena (1990) and Sood et al.
(2002); Somashekar et al. (2008) in Pseudoxytenanthera stocksii (Munro.) T.Q. Nguyen; Kabade (2009) in
Bambusa bambos and Dendrocalamus strictus (Roxb.) Nees; Negi & Saxena (2011) in Bambusa nutuns Wall.
ex Munro. This may be due to easy and faster uptake of nutrients and growth regulators from the liquid medium.
Precultured cut node explants compared to decapitated in vitro seedlings of Ochlandra wightii (Munro) C.E.C.
Fisch. where explants cultured onto half-strength of MS liquid supplemented with various concentrations of
sucrose for the induction of in vitro rhizomes studied by Bejoy et al.(2012). In rooting, similar to our study
Shirqurkar et al. (1996) observed in Dendrocalamus strictus where spontaneously during the rooting phase in
Borpuzari & Bisht 2019
vitro rhizome formation when plantlets were allowed to proliferate on MS medium supplemented with the low
concentration of BAP. Simultaneously, Bambusa bambos showed the highest multiplication that can be obtained
in the medium without kinetin but the lowest rooting was observed in medium without Kn by Nayak et al.,
(2010). Krishnamurthy et al. (2001) showed the longest root with the application of 0.5 mg L-1 BAP in
Polianthes tuberosa L. Here, we have successfully developed efficient plant regeneration and rhizome induction
in low hormonal concentration which may be a helpful study for the future course of the investigation.
CONCLUSION
Here we have successfully developed efficient plant regeneration for Dendrocalamus longispathus using
single step culture of the nodal segment along with rooting. The findings may lead the further studies of the
commercial production through liquid culture.
ACKNOWLEDGEMENTS
Author has duly acknowledged the Director, RFRI for providing the facility and financial support to ICFRE,
Dehradun for successful completion of the present investigation.
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Research article
Analysis of soil and tree productivity under high density planting

system in mango cv. Dashehari (Mangifera indica L.)
Tarun Adak, Dushyant Mishra, Kailash Kumar* and Vinod Kumar Singh
Division of Crop Production, ICAR-Central Institute for Subtropical Horticulture, Rehmankhera,
Lucknow-226101, Uttar Pradesh, India
*Corresponding Author: kailash1952@gmail.com [Accepted: 17 February 2019]
Abstract: Farmers have no alternative but to adopt high-density planting for enhancing fruit
productivity level and profitability with the shrinking land resources and smaller land holding
sizes. The highest yield of 16 t ha-1 followed by 10.0 t ha-1 and the least of 5.5 t ha-1 was recorded
from 400, 267 and 100 trees ha-1, respectively from 19 yrs old Dashehari mango orchard in sandy
loam soils at research farm of ICAR-CISH, Rehmankhera, Lucknow. Planting density systems
(1600, 800, 400, 266, 178 and 100 trees ha -1) also impacted soil properties. Water holding capacity
varied between 16.6 to 26.1 % across systems while porosity ranged from 46.2–71.5 %. Likewise,
bulk density and particle density had values of 1.2 to1.7 and 2.2 to 2.9 g cm-3 respectively. The
mean soil organic carbon content was 0.38%, pH of 7.2, available N, P, K was 66.92, 13.91 and
77.85 mg kg-1 respectively. Soil and leaf micronutrients analyzed across six different density
systems showed wide variations and indicated the need for optimum nutrient management.
Significant and positive correlations were recorded between soil organic carbon with other soil
properties. The study revealed that medium density system (400 trees ha -1) is to be practiced at
farmers’ field.
Keywords: High-density system - Traditional planting - Yield - Soil properties - Statistical
correlation.
[Cite as: Adak T, Mishra D, Kumar K & Singh VK (2019) Analysis of soil and tree productivity under high
density planting system in mango cv. Dashehari (Mangifera indica L.). Tropical Plant Research 6(1): 24–30]
INTRODUCTION
With the fast growth of urban development, industrial development, housing projects and other related land-
based projects, availability of farming land is shrinking. Farmers are searching for an alternative system of
planting fruit crops apart from traditional planting system in order to maintain their productivity level and
earning. In this backdrop, high-density planting system (HDP) emerged as alternative system of plantations for
sustaining the fruit production level (Singh et al. 2007). In general, traditional cultivation of mango (10.0 × 10.0
m spacing) can accommodate 100 trees ha-1 while the HDP aims at accommodating 1600, 800, 400, 266 and 178
trees ha-1. The yield potential of these orchards was targeted for at least 20 t ha-1 based on soil and tree
management system. Meland (2005) recorded the highest cumulative yield after 9 yrs of planting in high density
system of 5000 trees ha-1. Even, Zaman & Schuman (2006) also opined that in order to obtain desirable fruit
yield, site-specific nutrient management based on soil properties and tree performance should be the top priority.
Distribution of soil micronutrients should also be considered for quality production (Nagendran &
Angayarkanni 2010, Sharma et al. 2011, Mishra 2014). Singh et al. (2017) recorded the highest yield of 22.72
and 23.36 kg tree-1 from a 13 years old Amrapali mango in high-density system (2.5 × 2.5 m) under different
integrated nutrient management system.
For any production level, soil plays immense role for not only maintaining a satisfactory level of yield but
also to sustain it for a long-term basis. The key component being its physical, chemical and biological parts that
contribute significantly towards soil quality guarding the productivity level. Under normal/traditional system,
management of soil properties is crucial, however, in case of high-density systems, role of soil required more
emphasis as nutrient mining is obvious. Adak et al. (2015) observed that compaction in lower depth soils
Received: 15 September 2018 Published online: 28 February 2019
increased the bulk density and lowers porosity across six plantation systems. Water holding capacity was higher
in the system where less number of trees were accommodated than 1600 and 800 trees ha -1. Singha et al. (2016)
based on a study on 20 yrs old Guava HDP system (2200, 1100, 555 and 277 trees ha-1) concluded that medium
density systems is best suited from view point of microbial activities. The root systems may act as precursors of
nutrient release and spatio-temporal variations in soil properties in HDP system that determines the management
modules to be adopted. Establishment of HDP system may not be suitable in all types of soil. A detailed
analysis of clay, silt, sand contents are obvious as they impact the nutrient dynamics and storage. Adak et al.
(2016a) confirmed that Zn, Cu and Fe storage had inverse relationship with clay + silt content and there was a
tendency of decreasing levels of micronutrients in deeper soil depths (30–60 and 60–90 cm) as compared to the
top layer (0–30 cm). Thus, the present study was laid out to assess the tree performances considering soil
properties and foliar nutrients.

Mango cv Dashehari planted at a spacing of 2.5 × 2.5, 2.5 × 5.0, 5.0 × 5.0, 5.0 × 7.5, 7.5 × 7.5 and 10.0 ×
10.0 m accommodating 1600, 800, 400, 266, 178 and 100 trees ha-1 was selected for evaluating the performance
of different density plantations (planted during August 1992) in research farm of Central Institute for Sub-
tropical Horticulture, Rehmankhera, Lucknow, Uttar Pradesh, India (26.54º N Latitude, 80.45º E Longitude and
127 m above mean sea level) (Fig. 1). Uniform tree, soil, water management and plant protection measures were
applied during study periods. NPK @1.0, 0.50 and 1.0 kg per tree was applied during the month of September-
October each year in tree basin. Nutrient source in the form of Urea (2.17 kg), SSP (3.12 kg) and MOP (1.67 kg)
was used. Tree growth, productivity and other related information were recorded. Undisturbed core soil samples
were collected from tree basin in a randomized block design with four replications. Soil physical characteristics
were determined in laboratory as per the standard methodology. Mechanical analysis was done using
hydrometer method; sand, silt and clay of 26.2, 68.6, 5.2 percent in 2.5 × 2.5 m, 25.9, 68.7, 5.4 percent in 2.5 ×
5.0 m, 33.6, 58.0, 8.5 percent in 5.0 × 5.0 m, 27.2, 62.9, 9.8 percent in 5.0 × 7.5 m, 27.2, 62.9, 9.8 percent in
32.5, 58.2, 9.3 percent in 7.5 × 7.5 m and 38.4, 53.7, 7.9 percent in 10.0 × 10.0 m planting systems was
determined. Soil pH, soil organic carbon, available N (auto-N analyzer), available P (spectrophotometer) and
available K (atomic absorption spectrophotometer) were estimated. The DTPA extractable Zn, Cu, Mn and Fe
contents in the soil were also estimated (AAS). Recently mature leaf samples were collected and processed for
analysis. Nitrogen was estimated by micro-Kjeldahl method whereas P by vanado-molybdate colorimetric
method. Potassium and micronutrients Fe, Mn, Cu and Zn was analyzed in ICPE. Statistical analysis was
performed using SPSS. All data were subjected to a one-way analysis of variance (ANOVA). Relationships
between the parameters studied were quantified using Pearson’s correlation coefficients. Histograms and
correlation matrix were developed using SPSS software package. Required graphs were generated using MS
Excel software.
High density mango plantations
1800
1600
1600
1400
Number of tree ha -1
1200
1000
800
800
600
400
400 267
177
200 100
0
2.5×2.5 2.5×5 5×5 5×7.5 7.5×7.5 10×10
Density (m)
Figure 1. High density system in Mango cv Dashehari at Lucknow.
RESULTS AND DISCUSSION

Appraisal of vegetative growth and productivity level
A detailed study of vegetative growth pattern and productivity level was analyzed in different planting
systems. The maximum tree height was recorded as 5.20, 5.40 and 5.55 m in 16 th, 18th and 19th yrs old
Dashehari mango tree in higher density planting system (1600 trees ha-1). In case of 800 trees ha-1, the tree
height was 5.15, 5.30 and 5.45 m respectively while minimum tree height of 4.50, 4.80 and 4.90 m in
Adak et al. 2019
conventional planting system (100 trees ha-1) respectively. The canopy spread was also studied and it was
recorded that maximum canopy spread in north-south and east-west directions (5.47 m and 5.43 m) was
recorded in 178 trees ha-1 and least in 1600 trees ha-1 (3.29 m and 3.22 m). Highest fruit yield was recorded in
medium density planting (400 trees ha-1) and the lowest being in traditional plantings (100 trees ha-1). The
observed highest yield was 15.12, 15.55 and 16.0 t ha -1 and minimum of 4.15, 4.60 and 5.50 t ha -1 in 16th, 18th
and 19th yrs of old tree. Meland (1998) recorded one-third more yields in highest density as compared to the
lowest density plantations over 7 yrs of plantations. Tardaguila et al. (2011) inferred for considering variations
of soil properties in order to relate vegetative growth and yield components. Yadav et al. (2011) obtained fruit
yield of 25.00 and 26.72 q ha-1 in Amrapali under high density system. Sharma et al. (2016) recorded higher
productivity and profitability under HDP system in Amrapali through different nutrition sources. Similarly,
Kumar et al. (2017) recommended for precision farming strategies for achieving greater sustainable yield in 20
yrs Dashehari Mango based on soil physico-chemical, foliar nutrient analysis, yield and yield components.
Changes in soil physical properties as a function of orchard ground floor management system over a short or
long-term basis was recorded (Adak et al. 2017). A range of 19.2–24.6 % WHC, 39.3–54.3 % porosity, BD of
1.27–1.48 and PD of 2.39–2.78 was estimated across 0–30 cm soil depth with 10 cm interval. Even, different
micronutrient contents in Amprapali were observed among different planting systems (Raj et al. 2017). To
improve the orchard sustainability, the role of soil micronutrients is obvious (Adak et al. 2012).
Soil properties and foliar nutrients in HDP systems
Figure 2. Histographic distribution of soil physical properties.

The univariate statistics analysis of soil parameters and foliar nutrients was tabulated in table 1 and 2. The
estimated soil properties indicated that the soils of planting density systems had an average pH of 7.2 with 6.9 to
7.6 ranging values across six different spacing. Soil organic carbon is low and below critical level; with mean
value of 0.38%. Soil available N, P and K was also at lower side of 66.92, 13.91 and 77.85 mg kg-1 respectively.
Available Zn was deficient varying from 0.10–0.52 and average of 0.25 mg kg-1. Average value of available Cu,
Mn and Fe was 2.41, 6.48 and 5.71 mg kg-1 across HDP systems. Wide variations in terms of soil physical
properties were recorded. A range of 16.6 to 26.1% water holding capacity was noted across systems with mean
value of 21.2% while porosity ranged from 46.2 to 71.5% (mean 59.1%). Similarly, bulk density varied from
1.2–1.7 g cm-3 and particle density had values of 2.2–2.9 g cm-3, respectively. Higher density plantations showed
compaction in deeper depths; hence increased bulk density and lowers porosity. Histographic distribution
depicted widespread distribution among the soil properties (Fig. 2–4). The distribution chart indicated maximum
number of BD (10%), WHC (>5%), PD and SOC (>4%) and Porosity (>5%) frequency level. In case of
micronutrients, highest Zn, Mn and Fe had frequency level >15% while Cu up to 20%. Foliar nutrient analysis
showed need for proper nutrient management for achieving potential yield. The mean N, P and K content of 1.7,
0.14 and 1.30% was recorded. The micronutrients were found to be varied widely; average values around 10.33,
29.24, 96.17 and 190.24 mg kg-1 of Zn, Cu, Mn and Fe content was noted respectively.
Table 1. Univariate statistics of soil parameters under high density mango plantations of subtropical region of Lucknow, India.
Standard Standard Skewness Kurtosis
Soil parameters Range Mean CV %
deviations error of mean
pH 6.9–7.6 7.20 0.19 0.002 9.6 0.51 -0.13
Available N ( mg kg-1) 29.0–98.0 66.92 14.04 1.30 21.0 -0.17 0.56
Available P ( mg kg-1) 5.7–30.7 13.91 5.19 0.18 37.3 0.89 0.40
Available K ( mg kg-1) 32.9–115.5 77.85 18.07 2.20 23.2 -0.28 -0.01
Soil organic carbon (%) 0.12–0.66 0.38 0.13 0.001 34.6 0.22 -1.01
Available Zn ( mg kg-1) 0.10–0.52 0.25 0.09 0.001 36.6 0.63 -0.03
Available Cu ( mg kg-1) 0.58–10.16 2.41 1.87 0.024 77.7 2.33 6.32
Available Mn ( mg kg-1) 2.92–15.38 6.48 2.15 0.032 33.2 1.29 2.81
Available Fe ( mg kg-1) 2.76–12.08 5.71 1.65 0.019 28.8 0.85 1.04
Figure 3. Frequency distribution of SOC, available N, P and K across high density mango orchard soils.
Sustainability of high-density plantations depends on a number of factors associated with production levels;
some are directly while others are indirectly impacting the gap between observed vs. potential productivity.
Maintenance of optimum canopy architecture, tree brunch angle, pest protection and moreover declines in soil
organic matter levels, surface water quality degradation, reduced water infiltration rates and other soil physical
parameters may impact on its below satisfactory level of production (Mishra & Adak 2009, Kumar &
Adak et al. 2019
Ravishankar 2011). Management options play crucial role as the gap between supply and demand leads to the
nutrient deficiency and thereby yield sustainability and orchard health (Perry et al. 2010, Srikasetsarakul et al.
2011, Prakash et al. 2011). Ahmad et al. (2003) evaluated the performance of different nutrition sources under
high-density planting in Amrapali mango. Gucci et al. (2012) recorded variable yield performances and changes
in soil properties as a function of soil management system in high-density olive orchard. Adak et al. (2014)
recorded yield improvement in Dashehari mango under quality fertigation programme wherein soil organic
carbon, available P and K improvement in topsoil layer (0–30 cm) was noted. The role of soil compaction in
higher density plantation system (>400 trees ha-1) might have decreased the yield. Even the soil organic carbon
and micronutrient stocks played a significant role on nutrient transformations. It was estimated that these
systems could store <9.0 mg ha-1 soil organic carbon. Further, >50% Zn stock lies in the 1.1 to 1.5 kg ha -1,
>60% Cu stock in 11 to 20 ha -1 category, near to 70% Mn in 31 to 40 ha-1 and upto 50% Fe stock of 21-30 ha-1
category, which may utilize the reserve source for nutrient release (Adak et al. 2016b). A clear understanding of
the soil organic carbon dynamics indicates the potential benefits of quality fruit production. The storage of soil
organic carbon was observed higher in HDP system as compared to a conventional system, thus the role of
organic management becomes crucial in HDP system in terms of organic carbon mediated nutrient availability
and its dynamics (Adak et al. 2015). Based on a study in high-density guava plantation system having 555, 277
and 5000 trees ha-1, it was found that availability of P, K and organic carbon content was higher in 555 and 5000
trees ha-1 than conventional system (277 trees ha-1). Stocks of micronutrients were calculated and data showed
that HDP system could store more nutrients in root zone depth of 0–30 cm soil but proper precision farming is
needed for better productivity (Adak et al. 2016c).
Table 2. Univariate statistics of foliar nutrients in high density mango plantations of subtropical region of Lucknow, India.
Standard Standard
Soil parameters Range Mean CV % Skewness Kurtosis
deviations error of mean
N (%) 1.2–2.2 1.70 0.20 0.001 13.8 0.03 -0.09
P (%) 0.09–0.19 0.14 0.02 0.001 15.9 -0.10 -0.28
K (%) 0.66–1.96 1.30 0.36 0.003 27.5 -0.16 -1.06
Zn ( mg kg-1) 3.0–20.0 10.33 4.2 0.42 41.0 0.56 -0.33
Cu ( mg kg-1) 0.1–70.0 29.24 2.5 1.98 73.5 0.43 -0.89
Mn ( mg kg-1) 25.0–208.0 96.17 5.3 7.57 55.4 0.38 -0.96
Fe ( mg kg-1) 106.0–392.0 190.24 5.2 5.09 27.5 1.60 4.30
Figure 4. Histographic distribution of soil micronutrients.

Correlation among the soil properties
The correlation study indicated the positive and significant correlation of soil organic carbon with the other
soil nutrients (Table 3). It emphasizes the fact that there is an urgent need for optimum nutritional management
in the soils of the high density plantation system in order to sustain the productivity level as well as nutrient
release to plants. Soil organic C significantly correlated with N (0.328*), P (0.363**), K 90.287**), Zn
(0.453**), Cu (0.389**), Mn (0.339***) and Fe (0.438**). Likewise available P, K had a positive correlation
with the micronutrients. The positive correlation among these soil properties indicated the positive effect of each
nutrient on quality fruit production. Adak et al. (2014) observed positive correlation of yield with SOC
(r=0.994**) and soil nutrients (r=0.898* to 0.994**). Even available K and P was significantly correlated with
acidity, ascorbic acid (r=0.865* to 0.921**).
Table 3. Correlation matrix of soil nutrient parameters under high density mango plantations of subtropical region of
Lucknow, India.
Soil parameters SOC N P K Zn Cu Mn Fe
(mg kg-1) (%) (mg kg-1) (mg kg-1) (mg kg-1) (mg kg-1) (mg kg-1) (mg kg-1) (mg kg-1)
SOC (%) 1 .328(*) .363(**) .287(**) .453(**) .389(**) .339(**) .438(**)
Available N 1 .354(*) NS NS .330(*) NS NS
Available P 1 .477(**) .516(**) .597(**) .320(**) .425(**)
Available K 1 .670(**) .468(**) NS .486(**)
Available Zn 1 .587(**) .397(**) .557(**)
Available Cu 1 .218(*) .495(**)
Available Mn 1 .358(**)
Available Fe 1
Note: * = Correlation is significant at the 0.05 level, ** = Correlation is significant at the 0.01 level.
CONCLUSION
It was inferred from the present study that high density system of plantations are fruitful for getting higher
yields as compared to traditional cultivation. Among the HDP system, medium density is the best and may be
recommended at farmers’ field in order to maintain satisfactory yield level. Farmers could get Dashehari mango
yield of 15–16 t ha-1 or even higher based on canopy and nutrient management. Nutrient data indicated wide
variations across these density plantation systems. Impacts of density on soil physical properties were also
observed. Need for proper management system is still required for getting more yields.
ACKNOWLEDGEMENTS
Director, CISH, Lucknow is duly acknowledged for his tremendous support and facilities provided to carry
out the experiment. We also acknowledge all of our colleagues who helped directly or indirectly in carrying out
the study. We gratefully acknowledge the anonymous reviewers for their constructive suggestions for improving
the quality of the manuscript.
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Research article
Variability in yield and composition of oil from Indian Sandalwood

(Santalum album L.) trees grown in homogeneous conditions
Surendra Singh Bisht*, Mamata Ravindra and Divyashree N Gayathri
Chemistry and Bio-Prospecting Division, Institute of Wood Science and Technology,
18th Cross Malleshwaram, Bangalore-560003, Karnataka, India
*Corresponding Author: ssbchem@gmail.com [Accepted: 20 February 2019]
Abstract: The study evaluated the variability in yield and composition of oil from the heartwood
of Indian Sandalwood (Santalum album) trees grown in homogeneous condition. Trees grown at
Institute of Wood Science and Technology campus, Bengaluru were considered in this study.
Wood samples were collected from trees having different girth size (47.1, 53.4, 61.2, 69.1, 72.2,
74, 75.4, 81.6 and 82.4 cm). About 500 g of heartwood powder per sample was extracted by
hydro-distillation method using Clevenger's apparatus. Oil yield extracted from these samples
varied from 1.6–3.6 % of heartwood. Chemical profiling of the oil samples was carried out by gas
chromatography-mass spectrometry (GC-MS-QP-2010 Ultra Auto Sampler). A chemically
diversified alkanes, sesquiterpenoids, sesquiterpene, fatty acids, and alcohols, were detected. The
major constituents were α-santalol (41.7–53.67 %), β-santalol (18.2–27.9 %), epi-β-santalol (2.7–
7.18 %), β-santalene (1.39–5.30 %), α-santalene (0.4–4.87 %), and α-bergamotol (3.1–9.3 %). In
this study, it was concluded that the oil yield and its composition varies among the trees with
different girth. But no particular trends were observed between the girth size and oil yield.
Keywords: Santalum album L. - Indian Sandalwood oil - Composition - Girth - GC-MS.
[Cite as: Bisht SS, Ravindra M & Gayathri DN (2019) Variability in yield and composition of oil from Indian
Sandalwood (Santalum album L.) trees grown in homogeneous conditions. Tropical Plant Research 6(1): 31–
36]
INTRODUCTION
Santalum album L. is a small to medium-sized, evergreen semi parasitic tree of family Santalaceae (Fox
2000, Chaudhary et al. 2016). S. album commonly known as Indian Sandalwood is one of the oldest and
precious sources of natural fragrance with immense medicinal and commercial significance (Burdock & Carabin
2008, Hansda 2009, Bajpai et al. 2016). Sandalwood has been esteemed all over the world for its peculiar,
sweet, long lasting and medicinally valued fragrant oil. Indian Sandalwood oil derived from heartwood of the
tree have been used in various traditional systems of medicine, like Ayurveda, Siddha and Unani medicine in the
treatment and prevention of wide range of ailments (Sensarma 1989). The value of Indian Sandalwood tree
mainly depends on the volume of its heartwood and the quantity and quality of the heartwood oil. The quality of
SW oil mainly because of the concentration of oxygenated sesquiterpenes i.e. α-santalol and β-santalol, due to
which it has a pleasant characteristic aroma. Earlier reported main sesquiterpene and sesquiterpene alcohols of
Indian Sandalwood (Santalum album L.) oil were α-santalol (1), β-santalol (2), α-bergamotol (3), epi-cis-β-
santalol (4), cis-lanceol (5) and α-bisabolol (6) (Fig. 1). The hydrocarbons, α-santalene (7), β-santalene (8), epi-
β-santalene (9), α-bergamotene (10), β-bisabolene (11) and α-curcumene (12) were also present in the oil (Fig.
1) (Verghese et al. 1990).
Various studies have been carried out for documenting variation in oil content and its composition. The
study conducted on heartwood oil yield in Santalum yasi Seem and S. album by Doran et al. (2005) and Xiaojin
et al. (2011). The variation of oil content in 6 year old S. album was estimated from 0.64% to 1.78%. The
studies carried out in Sri Lanka by Subasinghe et al. (2013) on S. album showed a higher variation of oil content
and they found high oil content, i.e. up to 6.36%. Further, the research was conducted on the estimation of alpha
and beta santalol levels in S. album, Santalum spicatum, S. yasi, Santalum austrocaledonicum by some other
Received: 05 November 2018 Published online: 28 February 2019
Bisht et al. 2019
researcher as well (Brand et al. 2006, 2007, Xiaojin et al. 2011). Moreover, the content and composition of oil
from the central and transition zones of the Sandalwood disc (Shankaranarayana et al. 1998), analysis of growth
and oil composition (Xiaojin et al. 2011) and solvent extractable volatile profiling (Misra et al. 2013) from
heartwood of East Indian sandalwood tree are the few studies in this direction.
OH OH HO
 Santalol (2)  Bergamotenol (3)

 Santalol (1)
OH
H
OH
HO
epi-cis- -Santalol (4) cis-Lanceol (5)  Bisabolol (6)
Santalene (7) Santalene (8) epi-Santalene (9)
Bergamotene (10) Bisabolene (11) -curcumene (12)

Figure 1. The volatile compounds reported from Indian sandalwood oil.
It has been found that in most of these studies the growing conditions or environments were not similar.
Moreover, the quantities of the heartwood for extracting oil were not sufficient, which also plays an important
role in estimating oil yield and its composition. Variations in the composition of oil from trees grown in
homogenous condition may have an effect on the quality of SW oil, which could lead to inconsistency in
medicinal and aromatherapy properties. In this connection, it is imperative to understand and estimate the yield
and chemical composition of Sandalwood oil from mature Sandalwood tree grown in homogenous conditions.
Therefore, the aim of this study was to analyze the variation in yield and composition of oil from Sandalwood
trees grown in uniform condition.

General experimental methods
Heartwoods were collected from Sandalwood trees grown in Institute of Wood Science and Technology
campus. SW oil was extracted by hydro-distillation method using Cleavenger’s apparatus. Refractive index was
measured by Abbe 5 Refractometer. Relative density was measured gravimetric method using glass specific
gravity bottle. The instrument Buchi Rota Vapor used for evaporation of excess amount solvents. The resulting
extracted oils were analyzed by GC-MS (GCMS-QP 2010 Plus, Shimadzu make).
Plant materials and chemicals
SW samples were collected from different girth size sandalwood trees growing in the campus of Institute of
Wood Science and Technology, Bengaluru, India. Collected wood samples were processed for separation and
chipping of heartwood by chopping method. Heartwood chips were pulverized into powders and air dried for 24
h, prior to hydrodistillation. The LR and HPLC grade solvents (n-hexane, chloroform, diethyl ether etc) were
purchased from Merck Specialties Pvt. Ltd. and HiMediaLaboratories Pvt. Ltd. Distilled water was used during
the experiment.
Extraction of SW oil
Total 500 g of heartwood powder was hydro distillated for 14–15 hr by using Clevenger’s apparatus
(Hettiarachchi 2008). The hydrodisttiled oil was separated from water by separating funnel using organic
solvent (petroleum ether). Collected oil and solvent mixture was dried over Na 2SO4 and concentrated under
reduced pressure using rotovapor at 35–40 °C to get oily fractions. The resulting oily mass was subjected to GC-
MS analysis (Bisht & Hemanthraj 2014).
Physicochemical analysis
Refractive index was recorded by Abbe 5 Refractometer at room temperature. Relative density was
calculated by the ratio of the mass of a given volume of the oil to the mass of an equal volume of distilled water
at room temperature.
The relative density was calculated by the following equation:
Relative Density = m2 – mo/m1-m0
Where,
 m0 was the mass in grams of the empty specific gravity bottle,
 m1 the mass in grams of the specific gravity bottle filled with water,
 m2 is the mass, in grams, of the specific gravity bottle filled with the essential oil.
Equal volumes of the essential oil and water were weighed successively in a glass specific gravity bottle at
room temperature.
GC-MS analysis
The resulting oily masses were analyzed by GC-MS, equipped with RTX-Wax capillary column, connected
to anion trap quadrupole (ITQ) mass selective detector, with a unit mass resolution. The split was 1:90, with
helium as the carrier gas at a flow rate of 1 ml/min, while the damping gas flow was 0.3 ml min -1. The GC oven
temperature program was as follows: 40°C to 300°C., by ramping at 3°C, and held at 220°C for 20 min. The
injector temperature was maintained at 220°C and the transfer line was held at 220°C. The detection was
performed by a Thermo ITQ 900 mass spectrometer in the EI mode (ionization energy of 70 eV, ion source
temperature of 180°C, emission current of 220 mA). The acquisition was made in full scanning mode (mass
range 50–900 m z-1; 3 scans s-1). Maximum ionization time was 25 ms. A solvent delay time of 5 min (setoff)
was used to avoid overloading the mass spectrometer with solvent. The resulting GC-MS profile was analyzed
using National Institute of Standards and Technology (NIST-2011, Washington DC, USA) and Dr. Duke’s
Phytochemical and Ethnobotanical Database (http://www.ars-grin.gov/duke/). Qualitative analysis of metabolite
was done using its percentage peak area appeared at the total ion chromatogram in GC-MS analysis (Bisht &
Hemanthraj 2014).

Wood samples were collected from trees having different girth size i.e., 47.1, 53.4, 61.2, 69.1, 72.2, 74, 75.4,
81.6 and 82.4 cm. Oil yield from these samples varied from 1.6 to 3.6 % of heartwood (Table 1). Highest %
yield of oil (3.6%) was observed in tree with girth 72.2 cm while the lowest % oil yield i.e. 1.7 and 1.6% in tree
with girth size 69.1 cm and 72.2 respectively. The values of physical parameters such as relative density and
specific gravity of the oils were observed within the limit of high-quality SW oil (ISO 2002). The colour were
observed as colourless to pale yellow and refractive index (1.501–1.5025) of the oil were almost consistent with
the all samples while specific gravity was varies from 0.9435-0.974 (Table 1).
Table 1. Oil yield and physical parameter of extracted SW oil. [± standard error]
Girth size Refractive
Sample Oil yield (%) Oil Colour Specific gravity
(cm) index (RI)
T1 47.1 2.5±0.1 Colourless 1.5010 0.960 (±0.01)
T2 53.4 2.0±0.1 Pale yellow 1.5015 0.964 (±0.02)
T3 61.2 2.2±0.2 Pale yellow 1.5020 0.963 (±0.01)
T4 69.1 1.7±0.2 Pale yellow 1.5025 0.957 (±0.03)
T5 72.2 3.6±0.1 Pale yellow 1.5010 0.962 (±0.02)
T6 72.2 1.6±0.1 Colourless 1.5020 0.942 (±0.02)
T7 74.0 2.0±0.2 Colourless 1.5020 0.9435 (±0.01)
T8 75.4 2.0±0.2 Pale yellow 1.5020 0.974 (±0.03)
T9 81.6 2.6±0.1 Colourless 1.5010 0.953 (±0.01)
T10 82.4 3.2±0.2 Pale yellow 1.5025 0.963 (±0.01)
Bisht et al. 2019
Figure 1. Variation trend of % SW oil yield in different girth SW trees.

Total 35 volatile metabolites were detected with significant area percentage in total ion chromatogram (Fig.
2). Diversified phytochemicals such as alkanes, sesquiterpenoids, sesquiterpene, fatty acids, and alcohols were
observed in extracted Sandalwood oil. The major constituents were α-santalol (41.7–53.67 %), β-santalol (18.2–
27.9 %), epi-β-santalol (2.7–7.18 %), β-santalene (1.39–5.30 %), α-santalene (0.4–4.87 %), and α-bergamotol
(3.1–9.3 %) (Table 2 & 3).
Figure 2. GC-MS Chromatogram of Heartwood Oil.

Table 2. Mass fragmentation of GC-MS identified metabolites.
tR Molecular Mass fragmentation
Metabolites Mol. Wt
(Min) formula (mass m z-1 values)
11.81 α-Santalene C15H24 204 204(M+), 189, 161, 121, 107, 69, 41
14.26 β-Santalene C15H24 204 204(M+), 122, 94, 79, 67, 55, 41
30.44 α-Santalol C15H24O 220 220 (M+), 187, 121, 107, 93, 79, 41
30.6 Z-α-trans-Bergamotol C15H24O 220 220(M+), 132, 119, 107, 93, 79, 41
31.55 E-cis-epi-β-Santalol C15H24O 220 220(M+), 122, 107, 94, 79, 67, 41
32.50 trans-β -Santalol C15H24O 220 220 (M+), 189, 122, 94, 79, 67, 41
The quantity of metabolites varied among all the samples and the metabolites which were detected in area
percentage ≥0.50 of total ion chromatogram considered as major metabolites (Fig. 2; Table 2). The results
showed that the extracted oils contain high concentration of α-santalol and β-santalol at retention time (tR)
30.44 min and 32.5 min respectively. The concentration of α-santalol varied from 41.7–53.67 %, the lowest
being in T-2 and highest in T-3. The content of β-santalol ranged from 18.2–27.9 %, the lowest being in T-8 and
highest in T-10.
Table 3. Variation of metabolites (area %) in extracted SW oil.

Area %
Metabolites
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
α-Santalene 0.6 4.8 1.3 1.2 1.0 0.3 0.9 1.1 0.4 0.8
β-Santalene 2 4.9 3.9 3.1 2.6 1.4 2.9 3.1 1.5 2.7
α-Santalol 53.2 41.7 53.7 45.6 52.9 47.4 51.0 47.4 47.9 46.2
Z-α-trans-Bergamotol 6.8 6.3 5.2 8.4 6.6 7.2 6.2 4.9 9.3 7.5
E-cis-epi-β-Santalol 3.8 4.1 2.7 4.2 3.4 4.9 3.7 2.8 5.5 5.5
trans-β-Santalol 25.0 24.1 25.9 25.1 26.8 25.2 25.8 18.2 27.0 27.9
The six metabolites were present in significant amount in all samples with little variation in concentration of
α-santalol and β-santalol. While α-santalene, β-santalene, epi-β-santalene, α-bergamotol and epi-β-santalol were
detected with high deviation. Further the trend in variation of major metabolites among the different girth trees
was analyzed. In general the relative variation of the α-santalol and β-santalol among the all samples was almost
same (Fig. 3). Although there was no particular relationship observed between the girth sizes and oil yield of
Sandalwood trees grown in the same location. There may be a strong influence of genetic characteristics to the
quantity and quality of sandalwood oil variations of S. album trees of similar size growing under similar soil and
climatic conditions.
Figure 3. Variation trend of SW oil composition in different girth SW trees.
CONCLUSION
Metabolite profiling of the heartwood oil by using GC-MS techniques has provided a vast array of
metabolites, which included n-alkanes, sesquiterpene, sesquiterpenoids, fatty acids, alcohols and other
hydrocarbons. Total % oil yield from trees having different girth size (47.1–82.4 cm) were observed from 1.6–
3.6 % of heartwood. Although, there were no particular trends observed between the girth sizes and % oil yield.
But in general, almost similar type trends in the relative ratio of α-santalol and β-santalol content were observed.
The physical parameters such as relative density and specific gravity of the oils were observed within the limit
of high quality SW oil. These variations can be suspected that the genetic factors may play a significant role in
oil formation. The unknown causes of the variation of sandalwood oil compounds show the importance of
conducting further research on sandalwood oil formation with consideration of the external and internal
parameters.
ACKNOWLEDGEMENTS
Authors thank the Director and Group Coordinator Research of Institute of Wood Science and Technology,
Bangalore-560003 for encouragement and support.
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Brand JE, Fox JED, Pronk G & Cornwell C (2007) Comparison of oil concentration and oil quality from
Santalum spicatum and S. album plantations, 8–25 years old, with those from mature S. spicatum natural
stands. Australian Forestry 70: 235.
Burdock G A & Carabin I G (2008) Safety assessment of sandalwood oil (Santalum album L.). Food and
Chemical Toxicology 46(2): 421–432.
Chaudhary LB, Kushwaha AK & Bajpai O (2016) Santalum album L. In: Trees of Uttar Pradesh (Part 1).
Bennett, Coleman & Co. Ltd., Lucknow, India, pp. 296.
Doran JC, Thomson L, Brophy JJ, Goldsack B, Bulai P, Faka’osi T & Mokoia T (2005) Variation in heartwood
oil composition of young sandalwood trees in the south Pacific (Santalum yasi, S. album and F1 hybrids in
Fiji, and S. yasi in Tonga and Niue). Sandalwood Research Newsletter 20: 3–7.
Fox JE (2000) Sandalwood: the royal tree. Biologist 47(1): 31–34.
Hansda R (2009) The outlook for non-wood forest products in Asia and the Pacific. In: Asia-Pacific Forestry
Sector Outlook Study II Working Paper Series. p. 89.
Hettiarachchi DS (2008) Volatile oil content determination in the Australian sandalwood industry: Towards a
standardised method. Sandalwood Research Newsletter 23: 1–4.
ISO (2002) International Organisation for Standardisation, 2nd edition. ISO 3518.
Misra BB, Das SS & Dey S (2013) Volatile profiling from heartwood of East Indian sandalwood tree. Journal
of Pharmacy Research 7(4): 299–303.
Sensarma P (1989) Plants in the Indian Puranas. Naya Prokash.
Shankaranarayana KH, Ravikumar G, Rangaswamy CR & Theagrajan KS (1998) On sandalwood HESP and
ESPO oils from heartwood of sandal (sandal & its products). Australian Centre for International
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Verghese J, Sunny TP & Balakrishnan KV (1990) (+)‐α‐santalol and (−)‐β‐santalol (Z) concentration, a new
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Xiaojin L, Daping X, Zengjiang Y, Ningnan Z, Lijun Y (2011) Preliminary analysis of growth and oil
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Review article
Propagation methods in Babaco plants (Vasconcella x helbornii)

M. G. Jadán* and C. Dorca-Fornell
Universidad de las Fuerzas Armadas-ESPE, Av. General Rumiñahui s/n, Sangolquí- Ecuador,
P.O.Box:171-5-231B
*Corresponding Author: mbjadan@espe.edu.ec [Accepted: 25 February 2019]
Abstract: Babaco, (Vasconcellea x helbornii syn Carica pentagona), is an endemic fruit to South
of Ecuador and North of Peru, which is becoming very popular on the continuously expanding
subtropical fruit market for a few years now. From the nutritional point of view, this fruit is very
rich in fiber, it also has a good amount of vitamins, minerals and papain, an enzyme with great
digestive and anti-inflammatory properties. However, due to the diseases that attack babaco plants
and the sterile character of their fruits, exports have been significantly reduced being considered as
an unprofitable agricultural production. New techniques in plant biotechnology as in vitro culture
could make possible a massive production of free-pathogens babaco plants. In particular, somatic
embryogenesis is a rapid way of obtaining genetically identical individuals which is also used in in
vitro conservation of germplasm and plant genetic improvement. The present document aims to
summarize the techniques of obtaining new plants of babaco and evidences the achievements
obtained in somatic embryogenesis in the Caricaceae family up today.
Keywords: Babaco - Vasconcellea x heilbornii - Cárica pentágona - Caricaceae - in vitro culture
- Somatic embryogenesis.
[Cite as: Jadán MG & Dorca-Fornell C (2019) Propagation methods in Babaco plants (Vasconcella x helbornii).
Tropical Plant Research 6(1): 37–45]
INTRODUCTION
Babaco, [Vasconcellea x helbornii (Badillo) Badillo syn. Carica pentagona] (Badillo 1993, 2001), is an
endemic plant to southern Ecuador and northern Peru, which belongs to the Caricaceae family, that is formed
by six genera and 35 species (Carvalho & Renner 2012). Probably under human influence, V. stipulata (Badillo)
was hybridized with V. cundinamarcensis (Badillo) resulting in V. x heilbornii hybrids including babaco and
jigacho (Van Droogenbroeck et al. 2004, Coppens d'Eeckenbrugge et al. 2014). Babaco is among the so-called
mountain papayas, which develop at elevation of 2000 m asl, (Scheldeman et al. 2003). It is a shrub plant with
semi-perennial culture and a parthenocarpic fruit (it does not have seeds); an elongated and pentagonal berry
type, whose size varies from 30 cm long to 15 cm in diameter (Fabara et al. 1985).
In recent years, the export of fresh fruits, such as babaco, has been strengthened, so product quality becomes
increasingly demanding in terms of organoleptic and phytosanitary characteristics. However, due to the high
sensitivity of this crop to temperature and humidity (Janick & Paull 2008), along the diseases produced by the
various pathogens that tend to affect the plant and the sterile nature of its fruit, the best yields are obtained under
greenhouse conditions since open sky culture result in a not profitable production (Robles-Carrión et al. 2016).
Although, the production of babaco fruits in greenhouses is a challenge as well. One of the pathogens that
causes the greater economic losses in babaco plants is the Fusarium oxysporum Schltdl., which produce 'babaco
vascular wilt' in fields and greenhouses which can achieve losses of 100% of production (Robles-Carrión et al.
2014). To all these reasons, many scientists have attempted to overcome all the limitations regarding babaco
plants propagation by the use of alternative techniques as biotechnological techniques with successful results. So
far, biotechnological methods have lead to the production of pathogen-free plants, massive propagation (Vasil
1994, Kitto 1997), genetic improvement through mutation induction, in vitro selection (Predieri 2001), and
genetic engineering (Herrera-Estrella et al. 1983), in several plant species including babaco. In the present
review, we aim to summarize the state-of-the-art of babaco propagation methods by studying the most relevant
works of this plant and its Caricaceae relatives.
Received: 07 June 2018 Published online: 28 February 2019
Jadán & Dorca-Fornell 2019
Propagation by Stakes
Since, babaco plants have a parthenocarpic fruit (seedless) is limited to asexual reproduction by cuttings,
buds, grafts or stakes, which leads to the attainment of a new mature plant at approximately two years (AAIC
2003). For this reason, the spread of diseases increases. Among the most important diseases are the massive
bacterial infections of the genus Erwinia caratovora (Jones) Waldee and Agrobacterium sp. and the fungus
Fusarium oxysporum. Vasconcellea that produces 'babaco vascular wilt' and generates large losses (Scheldeman
et al. 2003). As a preventive measure, farmers use a large amount of agrochemicals that affect the quality of the
fruit, pollute the environment and harm human health (AAIC 2003). Nowadays, this is one of the major
problems around babaco production being pointed out by several researches (Scheldeman et al. 2003, AAIC
2003, Falconí et al. 2006, Chávez 2006, Robles-Carrión et al. 2014, 2016). Some efforts have been done on a
genetical improvement of babaco by grafting on three varieties of papaya patterns; Hawaiian, Criolla and
Maradol papaya, tending to the production of disease-free plants tolerant to Fusarium oxysporum. According to
Chávez (2006), the best yields, with 85% of successful plant grafted, were obtained by the combination of
Hawaiian papaya and babaco plants, followed by papaya “criolla” with 73% of successfully grafted plants,
while Maradol papaya and babaco grafts presented the lowest values. Successfully grafted plants showed
increased resistance to babble diseases such as 'babaco vascular wilt', contributing to increasing its production.
According to Falconí et al. (2006), the length of the babaco stakes to propagate should be between 25 to 30 cm
and have a diameter of about 4 to 6 cm. They must have a superior cut in bevel to avoid the accumulation of
rainwater and a transverse basal cut to have a greater surface of rooting by applying growth regulators. By
means of this method and after several forms of disinfection, a new plant can be obtained at an overall of 10 or
12 months. Soft buds are a widely used form of propagation, which are extracting from growing plants. Shoots
can be also produced for this purpose being of a length of 10 cm and a diameter of about 1.5 to 2.5 cm. Then,
the upper part of the shoot is cut out to stimulate sprouting to finally induce rooting (AAIC 2003). Although,
this technique for obtaining new plants of babaco takes too long time and does not ensure the acquisition of a
new plant without infections. Currently, new techniques such as in vitro culture are intended to improve these
times for the production of the species and to obtain disease-free and genetically improved plants (Soria & Viteri
1999).
Somatic embryogenesis propagation
i. Somatic embryos development: Somatic embryogenesis is a process by which somatic cells are transformed
into somatic embryos, which resemble zygotic embryos morphologically, since are carriers of typical
embryogenic organs; however, they develop in a different way (Adobkar et al. 2012). The greater interest in
somatic embryos focuses on their practical applications for large-scale vegetative propagation, particularly
due to the possibility of expanding propagation through the use of bioreactors (De Feria 2000). Most
probably, somatic embryogenesis can be achieved for all plant species in which an explant, a culture media
and the appropriate environmental conditions are used. Some examples are found in forest species,
(Celestino et al. 2005), fruit trees, (Jordán 2011, Bao-Fundora et al. 2013) and ornamental plants (Ribero-
Bautista et al. 2008). In addition, in the majority of cases, somatic embryos or embryogenic cultures may be
cryopreserved, which makes it possible to establish gene banks, constituting a pathway for cryopreservation
and regeneration (Engelmann et al. 1997). Moreover, embryogenic cultures are used as targets suitable for
genetic transformation (Peña & Séguin 2001) and functional genomics, allowing the evaluation of the
function of specific genes (Campbell et al. 2003).
Somatic embryos can be obtained from different parts of the plant, like as young leaves, hypocotyls,
stem, immature seeds, and suspension cell cultures. The cells of this tissue being organized cells are inducted
to the disorganization and again to the reorganization of them (Freire 2003). Caricaceae somatic embryos
have been obtained from different sources of explants, as for example in C. papaya have been obtained from
leaves, immature seed integuments, mature and immature zygotic embryos and many others (Fitch &
Manshardt 1990, Cabrera-Ponce et al. 1995, Monmarson et al. 1995, Posada-Perez & Koski 2007, Malabadi
et al. 2011). Somatic embryos of C. stipulata were also obtained from peduncle calli (Litz & Conover 1980)
and C. pubescent from germinated hypochromic callus in vitro (Jordán 1986). Although, several studies have
been carried out in other Vasconcellea, such as Jordán (1986), who worked to achieve the regeneration of
plants via somatic embryogenesis of V. cundinamarcensis without satisfactory results. Results of Jordán et
al. (2011), achieved only the formation of multiple buds from nodal sections of V. chilensis adult plants.
However, babaco has only been in vitro propagated from nodal sections (Cohen & Cooper 1982), buds,
apical and lateral buds (Cossio 1985). Some attempts have led to the formation of calli from ovules and
leaves (Vega de Rojas & Kitto 1991, Vega de Rojas et al. 1993, Quillay 2011), the formation of buds via
organogenesis (Jordán & Piwanski 1997), and somatic embryos from cell suspensions obtained from calli
(Jordán & Velozo 1996). In addition, an effective protocol for the isolation and fusion of babaco and jigacho
protoplasts from callus and leaf respectively (Rivera & Jadán 2010) has been standardized.
One of the challenges, when intend to propagate babaco plants by tissue culture techniques, is the plant
material used, which come from plants growing in field conditions, without any sanitary control and exposed
to the environment, rending this technique unsuccessful. Newly findings of Jadán et al. (2016a), showed a
stable protocol to create a bank of donor plants of the babaco hybrid from cuttings, under phytosanitary
semicontrolled conditions by the use of Carbendazim fungicide and biostimulant (GERMO-TB01)
application to increase the number of axillary shoots. The results of this work allowed to achieve 100%
sprouting and root formation in the different stem sections, which could be used in in vitro propagation
protocols ensuring a high genetic and phytosanitary quality. Furthermore, aiming to solve the problem of
microbial contamination of bacteria, which also impede the proliferation of in vitro tissues, Jadán et at.
(2016b), reported the establishment of 68.5% of bacteria-free explants from babaco buds using 1.5% Sodium
Hypochlorite for 10 minutes and immersion in a solution of Gentamicin 50 mg l-1 and Streptomycin 25 mg l-
1
for 3 hour.
In the process of induction of somatic embryogenesis, not only the type of explants and genotype must be
taken into account, but the effect of the composition of the medium at each stage of the tissue culture is
equally very important, since optimal media conditions vary depending on the type of explant and genotype.
Although the, (NN) medium (Nitsch & Nitsch 1969) is widely used, but, the most commonly used culture
medium for the development of somatic embryogenesis is (MS) Murashige & Skoog (1962) with some
modifications. Most researchers working on the development of somatic embryogenesis in species of the
Caricacea family use half the concentration of the MS salts and 2,4-diclorofenoxiacético, (2,4-D), as a
growth regulator, in concentrations from 1–10 mg l-1 (Zhu et al. 2004). However, other authors have used
growth regulators 1-Naphthaleneacetic acid, (NAA), indolacetic acid, (IAA), 6-Benzylaminopurine, (6-BAP;
cytokinin), Kinetin and Zeatin (Jordán & Velozo 1996, Cabrera-Ponce et al. 1996). In addition, Woody Plant
Medium, (WPM), culture medium (Lloyd & McCown 1981) has been used in in vitro culture of babaco as
well (Jordán & Piwanski 1997).
Recently, Cornejo-Rodriguez et al. (2009), evaluated four auxins (NAA, Picloram, 2,4-D and 2,4,5-
Trichlorophenoxyacetic acid, (2,4,5-T)) in combination with different concentrations of 6BAP for the
induction of papaya calli and different concentrations of 2,4,5-T and Thidiazuron, (TDZ; cytokinin), for the
induction of papaya somatic embryos. Concentrations between 1 and 5 mg l-1 of auxins such as NAA and
2,4,5-T combined with 1 and 2 mg l-1 of 6BAP were found to be the most suitable for callus induction.
Cultures pretreated with Picloram during the callus induction phase show greater efficiency in the induction
of embryos at concentrations of 1 mg l-1 of 2,4,5-T. Further, the use of 1 mg l-1 of 2,4,5-T in combination
with 2 mg l-1 of TDZ, resulted in 81% of embryo induction.
ii. Maduration of somatic embyos: The maturation phase of somatic embryo development is essential to promote
germination. In this phase, cell expansion, the accumulation of reserve substances, and tolerance to
desiccation are achieved (Parrott 1993). The correct accumulation of reserves leads to an increase in the dry
mass of the somatic embryos, which indicates a high quality in the vigor and positively influences its later
germination (Fuji et al. 1990).
Although, it has been reported embryo maturation is deficient in the absence of ABA (abcisic acid),
leading to abnormal morphology and early germination (Fernando & Gamage 2000), Vega de Rojas & Kitto
(1991), obtained mature somatic embryonic structures from nodule structures of babaco ovules by
transferring them to media with GA3 (0.1 mg l-1) in addition to activated carbon (2.0 g l-1) or casein
hydrolysate (200 mg l-1) plus IAA (0.5 mg l-1). Nevertheless, according to Malabadi et al. (2011), some
varieties of papaya have greater effectiveness in maturation starting from partially dried tissue combined
with the addition of ABA.
Somatic embryo germination
Germination of somatic embryos is defined as the radicle and bud developmental process. In many plant
species, somatic embryo germination and in consequence the subsequent plant recovery is relatively poor and
difficult. Some of the handicaps, which influence low embryo germination, are poor somatic embryo quality,
lack of proper maturation desiccation tolerance and dormancy or inhibitors(s) within the embryo (Rtienne et al.
1993, Mujib et al. 1998). In recent years several innovated methods, such as an addition of osmotic agents,
carbohydrates, sugar alcohols etc., have been used to improve embryo quality before germination (Xing et al.
1999, Lipavská & Konrádová 2004, Robichaud et al. 2004). Although the production and quality of somatic
embryos have been improved in several crop species by using the temporary immersion systems known under
the trade name RITA, no many researchers have used the temporary immersion system for the propagation of
Caricaceae family so far. Some successful results have been observed by Vegas-García et al. (2015), which
standardized the conditions of initiation, multiplication, rooting and acclimatization of hermaphrodite papaya
plants from axillary shoots produced in RITA® temporary immersion vessels. Stem cuttings were obtained by
direct treatment of immersion with the subsequent maintenance of cuttings in water with aminoisobutyric acid,
(AIB), and 50 mg l-1 for the formation of roots (Jordán 2009).
Germination phase has been also improved by the trial out of many protocols by using several combinations
of growth regulators and growth media, thus currently; there are many studies which show promising results in
Caricacea family. Some of them are listed below:
For example, Yie & Liaw (1997), used two methods of in vitro cultivation to regenerate papaya plants: from
callus and individual apical buds. Callus was induced from stem sections of papaya seedlings in a medium
consisting of 1 mg per 1 NAA and 0.1 mg 1-1 kinetin. The authors observed that calli regenerated
shoots/embryos when transferred to medium with lower concentrations of auxin, 0 to 0.05 mg 1-1 IAA, and
higher cytokinin concentration, from 1 to 2 mg 1-1 kinetin. They also observed the production of multiple
outbreaks from decapitated apical buds grown in an average medium supplemented with 0.05 mg 1-1 IAA and/or
5 mg 1-1 kinetin or 0.5 to 1.0 mg 1-1 benzyladenine. The root formation of shoots and embryos derived from
callus was produced in a medium with 5 mg 1-1 IAA and at a light intensity of 3,000 to 4,000 lux. Plants with
roots obtained were established successfully in the soil and in standard conditions of greenhouse effect after
passing a phase of acclimatization in which their initial development was induced on wet vermiculite in covered
polyethylene pots.
It has been reported, the formation of buds in axillary buds and the formation of calli in leaf sections derived
from seedlings through the use of NN medium supplemented with NAA and BAP as well as the increase of the
germination time and the percentage germination up to 53% in the presence of hydrogen peroxide in in vitro
conditions in V. stipulata (Vélez-Mora et al. 2015).
For rhizogenesis of shoots, a subculture AIB 2.0 mg l-1 (Jordán & Piwanski 1997), has been used, although
roots have also been induced in the presence of NAA 0.01 mg l-1 and BAP 0.1 mg -1 and NN nutrients from
axillary buds (Jordán et al. 1999). In addition, the inclusion of activated charcoal 0.6 g l-1 and casein and
cysteine hydrolysate helps avoid browning in WPM and MS media, besides improving root proliferation in
babaco axillary bud cultures. According to Vaca-Suquillo (2008), buds was obtained by the use of 0.167 ppm
IAA and a 50% rooting percentage was obtained by using 0.5 ppm AIB and 1ppm NAA in babaco explants.
Studies of Jordán (2011), showed a high number of outbreak formation from in vitro induction in the
presence of relatively high levels of TDZ, IAA and organic additions in WPM media, including casein
hydrolysate, adenine sulphate and cysteine, in V. chilensis ex C. chilensis nodal sections. Somatic shoots and
embryos have been obtained by using high concentrations of TDZ, in addition to IAA and gibberellic acid
(AG3), from foliar leaf sections in babaco. Likewise, leaf section shoots have been induced in MS medium with
TDZ neat or in combination, including casein hydrolysate and adenine sulfate. It has been pointed out that the
application of GA3 promotes the germination of seeds in V. stipulata (Vélez-Mora et al. 2015), V.
cundinamarcensis, V. x heilbornii, by accelerating the transport of nutrients via the endosperm (Scheldeman et
al. 2003). 0.1 mg l-1 BAP, 0.1 mg l-1 and GA3 126 mg l-1 floroglucinol have been used for the multiplication of
axillary shoots in Carica papaya L. (De Winnaar 1988) and in Carica pubescens (Jordán 1992), as well as
combinations of IBA and floroglucinol for the induction of rooting in apical buds in babaco (Jordán & Piwanski
1997), although this combination was not successful as a promoter of Vasconcellea Chilensis ex Carica chilensis
root (Jordán 2011). Several in vitro studies in which floroglucinol has been used have shown that this compound
is much more potent than other growth regulators in inducing and enhancing different events of plant
development, especially in plants where, not yet, an effective in vitro propagation protocol has been
standardized (Teixeira da Silva et al. 2013). The use of other bioregulatory growth compound, such as
Pectimorf®, used as auxins and cytokinins substitute or complement, have been successfully applied in the
germination of Nicotiana tabacum (Acosta et al. 2004), as well as to increase the production of citrus fruits,
such as Citrus macrophyla Western by the use of somatic embryogenesis techniques (Bao-Fundora et al. 2013).
Lately, Posada-Pérez et al. (2016), had a positive effect on rooting and ex vitro acclimatization of papaya shoot
(Carica Papaya) by the use of Pectimorf®.
Since somatic embryo plants are smaller and weaker than those of zygotic or seed embryos, the conversion
rates are much lower in somatic embryos compared to natural seed (Lai et al. 1995). One way to optimize
maturation and conversion in somatic embryos plants is to try to simulate the natural processes of plant
development. In order to obtain an efficient result in the production of plants, traits that take into account the
time and type of application of growth regulators, low oxygen concentration and desiccation of mature embryos
can be used (Carman 1989). In some Vasconcellea species, the use of MS medium salts and/or media with low
nitrate levels as NN and woody plant medium allowed survival and good quality of the plants (Jordán 1986,
Jordán & Piwanski 1997). Furthermore, the use of treatments to improve the post-germination, such as the
application of low temperatures to activate cell development; (Das et al. 2002), are highly recommended, since
depending on the species, direct induction of somatic embryos germination results in a low percentage of
positive results (Parrott et al. 1988, Senaratna et al. 1990).
Ex vitro acclimatization
A large number of plants produced in vitro do not survive the transfer from in vitro conditions to an ex vitro
environment under greenhouse or field conditions (Hazarika 2003, Hazarika & Bora 2010). Anomalies in the
morphology, anatomy and physiology of seedlings cultured in vitro can be repaired after transfer to ex vitro
conditions (Maene & Debergh 1987). Hyperhydricity can be controlled in a number of ways including better
aeration of the vessel used in vitro (Rossetto et al. 1992), by reducing cytokinin levels (Williams & Taji 1991),
increasing agar concentration (Brand 1993), and by changing the concentration of the components of the
medium (Ziv et al. 1989). Since the photosynthetic activity is scarce in the in vitro cultures, it is necessary to
adapt the foliar system to become an active photosynthesis system. Several strategies have been proposed for
this adaptation, such as the elimination of carbon sources, the mechanical defoliation of seedlings, the induction
of storage organs and/or the use of growth retardants to inhibit leaf growth (Ziv & Lilien-Kipnis 1990). Many
efforts have been done on amending this scenario. For example, studies focused on obtaining elite plants of
Vaconcellea Stipulata resulted in the significant reduction (50%) of hyperhydricity under in vitro conditions
when gibberellic acid was added at low concentrations in the NN media, allowing to recover up to
approximately 80% of viable seedlings (Vélez-Mora et al. 2015). Cruz et al. (2008) obtained the in vitro rooting
of transgenic papaya plants by using 2 mg.l-1 of indole-3-butyric acid in the culture medium, as well as ex vitro
rooting with high percentages of survival. The synergistic action of AIB with 9.0 mg l-1 Pectimorf® allowed to
obtain in vitro plants with greater leaf area, fresh weight, number of roots, photosynthetic rate and stomatal
conductance, which together with a high percentage of rooting and less percentage of open stomata allowed to
reach a 76.2% survival ex vitro conditions in Carica Papaya seedlings (Posada-Pérez et al. 2016).
Genomic aspects of somatic embryogenesis propagation
It is important to take into account plants been regenerated by tissue culture are prone to somaclonal
variations with genetic alterations events due to the stress induced by the in vitro culture conditions and the
mode of regeneration. Genetic stability is an essential requirement for its use (Bhowmik et al. 2016). To this to
solve, ISSR markers, which have only been used to characterize genetic diversity and gene flow between
populations in babaco (Kyndt et al. 2005), it has been used to determine the genetic stability of babaco plants
propagated in vitro, resulted in no differences at the molecular level between the in vitro plants and the mother
plants (Jadán et al. 2017). Although, somaclonal variations events could be an alternative to improve the quality
of fruits, either in size (smaller units for export), compact fruits or resistant to pests and/or other diseases (Vega
de Rojas & Kitto 1991).
Nowadays, Vasconcellea genomic research is very poor, just counting with the development of some BAC
libraries of few species. Although, genome sequencing and genetic mapping technology will make possible the
identification of genes of interest in Vasconcellea, which will open a door in the expansion of new species of
this genus (Scheldeman et al. 2011). Recently, the first papaya callus transcriptiome profile analysis revealed the
presence of highly expressed somatic embryogenesis genes, such as SERK and LEA, hormone-related genes,
stress-related genes, and genes involved in secondary metabolite biosynthesis pathways, as well as, transcription
factors families as, NAC, WRKY, MYB, WUSCHEL, Agamous-like MADS-box protein and bHLH, which are
known to play a important role in somatic embryo development in other species (Jamaluddin et al. 2017). In
addition, recent studies by Moura-Vale et al. (2014), in which they analyzed the differential expression of 6%
Polietilenglycol induced treatment of three proteins (enolase, esterase and ADH3), concluded that these three
proteins could play an important role in the maturation of the somatic embryos, being able to be used as
candidate biomarkers of somatic embryogenesis in papaya. The discovery of genes and proteins expressed in
papaya somatic embryogenesis development provides important information to improve our understanding on
genes and proteins associated with somatic embryogenesis in other relative plant species as babaco, which could
lead to the optimization of protocols allowing the use of biotechnology for babaco propagation.
CONCLUSION
Somatic embryogenesis is making its way as the most suitable via of plant regeneration to complement
classical improvement techniques in various agricultural species such as forestry (Celestino et al. 2005),
ornamentals (Ribero-Bautista et al. 2008), fruit trees (Jordán 2011, Bao-Fundora et al. 2013), among others.
Many efforts are being devoted to the application of this technique to obtain effective results based on the clonal
evaluation and cryopreservation of embryogenic lines to be used in each place and suitable purpose (Engelmann
et al. 1997). In addition, the latest advances in genomics are being applied to elucidate the metabolic pathways
of regeneration processes (Campbell et al. 2003, Scheldeman et al. 2011, Jamaluddin et al. 2017). In light of the
low regeneration of babaco plants by conventional methods as stakes, some efforts need to be done in the
production of babaco by in vitro techniques. To date, the studies carried out in Caricaceae family and
specifically in babaco plants are scarce, but the emerging possibility of cloning by somatic embryogenesis this
species is an idea started by many researchers who have as a purpose the production of high quality and
pathogen-free plants. The use of biotechnology as a tool in the propagation of elite plants of this crop can be an
alternative via for rapid multiplication, as well as the introduction on a commercial scale of new varieties or
hybrids.
ACKNOWLEDGEMENTS
To the Institute of Biotechnology of the IBP Plants, Dr. Idlamis Bermudez- Caraballoso and Dr. Rafael
Goméz Kosky.
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Short communication
Annual flowering of Dendrocalamus longispathus (Kurz) Kurz

in Mizoram
Sandeep Yadav*, Hans Raj and Lalnunmawia
Forest Research Centre - Bamboo and Rattan, Aizawl-796007, Mizoram, India
*Corresponding Author: sandeep080987@gmail.com [Accepted: 10 March 2019]
[Cite as: Yadav S, Raj H & Lalnunmawia (2019) Annual flowering of Dendrocalamus longispathus (Kurz) Kurz
in Mizoram. Tropical Plant Research 6(1): 46–48]
INTRODUCTION
Bamboo belongs to the Poaceae family and it has about 90 genera with over 1200 species distributed all
over the world (Lobovikov et al. 2007). It is naturally distributed in the tropical and subtropical belt between
approximately 46° north and 47° south latitude, and is commonly found in Africa, Asia and Central and South
America. There are few species which can grow in temperate zones also such as Arundinaria gigantean (Walter)
Muhl. Among asian countries, India and China are the two major bamboo producing countries (Maxim et al.
2005).
The bamboo bearing area in India is estimated to 15.69 million hectares (Anonymous 2017). There are about
125 indigenous and 11 exotic species of bamboo belonging to 23 genera reportedly found in India (Anonymous
2017). About 66% of the growing stock is concentrated in the North Eastern states of the country (Adkoli 2002).
The principal bamboo genera occurring in India are Arundinaria, Bambusa, Chimonobambusa, Dendrocalamus,
Dinochola, Gigantochloa etc.
Mizoram is situated in the north eastern part of India, and it shares international borders with Bangladesh in
the west and Myanmar in east and south. The geographical area of the state is 21,081 km2 which constitutes
0.64% of the total area of the country. The recorded forest cover in the state is 18,186 km2 which works out to
be 86.27% of its geographical area (Anonymous 2017). The extent of bamboo bearing area in the forests of the
state is 3,267 km2 (Anonymous 2017). A number of bamboo species grow very luxuriantly in the state, most
notably is the Melacanna beccifera (Roxb.) Kurz, which constitutes approximately 70% of the bamboo cover.
Dendrocalamus longispathus (Kurz) Kurz, D. hamiltonii Gamble, D. strictus (Roxb.) Nees and Bambusa tulda
Roxb. are the other notable bamboo species of the state having populations distributed in small patches across
the state.
Dendrocalamus longispathus is a large caespitose bamboo; culms up to 20 m tall, up to 10 cm in diameter;
nodes swollen; internode swollen, 25–60 cm long. It occurs in moist hill slopes and along streams in the moist
fertile loamy soil and particularly shaded fringes of the forest cover. It is distributed in India (Assam, Manipur,
Mizoram and Tripura), Bangladesh and Myanmar. It is called as Rawnal in Mizo language. Mature culms are
harvested for its use as construction material and tender shoots are harvested during rainy season for local
consumption. Apart from these two most common uses, it is also being utilized for handicraft industries, mats,
and matply industries. To sum up, it can be argued that after Melocanna baccifera (Roxb.) Kurz, it is the second
most utilized bamboo to sustain rural livelihood of the state.
According to Gamble (1896), D. longispathus flowered during 1876 and 1879–80 in Chittagong
(Bangladesh) and during 1871 and 1891 in Myanmar. Gupta (1972) reported it in flowering from Assam in 1968
and in Mizoram during 1966–67. Bahadur (1979) stated that it was flowering in Forest Research Institute,
Dehradun in 1979. Sharma et al. (2014) reported its sporadic flowering in Kolasib and Mamit districts of
Mizoram.
In this present communication, it is reported that since 2014, small and segregated populations of this
species flower annually across the state (Table 1). Flowering and non-flowering clumps can be found within the
same site (Fig. 1). Few individual in the same population did not flowered at all. This peculiar flowering pattern
of D. longispathus is a cause of concern for all those who are associated with it. Localized arid conditions can
Received: 11 July 2018 Published online: 30 April 2019
be one of the factors triggering flowering. Usually this species is present in gradually sloping hills where few
natural water sources are present at some places. There is a very less rainfall in Mizoram from October till
March. The observed sporadic and segregated flowering pattern is may be due to localized unavailability of
water. Aftermath of flowering in D. longispathus includes death of flowering culms and increase in rodent
population. Since jhumming or slash and burn practice is quite prevalent in Mizoram, therefore, dead flowering
clumps are very susceptible to fire.
Table 1. Flowering in Dendrocalamus longispathus (Kurz) Kurz in Mizoram since 2014.
S.N. Year Month of flowering Place of flowering Remarks
1. 2014 October-November Kolasib and Mamit district Clumps died after
2. 2015 October-December Kolasib, Mamit, and Sercchip flowering and seed setting.
district Population of wild rodents
3. 2016 October-November Lengpui, Aizawl and lunglei and other small mammals
district increased due to the
4. 2017 October-November Lengte, Lawngtlai, Siaha and abundant availability of
Mamit district bamboo seeds.
5. 2018 October-November Lengpui, Sercchip, Kolasib, Mamit
Figure 1. Dendrocalamus longispathus (Kurz) Kurz: A, Flowering population, Mizoram in 2017 (Note the non-flowering
green clumps amongst flowering brown clumps); B, Individual clump in flowering stage; C, Inflorescence; D, Immature
seeds.
Yadav et al. 2019
REFERENCES
Anonymous (2017) India State Forest Report. Forest Survey of India, Dehradun, India.
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longispathus (Kurz) Kurz in Mizoram, India. Tropical Plant Research 1(1): 26–27.
Research article
Phytochemical study of medicinal plants used against buruli ulcer

by Ntandu people in Kongo Central, DRC
Idrissa Assumani Zabo1*, Ndombe Tamasala1 and Lusala Diakedika2
1
National Pedagogical University (Université Pédagogique Nationale)
2
Kasangulu High School (Lycée Kasangulu)
*Corresponding Author: idrissa.assumani@upn.ac.cd [Accepted: 15 March 2019]
Abstract: In Africa, ancestors are known to possess phytotherapy knowledge. This knowledge is
transmitted from one generation to another through oral tradition. Based on experience, this
knowledge is unaware of the chemical composition of the plants used. The study is to justify its
scientific basis in the treatment of Buruli ulcer. Ethnobotanical data are collected from older men,
traditional healers, herbalists, practitioners and patients who have suffered from Buruli ulcer. The
species mentioned in the recipes were screened for the detection of major chemical groups. Aloe
tenuifolia, Annona senegalensis, Brillantaisia owariensis, Vernonia amygdalina and Strychnos
icaja are involved in the management of Buruli ulcer. Chemical screening has revealed the
presence, to varying degrees, of the following secondary metabolites: tannins, alkaloids,
saponosides, free quinones, anthocyanins, bound quinones, terpenoids, polyphenols, steroids,
coumarins and reducing sugars. The presence of these metabolites provides a scientific basis for
Ntandu endogenous knowledge. These findings give credence to the ethnomedical use of in the
treatment of Buruli ulcer in Ntandu people.
Keywords: Endogenous knowledge - Ntandu people - Mycobacterium ulcerans - Medicinal plants
- Phytochemistry.
[Cite as: Idrissa AZ, Ndombe T & Lusala D (2019) Phytochemical study of medicinal plants used against buruli
ulcer by Ntandu people in Kongo Central, DRC. Tropical Plant Research 6(1): 49–53]
INTRODUCTION
In order to restore disturbed health, the population uses medicinal plants. Thus, important data are collected
in the Democratic Republic of the Congo (DRC) (Ngbolua et al. 2013, Ngbolua et al. 2016). In Africa, ancestors
are reputed to possess phytotherapeutic knowledge. They transmit it from one generation to another through the
oral tradition. Qualified as empirical, this knowledge is not based on any theory and is unaware of the chemical
composition of drugs used for health care (Nacoulma 1996).
Nowadays, the medicinal virtues of plants are, more and more recognized. Indeed, various authors have
reported the presence of secondary metabolites that may act actively against certain pathogens, or even proven
their effectiveness in the laboratory against certain diseases (Kikakedimau et al. 2012, 2013, Mbaya et al. 2014,
Wanzala et al. 2017).
Buruli ulcer was recorded for the first time in 1897 at Buruli village in Uganda. The first sightings were
published in Australia in 1937 as Bairnsdale ulcer and then in the former Belgian Congo in 1942 (Aubry 2017).
Buruli ulcer caused by Mycobacterium ulcerans, a pathology that causes large and deep skin lesions causing
serious functional sequelae in 25% of cases. Although it is the third most common mycobacteriosis in the world,
after tuberculosis and leprosy, Buruli ulcer remains a shadow disease due to the localization of endemic foci in
remote rural areas. There has been a dramatic increase in cases worldwide since the 1980s. (WHO 2002, 2003,
Ouattara et al. 2002, Mougin 2009, Abgeguen et al. 2010).
To date, 36 countries, mostly in tropical hot and humid climates, have reported Buruli ulcer in Africa, South
Asia and the Western Pacific. Although other outbreaks exist in Australia, French Guinea, Peru, Papua New
Guinea and Japan, Africa appears to be the most affected region (WHO 2012). This massive increase in the
number of cases and countries affected by this scourge over the last 30 years led the World Health Organization
Received: 07 August 2018 Published online: 30 April 2019
Idrissa et al. 2019
(WHO) to launch a specific control program in 1998 which was intensified in 2004 (Josse et al. 2004, WHO
2004, 2008, 2009, Quentin et al. 2014).
In the DRC, Van Oye & Baillon (1950) claimed to find the first case of Buruli ulcer but, the disease existed
in the Kongo Central province well before the 1935s. Since then, many cases were more and more registered
and Songololo territory in Kongo Central Province remains the main focus (Meleney & Johnson 1950).
The antiquity of this mycobacteriosis in this province raises the following questions:
• Is there any endogenous knowledge against Buruli ulcer in Ntandu people?
• If so, is its effectiveness based on any foundation?
The plausible hypotheses can be summarized as follows: The Ntandu people, has for many years, faced
Buruli ulcer, otherwise known as "mbasu", by developing endogenous knowledge. For its anchoring in the local
culture, herbal medicine must has proven its effectiveness.
The study aims to promote Congolese biodiversity and promote the green economy through the census and
popularization of plant species likely to treat Buruli ulcer. It consists in verifying the scientific value of
information collected from traditional healers, herbalists and other former Buruli ulcer patients.
MATERIAL AND METHODS

Material (plant species) selection
Inquiry questionnaires were sent to traditional healers, herbalists, and former Buruli ulcer patients to collect
data from the field. Traditional healers cited five species: Aloe tenuifolia Lam., Annona senegalensis subsp.
oulotricha Le Thomas, Brillantaisia owariensis P. Beauv, Vernonia amygdalina Delile and Strychnos icaja
Baill. Three species were collected in Kasangulu from the Sisters of St. Mary of Kisantu in Kasangulu and the
other two in Kimpese in the territory of Songololo.
Method of preparation
The aerial parts (leaves and stems) and the underground part (root) were dried at 40°C in an oven, after
drying, the samples were milled at the Thomas brand mill and sieved in order to collect the powder.
Preparation of the aqueous and organic phases
Five grams of the powder of each species were introduced into an Erlenmeyer flask containing 200 mL of
distilled water and then refluxed for 15 minutes. The mixture was cooled for 24 minutes and placed in a
separating funnel in which five mL of dichloromethane (CH 2Cl2) were added thereto. After manual stirring for 3
to 5 minutes, the gas escapes from the separating drum until exhaustion.
Phytochemical Screening
The roots decoctions were separately prepared for each species. These analyzes were used to identify
secondary metabolites: alkaloids, saponosides, tannins, anthocyanins, quinones, polyphenols, reducing sugars,
terpenoid, steroids and coumarins. The different chemical groups are characterized according to the techniques
described in Wagner (1983), Békro et al. (2007) and N'Guessan et al. (2009).
RESULTS
Ethnobotanical data
Ethnobotanical data on families, plant species and parts or organs used in Buruli ulcer herbal medicine are
presented in table 1.
Table 1. Plant species used against buruli ulcer by Ntandu people.
Family Species Part of the plants used
Asparagaceae Aloe tenuifolia Lam Leaves
Annonaceae Annona senegalensis subsp. oulotricha Le Thomas Leaves
Acanthaceae Brillantaisia patula P. Beauv. Leaves
Asteraceae Vernonia amygdalina Delile Leaves
Loganiaceae Strychnos icaja Baill Stem
The analysis of this table reveals that 5 plant species belonging to 5 families are used in the phytotherapy of
Buruli ulcer among the Ntandu of the Lukaya District in Kongo Central. It appears that the leaves predominate
in the recipes compared to the stems.
Phytochemical Screening
The phytochemical screening findings of the studied plants are given below (Table 2).
Table 2. Different major groups of compounds found after screening.

Extracts Plant species
Secondary Aloe tenuifolia Brillantaisia Annona senegalensis Vernonia Strychnos
metabolites owariensis subsp. oulotricha amygdalina icaja
Leaves Roots Leaves Roots Leaves Roots Leaves Roots Stem
Alkaloids + + + + - - + + +
Saponosides - - - - + - + + +
Tannins + + + - + + + + +
Aqueous
Anthocyanins + + + + - + + + -
Free quinones + + - - - + - - -
Polyphenol + + + + + + + + +
Reducing sugars + + + + + + + + +
Quinone related + + - - + + + - +
Organic
Terpenoids + + - - - + + - +
Steroid + + + + + + + + -
Coumarins - + + + + + + + +
Note: +, Presence of the active ingredient; -, Absence of the active ingredient.
In view of the results obtained after the phytochemical screening, it emerges that:
 Alkaloids were found in the aqueous extracts of the plant species studied with the exception of Annona
senegalensis subsp. oulotricha Le Thomas.
 Saponosides are absent in aqueous extracts of Aloe tenuifolia Lam and Brillantaisia owariensis P. Beauv.
 The tannins are present in the aqueous extracts of the plant species studied with the exception of the roots of
Brillantaisia owariensis P.Beauv.
 Anthocyanins were detected in the aqueous extracts of the plant species studied with the exception of leaves
of Annona senegalensis subsp. oulotricha Le Thomas and stalks of Strychnos icaja Baill.
 Free quinones are found in the aqueous extracts of some species while those bound are identified in organic
extracts;
 Polyphenols and reducing sugars are ubiquitous in the aqueous extracts of the species studied.
 Steroids and coumarins are identified in organic extracts of different species. They are respectively absent in
the Strychnos icaja Baill stem and Aloe tenuifolia Lam leaves.
DISCUSSION
In 2004, the WHO issued a recommendation to use antibiotic therapy in the treatment of Buruli ulcer, while
surgical interventions were the basis of treatment. An 8-week antibiotic therapy reduces the size of the lesions
and reduces the extent of surgery, if necessary (Lagarrigue et al. 2000, Kanga et al. 2003, WHO 2004).
In practice, the medical prescription includes rifampicin (10 mg kg -1) once a day orally and streptomycin (15
mg kg-1) once a day by intramuscular injection for four weeks. The evaluation takes place after four weeks. In
case of regression or stabilization, treatment with antibiotic continues for eight weeks. In case of extension, the
surgery is associated with the anatomo-pathological examination. Antibiotic therapy is not very effective on
bone lesions (Darie 2003, Aubry 2017).
Herbal medicine could be an alternative to this treatment. Indeed, Bi et al. (2016) demonstrated the anti-
ulcer effects of herbaceous ingredients on animals while Choi et al. (2015) shows the use of Brillantaisia
madagascariensis T. Anderson ex Lindau in Tanzania for its anti-proliferation effects.
Some listed species are already tested by various authors. Ibrahim et al. (2007) provided information on the
toxic and anti-inflammatory activities of Vernonia amygdalina while Ezeonu et al. (2016) reported the
immunomodulatory properties of the same species.
Alembert et al. (2014) isolated from the roots of Strychnos icaja an asymmetric alkaloid called
"Strycnobaillonine". Further, Philippe et al. (2003) isolated from the roots five alkaloids: three monomers,
protostrychnine and genostrychnine, pseudostrychnine already found in the leaves of the plant, strychnogucin C,
a new bisindolic alkaloid and strychnohexamine, the first indolomonoterpenic trimeric alkaloid of natural origin.
The latter showed antiplasmodial activity against the Plasmodium falciparum FCA strain close to 1 μm. The
use of Strychnos icaja in the treatment of Buruli ulcer may be justified by the presence of alkaloids which have
proved themselves in other conditions.
Idrissa et al. 2019
CONCLUSION
The aim of the study was to promote Congolese biodiversity and promote the green economy through the
identification and dissemination of plant species likely to treat Buruli ulcer. This valorization passes
explanations based on recognized and proven scientific evidence.
According to ethnobotanical surveys, the Buruli anti-ulcer floret of Ntandu endogenous knowledge is
composed of 5 species. These are: Aloe tenuifolia Lam.; Brillantaisia owariensis P. Beauv.; Annona
senegalensis subsp. oulotricha Le Thomas; Vernonia amygdalina Delile; Strychnos icaja Baill
The phytochemical analysis of the aqueous and organic leaves and root extracts revealed that these species
secrete secondary metabolites recognized as being effective against bacterial germs.
These results confirm the hypotheses that the Ntandu people, confronted with Buruli ulcer problems, in other
words "mbasu" for many years, have developed endogenous knowledge. The plants used secrete secondary
metabolites capable of acting actively against Mycobacterium ulcerans.
ACKNOWLEDGMENTS
The authors thank all those who agreed to participate volunteer in the survey and provided information on
the herbs used to treat Buruli ulcer. Special thanks to Lusadisu Zolana Alphonse, Kibunga Lutumba Albert,
Lema Lufulu and Masunda Nsanda Jean for their dedication for this survey.
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Research article
Selection of suitable digital elevation model for analysis of forest

cover in different agro-climatic zones of Jharkhand, India
Shambhu Nath Mishra, Dharmendra Kumar and Sharad Tiwari*
Institute of Forest Productivity, ICFRE, Lalgutwa, Ranchi- 834005, Jharkhand, India
*Corresponding Author: tiwaris@icfre.org [Accepted: 19 March 2019]
Abstract: Digital Elevation Model (DEM) has wide ranging application in the study and analysis
of various environmental and biodiversity conservation issues. Due to geographical variations, the
accuracy of DEMs generated from different satellite sources needs to be ascertained for choosing
the best suitable DEM for a particular study area. In the present study, the performance of DEM
datasets of Cartosat-1 and Shuttle Radar Topography Mission (SRTM) has been evaluated on the
basis of slope, aspect, altitude and hill-shade map generated through these DEMs for different
agro-climatic zones of Jharkhand. Elevation values deduced through Cartosat-1 and SRTM
datasets were compared with actual Ground Control Points (GCP) recorded using Global
Positioning System (GPS) for testing their accuracy. The forest cover map was created by Landsat
7 ETM+ data and subsequently superimposed on altitude map, generated using SRTM and
Cartosat-1. Further, it was visually compared with the Survey of India topo-sheet (1:50000) for
analyzing undulating topography and forest cover of Jharkhand. The comparative study based on
different parameters for DEM dataset from Cartosat-1 and SRTM, reveals that SRTM data
performed better than Cartosat-1 for the study of forest cover in different agro-climatic zones of
Jharkhand.
Keywords: DEM - Cartosat-1 - SRTM - LULC - Forest - GCP.
[Cite as: Mishra SN, Kumar D & Tiwari S (2019) Selection of suitable digital elevation model for analysis of
forest cover in different agro-climatic zones of Jharkhand, India. Tropical Plant Research 6(1): 54–62]
INTRODUCTION
A DEM offers the most common method for extracting topographic information. It is highly used for ortho-
rectification of satellite or aerial images. It is also used for creation of slope, aspect, hill shade, stream, altitude
and view shade maps. Further, these maps can be used for morphologic interpretation, drought monitoring, soil-
erosion, flood analysis, bioclimatic analysis, landslide studies, water shade lineament, groundwater depth and
rock strata thickness and geomorphic analysis. DEM data are widely used in various geological and
geomorphologic studies, substructure planning, visual analysis of topography, landforms, as well as modeling of
surface processes. Over the years, the availability of high-resolution satellite imagery has unfolded new vistas
for studying many geographical features and the use of Digital Elevation Model (DEM) is providing excellent
data component support in the study of forestry and ecological issues. It was quite a tedious task to choose the
better open source DEM dataset for the assessment of probability distribution mapping of forest species in the
state of Jharkhand through Spatial Distribution Modeling approach.
Over the years, many researchers have made effort to evaluate the performance of Cartosat-1, SRTM and
ASTER based products, in various application fields. Sugandh & Srinivasan (2010), used DEM datasets derived
from Cartosat-1 and SRTM to study the spatial relationship between landslide-occurrence and the influencing
factors in the hills of Ooty, Nilgiri District, Tamilnadu. In the study of landslides, the results of Carrtosat-1 are
found better than SRTM (Sugandh & Srinivasan 2010). Forkuor & Maathuis (2012) compared ASTER and
SRTM data to study implications on hydrological and environmental modeling in two regions of Ghana and
found SRTM accuracy being closed to 1:50,000 topographical map series. Patel (2012) carried out comparative
study for evaluating the performance of digital elevation models derived through Cartosat-1, ASTER, SRTM
and SOI toposheet (1:50,000) and reported the accuracy achieved through Cartosat-1 was better as compared to
Received: 30 March 2018 Published online: 30 April 2019
other datasets and found that the altitude map derived from Cartosat-1 was similar to that of SOI toposheet.
Yarrakula et al. (2013) used Cartosat-1 and SRTM data for digital elevation modeling in Kharkai river area,
Jamshedpur of Jharkhand state. Gajalakshmi & Anantharama (2015) evaluated the performance of Cartosat-1
DEM and SRTM-DEM for elevation data and terrain elements in South Pennar, Karnataka. Al-Fugara (2015)
carried out study to assess the accuracy of SRTM and ASTER DEM models in calculation of elevation data of
“humrratessahan” watershed, having highly varied topography on the shore of Dead Sea and reported that
ASTER performed consistently lower values than SRTM for the earth’s lowest elevation regions. Patel et al.
(2016) performed study to evaluate the accuracy performance of Cartosat-1, ASTER and SRTM derived DEMs
for elevation calculation of Bhopal, MP at known elevation points measured using Differential Global
Positioning System (DGPS) found Cartosat-1 data product deducing better result than SRTM and ASTER in
their evaluation using known DGPS points. The spot height accuracy of SRTM-DEM of 1 arc second seems to
be good for all kinds of terrain modeling, Baral et al. (2016). SRTM DEM performs better than the ASTER and
Cartosat-1 DEMs in assessing the accuracy of soil erosion Mondal et al. (2016) used Cartosat-1, SRTM and
ASTER derived DEMs to assess the soil erosion uncertainty for Narmada river basin in Central MP. Bhadoriya
(2017) attempted to study the quality of Cartosat-1, SRTM and ASTER DEM, at sub-catchment level and
sensitivity of these datasets in hydrological modeling, reported that Cartosat-1 DEM appears to be fine for the
requirements of the water resource management for the grass root level mapping. Subbu Laxmi & Yarrakula
(2017) attempted the accuracy assessment of DEMs derived from Cartosat-1, SRTM, Google earth and Survey
of India toposheet for plain and hilly terrain sites around Madduleru river.
The aim of the present study was to evaluate suitability performance of DEM generated from two different
datasets one is from Indian Space Research Organization’s satellite Cartosat-1 and another is international
research effort satellite Shuttle Radar Topography Mission (SRTM) and to find out which one is more suitable
for analyzing forest cover in different agro-climatic zones of Jharkhand, and ultimately can provide more
suitable and accurate set of database for carrying out species distribution mapping studies in Jharkhand.
STUDY AREA
Figure 1. Study area.

The state of Jharkhand lies between latitude 22º 00′ N to 24º 37′ N and longitude 83º 15′ E to 87º 01′ E. The
state is located in eastern India (Fig. 1) having 24 districts and its capital is Ranchi. The state has an area of
79,714 km2 and is home to 32.97 million people (414 persons km-2) (Anonymous 2011). The state has recorded
forest of about 23,605 km2 which is 29.61 per cent of its total geographical area based on Forest Survey of India
Report (http://fsi.nic.in/forest-report-2015). Forest type mapping using satellite data shows that the state has five
forest types which belong to two forest type groups, viz. Tropical Moist Deciduous and Tropical Dry Deciduous
Forests (http://fsi.nic.in/forest-report-2015). The annual average rainfall in the state is 1400 mm; however, it
varies from 1200 mm to 1800 mm. The temperature varies from 5°C to 40°C. The state possesses lateritic soil
Mishra et al. 2019
spread all around. In the present work, two post processed datasets known for their global coverage i.e. Cartosat-
1-DEM and SRTM-DEM have been studied and compared for suitability between DEM to DEM and Ground
Control Points (GCPs) to DEM for following agro-climatic sub zones of Jharkhand (Singh 2014, Petare et al.
2016):
a) Central and North eastern plateau sub zone (Zone-IV- Dumka, Deoghar, Godda, Sahebganj, Pakur,
Hazaribagh, Koderma, Jamtara, Chatra, Giridih, Dhanbad, Bokaro and two-third of Ranchi),
b) Western plateau sub zone (Zone-V- Palamu, Latehar, Lohardaga, Garhwa, Gumla, Simdega and one-
third of Ranchi) and
c) South eastern Plateau sub zone (Zone-VI- East Singhbhum, West Singhbhum and Saraikela
Kharsawan).
DATA USED
Cartosat-1 DEM
The Indian Space Research Organization (ISRO) has launched Cartosat-1 also named as IRS P5 DEM in
2005, dedicated to stereo viewing for large-scale mapping and terrain modeling applications. Cartosat-1 has
panchromatic camera with spatial resolution of 2.5 m and swath about 27 km. Cartosat-1 has a GPS receiver for
the position and reference for the elevation determination. The features in this dataset incorporate very advanced
techniques are employed for mapping of urban areas and updating the topographic maps for various features
such as local roads, parks, small ponds, play grounds, swimming pools, crop field boundaries, hydrological
features, flow accumulation and stream network, for observing terrain conditions that is slope and aspect etc.
(NRSC 2011). In this study a Cartosat-1 stereo-pair of 2.5 m spatial resolution covering Jharkhand State in 15
tiles (Cartosat-1 Tiles no: - F44F, G44X, F45G, F45A, G45S, F45N, F45H, F45B, G45T, F45I, F45C, G45U,
F45D, G45V, G45P) have been acquired on 24.09.2014 from www.bhuvan.nrsc.gov.in website and tested for
DEM creation (Fig. 2). For DEM generation Leica Photogrammetric suite (LPS) version 2011 package was
used.
Figure 2. Maps showing Cartosat-1 and SRTM DEMs of the study area.
SRTM DEM: Shuttle Radar Topography Mission-Digital Elevation Model
SRTM is the first space-born single pass interferometric Synthetic Aperture Radar (SAR) which produces
high resolution, digital elevation maps (Anonymous 2017). For the present study, SRTM world DEM was
downloaded on 24.09.2014 from www.usgs.gov website and was clipped covering the Jharkhand State using
ArcGIS 10.0 software (Fig. 2).
Landsat 7 ETM
The forest cover maps were created using Landsat 7 ETM data (path-row: 139–43, 139–44, 139–45, 140–43,
140–44, 140–45, 141–43, 141–44, 141–45, 142–43) for the year October, 2010. Proper season for studying
Arid, semi-arid and dry deciduous natural forest and scrub vegetation in India is October to December. The tiles
were downloaded from the USGS website http://earthexplorer.usgs.gov on 24.09.2014.
Topographic Map of Survey of India

Survey of India topographic map of toposheet number G-44, G-45, F-44 and F-45 at 1:50,000 scales have
been used in the present study. A topographic map is a detailed and accurate graphic representation of cultural
and natural features on the ground. Topographic maps render three-dimensional ups and downs of the terrain on
a two-dimensional surface.
Field verification (Ground truthing)
Total 30 Ground Control Points (GCP) has been recorded, covering 10 GCP each from all the three agro-
climatic zones of Jharkhand. The administrative boundary of Jharkhand state was downloaded from the website
www.diva-gis.org.
METHODOLOGY
Study was carried out on two identical Digital Elevation Model (DEM) datasets one is Indian Remote
Sensing Satellite Cartosat-1 and another is NASA’s Global SRTM as per flow chart (Fig. 3). These satellite
imageries were geometrically corrected with reference to SOI topo sheets at 1:50,000 scale and acquired in the
standard projection system (UTM 45N, WGS84 datum). The two different DEM datasets were compared in
three agro-climatic zones of Jharkhand on the basis of slope, hill-shade, aspect and altitude map derived from
DEMs.
Agro-climatic Zone wise Remote Sensing Data of Jharkhand
Cartosat-1 SRTM
Aspect Slope Hillshade Altitude Aspect Slope Hillshade Altitude
Forest cover map was created by Landsat 7 ETM+ data

and subsequently superimposed on altitude map
Visual Interpretation
Statistical /Visual comparison Field verification

comparison
Result showing best data for analyzing forest cover in

different Altitude and Agro-climatic Zones of Jharkhand
Figure 3. Methodology flow chart.
Mishra et al. 2019
Image processing and GIS analysis was performed using ArcGIS 10.0 on the SRTM and Cartosat-1 DEM
dataset. The slope, aspect, hill-shade and altitude maps were prepared using surface tool under Spatial Analyst
in ArcGIS software. The slope map was prepared in ArcGIS software, describes the slope for each raster cell in
degrees based on the elevation at each point. The surface analysis aspect tool was used to generate aspect map
and hill-shade map from SRTM and Cartosat-1 data. The aspect map displays each raster cell grouped into
compass directions (north, north-west, etc.); however, the hill-shade map shows shade effect based on the input
parameters that are entered in the tool. The resulting map is easier to visually interpret than the original DEM
because some topographic features are better visible on small scale (Sitabi 2015).
RESULT AND DISCUSSION

Present study provides an insight into various aspects of data products generated using Cartosat-1 and SRTM
datasets with reference to slope (rate of maximum change in z-value from each cell), hill shade (grayscale 3D
representation of the surface), aspect (identifies the down slope direction of the maximum rate of change in
value from each cell to its neighbors) and elevation (the relief features of the earth's surface). Following
observations are made from the study:
Visual comparison
In the present study, same protocol of remote sensing technique has been applied for both Cartosat-1 DEM
and SRTM-DEM. On minutely observing the Altitude, Aspect, Slope and Hill-shade maps (Fig. 4), many visual
variations are observed.
On aspect wise parameters (Flat, North, Northeast, East, Southeast, South, Southwest, West, Northwest)
both SRTM and Cartosat-1 DEM show similar value (Fig. 4C, D). The minute observation of Slope map shows
prominent difference in values for Flat (Green colour representation), Shallow (Yellow colour representation),
Moderate (Brown colour representation) and Steep (Red colour representation), where the value varies from 0-
15.27 for Cartosat-1 and 0-21.42 for SRTM DEM (Fig. 4E, F). The hill-shade map (Fig. 4G, H) of the study
area also shows prominent variation in values, where it is in the range of (135–214) for Cartosat-1 and (118–
222) for SRTM.
The deviation in case of SRTM-DEM for highly rugged terrains of Central and North eastern plateau
climatic sub zone of Jharkhand is significant. However, for rest two climatic sub zones of Jharkhand, the
deviation was identical.
Statistical comparison
Table 1. Altitude wise total area and forest cover of Jharkhand.
Range Total area in km2 Total area in Forest Cover in Forest Cover in Standard
(Cartosat-1) km2 (SRTM) km2 (Cartosat-1) km2 (SRTM) deviation
0–250 35953.8 26875.4 6356.2 3944.3 1705.5
251–500 30071.8 34638.7 13041.9 13620.2 408.9
501–750 12508.7 16385.7 3369.9 4973.3 1133.8
751–1500 1406.6 2029.2 1144.1 1354.7 148.9
TOTAL: 79940.8 79928.9 23912.1 23892.5 13.9
Table 2. Agro climatic zone wise distribution of forest cover in Jharkhand in km2.
Agro climatic zone Total Forest Cartosat-1- DEM SRTM –DEM Difference in Standard
Cover Elevation (m) Elevation (m) Elevation (m) Deviation
(km2) Max Min Max Min Max Min Max Min
Zone-IV- Central and North
eastern plateau sub zone 9112.8 879 -41 1355 20 -476 -61 336.6 43.1
Zone-V- Western plateau
sub zone 7719.1 1095 62 1152 118 -57 -56 40.3 39.6
Zone-VI- South eastern
7086.9 858 -11 898 44 -40 -55 28.3 38.9
Plateau sub zone
Figs. 5 & 6 shows altitude (Table 1) and Agro-climatic zone wise distribution of forest cover (Table 2) in
different ranges from 0–250 m, 251–500 m, 501–750 m and 751–1500 m. Results show that, there is a
prominent standard deviation in each range, however, SRTM showing similar value in each ranges of data
obtained from Bhuvan Portal. Altitude wise analysis for forest area shows maximum variation for the range 0–
250 m and minimum for the range 751–1500 m, and the standard deviation for these range stands at 1705.46 and
148.93 respectively. However, total area calculated from these DEMs are almost same and the standard
deviation for total forest area is found to be 13.9 (Table 1; Fig 5).
Figure 4. Altitude, Aspect, Slope and Hill-shade maps of Jharkhand, India.

The agro-climatic zone wise distribution of forest cover for the Cartosat-1 and SRTM-DEM shows that for
Zone-IV- Central and North Eastern plateau sub zone, the maximum and minimum variation in standard
Mishra et al. 2019
deviation is 336.58 and 43.13; for Zone-V- Western plateau sub zone the maximum and minimum variation in
standard deviation is 40.30 and 39.59 and for Zone-VI- South Eastern plateau sub zone, the maximum and
minimum variation in standard deviation is 28.28 and 38.89 (Table 2; Fig. 6).
Figure 5. Forest Cover Maps of Jharkhand using Cartosat-1 and SRTM DEM.
Figure 6. Agro-climatic Zone Map of Jharkhand & Agro-climatic Zone wise Forest Cover Map of Jharkhand.
Control Points
Total 30 numbers of GPS points were recorded in all the three agro-climatic zones of Jharkhand, comprising
of 10 control points in each agro-climatic zone, and elevation data were recorded on these points, to validate the
elevation accuracy achieved using SRTM and Cartosat-1 dataset. A comparison of elevation variation in SRTM
and Cartosat-1 dataset with GPS points collected in all three agro-climatic zones has been shown in figure 7.
The comparative charts show that, the elevation output achieved through SRTM data, is closer to elevation data
recorded at various GPS points, in each of the agro-climatic zones. However, in another similar study conducted
by Yarrakula et al. (2013) around Kharkai river area, Jamshedpur, where Cartosat-1 and SRTM dataset were
used for digital elevation modeling, it was reported that the accuracy of Cartosat-1 DEM will be higher if proper
ground control points are collected for undulating region. This variation in findings may be due to the variation
in size of the study area. In the present study, we have applied the technology to a very large area i.e. for the
entire Jharkhand state, whereas the Kharkai river area, Jamshedpur is a very small part of Jharkhand.
CONCLUSION
In the present study, various output products were derived for different agro-climatic conditions of
Jharkhand using SRTM and Cartosat-1 datasets. These outputs were further verified with, ground truth data sets
and comparative study reveals that as compared to Cartosat-1, the results obtained through SRTM datasets are
closer to ground truth dataset. Thus, in general, the accuracy achieved from SRTM DEM datasets would provide
better results for the studies of forest cover mapping or species distribution modeling in different agro-climatic
conditions of Jharkhand which is in the line of Das et al. (2016); although the SRTM datasets provide
substantial results, the Cartosat-1 dataset does not provide reliable information, Baral et al. (2016); the spot
height accuracy of SRTM DEM seems to be good for all kind of terrain in comparison to Cartosat-1 DEM in the
study of three different terrain of India and Forkuor & Maathuis (2012); in the study of over two regions in
Ghana for Hydrological and Environmental Modeling has revealed that SRTM is “closer” to the reference DEM
than ASTER. For elevation parameter, study indicates that, SRTM gives better accuracy than Cartosat-1. Thus,
the observation of present study suggests that, for analyzing forest cover in different agro-climatic zones of
Jharkhand, the use of SRTM datasets could give better output performance and accuracy as compared to
Cartosat-1.
Figure 7. Elevation variation in agro-climatic zones: A, Zone IV- Central and North Eastern Plateau Sub Zone; B, Zone V-
Western Plateau Sub Zone; C, Zone VI- South Eastern Plateau Sub Zone.
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Research article
Comparative study of the floristic and structural diversity of

three lowland forests of the former Province Orientale,
Democratic Republic of the Congo
Jean-Léon K. Kambale1, Bernard T. Malombo1, Eric W. Katembo2, Reddy E. Shutsha1,
Judith M. Tsongo1, Elie P. Bugentho1, Patrick K. Mutombo1, Roger A. Angoyo1,
Hyppolyte S. Nshimba2, Gédéon N. Bongo1* and Koto-te-Nyiwa Ngbolua3
1
Centre de Surveillance de la Biodiversité, Université de Kisangani, B.P. 2012 Kisangani, RD Congo
2
Faculty of Sciences, University of Kisangani, P.O. Box. 2012 Kisangani, Democratic Republic of the Congo
3
Department of Biology, Faculty of Sciences, University of Kinshasa, P.O. Box. 190, Kinshasa XI, Democratic
Republic of the Congo
*Corresponding Author: gedeonbongo@gmail.com [Accepted: 23 March 2019]
Abstract: The present study was carried out in the aim of comparing the floristic diversity, the
areal richness and the structure of three lowland forest types of the former Province Orientale
notably Rubi Télé in Buta territory, UMA in Ubundu territory and Wela in Aketi territory. This
comparative study is a contribution to the improvement of knowledge on the influence of soil type
and the remoteness of forest types on the floristic composition and distribution of species in the
lowland forests of the former Province Orientale. To achieve this, an inventory of all trees with
dbh ≥ 10 cm (all species combined) in 36 plots of 0.25 ha was performed. At the end of this
floristic and structural study conducted in the forests, we counted a total of 1120 individuals
distributed to 191 species in 40 families in these three different sites. The average basal area for
these three sites is 25.40 m2 ha-1. In terms of group diversity, the ANOVA test showed no
significant differences, because F = 1.844, p value is greater than 0.05, either 0.213. In terms of
diametric structure, the chi-square was calculated Chi-square = 35.5599, df = 18, p value =
0.00803. The density for extrapolation (per hectare) with ANOVA the value for F = 7.91, p value
= 0.0104 this shows that there was a significant difference.
Keywords: Diversity - Lowland forest - Province Orientale - DRC.
[Cite as: Kambale J-JK, Malombo BT, Katembo EW, Shutsha RE, Tsongo JM, Bugentho EP, Mutombo PK,
Angoyo RA, Nshimba HS, Bongo GN & Ngbolua K-t-N (2019) Comparative study of the floristic and structural
diversity of three lowland forests of the former Province Orientale, Democratic Republic of the Congo. Tropical
Plant Research 6(1): 63–73]
INTRODUCTION
In the current situation in which human society seeks to develop from environmental resources, the major
challenge is to know the place as well as the importance of terrestrial biodiversity in this environment. The
environment is threatened following the search for the livelihoods of living beings, particularly man. Threats to
biodiversity, including deforestation, environmental degradation and crop unification, have re-actualized the
issue of the origin and maintenance of biodiversity (Carpentier 1999).
In recent decades, deforestation and degradation of tropical rainforest have attracted considerable
international attention. Therefore, the Rio Conference in 1992 called for the sustainable management of these
forests as the most effective means of combating their accelerated degradation (CBD 2012). These tropical
forests host many species and are among the richest ecosystems on our planet (Omatoko et al. 2015, Asimonyio
et al. 2015a,b). The multiple interactions of species with each other and with their environment make these
forests extremely complex ecosystems (Kambale et al. 2016a). At the phytoecological level, the population of a
region can be considered from two different points of view; either its floristic groupings (dynamism) or their
Kambale et al. 2019
structure (physiognomy) (Kambale et al. 2016b, Bajpai et al. 2017). Plant formations are sensitive to local
environmental conditions such as soil, topography, sunshine, humidity and it is generally observed that the
composition and structure of an ecosystem vary from a location to another depending on the local values of
these factors (Kambale et al. 2016c). Despite this complexity, the ecological richness of tropical rainforests
stimulates several topics of research in order to better understand its origins and the mechanisms of its
maintenance in order to have a better conservation (Blanc et al. 2003). The complexity of the forest structure
observed at the scale of a station (floristic and three-dimensional structure) comes from a large number of plant
species, their varied architecture and of the coexistence of different phases of development at a specific moment
(Trichon 1997). This complexity is also explained by the lack of a non-mastery of all the keys of identification
of the species on the field and this identification remains a puzzle in botany. The immense richness of tropical
rainforests equals the complexity of their ecological mechanisms.
Tropical forests are exceptional reservoirs of biodiversity, as many researchers in the world have
experienced. For several decades, they have been at the heart of international issues on climate change
(Marquant et al. 2015). The protection of biodiversity has become a major issue in forest policy but before
getting there, it is imperative to know the composition of these ecosystems which deserves particular emphasis
today for their valorization (Timilsina et al. 2007, Sambare et al. 2011, Bajpai et al. 2015, Masens et al. 2017).
A very crucial step in the negotiation of carbon credit for the countries which still conserve their forests as a
carbon sink.
The Democratic Republic of Congo (DRC) is a region that hosts enormous biodiversity and its forests are
classified among the richest ecosystems on the planet. However, its forest is among the least studied ecosystems
in Africa. With climate change, there is a risk of seeing the disappearance of several species that are bad or not
yet known. Also, DRC has a great wealth of watercourses by its river that runs from South to North and from
East to West, and its affluents including Itimbiri, Tshopo, Lindi, Aruwimi, Kwango and Kwilu rivers. In a
botanical point of view, there are few studies conducted along these rivers in the country (CSB 2014). From this,
it is necessary to provide other researchers with information on the structure and floristic diversity of three forest
types, including one along the large river in the province of Bas Uélé called ITIMBIRI. In addition, research in
the Province Orientale in general and that of the Tshopo, in particular, are numerous, nevertheless, it would also
be better to compare them with the forests of other parts of the Province Orientale still little known. Therefore,
we tried to compare three forest types including UMA in the territory of Ubundu in Tshopo province, Rubi-Télé
in the territory of Buta and Wela in Aketi territory, both in Bas-Uele province. It is in this context that a
multidisciplinary team undertook a survey in Aketi, UMA and Wela in order to contribute to the better
knowledge of the biodiversity of the former Province Orientale so that its biological resources might be known
at the scientific level and this would help the policy-makers in decision-making. In order to spread its biological
(forest) resources on a scientific level and this will help the decision-makers in decision-making. Due to the
remoteness, soil types and ecological barriers including rivers between the three sites which are totally different
in all aspects of the florule constitute the main topic of this study.
MATERIAL AND METHOD

Study area
This research was carried out in three sites of the former Province Orientale. The first site is located in Aketi
territory in the village of Wela, Bas Uele province which has the following geographical coordinates: latitude
024° 01′35.1″ & longitude 03° 19′27.6″; Rubi-Télé 02° 19′072″ & 024° 58′36.8″ and so UMA 00° 33′15.2″ &
025° 55′46.2″. They are under the Equatorial climate of continental type (hot and humid).
Material
The biological material of this study was trees which were used for different measures.
Data collection
This study was conducted in the former Province Orientale and plots of 50 m × 50 m at each site was
installed. All species having circumferences with a diameter at the height of 1.30 m (dbh) ≥ 10 cm were
inventoried. In addition, the botanical flora and works of Tropical Africa along with updated taxonomic catalogs
were used for species identification. Some control samples were kept in the collection room of the Biodiversity
Surveillance Centre of the University of Kisangani.
Data treatment
The treatment of data which was focused on the floristic similarity between the three sites was assessed
using Morisita-Horn's similarity indices. This index corresponds to the ratio of the probability that two
individuals randomly selected from two samples belong to the same species. The BiodivR program
automatically calculates the values of this index according to k. It varies from 0 to 1, two plant communities are
different if MH tends towards 0 (either all values < 0.5) and identical if MH tends towards 1 (or all values ≥ 0.5;
value close to 1).
Figure 1. Map of the study area.

Areal richness
This analysis indicates the number of species collected per area unit and it is one of the most common
measures of biodiversity.
Indices of taxa importance values (IVI)
It is the sum of the relative density, relative dominance for a taxon (Nshimba 2008).
IVI = AD + RDom + RF
Where: IVI is the index of importance values of taxa; AD: Absolute Density of taxa; RDom: Relative
Dominance of Taxa, RF: Relative frequency.
Relative frequency
According to Curtis & McIntosh (1950), the frequency of a species is equal to the number of occurrences of
this species in the inventory area. The relative frequency of a species is equal to the quotient of frequency by the
sum of frequencies of all species and multiplied by 100 (Nshimba 2008).
Frequency of a species
Relative Frequency of a species = ∑ × 100 (1)
Frequency of all species
The area-species curve

It expresses the increase in the species number of species (Y axis) according to the increasing surface (X
axis). The area-species curve helps to determine the minimum area to be inventoried.
Diversity indices
Diversity indices used in this study were the following:
Shannon diversity Index (H’): indicates the species richness in a local ecosystem (Marcon 2015).
𝑁𝑖 𝑁𝑖
𝐻′ = ∑ ) ∗ 𝑙𝑜𝑔2 ) (2)
𝑁 𝑁
Where, Ni: the total number of individuals of a given species (i ranging from 1 to ∑),
∑: the total number of species and
N: the total number of individuals.
It has to be noted that when H '= 0 i.e. all individuals in a population belong to the same species.
Kambale et al. 2019
Piélou's equitability (EQ):

It is the ratio of the diversity of a population or sample and the number N of species present. It expresses the
regularity, the equitable distribution of individuals within the plots.
𝐼𝑆𝐻
𝐸𝑄 = (3)
𝑙𝑜𝑔2 (𝑁)
Simpson diversity index

Simpson diversity index (D) allowed from the abundance data to analyze local biological diversity of
different vegetal communities studied.
This index measures the probability that two randomly selected individuals belong to the same species:
𝑆 = ∑ 𝑓𝑖 𝑒𝑡 𝑓𝑖 = 𝑁𝑛𝑖 (4)
Where, Ni: the number of individuals of a given species,
N: the total number of individuals.
This index will have a value of 0 to indicate the maximum diversity, and a value of 1 indicates the minimum
diversity. In order to obtain "more intuitive" values, the Simpson diversity index represented by 1-D may be
preferred, the maximum diversity maximum being represented by the value 1, and the minimum diversity by the
value 0. It should be noted that this diversity index gives more weight to abundant species than to rare species.
Adding rare species to a sample hardly changes the value of the diversity index.
Data analysis
Following floristic parameters analyzed were: abundance of species and family, indices of diversity and rare-
species curve. For the structural parameters, the terrial area variability and diametric structure were used.
ANOVA test was used for the data that we had. These parameters were calculated using PAST version 2.17 and
R version 3.4.0 softwares respectively.

Floristic composition
A total of 1120 individuals distributed in 191 species and 40 families in these different sites was inventoried.
The average basal area of these sites was of 25.40 m2 ha-1. In Wela, 346 individuals gathered into 72 species and
distributed in 30 families were inventoried while in Rubi-Télé, a total of 451 individuals grouped into 79 species
and 29 families were listed and in UMA a total of 323 individuals was grouped into 59 species distributed into
24 families.
Significance value indices of species (IVI)
The significance index value of species of different sites are presented in the figures below.
1. Significance value index of Wela specie
For Wela species, the significance value index is presented in figure 2 below.
Figure 2. Significance value index of Wela species.

From the figure above, it can be observed that following species characterize Wela flora namely:
Gilbertiodendron dewevrei (De Wild.) J.Léonard, Pseudospondias microcarpa (A. Rich.) Engl., Diospyros
boala L., Pancovia harmisiana Gilg., Strombosia grandiflora Hook. f. ex Benth, Desplatsia dewevrei (De Wild.
& Th. Dur.) Burret, Treculia africana Decne, Nauclea diderrichii Merr., Panda oleosa P. and Ceiba pentandra
(L.) Gaertn.
2. Significance value index of Rubi-Télé species
The significance value index of Rubi-Télé species is presented in figure 3.
Figure 3. Significance value index of Rubi-Télé.

From the figure above, it can be observed that following species characterizing the Rubi-Télé flora are
notably: Julbernardia seretii (De Wild.) T., Anthonotha macrophylla P. Beauv., Diospyros crassiflora Hiern.,
Diospyros boala De Wild., Greenwayodendron suaveolens Engl., Alstonia boonei De Wild., Celtis milbraedii
Engl., Strombosiopsis tetrandra Engl., Drypetes likwa (Wight & Arn.) Pax & K.Hoffm. and Gilbertiodendron
dewevrei (De Wild.) J.Léonard.
3. Significance value index of UMA species
The significance value index of UMA species is presented in figure 4.
Figure 4. Significance value index of UMA.

From the figure above, it can be observed that following species characterize UMA flora are namely:
Gilbertiodendron dewevrei (De Wild.) J.Léonard, Anonidium mannii (Oliv.) Engl. & Diels, Cola griseifolia De
Wild., Diogoa zenkeri Engl., Staudtia kamerounensis Warb., Polyalthia suaveolens Engl. & Diels, Diospyros
boala (H. Perrier) G.E. Schatz & Lowry, Phyllocosmus africanus Hook.f, Celtis tessmanii Rendle and
Diospyros sp.
Significance value indices of species for the three sites
1. Index of Morisita
The index of Morisita of different communities of the study sites is presented in figure 5.
Kambale et al. 2019
2. Area-species curve
The area-species curve expresses the increase in the number of species according to the increasing
surface. It is often established to show the increase in the number of species with increasing surface area
sampled. The figure 6 shows the area-species curve for selected three forests.
Figure 5. Index of Morisita. [Rbt- Rubi Télé]
Figure 6. Area-species curve. [Rbt- Rubi Télé]

From the figure, it is clearly shown that the three sites are diversified though Rubi Télé is more diversified
than the two other. Considering the trend of the curve, it is shown that the number of species increase with the
sampled area for each site i.e. there is a probability to have new species for all the sites if an inventory was
performed in a large area even when the number of plots is enlarged.
Similarity between communities
The similarity between communities is given in the table 1.
Table 1. Similarity between communities.
Rubi-Tele UMA Wela
Rsubi-Tele 79
UMA 0.154336 59
Wela 0.005349 0.000457 72
From this table, it is shown that Rubi-Télé is similar to UMA and Wela. This is due to the to environmental
conditions, soil as well as the topography of UMA
Diametric structure
The density of the three sites is presented in the figure 7. The plots of Rubi Télé have a high density versus
to UMA. It is important to note that environmental factors play a predominant role in this aspect. The two sites
Rubi-Télé and UMA were located on land whereas Wela was located on the hydromorphic ground along the
river called Itimbiri which stands out in diversity compared to other sites.
Figure 7. The density of the three sites.

Distribution of species in diametric classes
The distribution of species following the diametric classes is presented in the figure 8.
Figure 8. Distribution of species following the diametric classes. [Rbt- Rubi Télé]
From the figure, it can be observed that among the 10 diametric classes, the one ranging between 10 and 19
cm has a high number of individuals while other sites have fewer individuals. There was no statistical
significance concerning the diversity of communities because the F value is higher than the p value. Regarding
the density of extrapolation (per ha), there was a significance difference because the p value was less than 0.5.
Factorial Correspondence Analysis
1. The classification
The factorial correspondence Analysis for different species is given in the figure 9. It emerges from this
figure that the three communities are not different and have common species for each group, it has to be
observed that the grouping of Wela has no common species with a long distance between the two other
groups, But also the group of Wela was along the Itimbiri river while UMA was on a firm ground where the
water does not influence the distribution of species. The identified plant communities share many species in
common. From a deterministic point of view, this situation may be associated with low environmental
heterogeneity. This hypothesis is however not validated in most tropical forests (Kouob 2009).
Kambale et al. 2019
The dendrogram displaying the hierarchical classification of the three vegetal communities is shown in
figure 10.
Figure 9. Factorial Correspondence Analysis.
Figure 10. Dendrogram of three vegetal groups.

The classification of the surveys on the basis of the similarity index makes it possible to observe from 20%
of similarity, three groups have been split. This classification reveals Wela's restriction to Rubi Télé and UMA.
Initially the idea was to observe Rubi Télé and Wela grouped together because they are two sites located in the
same axis but the above figure shows the contrary for Rubi Télé becomes a neighbor of UMA, it is up to us to
report that these groups had a trend of a mono dominance, it is not really real mixed forests unlike Wela which
shows its heterogeneity very advanced and away from these two groups.
DISCUSSION
Plant diversity and ecosystem functioning
In Tropical rainforests, the great diversity of plants generally requires that inventories can be limited
according to the problem and the type of vegetation. In this study, the analyses focused on the floristic and
structural diversity of three groups. To achieve this goal, data were collected in plots of 50 m × 50 m. Therefore,
the results of this study may be different from those obtained in other surveys of different sizes and shapes. The
number of individuals identified for the specificity or attributed to a morphospecies depends on the experience
of the botanist who conducted the inventory, the time spent on taxonomic analyzes and the collection of
herbarium samples (Parmentier et al. 2007).
The richness of temperate forests ranges between 15 and 25 species, that of dry tropical forests between 50
and 60, it reaches about 150 species in tropical rainforests and 250 species in equatorial forests where the dry
season is non-existent (Gentry 1992) and the current results are in the same range because 191 species were
identified in the different plots used in this study. The diversity of the elements of a community is a key concept
which is vital for the analysis.
The concept of diversity has two aspects namely: the number of element categories, the number of distinct
taxa, then we talk about specific diversity for species and generic diversity for genera, and on the other hand, the
regularity, which is the way in which individuals are divided among different taxa categories (Marion et al.
2003). Of the total of 90 families surveyed, 80 families were recognized in the dry land forest, 67 in the
periodically flooded forest and 64 other families in the swamp forest. The Euphorbiaceae, Rubiaceae, and
Caesalpiniaceae families had higher specific and generic diversity compared to others. Sonke (1998) reported
that the compiled floristic data from three surveys showed the dominance of Rubiaceae (37%) and Fabaceae
(16.7%), followed by Apocynaceae and Euphorbiaceae with 11.1% each. As for the weighted spectrum of the
flora, a preponderance of Ochnaceae (28.9%) and Rubiaceae (26.3%) was noted. They were followed by
Bignoniaceae (10.5%), Euphorbiaceae (6.9%) and Achariaceae (6%). For the current work, two plots were
settled consecutively on the mainland and the other one was along the Itimbiri river. The finding of the current
study is different from this author quoted above.
Kouob (2009) reported that the classification of the surveys on the basis of the Sorensen similarity index
makes it possible to observe from 40% of similarity, 2 groups of 34 and 32 surveys, as well as 4 marginal
surveys. From 45% of similarity, the 34 western records of the RBD (FO) form 3 subgroups, while the 32
eastern records of the RBD (FE) form 2 subgroups. This classification revealed the restriction of
Distemonanthus benthamianus Baill., Dacryodes buettneri (Engl.) H.J.Lam and Dichostemma glaucescens
Pierre in the western groups (FO) of the RBD, and Scorodophleus zenkeri Harms, Afrostyrax lepidophyllus
Mildbr., Pausinystalia macroceras (K.Schum.) Pierre in the eastern groups of the RBD (FE).
Kambale (2016b,c), Nshimba (2008) and Sonke (1998) reported that its Sorrensen similarity coefficients
ranged between 0 and 1 in his research on three different sites located at the Mbiye Island. The UPGMA method
(Unweighted pair group median average) gives equal weight to each point in each forest. By presenting his
results in a dendrogram, the above authors found that the presence of certain species is limited in certain types
of forests. The floristic distances are small in these three sites, but these results show a good classification in the
ordination of heterogeneity in the contact zone. In the present work, the classification of the statements on the
basis of the index of similarity allows the observation of 20% of similarity of the three groups studied. This
classification reveals Wela's restriction to Rubi Télé and UMA.
CONCLUSION
The floristic characterization of three groups of which two on land and one on the hydromorphic ground
(along the Itimbiri river) shows a richness as well as a similarity between them. It is worth pointing out that
UMA is more similar to Rubi télé than Wela and it is hoped that it is due to environmental factors but also to the
edaphic factors.
It was found that the two plant formation has in common the most species. A significant part of this diversity
to Rubi-Télé reveals such a strong heterogeneity of species which demonstrates the presence of a good non-
enthropized mature forest than Wela. To this end, the solutions currently envisaged for the conservation of
tropical rainforests, in particular, the establishment of biological reserves, sustainable forestry, the restoration of
degraded forests or the creation of forest plantations, require a deepening of knowledge of the ecology and
dynamics of regeneration of tree species in ecosystems.
ACKNOWLEDGMENTS
We express our gratefulness to the Centre de Surveillance de la Biodiversité (CSB, Kisangani) for its support
to the realization of this study.
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Research article
Assessment of liana diversity and carbon stock in differently

disturbed tropical dry evergreen forests of southern India
K. Naveen Babu and N. Parthasarathy*
Department of Ecology and Environmental Sciences, School of Life Sciences, Pondicherry University,
Puducherry-605014, India
*Corresponding Author: parthapu@yahoo.com [Accepted: 07 April 2019]
Abstract: Lianas are important components of tropical forests that play a crucial role in forest
dynamics. We investigated biodiversity and carbon stock of lianas in two tropical dry evergreen
forest (TDEF) sites, the relatively undisturbed Sendhirakillai (SK) and disturbed Palvathunnan
(PT) by establishing one-hectare plot in each site. All lianas ≥ 1cm diameter (measured 1.3 m from
rooting point) and trees ≥ 10cm girth (measured at 1.3 m from ground) were enumerated to
ascertain species richness and stem density of lianas and trees with respect to site disturbance.
Liana diversity totaled 24 species (16 in SK and 20 in PT) in the two studied sites. Liana density
totaled 1182 individuals (744 ha-¹ in SK and 438 ha-¹ in PT). Fifty percent of species were shared
between the two sites. A considerable variation in the density, dominance and basal area of
different species was found between the sites. No dominant family was common to both the sites.
Stem twining and zoochory was predominant in both disturbed and undisturbed sites. Lianas
comprised 36.4% (SK) & 48.8% (PT) of the total woody species richness (lianas + tress) and 37%
(SK) and 38.3% (PT) of the total woody species density. The mean aboveground biomass and
carbon stock of lianas were 7.2 Mg ha-1 and 4.5 Mg ha-1, accounting for 1.94% of total woody
species community (trees + lianas). The aboveground biomass and carbon stocks were highest in
undisturbed site SK compared to disturbed site PT. The extent of liana diversity and their
contribution to the total woody plant abundance and biomass highlight the importance of lianas in
forest functioning, dynamics and mitigating climate change. Considering the biodiversity TDEFs
hold and the current level of human disturbance, a holistic approach in conservation is
emphasized.
Keywords: Liana - Biomass - Disturbance - Wood specific density - Allometric equation - Woody
species diversity - Conservation.
[Cite as: Babu KN & Parthasarathy N (2019) Assessment of liana diversity and carbon stock in differently
disturbed tropical dry evergreen forests of southern India. Tropical Plant Research 6(1): 74–89]
INTRODUCTION
Lianas (woody climbers) represent one of the dynamic life-forms, which are prominent and abundant
throughout the world but they are more conspicuous and characteristic in tropical forests of three continents
(Tropical America, Africa and Asia), having high taxonomic diversity and species richness (Gentry 1991,
Schnitzer & Bongers 2002, Mascaro et al. 2004). Like other life-forms, these woody plant climbers germinate at
the ground, root permanently in soil and possess the capacity to climb host plants (trees) by their specialized
climbing structures to reach the forest canopy (Gerwing 2004). Lianas have relatively little structural support
and typically have a very high canopy, stem biomass ratio, resulting in a higher proportion of assimilating
biomass than other woody plants (Schnitzer & Bongers 2002).
Lianas comprise up to 35% of species diversity and between 10%–45% of total woody stem density (Gentry
1991, Senbeta et al. 2005, Addo-Fordjour et al. 2009a, DeWalt et al. 2015). They contribute about 10% of
tropical forests aboveground biomass due to their thin stems and low wood density with up to 30 % of total
aboveground biomass in liana-dense areas (DeWalt & Chave 2004) that help in the ramification of tropical
forest dynamics and functions (Schnitzer & Bongers 2002). Lianas have potential to reduce aboveground carbon
storage by up to 50 % (Duran & Gianoli 2013). Although lianas account for less than 10% of total woody
species biomass they contribute up to 40% of forest leaf biomass and canopy leaf cover (Putz 1984, Rodriguiez-
Ronders et al. 2016).
Lianas can rapidly colonize the open habitats such as canopy gaps, forest edges and clearings in the forests
that provide more light for their growth and proliferate in heavily disturbed areas (Putz 1984, Laurance et al.
2001). Lianas depend on trees to support their biomass and compete for both above and below ground for
resources. Trees with heavy liana loads often show decreased growth and increased mortality, reduced biomass,
fecundity (Kainer et al. 2006) and survivorship. It is estimated that lianas could reduce tree growth as much as
84% and increase tree mortality risks by two to three folds (van der Heijden et al. 2013). They act as key
ecological components of the whole forest in transpiration, carbon sequestration and forest regeneration.
With the exacerbation of forest fragmentation, natural and anthropogenic disturbances and global
environmental changes, an increase in liana abundance and biomass are witnessed across all tropical regions
(Wright et al. 2004, Schnitzer & Bongers 2011) which will consequently influence forest structure and
composition, and functions especially carbon sequestration potential. For example in the seasonal tropical
rainforests of Panama, liana biomass and abundance have been increasing over the past 40 years (Ingwell et al.
2010). Moreover, reduction in forest-level carbon in the presence of lianas was also found (van der Heijden et
al. 2015), as lianas themselves do not seem to compensate for the loss in tree biomass that they cause (Roels
2016).
Evidences are still accumulating about the effect of disturbance on liana community. Some studies suggest
that density, abundance and biomass of lianas increase with an increase in disturbance (Schnitzer & Bongers
2011) and sometimes liana basal area and aboveground biomass also depend on the structural characteristics of
the forest and physical environment (van der Heijden & Phillips 2008). Moreover a significant increase in
human disturbance by various means especially logging lead to a reduction in liana abundance (Addo-Fordjour
et al. 2008). It is also reported that liana density and basal area are positively related to disturbance particularly
on the local scale (Putz 1984) and frequently correlated with trees having low basal area and wood density
(Laurance et al. 2001).
Similar to many tropical forest ecosystems, lianas form one of the major structural components of tropical
dry evergreen forest (TDEF) in India. As per the recent reports (DeWalt et al. 2015), TDEFs are one of the
liana-dense forests in Asian tropics. Although there are studies carried out in TDEFs on lianas, an integrated
research on liana diversity and their contribution to woody species abundance and carbon stocks are negligible.
And many researchers quantified the biomass and carbon stock potential of trees in tropics but lianas have got
very less importance and there exist very few studies. Hence, this study is aimed at investigating liana
biodiversity, carbon stock, and its share to total woody species abundance and biomass in selected tropical dry
evergreen forests experiencing varied disturbance regimes.

Study area
Table 1. Characteristics of two tropical dry evergreen forest sites on the Coromandel Coast of India. [Climate data source:
Meteorological observatory, Cuddalore, Tamil Nadu, India]
Study area
Variable
Sendhirakillai (SK) Palvathunnan (PT)
Location (Lat. & Long.) 11° 30' 07'' N 79° 41'49'' E 11° 31' 58'' N 79° 41'51'' E
Forest area 3 ha 1.4 ha
Mean annual rainfall 1184 mm 1184 mm
Mean annual temperature 28.85°C 28.85°C
Soil Alluvial; sandy loam Alluvial; sandy loam
Distance to human settlement ~100 m ~ 300 m
Forest stature Tall (~ 8 m); 3-layered Short (~ 5 m); 2-layerd
Disturbance level Relatively Undisturbed (Score- 13) Much disturbed (Score- 30)
Two 1-ha plots were surveyed for liana species in two tropical dry evergreen forest sites Sendhirakillai (SK)
and Palvathunnan (PT) located in Cuddalore district ( lat. 11° 44’ 40.91”N and long. 79°46’ 4.87”E) of Tamil
Nadu, south India (Fig. 1; Table 1). These sites are situated at 54 km south of Puducherry town on the
Coromandel Coast. Though the inter-site distance is 6 km, the two study sites are unique in terms of dominant
woody species, forest stature, level of disturbance and cultural aspects. These are sacred groves and encompass
temples within the forest and protected by the local communities. Both the study sites are prone to varying level
Babu & Parthasarathy 2019
of anthropogenic disturbances (Table 1). The climate here is tropical dissymmetric with heavy rains received
during north-east monsoon followed by dew formation for six months (mid-October to mid-March) and then a
little inconsistent rainfall during south-west monsoon (Parthasarathy et al. 2008). The mean number of rainy
days in the annual cycle is 55.5. Both the sites’ vegetation is characterized as tropical dry evergreen forest
[TDEF; Type 7/CI of Champion & Seth (1968)] which is short-statured, 2- to 3- layered form, tree-dominated,
liana-dense with sparse ground flora (Parthasarathy et al. 2008).
Figure 1. Map showing the location of study sites (SK- Sendhirakillai; PT- Palvathunnan) along the Coromandel Coast in
Tamil Nadu state of India and the Google earth image of SK and PT sites.
Field inventory
Two 1-ha plots, one in each study site were entrenched. Each one hectare plot was further subdivided into
one-hundred 10 m × 10 m quadrats to facilitate liana species inventory. All lianas ≥ 1 cm diameter (measured
1.3 m from rooting point) were enumerated systematically within the 10 m × 10 m quadrat. All trees ≥ 10 cm
girth (measured at 1.3 m from ground) were also enumerated to ascertain lianas’ share to total woody richness
(trees + lianas), density and carbon stock. In the case of trees with multi-stems, their girth was measured
separately and the basal area was calculated and added up. The specimens were identified on site and using
regional flora (Gamble & Fisher 1915–1935), then cross-checked with herbarium lodged in the Department of
Ecology and Environmental Sciences, Pondicherry University. The climbing mechanism and dispersal mode of
liana species were noted down.
Human disturbance was determined for both the sites SK and PT, and the disturbance score was calculated
using standard protocol (Veblen et al. 1992), with modifications (Mani & Parthasarathy 2005) by on-field
observations and also by participatory rural appraisal tools. The scores were summed up to validate the level of
disturbance in both the sites. High scores indicate the high level and low score signifies a low level of
disturbance.
Data analysis
Diversity indices (Shannon, Simpson index and Fisher’s) were computed using PAST 3.1 software (version
3.1; Øyvind Hammer, Natural History Museum, University of Oslo) (Naveenkumar & Sundarapandian 2018).
The modified importance value index (IVI) and family importance value (FIV) were calculated (Mori et al.
1983). The diameter values were considered to calculate aboveground and belowground biomass (AGB, BGB).
Liana contribution to the forest structure was determined by calculating the ratios of woody species (trees
+liana) richness and density as well.
Liana above ground biomass was estimated following allometric equation developed by Schnitzer et al.
(2006):
AGB = exp [– 1.484 + 2.657 ln (D)]
Where, D is the liana diameter
Tree aboveground biomass was estimated following allometric equation provided by Chave et al. (2005)
using two variables, wood specific density (WSD) and diameter:
AGB est = ρ × exp (-1.499 + 2.148 ln (D) + 0.207(ln (D))2 - 0.0281(ln (D))3)
Where, D is the diameter of tree species and ρ is their wood specific density (WSD)
The WSD of each tree species was obtained from the global wood database (Zanne et al. 2009) and the
available literature (Mani & Parthasarathy 2007). The woody species for which wood specific density value was
not available; a generalized allometric equation (Pearson et al. 2005) was used using D as the only variable.
The belowground biomass for both the life-forms was determined by multiplying aboveground biomass with
0.26 (IPCC 2003) and the carbon stock (CS) was computed to be 50% of the total biomass (AGB+BGB) (IPCC
2005).
The liana diversity data of the present two sites were compiled with the data of eighteen other tropical dry
evergreen forest sites (Parthasarathy et al. 2015) to compare liana richness, density, basal area and carbon stock
against disturbance. The scores that had 1–9 designated as undisturbed sites (UND), 10–18 as relatively
undisturbed (RUD), 19–27 as moderately disturbed (MOD), and 28–36 as heavily disturbed (HD) (Table 5). A
simple regression was performed to test the relationship between the disturbance score with the liana species
richness, tree density, and basal area.
RESULTS
Liana diversity
A total of 1182 liana individuals (stems ≥ 1 cm dbh), that belonged to 24 liana species, 22 genera and 18
families were inventoried in two tropical dry evergreen forest sites SK and PT (Table 2). The mean species
richness was 18 species per one-hectare plot. Site PT had greater liana species richness (20 species, 18 genera
and 14 families) than site SK (16 species, 15 genera and 13 families; Table 2). Twelve species (50%) were
common to both the sites. The diversity indices varied between the two sites and the values increased with
increase in site disturbance (Table 2).
Table 2. Summary of liana diversity inventory in two tropical dry evergreen forest sites (SK-
Sendhirakillai; PT- Palvathunnan) on the Coromandel Coast of India.
Study site Total for 2 ha
Variable
SK PT (average)
Species richness (number ha-¹) 16 20 24 (18.00)
Number of genera 15 18 22 (16.50)
Number of families 13 14 18 (13.50)
Liana contribution to total woody species 36.4 48.8
(trees + lianas) richness (%)
Liana density ( stems ha-¹) 744 438
Liana contribution to total woody species 37 38.3 1182
(trees + lianas) density (%)
Basal area (m² ha-¹) 1.007 0.506 1.50 (0.75)
Diversity indices
Shannon index 1.99 2.22
Simpson index 0.81 0.86
Fisher’s alpha 2.88 4.32
Liana density, dominance and basal area
The density, dominance and basal area of different liana species varied between the two study sites. Less
disturbed site SK harbored more than one and a half times greater liana density (744 individuals) and basal area
(1.007 m² ha-¹) than heavily disturbed site PT (438; 0.506) (Table 3). Site SK was dominated by Strychnos
lenticellata (Dennst.) S.Hill with 240 individuals (32% of total liana stems) followed by Reissantia indica
(Willd.) Halle (164; 22%), Tinospora cordifolia (Willd.) J.D.Hook.& Thoms. (79; 10%) and Combretum
albidum G.Don (74; 9.9%), whereas site PT was dominated by Coccinia grandis (L.) Voigt (97; 22%) followed
by Tinospora cordifolia (73; 16%), Grewia rhamnifolia Heyne ex Roth (68; 15.5%) and Pachygone ovata
(Poir.) Miers ex Hook. (68; 15.5%) (Table 3). Among these dominant species Tinospora cordifolia was found in
both the sites. The dominant species accounted for a remarkable share of 75% of total stems and 79% of total
basal area in site SK; while in site PT they made up to 70% of total stems and 56.4% of total basal area.
Interestingly, the two highest contributors in terms of density and basal area namely Strychnos lenticellata and
Combretum albidum at site SK were not represented in site PT.
Table 3. List of liana species enumerated in two tropical dry evergreen forest sites (SK- Sendhirakillai; PT- Palvathunnan), with their
stem density, important value index (IVI), aboveground biomass (AGB) in kg, carbon stock (CS) in kg, climbing mechanism (CM),
and dispersal mode (DM).
SK PT
Species Family CM DM
Density IVI AGB CS Density IVI AGB CS
Acacia caesia (L.) Wild. Mimosaceae SA AU 1 1.16 73.73 46.45 0 0 0.00 0.00
Aganosma cymosa Apocynaceae ST W 2 0.89 0.54 0.34 3 2.19 1.23 0.77
(Roxb.) G.Don var.
cymosa Hook. f.
Azima tetracantha Lam. Salvadoraceae SA A 1 0.46 0.56 0.35 0 0 0.00 0.00
Cansjera rheedii Gmel. Opiliaceae ST A 30 14.73 198.03 124.76 4 3.46 51.51 32.45
Capparis brevispina DC. Capparaceae SA A 3 1.83 41.55 26.18 1 0.81 1.96 1.23
Capparis zeylanica L. Capparaceae SA A 11 5.81 93.10 58.65 26 22.58 485.98 306.17
Carissa spinarum L. Apocynaceae SA A 3 1.40 3.18 2.00 1 0.77 1.38 0.87
Cayratia pedata (Lam.) (Vitaceae) TC A 0 0 0.00 0.00 2 1.45 0.68 0.43
Juss.ex Gagnep.
Cissus quadrangularis L. Vitaceae TC A 0 0 0.00 0.00 5 3.78 5.39 3.39
Cissus vitiginea L. Vitaceae TC A 23 11.63 387.05 243.84 23 24.85 666.45 419.87
Coccinia grandis (L.)
Cucurbitaceae TC A 23 10.55 91.41 57.59 97 56.24 199.49 125.68
Voigt
Combretum albidum Combretaceae ST W 74 45.85 2994.98 1886.84 0 0 0.00 0.00
G.Don
Derris scandens (Roxb.) Papilionaceae ST AU 0 0 0.00 0.00 4 15.28 1434.90 903.99
Benth.
Grewia rhamnifolia Tiliaceae SU A 14 14.08 993.76 626.07 68 67.8 1804.82 1137.04
Heyne ex Roth
Hugonia mystax L. Linaceae HC A 0 0 0.00 0.00 1 0.77 1.17 0.74
Lantana camara L. Verbenaceae SA A 0 0 0.00 0.00 2 1.48 1.04 0.65
Leptadenia reticulata Asclepiadaceae ST AU 0 0 0.00 0.00 4 2.72 9.43 5.94
(Retz.) Wight & Arn.
Pachygone ovata (Poir.) MenispermaceaeST A 10 3.37 29.26 18.43 68 32.37 202.97 127.87
Miers ex Hook.
Pyrenacantha volubilis Icacinaceae ST A 66 20.01 28.61 18.03 30 15.73 12.64 7.97
Wight
Reissantia indica (Willd.) Celastraceae SU W 164 67.99 2229.26 1404.43 23 15 123.17 77.59
Halle
Strychnos lenticellata Loganiaceae ST A 240 78.48 1897.86 1195.65 0 0 0.00 0.00
(Dennst.) S.Hill
Tinospora cordifolia MenispermaceaeST A 79 21.77 206.41 130.04 73 30.27 109.69 69.11
(Willd.) J.D.Hook. &
Thoms.
Toddalia asiatica (L.) Rutaceae SA A 0 0 0.00 0.00 2 1.65 4.93 3.11
Lam.
Trichosanthes tricuspidata Cucurbitaceae TC A 0 0 0.00 0.00 1 0.73 0.40 0.25
Lour.
Note: SA- Scrambler-armed; ST- Stem twinner; TC- Tendril climber; SU- Scrambler-unarmed; HC- Hook climber; A- Animal; W-
Wind; AU- Autochorus.
Importance value index (IVI) and family importance value (FIV)
In site SK, Strychnos lenticellata with an IVI of 78.48 was the most important species whereas Grewia
rhamnifolia (IVI – 67.8) was the most important species in site PT. Nine families (50%) were common to both
the study sites and four families were exclusive to site SK and five families were exclusive to site PT. The three
dominant and important families in terms of density and FIV were Loganiaceae, Celastraceae and Combretaceae
in site SK, while in PT Menispermaceae, Cucurbitaceae and Tiliaceae were the most dominant families (Fig. 2).
The Menispermaceae was taxonomically diverse and formed the most speciose family, represented by two
genera and two species each in both the sites. The three dominant families contributed 64.2% of total liana
stems and 75.4% of the total basal area in site SK, while in PT they contributed 70% of total stems and 55.4% of
the total basal area.
Figure 2. Family importance value (FIV) of liana families in two tropical dry evergreen forest sites (SK- Sendhirakillai; PT-
Palvathunnan).
Diameter class distribution of lianas
In both the sites liana density decreased with increase in diameter class. In less disturbed site SK, 89 % of
liana density was contributed by 1–6 cm dbh class and 58% of liana stems were restricted to the lowest diameter
class of 1–3 cm. The lower and middle diameter classes (1–3 cm and 3–6 cm) contributed 13.9% (0.14 m2 ha-1)
and 37.7% (0.38 m2 ha-1) to their respective total basal area. In the highly disturbed site PT, 93.4% of liana
density fell within 6 cm dbh and 77.6% of liana individuals were represented in the lowest diameter class 1–3
cm. The lower and middle diameter classes (1–3 cm and 3–6 cm) contributed 21.73% (0.11 m2 ha-1)
respectively, to their total basal area. The density of large lianas (≥ 6 cm) was maximum in less disturbed site
SK (with 80 stems) than the highly disturbed site PT (with 29 stems). In the highest diameter class (>15 cm),
basal area was notably higher in site SK than PT (Fig. 3).
Figure 3. Liana density and basal area by diameter classes in two tropical dry evergreen forest sites (SK- Sendhirakillai; PT-
Palvathunnan).
Climbing mechanisms and dispersal modes

Among the 24 liana species recorded in the two study sites, four climbing mechanisms (Fig. 4) and three
dispersal modes were identified (Table 3). Stem twining was predominant in both the sites in terms of richness
(7 species) and density (67.3% of total density in SK and 42.6% in PT); whereas in terms of richness alone, both
stem twiners and scramblers were dominant (Table 3). The highly disturbed site PT had a higher proportion of
species richness (6 species) and density (29%) of tendril climbers than the undisturbed site SK (2 species; 6%).
The lone hook climber Hugonia mystax L. was found only in the disturbed site PT. Zoochory was predominant
over autochory and anemochory in both the sites. At site SK, 75% (12 species) of lianas are dispersed by
animals followed by wind dispersed (18% - 3 species) and self dispersed (6% - 1 species). At PT, the proportion
of lianas with wind and self dispersed diaspores are similar to site SK (3 and 1 species), but the number of lianas
dispersed by animals are greater (80% -16 species) than PT. (Table 3).
Figure 4. Climbing mechansims possessed by lianas in two tropical dry everegreen forests (SK- Sendhirakillai; PT-
Palvathunnan): A, Stem twiner - Strychnos lenticellata - anti-clockwise twining around Drypetes sepiaria (host tree); B,
Hook climber - Hugonia mystax with paired hooks; C, Armed scrambler - Stem of Capparis zeylanica with recurved thorn
from bullate base; D, Tendril climber - Spirally coiled tendrils of Cisssus quadrangularis; E, Unarmed scrambler - multiple
stems of Grewia rhamnifolia.
Table 4. Basal area (BA), aboveground biomass (AGB), belowground biomass (BGB), total biomass (TB) and carbon stock
(CS) of trees and lianas in two tropical dry evergreen forest sites on the Coromandel Coast of India.
Study site
Variable
Sendhirakillai Palvathunnan
Basal area (m² ha-¹)
Trees 33.33 27.62
Lianas 1.007 0.506
AGB (Mg ha-¹)
Trees 386.33 358.66
Lianas 9.26 5.14
BGB (Mg ha-¹)
Trees 100.52 93.25
Lianas 2.41 1.33
Biomass (Mg ha-¹)
Trees 487.15 451.91
Lianas 11.67 6.47
Carbon Stock (Mg ha-¹)
Trees 243.57 225.95
Lianas 5.84 3.24
Liana share to woody-plant community

Lianas contributed 36.4% (SK) and 48.8% (PT) to the overall woody species richness (trees + lianas) and
37% (SK) and 38.3% (PT) to the overall woody species density (Table 4). Whereas lianas added just 2.93 %
(SK) and 1.8% (PT) to the total woody-plant basal area. Site SK registered highest liana contribution to total
woody-plant aboveground biomass and carbon stock (2.36%), while site PT registered the lowest (1.41%)
(Table 4).
Above ground biomass (AGB) and carbon stock (CS)
The mean AGB and CS of lianas were 7.2 Mg ha-1 and 4.5 Mg ha-1, accounting for 1.94% of total woody
species biomass (trees + lianas). The AGB and CS were greater in undisturbed site SK compared to disturbed
site PT (Table 4). In site SK the highest contributing species to carbon stock include Combretum albidum (1.88
Mg), Reissantia indica (1.40 Mg) and Strychnos lenticellata (1.19 Mg). Whereas at PT Grewia rhamnifolia
(1.13 Mg), Derris scandens (Roxb.) Benth. (0.90 Mg) and Cissus vitiginea L. (0.44 Mg) were the highest
contributors. Lianas ≥ 10cm diameter represented by 13 individuals accounted for 24.6 % of total AGB in SK
and in PT there were 8 stems contributing to 45.8 % of total liana AGB. Notably, in disturbed site PT Derris
scandens (4 stems) alone contributed approximately 28% to total liana biomass.
DISCUSSION
Lianas are considered to be an important life-form in tropical dry evergreen forest sites due to their moderate
species richness (11–31 species ha-1) and high density (408–1658 stems ha-1) (Reddy & Parthasarathy 2003,
2006, Vivek & Parthasarathy 2015a). Besides richness, the density, basal area, carbon stock values were higher
in site SK and this difference can be attributed to the uniqueness of the site in terms of forest stature, disturbance
level and human pressure (Table 1) and the presence of larger trees which supported large lianas (≥ 6cm) and
thus greater diameter and basal area (Reddy & Parthasarathy 2006).
Comparative analysis of lianas at 1-ha scale for stems ≥ 1 cm diameter inventoried in a total of 20 TDEF
sites revealed that species richness, density, basal area, carbon stock and set of dominant species varied across
the sites (Table 6), indicating that, no two sites can be considered as replicas. However, Strychnos lenticellata
was an exception, maintaining its dominance in the maximum number of sites. Across the 20 sites, species
richness, density, basal area and carbon stock ranged 11–29 species ha-1, 408–1658 stems ha-1, 0.20–1.76 m2 ha-1
and 1.42–33.88 Mg ha-1, respectively, and showed averages of 24 species ha-1, 820 stems ha-1, 0.87 m2 ha-1, and
5.29 Mg ha-1 respectively. The density and carbon stock contribution by lianas grossly varied as relatively un-
disturbed sites scored higher species density and carbon stock compared to disturbed sites. However, a high
variability is evident within a category, with 408 to 1455 lianas ha-1, 1.42 to 24.14 Mg ha-1 in undisturbed and
relatively undisturbed categories and 438 to 835 lianas ha-1, 3.24 to 18.46 Mg ha-1 in heavily disturbed sites. The
set of dominant and rare species varied across the 20 sites (Table 6, See Reddy & Parthasarathy 2003, 2006,
Vivek & Parthasarathy 2015a). A species rare in one site may be dominant in another site. Why does dominance
vary? Innate site variation with a pool of species which occur in a given category of site disturbance, forest
structure, and variation in the local ecological setting, edapho-microclimatic conditions and the edge effect
could contribute to the prevailing species composition of a site. Sites that are more exposed may harbor a higher
proportion of thorny stragglers as is evident at the disturbed PT and other TDEF sites.
Although all the 20 sites fall under the same forest type- TDEF, the stature, set of dominant tree species, tree
density variation, soil sub-type and the extent of human disturbance including edge effect could have
contributed to varying liana species composition. Species richness of 20 sites showed a weak negative
correlation with disturbance score (Fig. 5). Liana basal area of 20 sites showed a significant positive relationship
with disturbance but, density did not show any relation (Fig. 5). This could be ascribed to the presence of large
lianas in disturbed forests and removal of small size lianas by the local people as they are easy to cut. Therefore
medium and large lianas attain higher stem diameter and contribute to the high basal area. Pandian &
Parthasarathy (2016) also found that increasing AGB with an increase in disturbance in four inland tropical dry
evergreen forest sites.
Comparison of species richness and density across the tropics is difficult due to the variation in methods
used, area sampled and diameter limit considered. At the scale of 0.80 to 1.25 hectare area and for stems ≥ 1cm
diameter, our results are comparable with the other studies across the tropics (Table 5). An average richness 18
species ≥ 1cm diameter ha-1 in our study sites coincides with the other studies reported from Indian tropics:
Western Ghats- 17–23 species 1.08 ha-1 in two montane evergreen forests (Mohandass et al. 2015), 19–22
species ha-1 in two tropical forests of northeastern India (Barik et al. 2015). However, the average liana richness
of this study is much lower than the results obtained from other parts of Asia: India (Muthuramkumar &
Parthasarathy 2000, Padaki & Parthasarathy 2000, Muthumperumal & Parthasarathy 2010), South China (Ding
& Zang 2009), Africa: Ethiopia (Senbeta et al. 2005), Neotropics (Ibarra-Manriquez & Martinez-Ramos 2002,
Burnham 2004), Panama (Putz 1984), Australia (Chalmers & Turner 1994) and New Caledonia (Bruy et al.
2018). This could be attributed to variation in total forest area (TDEFs occurs as patches), forest type and
dynamics, stature, macro and micro-climatic condition, and disturbances by various means (overexploitation of
resources, illegal cutting of host plants and lianas and grazing by animals, etc.).
Figure 5. The correlation between disturbance score and liana species richness, density and basal area pooled for twenty
tropical dry evergreen forest sites on the Coromandel Coast of India.
Table 5. Compilation of liana diversity inventories carried out in various selected tropical forests of the world at the unified scale
for stems ≥ 1 cm dbh.
Area Total number of
Forest type Site location Reference
Inventoried Individuals Species
Dry, mesic and rain New Caledonia 1.08 ha 992 71 Bruy et al. (2018)
forests
Tropical dry evergreen Cuddalore, Nagapattinam, 10 ha 9237 52 Vivek et al. (2015)
Forest Pudukottai dist. of Tamil
Nadu
Tropical montane forest Nilgiri biosphere reserve, 13.58 ha 1658 33 Mohandoss et al.
India (2015)
Tropical rain forest Atewa range Tropical rain 2.5 ha 429 40 Addo-Fordjour et al.
forest Reserve (2012)
Tropical lowland forest Pasoh forest reserve, 2.5 ha 1623 167 Nurfazliza et al. (2012)
Malaysia
Lowland tropical forest Barro Colorado Island, 50 ha 47183 162 Schnitzer et al. (2012)
Panama
Tropical lowland forest Hainan Island 3 ha 1594 70 Ding & Zang (2009)
Tropical evergreen Varagalaiar, W.Ghats, India 30 ha 11200 75 Muthuramkumar &
forest Parthasarathy (2000)
Tropical, sub tropical & Eastern himalayas & N.E. 94 ha 3586 196 Barik et al. (2015)
temperate forests India
Evergreen, semi- Six hills, Eastern Ghats 55 ha 32033 143 Muthumperumal &
evergreen deciduous and Parthasarathy (2010)
thorny type
Subtropical wet forest Puerto Rica 0.8 ha 419 6 Rice et al. (2004)
Tropical rain forest Yasuni national park, 2.4 ha 4348 311 Burnham (2004)
Ecuador
Tropical rain forest Lacandon, Mexico 1.2 ha 2510 90 Ibarra-Manriquez &
Martinez-Ramos
(2002)
Tropical rain forest Albertine rift, Uganda 6 ha 2783 35-62 Eilu (2000)
Rain forest Lower Hunter valley, 1 ha 520 27 Chalmers & Turner
Australia (1994)
Semi-decidious forest Barro Colorado Island, 1 ha 3165 65 Putz (1984)
Panama
The average density of lianas (591 stems ha-1) found in this study is higher than the values obtained from
other Indian tropics (Muthuramkumar & Parthasarathy, 2000, Muthuramkumar et al. 2006, Mohandas et al.
2015, Barik et al. 2015,), Asian tropics (Ding & Zang 2009), Neotropics (Burnham 2004) and Australia
(Chalmers & Turner 1994). Whereas liana density is slightly lower than some other peninsular Indian sites
(Padaki & Parthasarathy 2000, Muthumperumal & Parthasarathy 2010) and two to four-fold lower than the than
the results from New Caledonia (Bruy et al. 2018), Neotropics (Putz 1984, Ibarra-Manriquez & Martinez-
Ramos 2002), Africa (Senbeta et al. 2005).
Table 6. Comparison of liana species richness, density, basal area and carbon stock (CS) at the unified scale of 1-ha for
stems ≥ 1cm dbh , and list of top 3 dominant species along with site disturbance scores and categories (UN- undisturbed
RUN- relatively undisturbed; MOD- moderately disturbed; HD- heavily disturbed) of various tropical dry evergreen
forest sites. (SK- Sendhirakillai; PT- Palvathunnan; OR-Oorani; AK- Arasadikuppam; KK- Kuzhanthaikuppam; PP-
Puthupet; AP- Araiyapatti; KR- Karisakkadu; SP- Shanmuganathapuram; MM- Maramadakki; KA- Karukkai; KT-
Kothattai; MK- Manadikuppam; PC1- Point Calimere 1; PC2- Point Calimere 2; PR- Purangani; SL- Silattur, SN-
Sunayakadu; SV- Suran Viduthi; VV- Vanniyan Viduthi)
Site Species Density Basal area CS Disturbance
Top 3 dominant species Ref.
code richness no. ha-1 m2 ha-1 Mg ha-1 score category
SK 16 744 1 5.84 13 RUN Strychnos lenticellata A
Reissantia indica
Tinospora cordifolia
PT 20 438 0.5 3.24 29 HD Coccinia grandis A
Tinospora cordifolia
Grewia rhamnifolia
AK 29 1163 0.58 11.01 19 MOD Strychnos lenticellata B
Derris ovalifolia
Secamone emetica
OR 24 812 1.85 12.96 23 MOD Strychnos lenticellata B
Reissantia indica
Combretum albidum
KK 28 497 1.37 41.85 24 MOD Combretum albidum B
Derris scandens
Reissantia indica
PP 28 835 0.99 3.63 33 HD Strychnos lenticellata B
Jasminum angustifolium
Gymnema sylvestre
AP 26 792 1.09 9.15 13 RUN Combretum albidum C
Derris scandens
Cissus quandrangularis
KR 23 515 0.67 33.88 23 MOD Combretum albidum C
Reissantia indica
Coccinia grandis
SP 27 775 0.44 30.44 26 MOD Combretum albidum C
Strychnos lenticellata
MM 21 596 1.68 18.46 32 HD Combretum albidum C
Derris scandens
PC1 28 672 0.68 6.56 4 UN Jasminum angustifolium D
Carissa spinarum
PC2 31 720 0.44 5.21 5 UN Carissa spinarum D

Zizyphus oenoplia
PR 22 919 0.65 6.29 13 RUN Strychnos lenticellata D
Hugonia mystax
Secamone emitica
SN 28 701 0.95 24.14 13 RUN Combretum albidum D
Acacia caesia
Carissa spinarum
MK 11 408 0.20 1.42 15 RUN Reissantia indica D
Combretum albidum
SL 25 1213 0.77 8.43 15 RUN Strychnos lenticellata D
Combretum albidum
Carissa spinarum
SV 27 1455 1.06 10.31 16 RUN Strychnos lenticellata D
Derris ovalifolia
Hugonia mystax
VV 20 1658 1.76 26.55 19 MOD Strychnos lenticellata D
Reissantia indica
Grewia rhamnifolia
KA 22 941 0.61 5.77 23 MOD Strychnos lenticellata D
Combretum albidum
Grewia rhamnifolia
KT 20 552 0.23 2.21 27 MOD Ichnocarpus frutescens D
Pyrenacantha volubilis
Note: Ref.- Reference; A- Present study; B- Reddy & Parthasarathy (2003); C- Reddy & Parthasarathy (2006); D- Vivek
& Parthasarathy (2015).
Many studies have confirmed that liana diversity and density have been shown to be higher in disturbed
forests than the undisturbed forests (Schnitzer & Carson 2010, Anbarashan & Parthasarathy 2013, Roels 2016)
as disturbed forests have the ability to promote liana success by providing more favorable conditions than the
less disturbed forests (DeWalt et al. 2000, Lawrance et al. 2001, Ibarra-Manriquez & Martinez-Ramos 2002).
However, in the current study species richness was greater in the disturbed forest supporting the view of the
above studies. But, density has shown a reverse trend with high density in undisturbed forest and this is
supported by few other studies conducted in tropics (Chittibabu & Parthasarathy 2001, Addo-Fordjour et al.
2009a, 2012, Rahman et al. 2010).
The diversity indices were higher in disturbed site PT than the undisturbed site SK which is in conformity
with the other findings from Indian tropical dry evergreen forests (Anbarashan & Parthasarathy 2013) and
Panamanian lowland forests (DeWalt et al. 2000). The species composition varied between the two sites
indicating that the forest stature and the level of disturbance coupled with micro-environmental conditions
within the sites play an important role (Parthasarathy et al. 2015). Tinospora cordifolia was the only dominant
species common to both the sites and that shows its ability to tolerate disturbance. Interestingly the density of
top five lianas of both the sites differed with contrasting density values. Though disturbance might be the reason
for this variation as it may respond differently to depending on the intensities, there may be different ecological
demands and possible habitat associations among the liana species attributed to this change (Yuan et al. 2009,
Addo-Fordjour et al. 2012). The presence of higher tendril climbers and four times higher density of Coccinia
grandis in disturbed forest attests that site PT is highly disturbed. This is in agreement with the results of Vivek
& Parthasarathy 2015a who reported greater abundance of tendril climber in disturbed forests which can also be
related to the low canopy height of trees and the extent of forest disturbance (Ding 2006, Parthasarathy et al.
2015).
Two species Strychnos lenticellata and Combretum albidum have ability to exploit site resources and survive
in different environmental conditions including disturbed forests (Vivek & Parthsarathy 2015a) but they were
not represented in the disturbed site in our study area. One possible reason could be that undisturbed site SK is
tall statured with a dense tree canopy which favored the shade tolerant species Strychnos lenticellata. This
finding is similar to the study conducted in few other tropical dry evergreen forest sites (Anbarashan &
Parthasarathy 2013). Another reason could be the vigorous vegetative sprouting capacity and dispersal strategy
(Ewango 2010). Even though the distance between the two sites is hardly six km and faunal diversity
particularly of avifauna is high similar species composition could have been expected but what prevents
diaspore dispersal/species establishment remains unclear thus ultimately making each TDEF site unique like
this. The Menispermaceae was abundant in undisturbed as well as in disturbed sites supporting previous study
pointing out that Menispermaceae was one of the dominant families represented in all four disturbance
categories (Anbarashan & Parthasarathy 2013).
The diameter class distribution of lianas followed a typical reverse J-shaped curve in both the sites. The
small stem lianas were relatively lesser in undisturbed site. Anbarashan & Parthasarathy (2013) also found that
undisturbed forests had less small stems than the heavily disturbed forest. The greater number of lianas in small
diameter class may be assigned to their extremely slow stem diameter increments by an inverse allocation of
resources as compared with trees within the same forest (Muthumperumal & Parthasarathy 2010). But also
disturbance contributes to this, by creating gaps where lianas can recruit and proliferate best and therefore lead
to a higher abundance of small lianas (Roels 2016).
Stem twining formed the predominant climbing mechanism in terms of richness and density in both the sites,
as reported from other tropical forests (Muthuramkumar & Parthasarathy 2000, DeWalt et al. 2000, Cai et al.
2009, Anbarashan & Parthasarathy 2013, Mastan et al. 2015, Vivek & Parthasarathy 2015a, Srinivas &
Sundarapandian 2017, Bruy et al 2018). Zoochory formed the most common dispersal mode in both the sites
revealing the importance of lianas in providing resources and shelter which can also increase animal population
densities. This result coincides with many other studies from Indian tropics (Muthuramkumar & Parthasarathy
2000, Reddy & Parthasarathy 2003, 2006, Anbarashan & Parthasarathy 2013, Vivek & Parthasarathy 2015a)
and Asian tropics (Yuan et al. 2009). On a comparative basis greater number of liana species are wind dispersed
in Neotropics as Bignoniaceae is one of the dominant families whose winged seeds in follicles are wind
dispersed which increasing the possibility of circulating to more areas, which might offer the reproductive
advantage over other dispersal modes for liana species.
In tropical forests, lianas commonly compose 8–45% of the woody stems (Schnitzer & Bongers 2002,
Parthasarathy et al. 2004, Schnitzer et al. 2006, 2012, DeWalt et al. 2015, Addo-Fordjour et al. 2016) and 20%
of woody stems in dry forests of Central America (Gillespie et al. 2000). Lianas in the present study contributed
42.6% to the total woody species density and this is in agreement with other studies (Vivek & Parthasarathy
2015a, Naveenkumar et al. 2017). Perez-Salicrup (2001), Parthasarthy et al. (2004, 2015) and DeWalt et al.
(2015) found that lianas comprise up to 35% of woody species richness in tropical forests. An important finding
is that across the sites the liana share to the total woody species (trees + lianas) richness, density and carbon
stock ranged 36.4–53.8 %, 24.5–62 %, 1–21.4 % respectively (Table 7). Parthasarathy et al. (2004) and Vivek &
Parthasarathy (2015a) reported that lianas contributed respectively 55.2% and 52% of total woody species
richness and density in different tropical dry evergreen forest sites. Overall, considering 20 sites together lianas
contributed 47%, 42% and 6.3% to the total woody species richness, density and carbon stock (Table 7).
Table 7. Species richness, stem density and carbon stock of lianas and trees by sites and percentage (%) contribution of lianas
to these three variables in 1-ha plots of 20 different tropical dry evergreen forest sites. (For site codes see Table 6)
Species richness Density Carbon Stock
Liana Liana Liana
Site code
Lianas Trees share (%) Lianas Trees share (%) Lianas Trees share (%)
SK 16 28 36.4 744 1264 37.0 5.84 243.57 2.4
PT 20 21 48.7 438 707 38.3 3.24 225.95 1.4
AK 29 31 48.3 1163 2815 29.2 11.01 213.25 4.9
OR 24 30 44.4 812 1619 33.4 12.96 322.7 3.8
KK 28 26 51.8 497 1459 25.4 41.85 153.15 21.4
PP 28 24 53.8 835 1567 34.8 3.63 370.70 1.0
AP 26 35 42.6 792 807 49.5 9.15 204.50 4.2
KR 23 30 43.4 515 596 46.4 33.88 189.20 15.2
SP 27 26 51.0 766 1663 31.5 30.44 196.20 13.4
MM 21 28 42.8 596 724 45.2 18.46 160.20 10.3
PC 1 28 37 43.0 672 790 45.9 6.56 358.84 1.8
PC 2 31 27 53.4 720 803 47.2 5.21 193.66 2.6
PR 22 20 52.3 919 948 49.2 6.29 80.73 7.2
SN 28 27 50.9 701 841 45.4 24.14 222.50 9.8
MK 11 18 37.9 408 786 34.1 1.42 60.80 2.3
SL 25 22 53.1 1213 1211 50.0 8.43 161.80 4.9
SV 27 37 42.1 1455 888 62.0 10.31 235.50 4.2
VV 20 30 40.0 1658 1693 49.4 26.55 215.20 10.9
KA 22 20 52.3 941 845 52.6 5.76 165.20 3.3

KT 20 25 44.4 552 661 45.5 2.21 187.00 1.1
Average 24 27 47.0 820 1134 42.0 13.4 207.70 6.3
The effect of disturbance is clearly reflected in the present study on basal area and aboveground biomass
(AGB) and carbon stock (CS), with higher values in the undisturbed site than the disturbed site. These results
are in conformity with the other studies which demonstrated a decrease in the basal area of lianas with an
increase in disturbance (van der Heijden & Phillips 2008, Addo-Fordjour et al. 2012, Anbarashan &
Parthasarathy 2013, Mohandass et al. 2015). In contrast, Addo-Fordjour et al. (2009b) found a contrary result
that is increasing basal area with increasing disturbance. Overall, decrease in basal area, AGB and CS of lianas
in site PT can be explained by the fact that, frequent removal of lianas and available host trees by the local
communities thereby affecting their diversity, composition, basal area, AGB and CS potential which is also
noticed in other studies (Chittibabu & Parthasarathy 2001, Addo-Fordjour et al. 2012). It was also found that, a
10-year change in tree diversity in disturbed site PT resulted in loss of 44% of total tree stems (Babu &
Parthasarathy, unpublished) which underline fact that in long run lianas always require supportive host trees for
their very survival, and a substantial reduction in tree density as in the case of site PT is deleterious to liana
community.
The average above ground biomass of lianas in the present study is 7.2 Mg ha-1. This value is comparable
with other studies 3.7 to 12.3 Mg ha-1 (Laurance et al. 2001), 3.39 Mg ha-1 (Lu et al. 2009), 43 Mg ha-1
(Gerwing & Farias 2000), 3.6–9.33 Mg ha-1 (Naveenkumar et al. 2017), 2.24–42.13 Mg ha-1 (Vivek &
Parthasarathy 2015b), 15.05–49.08 Mg ha-1 (Pandian & Parthasarathy 2016). This indicates that lianas
contribute less biomass as compared to trees yet, they may play important role in reducing the total forest carbon
stock and carbon sequestration potential by competing with their hosts for above and belowground resources
(Schnitzer & Bongers 2011). The contribution of lianas to the total biomass rarely surpasses 5% in undisturbed
forests (Hegarty & Caballé 1991), but in case of Roels (2016) and our study, the lianas share was less than 5%
in both undisturbed and disturbed sites.
CONCLUSION
We conclude that human-induced disturbances would have resulted in varied species diversity, biomass and
carbon stock of liana species in the studied sites. TDEF sites are subjected to various anthropogenic disturbances
and loss of belief system over two decades resulted in rapid decline of trees and increase in liana community
(Pandian & Parthasarathy 2016). The data obtained and discussed from 20 sites provide valuable information to
researchers in understanding the importance of lianas in maintaining the present level of diversity, carbon stocks
and dynamics in this unique forest type. The extent of variation in species diversity and carbon stock between
these sites, call for the concerted effort by the management of TDEFs to control human disturbance in the forest
and embark on artificial regeneration of lianas. Data on carbon stocks and flux in tropical forests are urgently
required to understand how lianas would respond to climate change and anthropogenic disturbances.
ACKNOWLEDGEMENTS
The first author thanks Biswajith Harpal for help in data collection. NB also thanks Venkat Reddy and A.
Ravi for the financial support.
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Research article
Identification of soil microbial population

under different land use
G. M. Akande* and F. O. Adekayode
Department of Crop, Soil and Pest Management, Federal University of Technology, Akure, Nigeria
*Corresponding Author: monisolagladys@gmail.com [Accepted: 10 April 2019]
Abstract: Soil microorganism is important for the restoration, sustainability, balancing of soil
ecosystem and organic matter transfer. The diversity of the microbial community in soil is closely
related to the functions and structure of its surrounding ecosystem. The aim of this research work
is principally on the identification of microbial population under different land use types in Akure,
Nigeria. The land use types were oil palm, teak plantation, unclear forest, and cassava and sugar
plantations. The soil samples were collected at depth of 0–15 cm, 15–30 cm and 30–75 cm on each
land use area and were taken to the laboratory for microbial analysis. Microbial analysis was
carried out using the dilution spread plates techniques of identification of microbial population.
The bacteria isolates were identified by morphological and biochemical characterization using
taxonomy scheme of Bergey’s manual of determinative bacteriology. The fungal isolate were
stained with lactophenol cotton blue and observed under the microscope for identification. The
result showed that, there are 40 different species of bacteria and 10 fungal strains isolated from all
the land use types. Some of the isolated bacterial species were from phylumactinobacter,
bacteriodietes, firmicutes, proteobacter and that of fungi were representatives of phylum
Ascomycota and Zycomycota. The data on bacteria and fungi were analyzed using ANOVA. The
means of bacteria and fungi occurrence were separated using least significance difference at 5%
level. It was found that the cassava land showed higher diversity of microbial population, this
might be attributed to the effect of tillage on the land year by year which enhanced the free
movement of air and encourages the availability of microbial population due to the presence of
some microorganism in the tuber of cassava which had a great influence on soil organic matter
contents via mineralization and decomposition.
Keywords: Soil ecosystem - Microbial population - Bacteria - Fungi - Land use types.
[Cite as: Akande GM & Adekayode FO (2019) Identification of soil microbial population under different land
use. Tropical Plant Research 6(1): 90–100]
INTRODUCTION
Soil harbors over 109 microorganisms, soils are the most diverse habitat of microbes on earth and provide an
ideal reservoir for microorganism (Dischinger et al. 2009). It’s also a highly complex biological system that
influenced the physical-chemical environmental parameters. The microorganism, which is sensitive indicators of
the status of an ecosystem, can lose their resilience to ecosystem disturbances and become unable to perform
their nutrients cycling functions. The importance of microorganism in ecosystem functioning has led to an
increased interest in determining soil microbial population (Azam et al. 2003). The soil microbial communities
are the active component ecosystem. It had been revealed that microorganism varied in and between soil types
with bacteria being the most numerous (Girvan et al. 2003), It was reported that fungi in soil constituting a
significant part of the soil biomass and served several important functions as decomposition of organic material,
nutrient cycling, formation of soil aggregates and mycorrhizal symbiosis in soil (Kabir et al. 2003, Tahjib-Ul-
Arif et al. 2018).
The land use types had been reported to play important roles in regulating microbial communities in tropical
soils than in many temperate systems, where favorable climatic factors, combined with access to fertilizers and
other soil amendments, helped to buffer changes in the soil environment (Buckley & Schmidt 2001). Land use
Received: 24 November 2018 Published online: 30 April 2019
change affects the microbiota and their functions (Fierer et al. 2006). Change in land can affects the composition
and function of microbial communities. Analysis of changes in microbial communities in tropical soils had
hitherto been limited to a few land use types. These analyses had shown increases in microbial and fungal
biomass as a result of differing organic matter inputs.
Particular the planting of different crops on the land, has led to decreases in soil microbial population
throughout the world and has declined soil biodiversity of the microorganism (Liiri et al. 2012). Therefore, the
need for identification of soil microbial population is becoming a rapidly growing concern. The study of soil
microorganism through the world has yielded a lot of soil microbial species that are of a great value for the
ecosystem. Microorganisms are easy to isolate, culture and maintain to improve their strain, microbes are
omnipresent and exist in the environment. Bacteria are predominant because of their resistance endospore
formation (Cafini et al. 2006). Therefore, determination of microbial population and identification of microbial
population is a valuable tool for interpretation of microbial information in an ecological contexts and to examine
how the abundances of major soil bacterial phyla corresponding to the biotic and abiotic characteristics of the
soil environment. The absence of available data indicating the microbial population of soil types has led to
assess the identification of soil microbial population in Akure, Nigeria.

Description of the study area
The study was conducted at the teaching and research farm of the Federal University of Technology Akure,
Nigeria. Akure is located in the tropical rainforest area of southwestern Nigeria with a mean annual rainfall
between 1300 mm and 1600 mm, which normally occurs between March to November and peaks in June/July.
The dry season lasts about 3 months (December–February). It has a mean annual temperature of 27ºC
(minimum) and 32ºC (maximum) with the elevation ranged from 255 to 381 meters above the sea level and the
relative humidity during the raining season is between 85 and 100% and less than 60% during the dry season
(Fasinmirin & Oguntuase 2008). The soils in these sites are classified as Alfisols by USDA soil taxonomy
system and are characteristics of the variety found in the intensively weathered areas of basement complex
formations in the tropical rainforest zone of south-western Nigeria (Onyekwelu et al. 2006).
Land use type
Land use types are oil palm, teak plantation, unclear forest, cassava and sugar. The teak plantation and
unclear forest land is a natural forest which has not been disturbed for at least 100 years. Although shrubs and
climbers are present in the unclear forest, the major plant species are trees such as bamboo (Bambusa vulgaris
Schrad. ex J.C.Wendl.), oil bean (Pentaclethra macrophylla Benth.), and oil palm (Elaeis guineensis Jacq.). The
arable and fallow land had been previously cultivated for 7 years with cassava (Manihot esculenta Crantz), and
maize (Zea mays L.) either as sole crops or as crop combinations in various plots. Some of the crops cultivated
during the previous years were cocoyam (Colocasia esculenta (L.) Schott), eggplant (Solanum melongena L.),
pepper (Capsicum spp.), sweet potato (Ipomoea batatas (L.) Lam) and yam (Dioscorea spp.). At the time of
sampling, both cassava and maize were cultivated on the land.
Soil sampling
Seven sample plots were demarcated on the ground in each land use. The size of the sample plots in oil
palm, teak plantation, uncleared forest, cassava and sugar cane were 20 m × 20 m, 10 m × 10 m, 10 m × 10 m, 5
m × 5 m and 1 m × 1 m respectively. The experimental design for the investigation was split plot with each land
use type as the main plot while the sub plots were soil depth at 0–15 cm, 15–30 cm and 30–75 cm.
Microbial Analysis
The soil samples were collected during the raining season at the depth of 0–15 cm, 15–30 cm and 30–75 cm
at four locations in each sample plots and the soil was thoroughly mixed to obtain a composite sample. The soil
samples were collected in separate sterilized samples bottles. Each sample bottle was labelled properly with land
use types, location collection date and was taken to the laboratory for microbial analysis. The number of soil
microorganisms was determined using the dilution spread plate technique. Nutrients agar (NA) and Potato
Dextrose agar (PDA) were the culture media of choice used for bacteria and fungi. One milliliter aliquot of
sample was pipetted into sterile test tube and serially diluted in another six set of test tubes each containing 9ml
of sterile distilled water to dilution ratio10-6., 0.1 ml portion of the diluents from the fourth (10 -4) and fifth (10-5)
dilution factors were pipette separately aseptically into different sterile petridishes and 20 ml of the cool (45ºC)
sterile molten agar media was added under aseptic condition, swirled gently for even distribution of the inocula,
Akande & Adekayode 2019
allowed to set and incubated at 30–37 ºC for 24 hours (for bacterial), and at 25–27 ºC for 72 hours for fungi. At
the end of incubation (24 hours) microbial colony were counted and recorded appropriately for bacteria while
after 72 hours microbial colony were counted and recorded appropriately for fungi.
Isolation and purification of bacteria strain
The strain of bacteria were selectively isolated by streak method to subculture the bacteria. An inoculating
loop was sterilized using hot flame, allowed to cool before it was used to take part of a grown bacteria colonies
from the cultured agar and streaked on the surface of fresh nutrient agar. The agar was incubated at 37ºC in an
inverted position for 24–48 hours. Individual bacteria colonies were identified by morphological and
biochemical techniques using the taxonomy scheme of Bergey’s Manual of Determinative Bacteriology (Holt et
al. 1994). The cultural characterization of bacteria colonies isolated was done by observing the colonies for
color, shape, edge, elevation and surface appearance displayed on Nutrient agar while the following biochemical
tests catalase test, motility test and oxygen- relation, methyl Red Tests, Fermentation of sugars (carbohydrates),
gram’s reaction, coagulate Test, were carried out for the identification according to Bergey’s manual of
determinative Bacteriology.
Isolation and purification of fungi strain
The fungi isolated were stained with lactophenol cotton blue and observed under the microscope for
identification of mycelia and spore structures. The fungi species were classified based on cultural and
microscopic spore characteristic.
Data Analysis
The soil biological properties were subjected to analysis of variance (ANOVA) using the SPSS version 15.
Treatments mean was separated using Duncan Multiple Range Test at p< 0.05.
RESULTS
Table 1. Colony forming units of microbial population under different land use types.
Bacteria population Fungi population
Land use
cfu g-1 Soil (104) cfu g-1 Soil (104)
Oil palm 6.72b 5.54c
Teak 6.57d 5.44d
Uncleared forest 6.64c 5.45d
Cassava 6.80a 5.59b
Sugar cane 6.78a 5.64a
Note: Mean followed by the same letter in each column are not significantly different from
each other by Duncan’s multiple Range Test (DMRT) at 5% level of probability.
Table 2. Colony forming units of microbial population with different soil depths.
Soil depths (cm)
cfu g-1 Soil (104) cfu g-1 Soil (104)
0–15 6.74a 5.61a
15–30 6.70b 5.52b
30–75 6.67c 5.46c
Note: Mean followed by the same letter in each column are not significantly different from each other by
Duncan’s multiple Range Test (DMRT) at 5% level of probability.
Colony forming units of microbial population under different land use types are shown in table1. Microbial
population varied significantly (p< 0.05) across the land use types, the number of soil bacteria in the cassava
land was significantly higher (p < 0.05) with 6.80×104 cfu g-1 compared to other land use types and lowest
bacteria population was observed in teak plantation with 6.57×104 cfu g-1. Significantly higher number of soil
fungi was present in the sugarcane with 5.64×104 cfu g-1 compared to other land use type. Colony forming units
of the microbial population with different soil depths was presented in table 2. Microbial population varied
significantly (p< 0.05) across various soil depths (Table 2). Maximum number of microbes was recorded from
surface soil (0–15 cm) as compared to other depths. The interaction effect of land use by soil depth was
recorded in table 3. The highest microbial population of 6.83×104 cfu g-1 was recorded at the surface soil layer
of the cassava land (0–15 cm), whereas the lowest 6.53×104 cfu g-1 was observed at subsoil layer of the teak
land (30–75 cm) (Table 3). The fungi population was significantly high at the surface layer of cassava (30–75
cm) with 5.72×104 cfu g-1 and lowest value was recorded at subsoil layer of uncleared forest (30–75 cm) with
5.43×104 cfu g-1.
Table 3. Colony forming units of microbial population under different land use types with different soil depths.
Land use Depths (cm)
cfu g-1 Soil (10-4) cfu g-1 Soil (10-4)
Oil palm 0–15 6.76bcd 5.66a
15–30 6.72de 5.53b
30–75 6.68ef 5.43bc
Teak 0–15 6.61gh 5.51bc
15–30 6.56hi 5.41d
30–75 6.53i 5.40d
Uncleared forest 0–15 6.69e 5.49bc
15–30 6.63fg 5.44cd
30–75 6.63fg 5.43cd
Cassava 0–15 6.83a 5.72a
15–30 6.81ab 5.57b
30–75 6.77bcd 5.48bc
Sugarcane 0–15 6.81ab 5.69a
15–30 6.78abc 5.66a
30–75 6.74cde 5.56b
Note: Mean followed by the same letter in each column are not significantly different from each other by Duncan’s
multiple Range Test (DMRT) at 5% level of probability.
The Morphological characteristics of bacterial isolates from oil palm land were presented in table 4. These
bacteria include Pseudomonas aeruginosa, Flavobacterium spp., Actinobactera nitritus, Moraxella lacunata,
Brucella spp., Bacteroides spp., Gemella spp. and Neisseria spp.
Table 4. Morphological characteristics of bacterial isolates from oil palm land.
Isolate Probable organism Macroscopic characteristic Microscopic characteristic
1 Pseudomonas aeruginosa Green, Pairs and Chain Gram negative, Rod
2 Flavobacterium spp. Yellow and Round Gram positive Cocci
3 Actinobacter anitritus Creamy, Pairs and Chain Gram negative, Cocco baccila
4 Moraxella lacunata Pink , Chain and Clusters Gram negative, Cocci
5 Brucella spp. Off white, Pairs and Chain Gram negative, Short rod
6 Bacteroides spp. Dirty white, Bundles and Chains Gram negative, Rod
7 Gemella spp. Dirty white, Pairs and Chain Gram negative, Cocci
8 Neisseria spp. Creamy and Round Gram negative, Cocco baccila
Table 5. Biochemical identification of bacterial isolates from oil palm land.

Isolates
Biochemical test
1 2 3 4 5 6 7 8
Coagulate test - - - - - - - -
Catalase + + + + + + + +
Oxidase - - - - - - - -
Mortility + - - - - - - -
Methy red + + + + - + - +
Starch hydrolysis - + + + + + + +
Sugar utilization
Glucose AG AG AG - - AG AG AG
Lactose - - AG - - AG AG -
Sucrose - - AG - - AG AG -
Maltose - - - - - AG AG -
Manitol AG AG - - AG AG AG -
Inositol AG AG - - AG AG AG -
Xylose - - AG - - AG AG AG
Flavobacterium
Bacteriodes spp.
Neissera spp.
Brucella spp.
Gemella spp.
Pseudomona
Actinobacter
Organism
Probable
aeruginosa
Moraxella
antitritus
lucunata
spp.
Note: +, Positive; -, Negatives; AG, Acid and gas fully produced; ND, Not indicate; A, Acid produce
only.
The Biochemical identification of bacterial isolates from oil palm land were shown in table 5. The genera
Flavobacterium spp., Brucella spp. are gram-positive bacteria while Pseudomonas aeruginosa, Actinobactera
nitritus, Moraxella lacunata, Bacteroides spp., Gemella spp. and Neisseria spp. are gram-negative bacteria.
Table 6. Morphological characteristics of bacterial isolates from teak land.
1 Bacillus cerus Creamy, Bundles and Chain Gram positive, Ova
2 Bacillus pumilus Creamy and Round Gram positive, Long rod
3 Bacillus subtilis Off white and Pairs Gram positive, Rod
4 Clostridium welchii Creamy, Chain and Pairs Gram positive, Rod
5 Clostridium septicum Dirty white, Chains and Clusters Gram positive, Rod
6 Clostridium carnis Off white, Pairs and chains Gram positive, Rod
7 Kurthia spp. Creamy and Round Gram positive, Rod
8 Corynebacterium murium Creamy, Pairs and Chains Gram positive, Rod
Table 7. Biochemical identification of bacterial isolates from teak land.

Isolates
Biochemical test
1 2 3 4 5 6 7 8
Catalase + + + + + + + +
Oxidase - - + - + + + -
Mortility + + - - - - - -
Methy red + + + - + + + -
Starch hydrolysis + + + + + - + +
Sugar utilization
Glucose AG AG + + + + + +
Lactose AG AG + - + + + -
Sucrose AG AG + + + - + +
Maltose AG AG + - + + + -
Manitol - AG + - + - + -
Inositol - AG + - + - + -
Xylose AG AG + - - + + -
Corynebacterium
Bacillus pumilus
Bacillus subtilis
Bacillus cerus
Organism
Probable
Kurthia spp.
Clostridium
Clostridium
Clostridium
septicum
murium
welchii
carnis
Note: +, Positive; -, Negatives; AG, Acid and gas fully produced; ND, Not indicate; A, Acid
produce only.
Table 8. Morphological characteristics of bacterial isolates from uncleared forest.

1 Vibrio spp. Dirty white, Bundles and Chains Gram negative, Long rod
2 Bordetella spp. Dirty white, Pairs and Chains Gram negative, Short rod
3 Acinetobacter pertussis Creamy, Bundles and Chain Gram negative, Short rod
4 Morganella iwoffi Creamy, Pairs and Chains Gram negative, Long rod
5 Chromobacterium morganii Creamy Chains and Clusters Gram negative, Short rod
6 Necromonas lividum Creamy and Round Gram negative, Long rod
7 Moraxella bovis Creamy and Ova Gram negative, Rod
8 Eubacterium letitum Creamy, Chains and Clusters Gram positive, Long rod
The Morphological characteristics of bacterial isolates from teak land were presented in table 6. These
bacteria include Bacillus cerus, Bacillu pumilus, Bacillu subtilis, Clostridium welchii, Clostridium septicum,
Clostridium carnis, Kurthia spp. and Corynebacterium murium. The Biochemical identification of bacteria
species isolated from teak land was shown in table 7. Bacillus cerus, Bacillu pumilus, Bacillu subtilis, Kurthia
spp. and Corynebacterium murium are gram-positive bacteria while the spore fo-rmers include Clostridium
welchii, Clostridium septicum, Clostridium carnis, Corynebacterium murium. Bacillus cerus, Bacillus pumilus
and Bacillus subtilis. The morphological characteristics of bacterial isolates from the uncleared forest were
presented in table 8. These bacteria include Vibrio spp., Bordetella spp., Acinetobacter pertussis, Morganella
iwoffi, Chromobacterium morganii, Necromonas lividum, Moraxella bovis and Eubacterium letitum. The
Biochemical identification of bacteria species isolated from the uncleared forest was shown table 9.
Table 9. Biochemical identification of bacterial isolates from uncleared forest land.
Isolates
Biochemical test
1 2 3 4 5 6 7 8
Catalase + + + + + + + +
Oxidase - + - - + + - -
Mortility + - - + + - - -
Methy red + - - + + + + +
Starch hydrolysis + + + + + + + +
Sugar utilization
Glucose + - - - - - - -
Lactose + - - - - - - -
Sucrose + - - - - - - -
Maltose + - - - - - - -
Manitol + - - - - - - -
Inositol + - - - - - - -
Xylose + - - - - - - -
Chromobacterium
Acinetobacter
Eubacterium
Necromonas
Organism
Probable
Morganella
Vibrio spp.
Bordetella
Moraxella
morganii
Pertussis
lividum
letitum
iwoffi
bovis
spp.
produce only.
Eubacterium letitum is the only gram-positive bacterium that was identified in the forest land while Vibrio
spp., Bordetella spp., Acinetobacter pertussis, Morganella iwoffi, Chromobacterium morganii, Necromonas
lividum and Moraxella bovis are gram-negative bacteria.
Table 10. Morphological characteristics of bacterial isolates from cassava land.
1 Peptococcus spp. Off white, Pairs and Chain Gram positive, Cocci
2 Streptococcus epidermicus Creamy, Clusters and Chains Gram positive, Cocci
3 Streptococcus equisimillis Creamy, Chains and Clusters Gram positive, Cocci baccila
4 Micrococcus roseus Creamy and Pairs Gram positive, Cocci
5 Micrococcus varius Creamy, Pairs and Chains Gram positive, Cocci
6 Salmonella pullorium Off white, Pairs and Chains Gram negative, Rod
7 Proteus murabillis Creamy and Round Gram negative, Long rod
8 Serratia rubidaea Pink and Pairs Gram negative, Rod
Table 11. Biochemical identification of bacterial isolates from cassava land.

Isolates
Biochemical test
1 2 3 4 5 6 7 8
Catalase + + + + + + + +
Oxidase - - ND ND - - - -
Mortility - - - - - + + +
Methy red + + + + - + - +
Sugar utilization
Glucose - - + + - - - +
Lactose - - - - - - - -
Sucrose - + + + - - + +
Maltose - - - + - - - -
Manitol - - - - - - - -
Inositol - - - - - - - -
Xylose - - - - + + - -
Streptococcus
Streptococcus
Micrococcus
Micrococcus
Peptococcus
epidermicus
equisimillis
Salmonella
Organism
murabillis
Probable
pullorium
rubidaea
Serratia
Proteus
roseus
varius
spp.
Note: +, Positive; -, Negatives; AG, Acid and gas fully produced; ND, Not indicate; A, Acid produce
only.
The Morphological characteristics of bacterial isolates from cassava land were presented in table 10. These
bacteria include Peptococcus spp., Streptococcus epidermicus, Streptococcus equisimillis, Micrococcus roseus,
Micrococcus varius, Salmonella pullorium, Proteus murabillis and Serratia rubidaea. The Biochemical
identification of bacteria species isolated from cassava land was shown in table 11. The bacteria, Peptococcus
spp., Streptococcus epidermicus, Streptococcus equisimillis, Micrococcus roseus and Micrococcus varius are
gram-positive bacterium while Salmonella pullorium, Proteus murabillis and Serratia rubidaea are gram-
negative bacteria.
Table 12. Morphological characteristics of bacterial isolates from sugar cane land.
1 Enterobacter cloacae Creamy, Chains and Clusters Gram negative, Rod
2 Proteus inconstsus Dirty white, Chains and Clusters Gram negative, Ova
3 Klebsiella oxytoca Off white, bundles and Chains Gram negative, Cocco baccila
4 Serratia marcescens Red, Pairs and Chains Gram negative, Cocci
5 Enterobacter aerogenes Off white, Pairs and Chain Gram negative, Short rod
6 Klebsiella aerogenes Creamy, Pairs and Chains Gram negative, Rod
7 Leuconostoc spp. Creamy, Bundles and Chain Gram positive, Cocci
8 Nocardia caviae Creamy, Pairs and Chains Gram positive, Ova
The Morphological characteristics of bacterial isolates from sugar cane land were presented in table 12.
These bacteria include Enterobacter cloacae, Proteus inconstsus, Klebsiella oxytoca, Serratia marcescens,
Enterobacter aerogenes, Klebsiella aerogenes, Leuconostoc spp. and Nocardia caviae.
Table 13. Biochemical identification of bacterial isolates from sugar land.
Isolates
Biochemical test
1 2 3 4 5 6 7 8
Catalase + + + + + + + +
Oxidase - - - - - - - -
Mortility + - - - - - + -
Methy red + - + - + + + +
Sugar utilization
Glucose + + + + + + + +
Lactose + + + + + + + +
Sucrose + - + + + + + +
Maltose + + + + + + + +
Manitol + + + + + + + +
Inositol + + + + + + + -
Xylose - - - - - - - -
Enterobacter
Enterobacter
Leuconostoc
marcescens
inconstsus
aerogenes
aerogenes
Klebsiella
Klebsiella
Organism
Nocardia
Probable
Serratia
cloacae
oxytoca
Proteus
caviae
spp.
produce only.
The Biochemical identification of bacteria species isolated from sugar cane land was shown in table 13.
Leuconostoc spp. and Nocardia caviae are gram-positive bacterium while Enterobacter cloacae, Proteus
inconstsus, Klebsiella oxytoca, Serratia marcescens, Enterobacter aerogenes and Klebsiella aerogenes are
gram-negative bacteria.
Characteristic of fungi isolated from the soil sample collected from all the land use areas were shown in
table 14. The fungi identified were as follows: Rhizopus stolonifer, Aspergillus funmigatus, Ulocladium
lanuginosum, Aspergillus ustus, Gliocladium rosum, Alternaria alternate, Aspergillus niger, Penicillium
italicum, Mucor spp. and Candida spp. Most of the fungi appeared in different colors on the agar media (PDA).
Rhizopus stolonifer appeared White cotton-like mycelia at 24 hrs, turning dirty with development of black
spores’ mycelium on Potato Dextrose Agar (PDA), Aspergillus funmigatus, appeared white base with brown
conidiophores which make it appears brown in color on PDA, Ulocladium lanuginosum, appeared Dark on
Potato Dextrose Agar with dictyosporus, usually without constriction at major septum, borne singly, apical and
on new sympodial growing points saprophytic, Aspergillus ustus appeared brown in color on Potato Dextrose
Agar with White base with brown conidiophores. Gliocladium rosum appeared Hyaline or brightly colored in
mass on the PDA, one-celled, produced successively apically and collecting in mucilaginous droplets,
saprophytic, common in soil, they also produced a verticillium state. Alternaria alternate have a dark
Conidiophores, mostly simple, rather short or elongate, typically bearing a simple or branched chain of conidia
on PDA. Aspergillus niger appeared on PDA in Black mycelium. Penicillium italicum appeared Yellowish
green to dark green hyphae on PDA. Mucor spp. appeared in a White to grayish brown colors on PDA, wooly to
cotton spreading colonies and darkens with time and Candida spp., appeared Creamy to white color, circular,
mucold, raised and opaque colonies with entire margin on PDA.
Table 14. Characteristic of some fungi isolated from soil sample collected from all the land use.
Name of
Spores/conidia arrangement under the
Isolate Cultural characteristics organism
microscope
present
01 White cotton-like mycelia at 24 hrs, Non-septate hyphae and coenecytic, thin Rhizopus
turning dirty with development of sporogiophores, sporangium have well stolonifer
black spores mycelium developed collumelium which is
umbrella like, spores are of various
shape but are generally oval.
02 White base with brown Upright conidiophores that terminated in Aspergillus
conidiophores which make it a clavate swelling bearing phialides at funmigatus
appears brown in color. the apex. Conidia are one-celled and
vesicles globose, non-septiate and non
collumella.
03 Dark, dictyosporus, usually without Conidiophores arising as upright Ulocladium
constriction at major septum, borne branches of mycelium, conidia lanuginosum
singly, apical and on new sympodial (porospores).
growing points saprophytic.
04 White base with brown Conidiophores upright, simple, Aspergillus ustus
conidiophores which make it terminating in aglubose or clavata
appears brown in color. swelling, bearing phialides at the apex or
radiating from the entire surface, conidia
(philalospores) one-celled globose, often
variously colored in mass in dry
basipetal chains.
05 Hyaline or brightly colored in mass, Corda, conidiophores portion bearing Gliocladium
one-celled, produced successively penicillate branches, forming a compact rosum
apically and collecting in ‘’brush” as in penicillum, conidia
mucilaginous droplets, saprophytic, (phialaspores).
common in soil. They also produced
a verticillium state.
06 Conidiophores dark, mostly simple, Conidia (porospores) dark, typically with Alternaria
rather short or elongate, typically both cross and longitudinal septa, alternate
bearing a simple or branched chain variously shaped,obclavate to elliptical
of conidia. or ovoid, frequently borne acropetally in
long chains.
07 Black mycelium Conidiophores were upright, unbranched Aspergillus niger
simple and terminating in a globose
swelling bore phialides at apex.
08 Yellowish green to dark green Conidiophores arranged singly form the Penicillium
hyphae mycelium, they were mostly ovoid or italicum
globose near the apex.
09 White to grayish brown, wooly to Broad non septate hyphae, Mucor spp
cotton spreading colonies and sporongiophores are long terminates in a (Scholer, 1983)
darkens with time round spore filled sporangium absence of
rhizoids.
10 Creamy to white, circular, mucold, Round to oval shaped ellipsoidal Candida spp
raised and opaque colonies with budding cells with blastoconidia and (C-P Robin
entire margin pseudohyphae, some cell appeared singly Berkhout, 1923)
and others in pairs.
Note: Some of the fungi identified in table .14 are similar to those identified by Olukunle et al. (2011) in determination of
degrading activity of fungi on kerosene, diesel and petrol using classical selective enrichment method.
DISCUSSION
The total microbial count in this study range from a low of 6.57×104 cfu g-1 in the teak plantation to 6.80×104
cfu g-1 in cassava land, with significantly higher number of soil fungi was present in the sugarcane with
5.64×104 cfu/g-1 compared to other land use type. The population of bacteria was higher in cassava land use
types, than in other land use area. and this suggested that microbial population variation in cassava land might
be attributed to the effect of tillage on the land year by year which enhanced the free movement of air, which
encourage the availability of microbial population and as a result of presence of some microorganism in the
tuber of cassava which had a great influence on soil organic matter contents through mineralization and
decomposition. This research was in agreement with research conducted by (Uzochukwu et al. 2001) on the
evaluation of microorganism from cassava waste water. He revealed that the presence of sugar and starch in
cassava tuber stimulate microbial growth.
The high population of fungi in sugarcane land might be as a result of longtime cultivation of the sugarcane
land, which might homogenize the soil resources and increases the fungi population in sugarcane land (Cotty et
al. 1994) The microbes in the soil also decreases with increase in soil depths, in which the counts were higher in
surface soil at 0–15 cm, this is due to the presence of more organics and nutrients at the surface layer of the soil.
Organic matter generally decreases from the surface to the bottom of the soil profile (Trumbore 2000). The
decreasing trend of microbial abundance in the downward direction is commonly seen and is naturally obvious
because the majority of soil microorganisms are heterotrophs.
From table 4 to table 13, eight isolates were obtained from each land use, make it up to 40 isolates in all the
land use area, this study was in line with Borneman et al. (1997) which suggested that, land use soils contained
high levels of microbial diversity composed of some unusual microorganisms. This same author also
investigated the effects of vegetative cover and land-use changes on microbial communities and he indicated
that distinct microbial communities were present under each form of land use. It was observed that, there were
more Gram-negative type bacteria in the land use than Gram-positive bacteria. Flavobacterium, Bacillus,
Clostrium, Kurthia, Corynebacterium, Eubacterium, Peptococcus, Streptococcus, Micrococcus, Leuconostoc
and Nocardia are gram-positive bacteria while Pseudomonas, Atinobacter, Moraxella, Bacteroides, Gemella,
Neisseria, Vibrio, Bordella, Acinetobacter, Morganella, Chromobacterium, Necromonas, Salmonella, Proteus,
Serratia, Enterobacter and Klebsiella are gram- negative bacteria. The spore forming bacteria include
Clostridium spp., Corynebacterium spp. and Bacillus spp.
The macroscopic and cultural characteristics of fungi isolated from all the land use area are presented in
table 14. They appeared in various colors’ of Yellow, black, brown, white and green on the Potato Dextrose
Agar (PDA). The fungi identified were as follows: Rhizopus stolonifer, Aspergillus fumigates, Ulocladius
lanuginosum, Mucor spp., Aspergillus ustus, Canadida spp., Gliocladium rosum, Alternaria alternate,
Penicillum italicum and Aspergillus niger. These results was in agreement with previous work of Amir &
Pineau (1998), That revealed the level of root colonization of soil microbial characteristics with highest biomass
and activity under land use type and vegetation. Results showed that the microbial population is higher in
cultivation area of the land use; this might be attributed to the fact that cropping appeared to affect microbial
communities positively, as bacterial and fungal diversity increased under cultivation. Knowles (1998) and
Ehiagbonare et al. (2009) discovered that the presence of bacteria and fungi diversity increased under
cultivation. The soil of the land use types contained more bacteria than fungi which could be corroborated by
the previous observation that soil usually contained greater amounts of soil bacteria than fungi. This was
because bacteria are less susceptible to changes in soil and environmental conditions, unlike fungi which are
easily restricted by soil pH, nutrient and harsh environmental conditions (Zhang et al. 2002).
CONCLUSION
Results from the present study demonstrate that land use types exert a profound influence on microbial
populations; the microbial population was greatly influenced by land use type. Among the land use types, the
cassava land harbors more microbes due to the availability of some microorganism in the tuber of cassava which
had a great influence on soil organic matter contents through mineralization and decomposition. The dilution
spread plates techniques of identification of microbial population enable the soil pathologist to understand the
basic principles of identification of microbiological and physiologic traits. The information from the techniques
employed in this study showed that there could be more novel bacterial and fungal species that could be
recovered in future studies by using the recently developed technique that allows quick and detailed analysis of
the microbial diversity in different land use types.
ACKNOWLEDGMENTS
I wish to express my deep appreciation to my supervisor, Professor F. O Adekayode is highly acknowledged.
My heartfelt thanks go to Mr. Hassan, Soil Laboratory technicians of Federal University of Technology, Akure
Nigeria, for offering his all possible helps in all aspects of the laboratory work. The management of Teaching
and Research Farm of Federal University of Technology is highly acknowledged .
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Research article
Qualitative and quantitative gas chromatography-mass

spectroscopy analysis and characterization of naturally isolated
mucilage in Hibiscus cannabinus L. (Malvaceae)
Sushma Chaudhary1,2, Manjul Pratap Singh2 and A. K. S. Rawat1*
1
CSIR-National Botanical Research Institute, Lucknow-226001, Uttar Pradesh India
2
Babu Banarasi Das University, Faizabad Rood, Lucknow, Uttar Pradesh-226028, Uttar Pradesh, India
*Corresponding Author: rawataks@76gmail.com [Accepted: 10 April 2019]
Abstract: Deccan hemp is rich in mucilage and of immense value. This study was performed to
examine the mucilage of Deccan hemp and its (Gas Chromatography-Mass Spectroscopy) analysis
was performed to identify the presence of sugar composition in mucilage for the development of
pharmaceutical formulation. Mucilage was found to be 9.54% w/w which was off white in colour,
tasteless and with characteristic odour. Physicochemical characterization revealed that mucilage
has enough moisture that is 9.34 % w/w and is of neutral pH. It was found to be soluble in hot
water and insoluble in organic solvents while in cold water mucilage swelled to form a gel. The
GC-MS analysis of mucilage showed the presence of glucose, fructose, sucrose, maltose and
xylose that scope to be of scientific relevance particularly plant polymer based excipient and
coating material in pharmaceutical products. The present investigation showed that Deccan hemp
mucilage has high pharmaceutical significance it can be used as excipient and coating material in
pharmaceutical formulation.
Keywords: Pharmaceutical products - Pharmacology - Photochemistry - Organoleptic - Swelling
index.
[Cite as: Chaudhary S, Singh MP & Rawat AKS (2018) Qualitative and quantitative gas chromatography-mass
spectroscopy analysis and characterization of naturally isolated mucilage in Hibiscus cannabinus L.
(Malvaceae). Tropical Plant Research 6(1): 101–105]
INTRODUCTION
Hibiscus cannabinus L. commonly known as Kenef native of Africa is a member of Malvaceae family. It is
an erect annual herb, grows up to 2 m in wild and 5 m in cultivars. The stem is cylindrical and slender. The
cultivar is unbranched and smooth while the wild is prickly, entirely green, green with red or purple
pigmentation or red sometimes lower half green and upper half pigmented. The tap root is well developed
measures up to 25cm deep with lateral roots spread horizontally to 1m and the adventitious root on lowest stem
section (Shamsuddin & van der Vossen 2003). It is a plant of great therapeutic value with its crude extract
exhibiting multidimensional activities which include antidiabetic activity (SundarRajan et al. 2011), anti-
inflammatory activity (Shaikh et al. 2016), fungiotoxic activity (Kobaisy et al. 2001), anti-hyperlipidemic
activity (Shivali & Pradeep 2010), anti-ulcer activity (Nyam et al. 2016) and besides these, it is a good source of
mucilage which makes it a drug of pharmaceutical value. Mucilage is a good source of natural polymer with
thickening and binding properties in different industrial applications such as food pharmaceutical and cosmetic.
Plant material and products are compatible with environmental safety and human health. The plant-based
polymer have been effectively applied in various pharmaceutical dosage forms like nanoparticles coating
material, film coating agents, matrix controlled system, suspensions, implants, buccal films and microspheres.
They have been used as viscosity enhancers, stabilizers, disintegrates, solubilizes, emulsifiers, bio adhesives and
binders (Clarke et al. 1979, Franz 1989, Zimmermann et al. 1994). Mucilage have been extensively used in the
field of drug delivery for their easy availability, cost effectiveness, eco friendliness, emollient and non irritant
nature, non toxicity, capable of multitude of chemical modifications, bio-degradable and compatible due to
natural origin (Durso 1980, Chang & Shukla 2003). Mucilage is intracellular physiological product of
Received: 27 October 2018 Published online: 30 April 2019
Chaudhary et al. 2019
metabolism (Geetha et al. 2009). Which is polysaccharide mixture having high molecular weight i.e., 20000 and
more (Narkhede et al. 2010); commonly found in various organs of many higher plant species (Hadley 1997)
and it is an amorphous polysaccharide, its composition was found to be D-Glucose, D-Fractose, L-
Galactouronic acid, D-Galactose, L- Rhamnose, Sucrose, Maltose and Xylose (Baveja et al. 1988). Mucilage
acts as a membrane thickener and food reserve in the plants (Banker & Anderson 1987), Hence the present study
was under taken with the aim to analyze the sugar composition present in mucilage along with
physicochemical properties (Fig. 1; Table 1).
Figure 1. Plant of Hibiscus cannabinus L. (Deccan Hemp). [Inset: Fruits]

Table 1. Organoleptic and physicochemical characteristic of Deccan Hemp mucilage
S.N. Properties of Mucilage Results
1 Swelling Index 9±0.52 % w/w
2 Solubility Soluble in hot water,
Insoluble in organic solvents and in cold water swells to form a gel
3 Loss on drying 9.34 %w/w
4 pH 7.2±0.2
5 Colour Off white colour
6 Odour Odour less
7 Taste Taste less
8 Texture Irregular
9 Fracture Rough
MATERIAL AND METHOD

Material
The plant with fruits was collected and authenticated by Dr. A. K. S. Rawat (Principal scientist and Head of
the department), Pharmacognosy and Ethenopharmacology divison CSIR-National Botanical Research Institute,
Lucknow (voucher field booklet no 254060).
Processing and extraction
100 g of hemp fruit was cut into pieces, soaked in 1 l distilled water for 3–4 hours followed by heating at
70ºC for 5–10 minute and crushed with mechanical blender which was filtered by muslin cloth after which
ethanol was added into the filtrate in (1:2) ratio to precipitate mucilage and dried in hot air oven at 40–45ºC. The
mucilage powder thus obtained was passed through sieve # 40 and stored in desiccator at room temperature
(Kirtikar & Basu 1991).
Organoleptic Evaluation: The isolated mucilage was characterized for organoleptic properties such as colour,
taste, odour, texture, and fracture (Baveja et al. 1989).
Solubility: 1 g dry mucilage powder was solubilized with polarity gradient solvents and the solubility was
determined (Baveja et al. 1989).
pH: The mucilage was weighed and dissolved in water separately to obtained 1% w/v solution. The pH of
solution was determined using digital pH meter (Baveja et al. 1989).
Swelling Index: The swelling index is the volume (in ml) taken up by the swelling of 1g of test material under
specified conditions (Baveja et al. 1989).
Gas Chromatogrphy Mass Spectroscopy Analysis

Procedure: Preparation of Trimethylsilyl ethers are a convenient way (TMS) to derivative, experimental
conditions. Trimethylsilyl ethers derivatives were prepared of several different extracts. Taken 5 mg of the
sample was suspended in 40 µl of the solution of methoxylamine hydrochloride in pyridine (20 mg ml-1). The
mixture was shaken for an hr at 37ºC before adding 70 µl of the 2,2,2-trifluoro-N-methyl-N-methyl-N-
Trimethylsilylacetamide (MSTFA). Shaking was continued for another 30 min. After that resulting derivatized
mixture of metabolites were subjected to analysis on Gas Chromatography-Mass Spectrometry (GC-MS) by
Thermo Fisher TRACE GC ULTRA coupled with DSQ II Mass Spectrometer instrument using a TR 50 ms
column (30 m × 0.25 mm ID × 0.25 × µl film thickness) carriergas, helium temperature programming 5 minutes
delay for solvent, at 50ºC temperature hold time 1.0 min, rising at 200ºC min-1 to 310ºC and finally held iso-
thermally for 15 min. The injector temperature was 230ºC and carrier flow was constant flow of 1 ml min-1 in
split mode (1:50) with injector volume 1 µl. The ion source temperature was set at 220ºC, transfer line
temperature was 300ºC and the ionization of the sample components were performed in EI mode at an
ionization voltage of -70 eV. The mass range was used from m/z 50 to 650 amu. The identification of individual
compound was made by comparison of their mass spectra with those of the internal reference mass spectra
library (NIST/ Wiley) with authentic compounds. (Pathak et al. 2014, Pandey et al. 2016).

The extracted and isolated mucilage was evaluated for various parameters like percentage yield of mucilage
was found to be 9.54% w/w, Organoleptic properties and Physicochemical parameters: solubility, moisture, pH,
colour, oduor, taste, fracture, and texture were shown in table 1. The chemical profiling of mucilage of Hibiscus
cannabinus L. after hydrolysis with the help of GC-MS analysis showed number of peaks out of which five
highest characteristic peaks were taken in to consideration. The various peaks in the chromatogram showed
various compounds of sugar with a retention time (RT), percentage area of the peaks and it was compared along
with the database spectrum of known components stored in the GC-MS library. The retention time (RT) was
found to be 37.39 min, 25.42 min, 21.82 min, 41.59 min, 27.56 min indicating the presence of Sucrose, D-
glucose, D-fructose, Maltose, D-xylopyronose respectively as shown in table 3 and figure 3. The mucilage
sample peaks were also compared to the standard peaks of the sugar shown in tables 2, 3 and figures 2, 3.
Table 2. GC-MS standards of sugar.
S.N. Retention Time Peak Area Peak Height Peak width Area %
1 22.90 121630590 264616 0.22 0.02
2 25.59 1528278979 91677442 1.12 14.20
3 26.44 942687997 74970078 0.92 8.76
4 27.46 2693765449 267116185 0.35 25.03
5 27.81 2519904925 247869616 0.51 23.42
6 37.33 89264436 8978875 0.51 2.83
6 38.08 78649343 64592351 0.98 7.11
7 40.79 520419979 31499910 1.20 4.84
Figure 2. GC-MS chromatogram of standard of sugars showing peaks and retention time indicating the presence of sugar.
Table 3. Sugar present in mucilage of Hibiscus cannabinus L.(Deccan Hemp).

S.N. Retention Time Peak Area Peak Height Peak Width Area %
1 37.41 12805627845 1277851121 1.08 23.45
2 25.42 14032948788 903750351 0.90 25.70
3 27.56 10973965269 777829934 0.84 20.10
4 24.89 4277049054 436178303 0.43 7.83
5 41.59 3647753787 371474985 0.88 6.68
6 21.82 44313511 2800628 0.47 0.08
Figure 3. GC-MS chromatogram of Hibiscus cannabinus L., mucilage showing peaks and retention time indicating the
presence of sugar
CONCLUSION
The obtained mucilage was as efficient as the known ones, so, Deccan hemp was selected for the extraction
and isolation of mucilage from natural sources. Inferred Studies that Deccan hemp is an economically important
plant with enough mucilage content of high functional value to be used as bio-polymers in pharmaceutical
formulations like binding agent super disintegrates and coating material that can be used as in place of currently
marketed synthetic polymer and provide a new way to future technologies in Novel Drug Delivery System.
ACKNOWLEDGEMENTS
Authors are very thankful to Director, Council of Scientific and Industrial Research- National Botanical
Research Institute Lucknow, (CSIR-NBRI) to provide all lab facilities for this research work. First author is
express special thanks to University Grants Commission - Rajiv Gandhi National Fellowship (UGC-RGNF) for
financial supports to this research work.
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Research article
Quantification of the effect of agriculture on forest carbon stock:

Case study of a Nigerian forest reserve
T. E. Ogana* and F. N. Ogana
Department of Social and Environmental Forestry University of Ibadan, Ibadan, Nigeria
*Corresponding Author: topeadeniyi2005@gmail.com [Accepted: 10 April 2019]
Abstract: The competition for land between forest and agriculture has been a long-time issue. The
tropical forest has been greatly reducing due to agricultural activities. There are few studies on the
quantification of forest carbon stock loss caused by encroaching agricultural activities. Therefore,
this study compared the biomass in the areas encroached by farming activities and forested areas;
and also analyzed the forest cover change in Cross River South Forest Reserve, Nigeria. Data were
obtained through forest inventory and satellite imageries. Eight sample plots of 0.25 ha were used
(plots were laid in the forested and the encroached parts of the reserve). Established allometric
equation was used to estimate the biomass. Satellite images from Landsat between 2002 and 2017
were used for the forest cover change. The results showed that there is a significant difference in
the mean aboveground carbon density of the forested part (108.6571 tC ha-1) and the encroached
part (44.1567 tC ha-1) of the reserve. The forest cover change of Forest Reserve showed that about
6,750 ha was deforested within the period with an annual rate of forest cover loss of 0.54%. It was
concluded that the farming activities have negatively impacted the quantity of carbon stock of the
forest reserve.
Keywords: Carbon stock - Forest cover change - Encroachment - Agriculture.
[Cite as: Ogana TE & Ogana FN (2019) Quantification of the effect of agriculture on forest carbon stock: Case
study of a Nigerian forest reserve. Tropical Plant Research 6(1): 106–114]
INTRODUCTION
Forest and agriculture are two major land use types that compete with each other from time immemorial. The
forest is usually seen as a reservoir of land to be pounced on when the lands used for the agricultural purpose are
no longer fertile. In Africa, Nigeria was identified to have the greatest net loss of forest area between 2010 and
2015 which was estimated to be 410,000 ha yr-1 (Keenan et al. 2015). Deforestation and degradation of tropical
forest account for 12+6 % of the total emission of CO2 in 2008 (Van der Werf et al. 2009). Kumar et al. (2010)
stated that agriculture is the proximate driver of about 80% of deforestation worldwide. According to Geist &
Lambin (2002), commercial agriculture contributes about 35% of the deforestation in Africa while local and
subsistence agriculture contribute between 27–40 % of the deforestation. The population growth which has also
been on the increase at an exponential rate as a result, demand for food has also increased. Amongst the
essential needs of man, food falls in the first category of these needs. Thus, the quest for satisfying this need
makes people encroach the forest to cultivate their food crops. According to Minnen et al. (2008), there have
been projections that most of the agricultural expansion will be in the tropics. The forest is also very vulnerable
because of the low economic value placed on them particularly in the developing countries. Not until recently
when programmes such as Clean Development Mechanism (CDM), Reducing Emissions from Deforestation
and forest degradation (REDD+) were initiated to pay for the environmental service like carbon sequestration
that the forest provides, that the forest began to increase in value. Programme like REDD+ is characterized with
its financial incentive for conserving carbon in the forest (Ramananantoandro et al. 2015).
The importance of carbon sequestration by the forest cannot be underestimated amongst the services
provided by the forest ecosystem (Thorsen et al. 2014). The ecological outcome of deforestation and forest
degradation caused by agricultural activities include reduction in canopy cover, forest quality, structure,
composition, productive capacity of the forest, loss of biodiversity and so on (Laestadius et al. 2011). The
negative impact of deforestation and forest degradation on climate cannot be overemphasized as developing
countries account for 5.8 Gigatonne CO2 emissions per year through deforestation and forest degradation
(UNFCCC 2007). The per hectare changes in carbon stocks due to land-use change account for half of the
estimates of the global carbon flux (Houghton & Goodale 2004). Shifting cultivation involves clearing forest for
cultivating agricultural crops for a period, abandoned and moving on to clear another area of the forest. This
often causes degradation of land as the fallow period are shortened due to land scarcity. It has been identified as
a carbon source in the tropics emitting about 0.22 Pg C yr-1 (Houghton & Goodale 2004).
In Nigeria, there are few studies that provide information on the quantification of the loss of carbon stock of
forest due to encroaching agricultural activities. However, this is important for proper forest management
planning and policy-making particularly for programmes such as REDD+. Therefore, the objectives of the study
were to compare the biomass in the areas encroached by farming activities and the forest areas of the forest
reserve and also to analyze the forest cover change between the period of fifteen years (2002–2017).
METHODOLOGY
Study area
This study was carried out in Cross River South Forest Reserve, Cross River state, Nigeria. Cross River
South Forest Reserve (CRS FR) was gazetted in 1956 (Fig. 1). The reserve lies in the rainforest region of the
state. The forest is a rich rainforest but has been observed to be reducing in size from 52,630 ha in 1991 to
50,432 ha in 2001 (CRSFC 2001 cited by Oyebo et al. 2011). The climatic condition of Cross River state was
described by Oates et al. (2007) to have an annual rainfall of 3,000 mm in the south and 2,500 mm in the north
of the state. The central parts of the forest receive about 4,000 mm rainfall. Rainy season is between March and
November with peaks in June/July and September. The average annual temperature is 27ºC and the mean
monthly relative humidity ranges between 78% and 91% with an average of 85% (Oates et al. 2007).
Figure 1. Map of Study Area.

Data collection
The data were obtained through forest inventory and satellite imageries. Forest inventory was carried out to
collect data for estimating the aboveground biomass and also remote sensing for analyzing the forest cover
change of the forest reserve.
Forest cover change
i. Satellite Imagery Analysis: Satellite images between the period of 2002 and 2017 were acquired from
Landsat 7 and 8 respectively from U.S. Geological Survey Global Visualization (www.glovis.usgs.gov).
Imageries with spatial resolution of 30 m acquired were between December and February (Dry Season) to
Ogana & Ogana 2019
get cloud free images as possible. Table 1 shows the attributes of the imageries acquired.
Table 1. Attributes of Landsat Imageries.
Sensor Path/Row Date acquired Band Composition
Landsat 7 188/056 01/08/2003 B1 - B5
Landsat 7 187/056 01/30/2002 B1 - B5
Landsat 8 188/056 01/06/2017 B2 - B6
Landsat 8 187/056 12/30/2016 B2 - B6
Landsat 7: Blue (1), Green (2), Red (3), NIR (4) and SWIR (5)
Landsat 8: Blue (2), Green (3), Red (4), NIR (5) and SWIR (6)
The analysis was done in QGIS (version 2.18.0) and R (R Core Team 2016). Training plots were defined to
determine the forest cover change between 2002 and 2017. The classes were Forest (FF i.e. area which was
forest in 2002 and also remained as forest in 2017), Deforestation (FN i.e. area which was forest in 2002 and
became non-forest in 2017), Regeneration (NF i.e. area which was non-forest in 2002 and became forest in
2017), Non-Forest (i.e. area which was non-forest in 2002 and also remained as non-forest in 2017), Water (W)
and Cloud (C). Random-Forest algorithm by Breiman (2001) was used to carry out a supervised classification
for the Forest cover change based on the Landsat bands in R. Fifteen validation plots (pixel size of 30 m × 30 m)
were randomly drawn for each of the classes and cross checked with the use of Google Earth.
ii. Rate of Forest cover change analysis: The rate of forest cover change was calculated using the formula of
FAO (2015) below:
⁄( )
Rate of forest cover change = ( ) (1)
Where, A1 = Area of the target forest cover at date 1, A2 = Area of the target forest cover at date 2, t1 and t2
= date 1 and date 2.
In this study, A1, A2 and t2-t1 are as follows:
Forest Inventory
Eight sample plots of 50 m × 50 m were laid in the forest reserve. Four plots in the encroached part of the
forest with farming activities and four plots were laid in the forested part of the reserve. The inventory included
the identification of tree species, measurement of diameter at breast height (DBH) and total height for trees with
DBH ≥ 10cm in the plots.
The wood density of all the tree species were gotten from Global wood density database (Zanne et al. 2009)
and African wood density database except the wood density of Treculia obovoidea N.E.Br. which was gotten
from the study of Reyes et al. (1992) who found out the wood densities of Tropical African trees to have a mean
of 0.5 g cm-3. The Above ground biomass (AGB) was computed using the below allometric equation model
(Chave et al. 2014).
× (ρ ) (2)
Where, ρ = Wood density, DBH= Diameter at Breast Height, H= Height
According to IPCC Guidelines for calculating national greenhouse gas inventories (Eggleston 2008),
Aboveground Carbon Density (ACD) was computed using the formula mentioned below:
ACD = (3)
Data were analysed using frequencies, percentages, charts and t-test. T-test was to compare the aboveground
carbon density in the encroached part and the forested part.

Quantification of the Carbon Stock
The stand density of the plots in the forested part of the reserve ranged between 168 and 312 trees ha-1
compared to the plots in the agriculture encroached part of the reserve which ranged between 60 and 148 trees
ha-1 (Table 2). Two plots of the forested part fell within the range of 245 and 467 trees ha-1 recommended for the
stand densities of tropical forests (Campbell et al. 1992).
Table 2. Basal area (m2 ha-1) and Stand densities (N ha-1) per hectare of the forest and the farm.
Basal area Stand densities
Plot No. State
(m2 ha-1) (N ha-1)
1 23.197 312 Forest
2 15.941 252 Forest
3 14.215 168 Forest
4 17.362 212 Forest
1 3.760 136 Farm
2 8.971 60 Farm
3 8.705 144 Farm
4 10.966 148 Farm
The diameter distribution of the encroached part (Farm) of the FR (Fig. 2) shows that 65% of the trees are in
the diameter class of 10–20 cm. The reason majority of trees of this class are left is because the tree crowns are
not so big to cast shade on the crops the farmers have planted. In the forested part (Fig. 3), less than 50% of the
trees are in the diameter class of 10–20 cm and 40.4% fell in 20–40 cm diameter class. The prominent inverse J-
shape of the diameter distribution curve of the forested part of the FR is an exact representative how a natural
forest should be. However, both the forest part and the encroached part of the FR have high regeneration
potential. According to the study of Baishya et al. (2009), the FR has potential for high carbon sequestration
because of the presence of large number of trees in the small DBH classes.
Figure 2. Diameter distribution of the Encroached part of the FR
Figure 3. Diameter distribution of the forested part of the FR

Ogana & Ogana 2019
The Aboveground Carbon Density (ACD) of the forest and farm depicts that the minimum and maximum
ACD estimated in the forest and farm plots are (80.01 and 138.20 tC ha-1) and (8.802 and 58.930 tC ha-1),
respectively (Fig. 4). The mean values of the forested part (forest) and the encroached part (farm) of the forest
were 108.6571 and 44.1567 tC ha-1, respectively. Comparing their mean values using t-test, it showed that there
is a significant difference in the ACD with a p-value of 0.0126. Comparing the carbon in the forested part and
the farm of the reserve, the difference between the mean value of the carbon stock is large (64.5 tC ha-1).
Although the ACD of the forested area is still quite lower than the aboveground carbon density for a West
African tropical forest which is about 143.35 tC ha-1 (Lewis et al. 2013). Baishya et al. (2009) reported the
aboveground carbon density of India’s natural forest to be about 153 tC ha-1. This indicates a very great impact
encroachment of farming activities have on the carbon stock of the forest thus contributing to the atmospheric
CO2 causing climate change.
ACD (tC ha-1)
Figure 4. A boxplot of the Aboveground Carbon Density of the Forested and Encroached part of the Reserve
Although the farmers do not establish a monoculture field of arable crops, they leave some trees on the farm.
This is not because of their love or the knowledge of the importance of the trees but rather their inability to cut
some of these trees (cost and energy). Most of these farmers set the trees on fire to kill the trees before they start
cultivation on land (Fig. 5). This is similar to the findings of Olarewaju et al. (2017), who reported that forests
are being degraded by communities to support their livelihood. Jose & Bardhan (2012) reported the total
estimated aboveground biomass carbon in agroforest plot was 20.8 tC ha-1. This is quite lower compared to the
mean value of carbon (44.1567 tC ha-1) of the farm plots of this study, but the latter will result into release of
more CO2 into the atmosphere as the encroachers will still get rid of the trees by burning them. It is obvious that
the difference in the basal area and ACD between the forested part and the farm is due to the farming activities
which has encroached the forest reserve.
Figure 5. Woman burning a tree on the cultivated land inside the forest reserve.
Forest cover change of the Forest Reserve

The validation matrix (Table 3) was used to calculate the accuracy of the classification. The values of the
specific index (figure of merit) calculated for each of the classes were: deforestation (0.61), forest (0.75),
regeneration (0.87), non-forest (0.75), water (0.87) and cloud (0.67).
Cohen Kappa’s statistics function was used to determine the accuracy of the classification (Kappa’s value is
either less than or equal to 1, where 1 corresponds to a perfect classification). The corrected accuracy (Cohen’s
Kappa) of 0.83 was obtained for the forest cover change to cross check if a good classification was done in this
study. Overall, the corrected accuracy shows a good classification.
Table 3. Confusion matrix for validated points.
C FF FN NF NN W Actual
C 10 0 0 0 0 0 10
FF 0 15 2 1 0 2 20
FN 3 0 11 0 0 0 14
NF 0 0 0 13 0 0 13
NN 2 0 2 1 15 0 20
W 0 0 0 0 0 13 13
Predicted 15 15 15 15 15 15 90
Note: C, Cloud; FF, Forest; FN, Deforestation; NF, Regeneration; NN, Non-Forest; W, Water.
The result shows that the Forest cover change between 2002 and 2017 indicates that a lot of deforestation
took place in CRS FR during the fifteen-year period (Fig. 6). About 8% of the total area of CRS FR was
deforested during the period (Table 3). Observation on field confirms this as there were a lot of farming
activities taking place within the forest reserve. The imagery analysis showed that there were areas in the forest
reserve that were not forested in 2002 but in 2017 it was obvious there was natural regeneration of about 1.6%.
The areas that were forested in 2002 and still remains as forest in 2017 occupies about 78.7% of the entire area
of the forest reserve area. The forest cover change of CRS FR showed that an area of about 6,750 ha was
deforested between 2002 and 2017, though there was natural regeneration of about 1,250 ha (Table 4).
Figure 6. Forest Cover Change Map of Cross River South Forest Reserve between 2002 and 2017.
The annual rate of forest cover loss was 0.54%. This shows that in the nearest future, there would be further
encroachment of farming activities in the forested area of the reserve if it is business-as-usual. Using this rate of
forest cover loss to forecast, the implication is that in another 20 years’ time the forest cover would decrease by
6977.08 ha or more. The rate of forest cover loss observed in this study is similar to that of India’s forest cover
loss which ranged between 0.2 and 0.65 (Reddy et al. 2017). The result is also similar to the global Gross Forest
Cover Loss estimated to be 0.6% per year (Hansen et al. 2010). FAO (2015) reported that the global annual rate
of forest cover loss to be 0.08%; this was attributed to the forest loss in the Tropics while there was forest
Ogana & Ogana 2019
expansion in the Temperate and Boreal regions (Sloan & Sayer 2015).
Table 4. Forest cover change of the forest reserve between 2002 and 2017.
FR Total area (ha) Forest (ha) Deforested area (ha) Regeneration (ha)
CRS 80534.07 63347.76 6750.72 1254.87
Although an indefinite moratorium on timber extraction was declared in Cross River state in 2008 (Ministry
of Environment 2014), it has not been able to curtail deforestation as more of the forest are being converted for
agricultural purpose in this FR. This agrees with the findings of Brandt & Agrawal (2015), who found out that
some forest policies aided deforestation in the Congo basin. The findings of Suleiman et al. (2017) showed that
the major factors causing forest cover change in Falgore game reserve, Nigeria are the increase of fuelwood
harvest, overgrazing and expansion of crop cultivation. However, Ochege & Okpala-Okaka (2017) generalized
that forest cover loss is as a result of human disturbance. The findings of Orimoogunje et al. (2009) in the study
on land use changes and forest reserve management of Oluwa forest reserve Nigeria, showed that a large portion
of the forest reserve is being depleted as a result of agriculture. In Southeast Asia, crop production has also been
a major cause of deforestation for the past three decades (Imai et al. 2018). The study of Hor et al. (2014) in
Ratanakiri Province Cambodia identified the expansion of agriculture especially swidden practice as a
significant factor contributing to the decrease of forest area.
During this study, interviews were conducted to get information from the local people. The people, however,
said they go mainly into the forest reserve for their farming activities because of the fertility of the forest soil.
They also confirmed that the encroachment of the reserve was aggravated as a result of the early starters who
did not face any penalty for their actions (moving into the forest to farm). However, population growth and
poverty is another factor that also enhanced the farmers encroaching the reserve. The issue of population growth
resulting to deforestation is consistent with previous studies (Imai et al. 2018).
CONCLUSION
Quantitative analysis of the ACD of the encroached part and the forested part of the FR showed that there is
a significant difference. It can be concluded that farming activities in FR have a negative impact on the carbon
stock of the forest. Also, there is a loss in the forest cover of the FR within the period of fifteen years. The
continuous forest cover loss will result to release of more greenhouse gases. This means the forest will be a
carbon source instead of a sink. The FR should not the neglected but should be well managed for its rich carbon
stock potential as the regeneration potential in both the encroached part and the forested part is high. Thus, it is
important that the state forestry department should plan for the restoration and management of this forest
reserve.
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Short communication
Hygrophila madurensis (N.P. Balakr. & Subram.) Karthik. &

Moorthy: An overlooked endemic species of Tamil Nadu, India
C. P. Muthupandi, R. Kottaimuthu#,* and K. Rajendran
Department of Botany, Thiagarajar College, Madurai-625 009, Tamil Nadu, India
#
Current Affiliation: Department of Botany, Alagappa University, Karaikudi-630 003, Tamil Nadu, India
*Corresponding Author: kottaimuthu@yahoo.co.in [Accepted: 11 April 2019]
[Cite as: Muthupandi CP, Kottaimuthu R & Rajendran K (2019) Hygrophila madurensis (N.P. Balakr. &
Subram.) Karthik. & Moorthy: An overlooked endemic species of Tamil Nadu, India. Tropical Plant Research
6(1): 115–118]
INTRODUCTION
The family Acanthaceae is positioned under the order Lamiales and belong to the core class Euasterids I of
Core Eudicots (Chase & Reveal 2009). According to the recent estimate (Karthikeyan et al. 2009) 593
Acanthaceae taxa (475 species and 118 varieties) are present in India. The genus Hygrophila R.Br. belongs to
the tribe Ruellieae of family Acanthaceae (Scotland & Vollessen 2000) and comprises about 100 species (Hu &
Daniel 2011). India is known to have 18 species (Karthikeyan et al. 2009, Sunojkumar & Prasad 2014), of these
H. madurensis and H. thymus are endemic to Tamil Nadu (Singh et al. 2015, Kottaimuthu et al. 2018).
During the course of our recent studies on the wetland plants of Madurai District, we have collected an
interesting species of Acanthaceae that is characterized by distinctly pedicellate flowers, pedunculate cymes and
linear–oblong capsules. Critical studies with pertinent literature; it is identified as Hygrophila madurensis (N. P.
Balakr. & Subram.) Karthik. & Moorthy (Balakrishnan & Subramanyam 1963, Balakrishnan 1988, Raja et al.
2015). A perusal of literature revealed that this species is listed as critically endangered and endemic to Tamil
Nadu, found along the foothills of the Eastern Ghats (Balakrishnan 1988, Nayar 1996, Walter & Gillett 1998,
Reddy et al. 2006, Arisdason 2011). However, this species was not included in Acanthaceae of Eastern Ghats
(Pullaiah et al. 2011). A detailed description, photographs, associated species and threat status are provided for
easy identification and conservation of this little known endemic species.
Hygrophila madurensis (N. P. Balakr. & Subram.) Karthik. & Moorthy, Fl. Pl. India 22. 2009. Raja et al. in J.
Threat. Taxa 7(9): 7582. 2015.
Santapaua madurensis N.P. Balakr. & Subram. in J. Indian Bot. Soc. 42: 411. 1963; G. R. Kumari in A. N.
Henry, G. R. Kumari & V. Chithra, Fl. Tamil Nadu, Ind., Ser I: Analysis 2: 160. 1987; N. P. Balakr. in Red
Data Book Indian Pl. 2: 7. 1988. (Fig. 1)
Herbs, 10–35 cm high; branchlets decumbent, arising from base; stems quadrangular, swollen at nodes.
Leaves opposite, decussate, thin, membranous, glabrous, lanceolate or oblong–oblanceolate, base cuneate,
margin minutely crenulate, acute or subacute at apex, lateral nerves 5–7 pairs, prominent below; rhaphides
scattered on upper surface, petioles 2–3 mm long. Flowers in axillary open dichasial cymes becoming sympodial
and unilateral, usually shorter than leaves; primary peduncle ca. 5 mm long; internodes ca. 3 mm long; bracts
linear, acute, 2–5 mm long; pedicels ca. 1 mm long. Calyx 5 lobes, free, sub equal, linear, acute, 4–5×1 mm.
Corolla purple, bi-lipped, 5–10 mm long; tube funnel-shaped, broad, pubescent inside; upper lip bifid,
emarginate, lobes rounded; lower lip trifid, lobes obtuse, rounded. Stamens 4, didynamous; filaments linear,
filiform, glabrous, 2–4 mm long; anthers oblong, ellipsoid, 1.0–1.3 mm long. Ovary pubescent, oblong or
oblong–ellipsoid, 2 mm long, 2-celled with many ovules; style linear, 4mm long, hairy; stigma simple. Capsules
linear-oblong, flat, sessile, 6–8 mm long; seeds bearing throughout the length of the capsule; retinacula minute,
conical, straight, slender. Seeds 20–40, small, ellipsoid or ovoid–ellipsoid, compressed, 107.5–108.6 × 163.24 –
164.34 μm, glandular puberulous, when wet; hairs 24–27.61 μm long
Flowering & Fruiting: November to March.
Muthupandi et al. 2019
Figure 1. Hygrophila madurensis (N. P. Balakr. & Subram.) Karthik. & Moorthy: A, Flowering twig; B, Calyx; C, Stamens;
D, Ovary; E, Capsule; F, Seed close up.
Specimens examined: INDIA, Tamil Nadu, Madurai Dis., 08.03.2018, Sakkimangalam, Ayyankulam and C. P.
Muthupandi 65 (Thiagarajar College Herbarium); 10.03.2018, C. P. Muthupandi & R. Kottaimuthu 112
(Thiagarajar College Herbarium).
Distribution: INDIA (Tamil Nadu), Endemic.
Biotic Association: A single population of about 20 individuals were observed in the present locality and it is
often associated with the following species, Bergia ammannioides, Ludwigia perennis, Ammannia baccifera,
Echinochloa colona, Vahlia dichotoma, Lindernia parviflora, Scoparia dulcis, Chloris barbata, Mollugo
pentaphylla, Desmodium triflorum, Fimbristylis sp., Leptochloa sp., Ludwigia hyssopifolia, Prosopis juliflora,
Coldenia procumbens, Euphorbia thymifolia, Cyanodon dactylon, Nothosaerva brachiata, Aeschynomene
indica, Sphaeranthus indicus, Heliotropium indicum, Cyperus difformis, Phyllanthus maderaspatensis,
Fimbristylis milliacea, Corchorus fascicularis, Epaltes divaricata and Eclipta alba.
Nomenclature: Hygrophila madurensis was originally described by Balakrishnan & Subramanyam (1963)
under the genus Santapaua as Santapaua madurensis. However, later authors (Heine 1962 & 1971, Cramer
1989 & 1998, Sidwell 1999), agreed to follow the broader generic concept and placed Adenosma, Asteracantha,
Cardanthera, Hemiadelphis, Nomaphila, Plaesianthera and Synnema under Hygrophila and this was accepted
in the classification of Acanthaceae (Scotland & Vollessen 2000). Following this broader concept, Karthikeyan
et al. (2009) reduced the genus Santapaua into Hygrophila.
The type specimen was collected by K. Subramanyam near Nallakulam in Alagar Hills. Later Ravikumar
(1993) relocated this species from the type locality and there after it was also collected by Arulappan in
Narthamalai in Pudukkottai district (Balakrishnan 1988). Now the population becomes endangered due to
narrow distribution and over-grazing in the natural habitat (Reddy et al. 2006).
CONCLUSION
The range extension of endemic species will provide a wealth of research opportunities for ecologists and
conservation biologists in understanding the key drivers of endemism (Kottaimuthu 2017, Kottaimuthu et al.
2018). Hitherto this species is known only to very few locations. Hence, periodical assessment in all the known
localities and searching for other sites of occurrence in Tamil Nadu is strongly recommended. Moreover, all the
known population is prone to maximum human interference hence urgent conservation measures are
recommended for H. madurensis.
ACKNOWLEDGEMENTS
We would like to thank the Management and Principal, Thiagarajar College, Madurai, for the help provided
during the study. We are thankful to retired Dr. G. V. S. Murthy, Scientist G, Botanical Survey of India,
Southern Regional Centre, Coimbatore for granting permission to consult the herbarium and library.
REFERENCES
Arisdason W (2011) Hygrophila madurensis. In: IUCN 2011. IUCN Red List of Threatened Species. Version
2011.2. <www.iucnredlist.org>. Downloaded on 10 April 2018.
Balakrishnan NP & Subramanyam K (1963) A new genus of Acanthaceae from Peninsular India. Journal of
Indian Botanical Society 42: 411–415.
Balakrishnan NP (1988) Santapaua madurensis. In: Nayar MP & Sastry ARK (eds) Red Data Book of Indian
Plants, Vol. II. Botanical Survey of India, Howrah.
Chase MW & Reveal J (2009) A phylogenetic classification of the land plants to accompany APG III. Botanical
Journal of Linnaean Society 161: 122–127.
Cramer L (1989) The Hygrophila complex (Acanthaceae) in India and Ceylon. Nordic Journal of Botany 9(3):
261–263.
Cramer L (1998) Acanthaceae. In: Dassanayakae MD & Clayton WT (eds) A Revised Handbook to the Flora of
Ceylon, Vol. X. Amerind Pub. Co. Pvt. Ltd., New Delhi.
Heine H (1962) Notes on Some West African Acanthaceae: The reduction of the genus Asteracantha Ness to
Hygrophila R. Br. Kew Bulletin 16: 171–173.
Heine H (1971) Notes sur Les Acanthacees Africaines: Hygrophila R.Br. Adansonia 11(4): 656–659.
Hu J & Daniel TF (2011) Acanthaceae: Hygrophila. In: Wu ZY, Raven PH & Hong DY (eds) Flora of China,
Vol. 19. Missouri Botanical Garden Press, St. Louis, USA.
Karthikeyan, S, Sanjappa M & Moorthy S (2009) Flowering Plants of India - Dicotyledons (Acanthaceae -
Avicenniaceae). Botanical Survey of India, Kolkata.
Kottaimuthu R (2017) Crotalaria heyneana (Fabaceae) in Eastern Ghats of India: A new distributional record.
Journal of Economic and Taxonomic Botany 40(3–4): 156–157.
Kottaimuthu R, Muthupandi CP & Rajendran K (2018) Ecbolium viride (Forssk.) Alston var. chandrasekariana
Remadevi & Binojk. (Ruellieae: Acanthaceae): A new report for Tamil Nadu. NeBIO 9(2): 215–218.
Nayar MP (1996) Hot spots of Endemic Plants of India, Nepal & Bhutan. TBGRI, Thiruvananthapuram.
Pullaiah T, Rani SS & Karuppusamy S (2011) Flora of Eastern Ghats, Vol. IV. Regency Publications, New
Delhi.
Raja P, Soosairaj S, Dhatchanamoorthy N & Kala A (2015) A new distribution record for the critically
endangered Madura swamp weed Hygrophila madurensis (N.P. Balakr. & Subr.) Karthik & Moorthy
(Acanthaceae). Journal of Threatened Taxa 7(9): 7581–7583.
Ravikumar K (1993) Systematic Studies on the Dicotyledonous Plants of Madurai District, Tamil Nadu, India,
(Ph.D. Thesis). Bharathiar University, Coimbatore.
Muthupandi et al. 2019
Reddy CS, Brahmam M & Raju VS (2006) Conservation prioritization of endemic plants of Eastern Ghats,
India. Journal of Economic and Taxonomic Botany 30: 755–772.
Scotland RW & Vollesen K (2000) Classification of Acanthaceae. Kew Bulletin 55(3): 513–589.
Sidwell K (1999) The taxonomic position of the Sri Lankan species Brillantaisia thwaitesii (T. Anderson) L. H.
Cramer. (Acanthaceae). Kew Bulletin 54(1): 215–219.
Singh P, Karthigeyan K, Lakshminarasimhan P & Dash SS (2015) Endemic Vascular Plants of India. Botanical
Survey of India, Kolkatta.
Sunojkumar P & Prasad MG (2014) Taxonomic reinstatement of an endemic Hygrophila (Acanthaceae)
subsequent to its rediscovery after 180 years from India. Rheedea 24(1): 12–15.
Walter KS & Gillett HJ (1998) IUCN Red List of Threatened Plants. Compiled by the World Conservation
Monitoring Centre. IUCN, Gland.
Research article
Tree height prediction models for two forest reserves in Nigeria

using mixed-effects approach
F. N. Ogana
Department of Social and Environmental Forestry, University of Ibadan, Ibadan, Nigeria
Corresponding Author: ogana_fry@yahoo.com [Accepted: 12 April 2019]
Abstract: Height-diameter models for predicting tree height are essential for routine forest
inventory. These models can be developed using fixed-or mixed-effects approach. Few studies
have applied the mixed-effect approach to developed height prediction model for the natural forest
in Nigeria. Therefore, in this study, the mixed-effect modelling approach was used to develop
height prediction models for Ikrigon and Cross River South (CRS) Forest Reserves, Nigeria. Data
consist of 776 and 438 height-diameter pairs from Ikrigon and CRS Forest Reserves, respectively.
Five 2-parameters and five 3-parameters height-diameter models were evaluated including Nalund,
Wykoff, Curtis, Meyer, Michaelis-Menten, Chapman-Richards, Ratkowsky, Korf, Logistic and
Gompertz. Model fitting was done in two stages: Fixed-effect approach was used in the first stage
wherein candidate models were selected and refitted in the second stage using mixed-effect
approach. Adjusted coefficient of determination, root mean square error, mean absolute bias,
Akaike information and Bayesian information criterion were used to assess the models. The results
showed that Gompertz and Meyer models were more consistent. Gompertz and Meyer had
adjusted coefficient of determination, root mean square error, mean absolute bias, Akaike
information criterion and Bayesian information criterion of 0.642, 4.457, 3.501 and 4591.487,
4638.028; and 0.638, 4.482, 3.541, 4592.008, 4619.933, respectively for Ikrigon and 0.724, 4.076,
3.215, 2536.148 and 2576.970; and 0.711, 4.176, 3.273, 2536.352 and 2560.845, respectively for
CRS. The mixed-effect approach improved tree height predicting of the forest stands. These
models are recommended for estimating tree height in the forest reserves.
Keywords: Ikrigon - Cross River South forest - Height-diameter models - Gompertz - Meyer -
Mixed-effect.
[Cite as: Ogana FN (2019) Tree height prediction models for two forest reserves in Nigeria using mixed-effects
approach. Tropical Plant Research 6(1): 119–128]
INTRODUCTION
Tree diameter and height are routinely measured in forest inventory because they are the fundamental
variables from which other stand characteristics are derived (Ogana 2018). Diameter and height are key
components for analysis of forest stand structure, estimation of volume (Gomez-Garcia et al. 2014), biomass,
basal area, density and may reflect the competitive position of a tree in a stand (West 2015). Also, the height of
the tallest trees in forest stand is the basis for assessing site productive capacity (Calama & Montero 2004, West
2015). Tree diameter at breast height (1.3 m above the ground) can easily be measured with diameter or girth
tape with accuracy and low cost (Ferraz-Filho et al. 2018). However, the measurement of tree height is tedious
and relatively complex especially in forest with contiguous canopy and rugged terrain (Krisnawati et al. 2010,
Mehtätalo et al. 2015, Ozcelik et al. 2018). In view of this, only subsample of trees is measured for height.
Most studies have relied on the use of height-diameter (h-d) models to estimate the height of trees. Some
common h-d models that have been used in quantitative forestry include: Logistic (Peer & Reed 1920), Korf
(Lundqvist 1957), Chapman-Richards (Richards 1959), Curtis (Curtis 1967), Wykoff (Wykoff et al. 1982),
Ratkowsky (Ratkowsky 1990) etc. Many published forestry literatures exist on h-d models (Mehtätalo 2005,
Trincado et al. 2007, Temesgen et al. 2008, Krisnawati et al. 2010, Coble & Lee 2011, Mehtätalo et al. 2015,
Eby et al. 2017, Ogana 2018) for plantation and natural forest stand.
Ogana 2019
Modelling h-d relationship could be achieved either by using a fixed-effect or mixed-effect approach. In
fixed-effect approach, the ordinary least square or nonlinear least square technique is used; but the assumption
of independence is violated due to hierarchical data structure, that is, tree in plot (Ferraz-Filho et al. 2018,
Ozcelik et al. 2018). Measurement of trees within plot cannot be said to be independent. Such assumption of
independence can be readily overcome by mixed-effect model approach. In this approach, both the within and
between plot variation of the tree variable of interest are accounted for. Mixed-effect model has fixed and
random parameters (factors). One major advantage of mixed-effect model approach is that it “allows prediction
of a typical response, using only fixed-effect, and a calibrated response where random effects are predicted and
included in the model using measurements of heights from a sample of trees” (Burkhart & Tomé 2012).
Different studies including Sharma & Parton (2007), Budhathoki et al. (2008), Paulo et al. (2011),
VanderSchaaf (2014), Mehtätalo et al. (2015), Zang et al. (2016), Ferraz-Filho et al. (2018) and Ozcelik et al.
(2018) have applied the mixed-effect approach to model h-d relationship especially in even-aged forest
plantation. However, few studies have applied the nonlinear mixed-effects modelling approach for constructing
h-d models for natural forest stand in Nigeria. Therefore, the main objective of this study was to apply the
mixed-effect technique to model the height-diameter relationship in the natural stands of Ikrigon and Cross
River South Forest Reserves in Nigeria.

Data
The data for this study came from Cross River South Forest Reserve (CRS FR) and Ikrigon FR located in
Cross River Sate, Nigeria. CRS FR lies between latitude 5°50.978′ to 5°51.029′ N and longitude 8°29.833′ to
8°29.424′ E and occupies an area of 80,534.07 ha. Ikrigon FR lies between latitude 6°17.597′ to 6°17.862′ N and
longitude 8°35.597′ to 8°35.276′ E and occupies an area of 542.7 ha (Adeniyi 2017). Data were obtained from
20 (10 from each reserve) temporary sample plots (TSPs) of size 0.25 ha using systematic sampling. The plots
were laid at alternate pattern along the transect line at 100 m interval. A diameter threshold of ≥ 10 cm was
used. Diameter and height were measured to the nearest 0.1 cm and 0.1 m with diameter tape and vertex II,
respectively. The descriptive statistics of the inventoried data are presented in table 1. Species composition of
the reserves are presented in appendix I and II.
Table 1. Descriptive statistics of the measured variables.
Statistics
FR Variables
Mean Max Min SD
CRS Diameter 25.3 103.1 10.0 16.4
N = 438 Height 20.4 44.1 6.4 7.8
Tree species = 63
Ikrigon Diameter 29.9 125.0 10.0 15.9
N = 776 Height 24.1 43.8 5.7 7.5
Tree species = 74
Note: SD, Standard deviation.
Height-Diameter models
Table 2. Height-Diameter Models.
Mode name H-D Model References
Naslund H= Näslund (1937), Mehtätalo et al. (2015)
( )
Wykoff ⁄( ) Wykoff et al. (1982)
H=
Curtis H= ( ⁄( )) Curtis (1967), Mehtätalo et al. (2015)
Meyer H= ( ) Meyer (1940)
Michaelis-Menten (MM) H= Menten & Michaelis (1913), Huang et al. (1992)
( )
Richards H= ( ) Richards (1959), Mehtätalo et al. (2015)
Ratkowsky ⁄( ) Ratkowsky (1990), Mehtätalo et al. (2015)
H=
Korf H= ( ) Lundqvist (1957), Krisnawati et al. (2010)
Logistic H= Mehtätalo et al. (2015), Ogana (2018)
( ))
Gompertz H= ( Gompertz (1825), Mehtätalo et al. (2015)
Note: a, b, c = model parameters; bh = 1.3 (a constant used to account that DBH is measured at 1.3m above the
ground); H = height (m); D = diameter (DBH); e, base of the natural logarithm
There are several height-diameter models that have been applied to forestry with varying degree of success.
No single height-diameter model is suitable for all data structure. In this study, ten height-diameter models were
fitted to the data from Ikrigon and CRS forest reserves. Five 2-parameters and five 3-parameters height-diameter
models were evaluated including Nalund, Wykoff, Curtis, Meyer, Michaelis-Menten (MM), Chapman-Richards
(Richards), Ratkowsky, Korf, Logistic and Gompertz models (Table 2).
Mixed-effects model
Model fitting was done in two stages. The first stage involved fitting the ten (10) models using fixed-effects
modelling approach. In this approach, the between plot-height variation was ignored. The nonlinear least square
estimation was used. Conversely, in mixed-effects modelling both the within and between plot-height variation
were accounted for. Mixed-effects model has both fixed parameters and random parameters. While the fixed
parameter estimates the average height of the stand, the random component provides information on the
variation of height across the plots. Mixed-effects could be linear mixed-effects (if the base model is in linear
form) or nonlinear mixed-effects (if the base model is nonlinear). In this study, the nonlinear mixed-effects was
adopted since all the base models were nonlinear. Only the best two height-diameter models from the first stage
that were refitted using mixed-effects approach. The nonlinear mixed-effect was of the form of Mehtätalo et al.
(2015):
( )
Where, is the height of tree j on plot i and corresponding diameter (DBH) represented by . The vector
is the parameters (a, b and c) for a typical plot in the entire population of plots; (αi, γi and λi) is the plot
effect – it expresses the difference in the parameters of plot i from the typical plot. “Plot effects are assumed to
have a common multivariate normal distribution with mean 0 and variance-covariance matrix var ( ) = D for all
values of i” (Mehtätalo et al. 2015); and is the residual errors which are assumed to be normal, identical,
independent with zero mean and constant variance var ( ) = σ2.
Model assessment was based on adjusted coefficient of determination (R2adj), root mean square error
(RMSE), mean absolute bias (MAB), Akaike information criterion (AIC) and Bayesian information criterion
(BIC). To decide on the candidate models, a rank was assigned to each H-D model based on each fit index
(Tewari & Singh 2018). The smaller the rank, the better the model. These ranks were thereafter summed up to
reach a final fit rank for each model which shows the individual model performance with respect to all fit
indices considered in this study. Residual analysis was also used to check if the models violate the assumption
of homoscedasticity i.e. constant variance. Normality test of the residual was carried out using Shapiro-Wilk at
5% level. All statistical analyses including model fitting, residual analysis and normality test were carried out in
R (R Core Team 2017).

The models for predicting tree height in Ikrigon and CRS Forest Reserves have been developed and the
result for the fixed-effect approach are presented in table 3. The result showed that 2-parameter Meyer and 3-
parameter Gompertz h-d models provided the best prediction of tree height in the reserves. In Ikrigon FR,
Gompertz h-d model had R2adj, RMSE, MAB, AIC and BIC of 0.581, 4.823, 3.815, 4652.301 and 4670.918; and
Meyer had 0.581, 4.825, 3.815, 4650.780 and 4664.742, respectively. Similarly, in CRS FR Gompertz h-d
model had R2adj, RMSE, MAB, AIC and BIC of 0.668, 4.474, 3.595, 2563.620 and 2579.949; and Meyer had
0.666, 4.490, 3.588, 2564.664 and 2576.910, respectively. Furthermore, Meyer and Gompertz had the smallest
rank sum of 8 and 14, respectively in Ikrigon FR. Both functions had 11 in CRS FR data.
The performance of Naslund, Wykoff, Curtis, Michaelis-Menten (MM), Richards, Ratkowsky, Korf and
Logistic h-d models were comparable to Gompertz and Meyer. In fact, only marginal differences exist in the fit
indices of the h-d models. As a rule of thumb, two models are said to be the same if the difference in their AICs
(∆AIC) values is less than 2 (Tewari & Singh 2018, Ogana 2018). The difference in AIC values for all models
were less than two in both Ikrigon and CRS forest reserves. Furthermore, Shapiro-Wilk (S-W) test of normality
showed the ten h-d models did not violate the assumption of normality at 5% level of significance. All models
had p-value > 0.05. Also, the models followed the expected h-d curve of monotomic increment, inflection point
and asymptotic value for both reserves (Fig. 1).
Mehtätalo et al. (2015) also found these models to be efficient in predicting tree height for a wide
geographic region. However, the authors concluded that the 2-parameter Curtis and Naslund were more
Ogana 2019
consistent than others. In this study, the Curtis and Naslund models performed relatively well. Though all
models were good in this study, but Gompertz and Meyer were selected as the candidate h-d models for Ikrigon
and CRS forest reserves. This is because there were wide differences in the rank sum of Gompertz and Meyer
models compared to the other function considered in this study. And as such, they were further fitted with
nonlinear mixed-effect modelling approach. So that between plot variation can be accounted for.
The result of the nonlinear mixed modelling approach for Gompertz and Meyer are presented in table 4. The
result showed that there was improvement in tree height prediction for Gompertz and Meyer in both reserves.
The estimated variance-covariance matrices of Gompertz and Meyer models were positive definite. The R2adj
increased considerably while the RMSE, MAB, AIC and BIC for the candidate h-d models decreased compared
to the fixed approach. For example, the RMSE of Gompertz decreased by 0.366 m and 0.398 m while Meyer
decreased by 0.343 and 0.314 in Ikrigon and CRS FR, respectively (the values were obtained by subtracting the
models’ fit indices in table 3 and 4). This gives an indication of the efficiency of the mixed-effect modelling
approach. This improvement is not far-fetch from the fact that this approach was able to capture both within and
between plot variations in tree height.
A B
Figure 1. Fitted height-diameter curves of the ten models for (A) Ikrigon and (B) CRS forest reserves.
Temesgen et al. (2008) used RMSE and bias to evaluate the fixed-effect prediction part of a mixed-effects
model and the traditional fixed-effect model. The authors observed relatively poor predictive performance with
the fixed-effect part of a mixed-effects model. This is acceptable if estimation of the mean height of trees is of
importance; though fixed effect models does not give plot-specific estimation for a plot of interest (Mehtätalo et
al. 2015). The plot-specific prediction is improved by random-effect part of the mixed-effect models. Also,
Ozcelik et al. (2018) found the mixed-effect model for Chapman-Richards to be better than fixed-effect and
quantile regression for two Turkish tree species. In this study, the Chapman-Richards function was not further
considered because its rank sum based on the fit indices was relatively large.
Table 4. Fitted nonlinear mixed-effects height-diameter models and the fit indices.
FR Model Parameter Estimates R2adj RMSE MAB AIC BIC
Ikrigon Meyer a 34.662 0.638 4.482 3.541 4592.008 4619.933
b 0.042
sd (αi) 2.948
sd (γi) 0.008
corr (αi, γi) -0.67
σ2 4.525
Gompertz a 32.666 0.642 4.457 3.501 4591.487 4638.028
b 1.896
c 0.063
sd (αi) 2.297
sd (γi) 0.404
sd (λi) 0.005
corr (αi, λi) -0.619
corr (γi, λi) 0.892
σ2 4.501
CRS Meyer a 34.315 0.711 4.176 3.273 2536.352 2560.845
b 0.038
sd (αi) 5.826
sd (γi) 0.006
σ2 4.217
Gompertz a 34.482 0.724 4.076 3.215 2536.148 2576.970
b 1.760
c 0.048
sd (αi) 4.845
sd (γi) 0.257
sd (λi) 0.006
Ogana 2019
corr (αi, γi) 0.203

corr (αi, λi) -0.584
corr (γi, λi) 0.676
σ2 4.14
The trend in residual was analysed by plotting the model residual against the predicted height in 10 classes
in the data set for Meyer and Gompertz h-d model. Thin and thick lines were added to the class-specific means.
The thin line corresponds to the class-specific standard deviations while the thick line corresponds to the 95%
confidence intervals (CIs) of class mean. Thick lines that do not intersect the horizontal line at y equal 0 i.e., y =
0 are highlighted in red colour (Fig. 2 & 3). This was used to ascertain the model accuracy and the assumption
of homoscedasticity. The graphs show that the Gompertz Meyer models for predicting tree height in the reserves
had all 95% CIs for means that is, the thick lines intersected the x-axis except for Ikrigon. The Meyer h-d
predicted poorly in the first class of Ikrigon data (highlighted in red colour). The implication is that Gompertz h-
d model fit the Ikrigon and CRS forest reserve data relatively well compared to Meyer h-d. Also, the vertical
thin lines that is, class-specific standard deviations did not show increasing variance as a function of tree height
in the two reserves; which implies that the assumption of constant variance (homoscedasticity) was not violated.
A B
Figure 2. Residual plots of the (A) Meyer h-d model and (B) Gompertz h-d model in the data set from Ikrigon Forest
Reserve.
A B
Figure 3. Residual plots of the (A) Meyer h-d model and (B) Gompertz h-d model in the data set from CRS Forest Reserve.
CONCLUSION
The application of nonlinear mixed models for fitting height-diameter remains an effective and efficient
technique for improving the approximation of tree height. This technique improved the tree height prediction in
Ikrigon and CRS forest reserve. The 3-parameter Gompertz h-d and 2-parameter h-d model had the best
prediction. One major limitation of this study is that the fitted functions do not account for species variation.
Nevertheless, the functions can be used as proxy for estimation of tree height in Ikrigon and Cross River South
forest reserves.
ACKNOWLEDGEMENTS
The author is thankful to Ogana T.E. for making her data available for this study. Special thanks to the
anonymous reviewers for their helpful comments which improved the quality of the paper.
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Appendix I: Tree species composition of Cross River South Forest Reserve.

S.N. Species S.N. Species
1 Afzelia bipindensis 38 Irvingia gabonensis
2 Albizia gummifera 39 Irvingia wombulu
3 Albizia zygia 40 Khaya ivorensis
4 Allanblackia floribunda 41 Klainedoxa gabonensis
5 Amphimas pterocarpoides 42 Lannae welwitschii
6 Baillonella toxisperma 43 Lecanodiscus cupanoides
7 Baphia nitida 44 Lophira alata
8 Barteria nigritana 45 Lovoa trichilioides
9 Berlinia confusa 46 Mammea africana
10 Blighia sapida 47 Mitregiana inermis
11 Brachystegia nigerica 48 Musanga cecropioides
12 Bridelia ferruginea 49 Myrianthus arboreus
13 Canarium schweinfurthii 50 Parkia bicolor
14 Canthium palma 51 Pausinystalia talbotii
15 Carapa procera 52 Pentaclethra macrophylla
16 Celtis zenkeri 53 Pentadesma butyracea
17 Chrysophyllum giganteum 54 Piptadeniastrum africanum
18 Cleistopholis patens 55 Pterocarpus mibreadii
19 Coelocaryon preussii 56 Pterocarpus osun
20 Cola acuminata 57 Pterocarpus santalinoides
21 Cola millenii 58 Pterocarpus soyauxi
22 Combretodendron macrocarpum 59 Pterygota macrocarpa
23 Cylicodiscus gabunnesis 60 Pycnanthus angolensis
24 Dacryodes klaineana 61 Ricinodendron heudelotii
25 Daniella ogea 62 Santiria trimera
26 Dialium africana 63 Spathodea campanulata
27 Dialium guineense 64 Staudtia stipitata
28 Diospyros crassiflora 65 Sterculia setigera
29 Dracaena mannii 66 Strombosia pustulata
30 Drypetes staudtii 67 Symphonia globulifera
31 Enantia chlorontha 68 Tetrapleura tetraptera
32 Entandrophragma cylindricum 69 Treculia obovoidea
33 Entandrophragma utile 70 Uapaca guineensis
34 Funtumia africana 71 Uapaca staudtii
35 Funtumia elastica 72 Vitex grandifolia
36 Garcinia kola 73 Xylopia aethiopica
37 Hylodendron gabunense 74 Zanthoxylum zanthoxyloides
Ogana 2019
Appendix II: Tree species composition of Ikrigon Forest Reserve.

S.N. Species S.N. Species
1 Afzelia africana 33 Irvingia wombulu
2 Albizia gummifera 34 Khaya grandifoliola
3 Albizia lebbeck 35 Lannea welwitschii
4 Albizia parinarii 36 Lecanodiscus cupanoides
5 Albizia zygia 37 Lovoa trichilioides
6 Alstonia boonei 38 Margaritaria discoidea
7 Anthocleista vogelii 39 Milicia excelsa
8 Antiaris toxicaria 40 Millettia thonningii
9 Baphia nitida 41 Olax subscorpioidea
10 Blighia sapida 42 Parinari hypochrysea
11 Borassus aethiopium 43 Parkia bicolor
12 Bridelia ferruginea 44 Pentaclethra macrophylla
13 Carapa procera 45 Pentadesma butyracea
14 Ceiba petandra 46 Pierreodendron africanum
15 Chrysophyllum giganteum 47 Pseudospondias microcarpa
16 Cleistopholis patens 48 Pterocarpus osun
17 Cola acuminata 49 Pterocarpus santalinoides
18 Cola gigantea 50 Pterocarpus soyauxi
19 Cola millenii 51 Pterygota macrocarpa
20 Cola nitida 52 Pycnanthus angolense
21 Combretodendron macrocarpum 53 Pycnanthus angolensis
22 Dacryodes klaineana 54 Rauvolfia vomitoria
23 Dialium africana 55 Ricinodendron heudelotii
24 Dialium guineense 56 Statodi campanulata
25 Diospyros crassiflora 57 Sterculia setigera
26 Distemonanthus benthamianus 58 Symphonia globulifera
27 Dracaena mannii 59 Tectona grandis
28 Ficus exasperata 60 Terminalia superba
29 Funtimia africana 61 Tetrapleura tetraptera
30 Gmelina arborea 62 Trema guineensis
31 Gossweilerodendron balsamiferum 63 Vitex doniana
32 Grewia mollis
Research article
Functional properties for formulation development

in mucilage of Deccan hemp (Java jute)
Sushma Chaudhary1,2, Manjul Pratap Singh2, Manjoosha Srivastava1 and
A. K. S. Rawat1*
1
CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow-226001, Uttar Pradesh, India
2
Babu Banarasi Das University, Faizabad Rood, Lucknow, Uttar Pradesh-226028, Uttar Pradesh, India
*Corresponding Author: rawataks@rediffmail.com [Accepted: 13 April 2019]
Abstract: Deccan hemp is rich in mucilage and of immense value. This study was performed to
examine mucilage of the plant and its functional group with the help of FT-IR Spectroscopy for
preparation and development of pharmaceutical formulation. Mucilage was found to be 9.54%
w/w which was off white in colour, tasteless and with a characteristic odour. Physicochemical
characterization revealed that mucilage has enough moisture i.e. 9.34 % w/w and is of neutral pH.
It was found to be soluble in hot water and insoluble in organic solvents while in cold water
mucilage swelled to form a gel. FT-IR analysis of mucilage showed the presence of - as major
markers that scope to be of scientific relevance particularly plant polymer based excipient and
coating material in pharmaceutical products.
Keywords: Deccan hemp - Excipient - FT-IR - Spectroscopy - Mucilage - Pharmaceutical
products.
[Cite as: Chaudhary S, Singh MP, Srivastava M & Rawat AKS (2019) Functional properties for formulation
development in mucilage of Deccan hemp (Java jute). Tropical Plant Research 6(1): 129–132]
INTRODUCTION
The plant-based excipient polymer have been effectively applied in various pharmaceutical dosage forms
like nanoparticles, film coating agents, matrix controlled system, suspensions, implants, buccal films and
microspheres (Durso 1980, Chang & Shukla et al. 2003). Mucilages have been extensively used in the field of
drug delivery for their easy availability, cost-effectiveness, eco-friendliness, emollient and non-irritant nature,
non-toxicity, capable of many of chemical modifications, bio-degradable and compatible due to natural origin
(Baveja et al. 1989, Kirtikar & Basu 1991). Mucilage is intracellular physiological product release without
injury of the plant (Geetha et al. 2009). It is polysaccharide mixture having a high molecular weight (20000 and
more) (Narkhede et al. 2010), commonly found in various organs of many higher plant species (Hadley 1997). It
is an amorphous biomaterial and its composition was found to be rich in D-glucose, D-fractose, L- galactouronic
acid, D-galactose, L- rhamnose, sucrose, maltose and xylose (Naveen et al. 2013). Mucilage acts as a membrane
thickener and food reserve in the plants (Banker & Anderson 1987). They have been used as viscosity
enhancers, stabilizers, disintegrants, solubilizers, emulsifiers, bioadhesives and binders (Baveja et al. 1988).
Hence the present study was undertaken with the aim to extract, evaluate and characterize mucilage of plant for
its physicochemical parameters, functional properties and application prospects. It is a good source of natural
polymer with thickening and binding properties in different industrial applications. Plant material and products
are compatible with environment safety and human health.
MATERIAL AND METHOD

Material
The plant with fruits was collected and authenticated by Dr. A.K.S Rawat (Scientist & HOD) at department
of Pharmacognosy and Ethnopharmacology divison CSIR-National Botanical Research Institute, Lucknow
(voucher field booklet no 254060).
Processing and extraction
100 g of hemp fruit was cut into pieces, soaked in 1 L distilled water for 3–4 hours followed by heating at
Received: 05 September 2018 Published online: 30 April 2019
70ºC for 5–10 minute and crushed into mechanical blender which was filtered by muslin cloth after which
ethanol was added into the filtrate (1:2) ratio was to precipitate mucilage and dried in hot air oven at 40–45ºC.
The mucilage powder obtained passed through sieve # 40 and stored in a desiccator at room temperature (Lohar
et al. 2008).
Organoleptic evaluation
The isolated mucilage was characterized for organoleptic properties such as colour, taste, odour, and texture,
fracture (Lala et al. 1981).
Solubility
1 g dry mucilage powder was solubilized with polarity gradient solvents and the solubility was determined
(Lala et al. 1981).
pH
The mucilage was weighed and dissolved in water separately to obtained 1% w/v solution. The pH of
solution was determined using digital pH meter (Lala et al. 1981).
Swelling index
The swelling index is the volume (in ml) taken up by the swelling of 1g of test material under specified
conditions (Malviya et al. 2010).
FTIR Spectroscopy Analysis

Infrared (IR) spectroscopy was conducted using FT-IR Spectrophotometer; the spectrum was recorded in the
wavelength region of 4000 to 400 cm-1. The sample was mixed with KBr in ratio (1:4) and compressed into
pellet with pressure of 7–8 tons in press. The pellet was then placed in the light path and the spectra was
obtained at a resolution of 2 cm-1 from 4000 to 400 cm-1 for intepretation through software (Thermo Scientific
S.No1630) (Harika et al. 2016).

Table 1. Organoleptic and physicochemical characteristic of Deccan hemp mucilage.
S.N. Properties of mucilage Results
1 Swelling Index 9±0.52 % w/w
2 Solubility Soluble in hot water, Insoluble in organic
solvents and in cold water swell to form a gel
3 Loss on drying 9.34 % w/w
4 pH 7.2±0.2
5 Colour Off white colour
6 Odour Odour less
7 Taste Taste less
8 Texture Irregular
9 Fracture Rough
The percentage yield of mucilage was 9.54% w/w, Organoleptic characteristics and physicochemical
parameters i.e. solubility, moisture, pH, colour, oduor, taste and texture were show in table 1. The chemical
profiling of mucilage conforms the presence of six major markers through FT-IR. IR spectra at 3350 cm-1
indicates broad peak stretching and the presence of OH group. The IR spectra at 2850 cm-1 indicates C=H
stretching of aliphatic hydrocarbon group and spectra at 2800 cm-1 indicates C=H stretching (primary aliphatic
hydrocarbon group). The characteristic sharp peak at 1750–1700 cm-1 represents the stretching mode of the
carbonyl group (carboxylic acid). The characteristic COO- peak at 1620–1635 cm-1 confirms the presence of -
NH2 bending (amide group), whereas the absorption band at 1050 cm-1 corresponds to the OH- primary aliphatic
alcohol (Fig. 1; Table 2). There are some reports available demonstrating the role of present major functional
groups in the plant species (Carboxylic acid exhibit hydrogen bonding with themselves especially in non-polar
solvents, to increase stabilization of compounds and elevates their boiling points, Carboxylic acid praticipate in
hydrogen bonding as both hydrogen acceptors and hydrogen donars) (https://courses.lumenlearning.com,
https://Chem.libretexts.org ). CH2 group represents primary aliphatic hydrocarbon at wavelength of 2800 cm-1
this functional group characterizes the presence of polysaccharide. Due to the presence of an OH functional
group at wavelength of 1050 cm-1 indicates polysaccharide nature of mucilage, which results to higher boiling
points compared as to their parent alkanes. It was observed that IR spectra of Opium powder was similar to
deccan hemp as investigated/found in our study (Figs. 1 & 2) the OH stretching, CH2 stretching, amide and
aliphatic alcohol groups were found in the IR spectra of Opium and deccan hemp.
Figure 1. IR sprectra of Deccan hemp mucilage at resolution from 1000 to 4000 cm-1.
Table 2. Functional groups and peak value of IR spectroscopy of Deccan hemp mucilage.
Range
S.N. Functional Groups Peak Value Vibration Intensity
(Wave No) cm-1
1 Primary Aliphatic Alcohal 1050 1050 OH Strong
2 Amide 1620–1590 1620–1635 -NH2 Bending Broad peak
3 Carbonyl 1750–1700 1750 C=O Stretch Strong
4 Aliphatic Hydrocarban 2962–2853 2850, 2800 C-H Stretch Medium
5 Hydroxyl alcohol 3600–25000 3350 OH Stretch Broad peak
Figure 2. IR sprectra of Deccan hemp mucilage and Opium powder at resolution from 1000 to 4000 cm-1.
CONCLUSION
Studies infer that Deccan hemp is an economically important plant with enough mucilage content of high
functional value to be used as bio-polymers in pharmaceutical formulations and coating material. The IR spectra
of opium powder were found to be very similar to IR spectra of Decan hemp mucilage. Opium shows
antimicrobial, anti-inflammatory, analgesic activity etc hence on the basis of IR spectra it may be possible that
Decan hemp with exhibit the similar action and provide a new way to future technologies in Novel Drug
Delivery System.
ACKNOWLEDGEMENTS
Authors are very thankful to Director, Council of Scientific and Industrial Research- National Botanical
Research Institute Lucknow, (CSIR-NBRI) to provide all lab facilities for this research work. First author is
express special thanks to University Grants Commission - Rajiv Gandhi National Fellowship (UGC-RGNF) for
financial supports to this research work.
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Mumbai, pp. 296–303.
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tablet dosage forms. Indian Journal of Pharmaceutical Sciences 50: 89–92.
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tablet dosage forms; part-II. Indian Journal of Pharmaceutical Sciences 51: 115–118.
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Excipients: Polymethacrylates. The Pharmaceutical Press and The American Pharmaceutical Association,
pp. 462–468.
Durso DF (1980) Handbook of Water Soluble Gums and Resins. McGraw Hill, Kingsport Press, New York, NY,
pp. 12.
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Indian Journal of Pharmaceutical Education and Research 43: 261–265.
Hadley EH (1997) Encyclopedia of science and technology. McGraw-Hill Inc New York, pp. 730–731.
Harika B, Shanmuganathan S & Gowthamarajan K (2016) Formulation and Evaluation of Controlled Release
Cefixime Nanoparticles Prepared using Basella alba Leaf Mucilage and Chitosan as Matrix Formers.
Journal of Pharmaceutical Science & Research 8(2): 92–99.
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65–67.
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Lohar DR, Sharma SK & Lavekar GS (2008) The Ayurvedic phamacopeia of India. Minisrty of health and
family welfare, Government of India Department of Ayurveda, Yoga-Naturopathy, Unani, Siddha and
Homeopathy (AYUSH) Edition І Part-ІІ, Volume -ІІ, pp. 166–167.
Malviya R, Srivastava P, Bansal M & Sharma PK (2010) Formulation, Evalution and Comparison of Sustained
Release Matrix Tablets of Diclofenac Sodium Using tamarind Gum as Release Modifier. Asian Journal of
Pharmaceutical and Clinical Research 3(3): 238–241.
Narkhede Sachin B, Vidyasagar G, Jadhav Anil G, Bendale Atul R & Patel KN (2010) Isolation and evaluation
of mucilage of Artocarpus heterophyllus as a tablet binder. Journal of Chemical and Pharmaceutical
Research 2: 161–166.
Naveen G, Naik VV, Kumar D & Samifer S (2013) Effect of mucilage Abelmoschus esculentus as Tablet binder
in Diclofenac Sodium matrix Tablets. International Journal of Pharmaceutical and Chemical Sciences 2:
1320–1323.
Research article
Preliminary studies on seed dormancy of

Schleichera oleosa (Lour.) Merr.
Maitreyee Kundu* and Nimisha Chaturvedi
Tropical Forest Research Institute, P.O. R.F.R.C., Mandla Road, Jabalpur-482021, Madhya Pradesh, India
*Corresponding Author: spalliwest@yahoo.co.in [Accepted: 15 April 2019]
Abstract: Schleichera oleosa is a tropical deciduous tree species distributed in south-east Asia.
The aim of the present study was to investigate the nature of seed dormancy of Schleichera oleosa
that have low germination under laboratory conditions. Seeds were treated by scarification,
Gibberellic acid (GA), and combined treatment of scarification and GA before allowing them to
imbibe in moist paper. Maximum water uptake was observed in seeds that were soaked in GA after
scarification. The moisture content of seed coat remained unchanged during imbibition that
suggests the water-impermeable nature of seed coat. Scarification accelerated germination, but it
could not fully eliminate dormancy. The highest germination was found in the combined treatment
of scarification and GA application. Dry storage at room temperature for 9 months broke
dormancy and allowed germination of untreated seeds at 28C. The results indicate that
Schleichera oleosa seeds exhibit both physical (for its water-impermeable seed coat) and
physiological dormancy and need afterripening for 9 months to overcome the dormancy. The
ecological perspective of dormancy of this tropical seed has been discussed.
Keywords: Dormancy - Imbibition - Seed pretreatment - Dry storage - Germination - Schleichera
oleosa.
[Cite as: Kundu M & Chaturvedi N (2019) Preliminary studies on seed dormancy of Schleichera oleosa (Lour.)
Merr. Tropical Plant Research 6(1): 133–138]
INTRODUCTION
Dormancy is protective mechanism of plant against unfavourable environment. It helps in successful
establishment and maintaining the plant population in natural environment. It is a complex heritable trait
regulated by plant growth hormones, affected by developmental and environmental factors before and after the
seed is formed (Koornneef et al. 2002, Finch-Savage & Leubner-Metzger 2006). After shedding, break in
dormancy is largely dependent on the environmental factor that enables the maximum number of seeds to
germinate in favourable conditions. Nature of dormancy in temperate species is well known, as huge number of
work have been published on seed dormancy of those species. On the other hand, limited knowledge in
dormancy and germination of tropical species especially in in-situ conditions is the main constraint of the
restoration program of the tropical forest that have already been disturbed significantly due to human
interference. It is well-known that most of the tropical species are non-dormant (Casasola 1976). This may be
attributed to the fact that seeds are programmed to mature in favourable season for their germination (i.e.
temperature and water availability). In spite of that some species are also remain dormant (Ng 1973) and the
cause and break of dormancy in natural environment is not known.
Schleichera oleosa (Lour.) Merr. belongs to the family Sapindaceae. Members of this family are distributed
in vast ecological conditions. Species of this family mostly have physical dormancy (Cook et al. 2010), others
have physiological or combined dormancy (Baskin & Baskin 2004). In Indian subcontinent, Sapindus mukorossi
Gaertn. had been reported to have physical dormancy and S. trifoliatus L. have physical and physiological
dormancy (Baskin & Baskin 2004). Radhamani et al. (2003) observed better germination in Schleichera oleosa
seeds by breaking of seed coat and soaking overnight in water followed by Gibberellic Acid (GA3) and opined
that the seed had seed coat dormancy. Thapliyal & Tewari (2011) also concluded that mechanical scarification
resulted maximum germination of S. oleosa seeds. They also observed after-ripening for 2 months could
increase the germination percentage. However, it is not known how germination of this species is regulated in
Received: 06 December 2018 Published online: 30 April 2019
Kundu & Chaturvedi 2019
tropical deciduous forest having precipitation during monsoon season and the nature of dormancy was not fully
understood.
The purpose of this present study is to reveal the nature of seed dormancy of this species by the following
tests: 1) comparison on the rate of imbibitions of treated and untreated seeds and change in moisture content of
seed coat and cotyledons after soaking; 2) effect of different pretreatments on germination of seeds and 3) effect
of dry storage on germination capacity of seed.

Scheleichera oleosa (Lour.) Merr. (family: Sapindaceae) occurs from the foothills of the Himalayas and the
western Deccan of India to Sri Lanka and China. It spreads spontaneously in dry deciduous forest and savannas
with an altitude up to 1200 m having normal rainfall of 750 to 2800 mm. The absolute maximum temperature is
3540C and minimum being 24C. Seeds are subglobular, about 12 mm × 10 mm × 8 mm; hilum is orbicular,
testa is brown, smooth, glabrous enclosed in a succulent yellow aril. Number of seeds per kg varies from 1400
to 2200 kg-1. It is an economically important plant, work as best host for the lac insect (Laccifer lacca). The
pinkish brown heartwood is very hard and durable, excellent to make cartwheels, tool handles, roller for sugar
mills etc. Fruits are edible and oil from seed is used for culinary purposes, lighting and preparing traditional
medicines. S. oleosa is deciduous, but completely leafless for a few days only. Leaves drop in December to
February. The racemes of greenish yellow flowers appear with the young leaves in FebruaryApril (Chaudhary
et al. 2016). The fruits fall quickly to the ground after they ripen in JuneAugust.
The experiments were carried out in the laboratory of Seed Technology, Tropical Forest Research Institute,
Jabalpur in India. Fully mature seeds were collected from 10 trees of Scheleichera oleosa growing in the
Mandla District of Madhya Pradesh in India. Seeds from this mass collection were extracted from the fruits,
dried, pooled and used in experiments with 15 days of collection. Seed moisture content was determined before
the start of the experimentation.
To determine the nature of dormancy three experiments were conducted:
Imbibition test: Four treatments were given in freshly harvested seeds,
1. Untreated seeds were soaked in water at ambient (2730 C) temperature for 24 hrs.
2. Scarified- The pericarp was nicked with a scalpel on the proximal end and soaked in water at ambient
temperature for 24 hrs.
3. Gibberellic Acid (GA) treated- Seeds were soaked in GA3 solution at the dose of 500 ppm for 24 hrs.
4. Scarified + GA treated- After scarification seeds were soaked GA3 500 ppm solution for 24 hrs.
The initial dry mass of three 25 seed replicates was measured. After treatment, the surfaces of the seeds were
blotted dry with a paper towel and fresh weight was determined. This was considered as 0 hrs. Seeds were then
placed into dishes between two sheets of moistened blotting paper-towel. The experiment was conducted at
ambient room conditions (2730 C). After 12, 24, 48, 72, 96, 144 hrs, seeds were removed from the Petri
dishes, blotted dry, weighed to nearest 0.01 g and returned to the moistened paper in the dish. Imbibition curves
(increase in seed mass (fresh weight basis) over time) for each treatment were constructed and compared.
Water uptake by the seed parts
To determine the water uptake, another two sets of 100 seeds were placed between blotted paper sheets after
following treatments: 1) scarified (individually with a single-edge razor blade) 2) scarified + GA- treated 3) GA
treated and of 4) untreated seeds. Sampling was done after fixed intervals for three replications of fifty seeds.
During each sampling, surface water of seeds (with and without seed coat) was dried up with blotting paper and
moisture contents of seed coat, cotyledon and intact seed were estimated by drying the entire seeds and their
parts separately at 103C for 17 hrs. Moisture content was estimated as percentage of fresh weight.
Germination
Seeds were treated in the following ways for enhancement of germination:
1. Untreated- Intact seeds without any treatment
2. Seeds treated with GA: Seeds were soaked in GA3 500 ppm for 17 hrs and sown for germination.
3. Scarification: Seeds were mechanically scarified individually with a single-edge razor blade at the distal end
of the embryo.
4. Seeds were scarified and soaked with GA3 500 ppm for 17 hrs before sowing.
The germination test was performed by placing three replicates of 50 seeds each on moist paper in Petri
dishes at 28±2 °C in darkness. The germination was counted daily and seeds were considered to be germinated
when the radicles grew at least 1 cm. The number of rotten, empty, good germinated and un-germinated seeds
was determined by cutting test after 30 days of sowing. Germination percentage was calculated from this data.
Effect of dry storage on dormancy
Freshly harvested seeds were stored at temperature sequences simulating temperatures that seeds experience
in nature according to the season (varies 1040 C) during the month of July 2011. Germination was assessed 0,
3, 6, 9 and 12 months after collection to note the change in germination with time. Besides untreated seeds, two
treatments were given to the seeds: scarification and scarification with GA3 500 ppm before germination test.
Three replicates of 50 seeds in each treatment were placed at 28±2 °C in darkness.
Data were analysed statistically using two-way analysis of variance (ANOVA) using SPSS software.
Fisher’s least significant difference test was used to determine significant differences (P 0.05) between
individual treatments.
RESULTS
Effect of different treatments on rate of water uptake
There was little increase in seed weight for non-treated seeds (Fig. 1). Only about 5% increase was observed
during 7 days in imbibed condition. 17.4% increase in mean was observed when the seeds were exposed to
scarification treatment. However, combined treatment of mechanical scarification and GA caused maximum
subsequent water uptake (about 45%). This treatment caused significant increase in mass within three days on
moist filter paper at room temperature in comparison to other treatments.
Figure 1. Percentage increase in fresh weight of untreated and treated seeds of Schleichera oleosa (Lour.) Merr. imbibed on
germination blotter. [-♦-, Untreated; -●-, Scarification; -▲-, GA treatment; -■-, Scarification with GA treatment; Error bars
represent ± standard error]
Effect of different treatment on moisture contents of seed coat and cotyledon
Fig. 2 shows that no significant of changes were observed in moisture content of seed coat in all treatments.
Moisture content of cotyledon was not changed in untreated and GA treated seeds. Scarification helped in
increase in moisture content of some seeds. Significant increase in moisture content was observed in the
scarified seeds treated with GA. Seeds with this combined treatment, water uptake started within one day and
moisture content of colyledon increased to 29.4% and 35.2% after 4 and 8 days respectively.
Effect of different treatments on germination percentage
Mechanically scarified seeds treated with gibberelic acid (GA3) at 500 ppm showed highest germination of
about 72.6%. Scarified seeds those were scarified, but not treated with GA achieved 29.6% germination. Seeds
soaked in GA 500 ppm showed only 11.5% germination whereas untreated seeds (not treated) had 5.8%
germination (Fig. 3).
Effect of dry storage on germination
Significant increase in germination was observed after 9 months of storage in all treated and untreated seeds
(Table 1). Though scarification caused increase in germination percentage, negligible change in germination
was observed in untreated and scarified seeds till 6 months of storage. Combined treatment of scarification and
soaking in GA showed the maximum germination of freshly harvested seeds, as did 9 months storage of
untreated seeds.
Figure 2. Changes in moisture content of seed coat, cotyledon and whole seed in untreated and treated seeds of Schleichera
oleosa (Lour.) Merr. [-♦-, Untreated; -●-, Scarification; -▲-, GA treatment; -■-, Scarification with GA treatment; Error bars
represent ± standard error]
Figure 3. Mean germination percentage (±standard error) of untreated and treated seeds of Schleichera oleosa (Lour.) Merr.
Table 1. Mean germination percentage of untreated and treated seeds of Schleichera oleosa
(Lour.) Merr. after different periods of dry storage.
Months of Treatments
storage Untreated Scarified Scarified+ GA
0 7.3a 35.3a 70.7a
3 6.0a 27.3a 56.0a
6 8.0a 32.7a 66.0a
9 74.0b 79.3b 72.7a
12 79.3b 74.0b 67.3a
Note: Different letters in the same column denote significant differences (P0.05) using LSD.
DISCUSSION
The imbibitions experiment demonstrated that the untreated seed of Schleichera oleosa imbibed very low
amount of water, whereas water uptake was more in scarified seeds than untreated seeds. The seed coat was
impermeable to water as was evident from the experiment 3 where moisture content of seed coat remained
unchanged in all of the four treatments including untreated one. Germination percentage of scarified seeds was
improved to some extent. This clearly indicates that seed coat imposed physical barrier in water absorption.
Though mechanical scarification can promote germination in physiological dormancy, imbibitions study can be
a best method to identify physical dormancy (Baskin & Baskin 2004). More increase in fresh weight was
observed in scarified in comparison to unscarified seeds during imbibitions in summer farewell [Dalea pinnata
(J.F.Gmel.) Barneby], a species with physical dormancy (Perez et al. 2009). About 75% increase in fresh weight
was noted in seeds scarified with hot water in six physically dormant genera of Rhamnaceae, compared with
non-treated seeds having less than 16% increase in fresh weight. Apart from scarification, GA plays an
important role in germination of Schleichera oleosa seeds. Significant uptake of water was observed in scarified
seeds treated with GA, whereas germination percent was very low in when the seeds were treated with GA only.
Therefore, GA was not absorbed due to impermeability of seed coat of freshly collected seeds. Also, combined
treatment of scarification and GA application had increased the germination to 72.6%. The result suggests that
this species has two types dormancy i.e. physical and physiological. Similar nature of dormancy was observed in
Koelreuteria paniculata Laxm. (Park & Rehman 1999) and Sapindus drummondii Hook. & Arn. (Munson
1984), two other members of the Sapindaceae. Cirak et al. (2004) observed combined treatment of GA and
H2SO4 increased the germination of black henbane (Hyoscyamus niger L.) seeds and concluded that the seeds
have double dormancy involving a hard seed coat and a partially dormant embryo. On the other hand, Pereira et
al. (2016) concluded that seeds of Schinus molle L. had physiological dormancy though maximum germination
occurred in the seeds those were treated with H2SO4 after 3 months of dry storage. They observed both scarified
and non-scarified seeds absorbed water in imbibition test.
Effect on dry storage on germination showed that no remarkable change in germination was observed till 6
months of storage. After 9 months of dry storage seeds achieved full germination capacity without any
treatment. It indicates that seeds have physiological dormancy in addition to physical dormancy. Fresh seeds
need scarification for absorption of water, for this reason they could not absorb GA that resulted very few
germination of GA treated fresh seeds. However, after-ripening or dry storage for 9 months helped to remove
the dormancy in such a way that enabled the seeds in absorption of water through the water-gap (Gama-
Arachchige et al. 2011) and growth of the embryo. Imbibition test and water uptake by seed parts after sowing
supports presence of physical dormancy in the seeds of this species. Thapliyal & Tewari (2011) also observed
maximum germination of Scheleichera oleosa seeds which were after-ripened and mechanically scarified.
Dormancy due to impermeability of seed coat and dormant embryo had been termed as combinational dormancy
(Baskin & Baskin 1998).
Dry storage acts an environmental cue for break of physical and physiological dormancy in several species
including Sapindaceae (Turner 2005) in temperate zones though it was not documented in tropical species. Seed
fall in Schleichera oleosa occurs during July–August (after the start of the monsoon rain). After this period
temperature starts to fall and dry period starts which are not favourable for seedling growth. Combined
dormancy in Schleichera oleosa acts a safety mechanism for survival that prevents the seeds to germinate in the
first season and provide protection for survival up to the next season. It is also important to note that seeds were
able to overcome dormancy at the commencement of summer months (23 months before rain) to get the
maximum benefit of rain for germination and consecutive seedling survival. The combination of hard seed coat
and dormant embryo was reported in arid regions and mediterranian climate that protects the seed from
germinating after out of season rains (Kigel 1995, Norman et al. 1998). Completion of seed maturation just
before or during monsoon is common feature of dry deciduous forests of Indian subcontinent and seeds
maturing during or just before rain do not generally posses dormancy. However the arrival of monsoon differs
in different region and rain does not appear at the same time in each year. Seeds do not get ample moisture for
germination and seedling survival. Also moisture requirement for growth and tolerance to desiccation varies
among species. So, development of dormancy appears to be a protective mechanism for their better survival.
The factors that break physical and physiological dormancy may vary. In Schleichera oleosa few fresh seeds
germinated in control environment (dark 28C) and germination percentage were not increased till 6m of
storage in the laboratory condition that imitate seasonal temperature and humidity. After 9 months of storage,
growth potential of the embryo of untreated seeds increased and cells of the water gap in the seed coat became
weak that resulted in radical protrusion or germination. It appears that change in temperature and humidity
during autumn, winter and spring was responsible for removal of dormancy in this species.
It was observed that the seeds of this species could germinate at fixed range of temperature of 2540 C and
unlike temperate species change in germination temperature was not able to break the dormancy (Kundu,
unpublished manuscript). But storage temperature could be an important factor for break of dormancy of
tropical species. Argel & Patson (1999) opined that it was an important factor for natural softening of seed coat.
The future work on storage temperature on dormancy break may indicate the factors working in dormancy-
breaking mechanism of the seeds of this species.
ACKNOWLEDGMENT
The work was financially supported by Indian Council of Forestry Research & Education, Dehra Dun, India.
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Research article
Floristic composition and vegetation structure of Ades forest,

Oromia regional state, West Hararghe zone, Ethiopia
Dereje Atomsa1* and Duguma Dibbisa2
1
Department of Biological Science, School of Biological Science and Biotechnology, College of Natural and
Computational Science, Haramaya University, Ethiopia
2
Department of Molecular Biology & Biotechnology, School of Biological Science and Biotechnology, College
of Natural and Computational Science, Haramaya University, Ethiopia
*Corresponding Author: datomsa@yahoo.com [Accepted: 16 April 2019]
Abstract: This study was conducted at Ades forest in West Hararghe Zone, Ethiopia, for
determining vegetations composition and structure. Systematic sampling method was used to
collect vegetation data from 48 (20 m × 20 m) main sample plots for woody species that was
established along a transect line. Sample plots were spaced at 10 m altitudinal drop from top to the
bottom of the natural forest. Diameter at breast height and height of all woody species were
measured. Species abundance, vernacular name and environmental variables were recorded in each
sample plot. A total of 48 woody plants belonging to 42 genera and 29 families were recorded and
identified. Fabaceae family had the highest number of taxa followed by Rosaceae and
Flacourtiaceae families. Although the overall population structure of woody plants of the Forest
revealed good regeneration status, the presence of anthropogenic disturbance in the area
necessitates the need for conservation action in order to ensure sustainable utilization and
management of the Forest.
Keywords: DBH class - Vegetation structure - Regeneration.
[Cite as: Atomsa D & Dibbisa D (2019) Floristic composition and vegetation structure of Ades forest, Oromia
regional state, West Hararghe zone, Ethiopia. Tropical Plant Research 6(1): 139–147]
INTRODUCTION
Assessment of forest species composition and structure is very helpful in understanding the status of tree
population, regeneration, and diversity for conservation purposes (Mishra et al. 2013). Forest structure mainly
depends on the nature of ecosystem, species composition and regeneration condition of the tree species in the
area. Understanding of woody species composition and structure in a given forest is crucial for planning and
implementation of conservation activities (Malik et al. 2014, Malik & Bhatt 2015).
Forest ecosystems are home for biodiversity and provide food and other important materials to survive on
land. But, they are threatened from time to time mainly as a result of anthropogenic activities such as
deforestation for agricultural activities, overgrazing, construction materials, timber production, fire wood, road
construction, charcoal production and medicinal values (Yonas 2001, Getachew & Demel 2005, Liaison 2013,
Bajpai et al. 2018). These temporary benefit oriented deforestation is followed by land degradation and soil
erosion which result in biodiversity loss (Feyera 2006, Feyera & Denich 2006, Tadesse 2008). Destruction of
vegetation coves causes environmental degradation, climate change, drought, depletion of natural resources and
food shortage. These are the major issues of national and global concern in recent years.
Studies reported by (Demel 2001, Yonas 2001, FAO 2007) indicated that there are continuous deforestation
and land degradation in Ethiopia. Due to low level of peoples’ awareness on the role that forests have in terms
of ecosystem services, less attention has been given to their conservation. Adequate awareness regarding wise
use of natural resources should be given to the whole society so that some multipurpose endogenous and
medicinally important plant species will be saved from extinction. So, as a conservation method, scientific
studies on floristic composition and vegetation structure of a given natural forest patch is needed for
determination of a forest status to take appropriate conservation measures. As a result, this study was aimed with
Received: 14 December 2018 Published online: 30 April 2019
Atomsa & Dibbisa 2019
following objectives; to assess the floristic composition and structure, to document the woody plant species in
the study area and to analyze the structure of the Forest

Description of the study site
The study was conducted on Ades natural Forest, located in Oromia Regional State at Western Harerge
Zone, 371 Km from Addis Ababa, to the Eastern part of Ethiopian. The area has an average altitude of 1600–
3100 meters above sea level, average annual rain fall of 250–900 ml and average annual temperature of 16–
18ºC. The area is mainly covered by an irregular topography with depressions, numerous chain mountains, flat
lands, gorges scattered trees and dense shrubs of patch natural vegetation.
Floristic and structural data collection
Reconnaissance survey was made across the natural forest in order to obtain vegetation patterns and
determine representative sampling sites. Vegetation data were collected using a systematic sampling method as
discussed by Kent & Coker (1992). Sampling was done along an altitudinal gradient from 3100 m to 1600 m
above sea level. The data of vegetation attributes were measured for trees and shrubs, and recorded using twenty
by twenty meter size plots which were established along a transect line, starting from top to the bottom of the
natural forest. All the woody plant species encountered in each sample plot were recorded using vernacular or
local names and code was given for unknown specimen. Sampling quadrats were arranged along transects line,
which were spaced at 10 m altitudinal drop, along the elevation gradient of the Forest. Environmental variables
such as altitude and geographical coordinates were also measured for each plot using Geographical Position
System (GPS) (Kent & Coker 1992).
In each sample plot, the circumferences of woody species at breast height (about 1.3 m) were measured and
recorded during sampling in the field and conversion to diameter at breast height (DBH) and basal area were
made later. The measurement of circumferences was taken for trees and shrubs with height >2 m and
circumference >7 cm (DBH= 2.5 cm) and above. Density calculation was made for trees, saplings and seedlings.
Plants were identified to the species level. Moreover, forest stand structure parameters that are assumed to have
influence on tree species regeneration variables such as; stem density (SD), frequency and basal area (BA) were
analyzed using Minitab 16 statistical software and Microsoft office excel. MAT-LAB software was used for
drawing map of the study area. Specimens were collected, pressed, dried and brought to the Haramaya
University Herbarium for identification and to National Herbarium (ETH), Addis Ababa University for further
authentication. The specimens were dried in the dryer, kept in a deep freezer for 72 hours and identified
referring to the volumes of Flora of Ethiopia and Eritrea and finally documented.
Data analysis method
All individuals of plant species recorded in all quadrants were used in the analysis of species composition
and vegetation structure of the forest. The diameter at breast height (DBH), basal area, tree density, relative
density, dominance, relative dominance, height, frequency, relative frequency and important value index (IVI)
were used for description of vegetation structure.
RESULTS AND DISCUSSIONS

The vegetation of the area shows the composition of different species. Some of the dominant species in this
natural forest were Juniperus procera Hochst. ex. A. Rich., Podocarpus falcatus (Thunb) R.B. ex. Mirb.,
Croton macrostachyus Del., and Maytenus sp. The vegetation varied with altitude, from high and dense forest
with dominant secondary generation of Podocarpus falcatus at lower and middle altitudes to Juniperus procera
and Croton macrstachyus with intermingled of other species at higher altitudes.
Species composition
A total of 48 plant species were recorded from Ades natural forest in current study. Out of these, 15
(52.08%) species were trees while 23 (47.92%) species were shrubs. The list of all species is given in table 1.
The identified species belong to 42 genera and 29 families. Fabaceae was the most dominant families,
contributed 5 (16.7%), followed by Rosaceae and Flacourtiaceae both represented by 4 (13.7%) families.
Density, frequency and importance value index
The total tree density was 1,452.9 stems ha-1 and the total basal area was 13.5 m2 ha-1. The tree species with
the highest basal areas were Podocarpus falcatus (Podocarpaceae) and Juniperus procera (Cuppressaceae).
About 75.8% of the stems had DBH ≤20 cm, 24.8% between 20 and 50 cm and 5.4% ≥ 50 cm (Fig. 1). The
largest diameter was recorded for P. falcatus (87.6 cm) followed by Juniperus procera (47.5). Trees with
heights ≤ 20 m made up 48.8% of the total height class while individuals greater than 20 m, represented by
14.6% of the total height classes.
Table 1. List of Woody Species Collected from Ades Forest per all sampled plots.
Scientific name Family Local name H
Allophylus abyssinicus (Hochst.) Radlk. Sapindaceae Embis (Amh.) T
Apodytes dimidiate E. Mey. ex Am Icacinaceae Ararsaa (Or) S
Asparagus africanus Lam. Asparagaceae Sariiti (Or.) S
Bersama abyssinica Fresen. Melianthaceae Waakkaa (Or) T
Brucea antidysenterica J.F. Mill. Simaroubaceae Qommongo (Or) T
Calpurnea aurea (Ait.) Benth. Fabaceae Ceekaa (Or.) S
Carissa spinarum L. Apocynaceae Agemssa (Or.) S
Combretum molle G.Don Combretaceae Maldhisaa (Or.) S
Crotalaria laburnifolia L. Fabaceae - S
Croton macrostachyus Del. Euphorbiaceae Bekenissa (Or.) T
Dombeya torrida (J.F.Gmel.) P.Bamps Sterculiaceae Daanisa(Or.) T
Dovyalis abyssinica (A. Rich.) Warb. Flacourtiaceae Shimbirqoli (Or.) S
Dovyalis verrucosa (Hochst.) Warb. Flacourtiaceae Liqqimme (Or) T
Ekebergia capensis Sparrm. Meliaceae Sombo (Or.) T
Euphorbia ampliphylla Pax. Euphorbiaceae Adaamii (Or.) T
Euphorbia tirucalli L. Euphorbiaceae Caadaa (Or.) T
Ficus sur Forssk. Moraceae Harbuu (Or.) T
Hagenia abyssinica (Bruce) J.F. Gmel. Rosaceae Hexoo (Or.) T
Halleria lucida L. Scrophularaceae - S
Indigofera rothii Baker Fabaceae Ooshee (Or.) S
Juniperus procera Hochst. ex A. Rich. Cuppressaceae Getera (Or.) T
Lepidotruchilia volkensii (Gurke) Ler’y Meliaceae Miixoo (Or.) S
Maesa lanceolata Forssk Myrsinaceae Abbayyi (Or.) T
Maytenus sp. Celasteraceae Qaxamme (Or.) S
Maytenus undata (Thunb.) Blackelock Celasteraceae Wontofulasa (Or.) T
Millettia ferruginea (Hochst.) Bak. Fabaceae Birbirraa (Or.) T
Myrica salicifolia Hochst ex A. Rich. Myricaceae Macheensoo (Or.) S
Myrsine africana L. Myrsinaceae Kechemo (Amh.) S
Nuxia congesta R. Br. ex Fresen. Loganiaceae Machalo(Or.) T
Olea europaea L. subsp. cuspidata (Wall. ex G. Don.) Cif. Oleaceae Ejerssa (Or.) T
Oncoba spinosa Forssk Flacouriaceae Garabagush (Or.) T
Osyris quadripartita Decn. Santalaceae Watto (Or.) S
Pittosporum viridiflorum Sims Pittosporaceae dhamaye (Or.) T
Podocarpus falcatus (Thunb) R.B. ex Mirb. Podocarpaceae Birbirssa (Or.) T
Prunus africana (Hook. f.) Kalkm. Rosaceae Muka gurach(Or.) T
Psydrax schimperiana (A.Rich) Bridson Rubiaceae Gallee (Or.) S
Pterolobium stellatum (Forssk.) Brenan Fabaceae Kuntir (Amh.) S
Rhamnus staddo A. Rich Rhamnaceae Sibiillo (Or.) T
Rhus glutinosa A. Rich. Anacardiaceae Tatessa (Or.) T
Rhus natelensis Meikle Anacardiaceae Nanfaree (Or.) S
Rosa abyssinica Lindley Rosaceae Qajima (Or.) S
Rubus steudneri Schweinf. Rosaceae S
Schefflera abyssinica (A.Rich.) Harms Araliaceae Habaratuu (Or.) T
Scolopia theifolia Gilg. Flacourtiaceae Qillisaa (Or.) T
Teclea nobilis Del. Rutaceae Hadheessa (Or.) S
Vangueria madagascariensis Gmel. Rubiaceae Ababunee (Or.) S
Vernonia amygdalina Asteraceae - S
Vernonia urticifolia A. Rich. Asteraceae Reji Or.) S
Note: H= habit, T= Tree, S= Shrub.
Frequency is defined as the probability or chance of finding a plant species in a given sample area or
quadrant (Kent & Coker 1992). The frequencies of the tree species in all quadrants were computed. Podocarpus
falcatus was the most frequent species in the forest (10.2%) and followed by Pittosporum viridiflorum (8.38%)
and Juniperus procera (6.89%). Frequency is the number of times a plant species is present in a given number
of quadrats of a particular size or at a given number of sample points. The concept of frequency refers to the
uniformity of a species in its distribution over an area. A better idea of the importance of a species with the
frequency can be obtained by comparing the frequency of occurrences of all the tree species present. Relative
frequency, density and dominance of species were used to calculate the important value index (IVI) of all
woody species. Important value index is the degree of dominancy and abundance of a given species in relation
to the other species in the area (Kent & Coker 1992). The result of IVI calculated from relative density, relative
dominance and relative frequency was shown in table 2. The value of important value index (IVI) can be used to
compare the ecological impact of species in a given area (Lamprecht 1989).
In current study, the output of importance value index analysis showed that Podocarpus falcatus
(Podocarpaceae), Juniperus procera (Cuppressaceae), Vangueria madagascariensis (Rubiaceae), Maytenus sp.
(Celastraceae) and Scolopia theifolia (Araliaceae) were the top five IVI species in the study area. These five
species were contributed to 54.97% of the total IVI of all species. Few species have been reported to have high
IVI in tropical and subtropical forests (Derero et al. 2003). However, the high IVI is not always attributed to the
same structure parameter. The high IVI of Podocarpus falcatus was attributed to its high density and dominance
in the forest, that of Juniperus procera was due to its high dominance, and that of Vangueria madagascariensis
was attributed to its high density.
Table 2. Density, dominance, frequency and importance value index of woody species.
Scientific name Family D RD% Fr RFr DO RDO IVI
Allophylus abyssinicus (Hochst.) Radlk. Sapindaceae 1.04 0.07 4.17 0.58 0 0 0.65
Apodytes dimidiate E. Mey. ex Am Icacinaceae 22.4 1.54 18.75 2.62 0 0 4.16
Asparagus africanus Lam. Asparagaceae 1.04 0.07 2.08 0.29 0 0 0.36
Bersama abyssinica Fresen. Melianthaceae 4.69 0.32 2.08 0.29 0.32 2.37 2.98
Brucea antidysenterica J.F. Mill. Simaroubaceae 1.56 0.11 2.08 0.29 0 0 0.4
Calpurnea aurea (Ait.) Benth. Fabaceae 75.5 5.2 33.3 4.66 0 0 9.86
Carissa spinarum L. Apocynaceae 39.6 2.73 20.8 2.91 0 0 5.64
Combretum molle G. Don Combretaceae 0.52 0.04 2.08 0.29 0 0 0.33
Crotalaria laburnifolia L. Fabaceae 0.52 0.04 2.08 0.29 0 0 0.33
Croton macrostachyus Del. Euphorbiaceae 14.6 1 35.4 4.95 0.39 2.89 8.84
Dombeya torrida (J.F.Gmel.) P. Bamps Sterculiaceae 6.25 0.43 8.33 1.17 0.13 0.96 2.56
Dovyalis abyssinica (A. Rich.) Warb. Flacourtiaceae 58.3 4.01 31.3 4.38 0 0 8.39
Dovyalis verrucosa (Hochst.) Warb. Flacourtiaceae 0.52 0.04 2.08 0.29 0.01 0.07 0.4
Ekebergia capensis Sparrm. Meliaceae 21.9 1.51 27.1 3.79 0.03 0.22 5.52
Euphorbia ampliphylla Pax. Euphorbiaceae 1.56 0.11 4.17 0.58 0.01 0.07 0.76
Euphorbia tirucalli L. Euphorbiaceae 21.9 1.51 27.1 3.79 0 0 5.3
Ficus sur Forssk. Moraceae 1.04 0.07 2.08 0.29 0.09 0.67 1.03
Hagenia abyssinica (Bruce) J.F. Gmel. Rosaceae 2.6 0.18 2.08 0.29 0.48 3.56 4.03
Halleria lucida L. Scrophularaceae 12.5 0.86 2.08 0.29 0.2 1.48 2.63
Indigofera rothii Baker Fabaceae 1.04 0.07 2.08 0.29 0 0 0.36
Juniperus procera Hochst. ex A. Rich. Cuppressaceae 80.7 5.55 47.9 6.7 3.23 23.9 36.2
Lepidotruchilia volkensii (Gurke) Ler’y Meliaceae 19.8 1.36 25 3.5 0.03 0.22 5.08
Maesa lanceolata Forssk. Myrsinaceae 5.21 0.36 16.7 2.34 0.23 1.7 4.4
Maytenus sp. Celastraceae 103 7.09 43.8 6.13 0.29 2.15 15.4
Maytenus undata (Thunb.) Blackelock Celasteraceae 0.52 0.04 2.08 0.29 0 0 0.33
Millettia ferruginea (Hochst.) Bak. Fabaceae 1.04 0.07 2.08 0.29 0 0 0.36
Myrica salicifolia Hochst ex A. Rich. Myricaceae 29.7 2.04 16.7 2.34 0 0 4.38
Myrsine africana L. Myrsinaceae 44.8 3.08 18.8 2.63 0 0 5.71
Nuxia congesta R. Br. ex Fresen. Loganiaceae 1.04 0.07 2.08 0.29 0 0 0.36
Olea europaea L. subsp. cuspidata (Wall. Oleaceae 46.4 3.19 25 3.5 0.06 0.44 7.13
ex G. Don.) Cif.
Oncoba spinosa Forssk. Flacouriaceae 8.33 0.57 12.5 1.75 0.65 4.81 7.13
Osyris quadripartita Decn. Santalaceae 6.25 0.43 6.25 0.87 0 0 1.3

Pittosporum viridiflorum Sims Pittosporaceae 4.17 0.29 58.3 8.16 0.2 1.48 9.93
Podocarpus falcatus (Thunb) R.B. ex Podocarpaceae 263 18.1 70.8 9.91 6.28 46.5 74.5
Mirb.
Prunus africana (Hook. f.) Kalkm. Rosaceae 71.9 4.95 27.1 3.79 0.01 0.07 8.81
Psydrax schimperiana (A.Rich) Bridson Rubiaceae 0.52 0.04 2.08 0.29 0 0 0.33
Pterolobium stellatum (Forssk.) Brenan Fabaceae 1.56 0.11 2.08 0.29 0 0 0.4
Rhamnus staddo A. Rich Rhamnaceae 1.56 0.11 6.25 0.87 0.16 1.19 2.17
Rhus glutinosa A. Rich. Anacardiaceae 3.13 0.22 14.6 2.04 0.18 1.33 3.59
Rhus natelensis Meikle Anacardiceae 0.52 0.04 4.17 0.58 0.07 0.52 1.14
Rosa abyssinica Lindley Rosaceae 0.52 0.04 2.08 0.29 0 0 0.33
Rubus steudneri Schweinf. Rosaceae 6.77 0.47 8.33 1.17 0 0 1.64
Schefflera abyssinica (A. Rich.) Harms Araliaceae 0.52 0.04 2.08 0.29 0.3 2.22 2.55
Scolopia theifolia Gilg. Araliaceae 181 12.5 2.08 0.29 0.3 2.22 15
Teclea nobilis Del. Rutaceae 1.56 0.11 6.25 0.87 0.13 0.96 1.94
Vangueria madagascariensis Gmel. Rubiaceae 273 18.8 45.8 6.41 0 0 25.2
Vernonia amygdalina Delile Asteraceae 4.17 0.29 8.33 1.17 0.06 0.44 1.9
Vernonia urticifolia A. Rich. Asteraceae 3.13 0.22 4.17 0.58 0 0 0.8
Total 29 families 1452.9 100 714.6 100 13.5 100 302.5
Note: D= Density, RD%= Relative density, DO= Dominance, RDO%= Relative dominance, Fr= Frequency, RFr%= Relative
frequency, IVI= Importance value index.
Tree height and diameter classes
All woody plant individuals greater than 2 m recorded in the study area were classified into eleven height
classes (Fig. 1). There is higher number of tree individuals in the lower height class (2–20 m), 234 ha-1
individuals that accounts about (85.4%) of the total height classes while the rest height classes represented by
14.6 only. As the height class increases, the percentage number of individuals decreases. The diameter class
distribution of woody species in a given forest shows the general tendency of population dynamics of a given
species (Steininger 2000). The distribution of trees in different DBH classes was analyzed and classified into
eleven classes: 2.0–10.01, 10.01–20, 20.01–30, 30.01–40, 40.1–50, 50.01–60, 60.01–70, 70.01–80, 80.01–90,
90.01–100, >100cm (Fig. 1). The analysis of DBH class distribution revealed similar trends to that of height
class distribution. The majority of the tree individuals are distributed in the first and second DBH class (DBH <
20 cm) with 254 individual’s ha-1 (71.5%). The abundance of small diameter trees seemed to indicate that the
forest was disturbed recently or represented by secondary generation.
Figure 1. Diameter and height structure of the forest. [DBH classes (cm): 1= 2.0–10, 2= 10.1–20.0, 3= 20.1–30.0, 4= 30.1–
40.0, 5= 40.1–50.0, 6= 50.1–60.0, 7= 60.1–70.0, 8= 70.1–80.0, 9= 80.1–90.0, 10= 90.1–100, 11= >100; Height classes (m):
1= 2.0–10.1, 2= 10.1–20.0, 3= 20.1–30.0, 4= 30.1–40.0, 5= 40.1–50.0, 6= 50.1–60.0, 7= 60.1–70.0, 8= 70.1–80.0, 9= 80.1–
90.0, 10= 90.1–100, 11= >100]
The overall DBH class distribution shows an inverted j-shape like that of height class distribution. Similar
results were reported by Lulekal et al. (2008), Burju et al. (2013), and Gebrehiwot & Hundera (2014), from
Mana Angetu, Jibat and Belete forest respectively. However, this pattern does not describe the general trends of
population dynamics and recruitment processes of a given individual species in the forest. Analysis of
population structures for each individual woody species could provide more realistic and distinctive information
for forest conservation and management activities (Ensermu & Teshome 2008, Yineger et al. 2008, Dibaba et
al. 2014). The population structure of selected species of Ades forest followed four general diameter class
distribution patterns (Fig. 2), which indicated different population dynamics among species.
Size class representation
Most of woody plants in the forest showed inverted J-shaped curves regarding their height and size classes
that were indicative of good regeneration (Fig. 1). This might show that the reproductive capacity of the forest
must be sufficient to sustain the forest, and the population structures of most species have an inverted J-shaped
distribution pattern. However, population structure of the individual woody species revealed variable structural
patterns (Fig. 2): an inverted J curve type (Juniperus procera, Bersama abyssinica), Gauss type curve (Croton
macrostachyus ), interrupted Gauss type curve (Oncoba spinosa) and an irregularly interrupted curve or absence
of individuals in lower classes, higher classes or in lower and higher classes in some species like Rhus glutinosa.
Figure 2. Size classes representative patterns of woody species in Ades forest. [DBH classes (cm): I= 2.5–10, II= 10.1–20.0,
III= 20.1–30.0, IV= 30.1–50.0, V= 50.1–70.0, VI= 70.1–90.0, VII= >90]
Population structure is the distribution of individuals of each species in arbitrary diameter or size classes to
provide the overall profile of species under study. As it has been pointed out by Steininger (2000), the
population structural patterns could be interpreted as an indication of variation in population dynamics that may
happen from inherent traits or due to intervention of anthropogenic activities. This has significant implications
to their management, sustainable use and conservation (Simon & Girma 2004). The analysis based on relative
density distributions by diameter classes carried out for tree and shrub species in Ades forest showed different
patterns and the existence of four general patterns of population structure were recognized. Diameter class
distribution of woody species in the forest shows different patterns of population structure among species. These
were described using representative species in (Fig. 2).
Result showed that, the first pattern was a Guass distribution type which represented by Croton
macrostachyus (Fig. 2A). This pattern showed that the number of individuals were high in the middle classes
and decreased towards the lower and higher diameter classes. The tree species in this pattern of population
structure is Maytenus sp. and Croton macrostachyus. Species with such distribution pattern indicate a poor
reproduction and recruitment which may be associated with different factors that inhibit reproduction or the
presence of only few seed bearing individuals (Feyera 2006).
The second pattern is represented by Juniperus procera (Fig. 2B) which is an inverted J-shaped distribution
showed a pattern where species frequently had the highest frequency in lower diameter classes and a gradual
decrease towards the higher classes. Species such as Juniperus procera, Pittosporum viridiflorum, Scolopia
theifolia and Podocarpus falcatus were characterized by this distribution pattern in Ades natural forest. As
Ayalew et al. (2006), Ensermu & Teshome (2008), Yineger et al. (2008) and Dibaba et al. (2014) indicated in
different forest such pattern is normal population structure and shows the existence of species in healthier
condition good reproduction and recruitment.
The third population structural pattern consists of a species in the medium DBH classes and absent from the
lower and higher DHB classes. This pattern is represented by Hagenia abyssinica (Fig. 2C). The other species
showing this pattern include Rhamnus staddo, Dovyalis verrucosa and Ekebergia capensis. This might have
happened due to the stagnated and/or retarded reproductive capacities of the old-aged individuals of the species
and preference of species vegetative reproductive part at certain age of maturity.
The fourth pattern was formed by species having a broken an inverted J-shape, and inverted J-shape, U-
shape and irregular distribution over diameter classes and represented by Rhus glutinosa, Oncoba spinosa and
Bersama abyssinica (Figs. 2D–F). Some DBH classes had small or no number of individuals while other DBH
classes had large number of individuals. This irregular pattern of distribution might be due to selective cutting of
woody individuals by the local people for different purposes like construction, charcoal production, medicinal or
smoking purposes, firewood and others. Local people also use the forest as open grazing area. This may affect
the growth of seedling into young and matured individuals which could be one reason for forest structure
irregularities. Other species of examples for this pattern are Prunus africana, Bersama abyssinica, Oncoba
spinosa, Olea europaea subsp. cuspidata and Maesa lanceolata.
Population structure of a given forest or individuals of species can tell something what is or was going on in
that forest. Though the population structure of plants is described either by age, size or by their life stage, the
population structure of woody perennial species is often estimated by size class (Saxena & Singh 1984, Venter
& Witkowski 2010, Raj 2018). Information about population structure of a tree species indicates the history of
disturbance of species in the past and its environment, which in turn can be used to conjecture the future trend of
the population of particular species (Wale et al. 2012, Bajpai et al. 2015, Botelanyele et al. 2016, Iyagin &
Adekunle 2017). Study of population structure in tropical forests is ecologically noteworthy and it is useful and
functional in forest management practices (Sahu et al. 2010, Bajpai et al. 2012, Shiferaw et al. 2018).
Population structure, and consequently the regeneration status of a forest are influenced by an array of biotic and
abiotic factors. Several types of disturbances like logging, landslides, gap formation, litter fall, herbivores, fire,
grazing, light, canopy density, soil moisture, soil nutrients and anthropogenic pressure can affect the potential
regenerative status of species composing the forest stand spatially and temporally (Liang & Seagle 2002,
Ganesan & Davidar 2003, Pokhriyal et al. 2010, Sharma et al. 2014, Bajpai et al. 2017). Micro and macro
environmental factors and anthropogenic pressure affect the population structure and accordingly regeneration
condition of a forest ecosystem (Guarino & Scariot 2012).The population structure of a tree species and its
natural regeneration pattern are inter-connected to each other.
CONCLUSION AND RECOMENDATION

Assessment of floristic composition and vegetation structure of woody species in the forest is important for
their management, conservation, and sustainable utilization. The pattern of DBH and Height class distribution of
woody plant species in the Ades forest showed an inverted J-shaped distribution. However, analysis of
population structure of some species showed different patterns of population structure in the forest. The general
variability in vegetation structure and regeneration status indicates the history of the past disturbance to that
species and the environment. Therefore, appropriate management plan is required for their conservation and
sustainable utilization. Adequate awareness regarding wise use of this natural resource should be given to the
whole surrounding society so that some multipurpose endogenous and medicinally important plant species will
be saved from local extinction.
ACKNOWLEDGEMENTS
The authors are grateful to Haramaya University, Office of Research Affairs for granting this research
project, project team leader for his comments and suggestions. The authors are also highly thankful to Ades
Forest Gardner and local field guides for allowing us to collect plant samples and for their assistants during
sample collection.
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Research article
Chemical control of rice brown spot (Bipolaris oryzae)

in Paraguay
Marcelo Barúa1, Lidia Quintana2* and Aldo Ortiz2
1
Graduate Department Magister's Thesis in Crop Protection, FaCAF/UNI
2
School of Agriculture -National University of Itapua. Avenida Lorenzo Zacarías 255 y
Ruta VI. Encarnación, Paraguay
*Corresponding Author: lviedmaq@gmail.com [Accepted: 17 April 2019]
Abstract: Rice brown spot produced by Bipolaris oryzae is one of the most prevalent fungal
diseases in Paraguay and it is associated with rice brown spot, which can decrease yield and seed
quality. The research work was carried out in the 2016 crop season in the experimental plot of a
private rice producer, located on the district of San Juan Bautista, department of Misiones,
Paraguay with the aim of evaluating fungicides for the control of rice brown spot and its effect on
yield crop. The treatments consisted on the application of a mixture of fungicides, Azoxystrobin
20% + Difenoconazole 12.5% (400 cc ha-1) at different rice growth stages. All treatments with
fungicides decreased rice brown spot incidence significantly and increased yield compared with
non-sprayed control. Fungicides applied at R2+R4 and at R2+R3a+R4 resulted in a lower average
incidence of rice brown spot (17–15%).
Keywords: Oryza sativa - Bipolaris oryzae - Rice brown spot - Chemical control.
[Cite as: Barúa M, Quintana L & Ortiz A (2019) Chemical control of rice brown spot (Bipolaris oryzae) in
Paraguay. Tropical Plant Research 6(1): 148–151]
INTRODUCTION
Rice is affected worldwide by countless diseases produced by fungi, bacteria and viruses that have influence
in the production and sanitary quality of the seed (Ou 1985). In Paraguay, diseases produced by fungi that cause
foliar spots are Bipolaris oryzae (Breda de Haan) Shoemacker, Alternaria padwickii Ganguly M.B. Ellis,
Pyricularia grisea Cav, Cercospora sp., Microdochium oryzae Hashioka & Yokogi (Quintana et al. 2016).
Among the foliar diseases that occur in rice crops, the rice brown spot produced by Bipolaris oryzae is one of
the diseases with more prevalence in the different production regions of the country (Arriola 2017). The brown
spot is one of the main diseases that cause spots on grains and it can cause damage from 12–30 % on the weight
of the grains and from 18–22 % in the number of whole grains per panicle, besides causing broken grains,
reduction of viability, loss of quality, for producing dark plaster like grains (Prabhu & Filippi 1997, Guimaraes
2002). In favorable conditions, it can cause up to 50% of damage and up to 35% of loss of germination of the
seeds (Balardin & Borin 2001).
The grain spotting in the region has been rising in countries like Brazil, Argentina and Paraguay and the
majority of the commercial cultivars in use are susceptible to this disease. Farias et al. (2011) reported the
prevalence of Bipolaris spp. in spotted seed from Rio Grande do Sul, Brasil. In Argentina, it’s frequent to find
the presence of B. oryzae in rice seeds from commercial fields from the Northwest of Argentina (Guimaraes
2002). In our country, the seed analysis made between the years 2011–2013 reported that B. oryzae was one of
the species with major incidence on seeds (Quintana et al. 2017).
Chemical control against foliar pathogen has proven to be efficient in experiments made in different
countries (Ottoni et al. 2000). In this country published information on chemical control on rice crop is scarce
and the application of fungicides is recommended during the R3 state as a first application and the second
application at R4. The objective of this investigation was to evaluate a mixture of triazole fungicide + strobilurin
applied on different rice growth stages and to evaluate the effect of it on performance and grain quality.

The research work was carried out in the district of San Juan Bautista Department of Misiones (Paraguay),
during the 2016 crop cycle. The experimental design used was complete block randomized with four
replications, each experimental unit had 12 rows with a separation of 0.17 m between them. The variety
evaluated was IRGA 424. The treatments consisted in the application of a mixture of fungicides: Azoxystrobin
20% + Difenoconazole 12.5% in a dose of 400cc per hectare mixed with 400 cc of oil (Nimbus) applied in
different growth stages of the crop (Table1).
Table 1. Fungicide application scheme on different rice crop growth stage on IRGA 424 cultivar.
Treatments Growth Stages
T1azoxystrobin 20% + 0
-------------------
difenoconazole 15,5%
T2 azoxystrobin 20% + R2 Collar formation on flag leaf-booting
T3azoxystrobin 20% + R3 Panicle exsertion from boot, tip of panicle is above collar of
difenoconazole 15,5% flag leaf
T4 azoxystrobin 20% + R4 One or more florets on the main stem panicle has reached
difenoconazole 15,5% anthesis
T5azoxystrobin 20% + R4 + R6 Antesis + milkygrain
T6 azoxystrobin 20% + R2 + R3 Collar formation on flag leaf-booting + panicle exsertion from
difenoconazole 15,5% boot, tip of panicle is above collar of flag leaf
T7 azoxystrobin 20% + R3 + R4 panicle exsertion from boot, tip of panicle is above collar of
difenoconazole 15,5% flag leaf + anthesis
T8azoxystrobin 20% + R2+R3+R4 Collar formation on flag leaf-booting + panicle exsertion from
difenoconazole 15,5% boot, tip of panicle is above collar of flag leaf + anthesis
Source: Counce et al. (2000)
Extraction of samples to evaluate incidence and severity of rice brown spot
Samples of diseased plants were extracted 11 days after the last application of the fungicide (filled grain).
From each experimental unit 5 plants were collected from the central row which to calculate the percentage of
tillers affected with foliar spot in accordance with the Standard Evaluation System for Rice (IRRI 1988).
T
Incidence (%) = × 100
TT
Where, T= N° of tillers affected and TT= N° total of tillers.
To determine severity on the same samples the diagrammatic scale were used (Lenz et al. 2010).
Figure 1. Diagrammatic Scale of rice brown spot (Bipolaris oryzae) with different levels of disease severity (%).
Yield and weight of thousand seeds evaluation
To evaluate the crop yield and weight of 1000 seeds, 3 m2 of the central rows were harvested and processed.
The corresponding threshing was done manually and the weight corrected at 12 % of humidity and expressed in
kg ha-1. To evaluate the weight of 1000 seeds, 500 seeds were counted to be weighed and then multiplied by 2.
Barúa et al. 2019
Data Processing
For analysis of variance was using the statistics program Infostat version 2013 and the average comparison
was made through the Tukey test with 5 % of error probability.

Incidence and severity of the brown spot
ANOVA demonstrated high significance in the variables incidence and severity of brown spot (p<=0.05).
All treatments with fungicides decreased rice brown spot incidence significantly and increased yield compared
with non-sprayed control. The lowest levels of brown spot were produced with two (R3+R4) and three
applications of fungicide (R2+R3+R4). With one fungicide application (R4) and two applications (R4+R6 y
R4+R6 y R2+R3 reductions of 34, 36, 33 and 28 % of brown spot were obtained. These results are similar to the
works of Bordin et al. (2016) who reported a reduction of 50% in the incidence percentage and 2.7% of the
severity of brown spot with fungicide application. In this study, the lowest severity was produced on T7 and T8
(Table 2).
Table 2. Incidence and severity of brown spot on var. IRGA 424 with chemical protection in different rice growth stages.
Treatments Incidenc (%) Tukey (5%) Severity (%) Tukey (5%)
T1 Control 90.0 D 6.4 E
T2 Application at R2 65.7 C 2.4 B
T3 Application R3 72.2 C 2.8 B
T4 Application R4 33.5 B 2.4 B
T5 Application R4, R6 32.7 B 2.0 B
T6 Application R2, R3 35.5 B 2.4 B
T7 Application R3, R4 18.3 A 0.4 A
T8 Applic. R2, R3, R4 15.7 A 0.8 A
FC 42.2** 43.3**
CV 19.8 19.9
Note: FC- Calculated F in ANOVA; CV- coefficient of variation; **- Highly significant.
Different letters indicate significant differences (p ≤0.05).
Grain yield and thousand weight seeds
ANOVA analysis demonstrated a high significance in the variable yield (p<=0.05). All treatments with
fungicides increased yield compared with non-sprayed control. With three fungicide applications (R2+R3+R4)
the increments was of 23% (Table 3). These results are similar to those reported by Dallagnol et al. (2006), who
indicate that the application of fungicide results in an increase of the productivity of the grain between 6.1–
42.1%. Santos et al. (2009) report increments of 34% with two fungicide applications compared with non-
sprayed control. Celmer et al. (2007) indicate that with applications of triazole fungicides + strobilurin up to 52
% of increment in grains was obtained. In relation to thousand seeds weight, the analysis of variance resulted in
not significative differences, which does not match with the work made by Bordin et al. (2016), who found
increments in the weight of a thousand seeds after performing 3, 4 and 5 applications of fungicides at levels of
11.2 %, 13.8 % and 23.6 % respectively.
Table 3. Thousand seed weight and yield of var. IRGA 424 with chemical protection in different rice growth stages.
Thousand seed
Treatments Yield (kg ha-1) Tukey 5%
weight (g)
T1 Testigo 24 7576 A
T2 Aplicación R2 25 8436 BC
T3 Aplicación R3 25 8934 BC
T4 Aplicación R4 24 9000 C
T5 Aplicación R4, R6 26 9690 C
T6 Aplicación R2, R3 26 9616 BC
T7 Aplicación R3, R4 25 9556 C
T8 Aplicación R2, R3, R4 26 9816 BC
FC 2.17ns 9.8**
CV 4.17 5.6
Note: FC- Calculated F in ANOVA; CV- coefficient of variation; **- Highly significant.
Different letters indicate significant differences (p ≤0.05).
CONCLUSIONS
The application of fungicides in different rice growth stages provided significant reduction on the incidence
and severity of the brown spot on the cultivar IRGA 424, which was reflected in the performance of the grains
and was influenced by the period of application.
ACKNOWLEDGEMENTS
The authors are thankful to Prociencia/Conacyt for financial support.
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Research article
Germination and seedlings growth of Corn (Zea mays L.) to

allelopathic effects of rice (Oryza sativa L.)
Mohamad Hesam Shahrajabian1,2,3*, Mehdi Khoshkharam1,
Wenli Sun2,3 and Qi Cheng2,3
1
Department of Agronomy and Plant Breeding, Isfahan (Khorasgan) Branch,
Islamic Azad University, Isfahan, Iran
2
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing-100081, China
3
Nitrogen Fixation Laboratory, Qi Institute, Building C4, No. 555 Chuangye, Jiaxing-314000,
Zhejiang, China
*Corresponding Author: hesamshahrajabian@gmail.com [Accepted: 20 April 2019]
Abstract: Allelopathy is the direct or indirect effect of plants through chemical compounds
produced by the plants itself. . An experiment was conducted to study the allelopathic effect of
plant organs extract (leaf, stem, root and total), and different rice extract densities (0%, 25%, 50%
and 100%) on the germination and seedling growth of corn with four replications was used.
Treatments included Different plant organ extract showed significant impact on germination rate,
germination percentage, coleoptile weight, radicle weight, radicle length and coleoptile length of
corn. Rice extract densities effect on germination percentage, coleoptiles weight, radicle weight,
radicle length and coleoptiles length was significant. The interaction between rice extract and plant
organs had significant effect on radicle weight and coleoptiles length. Control treatment (0% rice
extract density) had obtained the highest germination rate, germination percentage, coleoptiles
weight, radical weight, radical length and coleoptiles length. All experimental characteristics
decreased with increase rice extract densities. The higher values for germination rate, germination
percentage, coleoptile weight, radicle weight, radicle length and coleoptiles length was related to
stem extract, followed by root, leaf and total extract. According to the results of this trial, it can be
concluded that one of the reasons of the decrease of cultivated plants operation after rice, is the
presence of allelopathic materials in herbaceous remains of this plant.
Keywords: Germination - Seedling growth - Corn - Rice - Allelopathy.
[Cite as: Shahrajabian MH, Khoshkharam M, Sun W & Cheng Q (2019) Germination and seedlings growth of
Corn (Zea mays L.) to allelopathic effects of rice (Oryza sativa L.). Tropical Plant Research 6(1): 152–156]
INTRODUCTION
Corn (Zea mays L.) is one the most important cereal crops grown in Iran, and its demand for food, animal
and industrial use is increasing rapidly as population increased (Khoshkharam et al. 2010, Esfandiary et al.
2011, Soleymani et al. 2011, Soleymani et al. 2012a,b). It is the third important food crop in the world
(Esfandiary et al. 2012, Soleymani & Shahrajabian 2012a,b, Soleymani & Shahrajabian 2013, Soleymani et al.
2016, Shahrajabian et al. 2017). Rice is also the most stable food for more than half of the world, and it is the
second most important food in Iran (Shahri et al. 2012, Yazdpour et al. 2012). Rice has allelopathic potential
against weeds and crops (Ma et al. 2014, AmirulAlam et al. 2018). In an ecological system, allelochemicals
produced by one crop species can influence the growth, productivity and yield of other crops of the same crop
(Chopra et al. 2017, Sitthinoi et al. 2017). Allelopathy has been considered not only as an environmentally
friendly approach for weed control but also as a potential reason causing autotoxicity in crop production (Ma et
al. 2014). A number of compounds, such as phenolic acids, fatty acids, phenylalkanoic acids, hydroxaicacids,
terpenes, indoles, and the labdane-related diterpenoidmomilactones, have been identified as potential rice
allelochemicals (Khanh et al. 2007, Kato-Noguchi & Peters 2013). RashedMohasel et al. (2009) reported that
the leaves and corms extract of saafron reduced plant height, leaf weight and stem weight of redroot pigweed
Received: 15 January 2019 Published online: 30 April 2019
and lambs quarter. Sitthinoi et al. (2017) demonstrated the allelopathic effect of jungle rice extracts on the seed
germination and seedling growth or rice. Therefore, the aim of this study was to survey the allelopathic effects
of rice (Oryza sativa L.) on germination and seedling growth of corn (Zea mays L.).

This research accomplished in seed technology laboratory of Faculty of Agriculture, Islamic Azad university
of Isfahan in 2018 (latitude 32° 40' N, longitude 51° 58' E, and 1570 m elevation). A factorial layout within
completely randomized design with four replications was used. Treatments included plant organs extract (leaf,
stem, root and total), and different rice extract densities (Lenjan Cultivar) includes 4 levels of 0%, 25%, 50%
and 100%. Rice plants were collected from experimental fields. The samples were cleaned, dried, ground and
then put into water for almost 48 hours to obtain the extract. The aqueous extract was prepared by adding 100 g
of ground rice straw into 1 L distilled water for 48 hours. The extract were filtered through muslim cloth and
then through Whatman No. 1 filter paper. The seeds of corn (SC 704) were put in sodium hypocoloid 5% during
10 minutes and then they were washed by distilled water. Seeds soaked in distilled water used as control
treatment. For germination test, 20 seeds were put in 12 cm petridishes on two layers of filter paper and 5 ml of
distilled water for control and 5 ml from levels of expected extract were added to it. The lids of containers with
the temperature 25°C were prepared (12 hours in the day and 12 hours in the night). Every day, the germinated
seeds were numbered in the certain time. The criterion of radical exit germination has been considered 1 mm. At
the end of germination test, the length of radical and coleoptiles were measured. Both radical length (root) and
plumule (shoot) length was measured using a ruler in cm. Also, at the end, the extreme percent of germination
and the rate of germination were accounted. For counting the length of radical and coleoptiles, 10 germinated
seeds were taken out from petridishes and measured. For accounting germination rate, from the second day, unit
when the seeds did not germinate, the germinated seeds were counted per 24 hours and on time.
The germination rate was defined as following equation,
GR = Σ
Where, n is the number of germinated seed on growth day and g is the number of germination seedsAnalysis
of variance (ANOVA) was used to determine the significant differences. The Multiple Range Test of Duncan
performed the separation means (P<0.05). All statistics was performed with the SAS statistical software.

The influence of rice extract was significant on germination percentage, coleoptile weight, radicle weight,
radicle length and coleoptiles length. Germination rate was not significantly influenced by rice extract. All
experimental characteristics, namely germination rate, germination percentage, coleoptiles weight, radicle
weight, radicle length and coleoptiles length was significantly influenced by plant organs. The interaction
between rice extract and plant organs had meaningful effect on radicle weight and coleoptile length. However,
germination rate, germination percentage, coleoptiles weight and radicle length was not significantly affected by
rice extract and plant organs interaction (Table 1). Soleymani & Shahrajabian (2012c) also reported the
significant influence of sesame extract density and plant organs on germination percentage, coleoptile weight,
radical and coleoptile length.
Table 1. Analysis of variance for experimental characteristics.
Germination Germination Coleoptile Radicle Radicle Coleoptile
S.O.V. d.f.
rate percentage weight weight length length
Replication 2 0.82 25.41 0.000023 0.00000004 0.09 0.12
Rice extract (a) 3 0.59ns 670.08** 0.000515** 0.00003736** 13.1** 10.42**
Plant organs (b) 3 11.53** 1121.28** 0.000314** 0.00011698** 1.2** 3.03**
a×b 9 0.56ns 61.11ns 0.000019ns 0.00000207* 0.22ns 0.56*
Error 30 1.3 48.54 0.000024 0.00000083 0.15 0.25
Note: ns- Non significant, *- Significant at 0.05 significant in F-tests, ** Significant at 0.001 significant in F-test, d.f.-
Degree of freedom.
The highest germination rate was related to control treatment (control treatment) (2.33%), which had no
significant differences with other treatments. Germination percentage was decreased significantly with increase
in rice extract density from 0% to 100% rice extract density. Germination percentage in 0%, 25%, 50% and
100% of rice extract density was 72.47%, 55.34%, 68.73% and 62.87%, respectively. Germination is the most
sensitive stage in the life cycles of plant and uniform germination is essential to having a good green area and
Shahrajabian et al. 2019
crop growth (Soleymani & Shahrajabian 2012c,d, Soleymani & Shahrajabian 2018). The higher values for both
coleoptile weight and radicle weight was obtained for control treatment (0%), followed by other treatments.
There were significant differences between 0% of rice extract with other treatments. The highest radicle length
and coleoptiles length was 6.00 mm, and 4.20 mm, which was obtained for control treatment. Both radicle
length and coleoptiles length was decreased significantly from application of 0% to 100% rice extract density.
Afridi et al. (2013) also found that rice straw extract reduced the root length, shoot length, fresh biomass of the
associated. The maximum and the minimum germination rate was related to stem (3.23%), and total plant
extract (0.95%), which had meaningful differences with each other. Germination rate in root and leaf extract
was 2.47% and 1.71%, respectively. Although, the maximum germination percentage was obtained for stem
extract (74.03%), its difference with root extract (71.36%) was not significant. The minimum germination
percentage was achieved in total plant extract (53.09%), which had significant differences with all other
treatments. Afridi et al. (2013) reported that rice straw extract activity was due to the synergistic effects of
various allelochemicals which inhibited and restricted the germination and growth of the plants. The highest and
the lowest value of coleoptile weight was related to stem (0.031 mg), and total (0.020 mg), which had
meaningful differences with each other. There was no meaningful difference between stem and root extract, and
also between leaf and total plant extract. The higher values for radicle weight was obtained for stem (0.011 mg),
followed by root (0.006 mg), leaf (0.005 mg) and total plant extract (0.004 mg), respectively. Furthermore, all
differences between treatments were significant. Stem extract had obtained the highest value of radicle length
(4.79 mm), and coleoptiles length (3.38 mm), which had significant differences with leaf and total plant extracts.
However, its difference with root extract was not significant. The minimum radicle length and coleoptiles length
which was 4.04 mm, and 2.32 mm, was related to total plant extract (Table 2). Sitthinoi et al. (2017) reported
that the extracts from the shoot part of jungle rice had a greater inhibitory effect on the root length and seedling
dry weight than those from the root part. The highest germination rate (3.57%), germination percentage
(81.21%), coleoptiles weight (0.041 mg), radicle weight (0.0151 mg), radicle length (6.40 mm), and coleoptile
length (5.03 mm) was related to interaction between control treatment (0% of rice extract density) and stem
extract (Table 2).
Table 2. Mean comparison of germination rate (%), germination percentage (%), coleoptile weight (mg), radical
weight (mg), radical length (mm) and coleoptile length (mm).
Germination Germination Coleoptile Radicle Radicle Coleoptile
Treatment
rate percentage weight weight length length
Rice extract density (E)
0% (E1) 2.33a 72.47a 0.034a 0.0092a 6.00a 4.20a
25% (E2) 1.87a 55.34c 0.019c 0.0050c 3.79b 2.18c
50% (E3) 2.22a 68.73a 0.026b 0.0060b 4.12b 2.98b
100% (E4) 1.93a 62.87b 0.024b 0.0050bc 3.87b 2.27c
Plant organs (O)
Stem (O1) 3.23a 74.03a 0.031a 0.011a 4.79a 3.38a
Root (O2) 2.47ab 71.36a 0.029a 0.006b 4.57a 3.26a
Leaf (O3) 1.71bc 60.93b 0.023b 0.005c 4.38b 2.66b
Total (O4) 0.95c 53.09c 0.020b 0.004d 4.04c 2.32b
E×O
E1O1 3.57a 81.21a 0.041a 0.0151a 6.40a 5.03a
E1O2 2.38abc 74.69ab 0.035ab 0.0074cd 5.84a 4.84a
E1O3 1.89abc 69.58abcd 0.031bc 0.0066de 5.82a 3.67b
E1O4 1.49abc 64.4bcd 0.030bcd 0.0061def 5.93a 3.24bc
E2O1 2.61abc 65.49bcd 0.028bcdef 0.0089bc 4.18bc 3.13bcde
E2O2 2.26abc 65.92bcd 0.023cdef 0.0048fghi 4.18bc 2.63cde
E2O3 1.9abc 47.77ef 0.013gh 0.0033hi 3.65cd 1.67fg
E2O4 074c 42.17f 0.010h 0.0029i 3.15d 1.29g
E3O1 3.14ab 72.61abc 0.03bcde 0.0099b 4.17bc 3.12bcde
E3O2 2.56abc 73.35abc 0.03bcde 0.0053efg 4.18bc 3.21bcd
E3O3 2.21abc 68.7abcd 0.026bcdef 0.0048fghi 4.19bc 3bcde
E3O4 0.96bc 60.25cd 0.020efg 0.0033hi 3.95bc 2.59cdef
E4O1 3.59a 76.81ab 0.027bcdef 0.0091bc 4.41b 2.25def
E4O2 2.68abc 71.46abc 0.026bcdef 0.0052efgh 4.07bc 2.37cdef
E4O3 0.85c 57.67de 0.021defg 0.0038ghi 3.86bc 2.28cdef
E4O4 0.6c 45.54ef 0.020fg 0.0029i 3.14d 2.17ef
Note: Common letters within each column do not differ significantly. E- Rice extract, O- Plant organ.
CONCLUSION
Alleopathy is any effect that is caused by plants and microorganisms on another plant as a result of the
release of chemical compounds which are named allelochemicals; futherallelopathic effects on plants have no
limitations because they may be direct or indirect, harmful or beneficial influence. Allelochemicals are produced
by plants as a result of secondary metabolism products. Control treatment (0% rice extract density) had obtained
the highest germination rate, germination percentage, coleoptiles weight, radical weight, radical length and
coleoptiles length. All experimental characteristics decreased with increase rice extract densities. The higher
values for germination rate, germination percentage, coleoptile weight, radicle weight, radicle length and
coleoptiles length was related to stem extract, followed by root, leaf and total extract. Rice may increase the
presence of secondary metabolites, all of which may have different effects on seed germination percentage.
Moreover, the allelochemicals which are responsible for germination and growth reduction of different crops,
should be identified in future studies.
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Chopra N, Tewari G, Tewari LM, Upreti B & Pandey N (2017) Allelopathic effect of Echinochloa colona L.
and Cyperus iria L. weed extracts on the seed germination and seedling growth of rice and soybean.
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Esfandiary M, Soleymani A & Shahrajabian MH (2012) Evaluation of yield and yield components of corn
cultivars in different planting methods under semi arid condition of Iran. Journal of Food, Agriculture and
Environment 10(2): 664–667.
Esfandiary M, Soleymani A, Shahrajabian MH & Darkhal H (2011) Effect of planting methods on grain yield
and leaf orientation at different stages of corn cultivars. Research on Crops 12(2): 336–340.
Kato-Noguchi H & Peters RJ (2013) The role of momilactones in rice allelopathy. Journal of Chemical Ecology
39: 175–185.
Khanh T, Xuan T & Chung I (2007) Rice allelopathy and the possibility for weed management. Annals of
Applied Biology 151: 325–339.
Khoshkharam M, Rezaei A, Soleymani A & Shahrajabian MH (2010) Effects of tillage and residue management
on yield components and yield of maize in second cropping after barley. Research on Crops 11(3): 659–666.
Ma Y, Zhang M, Li Y, Shui J & Zhou Y (2014) Allelopathy of rice (Oryza sativa L.) root exudates and its
relations with Orobanche cumana Wallr. And Orobanche minor Sm. germination. Journal of Plant
Interactions 9(1): 722–730.
RashedMohasel MH, Gharakhlou J & Rastgou M (2009) Allelopathic effects of saffron (Crocus sativus) leaves
and corms on seedling growth of redroot pigweed (Amaranthus retroflexus) and lambs quarter
(Chenopodium album). Iranian Journal of Field Crops Research 7(1): 53–61.
Shahrajabian MH, Soleymani A & Khoshkharam M (2017) Influence of green manuring from different cover
crops and farm yard manures on quantitative and qualitative characteristics of forage corn in low input
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Shahri MM, Yazdpour H, Soleymani A, Shahrajabian MH & Sharifianzadeh M (2012) Yield and yield
components of ratoon crop of rice as influenced by harvesting at different plant height and time. Research on
Crops 13(2): 408–411.
Sitthinoi P, Lertmongkol S, Chanprasert W & Vajrodaya S (2017) Allelopathic effects of ungle rice
(Echinochloa colona (L.) Link) extract on seed germination and seedling growth of rice. Agriculture and
Natural Resources 51: 74–78.
Soleymani A & Shahrajabian MH (2012a) Influence of nitrogen fertilizer on ash, organic carbon, phosphorus,
potassium, and fiber of forage corn intercropped by three cultivars of Berseem clover as cover crops in semi
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(Brassica napus) growth and germination. International Journal of Agriculture and Crop Sciences 4(4):
183–186.
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determination of recovery in rice varieties. International Journal of Biology 4(4): 23–30.
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Research article
Genetic diversity of Safflower (Carthamus tinctorius L.) genotypes

at Wollo, Ethiopia using agro-morphological traits
Anwar Kemal1,2* and Faris Hailu2
1
Department of Biology, College of Natural and Computational Science, Mizan-Tepi University,
P.O. Box: 121, Tepi, Ethiopia
2
Department of Biology, College of Natural and Computational Science, Wollo University,
P.O. Box: 1145, Dessie, Ethiopia
*Corresponding Author: ak.anwarkemal@gmail.com [Accepted: 22 April 2019]
Abstract: Safflower (Carthamus tinctorius) is an annual oilseed crop of the Compositae or

Asteraceae family commercially grown in Ethiopia and other countries. To study the genetic
variation and relationships among twelve Safflower genotypes an experiment was carried out
under field conditions in randomized complete block design with 3 replications in Wollo
University, Dessie campus using the agro-morphological traits that includes plant height, days to
flowering, Days to maturity, number of primary branches, number of secondary branches, number
of capitula per plant, number of seed per capitulum, thousand seed weight, yield per plant, and
yield per plot. Results of the analysis of variance showed significant differences among accessions
for traits like days to flowering, days to maturity at p ≤0.01, and for number of primary branches
and number of secondary branches at p ≤0.05. Seed yield per plant had significant correlations
with number of capitula per plant, number of seeds per capitulum, number of primary and
secondary branches. Cluster analysis grouped the 12 genotypes in 2 clusters and one of the
accessions remains ungrouped, according to their similarity in various traits studied. The first three
principal component analyses were found to explain 85% of the total variation that exists among
accessions. The results revealed the presence of a high level of genetic diversity that deserves
conservation attention and could be utilized in breeding program to improve Safflower varieties
with a high seed yield.
Keywords: Cluster analysis - Genetic variation - Genotype - Morphological character - Safflower.
[Cite as: Kemal A & Hailu F (2019) Genetic diversity of Safflower (Carthamus tinctorius L.) genotypes at
Wollo, Ethiopia using agro-morphological traits. Tropical Plant Research 6(1): 157–165]
INTRODUCTION
Safflower (Carthamus tinctorius L.) is an annual oilseed crop of the Compositae or Asteraceae family
commercially grown in Australia, Ethiopia, India, Mexico, United States, and several other countries (Hashemi
et al. 1994). Many countries grow Safflower around the world including Ethiopia. India and Ethiopia are the
countries with the longest tradition of growing Safflower as an oil plant (Weiss 2000). Vavilov (1951) proposed
Ethiopia, Afghanistan and India as centers of origin of cultivated Safflower. Safflower is one of the important
underutilized oilseed crops cultivated in a wide range of ecological environments, but it is generally regarded as
a crop for semi-arid regions (Johnson & Marter 1993). It is adaptable to conditions in wide ranging climate
zones from 60° N to 45° S it should therefore be suitable for cultivation in regions with a temperate climate
(Esendal 2001). Safflower production under excessive moisture and high humidity conditions is seriously
susceptible to diseases (Mündel et al. 2004). The crop has multipurpose crop of economic importance in a few
countries around the world (Knowles & Ashri 1995), and Ethiopia is one of the major producers of Safflower in
the world.
Traditionally, Safflower has been grown for centuries from China to the Mediterranean region and all along
the Nile valley up to Ethiopia (Weiss 1971). Safflower has been used in the Middle East, India and Africa for
purgative and alexipharmic (antidote) effects, as well as in a medicated oil, to promote sweating and cure fevers
Kemal & Hailu 2019
(Weiss 1971). In different parts of the world carpet-waving industries were used Safflower dies. Carthamin dye
was used widely to color cloth until the 19th century, when cheaper aniline dyes became available (Weiss 1983).
Traditionally, the crop was grown f.or its yellow-orange flowers, used for coloring and flavoring foods, and
making dyes (Knowles 1989).
In spite of the large volume of vegetable oils traded around the world, 75% of the production is from only
four crops: soybean, oil palm, rapeseed and sunflower (Khan et al. 2008). These four crops dominated over the
other crops (Murphy 1999) and many other oil crops are now underutilized or neglected, despite the fact that
these species provide opportunities through their great genetic diversity and diverse agro-ecological adaptation
(Thies 2000). Considering the increasing market demand for high polyunsaturated food products in developed
countries Safflower is an oilseed crop with great potential. In addition, there are signs of recent growth in the
market for Safflower, the price premium for Safflower oil and optimistic prospects have motivated a number of
countries to introduce Safflower as an oilseed crop (Johnson & Marter 1993). Safflower oil is thought to be one
of the highest quality vegetable oils, containing oleic acid and linoleic acid (Khan et al. 2008). As a rainfed
crop, Safflower is adapted to some regions and sown by growers when environmental and economic conditions
are suitable (Wachsmann et al. 2008).
Currently, Safflower is regarded as an important oilseed crop, and is grown commercially for its edible oil
rich in polyunsaturated fatty acids (Dwivedi et al. 2005, Singh et al. 2007). It is usually grown after cereals or
fallow in crop rotation and can be adapted in organic production systems (Bavec & Bavec 2007). Safflower is
rich in vitamin A, iron, phosphorus, and calcium. Young raw plants of Safflower are sold in markets in countries
such as India and other countries (Nimbkar 2002). Safflower has become an industrial crop production platform
based on low out-crossing and weediness habits, a different appearance from other oilseed crops such as canola
and excellent agronomic traits such as taproot architecture that accesses sub-soil water reserves (Markley et al.
2006).
The research and development on different aspects of Safflower, despite its adaptability to varied growing
conditions with very high yield potential and diversified uses of different plant parts, have not received due
attention. This probably is the main reason for its status as a minor crop around the world in terms of area and
production, compared to the other oilseed crops. However, interest in this crop has been rekindled in the last few
years due to three major reasons (Singh et al. 2007): (1) A huge shortfall in oilseed production in countries
having a sizable area with scanty rainfall, to which Safflower is most suited; (2) The preference of consumers
for healthy oil with less amounts of saturated fats, for which Safflower is well known; (3) The medicinal uses of
flowers in China and extraction of edible dyes from flowers have become more widely known.
The success of Safflower as a commercial oil seed crop in traditional areas and its expansion in new areas
will mainly depend on the level of improvement made in both its yield and oil content. The low oil content of
28–30 percent and low yield of 600 kg ha-1 makes Safflower a poor competitor. Safflower developed
considerable diversity after it was taken into cultivation for a long time across huge and diverse regions in the
Old World, and there is evidence of incipient genetic differentiation (Knowles 1989). Introduction is the
simplest way to facilitate crop improvement and has been used successfully to establish many oilseed crops in
new areas (Knowles 1983). Further domestication and development of underutilized crops is a possible solution
for the growing and diversified nutritional needs of humankind (Muhammad et al. 2001).
Safflower has received limited research resulting in only small improvements in production, a limited range
of cultivars and deficiencies in the awareness of adaptation and agronomic requirements (Wachsmann et al.
2008). Moreover, limited attention has been given to conserve, add value and improve the productivity of
Safflower in Ethiopia (Edwards 1991).
Ethiopian Safflower is neglected and underutilized so far, it is cultivated only as a minor oil crop with
inadequate information available on its genetic resources. In Ethiopia, Safflower cultivation is mostly done by
small farmers in well fertile and drained field, usually around homesteads. However, seeds harvested from the
plant are used for oil extraction, roasted seed and other traditional uses (seed is extracted into drinks used during
fast by people in Wollo region). But industrial applications of Safflower plants remain as low acreage crop
compared to other oilseed crops like sesame. This demonstrates a need for research examining the agronomic
performances of newly released Safflower genotypes in diverse environments (Öztürk et al. 2008).
Understanding the extent and distribution of genetic diversity within species help in the future breeding
program of Safflower (Padulosi et al. 1999). The diversity of Safflower in Wollo region could mainly be
attributed to different agro ecological conditions. The comparison of various agronomic characters of genotypes
under agro climatic condition of Safflower growing areas in Wollo region helps agronomists and the
local
farmers in the crop improvement.
The main objective of the study was to investigate the genetic diversity among the genotypes of Safflower in
Wollo, Ethiopia. The specific objectives were to assess the genetic variation of C. tinctorius genotypes using
agro-morphological traits and to identify high yielding, better adapted genotypes of Safflower for future use in
improving the crop.

Description of the study area
Wollo is located in the Amhara Regional State and the experiment was carried out in the field at Wollo
University, Dessie (11 8′ N; 39 38′ E; with an annual rainfall of 900–1400 mm). Dessie is located at about 401
km of Addis Ababa to the north. Wollo province comprises diverse agro-climatic condition with different
altitudinal ranges. Most of the region is characterized by mountainous, semi-arid environmental condition. The
accessions were collected from different major Safflower producing sites representing different landraces in the
region.
Plant material
Safflower (Carthamus tinctorius L.) is herbaceous, branched and thistle-like annual plant with strong
taproots which help them to survive in dry climates. Safflower is a highly branched, herbaceous, thistle-like
annual or winter annual, usually with many long sharp spines on the leaves. Plants are 30–150 cm tall with
many spines on the leaves, globular flower heads (capitula) and commonly, brilliant yellow, orange or red
flowers (Fig. 1). Achenes are smooth, four-sided and generally lack pappus (Le & Mündel 1996).
Figure 1. Safflower (Carthamus tinctorius L.): A, Vegetative stage; B, Orange flowers; C, Yellow flowers.
A total of twelve Safflower accessions collected from different parts of Wollo region were planted in the
experimental field at Wollo University, Dessie. For the 12 accessions seven of the seed samples were received
from EBI (Ethiopian Biodiversity Institute) and the remaining five were collected by the researcher (Table 1).
The accessions were planted in January 2014 and harvested in the early July 2014 manually at maturity before
the beginning of the main rain season. Several agro-morphological characters were recorded during the field
experiment.
Table 1. List of Safflower accessions and regions of collection used in this study.
No. Accession No./ Name Region Zone Woreda
1 51526 Amhara South Wollo Tenta
2 207477 Amhara South Wollo Ambasel
3 212588 Amhara South Wollo Ambasel,1440m
4 214915 Amhara North Wollo Wadla
5 241792 Amhara North Wollo Meket, 2045m
6 241793 Amhara North Wollo Bugna, 2070m
7 241794 Amhara North Wollo Bugna, 1990m
8 Kundi Amhara South Wollo Kutaber
9 Barkana Amhara South Wollo Kutaber
10 Kalu Amhara South Wollo Kalu
11 Argoba Amhara South Wollo Argoba
12 Kola Gerado Amhara South Wollo Kutaber
Field experimental procedures
Kemal & Hailu 2019
The field experiment was laid out using Randomized Complete Block Design (RCBD) with three
replications to plant the 12 accessions systematically and field heterogeneity was controlled. Each accession was
represented by a single 1.6 m × 1.6 m plot per replication with 0.5 m gap between plots. The spacing between
rows of each plot was 40 cm and spacing between plants was 20 cm. To study the quantitative data five
individual plants was tagged within each plot randomly for later morphological variation evaluation. Manual
thinning was used after emergence to obtain normal density. Fertilizers were applied at 100 kg N ha-1 and 100
kg P ha-1 prior to sowing and 50 kg N ha-1 after sowing and at shooting stage. The crop was irrigated as needed.
All accessions were provided with the same agronomical practices and weeds were manually controlled.
Data collection
Agro-morphological characters as described below were recorded from five randomly chosen plants per plot.
A. Plant height (PH): The main stem length in centimeter (cm) from ground level to the tip of mains stem at
maturity time
B. Days to flowering (DF): Total number of days taken from the date of sowing to the day of first flower
opening in each plant
C. Days to maturity (DM): Total number of days taken from the date of sowing to the day of the complete
maturation of the plant
D. Number of Primary branches (NPB): Total number of primary branches produced on the main stem by
individual plant
E. Number of secondary branches (NSB): Total number of secondary branches arising from the primary
branches in each randomly selected plant
F. Number of capitula per plant (NCP): Total number of capitula produced by a single plant at maturity
stage
G. Number of seeds per capitulum (NSC): Total number of seeds obtained from main capitulum
H. Thousand seed weight (TSW) (g): The weight of one thousand seeds from randomly selected seeds were
weighed
I. Yield per plant (YPP) (g): The individual plant was threshed and the yield of a single plant was measured
J. Yield per plot (YPPt) (kg): The total seed yield obtained from each plot was measured and recorded
Data analysis
The genetic diversity and relationships among Safflower genotypes were evaluated using quantitative data
analysis. All traits recorded in the data collection were statistically analyzed by applying SPSS version 20.
Morphological traits of each genotype were measured on five randomly chosen plants in each replication for
genetic diversity analysis of Safflower accessions from Wollo region.
i. Analysis of variance: Analysis of variance of data for agro-morphological characters was performed using
SPSS statistical package. The quantitative data for all the parameters were subjected to analysis using SPSS
statistical computer package version 20. Range, mean, standard deviation and variance were calculated for
each trait.
Data taken from five sample plants in each plot (from middle rows) was used in the analyses for traits
that required sampling. Total variation was partitioned in to known and unknown effects following the
standard procedures of ANOVA given in table 2 (Gomez & Gomez 1984).
Table 2. Source of variation, degree of freedom and mean squares for RCBD.
Source of variation Degree of freedom Mean square Expected mean square
Replications (r-1) MSr σ2e+ g σ2r
Genotypes (g-1) MSg σ2e+ σ2g
Error (r-1) (g-1) MSe σ2e
Note: r- Number of replications, g- Number of genotypes, MSe- Mean square of error, MSr- Mean square of
replication, MSg- Mean square of genotypes.
ii. Correlation coefficient (r): The associations among various agro-morphological traits were estimated using
simple Pearson’s product moment correlation coefficients by using SPSS statistical package software of
version 20, with appropriate degrees of freedom and probability level, significance of the correlation
coefficients was tested.
iii. Cluster Analysis: Cluster analysis was carried out to identify the traits that account for the genetic variation
and to determine pattern of genetic similarity of all accessions. The means of each geographical group for all
agro-morphological traits were used for cluster analysis to study the relationship between geographical
groups. Genetic distance and cluster analysis of agro-morphological data was used to reveal genetic
difference within the population of Safflower. The cluster analysis was conducted on average taxonomic
distance and a dendrogram was generated based on the genetic distance matrix using SPSS statistical
computer package version 20 (Samarajeewa et al. 2004, Seetharam et al. 2004).
iv. Principal Component Analysis: To understand which trait is responsible for the variation that existed among
the twelve accessions collected from different region of Wollo, all of the traits were grouped in to different
principal components. According to Johnson & Wichern (2002) it is possible to decide the importance of
traits in different principal components. Traits that accounted for the highest variation within each principal
component are also described.
RESULTS
Variance components, mean comparisons, range and standard deviation for plant height, days to flowering,
number of primary branches, number of secondary branches, days to maturity, number of capitula per plant,
number of seeds per capitulum, thousand seed weight, yield per plant, and yield per plot are presented in table 3.
Analysis of Variance
Table 3. Range, mean, standard deviation and variance of accessions used for the study based on agro-morphological traits.
PH DF NPB NSB DM NCP NSC TSW YPP YPPt
Std. Dev. 11.241 1.437 3.373 0.921 1.521 39.227 6.420 3.201 78.124 1.861
Mean 113.917 126.361 25.400 6.383 165.028 151.650 38.811 44.483 251.406 5.902
Range 48.600 6.000 13.400 3.900 6.000 137.700 24.200 13.300 289.630 6.750
Variance 126.357 2.066 11.377 0.848 2.313 1538.763 41.220 10.248 6103.361 3.463
Note: PH- Plant height (cm), DF- Days to flowering, DFF- Days to 50% flowering, DM- Days to maturity, NPB- Number of
primary branches per plant, NSB- Number of secondary branches per plant, NCP- Number of capitula per plant, NSD-
Number of seed per capitulum, TSW- 1000- seed weight (g), YYP- Yield per plant (g), YYPt- Yield per plot (kg).
The analysis of variance showed significant difference among the accessions in days to flowering, days to
50% flowering and days to maturity at P ≤0.001, and number of primary branches and number of secondary
branches at P ≤0.05 shown in table 4.
Table 4. Mean squares value for eleven agro-morphological trait of Safflower accession.
Replication Accession Error
Characters C.V.
(df=2) (df=11) (df=22)
Plant Height 123.86 143.64 117.94 9.87
Days to flowering 0.1944 5.4217** 0.5581 1.14
Number of primary branches 25.4908 17.0382* 7.2636 13.28
Number of secondary branches 0.54333 1.44202* 0.72052 14.43
Days to maturity 0.1944 6.5126** 0.4066 0.92
Number of capitula per plant 1033.0 2080.9 1313.7 25.87
Number of seed per capitulum 40.778 31.551 46.095 16.54
1000 seed weight 25.4800 8.7761 9.5988 7.20
Yield per plant 5504.2 8712.0 4853.5 31.07
Yield per plot 3.4630 4.7112 2.8392 31.53
Note: df- Degrees of freedom, **- Significant at P ≤0.01, *- Significant at P ≤0.05.
Correlation coefficient
Correlations of yield per plot with other traits shows that yield per plot is significantly correlated, positively
with number of capitula per plant (0.87), number of seed per capitula (0.477), plant height (0.772), number of
primary branches (0.799), number of secondary branches (0.695) and yield per plant (0.993) and negatively with
days to flowering (-0.432). Number of capitula per plant shows significant association with number of primary
branches (0.824) and number of secondary branches (0.825). Plant height was positively and significantly (p
≤0.05) correlated with number of secondary branches (0.605) and number of seeds per capitulum (0.308), but
correlated negatively and significantly (p ≤0.05) with days to flowering (-0.402). This may be due to the
environmental condition on the field at the experimental site affecting normal flowering of the plant. There is
also positive significant association (0.647) between number of primary branches and number of Secondary
branches (Table 5).
Cluster Analysis
As shown in figure 2, 12 Safflower accessions collected from different regions of Wollo were clustered in
two groups based on the similarity they reveal and one of the accessions (212588) from Ambasel remain
Kemal & Hailu 2019
ungrouped. The first group consisted of 241793 from Bugna, 51526 from Tenta, Argoba and Barkana. The
second group was the largest group and consisted of three accessions from North Wollo (241794 from Bugna,
24792 from Meket, 214915 from Wadla), 207477 from Ambasel, Kundi, Kola gerado and Kalu.
Table 5. Pearson correlation coefficients of Safflower accessions.
Characters PH DF NPB NSB DM NCP NSC TSW YPP YPPt
PH 1
DF -.402* 1
NPB .671 -.390* 1
NSB .605* -.423* .647** 1
DM -.379 .322 -.360* -.320 1
NCP .757 -.518** .824** .825** -.346* 1
NSC .308* -.027 .348 .112 -.280 .142 1
TSW .300 -.002 .071 .062 -.068 .106 -.045 1
YPP .772 -.429** .807** .724** -.490 .876** .513** .243 1
YPPt .772* -.432** .799** .695** -.476 .877** .477** .283 .993** 1
Note: *- Correlation is significant at the 0.05 level (2-tailed), **- Correlation is significant at the 0.01 level (2-tailed).
PH- Plant height (cm), DF- Days to flowering, DFF- Days to 50% flowering, DM- Days to maturity, NPB- Number of
primary branches per plant, NSB- Number of secondary branches per plant, NCP- Number of capitula per plant, NSD-
Number of seed per capitulum, TSW- 1000- seed weight (g), YYP- Yield per plant (g) and YYPt- Yield per plot (kg).
Figure 2. Dendrogram of cluster analysis for 12 Safflower accessions based on morphological traits.
Principal component analysis
Three principal components, PC1 to PC3 accounted for the 85.8 % of the total variation that exist among
accessions. Out of the total principal components retained PC1 and PC2 with values of 60.8% and 15.9%
respectively contributed more to the total variation. Among the vectors of PC1 which accounted more to the
total variation (60.8%), yield per plant and yield per plot, number of primary and secondary branches, number of
capitula per plant and plant height had higher values. In the second principal component (PC2), which explain
about 15.9% of total variation, originated mainly from days to flowering, number of seeds per capitulum and
thousand seed weight. Similarly, among the vectors of PC3, days to flowering, and thousand weight had higher
values (Table 6).
DISCUSSION
Assessing genetic diversity provides helpful information needed in the Performance of a long-term breeding
program for crop improvement strategies through selection and hybridization. The use of agro morphological
trait can reveal differences among accessions providing efficient tool for crop management and conservation.
In this study, the results showed a considerable genetic variation for different agro-morphological traits
among accessions of Safflower as self-pollinating species maintain high genetic diversity at their polymorphic
loci (Hamrick & Godt 1989). From the analysis of variance, traits like days to flowering, and days to maturity
showed significant variation at (p ≤0.01) and for number of primary branches and number of secondary
branches at (p ≤0.05) indicating that the accessions collected from Wollo region show considerable genetic
variation. Thus, the populations/accessions from the region need to be conserved and managed properly.
Table 6. Eigen values and Eigen vectors of important principal components (PC) for variation among accessions
from Wollo region.
Eigen vectors
Characters
PC 1 PC 2 PC 3
PH 0.335780 0.102058 0.063371
DF -.257142 0.379838 0.516803
NPB 0.361186 0.002921 0.082258
NSB 0.332277 -.190279 0.386095
DM -.219703 -.264872 0.271060
NCP 0.368481 -.102047 0.172887
NSC 0.268732 0.319742 0.094728
TSW -.009849 0.580866 -.505288
YPP 0.375925 0.159286 0.041671
YPPt 0.369466 0.183532 0.000648
Eigen value 6.68824870 1.75395965 1.00253491
Proportion 0.6080 0.1595 0.0911
Cumulative 0.6080 0.7675 0.8586
Significant correlation was recorded between yield per plot and most other traits including yield per plant,
number of primary and secondary branches, number of capitula per plant, number of seeds per capitulum, and
plant height indicated that the improvements of one trait will lead to yield improvement of Safflower in Wollo
region. On the other hand, yield per plot was not significantly correlated with days to maturity and thousand
seed weight. Roopa & Ravikumar (2008) also observed a strong positive correlation between seed yield per
plant and number of capitula per plant, number of branches per plant. Thus, improvement of the yield will
greatly be efficient via number of primary and secondary branches, number of capitula per plant and number of
seeds per capitulum based selection. However, 1000-seed weight was not significantly correlated with any of the
other traits. The highest correlation coefficient was obtained between yield per plant and yield per plot (0.993)
indicating that the average value of traits measured on five single plants, can be used as a plot representative
which is in agreement with (Omidi et al. 2009).
The cluster analysis of Safflower accessions collected from different regions of Wollo showed that the
accessions from different regions are located in the same group indicating the absence of distinct regional
grouping. This is possibly due to environmental condition that affects performance of Safflower favoring the
accessions collected from regions with same environment of the experimental site. This result is in agreement
with the result in Ethiopian tetraploid wheat described by Hailu et al. (2006). Thus, accessions could be more
effectively clustered when considering traits related to grain yield rather than with geographical origin.
Similarly, Omidi et al. (1999) in study of 100 Safflower cultivars also concluded that grouping based on traits
related to seed yield per plant is more effective than based on their origin.
As described by Chahal & Gosal (2002) characters with largest absolute value closer to unity within the first
principal component influence the clustering more than those with lower absolute value closer to zero. In this
study result from the principal component analysis indicate the presence of variation among different accessions
of Safflower in Wollo region identifying that from PC1 which accounted 60.8% of the total variation yield per
plant is the most important trait responsible for highest variation from the component. Thousand seed weight
and days to flowering are also important traits that accounted the highest variation from PC2 and PC3
respectively. Thousand seed weight were very important trait responsible for highest variation from all of the
three principal components.
In order to achieve effective improvement for seed yield increased number of primary and secondary
branches, number of capitula, and number of seeds per capitulum are very important. According to Knowles
(1969) the flower colour of cultivated Safflower of Ethiopian origin as red, however, in this study the researcher
found that the Safflower accessions collected from different regions showed yellow and orange in addition to
red colour (Fig. 1) which vary among and within accessions but white flower colour was not observed in any of
the accessions. This shows that in Ethiopia there is a high level of genetic diversity of Safflower that still require
more study for better breeding performance and germplasm conservation.
Kemal & Hailu 2019
CONCLUSION AND RECOMMENDATIONS

The results shown in this study indicates the presence of high genetic variation among twelve accessions
collected from Wollo, Ethiopia which is useful for breeding and conservation purposes. Depending upon the
results of the data analyzed, appropriate breeding strategies can be adopted for the improvement of target trait.
Future breeding strategies to improve Safflower need to exploit the results of this study to increase seed yield
potential and other desirable traits.
A lack of information about genetic diversity has been a barrier to improve Safflower in Ethiopia,
particularly in Wollo region. The results of this study provided a better understanding of Safflower populations
in Wollo region. Therefore, the wise use of results obtained in this study would facilitate the improvement of
Safflower through breeding and the in situ and ex situ conservation of Safflower genetic resources in this region.
Desirable genotypes in terms of seed yield have been identified in this study and the use of these genotypes in
the breeding program would lead to improved Safflower varieties with a high seed yield.
In Ethiopia, Safflower is grown in the field with low agricultural inputs, which resulted in more pests,
diseases, and a low seed yield in this crop. Seed yield in Safflower is influenced by different factors such as
planting dates, weather patterns, relative humidity, irrigation, fertilizers, and farmers’ practices. Cultivation of
Safflower under good growing conditions would result in a relatively high seed yield. Therefore, development
of high yielding and better adapted genotypes including the cultivation technique need to be improved.
The use of agro morphological traits for assessment of Safflower genetic diversity would help to reveal
variation among accession from different regions. Several varieties from agro-morphological evaluation were
found to have high seed yield potential, and the use of these varieties would lead to an increased Safflower
production in Ethiopia.
ACKNOWLEDGEMENTS
I am thankful for the continuous encouragement and support of my family in every walk
throughout my life. My foremost gratitude goes to Dr. Faris Hailu, for his unreserved help during my study. My
appreciation is also extended to Ethiopian Institute of Biodiversity (EBI) for providing the seed samples used in
this study.
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genotypes using SSR markers and morphological characters. African Journal of Biotechnology 8(10): 2050–
2059.
Singh V, Deshpande MB, Nimbkar N & Singh RJ (2007) The first non-spiny Genetic resources, chromosome
engineering and crop improvement. CRC Press, Boca Raton, USA. Safflower released in India. Sesame and
Safflower Newsletter 18: 77–79.
Thies E (2000) Promising and Underutilized Species Crops and Breeds. Eschborn, Germany: GTZ.
Vavilov NI (1951) The origin, variation, immunity and breeding of cultivated plants. Chronica Botanica 13: 1–
36.
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and suggestions to increase production levels. Proceedings of the VII International Safflower Conference,
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Weiss EA (1983) Safflower. In: Oilseed crops. Tropical agriculture series. Longman, London, pp. 216–281.
Weiss EA (2000) Oil Seed Crops. Blackwell Science Ltd., Oxford, UK.
SOCIETY FOR TROPICAL PLANT RESEARCH
Society for Tropical Plant Research (STPR) is a professional body of
researchers, educationists and extension professionals to promote the
different research activities in the tropical and sub-tropical region
(Taxonomy, Diversity assessment, Phenology, Ecology &
Environment, Climate Change, GIS/Remote Sensing, Socio Economic
& Forestry, Plant Breeding, Physiology, Genetics, Bio-chemistry,
Molecular aspects etc.) related with plants (Bryophytes,
Pteridophytes, Gymnosperms & Angiosperms), algae, lichenised
(lichens) as well as nonlichenised fungi. The society is registered under
Act No. 21, 1860E with the Registration No. K-48941.
The main aim behind the establishment of this society is to provide a
good platform for the researchers of tropical and sub-tropical regions
working in different aspects of science related with the plants and its
surroundings. As well as the implementation of research out comes in
the sustainable uplifting in the lifestyle of local people with the help of
agro-forestry, awareness programmes, training programmes etc.
Contact Us
Society for Tropical Plant Research
197 A-1, Sahab Nagar, Kalyanpur, Kanpur, U.P.-208 017, INDIA
E-mail : india.tpr@gmail.com
Phone : +91 9889847979 (President)
+91 9415462755 (Secretary)

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com ISSN (Online): 2349-1183

Tropical Plant Research

Published by AkiNik Publications

Phenotypic plasticity of Centella asiatica (L.) Urb. growing in

MATERIALS AND METHODS

Table 1. Collection sites and habitats of Centella asiatica.

Effects of chitosan on vegetative organs growth and peroxidases

MATERIAL AND METHODS

Yace 9620A TMS4(2)1425 TMS30572

Range extension of Actinodaphne bourneae Gamble (Lauraceae):

Enhanced rhizome induction and fast regeneration protocol in

MATEIALS AND METHODS

Figure 1. Mature Dendrocalamus longispathus in natural habitat.

Physiologia Plantaram 15(31): 473–497.

Analysis of soil and tree productivity under high density planting

MATERIALS AND METHODS

Figure 1. High density system in Mango cv Dashehari at Lucknow.

RESULTS AND DISCUSSION

Figure 2. Histographic distribution of soil physical properties.

Figure 4. Histographic distribution of soil micronutrients.

density planting. Indian Journal of Horticulture 60(4): 322–326.

Variability in yield and composition of oil from Indian Sandalwood

 Santalol (2)  Bergamotenol (3)

epi-cis- -Santalol (4) cis-Lanceol (5)  Bisabolol (6)

Santalene (7) Santalene (8) epi-Santalene (9)

Bergamotene (10) Bisabolene (11) -curcumene (12)

MATERIALS AND METHODS

RESULTS AND DISCUSSION

Figure 1. Variation trend of % SW oil yield in different girth SW trees.

Figure 2. GC-MS Chromatogram of Heartwood Oil.

Table 3. Variation of metabolites (area %) in extracted SW oil.

Figure 3. Variation trend of SW oil composition in different girth SW trees.

Propagation methods in Babaco plants (Vasconcella x helbornii)

Cell Tissue and Organ Culture 44: 189–194.

Annual flowering of Dendrocalamus longispathus (Kurz) Kurz

Phytochemical study of medicinal plants used against buruli ulcer

MATERIAL AND METHODS

Table 2. Different major groups of compounds found after screening.

Selection of suitable digital elevation model for analysis of forest

Figure 1. Study area.

Topographic Map of Survey of India

Agro-climatic Zone wise Remote Sensing Data of Jharkhand

Aspect Slope Hillshade Altitude Aspect Slope Hillshade Altitude

Forest cover map was created by Landsat 7 ETM+ data

Statistical /Visual comparison Field verification

Result showing best data for analyzing forest cover in

Figure 3. Methodology flow chart.

RESULT AND DISCUSSION

Figure 4. Altitude, Aspect, Slope and Hill-shade maps of Jharkhand, India.

Comparative study of the floristic and structural diversity of

MATERIAL AND METHOD

Figure 1. Map of the study area.

The area-species curve

Piélou's equitability (EQ):

Simpson diversity index

RESULTS AND DISCUSSION

Figure 2. Significance value index of Wela species.

Figure 3. Significance value index of Rubi-Télé.

Figure 4. Significance value index of UMA.

Figure 5. Index of Morisita. [Rbt- Rubi Télé]

Figure 6. Area-species curve. [Rbt- Rubi Télé]

Figure 7. The density of the three sites.

Figure 9. Factorial Correspondence Analysis.