Volume 2, Issue 2 (2015) Tropical Plant Research

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

2(2): 7277, 2015
Research article
In vitro antioxidant activity of selected Ganoderma species found

in Odisha, India
Ashutosh Rajoriya, Sushri Shant Tripathy and Nibha Gupta*
Plant pathology and Microbiology division, Regional Plant Resource Centre, Bhubaneswar, Odisha, India
*Corresponding Author: nguc2003@yahoo.co.in [Accepted: 25 April 2015]
Abstract: Four species of Ganoderma were collected from different forests of Odisha and
analysed for the presence of antioxidant components. Data revealed the presence of DPPH free
radical scavenging properties ranged between 91.6495.51 % in these species. Ganoderma
applanatum exhibited a studied for the same. Both the Ganoderma species exhibited higher
amount of tannin content too. Alkaloid content was ranged in 3.665.51 mg.gm-1 in these four
species. Present study exhibited the presence of good higher amount of total phenol, ascorbic acid,
carotenoid whereas lycopene and ergosterol content was found to be maximum in Ganoderma
lucidium as compared to other mushroom species amount of flavonoid content in Ganoderma
tsugae followed by G. lucidium and G. applanatum. This preliminary exploratory data of the
present study indicated that Ganoderma species are good source of antioxidant components.
Further extraction and purification of this component may throw light on its role as an
antioxidant in Ganoderma sp.
Keywords: Mushrooms - Ganoderma - Antioxidants - Flavonoids - Ergosterol.
[Cite as: Rajoriya A, Tripathy SS & Gupta N (2015) In vitro antioxidant activity of selected Ganoderma species
found in Odisha, India. Tropical Plant Research 2(2): 7277]
INTRODUCTION
Mushrooms are well known for their therapeutic properties like antibacterial, antifungal, antiviral,
antitumour, immunomodulating, antiallergic, antiatherogenic, hypoglycemic, anti-inflammatory and
hepatoprotective activities (Ferreira et al. 2010, Lindequist et al. 2005). These bioactivities are mainly due to -
glucans, phenolics, vitamins, organic acids and trace elements (Cheung et al. 2003, Khatuna et al. 2013,
Iwalokun et al. 2007).
Mushrooms are also considered as home remedy to protect from various diseases elicited by oxidative stress
or free radical stress (Chen et al. 2012). It is mainly due to the antioxidants which are widely distributed in
mushrooms (Jose & Janardhanan 2000, Liu et al. 1997, Barros et al. 2007). The family Ganodermataceae
presents polypore kind of Basidiomycetous fungi having a double walled basidiospore (Donk 1964).
Basidiocarps of this genus possess a shiny surface which is associated with the pilocystidia embedded in an
extracellular melanin matrix (Moncalvo 2000). Ganoderma species are ubiquitous in the world with varied
characteristics, such as different shapes, size and color (red, black, blue/green, white, yellow, and purple) of the
fruit body, specific host and geographical origin (Zhao & Zhang 1994, Woo et al. 1999, Upton 2000).
Ganoderma lucidum (Curtis) P.Karst. (Common names: Reishei, Lingzhi) is a species of Basidiomycetes
that belongs to Ganodermataceae of Polyporales (Yang et al. 2000). Smina et al. (2011) reported various types
of antioxidants from the Ganoderma which can reduce oxidative damage by directly scavenging free radicals
generated in the cell. Ganoderma lucidium shows bioactivity against hypertension, bronchitis, arthritis,
neurasthenia, hepatopathy, chronic hepatitis, nephritis, gastric ulcer, tumorogenic diseases,
hypercholesterolemia, immunological disorders, scleroderma, cardiovascular disease, AIDS and cancer (Sliva
2003), beside G. lucidium, G. tsugae and G. applanatum is also used for the bioactivity which in turn can be
well utilized for the health benefits, many reports are available regarding their anticancer, antioxidant,
www.tropicalplantresearch.com 72
Received: 27 December 2014 Published online: 30 June 2015
Rajoriya et al. (2015) 2(2): 7277
.
antibacterial, antiviral behavior (Mau et al. 2005a, Mau et al. 2005b, Jeong et al. 2008, Kim et al. 1998, Rym et
al. 1999).
The main objective of the present work is to explore the antioxidant properties of some Ganoderma species
found in Odisha.
MATERIALS AND METHODS

Collection and identification
Healthy, fresh and succulent macrofungi were collected from tropical moist deciduous and semi evergreen
forests from different forest divisions of Odisha Macroscopic and microscopic examination of different parts
such as pileus, stipe, veil, ring, volva, lamellae and gills etc. were focused to identify species. All of the assays
were carried out using the entire mushroom. Mushrooms were cleaned and subsequently dried in the oven at
50C for about 4 hrs. All of the dried mushrooms were ground to fine powder (100 mesh) and stored in air tight
plastic container at room temperature till all the analysis.
Antioxidant components
Total Phenolic Content: Total phenolic content in the wild mushrooms were estimated through folin phenol
method as described by Singleton and Rossi (Moradali et al. 2006). The optical density was measured at 765
nm using spectrophotometer (Analytic Jena).
Ascorbic acid content: The ascorbic acid content in the wild mushrooms was determined by volumetric method
(Singleton & Rossi 1965). The amount of ascorbic acid in mg 100 g-1 sample is calculated by using formula;
0.5 mg/V1 ml V2 /5100/ weight of the sample100, when V1 is the standard ascorbic acid consumed
against dye.
Flavonoids: The flavonoid content in dried sample was estimated by using aluminum chloride colorimetric
technique and expressed in terms of quercetin equivalents per gram (Harris & Ray 1935).
Beta carotene and Lycopene: The concentration of -carotene and lycopene in mushroom extracts was estimated
by spectrophotometer following Nagata and Yamashita (1992), Chang et al. (2002) & Barros et al. (2007).
Carotenoid: The carotenoid content was estimated in 500 mg of dried mushroom powder treated with 10 ml of
80% acetone and centrifuged at 3000 rpm for 10 minutes at 4C. The quantity of carotenoid was calculated
(Arnon 1949).
Tannins: Tannic acid was served as a standard and tannin content was estimated at 760nm according to
Schanderl (1970) and expressed in mg.gm-1.
Alkaloids: Total alkaloid was estimated after extraction with glacial acetic acid and ethanol and precipitated
with Draggendroffs reagent. The residue treated with sodium sulfide and thiourea solution and optical
density was measured at 435 nm for alkaloid estimation (Srividya & Mehrotra 2003).
Ergosterol: Total ergosterol was estimated with chloroform and methanol mixture followed by NaCl2 and glacial
acetic acid treatment. Finally sterol content was estimated by using ferric chloride reagent and measuring
absorbance at 550 nm (Sadasivam & Manickam 1996).
Antioxidant assay
Free radical scavenging activity: The DPPH free radical scavenging activity was estimated in the methanolic
extracts by colorimetric method (Chan et al. 2007). 1 ml of methanolic extract was added with 2 ml of
DPPH solution (1:2) and incubated for 30 min. in dark after vigorous shaking. Absorbance was measured at
517 nm and scavenging activity of each extract was calculated.
Reducing power ability: Each mushroom extract (0.54 mg.ml-1) in methanol (2.5 ml) was mixed with 2.5 ml of
200 mM sodium phosphate buffer (pH: 6.6) and 2.5 ml of 1% potassium ferricyanide, and the mixture was
incubated at 50C for 20 minutes. After 2.5 ml of 10% trichloroacetic acid was added, the mixture was
centrifuged at 2000 rpm for 10 minutes. The upper layer (5 ml) was mixed with 5 ml of deionized water and
1 ml of 0.1% ferric chloride, and the absorbance was taken at 700 nm (Analytik Jena) spectrophotometer.
Ec-50 value was calculated in mg.ml-1 at 0.5 optical density against reagent blank (Oyaizu 1986).
Ferric Antioxidant Reducing Power (FRAP): 100 l of the methanolic extract was mixed with 3 ml of FRAP
reagent and incubated in the room temperature in dark for 10 minutes and finally absorbance was read at 593
nm (Analytik Jena) spectrophotometer. FRAP value was expressed in terms of mg AEAC.gm-1 of sample
(Benzie & Strain 1996, Athavale et al. 2012).
Rajoriya et al. (2015) 2(2): 7277
.
RESULTS AND DISCUSSIONS
In the present study four species of Ganoderma (i.e. G. lucidium, G. tsugae, G. applanatum and Ganoderma
sp.) were collected and analyzed for the antioxidant properties. Radical scavenging activity was seen best in the
G. tsugae (95.51%) and Ganoderma sp. (94.43%) at the IC50 value of 12 mg.ml-1 and 10 mg.ml-1 respectively
(Table 1). Thus the assay used to test the radical scavenging activity shows that scavenging activity is directly
proportional to the concentration of the sample. Since free radicals are the causal agents for the oxidative stress
and different aliments Ganoderma samples used in the present studies can with stand for the health benefits.
Presence of ergosterol in range of 0.0280.45 mg.gm-1 in these Ganoderma species is also revealed in the
present studies (Mattila et al. 2002).
Table 1. Representing antioxidant activity of different wild Ganoderma species from Odisha, India.
Species DPPH (%) IC50 (mg.ml-1) FRAP (mg AEAC.gm-1)
Ganoderma lucidium 93.19 9 2.290.14
Ganoderma tsugae 95.51 10 1.820.01
Ganoderma applanatum 91.64 6 2.430.05
Ganoderma sp. 94.43 12 0.240.04
The phenolic content of Ganoderma sp. was ranged in 911.60 mg.gm-1 whereas highest flavonoid content
was observed in G. tsugae (0.840.37 mg.gm-1). A very high amount of Ascorbic acid i.e. 1.400.01 and
1.10.16 mg.gm-1 was found in G. applanatum and G. lucidium, respectively (Table 2). More or less similar
quantities of carotenoid content ranged in 3.484.65 mg.gm-1 were observed in all Ganoderma sp. studied
except G. applanatum (7.430.29 mg.gm-1). All the four species of Ganoderma showed a good amount of
alkaloid and tannin content.
Table 2. Analysis of antioxidant components of wild Ganoderma species from Odisha, India.
S. No. Parameters G. lucidium G. tsugae G. applanatum Ganoderma sp.
1 Total Phenolic 9.000.30 9.000.10 11.600.20 11.400.10
2 Flavonoids 0.630.15 0.840.37 0.620.13 0.380.06
3 Ascorbic acid 1.100.016 0.700.017 1.400.011 0.600.017
4 Carotenoids 4.47 0.05 4.650.73 7.430.29 3.480.49
5 -Carotene 3.630.19 1.660.16 3.300.77 1.600.43
6 Lycopene 0.2240.029 0.1880.002 0.1770.014 0.0430.004
7 Ergosterol 0.490.067 0.470.067 0.440.094 0.280.044
8 Alkaloids 3.660.10 4.501.15 4.400.25 5.510.16
9 Tannins 2.290.14 1.820.01 2.430.05 0.240.04
The presence of phenolic compounds in Ganoderma confirms the observations of Rawat et al. (2013) and
Celik et al. (2014). Phenolic compounds are responsible factors of antioxidant properties in many mushrooms
and plants (Ferreira et al. 2009, Barros et al. 2008). Data revealed good radical scavenging properties by these
mushroom species may be due to good phenolic content also (Table 2). Flavonoids are the group of phenolic
compounds which were assumed to be accumulated only in the plants but not by the animal or any fungi
(Ferreira et al. 2009) however a good amount of flavonoid content was recorded in present studies. Flavonoid
content in all the four species of Ganoderma ranged from 0.84 to 0.38 mg.gm-1 which is also supported by the
studies of Logananthan et al. (2010) and Barros et al. (2008) and can be compared with the edible mushroom
varieties. Due to the presence of flavonoid content these species may be considered as curing agent for various
cardiovascular, anti-proliferative, detoxification and anti-inflammatory type of diseases (Le 2002).
Findings of carotenoid content in the Ganoderma species in good amount made them important at par with
fruits and vegetables (Mangels et al. 1993). A higher amount of Beta carotene and lycopene was exhibited in
these species and corroborated with the reports of Robaszkiewicz et al. (2010), Pal et al. (2010) and Celik et al.
(2014). Ascorbic acid is considered to exert a protective role against many oxidative stress related ailments such
as cardiovascular, cancer, neurodegenerative problems and cataract (Halliwell 1996). Ascorbic acids ranged
from the 1.400.60 mg.gm-1 of the sample which is comparable to the reports of Barros et al. (2008). Tannins
are polyphenolic compounds responsible for various bioactivities viz. antimicrobial, antioxidative and antitumor
Rajoriya et al. (2015) 2(2): 7277
.
activities (Hatano et al. 2006, Yoshizawa et al. 1987, Okuda & Ito 2011). Data presented in table 2 regarding
tannin content in the four species of Ganoderma ranged between 2.290.24 mg.gm-1 is well compared with the
results of Puttaraju et al. (2006) and Onuoha et al. (2010).
The present study suggests the Ganoderma species are the good source of antioxidant compounds. Further,
elucidation and purification of these compounds may lead towards the findings of new class of antioxidants
useful for the development of health care agent.
ACKNOWLEDGEMENTS
The financial assistance obtained from Ministry of Environment and Forests, Govt. of India (Project no. 22-
24/2010 CS.I) is gratefully acknowledged.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(2): 7881, 2015
Research article
Additions to lichen flora of Jammu and Kashmir, India

Reema Goni and Namrata Sharma*
Department of Botany, Jammu University, Jammu & Kashmir, India
*Corresponding Author: phyllanthus@rediffmail.com [Accepted: 10 June 2015]
Abstract: The paper deals with the addition of 44 species to the knowledge of lichen flora of
Jammu & Kashmir state, India. The corticolous (25) lichens exhibit their dominance in the area of
study followed by saxicolous (16), lignicolous (2) and terricolous (1) forms.
Keywords: Diversity - Hot spot - Doda - Himalayas - Altitude.
[Cite as: Goni R & Sharma N (2015) Additions to lichen flora of Jammu and Kashmir, India. Tropical Plant
Research 2(2): 7881]
INTRODUCTION
State of Jammu and Kashmir lies between 32 17' and 36 58' north latitudes and 73 26' and 80 30' east
longitudes and exhibits varied climate associated with wide altitudinal deviations. As such it provides a wide
range of substrates for growth and colonisation of lichens. It is often called as Hot Spot for lichen diversity in
India (Sheikh et al. 2006a). Inspite of this, lichen collection started late here in early thirties of last century
(Smith 1931). Comprehensive accounts came later. First of this series was by Sheikh et al. (2006a, b). The
authors listed a total of 279 lichen species from the state. The collections made by these authors were from
diverse areas, which included Achabal, Pahalgam, Kukernag, Verinag, Baltal, Neh Nar Glacier, Mamal village;
Kangan, Shankaracharya Hill, Ganderbal, Zaberwan, Sonmarg, Harwan Garden, Prang Garden, Shalimar
Garden, Gulmarg, Baba Rishi, Yarikah Pine Forest, Tangmarg, Drang Forest, Khilanmarg, Ferozpur;
Awantipura, Pingalgam & Goosu, Yusmarg, Pulwama and Budgam all from Kashmir region; Nandini Hill,
Jammu University Campus, Mansar Lake, Nagrota, Vijaypur, Nud, Banihal, Kishtwar, Chinta valley, Ramtund
Forest area, Kaplash & Bhaderwah from Jammu region and Hemis National Park from Ladakh region. 30
species from Mansar-Surinsar wildlife Sanctuary of Jammu were added to this list later in 2009. Solan (2010)
enumerated 18 species belonging to 15 genera and 10 families from Ramnagar Wildlife Sanctuary, Jammu.
Later inclusions were 77 species from Kishtwar, Rajouri and Jammu districts by Sheikh et al. (2013), 18 species
from Nandini Wildlife Sanctuary by Goni et al. (2013), 24 species from Zanskar valley, Ladakh by Kumar et al.
(2014) and 25 species from Kargil district of Ladakh region by Rahim et al. (2014). A checklist of lichen flora
of Jammu and Kashmir by Goni et al. (2015) revealed the occurrence of 356 species of lichens belonging to 35
families and 91 genera.
These wide collections however do not include several areas of the state. One such area is district Doda of
Jammu region lying between 3308' to 3324' North latitude and 7523' to 7549' East longitude and covering
an area of 4,500 km2 with an altitude varying from 914 to 4267 meter above mean sea level. This wide district
characterized by a wide range of climatic and physiographic conditions, providing thereby different type of
habitats. Our survey of this area led us to a collection of about 500 lichen samples. Complete identification of
these resulted in the addition of 44 species to the lichen flora of J&K state. This communication depicts these
additions.

During the period 20112014, various localities in the Doda district were surveyed for the collection of
lichens. The specimens were collected from base to chest height of tree trunk and from all other available
substrates.
Identification of the collected specimens was done using relevant keys and literature (Awasthi 1991, 2000,
2007, Nayaka, 2004, Divakar & Upreti, 2005). In the laboratory, the specimens were investigated
Received: 12 February 2015 Published online: 30 June 2015
Goni & Sharma (2015) 2(2): 7881
.
morphologically, anatomically and chemically. Chemistry was studied with the help of colour spot tests and thin
layer chromatography. The colour tests were performed with the usual chemical reagents which included 5%
KOH solution (K-test), aqueous solution of calcium hypochlorite (C-test) and paraphenylene diammine (PD
test). Lichen substances were investigated with the thin layer chromatography (TLC) in solvent system A (180
toluene: 60 dioxane: 8 acetic acid) using the technique of Culberson (1972) and Walker and James (1980).

The paper deals with a survey of district Doda of J&K state and enumeration of 44 species belonging to 27
genera and 18 families which are new additions to the lichen flora of J&K state (Table 1). Among the various
growth forms collected presently crustose forms (29) were the dominant followed by fruticose (7), foliose (6)
and squamulose (2) forms. On the basis of substratum type, 25 species have been observed to be corticolous,
while 16 species have been recorded as saxicolous forms. Lignicolous and terricolous forms are represented by
2 and 1 species respectively. Graphidaceae (7 species) was the dominant family followed by Parmeliaceae and
Ramalinaceae (6 species each), Teloschistaceae (4 species); Lecanoraceae and Physciaceae (3 species each);
Cladoniaceae (2 species); Verrucariaceae and Lecideaceae (2 species); Acarosporaceae, Arthoniaceae,
Coniocybaceae, Lichinaceae, Megasporaceae. Pannariaceae, Peltigeraceae, Pyrenulaceae and Trapeliaceae (1
species each). Caloplaca appressa, Cladonia didyma, Dimelaena oreina, Lichinella cribellifera, Melanelia
tominii, Porpidia albocoerulescens, Verrucaria laevata, Verrucaria margacea and Xanthoparmelia
antleriformis are some of the major lichen species collected from the study area (Fig. 1).
Table 1. Lichens of Doda district (J&K State) enumerated with their growth forms and substratum from Doda district of J&K state.
S. No. Taxa Growth form Substrate Place of collection Altitude
1 Acaropspora tominiana Magnusson Crustose Saxicolous Nalthi 1795 m
2. Arthonia radiata (Pers.) Ach. Crustose Corticolous Sartingal 1700 m
3. Aspicilia dwaliensis Rsnen Crustose Saxicolous Khanpura 1594 m
4. Bacidia alutacea (Kremp.) Zahlbr. Crustose Corticolous Dandi 1603 m
5. Bacidia medialis (Tuck. ex Nyl.) Zahlbr. Crustose Corticolous Dandi 1607 m
6. Bacidia submedialis (Nyl.) Zahlbr. Crustose Corticolous Dandi 1606 m
7. Buellia disjecta Zahlbr. in Hand. Mazz. Crustose Saxicolous Drudoo 1329 m
8. Buellia palnienis S.R. Singh & D.D. Awasthi Crustose Saxicolous Batogra 1905 m
9. Caloplaca appressa Wetmore & Krnefelt Crustose Saxicolous Phigsoo 1350 m
10. Caloplaca granularis (Mll. Arg.) Zahlbr. Crustose Corticolous Khani 2124 m
11. Caloplaca lithophila H. Magn. Crustose Saxicolous Gath 1198 m
12. Caloplaca ochroplaca Poelt & Hinter. Crustose Saxicolous Chilli 1785 m
13. Cladonia didyma (Fe) Vain. Fruticose Lignicolous Traun 1975 m
14. Cladonia fruticulosa Kremp. Fruticose Lignicolous Traun 1990 m
15 Chaenotheca chrysocephala (Ach.) Th. Fr. Crustose Corticolous Traun 1994 m
16. Dimelaena oreina (Ach.) Norman Sqaumulose Saxicolous Bhella 1142 m
17. Graphis arecae Vain. Crustose Corticolous Tanta 2100 m
18. Graphis dendrogramma Nyl. in Cromb. Crustose Corticolous Dandi 1675 m
19. Graphis epimelaena Mll. Arg. Crustose Corticolous Khanpura 1594 m
20. Graphis leptocarpa Fee Crustose Corticolous Dandi 1687 m
21. Graphis lineola Ach. Crustose Corticolous Udrana 1580 m
22. Graphis longiramea Mll. Arg. Crustose Corticolous Dandi 1626 m
23. Graphis scripta (L.) Ach. Crustose Corticolous Tanta 2089 m
24. Fuscopannaria subgemmascans Upreti & Divakar Squamulose Corticolous Puneja 1900 m
25. Lichinella cribellifera (Nyl.) P. P. Moreno & Egea Fruticose Saxicolous Shiwa 908 m
26. Lecanora pseudargentata Lumbsch Crustose Corticolous Dareja 1780 m
27. Lecanora sulphurescens Fe Crustose Saxicolous Dareja 1780 m
28. Lecidella elaeochroma (Ach.) M. Choisy Crustose Corticolous Bhalla 1678 m
29. Melanelia tominii (Oksner) Essl. Foliose Corticolous Tanta 1980 m
30. Mycobilimbia hunana (Zahlbr.) D.D. Awasthi Crustose Saxicolous Seri 1271 m
31. Parmelia squarrosa Hale Foliose Corticolous Sartingal 1851 m
32. Parmelienella wallichiana (Taylor )Hale Foliose Corticolous Drudoo 1324 m
33. Parmotrema pseudotinctorum (Abbayes) Hale Foliose Corticolous Seri 1391 m
34. Peltigera collina (Ach.) Schrad. Foliose Terricolous Amira nagar 1723 m
35. Porpidia albocoerulescens (Wulfen) Hertel & Knoph Crustose Saxicolous Khanpura 1595 m
Goni & Sharma (2015) 2(2): 7881
.
36. Pyrenula mamillana (Ach.) Trevis Crustose Corticolous Khani 220 m
37. Ramalina farinacea (L.) Ach. Fruticose Corticolous Traun 2013 m
38. Ramalina intermedia (Delise ex Nyl.) Nyl. Fruticose Corticolous Puneja 1900 m
39. Ramalina subfarinacea (Nyl.) Nyl. Fruticose Corticolous Pranoo 1945 m
40. Trapelia placodioides Coppins & P. James Crustose Saxicolous Sartingal 1987 m
41. Usnea orentalis Motyka Fruticose Corticolous Traun 2034 m
42. Verrucaria laevata Ach Crustose Saxicolous Kursari 1857 m
43. Verrucaria margacea (Wahlenb.) Wahlenb. Crustose Saxicolous Kothi 1657 m
44. Xanthoparmelia antleriformis (Elix) Elix & J. Johnst. Foliose Saxicolous Shiwa 870 m
Figure 1. Some major lichens: A, Caloplaca appressa; B, Cladonia didyma; C, Dimelaena oreina; D, Lichinella cribellifera:
E, Melanelia tominii; F, Porpidia albocoerulescens; G, Verrucaria laevata; H, Verrucaria margacea; I, Xanthoparmelia
antleriformis.
ACKNOWLEDGEMENTS
The authors are grateful to the Head, Department of Botany, University of Jammu; Dr. C.S. Nautiyal,
Director and Dr. D.K. Upreti, Head, Lichen Lab, CSIR-National Botanical Research Institute, Lucknow for
providing necessary laboratory and library facilities. We also thank UGC for financial support for field trips
from SAP grant to the Department of Botany.
REFERENCES
Awasthi DD (1991) A key to Microlichens of India, Nepal and Srilanka. Biblioth. The Lichenologist 40: 1-337.
Awasthi DD (2000) Lichenology in Indian Sub-continent. Bishen Singh Mahendra Pal Singh, Dehradun, India.
Awasthi DD (2007) A Compendium of the Macrolichens from India, Nepal and Sri Lanka. Bishen Singh
Mahendra Pal Singh, Dehradun, India.
Culberson CF (1972) Improved conditions and new data for the identification of lichen products by a
standardized thin- layer Chromatographic method. Journal of Chromatography 72: 113-125.
Goni & Sharma (2015) 2(2): 7881
.
Divakar PK & Upreti DK (2005) Parmeloid Lichens in India (A Revisionary study). Bishen Singh Mahendra Pal
Singh, Dehradun, India.
Goni R, Raina AKP & Magotra R (2013) Lichen diversity in Nandini Wildlife Sanctuary, Jammu (J&K).
Phytotaxonomy13: 106108.
Goni R, Raina AKP, Magotra R & Sharma N (2015) Lichen flora of Jammu and Kashmir State, India: An
updated checklist. Tropical Plant Research 2(1): 6471.
Kumar J, Rai H, Khare R, Upreti DK, Dhar P, Tayade AB, Chaurasia OP & Srivastava RB (2014) Elevational
controls of lichen communities in Zanskar valley, Ladakh, a Trans Himalayan cold desert. Tropical Plant
Research 1(2): 4854.
Nayaka S (2004) Revisionary studies on Lichen genus Lecanora sensu lato in India, Ph. D. Thesis. Avadh
University Faizabad, Uttar Pradesh, India.
Rahim A, Raina AK & Hussan A (2014) Lichen diversity of Kargil town and its adjoining areas, J&K.
International Journal of Current Research 5(14): 14.
Sheikh MA, Upreti DK & Raina AK (2006a) An enumeration of Lichens from three Districts of Jammu and
Kashmir, India. Journal of Applied Biosciences 32(2): 189-191.
Sheikh MA, Upreti DK & Raina AK (2006b) Lichen Diversity in Jammu and Kashmir, India. Geophytology
36(1&2): 69-85.
Sheikh MA, Raina AK & Upreti DK (2009) Lichen Flora of Surinsar-Mansar Wildlife Sanctuary, Jammu and
Kashmir. Journal of Applied and Natural Sciences 1(1): 79-81.
Sheikh MA, Raina AK & Hussan A (2013) A preliminary observation of lichen flora in three districts of Jammu
& Kashmir. International Journal of Current Research 5(4): 96668.
Smith A L (1931). Lichens from Northern India. Trans. British Mycological Society 16: 128-132.
Solan S, Mehta, KA & Magotra R (2010) A catalogue of lichens of Ramnagar Wildlife Sanctuary, Jammu
(J&K). Phytotaxonomy 10: 134138.
Walker FJ & James PW (1980) A revised guide to the microchemical techniques for the identification of lichen
products. Bulletin of British Lichenology Society 46: 13-29.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(2): 8284, 2015
Research article
Two species of Zygnemopsis (Skuja) Transeau from West

Bengal, India
Nilu Halder
Department of Botany, Raja Peary Mohan College, Uttarpara-712258, Hooghly, W.B., India
*Corresponding Author: niluhalder1@gmail.com [Accepted: 12 June 2015]
Abstract: In the present paper, two species of the genus Zygnemopsis viz. Zygnemopsis
pseudolahaulensis and Zygnemopsis benghalensis of Zygnemaceae under the order Zygnematales
of Chlorophyta had been morpho-taxonomically described first time from Hooghly district in West
Bengal, India. These algal species had been collected from ponds of this district. The above
mentioned two taxa were new reports from the district and also the second report from state of
West Bengal, India.
Keywords: New report - Chlorophyta - Zygnemopsis - Hooghly district - West Bengal.
[Cite as: Halder N (2015) Two species of Zygnemopsis (Skuja) Transeau from West Bengal, India. Tropical
Plant Research 2(2): 8284]
INTRODUCTION
The members of Zygnemaceae are filamentous and prefer to grow in winter season and, form climax in early
summer season. Finally they form zygospores or aplanospores during reproductive phase in summer as free
floating condition. Zygnemopsis (Skuja) Transeau, contains about 55 species all over the world and all the
known species are isogamous (Yin-xin 1994). Previously, few works had also been described the members of
Zygnemaceae from the state and country. Martens (1869) first recorded the occurrence of Zygnemaceae from
Raniganj area of West Bengal. In the year 1959, a commendable taxonomic work had been carried out by
Randhawa on the members of Zygnemaceae and he published his findings in the monograph 'Zygnemaceae'
from India. Some other noteworthy publications on Zygnemopsis included: Das (1962), Patel & Kumar (1971,
1977), Prasad & Kumari Vijay (1977), Sharma & Kargupta (1986) and Chalotra et al. (2013). As since 1986 no
morpho-taxonomic report on Zygnemopsis was found from West Bengal, keeping view this paucity of
taxonomic information the present study was undertaken from this area. The aim of the present study was the
exploration of biodiversity of Zygnemaceae and documentation of green filamentous algal species to prepare
algal data bases of this state in future. Human anthropogenic activities, loss of algal habitats and increase of
pollution level in water bodies might be responsible for rare occurrence of this alga in this state. Therefore, a
proper sustainable management is required for functioning aquatic ecosystems and maintains biodiversity of
phyco-flora.

Algal samples were collected in plastic and glass containers from two places viz. ponds at Jirat (N 23-12' E
88-45') and Somrabazar (N 23-13' E 88-43') of Hooghly district, West Bengal. Detail study was made by
examining specimens under Olympus microscope (Model-CH20i) for determination of species. Samples were
preserved in 4% formalin. Identification of different taxa was accomplished with the help of authentic literatures
viz. Randhawa (1959), Patel & Kumar (1971, 1977) and Sharma & Kargupta (1986). Each currently accepted
name has been provided with its author(s) name. Water temperature (C) was recorded using Zeals (U.K.)
Mercury thermometer on the spot. pH of water was measured with the help of portable digital pH meter (Merck,
Germany, Model No. 320). NO3-N, PO43-, dissolve oxygen (DO), biological oxygen demand (BOD), chemical
oxygen demand (COD), SO42-, total soluble salts (TSS), total dissolve solids (TDS) and total alkalinity were
measured by using UV-VIS spectrophotometer (CECIL CE- 7200) according to the method of APHA (2005).
All parameters in ecological notes were expressed in mg l-1 except pH and temperature (C).
Received: 25 February 2015 Published online: 30 June 2015
Halder (2015) 2(2): 8284
.
A total number of two algal species of the genus Zygnemopsis (Skuja) Transeau, 1934 viz. Zygnemopsis
pseudolahaulensis Sarma & Kargupta and Zygnemopsis benghalensis Sarma & Kargupta of Zygnemaceae under
the order Zygnematales of Chlorophyta had been morpho-taxonomically described first time from Hooghly
district in West Bengal, India.
Morpho-taxonomic description
Order: Zygnematales
Family: Zygnemaceae
1. Zygnemopsis pseudolahaulensis Sarma & Kargupta in Hydrobiologia 139:249, figs. 1-16, 1986 (Fig. 1A)
Free floating, greenish-brown, vegetative cells 15.2 to 17.2 m broad and 54.2 m to 76.2 m long;
chloroplasts two, nearly rounded; pyrenoid single in each cell; zygospores not formed; aplanospores get swollen
and filled with pectic-cellulose materials; ovoid to sub-globose and almost filling the sporangium laterally; 20.0
to 25.0 m broad and 29.0 to 33.0 m long and, brown; outer spore wall smooth, thin and median spore wall
wrinkled or with wavy corrugations.
Habitat: Pond water at Jirat.
Collection No: 1001; Dated: 29.02.2011
Ecological Notes: Jirat, water temperature: 21C; pH: 7.6; NO3-N: 0.20; PO43-: 0.32; DO: 6.4; BOD: 4.8; COD:
110.0; SO42- : 6.0; TSS: 110.0; TDS: 162.0; Total alkalinity: 128.0
Occurrence: Rare
Significance: Primary producer in aquatic bodies.
Figure 1. A, Zygnemopsis pseudolahaulensis Sarma & Kargupta; B, Zygnemopsis benghalensis Sarma & Kargupta.
2. Zygnemopsis benghalensis Sarma & Kargupta in Hydrobiologia 139: 247, figs. 1-11, 1986 (Fig. 1B)
Free floating, greenish-brown, vegetative cells 11.5 to 21.5 m broad and 40.5 m to 96.5 m long;
chloroplasts two and stellate; pyrenoid single in each cell; zygospores not formed; aplanospores get swollen and
filled with pectic-cellulose materials; ovoid to cylindric ovoid and almost filling the sporangium laterally; 16.0
to 31.2 m broad and 33.2 to 35.0 m long and, brown; outer spore wall smooth and median spore wall
irregularly slightly wavy.
Habitat: Pond water at Somrabazar.
Collection No: 1003; Dated: 29.02.2011
Ecological Notes: Somrabazar, water temperature: 21C; pH: 7.4; NO3-N: 0.15; PO43-: 0.28; DO: 6.6; BOD:
4.8; COD: 120.0; SO42-: 6.4; TSS: 98.0; TDS: 156.0; Total alkalinity: 132.0
Halder (2015) 2(2): 8284
.
Occurrence: Rare
Significance: Primary producer in aquatic bodies.
Reporting new species from any area as new record or recollecting the species have its own importance in
floristic works (Singh et al. 2014, Srivastava et al. 2014). Das (1962) described a new species of Zygnemopsisas
Z. queensefrom Calcutta. Patel & Kumar (1971) made observation on morphological and cytological
characteristics of Zygnemopsis godwardense sp. nov. from Gujarat and followed by earlier work they (1977)
also recorded three new species of Zygnemopsis viz. Z. chohanensis, Z. dharampurense and Z. tricarinata while
studying of Zygnemaceae from Gujarat, India. Prasad & Kumari Vijay (1977) identified a new species of
Zygnemopsis as Z. vermaii from India. Sharma & Kargupta (1986) first described three species of Zygnemopsis
viz. Zygnemopsis benghalensis sp. nov., Zygnemopsis pseudolahaulensis sp. nov. and Zygnemopsis scorbiculata
sp. nov. from West Bengal, India. Chalotra et al. (2013) made morpho-taxonomic studies on this genus
occurring in fresh water bodies in Jammu and Kashmir and reported three species namely Z. splendens, Z.
tiffaniana and Z. minuta which were new to algal taxonomy of Jammu. Apart from the above mentioned studies,
it was the second report since its original description by Sarma & Kargupta (1986) from Birbhum district, West
Bengal, India. This study might be helpful to explore diversity and occurrence of these species in aquatic
ecosystems.
The morpho-taxonomic study of two species of Zygnemopsis viz. Zygnemopsis pseudolahaulensis and
Zygnemopsis benghalensis under the order Zygnematales of Chlorophyta will be provided valuable taxonomic
information in respect of systematic position, author citation, description, habitat, collection number along with
dates, ecological note, significance and occurrence for the first time from this area.
ACKNOWLEDGEMENTS
The author expressed his deepest sense of gratitude and sincere thanks to Dr. S.N. Sinha, Dept. of Botany,
University of Kalyani, Nadia, West Bengal for providing opportunity to work under his guidance.The author is
also grateful to Dr. R.K. Gupta, BSI, Howrah for his kind co-operation.
REFERENCES
APHA (2005) Standard methods for the examination of water and waste water (21st ed.). American Public
Health Association, Washington, DC, New York.
Chalotra P, Gaind M & Anand VK (2013) Morpho-taxonomic studies on the genus Zygnemopsis (Skuja)
Transeau, 1934 (Chlorophyta) occurring in fresh water bodies of Jammu and Kashmir. International Journal
of Innovative Research & Development 2(4): 266272.
Das CR (1962) A new species of Zygnemopsis (Skuja) Transeau, 1934 from Calcutta 1961. National agricultural
fair. Current Science 31: 255.
Islam AKMN (1972) New and rare species of some green algae from Bangladesh. Nova Hedwigia 23(4): 655
663.
Martens GV (1869) Beitrgezur Algen-Flora Indiens. Flora 52: 23338.
Patel RJ & Kumar CKA (1971) Morphological and cytological studies in Zygnemopsis godwardense sp. nov.
Phycological society of India 10(12): 1217.
Patel RJ & Kumar, CKA (1977) Zygnemataceae of Gujarat, India. III. Zygnemopsis (Skuja) Transeau.
Actabotanica Indica 5(1): 2024.
Prasad BN & Kumari Vijay CMRS (1977) Zygnemopsis vermaii a new species from India. Geophytology 7(1):
5860.
Randhawa MS (1959) Zygnemataceae. Indian Council of Agricultural Research, New Delhi, pp. 1478.
Singh A, Tiwari V & Mohan J (2014) Chroococcales in river Gabga at JajmauGhat, Kanpur. Tropical Plant
Srivastava N, Suseela MR & Toppo K (2014) Fresh water cyanobacteria of Sai River near Lucknow, Uttar
Pradesh. Tropical Plant Research 1(2): 1116.
Yin-xin W (1994) A new species of Zygnemopsis (Zygnemataceae) and its reproduction cycles.Chinese Journal
of Oceanology and Limnology 12(2): 174180.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(2): 8589, 2015
Research article
The effect of sodium silicate and silica nanoparticles on seed

germination and growth in the Vicia faba L.
Ghaffar Roohizadeh*, Ahmad Majd and Sedigheh Arbabian
Department of Biology, Faculty of Biological Sciences, Islamic Azad University, Tehran, Iran
*Corresponding Author: groohizadeh@yahoo.com [Accepted: 17 June 2015]
Abstract: Silicon is the second most common element in soil that has beneficial effects on living
and non-living increase stress tolerance in plants. It can lead to increased production and product
quality, reduce evaporation of perspiration, increased stimulation of some antioxidant enzymes and
decreased sensitivity to some of the fungus. The effects of silicon on seed germination and growth
of the bean (Vicia faba L.) were investigated. The seeds of plant were treated by 0 (as control), 1.5
and 3 mM of sodium silicate and silica nanoparticles. There were three repeats for all treatments.
The test results showed that seeds treated with sodium silicate concentration of 3 mM significant
difference in the percentage of germination than the control no significant difference in the rate of
germination of seeds treated compared to control was observed. Hypocotyl length and the
flowering of all treated plants were significantly different compared to control. The highest
flowering in plants treated silica nanoparticles was observed at a concentration of 1.5 mM. Only
plants treated silica nanoparticles with a concentration of 3 mM significant difference in diameter
than the control plants. According to the test results can be deduced the effect of silicon
nanoparticles in the form of sodium silicate and silica increases the percentage of germination and
growth of broad bean.
Keywords: Nano silica - Sodium silicate - Growth - Seed germination - Vicia faba.
[Cite as: Roohizadeh G, Majd A & Arbabian S (2015) The effect of sodium silicate and silica nanoparticles on
seed germination and some of growth indices in the Vicia faba L. Tropical Plant Research 2(2): 8589]
INTRODUCTION
Vicia faba L. is an annual plant of family Fabaceae with 80110 cm height. The flowers of broad bean are
white with black or purple spots. The seeds are sheathed and the fruits, seeds and flowers have medical usages.
Vicia faba L.is hetero fertilized with 2n=12. Because of possessing of high percentage of proteins (30 34%) it
is an important crop. After oxygen, silica is the second structural element in the earth which is non-mobile in the
plants. Although silica is not necessary for plants, higher plants need it to have optimum growth (Richmond &
Sussman 2003, Ma et al. 2004, Currie & Perry 2007). The most effect of silica on plants, is related to the
resistance against biotic and abiotic stress (Ma & Yamaji 2006, Liang et al. 2007). As the cell wall of plants
prevents the entrance of elements into cells, the Nano particles which have less diameter than the pores of cell
wall, therefore can easily cross the pores. Nano particles in the leavess surface enter the plants through the
stomata and or base of hairs and is then transported to the different organs (Nair et al. 2010). Silica plays
important role in the tolerance against salt stress (Zhu et al. 2003), manganese toxicity (Shi et al. 2005), boron
toxicity (Gunes et al. 2007) and cadmium toxicity (Vaculik et al. 2009, Shi et al. 2010) via changing the activity
of antioxidant enzymes. This study the effect of sodium silicate and silica nanoparticles on seed germination,
hypocotyle length, stem diameter, and amount flowering of the broad bean is done.

In order to assess ment the effects of silica nano particles and sodium silicate, on seed germination of broad
bean (Vicia faba L.), the samples were grown in greenhouse. Before cultivation, the impact seeds were sterilized
in 5% hypochlorite sodium solution. The seed then were washed up by deionised water. In each pot 2 seeds
were cultivated. Solution containing 0 (as control), 1.5 and 3 mM of nano particle of silica and sodium silicate,
Received: 10 March 2015 Published online: 30 June 2015
Roohizadeh et al. (2015) 2(2): 8589
.
were used in the experiment. The temperature of greenhouse was adjusted to 222 C (at night) and 252 C (at
day). The relative humidity was 44%. The samples were treated for 65 days. SPSS Ver.16 was used for
comparing of the means using Duncan test at P<0/05, level of significance. The diagrams were plotted using
Excel software.
RESULTS
Percentage of seed germination
The results showed that the changes in Vicia faba seed germination, seed germination, but at a concentration
of 3 mM sodium silicate plants treated with control plants showed no significant difference. The germination of
seeds of plants treated silica nanoparticles with a significant difference in the concentration of 3 mM sodium
silicate treated plants (Fig. 1A).
Germination rate
The results showed that the changes in germination rate of seeds germination rate in all treated plants
compared to control plants was reduced, but the reduction in the level of P <0/05 was not significa. The lowest
rate of germination of seeds treated with sodium silicate concentration of 3 mM, respectively (Fig. 1B).
Figure 1. The effect of sodium silicate and silica nanoparticles on germination: A, Percentage of germination; B, Seed
germination rate. (Means SE and P< 0.05)
Figure 2. The effect of sodium silicate and silica nanoparticles on hypocotyl: A, Hypocotyl length; B, Stem diameter.
(Means SE and P < 0.05)
Hypocotyl length
The results showed that the hypocotyls hypocotyls axis length changes in the concentration of silicon and
silica nanoparticles was significantly different from controls and the concentration of silicon and silica
nanoparticles were significantly increased during the hypocotyls. The most length of the hypocotyls of plants
treated with a concentration of 1.5 mM silica nanoparticles as compared to the control. Between plants trated
.
with different concentrations of silicon 1.5 mM silica nanoparticles with a concentration of 3 mM, no significant
differences were observed (Fig. 2A).
Stem diameter
Study results showed that Stem diameter silica nanoparticles stabilization period, hearts treated plants only 3
mM significant difference in Stem diameter than the control. The plants treated with concentrations of 1.5 and 3
mM sodium silicate significant difference from plants treated silica nanoparticles with a Stem diameter of 1.5
and 3 mM showed Highest and lowest Stem diameter of the treated plants at a concentration of 3 mM sodium
silicate and silica nanoparticles with a concentration of 3 mM compared to control plants (Fig. 2 B).
The amount of Bolting
The results are significant differences in the rate of flowering in plants treated with concentrations of 1.5 and
3 mM sodium silicate and silica nanoparticles as compared to the control the amount flowering in all treatments
was higher than control. The increasing concentration of 1.5 mM or nanosilica flowering of treatment (Fig. 3).
Figure 3. The effect of sodium silicate and silica nanoparticles the amount of Bolting. (Means SE and P < 0.05)
DISCUSSIONS
Germination and seedling establishment of the most critical stages in the life cycle of the plant is The most
important stages of germination, including water absorption, enzymatic activity, the growth of the embryo, seed
coat and tears seedling emergence is the results of the experiments, the researchers suggest that chemical
treatments can stimulate seed germination in many species of plants. Zhu et al. (2010) reported calcium,
gibberellic acid, ascorbic acid, ethanol to speed up germination and Mohammadi et al. (2009) described the
effect of salysyk acid and gibberellic acid on seed germination rate of lentil. The positive effects silicon
attributes tomato plant germination (Haghighi et al. 2012) and soybean (Li et al. 2004) have also been reported.
Mozaffarian et al. (2011) & Manzer et al. (2013) demonstrated that silica nanoparticles improve seed
germination in tomato plants. Increased percentage of soybean germination by combining nanoparticles of
silicon and titanium has also been observed (Lu et al. 2002) this study, the addition of silicon to form sodium
silicate and silica nanoparticles become improves seed germination.
Effects of sodium silicate and silica nanoparticles on growth indices
The present study, reported that the use of silicon improves the growth of root, stem and leaves plant. This
effect may be due to the prominent role of silicon in improving plant water status (Romero-Aranda et al. 2006).
The benefits of using silicon indirect effects such as increased capacity and efficiency of photosynthesis,
transpiration and thus reduce shoot growth related (Liang 2003). Samuels et al. (1993) showed that in the
presence of silicon increases plant growth by improving the mechanical strength of stems and leaves on light
absorption and photosynthetic capacity of the plant is increased. Kamindiou et al. (2010) observed the effects of
silicon on morphological characteristics and growth of gerbera flowers in the greenhouse cultivation, and the
positive effects of the use of silicon as the medium Na2Sio3 the plant height, the thickness of the shoot the size
flowers flowering reported. Reezi et al. (2009) found that adding 50 mg-l potassium silicate or nutrient Hot Lady
.
Rose cut increases the number of flowers. The results of our study showed that the use of sodium silicate and
silica nanoparticles increases the length of the hypocotyl, stem diameter and the amount of Bolting of the Vicia
faba.
CONCLUSION
The results of this study, sodium silicate and silica nanoparticles can affect the germination of seeds. Thus,
in cases such as seeds that grow with the problem of different concentrations of these substances can be used to
facilitate and accelerate the germination of seeds and increase the efficiency of their applications. It can also be
used these to help plant growth and ultimately better performance.
ACKNOWLEDGEMENTS
The authors are thankful of all laboratories personnel Mahmoodieh Islamic University Tehran North Iran,
who contributed to this research.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(2): 90100, 2015
Research article
Stem gall of Michelia champaca L. (Magnoliaceae) induced by

Podothrips sp.: Identification, histochemical and
phytochemical studies
Monnanda Somaiah Nalini1*, K. E. Shilpa1 and Sekharappa Basavarajappa2
1
Department of Studies in Botany, University of Mysore, Manasagangotri, Mysore570 006, Karnataka, India
2
Department of Studies in Zoology, University of Mysore, Manasagangotri, Mysore570 006, Karnataka, India
*Corresponding Author: nmsomaiah@gmail.com [Accepted: 18 June 2015]
Abstract: Galls are organized structures of plant tissues induced by the insects. The present study
deals with the identification of gall-maker on the stem/twigs of Michelia champaca. The gall-
maker was identified as belonging to the order Thysanoptera, and was confirmed as the genus
Podothrips. Histochemical staining was carried out in different portions of gall as well as healthy
twigs for the presence of alkaloids, fats, starch, tannins and proteins. The staining varied in
different tissues. The galls at different development stages (young and mature) were tested for the
presence of total phenolic as well as carbohydrate content. The results revealed high phenolic
content in young galls where as low amount was found in a mature gall tissue, however, a 2.3-fold
difference was noted between the two. Very high concentration of carbohydrate content was
detected in young gall tissues. The gall serves both as a shelter and a food source, to the gall-
maker. The inner walls of the galls are moist and are usually rich in phenolics and carbohydrates.
Our work reports for the first time Podothrips sp. as gall-makers. Further studies on the ecology
of the insect would relate its occurrence on other host species and distribution.
Keywords: Gall - Michelia champaca - Histochemistry - Phenolics - Carbohydrate.
[Cite as: Nalini MS, Shilpa KE & Basavarajappa S (2015) Stem gall of Michelia champaca L. (Magnoliaceae)
induced by Podothrips sp.: Identification, histochemical and phytochemical studies. Tropical Plant Research
2(2): 90100]
INTRODUCTION
Michelia champaca L. is a medium to big sized tree with glossy leaves and yellow or orange flowers of the
family Magnoliaceae, native to the tropical and sub-tropical South and Southeast Asia, including Southern
China. The flower locally known as sampige or champa has great significance in Hindu mythology and has a
number of cosmetic, medicinal and economic uses. Fresh flowers are extracted into perfumes and medicinal
products which are used as cure for coughs and rheumatism. Cosmetic products such as Joy, Jadore and Dior
contain M. champaca fragrant extracts as ingredients in their composition (Armiyanti et al. 2010).
Michelia champaca is affected by biotic and abiotic factors. Among the biotic factors affecting the plants is
the presence of galls on the plant parts. Galls are abnormal growths on plants and are better understood as a suite
of adaptations to the inducing insect (Raman 2003). They are highly regulated growth manifestations on plants,
modified, symmetrical, natural-plant structures that are the outcome of messages from the inducing insects.
These structures develop as an extension of the host-plant phenotype (Raman 2012). Galls result from the
feeding of living organisms, such as insects, mites, and or mechanical stimulus. The attack of each species
results in a distinctive deformity on leaves, twigs or stem of a host plant. Such response also increases the
plants production of growth hormones such as auxins, cytokinins and gibberellins (Caron 2004).
Thrips are one class of well-known gall-forming insects, and belong to the order Thysanoptera. Thrips are
involved in several types of host relationship with plants, and their presence have been documented
(Ananthakrishnan & Raman 1979). Thrips (order-Thysanoptera) are tiny, slender insects with fringed wings.
They are very small, yellow, brown or black, slender insects ranging from 1/16 to 1/8 inch in length. Adults and
Nalini et al. (2015) 2(2): 90100
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larval thrips feed using a punch and suck technique. Their life cycle includes an egg stage, two larval instars,
two pupal stages, and an adult stage (Buss 2011).
Gall tissues are known to be sources of enzymes, phytochemical compounds such as alkaloids, fats, tannins,
proteins, starch, phenolics, carbohydrates etc. These accumulate in the tissues during gall development. Plant
phenolic compounds play an important role at different levels during plant-microbe interactions. A completely
different action of plant phenolic compounds lies in the defense of plants against pathogen attack (Vereecke et
al. 1997). Histochemistry is the branch of histology dealing with the identification of chemical components of
cells and tissues. Starch deposition occurs widely in the plant body, in the seeds, the parenchyma of the
secondary vascular tissue in the stem. Starch and protein are the principal ergastic substance of the protoplast.
Tannin is the heterogeneous group of phenol derivatives, usually related to glucosides. Fats are widely
distributed in the plant body and they probably occur in small amount of every plant cell. Many woody plants
contain medicinally important secondary products (Marmit & Sharma 2008) which can be analyzed using
staining techniques.
Various types of insect-plant galls exist in nature. Several plant species are reported to possess galls, yet the
occurrence of these in plants is to be documented. So far, no plant galls have been documented from Michelia
species. Since galls on the stem of Michelia was first noted in the Manasagangotri Campus, we undertook the
study to document and identify the gall-inducer and analyse the phytochemical and histochemical differences
between the healthy and galled tissue portions of the stem.

Collection of the sample and study area
Healthy and galled twigs of Michelia champaca L., were collected from the plants growing in
Manasagangotri campus (12o18' N & 76o12' E), Mysore, during the month of September to December, 2011.
The galled portions were excised from the stem with a metallic plier. The galls were separated according to their
size, as young and mature and photographed.
Identification of the gall-maker from the stem/twigs of Michelia champaca
Various stages of fresh gall materials such as the young and the mature (old) were collected. The collected
tissue was mounted on the clean glass slide and was kept on the field of Steriosome (Leica EZ4, MC234813,
China). The gall tissue was dissected by using clean micro dissection needles and observed for the presence of
the gall-maker. Different stages of insects were photographed by using different magnifications, 835X. These
were considered for the documentation studies.
Histochemical comparison between gall and normal tissues
Free hand cut sections of young twigs of Michelia champaca were taken by using clean and sharp blade and
floated in water contained in petriplates. Using pointed brush, fine transparent sections were selected and
immersed and placed in a cleaned glass slide and respective stains were added drop wise and covered with a
clean cover slip. Excess of stain was removed using a tissue paper and observed for the localization of viz.-
starch, tannins, proteins, fats and alkaloids under the microscope at various magnifications (4x, 10x and 40x)
and images were documented and compared.
Preparation of reagents
Reagents were prepared for histochemical studies according to the procedure of Momin & Kadam (2011).
Starch: 0.3 g of iodine and 1.5 g of potassium iodide were dissolved in 100 ml of distilled water. A drop of the
solution was added on the section, left for some time and washed water and observed under microscope.
Protein: Saturated aqueous solution of picric acid is an excellent precipitating agent for protein, staining them
an intense yellow. The sections were allowed to react with the reagent for 24 h. The sections were washed
with 60% alcohol and few drops of aqueous FeCl3 were added.
Tannins: Sections were treated with dilute acidic ferric chloride solution (0.5% to 1% of FeCl3 in 0.1 N HCl),
mounted in clove oil and observed under microscope for the presence of tannins.
Fats: 0.5 g of Sudan III and or Sudan IV is dissolved in 100 ml of 70% alcohol. Sections were immersed in the
stain for 20 min, rinsed quickly with 50% alcohol and mounted in glycerine for observations.
Alkaloids: Wagners reagent: One gram of iodine and 2 g of potassium iodide were dissolved in 50 ml of
distilled water.
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Estimation of total phenolic and carbohydrate content in the gall sample
Preparation of the extract: One gram each of young and mature gall samples were finely ground for few
minutes with 10 ml of 70% alcohol by using clean mortar and pestle. The extracts were taken in small
Eppendroff tubes and labelled. It was centrifuged at 5000 rpm for 10 min in a Spinwin centrifuge
(Tarsons, India). The supernatant was used for the estimation of total phenolic content.
Estimation of total phenolic content: The total phenolic content in the gall samples were determined by the
Folin-Ciocalteau (FC) method using Gallic acid as a standard (Volluri et al. 2011) with slight modifications.
Different concentration of extracts (50250 g.ml-1) was mixed with 1.0 ml of Folin-Ciocalteau reagent (1:1
dilution). 2.0 ml of Sodium carbonate (20%, w/v) was added and shaken well. The reaction mixture was
incubated for 45 min under dark. After the specified incubation period the absorbance was read at 765 nm in
UV/Vis Spectrophotometer (T 60, TTL Technologies, India). The absorbance of standard as well as the
samples was plotted. The amount of total phenolics in the samples was expressed in terms of Gallic acid
equivalents (g ml-1 GAE).
Estimation of total carbohydrate content: The total soluble carbohydrate content in the aqueous extracts of gall
tissues was determined using the phenol sulphuric acid method. Glucose was used as the standard
(Sadashivam & Manickum 2008). The aqueous extracts of the gall was obtained by boiling the known
amount of gall tissue (1 g.ml-1). The filtrate was used for the estimation of carbohydrates. A working
solution was prepared by using different concentrations (1 mg.ml-1) of standard (525 g). The samples were
aliquoted (50250 l). The volumes of aliquots were made 500 l with distilled water. A blank was set with
500 l using distilled water. One ml of liquefied phenol solution (5%) was added to all tubes followed by the
addition of 2.0 ml of concentrated sulphuric acid immediately and shaken well. The reaction mixture was
incubated for 30 min in dark place. The absorbance of the standard as well as the samples were read at 490
nm using Spectrophotometer. The absorbance values and the amount of total carbohydrate in the different
samples was calculated and represented.
RESULTS
Identification of the gall on stem/twigs of Michelia champaca L.
Morphological observations: Gall on M. champaca occurred on the main stem axis, as well as twigs, two feet
above the ground (Fig. 1B). The gall during initial development stages (young) appeared as snow-white out
growths (0.6 cm in diameter). They were formed at regular distance both on the stem as well as the twig
(Fig. 1C), as they matured, an increase in size (2.02.5 cm in diameter) was observed and later coalesced to
form large sub-spherical structures. One of the important observations made during the gall development is
the change from the initial snow-white colour (Fig. 1C) to slightly blackish structure at maturity (Fig. 1D).
Observation of mature galls by Steriotrinocular microscopy showed the presence of small black glandular
hairs filled with exudates (Figs. 1E & F).
Identification of the gall-maker: The gall-maker on the stem/ twigs of M. champaca was identified as belonging
to the order-Thysanoptera, which include Thrips. Upon periodical and successive observations it was
confirmed as the genus Podothrips. On the basis of the characters, and the availability of various stages of
development (Fig. 2), only one insect chamber was observed from the under surface of the gall. The body
size observed was 0.51.0 mm. The systematic position is as follows: Class: Insecta; Order Thysanoptera;
Family: Phlaeothripidae; Genus: Podothrips.
Histochemical studies: A comparison of section of galled, gall normal and healthy portion showed remarkable
differences anatomically (Table 1). In healthy portion, a conspicuous pith and sub-spherical outline of
vascular bundles with the larger bundles alternating with the smaller bundles was observed (Fig. 3A-a). In
gall normal, pith is conspicuous and the outline of vascular bundles is sub-spherical, the larger bundles
alternate with smaller bundles (Fig. 3A-b). The section resembles the healthy plant. In galled portion,
inconspicuous pith, radial growth of vascular bundles and conspicuous sclerenchyma cap was observed (Fig.
3A-c). The following are the results of the tests.
Alkaloids: A golden yellow colour revealed the presence of alkaloids (Figs. 3B-ac). In healthy tissue,
sclerenchyma cap and metaxylem and vascular bundles and entire pith region were lightly stained. In gall
normal stem tissue, an intense stain was observed in sclerenchyma cap and cells of metaxylem showed less
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intensity of stain and in pith region only centre portion were stained. In gall stem tissue, the sclerenchyma
cap, vascular bundles, medullary rays and metaxylem cells showed the high intensity of stain and outer cells
of the pith region was stained less intensely.
Figure 1. Galls on M. champaca L.: A, Healthy plant; B, Galls observed two feet above the ground level; C, Stem and twigs
showing galls; D, Close up indicating slightly blackish colour; E, Increased size with black glandular hairs filled with
exudates; F, Close up.
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Figure 2. Developmental stages of the gall-maker: A, Healthy plant; B, Infected plant; C, galls on twig; D, Single gall; E,
Dorsal view of gall; F, Ventral view of gall; G, Young thrip; H, Adult thrip.
Fats: Blue colour indicated the presence of fat. In healthy tissue, high intensity stain was observed in
sclerenchyma cap, vascular bundles and medullary rays and less intensity in pith cells, cortex and epidermis.
In gall normal stem tissue, sclerenchyma cap is continuous and forming a ring like structure. In these
vascular bundles, sclerenchyma cap and cortex showed the high intensity and less in pith cells. In galled
stem tissue, sclerenchyma cap was discontinuous. Sclerenchyma cap, medullary rays and vascular bundles,
showed the high intensity of stain, very less in pith regions (Figs. 3C-ac).
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Tannins: Blue colour indicated the presence of tannins. In healthy tissue, in xylem elements dark stain was
observed, high intensity of stain was observed in the sclerenchyma cap, vascular bundle and medullary rays.
The pith region showed very high intensity of stain. In gall normal stem tissue, the sclerenchyma cap,
vascular bundle and medullary rays showed the high intensity of stain and in pith region showed the very
high stain. In galled tissue, except the sclerenchyma cap all cells showed the high intense stain (Figs. 3D-a
c).
Table 1. Histochemical differences in the healthy and galled twig of M. champaca L.
Histochemical Histological components stained
Color
tests Healthy twig portion Galled twig portion
Alkaloids Golden Sclerenchyma cap, vascular bundles & Sclerenchyma cap, vascular bundles,
yellow entire pith (+) medullary rays (+++) & outer pith (+)
Fats Cobalt Sclerenchyma cap ( Continuous ring), Discontinuous sclerenchymatous cap,
blue vascular bundles, medullary rays vascular bundles, medullary rays (+++)
(+++); pith cells, cortex, epidermis (+)
Tannins Prussian Sclerenchyma cap, vascular bundles, Sclerenchyma cap (+); vascular bundles,
blue medullary rays & pith region (+++); medullary rays & pith region (+++)
Starch Blue Cortex, medullary rays & pith (+++); Sclerenchyma cap, metaxylem elements
& pith region (+++); cortex (+)
Proteins Intense Sclerenchyma cap, vascular bundles, Sclerenchyma cap, vascular bundles,
yellow medullary rays & pith region(+) medullary rays (+++);pith region (+)
Note: +, indicates light staining; +++, indicates moderate staining; +++, indicates intense staining.
Starch: Blue colour indicated the presence of starch, in healthy tissue, high intensity starch was observed in
cortex, medullary rays and pith cells, pith is conspicuous (Fig. 3E-a). In gall normal tissue, cortex,
metaxylem cells, medullary rays showed high intensity of stain. In pith region very high intensity of stain
was observed (Fig. 3E-b). In galled tissue, sclerenchyma cap, metaxylem elements and pith region showed
the intense stain, more in vascular region and less in cortex (Fig. 3E-c).
Protein: An intense yellow colour indicated the presence of proteins; almost all the tissue showed intense
staining. In healthy tissue, less intensity of stain was observed in the sclerenchyma cap and very less in all
parts of the sections (Fig. 3F-a). In gall normal tissue, high intensity of stain was observed in sclerenchyma
cap and metaxylem cells and less in vascular bundles and medullary rays and pith region showed less
intensity (Fig. 3F-b). In galled tissue, sclerenchyma cap, metaxylem elements, medullary rays and vascular
bundles showed the very high intensity and less intensity in the pith region (Fig. 3F-c).
Total phenolic and total soluble carbohydrate contents
Total phenolics: The phenolic contents of young and mature gall tissue having differences in the growth stages
were compared with Gallic acid as standard. In young and mature galls, the phenolic content of 240 g.ml-1
GAE and 225 g.ml-1 GAE were observed (Fig. 4). Both did not differ much in their phenolic contents.
However, a 2.3-fold difference was noted, when expressed in terms of weight of the gall tissue.
Total soluble carbohydrates: The total soluble carbohydrate content of young and mature gall tissues showing
much difference in the growth stages were compared with glucose as standard. The total soluble
carbohydrate was 9.4 g.ml-1 in young galls, whereas the mature gall contained 0.5 g.ml-1 (Fig. 5).
However, an 18.8-fold increase was noted in the concentration in the young galls when compared to mature
galls.
DISCUSSIONS
Insect galls are organized structures of plant tissue which form in response to either feeding by the gall-
maker (phytophagy) or egg-laying (oviposition) on plant tissue. There are number of galls types induced by the
insects on leaf, stem, twig, bud or flower and fruit. In the present investigation, galls on Michelia champaca
(Magnoliaceae) were noticed for the first time on the stem/twigs. So far, a stipular scar forming a ring around
the stem (node) in Magnolia virginiana has been reported to be caused by a psyllid, Trioza magnolia (Ashmead)
(Hall 2009).
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Figure 3. A, Healthy sections stained with safranin stain; Histochemical tests for: B, Alkaloids; C,
Proteins; D, Fats; E, Tannins; F, Starch (a, Healthy portion; b, Gall normal portion; c, Galled portion).
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Figure 4. Determination of total phenolic content in the gall tissues of M. champaca; Ethanolic extracts of young and mature
gall tissues were prepared (1g 10 ml-1). Concentrations ranging from 50250 g.ml-1 were tested for the total phenolic
content by Folin-Ciocalteu method using gallic acid as standard. The absorbance of the samples as well as the standard was
read at 765 nm and the values of the samples were calculated from the standard graph and represented in terms of g.ml-1
GAE.
Figure 5. Determination of total soluble carbohydrates in gall tissues of M. champaca: The total carbohydrate content of the
aqueous extracts of young and mature galls were determined by phenol-sulphuric acid method using glucose as standard.
Concentrations ranging from 50250 g.ml-1 were tested for the carbohydrate content. The absorbance of the samples and
the standard was read at 490 nm and the values of the samples were calculated from the standard graph and represented.
Galls are made up of cells that are more numerous or larger than the normal plant cell or plant organs whose
growth and development have been altered into unusual shapes. An insect gall is initiated because of the plants
response to the insects egg laying (oviposition) or presence of the egg, and/or feeding stimulation by the larva
(phytophagy). Plant cells are usually modified and enlarged, the plant tissue surrounds the egg or larva, and the
gall protects and feeds the gall-inducer. Galls are located on rapidly growing plant parts-on catkins, seeds,
flowers, petioles, branches and stems; most occur on leaves and buds. Some galls are single-chambered
(monothalamous) and contain only one gall-maker, and others are multi-chambered (polythalamous) which
contain many gall-makers (Buss 2011).
Gall on M. champaca occurred on the main stem axis, as well as lateral twigs, two feet above the ground.
The gall during initial development stages (young) appeared as snow white out growths (0.6 cm in diameter).
Nalini et al. (2015) 2(2): 90100
.
They were formed at regular distance and as they matured galls increased in size (2.02.5 cm diameter) and
coalesced to form large sub-spherical structure. Gopinathan & Suresh (1985) worked on the solid, indehiscent
galls on the stem of Pongamia glabra induced by an undescribed agromyzid (Diptera). They reported, that
young galls (815 612 mm) were relatively soft and green and with maturation, they became harder and
developed white patches and is directly proportional to the number of larvae residing inside. Mature galls
showed generally, a single gall chamber extending along the pith region.
The gall-inducer was identified as belonging to the genus Podothrips of the order- Thysanoptera, family
Phlaeothripidae. Only a single insect chamber was observed from the under surface of the gall. The body size
observed was 0.51.0 mm. The genus Podothrips are reported for the first time as gall-inducers. Furthermore,
thrips as gall-makers have been reported on plant species (Raman & Ananthakrishnan 1989). Thrips such as
Gynaikothrips uzeli induced galls on the leaves of Ficus bengalensis (Borbon 2011). Gynaikothips ficorum
caused leaf gall on Ficus laevigata (Santis 1980). Although, we have documented the evidence of this genus as
a gall-inducer, worldwide Podothrips are reported to cause huge losses to crops such as chilli (Scirtothrips
dorsalis), which is an important pest of crops in tropical and subtropical regions in Florida (Ludwig & Bogsan
2007).
A comparison of sections of galled, gall normal and healthy portion showed remarkable differences
anatomically. The following tests were conducted to localize viz., alkaloids, fats, starch, tannins and proteins in
tissues. In healthy portion, pith was conspicuous and the outline of vascular bundles was sub-spherical, the
larger bundles alternated with the smaller bundles. In gall normal, pith is conspicuous and the outline of vascular
bundles is sub-spherical, the larger bundles alternate with smaller bundles. The section resembles the healthy
plant. In the galled portion, inconspicuous pith, radial growth of vascular bundles and conspicuous
sclerenchyma cap was observed.
In the present study alkaloids, tannins, fat, proteins and starch were detected in various tissues and differed
considerably in healthy and galled portions of the sections. Our observations are supported by the studies of
Gopinathan & Suresh (1985) who worked on solid, indehiscent galls on the stem of Pongamia glabra induced
by an undescribed agromyzid (Diptera). In the histochemical studies, maximum concentrations of proteins were
observed in the nutritive cells of medullary region and phloem cells. Starch deposits occurred in the pith cells,
medullary rays in normal stem sections, whereas in gall portions they were located in the outer region of pith of
the nutritive zone. Tannins were present less in gall tissues, while they were totally absent in normal stem.
Kumar & Mathur (2009) reported the histochemical localizations in stem gall of Terminalia arjuna caused by
unknown Itonididae (Diptera). According to the authors, in normal stem sections, cortex, medullary rays and
pith cells contained starch as high intensity of staining was observed. It was present in high quantity in nutritive
zone and vascular region and less in cortical parenchyma cells of stem gall tissue. High intensity of proteins was
observed in the cortical zone. In normal stem, tannins were observed in cork, cortex, vascular and pith regions.
High amount of tannins were observed in cortical region and tannin filled cells were observed near the nutritive
zone of the stem gall. High protein was found in gall tissue as compared to normal tissue from the leaf gall of
Pongamia pinnata induced by Aceria pongamiae (Kumar 2012). The presence of starch near the gall cavity
suggests that the insects are utilizing starch as food materials as such. Increased amount of proteins were noticed
in the nutritive tissues, which helps the gall-makers growth and development.
An increased amount of tannins in gall tissue could be attributed to the higher incidence of polyphenol
activity. Phenolics are one of the much interest in phytochemical as bioactive components of food. It is now
possible to establish the antioxidant activities of plant derived phenolics in the aqueous and lipophilic phases.
The inner walls of the galls are moist and are usually rich in phenolics and carbohydrates. In the present study,
phenolic contents of young and mature gall tissue with differences in the growth stages were compared. In
young galls, 240 g.ml-1 GAE phenolic content and 225 g ml-1 GAE were observed in mature galls, suggesting
fewer differences in the content among young/mature gall tissues. Ramani & Kant (1989) reported that phenolic
content in the galls of Prosopis cineraria (L.) induced by Lobopteromyia were higher in the normal compared to
gall tissue both in vivo and in vitro. Abrahamson et al. (2003) investigated that rapidly growing galls in three
additional susceptible clones confirmed the increase in phenolics and showed that phenolic level increases as
much as five-fold in galls near their peak growth period. Akhlaq & Mohammed (2011) reported that the stem
gall of Tamarix aphylla was rich in phenolic compounds exhibited antioxidant activity.
Nalini et al. (2015) 2(2): 90100
.
The total soluble carbohydrate content was 9.4 g.ml-1 in young galls, whereas the mature gall contained 0.5
g.ml-1, which is negligible. However, 18.8-fold increase was noted in the concentration in young galls when
compared to mature galls. It may be inferred that high levels of carbohydrates are required during early stage of
gall development as a source for the development of insects. Still, more sampling and assessment of antioxidant
activity in the extracts may reveal that galls are needed good sources of antioxidants. To conclude, the gall
serves both as a shelter and a food source, also the gall-maker is partially protected from parasites and predators.
Further investigation to identify the species of the gall-maker of M. champaca would be beneficial, to elucidate
the life cycle as well as ecological studies.
ACKNOWLEDGEMENTS
The authors are grateful to the Chairman, DOS in Botany, and DOS in Zoology, University of Mysore,
Mysore, Karnataka, India for providing the necessary facilities during the study. The paper is the sincere
outcome of the second author KES during the MSc project work at the DOS in Botany, University of Mysore.
This work has been supported by funds from the University of Mysore.
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2(2): 101107, 2015
Research article
Assessment of land use land cover change in Chakrar watershed

using geospatial technique
Sandeep Soni1*, P.K. Garg2, Ashutosh Singh3 and Abhishek K. Maurya4
1
G.B. Pant Institute of Himalayan Environment and Development, Kosi Katarmal Almora Uttarakhand, India
2
Wadia Institute of Himalayan Geology, Dehradun, Uttarakhand, India
3
Forest Survey of India, Dehradun, Uttarakhand, India
4
MGCGV, Chitrakoot Satna, Madhya Pradesh, India
*Corresponding Author: sandeepsoni80@gmail.com [Accepted: 22 June 2015]
Abstract: Rapid urbanization, anthropogenic and socioeconomic activities and environmental

changes, in local and regional area, are important component responsible for extensive land use
land cover changes (LULCC). Now a day, it is important to determine LULCC at appropriate scale
to determine impacts of above mentioned components. In this study an attempt has been made to
analyze LULCC in the Chakrar watershed, Madhya Pradesh which is an important tributary of the
Narmada River. The watershed is covered by natural forest that is out skirt forest of Achanakmar-
Amarkantak Biosphere reserve of Central India. A series of systematically corrected orthorectified
Landsat imageries of 1990, 2000, 2005, 2011 and 2013 were used for classification. Geospatial
technique was used to assess LULCC and accuracy assessment was also done. It was obtained that
natural forest cover decreased by 24.11 Km2 and human settlement increased by 7.78 Km2.
Therefore, the study was important to assess the loss in natural vegetation that can affect natural
biodiversity, ecology and biosphere reserve including environment.
Keywords: Geospatial Technology - LULCC - Chakrar Watershed - Forest degradation.
[Cite as: Soni S, Garg PK, Singh A & Maurya AK (2015) Assessment of land use land cover change in Chakrar
watershed using geospatial technique. Tropical Plant Research 2(2): 101107]
INTRODUCTION
Change detection analysis is important for development planning and management activities and considered
as essential aspect for modeling the earth system. Change detection is useful for a wide variety of subjects for
example monitoring deforestation, coastal dynamics, shoreline change and river transportation on multi-
temporal satellite images (Muttitanon & Tripathi 2005, Fromard et al. 2004, Duncan et al. 1993). Land use Land
cover (LULC) mapping is essential for change detection analysis. Landuse refers to the way in which land has
been used by humans and their habitats, usually with accent on the functional role of land for economic
activities. Land cover refers to the physical characteristics of earths surface, natural vegetation, water bodies,
soil/rock, artificial cover and others resulting due to land transformation. The landcover changes occur naturally
in a progressive and gradual way, however sometimes it may be rapid and abrupt due to anthropogenic
activities. LULC changes especially those caused by human activities is the most important component of global
environmental change with impacts possibly greater than the other global changes (Turner et al. 1994, Jensen
2005). Land cover change is occurring from the conversion of forests to agricultural lands and built-up lands
(Delang 2002, Duram et al. 2004, Shalaby & Tateishi 2007, Turner et al. 2007, Munoz-Villers & Lopez-
Blandco 2008).
Now-a-day deforestation is the main problem for biodiversity and society. Deforestation affects not only the
ecosystem but also the usefulness of the forest as a resource. It involves multiple interrelated factors of people-
forest interaction and results in significant environmental and social consequences (Gessesse 2007). Forests are
valuable resources that provide enormous benefits for example wood and other products. Forests regulate
climate and fresh water flow, protect and enrich soils, control pests and disease, maintain biodiversity, safeguard
water quality offer beautiful landscapes and enrich humans spiritually (Byron & Arnold 1999, Kaimowitz &
Soni et al. (2015) 2(2): 101107
.
Angelson 1999, FAO 2005). Forest covers reduction through deforestation and conversion for agricultural
purposes can alter a watersheds response to rainfall events, that often leads to increased volumes of surface
runoff and greatly increase the incidence of flooding (McColl & Aggett 2007, Cebecauer & Hofierka 2008).
Forests have a pervasive influence on the ecosystems, environmental and the lives of people. They support
millions of local residents directly, while they are indirectly vital to many more that depend upon the water
cycle regulated by them (Bruijnzeel & Bremmer 1989, Hamilton 1987). Forest cover change is a process in
which the level of diversity and the density of individual species that makes up the natural vegetation structure
are altered as a result of natural/internal and external factors (Belaynesh 2002). The basic cause of forest
degradation is high population density and population growth rate (Dien 2004), climate change and over
consumption of ecosystem services which treat and tends to the biggest challenge for the society (Sachs et al.
2009). Global warming is common impact of deforestation including loss of biodiversity, reduced water cycling
(Dewi 2009) and loss of fuel, fodder, food (Gibson et al 2000, Wang et al. 2006). Logging, grazing and new
settlements establishment have contributed to forest degradation and depletion (Bekele 2001, Nair & Tieguhing
2004).
Remote sensing and GIS (Geographical Information System) technique (combined called as Geospatial
Technology) have been used extensively to provide accurate and timely information describing the extent of
LULC over time. It explores new ways to detect, characterize and monitor forest change (Kasischke et al. 2004).
Change detection analysis, employing both GIS and remotely sensed data, has been used to assess forest
depletion (Jacobberger-Jellison 1994, Peters et al. 1993, Prenzel 2004) in the Chakrar watershed. The watershed
is an important tributary of the Narmada River and is covered by natural forest which is out skirt forest of
Achanakmar-Amarkantak biosphere reserve of Central India.

Study Area
For the present study forest around Chakrar watershed was selected. The Chakrar watershed rises towards
south at an altitude of 980 meter of Satpura hills of Dindori district of Madhya Pradesh and flows to the north to
meet the Narmada River at an altitude of 700 meter. It is bounded by 2203112.24"N to 2205244.93"N latitude
and 8101441.23"E to 8102829.42"E longitude (Fig. 1). Total catchment area of the Chakrar watershed is 415
km2. Geologically the area is characterized by dominant basaltic lava flow of Deccan trap of Cretaceous to
Palaeogene age and in the high altitude area or margin area laterite of Cainozoic age is found. Rock type is hard
and compact. The study region is characterizes by high level plateau and half part by middle level plateau.
Average annual rainfall is 12001300 mm.
Figure 1. Location map.
Soni et al. (2015) 2(2): 101107
.
The watershed is rich of biodiversity. Shorea robusta (Sal) is common tree of the forest including
Lagerstroemia parviflora, Amla, Mahua and other tree species with different varieties of herbs and some
medicinal plant. The fauna comprises tiger, nilgai, sambar, chital, leopard, wild dog, bison (gour), black buck
and many others. Wheat, Paddy, Maize, Kodo-Kutki, Ramtil, Mustard, Masoor, Matar, Gram, Alsi, Soyabean
are the main crops of this district.
Experiment designing
In this study Survey of India digital topographic maps (64F/5, 6, 7 & 10) were used to extract watershed
boundary with a scale 1:50,000. For LULC mapping of the watershed, Landsat 5, 7 and 8 TM (path 143, row
44) satellite images of 5 different years (1990, 2000, 2005, 2011 & 2013) were used. Satellite data was procured
from USGS websites (www.glovis.usgs.gov). The satellite data and collateral data were processed and field
survey was conducted for ground truthing to perform supervised classification. The satellite data was processed
in ERDAS Imagine 9.2 software for geometric correction to remove geometric distortions, introduced by the
sensor system. It is needed to georeference the distorted data to a coordinate system. The imageries were
georeferenced using ground control points with a root mean square error (RMSE) of less than one pixel. The
Universal Transverse Mercator (UTM) geographic projection, WGS84 spheroid, and zone 44 North datum were
used in georeferencing the images. All the imageries were resampled to a 3030 pixel size using the nearest
neighbor resampling technique (Serra et al. 2003). Pixel based supervised image classification with maximum
likelihood classification algorithm was used to map the LULC classes. Eight LULC classes viz; settlement, low
dense vegetation, high dense vegetation, fallow land, open land, water bodies and agriculture were identified for
classification.
RESULT AND DISCUSSION

Accuracy assessment of the supervised classification of the satellite imagery was derived by using a
reference template from the margining data with 40 randomly selected samples on the latest imagery, from
which overall accuracy and Kappa statistics were derived. The Kappa statistics incorporated the diagonal
elements of the error matrices (Yuan et al. 2005). Satellite imageries of 1990, 2000, 2005, 2011 and 2013 were
classified and validated using error matrix and Kappa statistics. The overall accuracy was found to be 91 percent
whereas overall Kappa statistics was 0.8898. The statistics shows that the result was overall good.
Table 1. Land use land cover changes from 19902013.

1990 2000 2005 2011 2013
Class
Area Area Area Area Area Area Area Area Area Area
(Km2) (%) (Km2) (%) (Km2) (%) (Km2) (%) (Km2) (%)
Sattlement 4.91 1.18 6.41 1.54 10.96 2.64 12.29 2.96 12.69 3.06
River 2.67 0.64 2.71 0.65 2.73 0.66 2.73 0.66 2.72 0.66
Waterbodies 0.24 0.06 0.26 0.06 0.34 0.08 0.43 0.10 0.48 0.11
High Dense 139.83 33.69 132.15 31.84 131.73 31.74 96.25 23.19 95.85 23.10
Vegetation
Low Dense 54.54 13.14 58.46 14.09 54.92 13.23 75.55 18.20 74.37 17.92
Vegetation
Fallow Land 143.04 34.46 141.15 34.01 159.25 38.37 162.56 39.17 180.33 43.45
Open Land 16.40 3.95 11.83 2.85 17.74 4.28 16.63 4.01 17.29 4.17
Agriculture 53.39 12.86 62.07 14.95 37.35 9.00 48.60 11.71 31.29 7.54
Total 415.03 100.00 415.03 100.00 415.03 100.00 415.03 100.00 415.03 100.00
Significant changes were found in the LULC maps, prepared from Landsat imageries of 1990, 2000, 2005,
2011, and 2013 (Fig. 2) and trend analysis was carried out to compare the land cover type (Fig. 3). Regardless of
the proportion of changes in size of the cover types, significant changes have been observed between 1990 and
2013. The various land cover types; settlement, low land vegetation, fallow land show almost a similar trend
with dramatic increase in their areas (Table 1). In this period of 23 years, High dense vegetation (HDV) was
found as most affected land cover class. This class was degraded by 10.59%. In 1990, HDV was spread over
Soni et al. (2015) 2(2): 101107
.
139.83 sq km and remain to 95.85 km2 in 2013. It revealed a decreasing trend with highly positive correlation
coefficient (r2 = 0.8419). The cause of this degradation is the converting HDV into low dense vegetation (LDW)
and settlement. Low dense vegetation was increased to 17.92% (2013) from 13.14% (1990). It revealed an
increasing trend with positive correlation coefficient (r2 = 0.7274). In the watershed, reason of the forest
degradation is human interferences. The settlement was increased from 1.18% (1990) to 3.06% (2013). This
class revealed an increasing trend with highly positive correlation coefficient (r2 = 0.9066). Fallow land was
increased from 34.46% (1990) to 43.45% (2013) with increasing trend and high correlation coefficient (r2 =
0.8981). Agriculture was decreased from 12.86% (1990) to 7.54% (2013). This class revealed an increasing
trend with positive correlation coefficient (r2 = 0.5458).
Figure 2. Land use land cover change map of 1990, 2000, 2005, 2011 and 2013.
CONCLUSION
It is concluded that the forest within the watershed is degraded from 46.83% to 41.02% in 23 years i.e.
5.81%. Trend of deforestation is 0.35% area per year. It is happening due to rapid urbanization, increase in
settlement so. The study reveals that Remote Sensing and GIS are powerful for earth observation and
environmental degradation study. They provide LULC classification schemes, land changes and dynamics
characterization. The results from image classification showed that the major LULC changes in the Chakrar
watershed in three decade was forest. There were decrease in high dense vegetation and increase in settlement,
low dense vegetation agriculture and open land. The main transitions were observed among high dense
Soni et al. (2015) 2(2): 101107
.
Figure 3. Trend of land use land cover classes.

vegetation that converted to low dense vegetation settlement and open lands. It is considered that deforestation
in the Chakrar watershed was caused primarily due to agricultural activities and urban and rural development.
Reforestation and agroforestry practices can be followed over open land and deforested land to save forest,
biodiversity, biomass and natural habitat of flora and fauna.
ACKNOWLEDGEMENTS
Authors are thankful to Remote Sensing and GIS Lab, MGCGV Chitrakoot for providing the lab facility for
the present study.
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2(2): 108111, 2015
Research article
A rapid and economical method for the maceration of wood fibers

in Boswellia serrata Roxb.
Shashank Mahesh1*, Pramod Kumar1 and S. A. Ansari2
1
Tropical Forest Research Institute, PO-RFRC, Mandla Road, Jabalpur, Madhya Pradesh, India
2
Institute of Forest Productivity, Ranchi, Jharkhand, India
*Corresponding Author: shashank_maheshjbp@yahoo.com [Accepted: 07 August 2015]
Abstract: Boswellia serrata Roxb. (Burseraceae) is endemic to India and naturally distributed to
Chhattisgarh, Madhya Pradesh, Maharashtra, Odisha and parts of Rajasthan. It is drought tolerant
and resists fire. It has high pharmaceutical value due to its gum-resin. A rapid, convenient and
economical method for complete maceration of its wood fibers was developed during the process
of fiber analysis. Wood core samples were collected from the trees of B. serrata growing in
Institutes' campus and their fibers were macerated using different concentrations of macerating
agent (nitric acid). 50% nitric acid was found very efficient to separate out all the fibers. This
maceration protocol resolves clearly all the compact fibers and made their measurement
convenient. The method may be useful for studies of wood fibers for exploration of better
populations and may be used for quality paper production.
Keywords: Boswellia serrata - Nitric acid - Wood fiber - Maceration.
[Cite as: Mahesh S, Kumar P & Ansari SA (2015) A rapid and economical method for the maceration of wood
fibers in Boswellia serrata Roxb. Tropical Plant Research 2(2): 108111]
INTRODUCTION
Since ancient times, wood of many trees is regularly in use for quality furniture and in pulp and paper
industry. Durability and strength of products depends upon tensile strength of wood fibers. Cellulosic fibers are
normally longer in sapwood than heartwood and contribute 4045% wood's dry weight of fiber wall which
makes it suitable for pulping. Sapwood of Boswellia serrata is deficient in lignin, hemicelluloses and other
extractives and thus widely used in paper pulp process. Pulp formation involves mechanical and chemical
processing. In mechanical process wood is chopped whereas in chemical process fibers are capable of blending
easily without breaking. Besides other properties, the physical dimensions of the fiber are among the most
important factors in pulping and other commercial purpose. Fiber length is one of the quality parameters for
pulpwood, and it has been extensively studied in relation to tree age and within-tree position (Hudson et al.
1995, Sandercock et al. 1995). With increasing interest in non-wood pulping, it is essential to know the fiber
length for interpretation of variability also (Han et al. 1999).
Anatomical studies of plant stems or other parts, rarely convey an accurate picture of the real nature of the
cells of which they are composed. One method which reveals cells in their cellular structure is the dissociation
method. The target plant is treated with chemicals which dissolve the middle lamella and allow the cells/fibers
to become separated from one another. However, in some plants, the mild maceration process will not
completely dissociate to a single fiber unit, resulting in an aggregate of fibers. These aggregates of fibers have
the appearance of a single fiber. The maceration process is actually small-scale pulping and sometimes referred
test-tube pulping. Some processes result in maceration as well as bleaching.
Boswellia serrata (Burseraceae) is an excellent source of pulp for production of very high quality paper.
Species is endemic to India and naturally distributed in central Indian deciduous forest. Species has been heavily
exploited in past and therefore require conservation and field plantations of its better genotypes in terms of its
fibers to overcome its demand in pulp and paper industry. In order to make an analysis of wood fibers of B.
serrata, an economical, simple and rapid method for maceration of wood fibers was developed which may be
Received: 15 May 2015 Published online: 31 August 2015
Mahesh et al. (2015) 2(2): 108111
.
useful for other species also. Fiber maceration technique described by Jorge et al. (2000) was also compared in
this study.

The wood core samples were collected by increment boring from the trees of about 20 years old B. serrata
stand at Institute's campus. Collected wood core samples were submerged in 37% formaldehyde solution. Prior
to start the maceration process, samples were drained for formaldehyde solution to avoid more evaporation of
fumes. Three concentrations of nitric acid viz. 40%, 50% and 60% were used. Wood core samples were taken in
test tubes, dipped them completely in nitric acid solution and kept in a water bath at 70C. Maceration process
completes in 56 hrs with separation of white colored fibers. Test tubes containing macerated fibers were
removed from water bath and allowed to cool at room temperature. After cooling, nitric acid was drained and
macerated fibers were washed thrice with distilled water and filtered using Whatman Grade 1 filter paper for
separation of fibers. Precautions for well drain of formaldehyde solution is necessary prior to immersion in nitric
acid and after immersion also as the fumes contains nitrogen oxide, carbon dioxide and nitric acid (when
formaldehyde preserved samples immersed in nitric acid, the exothermic reaction occurs due to oxidation of
formaldehyde).
For slide preparation fibers were stained with 20% safranin solution and again washed with distilled water
for destaining of excess safranin. Placed some amount of fiber suspension on a standard glass slide with the help
of ink/medicine dropper and allowed for air drying. Mounting was done in canada balsam using a cover glass.
Use of glycerol enhances visibility of fibers.
RESULTS AND DISCUSSION
Figure 1. Macerated fibers with: A, 1:1 glacial acetic acid: hydrogen peroxide solution; B, 40% nitric acid;
C, 60% nitric acid; D, 50 % nitric acid.
In present study, partial maceration was observed in 40% nitric acid (Fig. 1B) whereas in 50% and 60%
nitric acid complete maceration occurs. However, observations recorded under microscope (5X magnification
using Leica microsystem EC 3, Switzerland with software LAS 4.3.0) reveal splitting of fibers in 60% nitric
acid (Fig. 1C) whereas, 50% nitric acid exhibited separation of fibers without their splitting (Fig. 1D).
Mahesh et al. (2015) 2(2): 108111
.
Maceration of B. serrata samples by the method of Jorge et al. (2000) reveals no separation of fibers even after
24hrs dipping in prescribed solution (glacial acetic acid : hydrogen peroxide in 1:1). Only partial maceration
observed in samples incubated at 70C for 3 hrs. Therefore, this method was not found efficient to macerate B.
serrata fibers and comparatively time consuming and costly also.
Nitric acid acts as an easy and fast resolving agent to break down the middle lamella for separating the cells.
Boiled nitric acid separates organs/cells much faster. Results reveal that 50% nitric acid is not only convenient
for maceration in hot condition but dissolves other extractives also. This protocol helps to reduce the time and
chemicals cost. Nitric acid in combination with other chemicals has been used for maceration of fibers in
different species. Schultzes method describes the application of a combination of various concentrations of
nitric acid with a small quantity of potassium chlorate and allowed to stand at room temperature or heated
slightly to initiate the reaction (Chamberlain 1915). There are some variations of Schultzes method. Jeffrey
(1917) proposed maceration by using mixture of equal portions of freshly combined 8 to 10% nitric acid and
chromic acid. Slightly heating hastens the reaction and macerates wood samples. Schmid (1982) made
sonification and other improvements on Jeffreys method. Large (up to pencil-size) pieces of wood are
macerated in Jeffreys solution. Franklin (1945) used acetic acid and hydrogen peroxide. Spearin and Isenberg
(1947) used sodium chlorite and acetic acid for maceration and found considerable damage in fiber length.
Burkart (1966) described treatment with triethylene glycol containing an organic catalyst, such as phenol
sulfonic acid or p-toluene sulfonic acid at 130C. Hall et al. (1986) used nitric acid and formaldehyde for
maceration of fibers. Han et al. (1999) compared fiber length measurement techniques such as digitizing, the
Kajaani procedure and NIH image in Kenaf (Hibiscus cannabinus L.) and developed a relationship between
fiber length, growth and pulping condition. Araujo et al. (2002) performed microwave assisted acid digestion
(2.0, 3.0, 5.0, 7.0 and 14.0 mol-1 with H2O2 (30% v/v) for evaluation of residual carbon content (RCC) using
inductivity coupled plasma optical emission spectrometry (ICP-OES) with axial viewing.
CONCLUSION
It reveals through our study that the maceration of wood fibers in 50% nitric acid consuming less time and
also economical than the other methods. Procedure is not only rapid and economical but also yielded complete
maceration which is a prime requirement for wood fiber analysis. Since, the pulp of B. serrata provide good
strength and quality in paper industry when mixed with 2540% long fibred bamboo pulp, the procedure may be
employed for evaluation of better genotypes and their conservation and multiplication for afforestation
programmes to meet out the demand of pulp-paper industry and pharmaceutical industry for better economic
returns.
ACKNOWLEDGEMENTS
Authors are thankful to the Indian Council of Forestry Research and Education, Dehradun for providing the
grant for the study and to the Director, Tropical Forest Research Institute, Jabalpur for necessary facilities and
suggestions.
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Science 105: 214.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(2): 112119, 2015
Research article
Floristic composition, life-forms and biological spectrum of

tropical dry deciduous forest in Sagar District,
Madhya Pradesh, India
A. S. Thakur
Department of Botany, Govt. College Khurai, Sagar, Madhya Pradesh-470117, India
*Corresponding Author: dr_asthakur@rediffmail.com [Accepted: 09 August 2015]
Abstract: Floristic studies were conducted in Baraytha forest. A total of 82 species belonging to
72 genera and 33 families of angiosperm were recorded during the sampling of vegetation. Based
on species contribution Fabaceae, Asteraceae, Rubiaceae, Combretaceae, Malvaceae, Mimosaceae
and Euphorbiaceae were found as dominant families. Life-forms in order of importance were
Phanerophytes (55%), Therophytes (32.5%), Chamaephytes (6.25%), Geophytes (3.75%),
Hemicryptophytes and Epiphytes both (1.25%). The dominance of phanerophytes and therophytes
reveals that phytoclimate of the area as phanero-therophytic.
Keywords: Floristic composition - Life-forms - Biological spectrum - Phytoclimate.
[Cite as: Thakur AS (2015) Floristic composition, life-forms and biological spectrum of tropical dry deciduous
forest in Sagar District, Madhya Pradesh, India. Tropical Plant Research 2(2): 112119]
INTRODUCTION
Floristic richness of an area gives the design and functioning of the natural communities and also adds to
complete understanding of the pattern and process of their structure. The floristic richness of an area depends
upon the type, quality and stratification of its vegetation Whittaker (1972). Quantitative floristic inventories of
forest ecosystems provides necessary context for understanding, planning and interppreting long-term ecological
research (Phillips et al. 2003, Baithalu et al. 2013). The information resulting from forest inventories not only
provides data on the floristic composition and abundance of individual species, but also on detailed structural
attributes of the vegetation (Palomino & Alvarez 2009). The information also serves as an invaluable research
base for diverse aspects of tropical ecology while providing information crucial for their conservation and
management (Ayyappan & Parthasarthy 1999).
Raunkier (1934) defined the life-forms as the sum of adaptions of plants to climate and Raunkiers system of
classification of life-forms is the most widely accepted one and has been universally followed. The ratio of life-
forms of different species in term of number or percentage in any floristic community is called Biological
spectrum or the spectrum of life-forms (Milne & Milne 1971), which can be used to indicate the stratification
and layering pattern of the community (Rao 1968, Krebs 1994), to indicate the prevailling environment (Kotiwar
et al. 1996), its aridity or humidity (Meher-Homji 1964), to monitor the impact of ambient stress factors on
climate (Palit et al. 2002), and to determine the nature of bioclimate or phytoclimate (Malik et al. 2006).
Several workers have studied floristic composition and biological spectrum of different regions in India
(Meher-Homji 1964, 1981, Pandey & Parmar 1993, Sharman & Dhakre 1993, Singh & Arora 1994, Reddy et al.
1999, 2002, Rana et al. 2002, Thakur 2003, Jamir et al. 2006, Shukla & Mishra 2006, Patel et al. 2010,
Pharswan et al. 2010, Thakur & Khare 2011, Reddy et al. 2011, Bajpai et al. 2012, Desai & Ant 2012, Thakur
et al. 2012a, Thakur et al. 2012b, Sindhuja et al. 2012, Vediya & Kharadi 2012, Sarkar & Devi 2014, Ashwini
et al. 2014, Radha 2014, Chauhan et al. 2014, Kargjam 2014, Kensa & Pramila 2014, Sharma et al. 2014,
Mohammad & Joshi 2015 and Sundarapandian & Subbiah 2015).
Thakur (2015) 2(2): 112119
.
The present study was conducted in Baraytha located 62 Km in north of Sagar. It lies at 24o17'N latitude and
o
78 56' E longitude. Altitude ranges from 400 to 425 meter and highest peak is 455 meters. On the basis of rock
formations and their characteristics, the site subdivided into four sub sites. i. Sandstone quartzite ii. Bijawar
sedimentary iii. Bundelkhand granite and iv. Banded iron formation. The area enjoys a typical monsoonic
climate with three well recognized seasons viz.-rainy, winter and summer. Average annual rainfall is about 1200
mm, chiefly received during the rainy season from the month of late June to early September. Winter rains are
of common occurrence. Summer season is from March to mid-June and very hot with maximum temperature of
45oC. Winter season is pleasant and dry with mean minimum temperature of 12.5oC.
Forest communities were analysed by selecting uniform stands at study sites. Specimen of all species
occurring in these plots belonging to trees, shrubs, herbs, climbers and epiphytes were collected and identified.
These species provided a general floristic view of the vegetation. However, the collections are underestimate the
floristics as the species occurring outside the sample plots were not considered. The generic coefficient of flora
was calculated according to Jacord (1912). Biological spectrum was prepared on the basis of percentage species
composition in each life-form following Raunkier (1934) and Muller-Dombois & Ellenberg (1974).
RESULTS AND DISCUSSION

Table 1. Habit classification and life forms of plants observed at Baraytha forest.
Life Sub-sites Baraytha
S. No. Name of plant species Family Habit
form I II III IV forest
1 Acacia catechu (L. f.) Willd. Mimosaceae Tree Ph + +
2 Acacia leucophloea Willd. Mimosaceae Tree Ph + +
3 Achyrathes aspera Linn. Amranthaceae Herb Th + + +
4 Adina cordifolia Hook. f. Rubiaceae Tree Ph + + + +
5 Aegle marmelos Correa. Rutaceae Tree Ph + + + + +
6 Ageratum conyzoides Linn. Asteraceae Herb Th + + +
7 Albizia lebbek Benth. Mimosaceae Tree Ph + +
8 Albizia odoratissima (L. f.) Benth. Mimosaceae Tree Ph + +
9 Alysicarpus monilifer DC. Fabaceae Herb Th + + + + +
10 Anogeissus latifolia (Roxb. ex DC) Wall. Combretaceae Tree Ph + + + +
11 Anogeissus pendula Edgew. Combretaceae Tree/Shrub Ph + + +
12 Barleria prionitis Linn. Acanthaceae Herb Ch + +
13 Bidens biternata (Lour.) Merr. & Sherff. Asteraceae Herb Ch + + +
14 Biophytum sensitivum DC. Geraniaceae Herb Th + + + +
15 Blepharis boerhaaviaefolia Pers. Acanthaceae Herb Ch + +
16 Boerhaavia diffusa Linn. Nyctaginaceae Herb G + +
17 Borreria stricta Linn. F. Rubiaceae Herb Th + + + + +
18 Boswellia serrata Roxb. ex Colebr. Burseraceae Tree Ph + +
19 Bridelia retusa (L.) Spreng. Euphorbiaceae Tree Ph + + +
20 Buchanania lanzan Spreng. Anacardiaceae Tree Ph + + +
21 Butea monosperma (Lamk.) Taub. Fabaceae Tree Ph + + +
22 Carissa spinarum Linn. Apocynaceae Shrub Ph + + + +
23 Casearia graveolens Dal. Bixaceae Tree Ph + +
24 Cassia fistula Linn. Caesalpiniaceae Tree Ph + + + + +
25 Cassia pumila Lamk. Caesalpiniaceae Herb Th + + +
26 Cassia tora Linn. Caesalpiniaceae Herb Th + + + +
27 Celosia argentea Linn. Amaranthaceae Herb Th + +
28 Corchorus actangulus Lam. Tiliaceae Herb Th + + + + +
29 Cordia vestita Hook. f. & Thoms. Boraginaceae Tree Ph + +
30 Crotalaria prostrata Roxb. Fabaceae Herb Th + +
31 Desmodium gangeticum DC. Fabaceae Herb Ch + +
32 Desmodium triflorum DC. Fabaceae Herb Th + + + + +
33 Diospyros melanoxylon Roxb. Ebenaceae Tree Ph + + + + +
34 Ehretia laevis Roxb. Boraginaceae Tree Ph + +
35 Elaeodendron glaucum Pers. Celastraceae Tree Ph + + + +
36 Elephantopus scaber Linn. Asteraceae Herb G + + + + +
37 Eragrostis pilosa Poaceae Herb (Grass) Th + + + +
38 Erythrinia variegata L. Fabaceae Tree Ph + +
Thakur (2015) 2(2): 112119
.
Life Sub-sites Baraytha
S. No. Name of plant species Family Habit
form I II III IV forest
39 Euphorbia hirta Linn. Euphorbiaceae Herb Th + + + + +
40 Flacourtia indica (Burm. F.) Merr. Bixaceae Tree Ph + + + + +
41 Gardenia latifolia Aiton. Rubiaceae Tree Ph + + + + +
42 Helicteres isora Linn. Sterculiaceae Shrub Ph + +
43 Hemidesmus indicus (Linn.) Schultz Asclepiadaceae Climber Ph +
44 Hibiscus solandra L. Herist. Malvaceae Herb Th + + + + +
45 Holarrhena antidysentrica Wall. Apocynaceae Shrub Ph + + + +
46 Ipomaea coccinea Linn. Convolvulaceae Climber G +
47 Iseilema anthephoroides Hack. Poaceae Herb (Grass) H + +
48 Justicia simplex Don. Acanthaceae Herb Th + + + + +
49 Kydia calycina Roxb. Malvaceae Tree Ph + +
50 Lagascea mollis Cav. Asteraceae Herb Th + + + +
51 Lagerstroemia parviflora Roxb. Lythraceae Tree Ph + + + + +
52 Lannea coromandelica (Houtt.) Merr. Anacardiaceae Tree Ph + + + + +
53 Lantana camara Linn. Verbenaceae Shrub Ph + + + +
54 Loranthus longiflorus Desr. Proteaceae Epiphyte E +
55 Madhuca indica Gmel. Sapotaceae Tree Ph + + +
56 Malvastrum tricuspidatum A. Grey. Malvaceae Herb Th + +
57 Miliusa tomentosa (Roxb.) J. Sinclair. Annonaceae Tree Ph + + +
58 Mitragyna parvifolia (Roxb.) Korth. Rubiaceae Tree Ph + + +
59 Mitreola oldenlandioidea Wall. Loganiaceae Herb Th + + + +
60 Ophlismemus burmannii (Retz.) P. Beauv. Poaceae Herb (Grass) Th + + + + +
61 Ougeinia oojeinensis (Roxb.) Hochr. Fabaceae Tree Ph + +
62 Phyllanthus debilis Ham. Euphorbiaceae Herb Th + + + +
63 Phyllanthus urinaria Linn. Euphorbiaceae Herb Th + + + + +
64 Randia spinosa (Thumb.) Keay. Rubiaceae Shrub Ph + + + +
65 Schleichera oleosa (Lour.) Oken. Sapindaceae Tree Ph + + + +
66 Sida spinosa Linn. Malvaceae Herb Th + + + + +
67 Sida veronicaefolia Lamk. Malvaceae Herb Ch + + + + +
68 Sporobolus diander (Retz.) P. Beauv. Poaceae Herb (Grass) Th + + + + +
69 Tectona grandis Linn. Verbenaceae Tree Ph + + + + +
70 Terminalia arjuna W. & A. Combretaceae Tree Ph + +
71 Terminalia belerica (Gaearth.) Roxb. Combretaceae Tree Ph + + + +
72 Terminalia tomentosa (DC.) W.& A. Combretaceae Tree Ph + + + +
73 Tridax procumbens Linn. Asteraceae Herb Th + + + + +
74 Ventilago maderaspatana Gaerth. Rhamnaceae Climber Ph +
75 Vernonia cinerea Linn. Asteraceae Herb Th + + + +
76 Wendlandia puberula DC. Rubiaceae Tree Ph + +
77 Xanthium strumarium Linn. Asteraceae Herb Th + +
78 Xylia xylocarpa (Roxb.) Taub. Fabaceae Tree Ph + +
79 Zizyphus oenoplia Mill. Rhamnaceae Shrub Ph + + + + +
80 Zizyphus xylopyrus Willd. Rhamnaceae Tree/Shrub Ph + +
Note: + = Presence, Ph = Phanerophyte, Ch = Chamaephyte, H = Hemicryptophyte, G = Geophyte, Th = Therophyte, E =
Epiphyte
A total of 82 species belonging to 72 genera and 33 families of angiosperm were encountered during the
sampling of vegetation (Table 1). Out of these total 36 tree species belongs to 31 genera; 08 shrubs species to 07
genera and 34 herbs species to 30 genera. Three climbers and one of epiphyte species also recorded. The
number of herb species is less than expected it is due to that only those species included in sampling that fell
within the sampling unit and sampling was done after the rainy season when dry period began. The dicotyledons
comprise 32 families 68 genera and 78 species and monocotyledons comprise 01 family 04 genera and 04
species. Out of the total 82 species dicotyledons represents 96.96% and monocotyledons 03.03%. The ratio of
monocotyledons to dicotyledons family, genera and species were 1:03, 1:05 and 1:05 respectively (Table 2). Out
of total 33 families of angiosperms in study area, the dominant families were Fabaceae (08species), Asteraceae
(07), Rubiaceae (06), Combretaceae (05), Malvaceae (05), Mimosaceae (04) and Euphorbiaceae (04) accounted
for 39 (47.56%) species and 31 (43.05%) genera. Among the total families 17 families were monogeneric (Table
3).
Thakur (2015) 2(2): 112119
.
Table 2. Comparative account of floristic composition of Baraytha forest.
Dicotyledons Monocotyledons Monocots to Dicots to
Category Total
Number % Number % dicots Ratio Monocots (%)
Family 32 96.96 01 3.03 1:03 96.96 33
Genera 68 94.44 04 5.55 1:05 94.44 72
Species 78 95.12 04 4.87 1:05 95.12 82
Hemicryptophyte Geophyte Epiphyte

1.25% 3.75% 1.25%
Chamaephyte
6.25%
Therophyte
32.5% Phanerophyte
55%
Phanerophyte Therophyte Chamaephyte

Hemicryptophyte Geophyte Epiphyte
Figure 1. Biological spectrum of Baraytha forest.
Table 3. Arrangement of taxa of Baraytha forest.

S.No. Family Genera Species S.No. Family Genera Species
Dicotyledons 17 Annonaceae 01 01
1 Fabaceae 07 08 18 Asclepidaceae 01 01
2 Asteraceae 07 07 19 Burseraceae 01 01
3 Rubiaceae 06 06 20 Celastraceae 01 01
4 Malvaceae 04 05 21 Ebenaceae 01 01
5 Euphorbiaceae 03 04 22 Geraniaceae 01 01
6 Acanthaceae 03 03 23 Convolvulaceae 01 01
7 Combretaceae 02 05 24 Loganiaceae 01 01
8 Mimosaceae 02 04 25 Lythraceae 01 01
9 Rhamnaceae 02 03 26 Nyctaginaceae 01 01
10 Anacardiaceae 02 02 27 Proteaceae 01 01
11 Amaranthaceae 02 02 28 Rutaceae 01 01
12 Apocynaceae 02 02 29 Sapindaceae 01 01
13 Bixaceae 02 02 30 Sapotaceae 01 01
14 Boraginaceae 02 02 31 Sterculiaceae 01 01
15 Verbenaceae 02 02 32 Tiliaceae 01 01
16 Caesalpiniaceae 01 03 Monocotyledons
33 Poaceae 04 04
Thakur (2015) 2(2): 112119
.
Present study area falls in a comparatively drier climate and most of the species shed their foliage during the
winter season, render these forests naked. Comparative analysis of floristic composition with other studies done
in Central India (Prasad & Pandey 1992, Thakur & Khare 2009) envisaged that in this region floristic
composition is poor. On the whole, it appears that long dry spell is perhaps the one of the major reason for the
poor floristic structure.
Generic coefficient as 87.80% was determined for the vegetation of Baraytha forest. On the basis of high
percentage of generic coefficient, it can be inferred more intergeneric competitions exist in the area.
The vegetation of Baraytha forest showed highest percentage of Phanerophytes (55%), other groups of Life-
forms in order of importance were Therophytes (32.5%), Chamaephytes (6.25%), Geophytes (3.75%),
Hemicryptophytes and Epiphytes both (1.25%) (Table 1 & Fig. 1). The Phanerophytes and Therophytes together
constitute 87.5% of the life-forms proportion. Phanerophytes showed maximum divergence from the normal
spectrum as given by Raunkier, accordingly the phytoclimate of the area may be termed as phanero-therophytic.
Similar phytoclimatic association has also been reported by other workers (Rajendraprasad et al. 1998,
Lakshmanan 1962, Misra et al. 1979, Saxena 1980, Saxena et al. 1982, Khatri 2000, Thakur & Khare 2011).
According to Meher-Homji (1964) the life-forms are reflected by bioclimate of the area. Thus in humid
regions the bioclimate should be phanerophytic, and in arid regions therophytic. Jamir et al. (2006) observed
that the montane humid forests of Meghalaya receives annual rainfall of 5500mm and represent 51% of
phanerophytes, so rainfall appears to be most important operative factor in the evolution of biological spectrum.
Structurally and floristically the tropical dry forests are less complex than wet forests, comprising about half or
less of the tree species of wet forest (Murphy & Lugo 1986). In this regard the study area is floristically poor.
However, it may be due to the area sampled being very small. In the present study, both phanerophytes and
therophytes share considerably importance in depicting the phytoclimate of dual character extremes i.e. warm-
moist and warm-dry climate. The study area experiences alternation of long dry period with moderate rainfall,
warm dry and warm moist climate are characteristics of this area and these climatic condition are responsible for
phanero-therophytic phytoclimate.
In the present study therophytes stand next to phanerophytes. The predominance of therophytes due to
grazing is a common phenomenon (Yadav & Singh 1977). It is also an indicator of biotic pressures (Barucha &
Dave 1944). Pandeya (1964) observed that the life-forms of the flora of different grassland association of Sagar
were maintained by intensity of grazing. The high percentage of therophytes in grassland is due to overgrazing
resulting in the introduction and spread of weedy grasses (Cain 1950, Down 1973) also recorded higher number
of therophytes in bare and early successional stands. According to Dansereau (1957) the predominance of
therophytes indicates warm climate. The growth of therophyte was much favoured in disturbed areas (Keeley &
Albert 1977, Vora & George 1987).
It is experienced that vegetation in a stress of biotic pressure gradually increase the percentage of
therophytes. It is pertient to state that composition of phanerophytes and therophytes is close in this area, an
increase in biotic pressure would change the biological spectrum totherophytic-phanerophytic.
ACKNOWLEDGEMENTS
Author is thankful to Central Regional Office of U.G.C. Bhopal, for providing Teacher Research Fellowship
and Department of Higher Education M.P. for the study leave to carry out this work. Authors do not forget to
pay his compliments to Professor P.K. Khare, Department of Botany, Dr. H.S. Gour University, Sagar, Madhya
Pradesh, India for excellent guidance.
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2(2): 120126, 2015
Research article
Estimation of genetic divergence, association, direct and indirect

effects of yield with other attributes in cotton (Gossypium hirsutum L.)
using biplot correlation and path coefficient analysis
Asif Latif1, Muhammad Bilal2*, Syed Bilal Hussain1 and Farah Ahmad3
1
Department of Plant Breeding and Genetics, Bahauddin Zakariya University, Multan, Pakistan
2
Department of Plant Breeding and Genetics, PMAS Arid Agriculture University, Rawalpindi, Pakistan
3
Department of Biochemistry, PMAS Arid Agriculture University, Rawalpindi, Pakistan
*Corresponding Author: m_bilal788@hotmail.com [Accepted: 10 August 2015]
Abstract: Genetic variation, Principle component analysis and Path analysis were analysed in
sixty cotton (Gossypium hirsutum L.) genotypes and Randomized Complete Block Design was
followed with three replications. A wide spread variation was observed among all the investigated
attributes. Principle component analysis was worked out to identify the diverse genotypes. Bolls
per plant, sympodia per plant and boll weight showed noteworthy positively association with seed
cotton yields. Path analysis exposed uppermost positive direct consequence of number of bolls per
plant on total yield whereas sympodia per plant and boll weight had maximum indirect positive
effect through number of bolls on seed yield. The correlation analysis and path coefficient analysis
and Principle component analysis together suggested that these attributes and genotypes should be
given prime emphasis in effective selection to get better cotton seed yield.
Keywords: Cotton - Path analysis - Principle component analysis - Seed yield.
[Cite as: Latif A, Bilal M, Hussain SB & Ahmad F (2015) Estimation of genetic divergence, association, direct
and indirect effects of yield with other attributes in cotton (Gossypium hirsutum L.) using biplot correlation and
path coefficient analysis. Tropical Plant Research 2(2): 120126]
INTRODUCTION
Cotton (Gossypium hirsutum L.) referred as White gold is a premier cash and fibre crop. It is major
contributor towards financial stability of more than 80 countries with annual production of 20 million tons
(Farasat et al. 2014). Characterization and conservation of genetic material in form of genotypes, best
performing breeding lines, landraces, wild lines is of significance value in evolutionary breeding and genetic
process (Haidar et al. 2012). Exploitation of genetic diversity through various morphological and agronomic
characters have been done for triumphant hybridization program which need perfect selection because
environment greatly influence on these traits and selection process (Ahmad et al. 2012). Therefore, considerable
emphasis should be given to evolve highly productive cotton varieties. Cotton production either in seed yield or
lint depends on characters like plant height, direct fruit bearing branches (sympodial) and indirect fruit bearing
branches (monopodial), boll weight (BW), bolls per plant, seed index, ginning out tern (Salahuddin et al. 2010).
Comprehensive understanding about the crop nature, performance level and association of numerous agronomic
attributes with yield is necessary for plant researcher to tackle the cotton yield limiting constraints.
Being an important asset to cotton breeders, correlation between yield and yield contributing attributes, is
principal endeavour to explore relation among traits and which plant parameters should be selected first to boost
up cotton seed and lint yield (Khakwani et al. 2012). Path analysis, handy technique, permits plant researchers
to select particular attributes that helps in the understanding of contribution of numerous traits in total variation
by partitioning the total correlation into direct and indirect effects (Shahriari et al. 2014). Multivariate statistical
technique mainly principal component analysis (PCA) utilized by breeder to assess genetic variation, which
leads to exploration of elite and phenomenal genotypes that could be used as diversified genotype for a future
cotton hybridization program (Rehman et al. 2015).
Latif et al. (2015) 2(2): 120126
.
Keeping in view the above facts about diversity in cotton, the present investigation was to explore genetic
diversity, notify extraordinary genotype and direct and indirect effect of important yield contributing traits on
yield.

The present research study was investigated during 2013 at agricultural experimental area of Bahauddin
Zakariya University, Multan. Experimental research material was comprised of sixty Cotton (Gossypium
hirsutum L.) genotypes and sown in 3 replications following Randomized Complete Block Design (RCBD) with
row to row (RR) and plant to plant (PP) distance of 2.5 feet and 1 feet respectively. All recommended
agricultural implementations like spraying, fertilization; weeding, irrigation and cotton production technology
was adopted in every replication. The data were collected from five tagged plants of each replication for plant
height (PH), number of monopodial branches, number of sympodial branches, boll weight (BW), number of
bolls per plant , seed index (SI), yield per plant (YPP) and ginning out turn (GOT%).
Collected data was subjected to analysis of variance using Statistix 8.1 (Umar et al. 2014). Principle
component analysis (PCA) was performed on the mean data using XLSTAT software (Khodadadi et al. 2011)
and path analysis was done by following the method of Dewey & Lu (1959) through SPSS Amos v20.0.0.
RESULTS
Analysis of variance (ANOVA) and heritability for various plant attributes
Analysis of variance depicted widespread significant variation (P<0.01) and suggested high level of genetic
variability among all yield contributing attributes in studied traits (Table 1). Among all attributes yield per plant
presented highest heritability followed by bolls per plant and sympodial branches as shown in table 1.
Table 1. Analysis of variance and heritability values of yield and other quantitative attributes.
S.O.V DF PH MOP SYP BPP BW SI GOT YPP
Reps 2 54.80__ 0.34__ 0.82__ 1.51__ 0.076__ 0.44__ 2.71__ 5.20__
** ** ** ** ** ** **
Genotypes 59 3454.2 0.61 71.29 337.9 0.39 2.72 13.16 1750.46**
Error 118 146.59__ 0.19__ 1.31__ 3.92__ 0.061__ 0.24__ 2.49__ 17.69__
2
h (%) 88.26__ 43.2__ 94.68__ 96.59__ 64.02__ 77.26__ 58.82__ 97.02__
Note: DF, Degree of Freedom; PH, Plant height; BPP, Bolls per plant; MOP, Monopodia per plant; YP,
Sympodia per plant; BW, Boll weight; SI, Seed Index; GOT, Ginning out turn; YPP, Yield per plant.
**, Highly Significant.
Principal component analysis (PCA)
Mean data of sixty cotton genotypes were calculated and PCA was applied to sum up momentous variation
from collected mean data. Out of eight principal components, three principal components (PCs) depicted more
than one eigen value so these three components have given due consideration for further explanation (Table 2).
First two principle components PC1 and PC2 explained 47.57% and 16.78% of variation respectively with
63.36% cumulative variance among all attributes. The attributes of worthy importance in PC1 were sympodial
branches, number of bolls per plant, boll weight (BW) and yield per plant (YPP) with maximum scores,
depicted more contribution towards total variation. The second PC was more associated with plant height and
seed index attributes (Table 2).
Relationship among Attributes
Correlation of all recorded data of cotton genotypes presented positive and significant as well as non-
significant association among different characters. By keeping the yield as supreme importance we observe the
relation of all other traits with yield. Outcome of Biplot graph depicted that sympodial branches per plant, bolls
per plant and boll weight (BW) had highly momentous and positive association with cotton yield. Ginning out
turn (GOT) and monopodia per plant exhibited positive relationship with cotton yield. In this study bolls per
plant have a direct influence and positive correlation with yield. In our study positive and significant correlation
was observed between boll weight (BW) and cotton seed yield (Fig. 1).
Genotype by trait analysis
Spread out graph termed as polygon created in PCA showed wide spread variation in cotton genotypes.
Genotypes which are present at vertex of polygon in biplot graph has longest distance from the origin, illustrated
Latif et al. (2015) 2(2): 120126
.
maximum variation for quantitative attributes and could be utilized as parental lines in broadening the genetic
base of cotton through breeding program. FH-1000, S-120, MG-6, MG-14 RH-1, MNH-147, CIM-20 and CIM-
70 present at the vertex of polygon and farthest from origin depicted maximum diversity while CIM-240
followed by CIM-448 and MNH-554 nearest to origin depicted lowest genetic diversity (Fig. 1).
Table 2. Eigen values, proportion of variability and quantitative traits that contributed to the eight principal components.
Statistical Variables PC1 PC2 PC3 PC4 PC5 PC6 PC7 PC8
Eigenvalue 3.726 1.343 1.028 0.855 0.664 0.333 0.043 0.008
Variability (%) 46.572 16.784 12.854 10.691 8.302 4.157 0.535 0.105
Cumulative (%) 46.572 63.356 76.210 86.901 95.203 99.360 99.895 100.000
Traits
PH 0.019 0.406 0.323 0.155 0.066 0.033 0.000 0.000
MOP 0.094 0.027 0.535 0.326 0.000 0.017 0.000 0.000
SYM 0.915 0.000 0.004 0.000 0.028 0.025 0.027 0.000
NOB 0.943 0.009 0.000 0.002 0.021 0.015 0.006 0.004
SI 0.002 0.696 0.007 0.023 0.246 0.025 0.000 0.000
BW 0.639 0.002 0.003 0.093 0.056 0.207 0.000 0.000
YPP 0.951 0.009 0.002 0.001 0.019 0.006 0.010 0.004
GOT 0.162 0.194 0.155 0.256 0.228 0.006 0.000 0.000
Note: PH, Plant height; BPP, Bolls per plant; MOP, Monopodia per plant; SYP, Sympodia per plant; BW, Boll
weight; SI, Seed Index; GOT, Ginning out turn; YPP, Yield per plant.
Bold values of each attribute depicted higher squared cosine value in eight principle components.
Figure 1. Biplot graph using mean data of sixty cotton genotypes.

Path analysis
Path analysis partitioned the observed correlation into undeviating and deviating effect of cotton variables
presented in figure 2. Path analysis depicted that bolls per plant exhibited highest positive and undeviating effect
on cotton yield. While other attributes depicted low direct effects on yield. Negative direct effect was observed
for plant height and seed index on yield (Table 3). Whereas, sympodia per plant and boll weight (BW) depicted
maximum indirect positive effect on seed yield via number of bolls per plant. In our study monopodial branches
put undeviating and highly positive effect on yield via number of bolls per plant. In our investigation ginning
out turn illustrated good positive indirect effect on yield via bolls per plant.
Latif et al. (2015) 2(2): 120126
.
Figure 2. Diagrammatic illustration of undeviating (direct) and deviating (indirect) consequences of independent attributes
on dependent attributes. (PH, Plant height; BPP, Bolls per plant; MOP, Monopodia per plant; SYP, Sympodia per plant; BW,
Boll weight; SI, Seed Index; GOT, Ginning out turn; YPP,Yield per plant)
Table 3. Path analysis of numerous attributes in (Gossypium hirsutum L.).

Traits PH MOP SYM NOB SI BW GOT
PH -0.0611 0.00456 0.00218 0.05701 0.00954 0.02015 0.00089
MOP -0.0096 0.02892 0.00989 0.28861 -0.0058 0.01289 0.00094
SYM -0.0038 0.0081 0.0353 0.84836 0.0001 0.06647 0.00547
NOB -0.004 0.0095 0.03409 0.87857 -0.0027 0.07004 0.00442
SI 0.01722 0.00495 -0.0001 0.07074 -0.0338 0.0087 -0.003
BW -0.0133 0.00401 0.02527 0.66274 -0.0032 0.09285 0.0052
GOT -0.0038 0.00189 0.01337 0.26886 0.00713 0.03343 0.01444
Note: Bold values representing direct effects.
DISCUSSION
It is evident from the Table 1 that all the genotypes in present study depicted considerable variation for all
investigated traits. This variation has ample scope for plant breeder and high magnitude of variability is pre
requisite for successful breeding programs to improve numerous traits (Ahsan et al. 2015). Judgment of
heritability (h2 B.S) values of various characters is of worth significance because higher heritability (h2) makes
selection process easier which lead to greater response of selection (Khan et al. 2010). Above 80% heritability
(high heritability) permits plant breeder to select traits in earlier generations and less than 60% heritability
considered as low heritability illustrated selection of traits lead to unfruitful results (Rehman et al. 2015). In this
study mostly traits showed higher heritability, hence these attributes are heritable and favourable for selection of
superior genotypes for genetic improvement of cotton yield (Kaydan & Yagmur 2008). Similar results of study
were in complete conformity with (Khan et al. 2015).
Latif et al. (2015) 2(2): 120126
.
To get better knowledge about associations among all genotypes multivariate analytical techniques i.e.
principal component analysis (PCA) was executed to discover diverse genotypes for successful hybridization
program (Tarighaleslami et al. 2012). In this study PCA abridged the total variation into eight principle
components. Out of which three principle components depicted more than one eigen values and these outcomes
were supported by the findings of Ahmad et al. (2015). Biplot analysis based on first two principle components
revealed genetically diverse genotypes via pattern of scattering (Fig. 1). The division of all genotypes in biplot
exposed the presence of considerable genetic variation (Mafakheri et al. 2010). In biplot graph the distance
between base of graph to traits presented the diverse nature of studied genotypes. Greater distance revealed
greater diversity among genotypes and provided significant information about the genotypic performance (Rana
et al. 2013). Genetic variation not only depends on geographical distribution of genotypes but this may be due to
other components like genetic flow, environmental variability, natural and non-natural selection and interchange
of hereditary materials. Thus for cotton breeding hybridization parental lines should be based on genetics rather
than geographical distribution (Bates et al. 1973).
The straight line connecting marked point of any attribute and base point of a biplot graph is mentioned as
traits vector and the cosine angle between parameters illustrated relationship among all the attributes.
Attributes having < 90o presented positive correlation while attributes with >90o showed negative correlation
and attributes at right angle (90o) presented independent behavior. Correlation through biplot graph depicted
momentous and positive association between sympodial branches per plant, bolls per plant and boll weight
(BW) and yield because these characters showed < 90owith cotton yield. Haidar et al.(2014) outlined similar
positive association for sympodial branches per plant, bolls per plant and boll weight (BW) with total yield. Boll
weight (BW) is considered as major contributor followed by bolls per plant in improvement of cotton seed yield
because it is supposed that greater the boll weight higher will be the cotton yield (Baloch et al. 2014). Our
findings are in confirmatory with (Ahmad et al. 2008) who also assumed direct effect of boll weight (BW) on
cotton seed yield but observed its significant convincing effect by means of number of bolls per plant. Our
results showed direct influence of number of bolls on yield. Therefore it is suggested that increasing number of
bolls and sympodia per plant lead to enhancing cotton seed yield. Similar results of authors were also supported
by the findings of Qayyum et al. (2012).
A straightforward association does not provide ideal picture of the contribution of numerous traits in total
yield. Therefore, Path coefficient analysis a practical biometrical tool utilized to partitioned the correlation into
direct and indirect effects on yield using genotypic correlation values (Dinakaran et al. 2012). In this present
study, bolls per plant exact direct effect on yield, similar findings were shown by Rao & Gopinath (2013) who
also reported direct effect of bolls per plant on yield. Therefore it is suggested that selection based on bolls per
plant will help plant breeder in the improvement of cotton yield. Salahuddin et al. (2010) reported alike
outcomes of indirect effect of sympodia per plant and boll weight (BW) on cotton yield as driven in our study.
Karademir et al. (2009) reported positive indirect effect of ginning out turn on high yield via bolls per plant.
However Haidar et al. (2014) reported contradictory results regarding indirect effect of ginning out turn (GOT)
on yield by mean of bolls per plant. This might be due to utilization of different genotypes in investigation.
CONCLUSION
Analysis of variance (ANOVA) indicated the extensive amount of divergence of all the studied traits. The
outcome of principle component analysis confirmed that genotypes A-One, S-120, FH-1000, CRIS-134, MNH-
147, CIM-20 and NS-121 depicted humungous genetic variability in germplasm. Parallel outcomes of
correlation through biplot and path analysis showed that bolls per plant, boll weight (BW) and sympodial
branches per plant has positive significant correlation and direct effect on cotton yield. Therefore these positive
and significant association and divergent parental genotypes notify through principle component analysis (PCA)
would be more effective to get breakthrough in successful cotton breeding program.
ACKNOWLEDGEMENTS
The authors would like to express their appreciation and deep regards to the department of Bahauddin
Zakariya University, Multan for providing research area and all kind of support for the successful completion of
the research study.
Latif et al. (2015) 2(2): 120126
.
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2(2): 127133, 2015
Research article
Heavy metal and phytochemical screening of anti-jaundice and

anti-malaria concoctions and genotoxicity assessment of the
concoctions using Allium cepa assay
Oluwatosin A. Arojojoye1*, Olajumoke O. Alao1, Oluwabusayo A. Owoeye2,
Adekunle O. Adejuwon3, Olusegun O. John-Dewole1 and Yetunde J. Jemiseye1
1
Department of Biochemistry, Faculty of Information Technology and Applied Sciences, Lead City University,
Ibadan, Nigeria
2
Department of Pediatrics, University College Hospital, Ibadan, Nigeria
3
Department of Microbiology, Faculty of Information Technology and Applied Sciences, Lead City University,
Ibadan, Nigeria
*Corresponding Author: tosyne568@yahoo.com [Accepted: 12 August 2015]
Abstract: Increasing population world-wide is depending on herbal medicine as a source of

primary health care. However, many plants used in traditional and folk medicine have been shown
to be potentially toxic, mutagenic, and carcinogenic. In this study, the cytotoxic and genotoxic
potentials of anti-jaundice and anti-malaria concoctions commonly used in Nigeria in treating
jaundice and malaria were investigated using Allium cepa assay. The concoctions were also
analysed for the presence of some heavy metals and phytochemicals. The roots of Allium cepa
were exposed to 10%, 50% and 100% of both concoctions respectively for 96 hours for
macroscopic and microscopic analysis and Allium cepas exposed to tap water served as control.
The anti-jaundice and anti-malaria concoctions significantly (p<0.05) inhibited the root growth of
Allium cepa when compared with control with EC50 values of 5% and 6% respectively. Both
concoctions also caused concentration dependent decrease in mitotic index in Allium cepa cells
and induced chromosomal aberrations like vagrant chromosome, multipolar anaphase, c-mitosis,
sticky chromosomes and binucleus but there were no chromosomal aberrations in Allium cepas
exposed to tap water. Phytochemical screening of both concoctions revealed the presence of
alkaloids, saponins, cardenolides, flavonoids, tannins and anthraquinones and heavy metals like
cobalt, cadmium and nickel were detected in the concoctions. The results of this study show the
cytotoxic and genotoxic effects of the anti-malaria and anti-jaundice concoctions on Allium cepa.
Keywords: Allium cepa - Genotoxicity - Cytotoxicity - Anti-jaundice - Anti-malaria.
[Cite as: Arojojoye OA, Alao OO, Owoeye OA, Adejuwon AO, John-Dewole OO & Jemiseye YJ (2015) Heavy
metal and phytochemical screening of anti-jaundice and anti-malaria concoctions and genotoxicity assessment
of the concoctions using Allium cepa assay. Tropical Plant Research 2(2): 127133]
INTRODUCTION
Medicinal plants are used by over 70% of the world population for livelihood, primary health care, self-
medication and national health services (Akerele et al. 1991, Heinrich 2010, Mehra et al. 2014). Some of the
factors responsible for this include better accessibility, affordability, acceptability and the belief that medicinal
plants work (Heinrich 2010). However, the potential toxicity of herbs has not been recognized by the general
public or by professional groups of traditional medicine (Soetan & Aiyelaagbe 2009). Most of the traditional
herbal products have never been the subject of comprehensive toxicological investigations, which is required for
modern pharmaceutical products (Subramanion et al. 2013). Based on their traditional use for long periods of
time, they are often assumed to be safe.
Despite the profound therapeutic advantages possessed by some of the medicinal plants, some constituents
of medicinal plants have been found to be potentially toxic, mutagenic, carcinogenic and teratogenic. Interaction
of plant extracts with cells may produce unwanted cellular damage. This interaction may vary depending on the
Received: 03 June 2015 Published online: 31 August 2015
Arojojoye et al. (2015) 2(2): 127133
.
active ingredient present in the extract, as it may occur on the cell surface, within the cell body, DNA, or in the
tissues beneath as well as at the extracellular matrix. It has been shown that while some medicinal plants may
suppress the effects of mutagens, others may contain toxic substances or provoke mutagenic effects (Vicentini et
al. 2001).
Reports of unwanted side effects and potential toxicity of phytomedicines have been on the increase in
recent years (Effraim et al. 2001, Ruffa et al. 2002, Amida et al. 2007, Konan et al. 2007, Sowemimo et al.
2007, Nwafor et al. 2007). Many researchers have shown that numerous herbal products, which are used as food
ingredients or in traditional medicine, have in-vitro mutagenic or toxic properties (Cardoso et al. 2006, Dciga-
Campos et al. 2007, Mohd-Fuat et al. 2007). The use of some plants has also been correlated with high rate of
tumour formation in human (Ames 1986, Nguye et al. 1989, Brito et al. 1990). The genotoxic and mutagenic
potentials of some herb extracts have also been reported by several workers (Barcelos et al. 2007, Konan et al.
2007, Sowemimo et al. 2007, Patterson et al. 1987, Magda 2001, Gadano et al. 2000, 2002, 2006, Paes-Leme et
al. 2005).
In Nigeria, a large percentage of the populace cannot afford to pay for orthodox medical service (Fasola &
Egunyomi 2005, Obi et al. 2006) and therefore depend on herbal concoctions for treatment of most ailments
most of the time, even though there is little information on the potential risk to health of such herbs. These
extracts are prepared as a single herb or as concoctions of herbs. Some of them have genotoxic potential,
cytotoxic and mitodepressive effects as reported by several workers (Heinrich et al. 2010, Akintonwa et al.
2009, Oyedare et al. 2009, Akinboro & Bakare 2007, Williams & Omoh 1996). Investigation of cytotoxic and
mutagenic potentials of medicinal plants is necessary to ensure their safe use. Assessment of the potential
cytogenotoxicity of traditional medicines is indeed an important issue as damage to the genetic material may
lead to critical mutations and therefore also to an increased risk of cancer and other diseases. The present study
determined the levels of heavy metals and phytochemicals in anti-malaria and anti-jaundice concoctions and
also evaluated the genotoxic potential of the concoctions using Allium cepa chromosomal aberration assay.
These concoctions are among the Nigerian folk medicinal plants frequently used in the treatment of malaria and
jaundice in infants, especially among the less privileged. Malaria is a prevalent disease that affects about 60% of
Nigerians annually (Ekwebene 2012) and most people depend on herbs for the treatment of malaria. Brinkmann
& Brinkmann (1991) reported that only 8.25% of people with malaria visit health services, indicating that the
rest uses alternative medicine which involves the use of medicinal herbs with anti-malarial properties. Jaundice
is also prevalent especially in children; therefore it is necessary to investigate the genotoxic potential of these
concoctions. This is essential as a guide towards standardization, safety administration and an understanding of
mechanism of action of the concoctions to see if they could be used in alternative medicine to treat malaria and
jaundice.

Source of Allium cepa and herbs for the concoctions
Allium cepa, along with leaves and barks of other medicinal plants used for the preparation of the
concoctions were obtained from Bode Market in Ibadan, Oyo State, Nigeria.
Preparation of Anti-malaria and Anti-jaundice concoctions
The concoctions were prepared according to the traditional use in Nigeria. The anti-malaria concoction was
made up of mango leaves, pawpaw leaves, cashew leaves, bitter leaves, neem bark and morinda lucida root
while anti-jaundice concoction was made up of lemon grass, lime leaves, yellow pawpaw leaves and pods of
capsicum leaves. The leaves and barks were rinsed with water and heated together in water until the colour of
the water turned brown; the mixture was then filtered to remove particulate matter. The mixture was cooled and
stored in a refrigerator until use.
Allium cepa assay
The outer scales of the onion bulbs and the brownish bottom plate were first removed. The rings of the root
primordial were left intact. The onion bulbs were first sprouted in water to initiate the growth of the onions with
base of the onions touching the water as described by Friskesjo (1987). After 24hours, the best in terms of root
growth were selected. Thirty five onion bulbs were divided into seven groups with five onions in each group.
The onion bulbs were then placed on top of beakers each filled with 10%, 50% and 100% anti-malaria and anti-
Arojojoye et al. (2015) 2(2): 127133
.
jaundice concoctions respectively and fresh tap water for control for 96hours. The beakers were kept in a dark
cupboard and the test samples were changed every 24hours, the root length of each bulb was also measured
daily. The EC50 values (effective concentration producing 50% growth inhibition) for the two concoctions were
determined by plotting a graph of concentration against root length. After 48hours, one root tip from each bulb
was harvested for cytological study. The root tips were fixed and macerated in 45% acetic acid-1M HCl (9:1)
solution and heated for 5 min at 50C (Odeigah et al. 1997a). Subsequently, the roots were placed on a slide and
the terminal root tips were cut off and used for further preparation. The rest of the material was removed from
the slide and the excess liquid was sucked up with a piece of blotting paper. Two drops of fresh filtrated 2%
orcein solution was added and mixed with the root tips by stirring and knocking with a blunt stick of stainless
steel. A cover slip was placed on the root cells and allowed to absorb stain for 5-10 min afterwards; the cells
were squashed by placing layers of blotting paper on the cover slip and pressing slightly down with the thumb.
The cover slip was fixed carefully to the slide with nail cortex. The slides were observed under the light
microscope and data on total cells, total dividing cells and cells carrying chromosomal aberrations were taken on
at least 5 slides prepared for each of the different concentration of the extracts and the control. The mitotic index
was calculated as, (No. of dividing cells / Total no. of cells) 100.
Phytochemical screening of the concoctions
The phytochemicals tested for are alkaloids, cardenolides, anthraquinones, saponins and tanins. The presence
or absence of these phytochemicals was carried out using standard methods; dragenduffs standard method for
alkaloid test (Oguyemi 1979), killer-killiani standard procedure for cardenolides test (Trease & Evans 1989),
chloroform or ammonia test for anthraquinones (Trease & Evans 1989), frothing test for saponin (Sofowora
1993), ferric chloride test for tannins (Jigna & Sumitra 2007) and ammonia test for flavonoids (Trease &
Evans1989, Sofowora 1993).
Determination of Heavy metals
The concentration of the following heavy metals: lead, chromium, cadmium, nickel and cobalt were
determined in both anti-malaria and anti-jaundice concoctions using atomic absorption spectrophotometer.
Statistical analysis
Results were expressed as Mean Standard Deviation. Differences between groups were determined. One
way ANOVA was performed using microcal origin software (Drug Discovery & Development magazine 2008)
and p-values < 0.05 were considered significant.
RESULTS
Table 1. Root lengths of Allium cepa exposed to various concentrations of anti-malaria and anti-jaundice concoctions.
Concentration Mean root length after Root length
24hrs 48hrs 72hrs 96hrs % of control
Control 0.20.18 1.30.5 2.80.4 5.80.8 100.0
10% 0.20.08 0.40.1 0.60.2** 0.70.2* 12.1
Anti-malaria
Concoction { 50%
100%
0.30.08
0.30.14
0.40.1*
0.40.1*
0.50.2**
0.50.2**
0.30.1**
0.50.1**
5.2
8.6
10% 0.30.09 0.40.05* 0.50.2** 0.70.1* 12.1
Anti-jaundice
Concoction { 50% 0.30.07
100% 0.10.04
0.30.08*
0.20.05*
0.40.1**
0.20.07**
0.40.1*
0.20.05**
6.9
3.4
Note: Significantly different from control *P<0.05, **P<0.001
There were significant (p<0.05, p<0.001 ) reductions in root lengths of Allium cepa after 24hours, 48hours,
72hours and 96hours exposure to 10%, 50% and 100% of anti-malaria and anti-jaundice concoctions
respectively when compared with control (Table 1), the root lengths of Allium cepa exposed to 10%, 50% and
100% of anti-malaria concoction after 96hours were 12.1%, 5.2% and 8.6% respectively of the root length of
control while the root lengths of Allium cepa exposed to 10%, 50% and 100% of anti-jaundice concoction after
96hours were 12.1%, 6.9% and 3.4% respectively of the root length of control (Fig. 1). The anti-malaria and
anti-jaundice concoctions altered phenotypic indices and also induced chromosomal aberrations in Allium cepa
cells (Table 2). Phytochemicals detected in the anti-malaria and anti-jaundice concoctions are alkaloids, tannins,
Arojojoye et al. (2015) 2(2): 127133
.
cardenolides, saponins and flavonoids (Table 3). Heavy metals detected in the anti-malaria and anti-jaundice
concoctions are cobalt, cadmium and nickel (Table 4).
Figure 1. Growth curve of Allium cepa roots exposed to various concentrations of: A, anti-malaria and B, anti-jaundice
concoction.
Table 2. Phenotypic indices and chromosomal aberrations in Allium cepa exposed to various concentrations of anti-malaria and
anti-jaundice concoctions.
Concentration No. of cells No. of dividing cells Mitotic index Binucleus Attached C-Mitosis
Control 425 56 13.17 - - -
10% 180 5 2.78 4 - 1
Anti-malaria
{ 50%
100%
120
78
-
-
-
-
-
-
-
-
-
-
10% 189 10 5.29 3 1 3
Anti-jaundice
{ 50%
100%
124
106
6
-
4.84
-
3
-
3
-
-
-
Table 3. Phytochemical screening of anti-malaria and anti-jaundice concoctions.

S.No. Phytochemical test Anti-malaria Concoction Anti-jaundice Concoction
1. Alkaloid Positive Positive
2. Cardenolides Positive Positive
3. Anthraquinones Negative Negative
4. Saponins Positive Positive
5. Tannins Negative Positive
6. Flavonoid Positive Positive
Table 4. Heavy metal analysis of anti-malaria and anti-jaundice concoctions.

S.No. Heavy metals Anti-malaria Concoction Anti-jaundice Concoction
1. Chromium ND ND
2. Lead ND ND
3. Cobalt 0.117 0.116
4. Cadmium 0.083 0.052
5. Nickel 0.232 0.377
Note: ND, Not detected
DISCUSSION
Allium cepa assay is a simple, cheap, reproducible and effective model for evaluating and monitoring
cytotoxicity and genotoxicity of chemicals and mixture substances (Fiskesjo 1993, 1997, Hoshina & Marin-
Morales 2009).
The results of this study showed that the concoctions significantly (p<0.05, p<0.001) inhibited root growth
in Allium cepa when compared with control. The anti-malaria concoction inhibited root growth with an EC50
value of 5% while anti-jaundice concoction inhibited root growth with EC50 value of 6%. This is an indication
that the concoctions are cytotoxic. The cytotoxic effects of some medicinal herbs such as Azadirachta indica,
Carica papaya, Cymbopogon citratus, , Mangifera indica, Ocimum gratissimum, Morinda lucida , Terapluera
Arojojoye et al. (2015) 2(2): 127133
.
tetraptera, Plumbago zeylanica, Xylopia aethiopica, Newbouldia laevis, Alstonia boonei, Enantia chlorantha
and Rauvolfia vomitoria have been reported as well (Oyedare et al. 2009, Akintonwa et al. 2009, Akinboro &
Bakare 2007). Root growth in Allium cepa like other plants is due to expansion of cells in the elongation zone of
the root tip where cellular differentiation occurs (Cordoba-Pedrosa et al. 2004). The observed inhibition of root
growth by the concoctions suggests that they contain toxic substances that impaired one or more other biological
processes which mediate cell expansion and differentiation at the elongation region of Allium cepa root tip.
Heavy metals such as lead, chromium, cobalt, cadmium and nickel detected in the concoctions might be
responsible for this effect.
The results of this study also show that the concoctions possess inhibitory, mito-depressive effects on cell
division and chromosome behaviour of Allium cepa which would have prevented DNA synthesis that resulted in
reduction in the number of dividing cells. The two concoctions caused reduction in mitotic index. The mitotic
index in Allium cepa exposed to 10% anti-malaria concoction was 2.78 compared with 13.17 of control; while
the mitotic index in those exposed to 10% and 50% anti-jaundice concoction were 5.29 and 4.89 respectively
compared with 13.17 of control. The decline of mitotic index below 22% in comparison to control can have
lethal impact on the organism (Antonsiewiez 1990). Reduction in the mitotic activity could be as a result of
inhibition of DNA synthesis or blocking of the G2 phase of the cell cycle, preventing the cell from entering
mitosis (Sudhakar 2001). There were no dividing cells in Allium cepa exposed to 50% and 100% anti-malaria
concoction and those exposed to 100% anti-jaundice concoction. This shows that the concoctions totally
inhibited cell division at these concentrations which is a sign of blockage of the cell cycle at the interphase
stage. Mitotic index is a measure of the total number of dividing cells in cell cycle; it gives a measure of the
proportion of cells in the mitotic phase of the cell cycle and its inhibition could be interpreted as cellular death
or a delay in the cell proliferation kinetics (Rojas 1993). Therefore, the decrease in the mitotic index of Allium
cepa cells exposed to anti-malaria and anti-jaundice concoctions shows that exposure of Allium cepa cells to the
concoctions resulted in cellular death. This shows direct genotoxic effect of these concoctions. The reduction of
the mitotic index might be as a result of the obstruction of the onset of prophase, the arrest of one or more
mitotic phases, or the slowing of the rate of cell progression through mitosis. The presence of alkaloids, tannins,
cardenolides, saponins and flavonoids in the concoctions might be responsible for their mitodepressive effects.
This is similar to the reports of Bernice et al. (2009) who also attributed the mitodepressive effects of Citrus
medica, Ocimum Gratissimum and Morinda lucida to the presence of alkaloids, tannins, flavonoids,
anthraquinones and saponoside. The mitodepressive effects of some plant extracts and their ability to block of
DNA synthesis have also been reported (Akinboro & Bakare 2007, Soliman, 2001, Askn & Aslanturk 2007,
Mercykutty & Stephen 1980, Schulze & Kirscher 1996).
Chromosomal aberrations are changes in chromosome structure resulting from a break or exchange of
chromosomal material. Chromosome aberrations observed in Allium cepa exposed to the two concoctions are
vagrant chromosomes, binucleus, attached and c-mitosis. These aberrations show that the concoctions affected
spindle formation which resulted in cell division disturbances. Vagrant chromosomes increase the risk for
aneuploidy (Leme & Marin-Morales 2009). Vagrant chromosomes may be caused by unequal distribution of
chromosomes with paired chromatids which could have resulted from non-disjunction of chromatids in
anaphase. Vagrant chromosomes and C-mitosis are usually attributed to the failure of the spindle apparatus to
organize and function in a normal way. Although c-mitosis may be regarded as indicative of a weak toxic effect
which may be reversible. However c-mitosis may induce the formation of polyploid cells when not reversed
(Odeigah et al. 1997b). Binucleated cells usually arise as a consequence of the inhibition of cell plate formation,
which could be as a result of the suppression of phragmoplast formation in the early telophase (Soliman 2001).
Induction of binucleated cells consequently leads to the formation of multinucleate cells in succeeding
generations (Oyedare et al. 2009). Phytochemical screening of anti-malaria and anti-jaundice concoctions
revealed the presence of alkaloids, tannins, cardenolides, saponins and flavonoids. These phytochemicals,
especially alkaloids may be responsible for chromosomal aberrations induced by the concoctions. Ene &
Osuala (1990) reported that alkaloids such as vincristins and vinblastine were responsible for chromosome
aberration observed in the water extracts of Borreria filiformis and Vinca rosea. Alkaloids inhibit mitosis by
binding with tubulin and prevent the formation of the mitotic spindle (Khakdan & Piri 2012). Maruta et al.
(1995) reported that Arctium lappa roots contained various alkaloids, tannins, saponins, anthraquinones and
cardiac glycosides and Khakdan & Piri (2012) also reported that spindle disturbances observed in Allium cepas
Arojojoye et al. (2015) 2(2): 127133
.
exposed to Allium lappa root extracts were due to the presence of alkaloids in the extracts. Oyedare et al. (2009)
also attributed chromosomal aberration (binucleated cells) observed in the cells of Allium cepa treated with an
aqueous extract of Ocimum gratissimum to the presence of alkaloids in the extract. Akinboro & Bakare (2007)
and Williams & Omoh (1996) also reported that the flavonoids (quercetin, rutin and kaempferol), tannins and
other phenolic compounds (hydrocinnamic acid) play roles in mitodepression and chromosomal aberrations in
Allium cepa exposed to extracts of Inula viscosa, Morinda Lucida, Carica papaya and Ocimum gratissimum.
Studies have shown that both tannins and flavonoids evoke free radical generation by complexing with cellular
proteins and DNA, which at the expense of a depleted antioxidant system could cause lipid peroxidation, DNA
damage and cell death (Iwalokun et al. 2011). However, the ability of tannins and flavonoids to induce
genotoxicity is based on their chemical composition and structure since some species of tannins and flavonoids
lack genotoxic effects. Heavy metals in the concoctions could also be responsible for the genotoxic effects of
the concoctions. Various heavy metals are known to induce chromosome breaks, fragments and micronucleus
formation in plants and mammalian test systems (Knasmuller et al. 1998), and their effects were emphasized to
be the result of formation of DNADNA and DNAprotein cross-links (De Flora et al. 1990, Costa 1991, Costa
et al. 1994). Chromium and nickel have been reported to affect the mitotic spindles thereby causing aberrant
mitotic stages (Anderson 1985).
CONCLUSION
The results of this study show the cytotoxic and genotoxic effects of the anti-malaria and anti-jaundice
concoctions on Allium cepa. Heavy metals and some phytochemicals detected in these concoctions as well as
other undetected toxicants in the concoctions could be responsible for these effects. Therefore, the use of these
concoctions in herbal medicine should be discouraged. However, there is need for evaluation of the genotoxic
effects of these concoctions in experimental animal models.
ACKNOWLEDGEMENTS
The Authors acknowledge the assistance of the laboratory technologists of Lead City University during the
practical aspect of this research.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(2): 134142, 2015
Research article
Effect of fluoride contaminated irrigation water on Eco-physiology,

biomass and yield in Gossypium hirsutum L.
Sanjay Kumar1 and Munna Singh2*
1
Department of Botany, University of Lucknow, Lucknow, Uttar Pradesh-226007, India
2
Chandra Shekhar Azad University of Agriculture & Technology Nawabganj, Kanpur,
Uttar Pradesh-208002, India
*Corresponding Author: sk.botanylkouniv@gmail.com [Accepted: 15 August 2015]
Abstract: The study was conducted to assess the response of a cash crop Gossypium hirsutum L.
to the irrigation of fluoride (F) contaminated water. Different concentrations of F i.e. 100, 200, 500
and 1000 ppm were used to study the effects of these concentrations of this element on CO2
assimilation, stomatal conductance, chlorophyll fluorescence, biomass and yield of G. hirsutum.
Photosynthesis (PN), stomatal conductance (gs), chlorophyll fluorescence (Fv/Fm), plant growth
and biomass accumulation in terms of fresh and dry weight were decreased significantly with the
application of F. The results were concentration dependent. However, a significant increase in
harvest index was noticed even at 200 ppm concentration of F. There was no any significant
phytotoxic effect noticed on morphology of plant in terms of necrosis, tip burning and curling of
leaf. Moreover, the parameters studied were decreased but the crop yield was increased slightly
cultivated with the application of F contaminated water, the studied cultivar of this crop may be
recommended for the cultivation in the fluoride contaminated zones.
Keywords: Biomass Production - Photosynthetic CO2 Assimilation - Stomatal Conductance -
Chlorophyll Fluorescence - Harvest Index.
[Cite as: Kumar S & Singh M (2015) Effect of fluoride contaminated irrigation water on Eco-physiology,
biomass and yield in Gossypium hirsutum L. Tropical Plant Research 2(2): 134142]
INTRODUCTION
Fluoride (F) occurs naturally in the earths crust in a very minute amount, but frequently acts as an
environmental pollutant (Jacobson et al. 1966). It is a common phytotoxicant, frequently present in air, water
and soil. It has been commonly released to the environment by a number of anthropogenic activities (EPA 1978,
Kamaluddin & Zwiazek 2003). Fluoride has been reported to cause dental and skeletal fluorosis in animals
(Raju et al. 2009). In case of plant certain physiological processes are known to be markedly affected by F e.g.
decreased plant growth (Elloumi et al. 2005, Jacobson et al. 1966 ), chlorosis (Mcnulty & Newman 1961),
leaf tip burn and necrosis Hadujue (1966). The importance of seed germination is widely recognized and its
study has been used as a model for investigating F toxicity by various authors (Elloumi et al. 2005, Gulzar
& Khan 2001, Gupta et al. 2009, Rubio-Casal et al. 2003, Wang et al. 1991, Wilde & Yu 1998).
Fluoride contamination is normally occurred in ground water due to the weathering of parental rocks such as
fluorapatite (Ca5(Po4)3F), Fluorite (CaF2), Cryolite (Na3AlF6), and fluorophlogopite etc. (Kabata-Pendias &
Pendias 1984, Elrashidi & Lindsay 1986, Fuge & Andrews 1988). Ground water is the main source of irrigation
for the crop production. Application of fluoride contaminated ground water for irrigation is widespread in many
fluoride contaminated areas which can affect growth and productivity of the crops. A number of studies have
been reported to evaluate the effects of fluoride on growth, productivity, yield and tolerance of plants (Elloumi
et al. 2005, Datta et al. 2012, Gadi et al. 2012, Singh & Verma 2013). Many workers have reported the effect of
F on various cereal crops and vegetables using concentration up to 200 ppm. Owing to high adaptability to arid
and semi-arid area climatic conditions and because of its high commercial value and widening market of its
product, G. hirsutum is receiving increase agricultural attention in North India. There is no any study has been
conducted on this plant as per our review literature. In the present study, the efforts have been made to evaluate
the efficacy of G. hirsutum towards the application of fluoride contaminated water used as irrigation. Moreover,
Kumar & Singh (2015) 2(2): 134142
.
the effects of fluoride on photosynthesis, growth, biomass and yield of cotton (G. hirsutum) crop have also been
studied.

Plant material
Cultivars PUSA-S-6 of Gossypium hirsutum was collected from Krishi Vigyan Kendra, Bulandshahr of
Sardar Vallabh Bhai Patel University of Agriculture Technology, Meerut (U.P.), India. The cotton seeds are
grown in soil bed and the next 10 days the germination pattern was observed. On 11 th days of germination, the
saplings were transferred to earthen pots having size 12" filled with 8 kg of soil having 3:1 ratio of soil and
compost respectively. The saplings were provided with adequate amount of water and natural condition for next
30 days to get established. Now, the treatment was continued for next 8090 days and consequently, the
observations were made. The 40 days old plants are exposed to Fluoride treatments viz., 100, 200, 500 and 1000
ppm of fluoride contaminated water upto field capacity (1 liter) once a week for six weeks. The control
seedlings were raised identically except without F in the irrigation water. Ten independent replications were
made for each treatment.
Measurement of physiological responses
The infrared gas analyzer (IRGA-6400, portable photosynthesis system, USA) was used for monitoring
photosynthetic CO2 assimilation (PN), stomatal conductance (gs), and chlorophyll fluorescence yield (Fv/Fm) in
fluoride irrigated cotton plants. These 84-day-old plants were used to obtain all physiological responses. Fv/Fm
was measured using plant efficiency analyzer (PEA, Hansatech, UK) to assess the functionality of PS II to
establish correlation with photosynthetic CO2 assimilation.
Electrolyte leakage
Measurement of membrane leakage in leaf tissues was made by following Valentoric et al. (2006). The
central portions of the leaf were cut into pieces of 1 cm, and 1 gm of these from each treatment was placed in
test tubes containing 15 ml of deionized water. The tubes were then tightly capped and shaken for 24 hr at 25
30 C. The electrical conductivity (EC1) of the solution was measured with an electrical conductivity meter (HI
8733, Hanna Instruments Inc., Woonsocket, USA). Afterward, the samples were autoclaved at 120C for 20 min
and allowed to cool (25C) to record the electrical conductivity (EC2) of the solution. The electrolyte leakage is
expressed as,
EC1
EL (%) 100
EC 2
Shoot and root length, total leaf area and number
The leaf area expansion (cm2), number and shoot and root length (cm) were measured after 50 days of F
treatment. The leaf area was calculated by the paper-weight method with the help of a calibration curve. The
harvest index was calculated by harvesting at least 10 seedlings of each treatment.
Fresh and Dry mass
The plants were removed from pots on 90 day and dipped in water filled bucket. The roots were washed
carefully to remove the adhering soil particles. Fresh weight of roots and shoots was taken by digital single pan
balance. The same plants parts were then placed in an oven at 70C till the weight become constant. The dried
plant parts were weighed to record the dry mass of roots and shoots.
Average biomass production
The average fresh and dry biomass production/day/plant was measured as follows,
Average biomass production (g/plant/day) Biomass of 90 days old plant/90
Harvest index
Harvest index (HI) was calculated as,
Economic yield (seed cotton dry weight)
HI
Biological yield (leaf stem dry weight)
Data (n=5) were analyzed by statistically by one way analysis of variance (SPSS, Statistical package and MS
Excel) using Duncans Multiple Range Tests (DMRT) to determine the significant differences among treatments
at probability (p) 0.05.
Kumar & Singh (2015) 2(2): 134142
.
RESULTS
Physiological responses
The effect of fluoride contaminated irrigation water on photosynthetic CO2 assimilation (PN), stomatal
conductance (gs) and chlorophyll fluorescence (Fv/Fm) variable yield per maximum yield was shown in figure
1(A, B & C) which was found to be decreased with increase the concentration of F in irrigatiuon water. The
decrease in PN, gs and Fv/fm was ~738, 838 and 427% respectively on the exposure of 1001000ppm F as
compare to their respective controls (Fig. 1. D, E & F).
Figure 1. Effect of fluoride contaminated irrigation water on photosynthetic CO2 assimilation (A), stomatal conductance (B)
and chlorophyll fluorescence (Fv/Fm) variable per maximum yield (C) were as (D), (E) and (F) are the respective loss
percentage. Observations were recorded with 4th leaf throughout by using 50 days old G. hirsutum plants after fluoride
contaminated irrigation (n=6, weekly-1). Each data point represents mean (n=5) with S.E.
Electrolyte leakage (EC)

Under the influence of fluoride toxicity the electrolyte leakage (EC) of leaf was affected very drastically. As
the concentration of fluoride increased, the monotonic increase in EC was obsrved with significatdifferenceat
p=0.05 (Table 1). The percent incecrease in EC at 100 ppm F concentratoin was 19%. Simillarly, the EC was
increased 31, 51 and 67% on the exposure of 200, 500 and 1000 ppm F respectively.
Kumar & Singh (2015) 2(2): 134142
.
Table 1. Effect of fluoride contaminated irrigation water on electrolyte leakage in cotton cultivar
PUSA-S6. The observations were made after terminating the F treatment (n=6, weekly -1). Each data
point represents mean (n=5) with S.E. ().
Leaf EC (%) Leaf EC (% increase)
Treatments (ppm)
PUSA-S6 PUSA-S6
0 0 -
100 19 35.71
200 31 121.43
500 51 264.29
1000 63 350.00
Note: F value at p=0.05 212.486
Shoot-root length, total leaf area and number

The phytotoxic effect of fluoride contaminated irrigation water upto 1000ppm was observed in the form of
reduction in leaf area expansion (50% as compare to the control), leaf number (48.43%) and plant height.
Interestingly, number of cotton ball was found highest in plants treated with 200 ppm F with improved harvest
index (0.92). The cotton plant was found to be tolerant towards the exposure of fluoride in terms of yield of the
crop. The effect of fluoride contaminated water irrigation on shoot and root length of cotton ciltivar (PUSA-S6)
was represented in figure 2 (A & B). A finding of morphological (growth) experiment showed a decreasing
trend in root length and shoots length with increasing sodium fluoride concentration. At 100 ppm F
concentration, root and shoot length were ~4% less than that of respective controls. At 200ppm fluoride
concentration, root and shoot length was decreased 12 and 18% respectively than of the respective controls.
Similarly, at 500 and 1000 ppm F concentration root and shoot length was decreased very significantly (p0.05)
as shown in figure 2 (C & D).
Figure 2. Effect of fluoride contaminated irrigation water on growth characteristics in G. hirsutum. The irrigation of
contaminated water of various fluoride levels (n=6, weekly-1). Each data point represents mean (n=5) with S.E. ().
Kumar & Singh (2015) 2(2): 134142
.
Fresh and Dry mass
The biomass of G. hirsutum was negetively correlated with the F treatments i.e., significant decrease in
shoot fresh and dry weight was observed. The decrease was reported to be maximum at 1000 ppm concentration
of F as compared with their control. Decrease in stem fresh and dry mass at 1000 ppm was found to be 60.43
and 43.38% were as in root it was 61.70 and 73.18% (Fig. 3AD) with respect to their coresponding controls.
30 16
A C
14
25
Stem dry weight (g)

Stem fresh weight (g)
12
20
10
15 8
6
10
4
5
2
0 0
12 4.5
B D
4
10
3.5
Root fresh weight (g)
Root dry weight (g)
8 3
2.5
6
2
4 1.5
1
2
0.5
0 0
0 100 200 500 1000 0 100 200 500 1000
Fluoride concentrations (ppm) Fluoride concentrations (ppm)
Figure 3. Effect of fluoride contaminated irrigation water on shoot and root fresh and dry mass of G. hirsutum. The
irrigation of contaminated water of various fluoride levels (n=6, weekly -1). Each data point represents mean (n=5) with S.E.
().
Average biomass production

The average biomass production per day per plant was found to be decreased with incease the F
concentrations (Table 2). The shoot average biomass production per day per plant was recorded to be 0.153,
0.138, 0.081 and 0.061 (g) with respect to the control, 100, 200, 500 and 1000 ppm respectively. The root
average biomass production per day per plant was also found to be decreased under the infuence of increased F
concentrations.
Table 2. Effect of fluoride contaminated irrigation water on shoot and root average dry mass accumulation of
cotton cultivar PUSA-S6. The irrigation of contaminated water of various fluoride levels (n=6, weekly -1).
Each data point represents mean (n=5) with S.E. ().
Shoots DW/day/plant (mg) Roots DW/day/plant (mg)
Treatments
PUSA-S6 PUSA-S6
0 0.153 0.0056a 0.041 0.0005a
100 0.138 0.0052b 0.037 0.0023b
200 0.102 0.0023c 0.029 0.0023c
500 0.081 0.0032d 0.021 0.0023d
1000 0.061 0.0017e 0.012 0.0017e
F value at p=0.05 100.193 35.948
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.
Harvest index
The harvest index of F irrigated plants favoured up-regulation (23%) for expressing plant productivity
particularly economic yield (cotton/fibre-seed). The intrinsic physiological and biochemical membrane
associated characteristics are yet to be revealed to diagnose possible logic of up-regulated plant productivity as
influenced by fluoride contaminated irrigation levels (Fig. 4). The F toxicity affects the plant performance by
reducing the shoot and root length. The harvest index showed the decreasing trend with increasing concentration
of F.
1 a a a
Harvest index (gg-1)
a
0.8 b
0.6
0.4
0.2
0
c 100 200 500 1000
Fluoride concentration (ppm)
Figure 4. Effect of F contaminated water irrigation on harvest index in G. hirsutum. Each data point represents mean (n=6)
with S.E.
The morphological and growth pattern of the plant irrigated with F contaminated water is depicted in figure
5. The plants showed decrease in height, leaf number and leaf area. It may be due to the phytotoxic effect of F.
At 100 ppm F concentration, the plant height was 8.22% less than that of control. At 200 ppm it was 16.43%
were as at 500 ppm it was 24.65% and finaly at 1000 ppm the loss in plant height was measured 32.88% than
that of control. Here the plants are arranged in order of fluoride treatment, i.e. C, 100, 200, 500 and 1000 ppm.
Figure 5. Effect of fluoride contaminated irrigation water on morphology of G. hirsutum plants. The photographs were
obained after terminating the treatment (50 days after application of fluoride contaminated water, n=6, weekly -1). The control
plants (C) were irrigated with normal tap water with treatments viz., 100, 200, 500 and 1000 ppm.
Kumar & Singh (2015) 2(2): 134142
.
DISCUSSION
Fluoride is a common pollutant in water and soil that inhibits seed germination, causes ultra-structural
malformation of chloroplast and mitochondria and alters membrane permeability with impaired photosynthetic
efficiency thereby affecting plant growth, productivity and biomass (Rubio-Casal et al. 2003, Elloumi et al.
2005, Sabal et al. 2006, Gupta et al. 2009, Gautam & Bhardwaj 2010, Datta et al. 2012, Singh & Verma 2013).
Due to bioaccumulation of F in the roots of the plants Zhang et al. (2013), it enters into the most efficient
photosynthetic zone, i.e. mesophyll cells, thereby resulting in loss of chlorophyll fluorescence yield (Fv/Fm) of
PS II. The reduction in photosynthetic CO2 assimilation is correlated with impaired CO2 diffusion (Karolewski
et al. 2000, Walna et al. 2007) and light harvesting chlorophyll pigments (Kamaluddin & Zwiazek, 2003, Pant
et al. 2008, Chakrabarti et al. 2013). It is also reported that NaF, presumebly block the dephosphorylation of
membrane protein (particularly the chlorophyll a/b protein complex) which is phosphorylated by light-activated
kinase. Thus fluoride facilitates the attainment of state 2 by interfering in state 1 to 2 transition Canaani et al.
(1984). And also the change in the PS-I / PS-II ratio have mostly assosiated with the rearrangment of
chlorophyll proein complexes in side thalakoid membrane Garstka et al. (2005).
The fluoride stress affects the plant physiology and growth adversely with increase in concentration of F
contamination irrigation water i.e. 100, 200, 500 and 1000ppm, the toxicity level increases which results the
decrease in biomass content of the plant (Fig. 2, 3). The fluoride affects photosynthesis by affecting the
membrane permeabilityintegrity and associated enzymes of Calvin cycle essential for carboxylation of
atmospheric CO2 in chloroplast (Garrec et al. 1981, Gautam & Bhardwaj 2010). The higher values of the
fluoride-contaminated irrigation water have also impaired physiological responses, i.e., PN, gs and Fv/Fm (Fig.
1) and also affected plant performance, productivity and biomass (Qingtao et al. 2002, Gautam et al. 2010,
Verma 2011, Singh & Verma 2013). The cumulative effect of above resulted in the reduction of leaf area
expansion and leaf number with depressed chlorophyll biosynthesis and its organization in the younger leaves.
With increased F concentration the leaf area expansion, leaf number was monotonically reduced. Thus, loss in
leaf area (Kamaluddin & Zwiazek 2003, Doley 1986, 2010) chlorosis and necrosis reduced canopy
photosynthesis (Doley 2012).
Under stress condition plants membranes are subjected to changes such as increase in permeability and loss
of integrity Blokhina et al. (2003). The degree of cell membrane injury induced by stress may be easily
estimated through measurements of electrolyte leakage from the cells (Bajji et al. 2002). Higher electrolyte
leakage indicates lower membrane stability index of plant cell. At lower F concentration, less electrolyte leakage
was observed. Hence, an increased electrolyte leakage with enhanced exposure to F, thereby apparently
impairing structural integrity of the biomembrane with loss in photosynthetic CO 2 assimilation (Zwiazek &
Skay 1988a). From the following it can be concluded that fluoride concentrations were inversly proportional to
the membrane intigity.
Fluoride causes reduction in root and shoots length due to unbalanced nutrient uptake by seedlings in
presence of fluoride (Sabal et al. 2006). Reduction in root length and shoot length has also been reported
by various researchers in Pisum sativum (Sabal et al. 2006, Prachi 2012), Cluster bean (Sabal et al. 2006).
Brassica juncea (Pant et al. 2008), Oryza sativa (Elluomi et al. 2005), Triticum aestivum (Bhargava &
Bhardwaj 2010) and Cicer arietinum (Dutta et al. 2012).
Fluoride causes reduction in root length and shoot length due to unbalanced nutrient uptake by seedlings in
presence of fluoride (Sabal et al. 2006). Fresh weight and dry weight decreased monotonically with increasing
fluoride concentration due to reduction of metabolic activity in presence of fluoride because germination is a
one kind of metabolism and fluoride acts as a metabolic inhibitor (Gulzar & Khan 2001, Gupta et al. 2009,
Sabal et al. 2006).
A significant elevation in activities of peroxidase, catalase, and ascorbic acid oxidase is also found to be
dependent on severity and duration of fluoride exposure (Karolewski et al. 2000, Walna et al. 2007, Kumar et
al. 2009) which may affect the quality and quantity of the plant produce. Thus, as seen in figure 1, 2, 3 and 5,
after F irrigation, loss in leaf area-expansion, leaf number, shoot length, chlorophyll, PS II efficiency, stomatal
conductance, and CO2 assimilation occurred with enhanced electrolyte leakage in the leaf cells, leading to a
reduction in harvest index (HI).
Kumar & Singh (2015) 2(2): 134142
.
CONCLUSION
Fluoride affect adversely to both animals and plants. But in this research it was found that, the cotton can
resist fluoride up to 200ppm in pot culture because the harvest index was slightly increased at this concentration.
Therefore, the cotton cultivation may be extended in F-affected (~200 ppm F) agro-climatic zones to sustain the
agro-socio economy.
ACKNOWLEDGEMENTS
This research was made possible by support from the University Grant Commission (UGC), New Delhi, by
an award of Rajiv Gandhi National Fellowship (RGNF) to Mr. S. Kumar and it is gratefully acknowledged.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
2(2): 143152, 2015
Review article
Biocomputation assisted data base of plant stress responsive traits:

Current progress and future challenges
Dibyendu Talukdar*
Dept. of Botany, R.P.M. College (University of Calcutta), Uttarpara, Hooghly, West Bengal-712258, India
*Corresponding Author: dibyendutalukdar9@gmail.com [Accepted: 16 August 2015]
Abstract: Plants are sessile organism and therefore, they are continuously exposed to diverse
types of biotic and abiotic stress factors. Understanding of plant stress response mechanisms and
application of knowledge derived from integrated experimental approaches gradually develop
holistic approaches in plant stress tolerance mechanism. The stunning advancements in Omics-
based technologies and information technology steered development of a broadening field of
science, termed bioinformatics or computational biology in which biological phenomena are
meaningfully explained through information technology. Plants respond to environmental stresses
through cascade of events, occurring at cellular, metabolic and molecular level but in fine tune
during tolerance mechanisms. During the last decade, the stress responsive factors have been
extensively studied in model and crop plants, but the global data is highly sparse in different data
bases. Present endeavor, thus, provides a comprehensive review on plant stress responsive
database generated through assistance of biocomputation in different plants. Furthermore, names
of different stress factors and data base links were also provided. The progress achieved, so far and
upcoming challenges of stress responsive data base were also discussed.
Keywords: Computational Biology - Transcription factors - Comparative genomics -
Bioinformatics tools - Arabidopsis thaliana.
[Cite as: Talukdar D (2015) Biocomputation assisted data base of plant stress responsive traits: Current progress
and future challenges. Tropical Plant Research 2(2): 143153]
INTRODUCTION
Plants are sessile organisms, and therefore, they have to withstand arrays of both biotic and abiotic stresses.
Stress response is the general term for defining the interaction between plants and the extreme environmental
conditions. The term Stress is difficult to define. However, it is a type of metabolic derailment which occurred
due to cellular and metabolic modulations in plant homeostasis (Kumar et al. 2014). Plants have developed
integrated and interactive mechanisms to detect precise environmental changes, allowing optimal responses to
adverse conditions (Atkinson & Urwin 2012). Stress response and tolerance towards these stresses are governed
by complex biological pathways and regulatory events involving multiple molecular components. The effects of
abiotic or biotic stress may occur singularly or in combination and induce cellular damage at multiple levels of
plant growth and development, and may induce varying degrees of alterations in cellular, metabolic and
molecular events, ultimately manifested in reduction of growth and development (Chinnusamy et al. 2004).
Prominent crop plants like rice, wheat, maize, grain legumes, vegetables, spices and other economically
important plants like oil-yielding crops, fiber-yielding crops, and plants with medicinal and aromatic values
suffered varying degrees of biotic and abiotic stresses (Mahajan & Tuteja 2005). This complex mechanism is
mediated through distinct signal transduction cascades, which in turn activate stress-responsive genes,
consequently leading to survival by transcriptional reprogramming during plant growth and development (Xiong
& Zhu 2001, Talukdar & Talukdar 2013, 2014). Understanding the molecular pathways and regulatory networks
which influence various facets of stress-responsive events in plants is crucial for developing stress-tolerant or
stress-adaptive varieties of plants. Furthermore, interactions of several biological signaling molecules
particularly, H2O2, nitric oxide and hydrogen sulfide with prominent antioxidant molecules like glutathione and
ascorbate and enzymatic defense components in modulation of stress response traits need to be studied
Received: 09 May 2015 Published online: 31August 2015
Talukdar (2015) 2(2): 143153
.
holistically for development of stress-tolerant crops (Chakrabarty et al. 2009, Talukdar 2011, 2012, 2013a, d,
2015b, Tripathi et al. 2012a, b).
The practice of integrating morphological, physiological, molecular, biochemical and genetic information
has long been applied to diverse fields of plant biological research (Yuan et al. 2008), inducing the origin of
system biology which landmarks the interactions among biological components using models and/or networks
to integrate genes, metabolites, proteins, regulatory elements and other biological components through data
mining, data modeling and visualization, meta-analysis, multivariate analysis, and dynamic as well as static
networks (You 2004, Yuan et al. 2008, Bhardwaj & Somvanshi 2015). Considering the large datasets
continuously generated through different omics technologies such as genomics, proteomics, transcriptomics,
interactomics and metabolomics, there is an urgent need for system integration in plant biology.
Biocomputatioal approaches offer a powerful platform of bioinformatics to gather information by integrating
various public data sets and sensitive prediction algorithms, helping to understand the major cellular and
molecular activities involved in stress response, adaptation and tolerance (Fernandez-Suarez & Birney 2008,
Perez-Rodriguez et al. 2010, Cramer et al. 2011, Kinsella et al. 2011, Sucaet & Deva 2011, Spooner et al. 2012;
Talukdar 2015a). While mathematical methods are based on description and analysis of intra and intercellular
processes and events through systematic mathematical equations, bio computation methods mainly serve for
creation of algorithms to simulate biological processes, and to construct and visualize them. The experimental
techniques employed in systems biology tend to have high-throughput capabilities by the employment of
techniques, such as proteinprotein interactions, transcriptional regulations (proteinDNA interactions) and
genetic interactions. This enables the researchers to determine the abundance or activity of numerous biological
components at the same time (Bhardwaj & Somvanshi 2015). In Arabidopsis thaliana, data base of biochemical
pathway has been developed (Caspi et al. 2010, Mueller et al. 2003), but stress responsive factors are not
distinct. The web-accessible database providing information on plant stress responsive traits at metabolic and
genomic levels proves to be an invaluable resource for crop improvement, particularly when the climate change
phenomena in the backdrop of immense population pressure are looming large. However, in most of the cases
the information is sparse and is not easily accessible. This review, thus, aims to integrate and reveal the current
progress and upcoming challenges of using biocomputational tools in developing data base of plant stress
responsive features.
BIOCOMPUTATION IN PLANT STRESS RESPONSIVENESS: The Arabidopsis and rice model

Genomics and proteomics technologies have revolutionized 21st century plant biology research with the
advent of ultra-high-throughput experimental platforms, optimized assay systems and advanced bioinformatics
approaches (Mochida & Shinozaki 2010). DNA microarray is a high-throughput technology extensively used to
investigate plant model organisms such as Arabidopsis and rice varieties to detect expression levels of multiple
transcripts quantitatively in parallel. On the other hand, tightly regulated protein interaction networks or
interactomics mediate cellular responses to environmental cues and direct the implementation of developmental
programs. The use of mathematical and computer modeling methods allows investigation of processes and
events that are difficult to study even using highly efficient experimental methods (Bhardwaj & Somvanshi
2015, Yuan et al. 2008). The signaling cascade of mitogen-activated protein kinase (MAPK) was analyzed by
biocomputer-based approaches while carbohydrates and N-glycosylation pathways were biocomputerized in
Pichia pastoris (Srivastava et al. 2013). There are few databases that have been designed for stress responsive
genes in plants. Arabidopsis thaliana (thale cress), a member of the Brassicaceae family, has become a widely
used model for the study of plant biology because of its small genome size, short generation time, facile
genetics, and ease of transformation. Since the completion of the genome sequencing in Arabidopsis
(Arabidopsis Genome Initiative 2000) and Oryza sativa L. genomes (Yu et al. 2002, Zhao et al. 2004), several
stress responsive data bases are being developed in both plant species, some of which are given below-
i) Arabidopsis Stress Responsive Gene Database (ASRGDB data base)
The Arabidopsis Stress Responsive Gene Database or ASRGDB database (http://srgdb.bicpu.edu.in/),
developed by School of Life Sciences, Pondicherry University, India as freely web-accessible platform, was
constructed and configured by using typical LAMP (Linux, Apache, MySQL, and PHP) server with data search,
use and retrieval facilities (Borkotoky et al. 2013). Database design and interface has been developed using
PHP and MySQL v. 5.5 whereas BLAST searches were carried out using PERL scripts. It listed around 44 types
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of different stress factors related to Arabidopsis thaliana, and contains 636 gene entries related to stress
response with their related information like gene ID, nucleotide and protein sequences, cross-response, and so
forth. The database is based exclusively on published stress responsive genes associated with the Arabidopsis,
and include BLAST search interface for both nucleotide and proteins. Genomic and proteomic data for the
collected gene have been obtained from the TAIR (The Arabidopsis Information Resources), while gene
expression data were obtained from Genevestigator (Zimmermann et al. 2004) and Arabidopsis Gene Family
Profiler (aGFP) (Duplakova et al. 2007). The database is freely web-accessible.
ii) STIFDB and STIFDB2 data base
The mechanism of stress-responsiveness in plants involves complex regulation of multiple genes and
transcription factors. About 10 specific families of transcription factors were found involved in A. thaliana
while six specific families of transcription factors were known to be participated in rice responding to diverse
abiotic stresses. Notable families involved are ABI3/VP1, AP2/EREBP, ARF, BHLH/myc, bZIP, HSF, MYB,
NAC, and WRKY for salinity, drought, abscisic acid, cold, excess light, and oxidative stress. STIFDB or the
Stress-responsive Transcription factor database is a database that curated information of stress-responsive genes
and transcription factor binding sites for abiotic stress-responsive genes in A. thaliana. STIFDB was developed
on a MySQL backend and the web interface was developed using HTML and JavaScript. Perl-CGI programs for
searching putative binding sites and for performing STIF prediction were used for the development of search,
query and retrieval system. Stress profiles have been created for each gene that indicates the associated stress
signals. Gene descriptions from TAIR (Lamesch et al. 2012), Rice Annotation Project (RAP) database (Ouynag
et al. 2007), rice DRTF (transcription factors) (Gao et al. 2006) and transcription factor-related information
from Database of Arabidopsis Transcription Factors (DATF) (Guo et al. 2005) were also added. The STIFDB
provides features like, 1. TFmap-a graphical representation of the upstream regions of the stress genes in
Arabidopsis thaliana with the predicted and the validated transcription factor binding sites marked along with
their Z-Scores; 2. TAIR ID; 3. Gene Ontology- Obtained from TAIR, gene ontology annotations are to help the
users to understand the known functional associations of genes in STIFDB; 4. Gene description- a short
description of genes along with predicted domain associations from InterPro database; 5. Gene names- using
standard gene names or its aliases reported in TAIR, users can access STIFDB; 6. Chromosome position-Users
can detect exact location of the relevant stress gene among the five A. thaliana chromosomes; 7. Reference-
publications and related resources; 8. Transcription Factor Family Name- Family whose binding site sequence
has been located/predicted on a given promoter sequence; 9. Binding Site Information-it refers to the core
binding sequence to which a transcription factor binds; 10. Orientation of Binding Sites- it refers to the DNA
strand on which the transcription factor-binding site has been located which is either on the forward strand or on
the reverse DNA strand; 11. Stress signals- type of stress, which regulates the transcription factor/s; 12.
Algorithms scores, etc (Shameer et al. 2009). Several studies involving plant stress-responsiveness (Kang et al.
2011, Sanghera et al. 2011, Babitha et al. 2013) and biocomputational approaches (Mishra et al. 2009, Georgii
et al. 2012) on stress-specific gene regulation have utilized the data compiled in STIFDB (Shameer et al. 2009).
The STIFDB2 is the up-dated large compendium of STIFDB, providing robust data platform of plant stress
regulome. New features such as: additional stress signals, new transcription factors and their binding sites,
additional stress-responsive genes from microarray experiments and orthologs recorded from other important
crop plants, such as maize, sorghum and soybean have been added in this data base (Naika et al. 2013).
STIFDB2 provides information on stress-responsive genes from A. thaliana and two rice subspecies (O. sativa
subsp. japonica and O. sativa subsp. indica). A total of 31 stress-responsive transcription factors such as
ABRE_ABI3_VP, AuxRE_ARF, C_ABRE_bZIP, GCC_box_AP2_EREBP, G_box2_bZIP, HSE1_HSF,
Myb_box1_MYB to Myb_box5_MYB, Nac_box_NAC, W_box_WRKY etc. identified for different stress
signals like cold, colddroughtsalt, drought, heat, ABA, aluminum, bacterial blight, high light, iron, NaCl,
osmotic stress, oxidative stress, UV-B and wounding was also compiled (Shameer et al. 2009, Naika et al.
2013). Six families of transcription factors were curated for subsp. japonica whereas three families such as
AP2/EREBP, NAC, and bZIP for salinity stress, ABA, cold and drought stress were catalogued in subsp. indica.
Transcription factor binding site was predicted by transcription factor family/subfamily, cis-element, consensus
binding site data and corresponding references (PubMed identifiers) using stress-responsive genes in rice
species. Using the genomic data mining approach, a data set of 3,150 unique stress responsive genes in A.
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thaliana, 1,118 genes in O. sativa subsp. japonica and 1,716 in O. sativa subsp. indica were identified and
annotated with stress signals. Chromosome location of genes in three taxa was also provided, and can be
browsed on the basis of chromosomal location. While ABA, bacterial blight, cold, drought, heat, iron and NaCl
stress signals were captured for subsp. japonica, cold, drought and NaCl-stress was signaled in subsp. indica
(Naika et al. 2013). STIFDB2 has 38,798 associations of stress signal, stress-responsive gene, transcription
factor binding site, orientation of binding site and z-scores predicted using the STIF (Stress-responsive
Transcription Factor) algorithm (Hidden Markov model). Thus, there are three categories of data compiled in
STIFDB2: A. predicted data, B. curated data which can be extracted, and C. annotation data compiled from
primary data base. Data can be browsed using one of the four key data sets: gene, transcription factors, stress
signals and chromosome, or using a variety of keywords including gene descriptions, stress signals,
transcription factors, Gene Ontology annotations, and nucleotide sequences (BLAST). STIFDB2 can also be
used to investigate the transcriptional regulatory cascade network underlying abiotic stress responses in A.
thaliana (Naika et al. 2013).
iii) The Arabidopsis thaliana salt responsive protein data base
Uniprot (Release 2011_11, http://www.uniprot.org) was used to select 292 A. thaliana salt-response
proteins. The UniProtKB GO annotation program, an integral part of UniProt biocuration, is powered by high-
quality Gene Ontology (GO) annotations to proteins in the UniProt Knowledgebase (UniProtKB). Its subsection
of GOTERM_BP_FAT provides information on the proteins response to various stresses. All expressed proteins
were extracted from Uniprot database to analyze GOTERM_BP_FAT. Subsequently, these proteins with key
word response to salt stress in the description of GOTERM_BP_FAT were referred as salt-response proteins
and their molecular function was classified following GOTERM_MF_FAT (Meili et al. 2012). The data base
harbors the largest number of proteins related to catalytic activity (18.5%), followed by proteins related to signal
transduction (17.5%), related to binding activity (12.0%), and related to ROS scavenging and defense (11.6%)
in relation to salt stress. Furthermore, out of 292 salt-response proteins, 146 were reportedly involved in the
response of cold, drought, and heavy metal stress in A. thaliana. Among them, 67 proteins for cold stress; 71
proteins for drought stress; and 54 proteins responded to heavy metal stress (Meili et al. 2012). Largest number
of proteins was involved in signal transduction, followed by catalytic activity (16.4%), and then ROS
scavenging and defense. Besides Uniprot, the cellular localizations and functions of different salt-response
proteins were further analyzed by Cytoscape v2.8.2, which is an open-source software package, widely used to
integrate and visualize diverse data sets in biology (Shannon et al. 2003). Available information on known
compound-target interactions in the context of a biological network of interest can be used by users to rapidly
identify novel avenues to perturb the system and potentially identify therapeutically relevant targets (Meili et al.
2012).
iv) QlicRice data base
Developed during the year 2011, QlicRice is an online data base and search engine for abiotic stress
responsive QTLs and annotated gene loci (MSU/TIGR) in rice. It is an interactive platform for collecting,
managing and searching particular rice QTLs along with user-friendly web-enabled data mining of genomic
information in rice, particularly in O. sativa subsp. japonica cv. Nipponbare. Rice QTLs can be searched for
abiotic stress, QTL identifier or locus identifier. About 974 abiotic stress related QTLs with 460 overlapping
TIGR/MSU loci were collected in the data base. Data mining for QTLs were performed by using Gene
Ontology and KEGG Orthology and the positions of QTLs have been identified relative to their overlapping
TIGR loci in comprehensively constructed physical and genetic map (Smita et al. 2011). A relevant glossary has
also been provided.
v) Rice Stress Gene Catalog tool
This is a global classification of genes from rice plant genome. This software was developed under the
adobe flex 3 platform while the central database was established by SQLight, and has been used only in the
portal. The MXML (www.adobe.com/) and Action Script 3.0 (www.actionscript.org/) was the major
programming languages. While MXML is used for mainly front end interface designing, Action Script is used
for data base establishment, online resource communication and in all programming logics. The system was
tested on computation node(s) with Linux, Windows XP and MAC platform (Ali et al. 2011). Facilities are
available for analysis of genomic and proteomic data, to find the homology between the sequences, profile
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searching, sequence comparison etc. Well-known packages like Clustal W and T-Coffee for multiple sequence
alignment, and FASTA and BLAST for sequence comparison have also been provided. There are four main
options for using the tool like browsing stress gene entries, search against tool by gene names or protein names,
and insert data and manipulations on FASTA sequence. Details about each stress responsive protein family, for
example, the list of Uniprot IDs, protein names, Gene Ontology terms and key publications for each protein can
be accessed through searching particular protein name against the Uniprot database (Mi et al. 2005). Search can
be facilitated by key words while users can modify it by inserting new data on stress gene/protein families.
Separate windows have been created for sequence alignment and comparison packages which can be run
independently (Ali et al. 2011).
BIOCOMPUTATION IN PLANT STRESS RESPONSIVENESS: The miRNAs

MicroRNAs (miRNAs) are endogenous, single-stranded, small (~2123nt), non-coding, regulatory RNA
molecules that manipulate messenger RNA degradation and translation repression by binding complementary
sites in the protein-encoding, or 3-untranslated regions, of target mRNAs (Bartel 2004, Zhang et al. 2009).
Accumulating evidences suggest that plant miRNAs play critical roles in plant physiology, such as floral organ
identity, leaf morphogenesis, cellular signaling, and stress responses (Lu et al. 2008, Chiou & SI 2011). Over
200 published studies representing 1038 regulatory relationships between 682 miRNAs and 35 different abiotic
stresses in 33 plant species have been reported. However, data from these individual reports has not been
collected into a single database. The lack of a curated database of stress-related miRNAs limits research in this
field, and thus a cohesive database system should necessarily be constructed for data deposit and further
application.
The PASmiR data base
PASmiR is a literature-curated and web-accessible database, developed to provide detail searchable
descriptions of miRNA molecular regulation in different plant abiotic stresses (Zhang et al. 2013). The database
was constructed using freely available and open source frameworks, such as Apache, Java Server Pages (JSP),
MySQL, Struts2, Spring, Hibernate and so on. The MySQL Server stored the curated miRNA-stress regulatory
relationships which are accessible through Structured Query Language (SQL) and web-hosted on Apache
Tomcat server and Java language and JSP scripts were combined to generate web page. Additionally, Hibernate
modules were applied to manipulate data and convert data formats (Zhang et al. 2013). The data base allows
users to browse, search, download, and update the data relevant to miRNA regulation in diverse abiotic stresses
such as heavy metal, salinity, drought, radiation, etc. through key words and/or through search fields like plant
species, abiotic stress, and miRNA. PASmiR also offers a fuzzy search engine, allowing querying of miRNA-
stress regulatory information, and feedback can be submitted. For each of regulatory relationship, detail
information regarding plant species name, type of abiotic stress, a miRNA identifier, change in miRNA
expression pattern under stress, detection method for miRNA expression (e.g. miRNA microarray, qRT-PCR,
northern blot, and deep sequencing, etc.), a reference literature, the target gene(s) of miRNA extracted from the
corresponding reference, and validated (predicted) target gene(s) derived from miRBase were collected (Zhang
et al. 2013). Data provided by PASmiR will facilitate improvements in the understanding of stress-focused
miRNA functional evolutionarily relationship in plant species. It has great potential in applications for
experimental biology aiming to improve crop performance and breeding characteristics. High connectivity for
different stresses in rice can be studied by studying 162 regulatory relationships between 113 miRNAs and 13
abiotic stresses in the crop. The PASmiR database is freely accessible at: http://hi.ustc.edu.cn:8080/PASmiR,
and http://pcsb.ahau.edu.cn:8080/PASmiR
BIOCOMPUTATION IN PLANT STRESS RESPONSIVENESS: Other data bases

i) Plant Stress-Responsive Gene Catalog data base (PSRGC data base)
The Plant Stress-Responsive Gene Catalog or PSRGC is designed and developed by Consultative Group of
International Agricultural Research (CGIAR) in the year 2011. This is a data base of orthologous and
paralogous relationships between different stress responsive genes for water and drought stress in different crop
plants. This data base is housed by NAR (Nucleic Acid Research) Molecular Biology (MB) Database Collection
with an entry number 1116. It has two main categories: 1. Nucleotide sequence data base with a sub-category of
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Transcriptional regulator sites and transcription factors, and 2. Plant data base with sub-category of General
Plant Data Base.
ii) Plant Stress Gene Database (PSGD)
Designed and developed through LAMP technology (Linux, Apache, MySQL, PHP) this data base
(http://ccbb.jnu.ac.in/stressgenes/) include 259 stress-related genes of 11 plant species along with all the
available information about the individual genes in model plant Arabidopsis thaliana, cereals like Oryza sativa,
Hordeum vulgare, Triticum aestivum, Zea mays, and Saccharum officinarum, legumes like Phaseolus vulgaris,
Glycine max, Arachis hypogaea, forage cereals like Pennisetum glaucum and vegetable crop Solanum
lycopersicum. Stress related ESTs were also found for Phaseolus vulgaris. Database also includes ortholog and
paralog of proteins which are coded by stress related genes (Prabha et al. 2011). About 33 genes such as
ABF3/DPBF5 (ABA responsive elements binding factor 3/DNA binding/protein binding), ABI4 (ABA
Insensitive 4), RAB18 (Responsive to ABA 18), CSD1, CSD2 & CSD3 (Cu/Zn Superoxide Dismutase 1, Cu/Zn
Superoxide Dismutase 2 & 3), FsD1, FSD2 and FSD3 (Fe-Superoxide Dismutase 1, 2 & 3), MSD1 (Mn
Superoxide Dismutase 1), DREB1A & DREB2A (Dehydration Response Element B1A & B2A), HOS10 (High
Response to Osmotic Stress 10), HSA32 (Heat Stress Associated 32), LOS4 (Low Expression of osmotically
responsive genes), SEP 1& 2 (stress Enhanced Protein 1, 2), SOS1 ( Salt Overly Sensitive 1) and STRS1 & 2
(Stress Responsive Supressor 1 and 2) have been catalogued in A. thaliana for stress like dehydration, salinity,
Abscisic acid, osmotic stress, and heat stress. Among cereals and millets, about 30 genes each in rice and wheat,
39 genes in Zea mays, 24 in Hordeum, and 03 each in P. glaucum, and Saccharum officinarum have been
catalogued in PSGD data base. Prominent genes involved in stress responsiveness documented here are for
drought/heat/temperature stress (putative receptor kinase, putative 1,4-benzoquinone reductase, auxin-
responsive GH3, Calmodulin-regulated ion channel, heat shock factor type, Superoxide dismutases, temperature
stress-induced lipocalin, WRKY transcription factor 20, 28, 36, & 41, Universal Stress Proteins), Oxidative
injury (Ascorbate peroxidase, Chitinase 1, 2 & 3), osmotic stress (glutathione S-transferase, NADPH oxidase,
LOX11-Lipoxygenase 11, betaine aldehyde dehydrogenase), cold (cold acclimation protein, dehydrin WZY1-2,
WCS120, WRKY transcription factor 20, 28, 29, 36), salt stress (Zinc-finger motif, salt-stress responding,
adenosine kinase, aldolase1 (preferred), aldolase1, ZmL54, gpm362, umc216, umc216 (ald1), ATP synthase
CF1 alpha subunit, beta glucosidase1, GPN1, GAPN, methionine synthase, rbcL, ribosomal protein S4, voltage
dependent anion channel protein), aluminium stress (wali2, 3 & 5 protein, PAL), endogenous hormone (LOC-
548234-alpha-hordothionin precursor, LOXA, LOX1.1), and in all stresses (sulfate transporter ST1). Among
legumes, two stress-related genes coding acetyl-CoA carboxylase carboxyltransferase beta subunit responsive to
wounding, Pseudomonas syringae infection, H2O2, jasmonic acid (JA), ethylene, or auxin treatment, and rps4
(ribosomal protein S4) along with 42 ESTs for drought stress responsiveness have been catalogued for
Phaseolus vulgaris (http://ccbb.jnu.ac.in/stressgenes/Phaseolus. html) while 62 proteins responding to different
abiotic stresses have been curated for soybean (http://ccbb.jnu.ac.in/stressgenes/glycinemax.html). Two genes
coding for DREB2A (DRE-BINDING PROTEIN 2A), DNA binding / transcription activator/ transcription
factor and ACC deaminase/D-cysteine desulfhydrase family protein have been catalogued for water stress and
salinity stress, respectively, in ground nut (http://ccbb.jnu.ac.in/stressgenes/Arachis_hypogaea. html). Among
the vegetables, 31 genes coding for proteins having roles in different stress conditions like salt stress and
potassium and iron deficiency signalling pathways, water-deficit, heavy metal, chilling tolerance, and other
abiotic stress conditions have been documented in tomato (http://ccbb.jnu.ac.in/stressgenes/solanum.html).
Notables proteins catalogued are abscisic stress ripening protein 1 (water-deficit), APX, TAPX (Ascorbate
peroxidase in all stresses), calcium-dependent protein kinase CDPK1, calmodulin-independent protein kinase
(salt and wounding), cat1, CAT2 (catalase-chilling tolerance, H2O2-mediated antioxidant defense), GPX,
phytoene synthetase, and rbcL (Rubisco Large sub-unit) for salt tolerance and other environmental stresses.
iii) Plant Stress Protein Database (PSPDB)
Plant Stress Protein Database (PSPDB) is a web-accessible (http://www.bioclues.org/pspdb/) resource which
covers 2,064 manually curated plant stress proteins catalogued from 134 plant species with 30 different types of
biotic and abiotic stresses. Functional and experimental validation of proteins responsive in biotic and abiotic
stresses has been employed as the sole criterion for inclusion in the database (Kumar et al. 2014). Proteins were
retrieved from UniProt through GO term search for various plant stress-related proteins. The data base is driven
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through MySql v. 5.0, PhP v. 5.2.4, and Perl v. 5.8.8. Seven abiotic and four biotic stress factors have been
curated in this data base which has facilities like multiple catalogue search, integrated tools loke NCBI,
BLAST, ClustalW, NJPLOT, etc, tutorials for search and updated version of proteins. Among abiotic stresses,
drought, salt, flooding, temperature, light, wounding, and oxidative stress are being curated while
phytohormone, antifungal, antibacterial, and antiviral protein factors have been catalogued for biotic stresses.
Notable plant taxa covered in the PSPDB data base are Arabidopsis thaliana, Avicennia marina (Grey
mangrove), Mesembryanthemum crystallinum (common ice plant), cereals/millets like rice, wheat, maize, rye,
sorghum, Avena sativa (oat), and Hordeum vulgare, vegetables such as tomato, potato, carrot, Solanum
cheesmanii (Galapagos island tomato), Spinacea oleracea, Allium cepa, A. sativum, Brassica napus, Benincasa
hispida, Sinapis alba (white mustard), Basella alba, Beta vulgaris, Cucurbita maxima, Cucurbita pepo,
Capsicum annuum, Brassica oleracea, Ipomoea batatas (sweet potato), Momordica charantia, Trichosanthes
anguina, and Cucumis sativus, legumes like Pisum sativum, Vigna radiata var. radiata, Vigna unguiculata var.
sesquipedalis, Vicia faba, Lens culinaris subsp. culinaris, ground nut, chickpea, Medicago sativa (alfalfa),
soybean, Phaseolus vulgaris, and Lotus japonicas, economically/commercially important plant such as
Sesamum indicum, Helianthus annuus, Hevea brasiliensis, Gossypium hirsutum, Vitis vinifera, Nigella sativa,
Citrus sinensis (sweet orange), Ginkgo biloba, Cycas revoluta, Pinus sylvestris, Dianthus caryophyllus,
Passiflora sp, Malva parviflora, Dahlia merckii, Zinnia violacea, Catharanthus roseus, Petunia hybrida,
Artemisia annua (sweet wormwood), Bryonia dioica, Asparagus officinalis, Calotropis procera, and Nicotiana
tabacum.
iv) PESTD data base
With 175976 entries, this data base was developed by International Crops Research Institute for the Semi-
Arid Tropics (ICRISAT), Hyderabad, India in 2005. This data base is curating transcripts with annotated
tentative orthologs from crop abiotic stress transcripts. ESTs from stress cDNA libraries across 16 crop species
including 6 important cereal crops (rice, wheat, maize, barley, sorghum, rye), 6 legumes (common bean,
soybean, cowpea, groundnut, chickpea, Medicago) and other dicots including Arabidopsis were systematically
collated and subjected to bioinformatics analysis such as clustering, grouping of tentative orthologous sets,
identification of protein motifs/patterns in the predicted protein sequence, and annotation with stress conditions,
tissue/library source and putative function (Balaji et al. 2006). With permission, this data base is available at
http://intranet.icrisat.org/gt1/tog/homepage.htm
v) GCP- comparative plant stress-responsive gene catalogue
Comparative biology highlights/predicts the evolution of gene families and biological processes, thus
providing valuable insights into organismal function and evolution (Koonin 2005, Brown & Sjolander 2006).
Accurate predictions of orthologous and paralogous relationships are essential in order to cross-reference genes
from one species to other related species. The Generation Challenge Programme (GCP: www.generationcp.org)
is a global crop research consortium of CGIAR. In order to enhance efforts in plant breeding for plant stress
tolerance, the consortium is relentlessly striving on comparative genomics and molecular analysis to plant
genetic resources. Primary goal of the project is to assemble tools for the compilation and visualization of
comparative information about stress-responsive genes (Wanchana et al. 2008). After Margaret Dayhoff, the
famous early pioneer in comparative analysis of sequences, the project was code-named as Dayhoff and hosted
on http://dayhoff.generationcp.org:8080/DayhoffWeb2/. Dayhoff is a MySQL database, designed to guide the
bioinformatics analysis and interpretation of research outputs obtained from comparative genomics experiments
with add-inns options to store protein family information such as protein multiple sequence alignments (MSA),
phylogenetic trees and supported stress evidence from experiments and the literature (Wanchana et al. 2008).
The web interface uses GCP- Java-based software technology (http://pantheon.generationcp.org) connected to
external tools like BLAST software and other GCP-funded comparative gene analysis resource called
GreenPhyl (http://greenphyl.cirad.fr/cgi-bin/greenphyl.cgi) for analysing and viewing the querys results related
to comparative genomics on plant stress responsive genes (Wanchana et al. 2008). The core data set in Dayhoff
consists of stress-related protein families characterized by a phylogenomic inference approach (Sjolander 2004).
Phylogenetic tree in the data base was constructed by 1) retrieving the homologous sequences for each stress
protein from Uniprot data base by using the FlowerPower tool (Glanville et al. 2007), 2) MSAs of homologous
proteins were constructed with the high-accuracy MSA program, MUSCLE v. 3.52 (Edgar 2004), and lastly, (3)
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functional subfamilies were identified for each group using the SCI-PHY web server (Glanville et al. 2007). The
data base has three main options for search: browsing protein families, query database by gene names or protein
names and BLAST search against protein families. Users can browse the entire set of stress-related protein
families by selecting the database from the main drop-down menu. The front page comprised of a list of protein
families, links for phylogenetic trees and MSA. Family ID links have been created for details about each protein
family, for example, the list of Uniprot IDs, protein names, Gene Ontology (GO) terms and key publications for
each Protein. Additional information is available from the drop-down list. For query, users can retrieve the
pertinent information by searching the database by keywords within Family name and Protein name. For
BLAST protein families, users can submit a protein or DNA sequence in Fasta or raw format in order to BLAST
in the Dayhoff database, interconnected with GreenPhyl database through GCP-compliant BioMOBY
(Wilkinson et al. 2005) client web service.
FUTURE CHALLENGES
Following recent advances in technology and the development of ultra-high-throughput technology, the field
of plant science is beginning to suffer from data overload. The biggest problems during constructing of genome-
scale models of biological systems are acquisition of over-crowded and noisy data which is an inherent property
of the high-throughput techniques. False-positive interactions in data sets due to different experimental or in
silico approaches are one of the main reasons behind this noise. The huge amount of information published daily
for any of the model or crop plant makes it almost impossible to store, classify and integrate the massive data in
biologically interpretative way. Although bioinformatics tools are extensively used to integrate these data into
meaningful data base/data sets, in many cases problems like false homology inference occurs. Besides, there are
some technological and computational limitations in calculation of the dynamics and structure visualization of
stress responsive pathway and gene to metabolic network study. Furthermore, most of these data are not
publicly available, and published paper often only provides limited data which may or may not be freely
accessible. Despite rapid advancements of NGS platforms in gene-sequencing methodologies and advantages of
metabolomics over transcriptomics and proteomics, the criticality of metabolome study has hindered the
progress of this newly introduced branch of omics technology in plant stress biology and data base
development. Therefore, integration of the stress-responsive gene/protein traits in different model and crop
plants will definitely enhance access to gene and protein data in a variety of bioinformatics analysis contexts.
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Short communication
Recollection of Stellaria congestiflora H. Hara (Caryophyllaceae)

with new distribution record from Western Himalaya
Satish Chandra*, D. S. Rawat and Jyotsna Rastogi
Department of Biological Sciences, College of Basic Science & Humanities, Govind Ballabh Pant University of
Agriculture and Technology, Pantnagar-263145, Uttarakhand, India
*Corresponding Author: satishchandrasemwal07@gmail.com [Accepted: 19 August 2015]
[Cite as: Chandra S, Rawat DS & Rastogi J (2015) Recollection of Stellaria congestiflora H. Hara
(Caryophyllaceae) with new distribution record from Western Himalaya. Tropical Plant Research 2(2): 153
155]
Stellaria congestiflora H. Hara (Caryophyllaceae) is an endemic herb found in alpine zones of Tibet and
Himalayan regions. The species was described by Hara (1977) from Taglung, South of Tukucha, Kali Gandaki,
Nepal and known to be common near the base of Mount Everest, Nepal. Globally the species has been recorded
in Xizang area (Tibet) China, Bhutan, Nepal and India (Grierson 1984, Press et al. 2000, Pusalkar & Singh
2010, Shilong & Rabeler 2001). In India this species is represented by only two specimens; one from Sikkim
(Below Phaklung rocky gully, Lasha Chhu, North district) and other from Uttarakhand (opposite Gothing,
Chamoli). From Uttarakhand this species was collected by B.D. Naithani in 1975 but he erroneously identified
specimens as Arenaria festucoides Benth. Later, Pusalkar & Singh (2010) correctly identified specimen as
Stellaria congestiflora and reported its presence in India from Uttarakhand and Sikkim. On account of only two
collections in Indian Himalayan region this species is rare, though it is reported common in Nepal Himalaya.
A routine plant exploration visit was conducted in Upper Nandakini valley and Roopkund area of Chamoli
district in AugustSeptember, 2014. Plant specimens of Stellaria congestiflora were collected from Homkundi
area (3018'30" N & 7944'16.2" E, 4000 m elevation) near Shila samudra glacier in district Chamoli,
Uttarakhand state, India. Species was identified with the help of protologue, description of species (Hara 1977,
& Rabeler 2001, Pusalkar & Singh 2010), specimen housed at BSD (Herbarium of Botanical survey of India
Northern Regional Circle Dehradun), images of isotype and other specimens at Royal Botanic Garden
Edinburgh (E) accessible through internet.
Species description
Stellaria congestiflora H. Hara in J. Jap. Bot. 52: 195. 1977; Stainton in Fl. Himalaya suppl. 7. Pt. 12.1988;
Shilong & Rabeler in Wu et al. Fl. China, 6: 27. 2001; Pusalkar & Singh in Ann. Forestry 18(1): 56. 2010.
Herbs, perennial. Stem tufted, branched at base, 510 cm tall, internode shorter than leaves, branches villous
on upper part, trichomes uniseriate, eglandular. Leaves sessile, ovatelanceolate, 8121.5 mm, greenish-
yellow, pungent, base obtuse, margin entire, apex acute, eglandular hairy at both surfaces and margins in
proximal half, distal half glabrous, mid vein conspicuous, ridged at abaxial surface, lateral veins obscure.
Flowers in dichasial cyme, forming corymbose tufts, 717 flowers in each corymb. Bracts foliaceous, pale
green, linearlanceolate, mid vein prominent, 6101.5 mm, glaborous. Pedicel slender, 0.31.0 cm long,
villous hairy. Sepal 5, ovatelanceolate, 581.52.0 mm, hairy toward base at abaxial surface, upper part
glabrous, margin scarious, apex acute, veins 3. Petal 5, white, 12(2.5) mm, bipartite up to base, lobes almost
equal, linear, apex obtuse, veins obscure. Stamen 10, equal to slightly longer than petals, antisepalous
comparatively longer and with prominent gland at base, filament 0.52.5 mm, anther brown. Ovary ovoid, 1.5
2.01.01.5 mm, style 3, equal to ovary. Capsule ovate, 2.02.51.5 mm, enclosed within persistent sepals,
opening by 6 valves to base. Seed brown, 48 per fruit, ca. 1 mm diameter, sub-orbicular, surface, rugose (Fig.
1).
Flowering & Fruiting: AugustSeptember.
Chandra et al. (2015) 2(2): 153155
.
Figure 1. A, Location near Silasamudra glacier in Western Himalaya from where species was collected, red circle indicate
area of specimen collection; B, Plant of Stellaria congestiflora H. Hara.; C, Flowers of Stellaria congestiflora H. Hara.
Specimens Examined: NEPAL, Taglung, South of Tuckucha, Kali Gandaki, 14500 ft., 16.07.1954, Stianton,
Sykes & William 1825 Isotype (E) (Image !). BHUTAN, Alooktha, 15000 ft., 12.08.1913, Ribu & Rhomoo 995
(E) (Image !). INDIA, Sikkim, North district, Lasha Chhu, below Phaklung, 4440 m, 19.07.1996, D.G. Long &
H. J. Noltie 343 (E) (Image !); Uttarakhand, Chamoli district, opposite Gothing, 12.09.1975, B. D. Nainthani
56215 (BSD)!; Homkundi area, near Shilasamudra glacier, 01.09.2014, D.S. Rawat & Satish Chandra 704
(Govind Ballabh Pant University Herbarium Pantnagar, Uttarakhand, India) !.
Habitat: Occasional among boulders along stream banks between 40004200 m elevation, with Stellaria
decumbens.
Present collection of Stellaria congestiflora from Shila Samudra Glacier area in upper Nandakini valley
(Chamoli district) represent the westernmost limit of this species in the Himalaya. Earlier report of its
occurrence in Gothing area, Chamoli district (Uttarakhand) by Pusalkar & Singh (2010) was considered as the
westernmost end of this species at 3050'40" N & 7948'25" E but the present collection at 3018'30" N &
7944'16.2" E (based on wikimapia.org) is further 4' westwards and more than 60 km (aerial distance) away
from Gothing. The earlier report by Pusalkar & Singh (2010) was based on a specimen collected in 1975 after
which it was never recollected from any area in Uttarakhand. No inclusion in Caryophyllaceae of India
(Majumdar 1993), in Flora of Sikkim (Srivastava 1998) and in Flowering Plants of Uttarakhand (Uniyal 2007)
clearly indicate it as a rare species in Indian Himalaya. Assessment of its populations as per the recent IUCN
criteria is suggested for ascertaining its status in India.
REFERENCES
Grierson AJC (1984) Stellaria L. In: Grierson AJC & long DG (eds) Flora of Bhutan. Riyal Botanical Garden
Edinburgh, UK. 1(2): 205209.
Hara H (1977) New or noteworthy flowering plants from Eastern Himalaya: 19. Journal of Japanese Botany
52(7): 193199.
Majumdar NC (1993) Caryophyllaceae. In: Sharma BD & Balakrishnan NP (eds) Flora of India. Vol. 2.
Botanical Survey of India, Calcutta, pp. 502595.
Press JR, Shrestha KK & Sutton DA (2000) Annotated checklist of the flowering plants of Nepal. Natural
History Museum Publications. Available from:
http://www.efloras.org/florataxon.aspx?flora_id=110&taxon_id=242318751 (accessed: 22 June 2015).
154
Chandra et al. (2015) 2(2): 153155
.
Pusalkar PK & Singh DK (2010) New plant record from western Himalaya. Annals of Forestry 18(1): 5562.
Shilong C. & Rabeler RK (2001) Stellaria L. In: Wu Z & Raven PH (eds) Flora of China (Caryophyllaceae
through Lardizabalaceae), Vol. 6. Science Press, Beijing, China, and Missouri Botanical Garden Press, St.
Louis, Missouri, USA. Available from: http:www.efloras.org/florataxon.aspx?flora_id=2&taxon_id=110972
(accessed: 22 June 2015).
Srivastava RC (1998) Flora of Sikkim (Ranunculaceae to Moringaceae). Oriental Enterprises, Dehradun, India.
Uniyal BP, Sharma JR, Choudhery U & Singh DK (2007) Flowering Plants of Uttarakhand (A Checklist).
Bishen Singh Mahendra Pal Singh, Dehradun, India.
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2(2): 156167, 2015
Research article
Comparative investigations on fruit microcharacters of four species

of Hieracium L. (Asteraceae) and their taxonomic significance
Tulika Talukdar*
Department of Botany, A.P.C. Roy Govt. College, Siliguri, Darjeeling 734010, West Bengal, India
*Corresponding Author: talukdartulip12@gmail.com [Accepted: 23 August 2015]
Abstract: In order to evaluate taxonomic implications of fruit or cypsela, detail macro as well as
micro-morphology and anatomy in four species belonging to the genus Hieracium L. of the tribe
Cichorieae, family Asteraceae have been investigated. Analysis revealed that in comparison to
shape, size and colour of cypsela, surface features like markings, presence or absence of rib and
their number along with carpopodium diversity were taxonomically more significant characters.
Among anatomical features, mesocarpic characters, distribution of vascular trace and vallecular
canal, and nature of testal layer were found to be diacritical for the studied species. These
morphological and anatomical features of cypsela can be used as species determining characters in
the genus Hieracium L. Finally, involving all these features of cypsela an artificial diagnostic key
to the four studied species namely Hieracium neopinnatifidum Pugsley, H. pilosella L., H.
semigothiciforme Zahn and H. umbellatum L. is constructed. This can be used as reference key to
identify taxa solely based on its fruit.
Keywords: Hawkweed - Carpopodium - Cypsela - Endosperm - Morphology.
[Cite as: Talukdar T (2015) Comparative investigations on fruit microcharacters of four species of Hieracium
L. (Asteraceae) and their taxonomic significance. Tropical Plant Research 2(2): 156167]
INTRODUCTION
The genus Hieracium L., commonly known as hawkweed is a member of the tribe Cichorieae of the
sunflower family (Asteraceae). Hieracium L. species are native to Asia, Africa, Europe and America (Strother
2007) and cytological analysis was carried out on Hieracium spp. from Indian Western Himalayas (Gupta et al.
2014). Being one of the largest genera with more than 10,000 species and subspecies Hieracium performs a
putative role in making Asteraceae the second largest family of flowering plants (Cokunelebi 2003).
Reproductive biology of the genus is very unique. They may reproduce exclusively by seeds, of either normal
sexual or asexual apomictic type, and alternatively by both seeds and runner like stolon. The apomictic
reproduction produces lots of minor geographic variations which in turn creates notorious difficulty in
classifying the genus Hieracium L. up to species level.
Generic and sub-generic circumscription of Hieracium L. remained controversial (Szelag 2014). Recent
treatments split Pilosella as a separate genus from Hieracium based on morphological, biochemical, cytological
and genetical features (Bremer & Anderberg 1996). Sennikov (2012) revised the Himalayan (India and
Pakistan) species of Hieracium. H. korshinskyi, H. kuusamoense and H. subramosum were found in place of H.
vulgatum, while H. robustum were observed in place of H. crocatum. He distinguished H. korshinskyi from H.
vulgatum and confirmed differences among rest of the taxa on the basis of morphological features such as leaf
texture, habit, nature of hairs, geographical distributions etc. The presence of H. umbellatum and H. virosum is
confirmed in South Asia (Sennikov 2012). In few taxonomic studies achene or cypsela characters along with
pollen grain and stolon are taken into consideration (Mrz et al. 2002, Sell & West 1975). Strategic demarcation
for Hieracium sensu lato and sensu stricto is not clear so far and reformation by shifting of species is still in a
flux (Cokunelebi & Beyazolu 2002, Cokunelebi 2003). Moreover, hybrid origin of many of the species as
evidenced by irregular meiosis, ploidy level variations, and apomictic development of embryos (Mrz 2003) put
some more pressure on the present taxonomic difficulty.
Talukdar (2015) 2(2): 156167
.
Micromorphological and anatomical features of different plant parts have enough potentiality for taxonomic
consideration in Angiosperms (Parveen et al. 2000, Ramayya 1972, Talukdar 2012, 2013). Cassini (1975), the
true founder of detailed and systematic studies in Asteraceae, believed that to understand a natural group like
Asteraceae it was necessary to study all the organs of a plant belonging to all the species in the family without
exception.
The fruit and seed characteristics in Asteraceae show marked variation that provides significant taxonomic
information (Kadereit & Jeffrey 2007, Nordenstam & Mukherjee 2010, Talukdar 2013). Perusal of literature
cited that there is very little comprehensive study on cypsela features in the genus Hieracium L. The aim of the
present investigation is to provide a detail account of fruit or achene microcharacters including broad range of
morphological and anatomical features of four species belonging to Hieracium s. lato. namely H.
neopinnatifidum Pugsley, H. pilosella L., H. semigothiciforme Zahn, and H. umbellatum L. For assessing their
taxonomic usefulness an artificial key to the studied species has also been constructed.

Plant materials
Plant materials (cypselas) for the present investigation were obtained (courtesy Prof. Sobhan Kumar
Mukerjee, FLS, Department of Botany, University of Kalyani, west Bengal, India) in the form of received
herbarium specimens from the Hortus Botanicus Hauniensis herbarium (DK), Denmark, of the world which is
mentioned in Index Herbarium (Holmgren et al. 1990). The list of the specimens with their collection number is
presented in table 1. Voucher specimens (cypselas) were maintained at Departmental Herbaria, University of
Kalyani, West Bengal, India.
Table 1. Source of materials with collection number.

S. No. Name of Taxa Locality Collection Number
1. Hieracium neopinnatifidum Pugsley DK Z S1973-1108
2. Hieracium pilosella L. DK W DK: 44PG7976
3. Hieracium semigothiciforme Zahn DK GE3026-0114
4. Hieracium umbellatum L. DK GE 3026-0038
Note: DK-Hortus Botanicus Hauniensis, Denmark.
Micromorphological analysis
For Micromorphological studies, mature cypselas were dipped in 15% NaOH solution for 27 days
depending upon the hardness and transferred into saturated chloral hydrate solution for few hours. After
repeated washing with water, cypselas were transferred into in 0.20.5% aqueous Safranin solution for 510
minutes and were placed in 70% phenol glycerine solution for studying different parts. Microphotographs were
taken using camera equipped Zeiss-stereo microscope.
Surface ultra-structural study
For scanning electron microscope (SEM) analysis, cypselas were mounted on labelled aluminium brass stubs
using double-sided adhesive tapes. All the carrying stubs were quick-dried using vacuum evaporator and
examined using FEI-QUANTA 200 Autoscanning Electron Microscope at Regional Sophisticated
Instrumentation Centre, Bose Institute, Kolkata, India.
For anatomical studies, mainly hand sections of cypselas from middle part were utilized. The cypselas were
softened by dipping in boiling water for 530 minutes, with a few drops of glycerol. After softening and
sectioning, the sections were dehydrated and stained using conventional method (Johansen 1940) with alcohol
grades. All the observed features of cross-section were documented with the aid of camera lucida drawings and
camera equipped microscope.
Terminology for the macro- and micro-morphological features, anatomical structures and SEM observation
primarily followed Ramayya (1972), Stearn (1973), Barthlott (1981) and partially improvised by the author
herself.
Measurement and statistical analysis
All measurements were taken from at least 10 intact and mature cypselas for each specimen and data were
obtained by Olympus stereo dissecting microscope. The length of the cypselas was taken as the length of the
Talukdar (2015) 2(2): 156167
.
body of cypsela from basal meristematic zone (carpopodium) up to apical end excluding pappus. The width of
the cypsela was measured from the widest point. The mean (M) and standard error (SE) of measurement were
calculated.
RESULTS
In the present investigation the cypselas of four species of Hieracium (H. neopinnatifidum, H. pilosella, H.
semigothiciforme, and H. umbellatum) were examined. Morphological features of cypsela like shape, size,
colour, surface markings, carpopodium, pappus etc. are represented by figures 13. All anatomical observations
are shown in figure 45.
Figure 1. Cypselas morphology: A-G, Hieracium neopinnatifidum; H-O, Hieracium pilosella. (A, ray cypsela; B, disc
cypsela; C, carpopodium of ray cypsela; D, base of disc cypsela; E, K, L, apex; F, M, surface; G, O, part of pappus bristle;
H, cypsela; I, J, base; N, margin)
Talukdar (2015) 2(2): 156167
.
Morphology of cypsela
Hieracium neopinnatifidum Pugsley (Fig. 1AG)
Cypsela was heteromorphic. Length of disc cypsela was ranged from 2.83.2 mm (excluding pappus) and
width varied between 0.2 and 0.5 mm. Cypsela was black, oblong cylindrical, straight in direction, truncate at
the apex and gradually tapered towards the base. Surface of cypselas was pubescent without any rib. After
clearing, surface showed striate markings (i.e. marked with a series of fine narrow parallel bands wider than the
lines of a lineate surface). A well developed, wide, tubular, hollow and broad based stylopodium with gradually
tapering ring like apical part was noted. Carpopodium was asymmetrical, smooth, ring like structure with visible
cells outline. Carpopodial cells were rectangular, vertically oriented, and present in two to five rows. Diameter
of carpopodium was lesser than the base of the body. Cypsela was basally inserted. Pappus was persistent but
fragile, represented by two rows of many multicellular terete and scabrous bristles. Bristles were free from one
another, unbranched, 1.22.3 mm in length, ivory white and somewhat coarser and rough due to projection tips
of the lateral cells.
Ray cypselas were more or less similar to disc cypselas, except the following characters,
1. Ray cypsela was larger than disc cypsela with 3.64.0 mm (excluding pappus) in length and 0.100.15
mm in width,
2. lorate in shape,
3. with symmetrical, smooth triangular ring like carpopodium and
4. diameter of carpopodium was same as the base of the body.
Hieracium pilosella L. (Fig. 1HO)
Cypsela was homomorphic. Length of cypsela was ranged from 2.02.2 mm (excluding pappus) and width
varied between 0.30.5 mm. Cypsela was black, narrow oblong, cylindrical and straight in direction. Apex of
cypsela was truncated wavy with two spiny projections and base was slightly narrower. Glabrous and ribbed
surface with muricated margin was noted; ribs were ten in number, prominent and straight. After clearing,
surface of cypsela showed lineate markings with transverse bands. A broad based stylopodium with upper
tubular solid part was present on the apex of cypsela. At the base an asymmetric, ring like carpopodium was
noted with one marked interruption. Carpopodial cells were visible and distinguishable from other cells of the
cypsela. They were elongated, thin walled, tangentially oriented, and were arranged in five to six layers.
Diameter of carpopodium was narrower than the base of the body. Cypsela was basally inserted and pappose.
Pappus was represented by many, persistent but fragile, multicellular, unbranched and scabrid-barbellate
bristles. Bristles were 0.231.00 mm. long, with conspicuous projection tips of the lateral cells. Base of pappus
was free from one another.
Hieracium semigothiciforme Zahn (Fig. 2AF)
Cypsela was homomorphic. Length was ranged from 2.02.4 mm (excluding pappus) and width varied
between 0.2 and 0.5 mm. Cypsela was black, narrow oblong, dorsiventrally compressed and straight. At the
apex cypsela was truncate and gradually narrower towards the base, with near about 10 lobes. Surface of cypsela
was glabrous, and after clearing it showed foveate (pitted) markings, arranged in vertical rows. A well-
developed stylopodium was noted at the apex. At the base carpopodium was symmetric, complete, smooth,
circular ring like with visible and distinguishable cells. Carpopodial cells were rectangular to elliptic, thick-
walled, tangentially oriented and arranged in five to six rows. Diameter of carpopodium was narrower than the
base of the body. Cypsela was inserted basally. Pappus was represented by persistent but fragile, multicellular,
terete and heteromorphic bristles. Bristles were many, free from one another, unbranched, 0.551.66 mm long.
Two types of bristles were noted,
a) Narrower, barbellate bristles with the tips of the lateral cells elongated and approximately as long as
width of the rachis of the bristles.
b) Broader scabrous bristles, with lateral projection that were narrower than the width of pappus bristles.
Hieracium umbellatum L. (Fig. 2GN)
Cypsela was heteromorphic. Disc cypsela was 2.32.7 mm (excluding pappus) long and 0.50.8 mm wide.
Cypsela was blackish brown, narrow oblong, dorsiventrally compressed and straight. At the apex cypsela was
truncate and towards the base was gradually tapered. Surface of the cypsela was glabrous with ten lobes and
showed lineate markings after clearing. A well-developed, narrow, tubular stylopodium was present at the apex.
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.
At the base carpopodium was symmetric, ring like with one marked interruption. Carpopodial cells were visible
and distinguishable. They were thick-walled, parenchymatous, square to rectangular, oriented vertically and
arranged in seven to eight layers. Diameter of carpopodium was narrower than the base of the body. Cypsela
was inserted basally and pappose. Pappus was represented by persistent but fragile, multicellular, terete and
barbellate bristles. Bristles were few, free from one another, unbranched, 0.10.2 mm. long with the tips of the
lateral cells as long as width of the rachis.
Ray cypsela was more or less similar with disc cypsela except in the following characters,
1. Lorate in shape with 3.03.2 mm (excluding pappus) in length and 0.20.4 mm in width.
2. Truncate at the apex and sharply tapered towards the base.
3. Carpopodium was symmetric, complete circular smooth ring like. Diameter of carpopodium was same as
the base of the body.
Figure 2. Cypselas morphology: A-F, Hieracium semigothiciforme; G-N, Hieracium umbellatum. (A, cypsela; B, base; C,
L, apex; D, M, surface (after cleaning); E, part of outer bristle; F, part of inner bristle; G, ray cypsela; H, disc cypsela; I,
base (ray cypsela); J, carpopodium (ray cypsela); K, base (disc cypsela); N, part of pappus bristle)
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.
SEM survey of cypsela
Hieracium neopinnatifidum Pugsley (Fig. 3AC):
Surface cells of cypsela were visible, rectangular, vertically oriented with straight anticlinal wall and shallow
periclinal wall. Surface was pubescent with sparsely distributed, triangular, and upwardly directed hairs. Pappus
was biseriate.
Hieracium pilosella L. (Fig. 3 DF)
Surface cells of cypsela were visible, rectangular, vertically oriented, with straight anticlinal and periclinal
wall. Surface was ribbed, muricate with short hard protuberances. Pappus was uniseriate.
Hieracium semigothiciforme Zahn (Fig. 3 GI)
Surface cells of cypsela were visible, rectangular, vertically oriented, with wavy anticlinal wall and straight
periclinal wall. Pappus was uniseriate.
Hieracium umbellatum L. (Fig. 3 JL)
Surface cells of cypsela were not visible. Very small triangular protuberances were noted on surface. Pappus
was uniseriate.
Figure 3. SEM analysis of cypselas: A-C, Hieracium neopinnatifidum; D-F, Hieracium pilosella; G-I, Hieracium
semigothiciforme; J-L, Hieracium umbellatum. (A, D, G, J, cypsela; B, E, G, K, base; C, F, I, L, apex)
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Anatomy of cypsela
Hieracium neopinnatifidum Pugsley (Fig. 4 A1A2)
Both ray and disc cypselas were same in anatomical features. Cypsela was oblate in transection with
convolute margin. Wall of cypsela was approximately 106.6 m wide in T.S. with 66.6 m thick pericarp.
Pericarp was found to be differentiated into two zones-epicarp and mesocarp. Epicarp was uniseriate, made up
of thin-walled, elliptic to rectangular, compactly arranged and tangentially oriented parenchymatous cells.
Mesocarp made up of continuous four to five layers of scelerenchymatous cells; cells were thick-walled,
compactly arranged, variously shaped, with large and round lumen. Three to four vascular traces and five to six
vallecular cavities were noted inside of sclerenchyma tissue in the mesocarpic zone. Testa/seed coat was
secondarily separated from pericarp, approximately 6.9 m thick, unilayered, cellular and compressed. Testal
cells were parenchymatous, thin-walled, elliptic, loosely arranged and tangentially oriented. Endosperm was
persisted in mature cypsela and biseriate. The two endospermic cell layers were dissimilar, of which the outer
cells were larger. Cells of both the layers were thin-walled, barrel shaped, tangentially oriented and
parenchymatous. The mature embryo occupied a major part of the cypsela. Cotyledons were two in number,
plano-convex in shape, anterior-posteriorly oriented (parallel to surface) with three secretory ducts in each
cotyledon, of which central one was larger than others.
Hieracium pilosella L. (Fig. 4B1B2)
Cypsela was circular in transection, with ten broadly semicircular ribs. Cypsela wall was 88 m and 41 m
wide at rib and furrow respectively with on an average was 58.5 m thick pericarp. Pericarp was differentiated
into two zones-epicarp and mesocarp. Epicarp was uniseriate, made up of thin-walled, rectangular, wavy,
compactly arranged and tangentially oriented parenchymatous cells. Epicarpic cuticle was noted which form
tubercle like outgrowths throughout the entire surface of epidermis. Mesocarp was sclerenchymatous with
variable thickness. At furrow mesocarp was uniseriate and was multiseriate at ridge. Mesocarpic cells were
thick-walled, oval to angular, compactly arranged and radially oriented with large elongated lumen. At each rib,
one central vascular trace and two lateral vallecular canals were noted just inside of sclerenchyma. Testa was
found to be adpressed with pericarp, approximately 4.13 m thick, unilayered, cellular and compressed. Testal
cells were parenchymatous, thin-walled, transversely elliptic, compactly arranged and tangentially oriented.
Endosperm was persisted in mature cypsela and biseriate. Cells of two endospermic layers were dissimilar with
larger and wider outer cells. Cells of both the layers were barrel shaped, thick-walled, parenchymatous,
compactly arranged and tangentially oriented. The mature embryo occupied more or less the entire part of the
cypsela. Cotyledons were two in number, plano-convex in shape, anterio-posteriorly oriented (parallel to
surface) with three secretory ducts in each cotyledon, of which central one was larger than others.
Figure 4. Cypselas anatomy: A1-A2, Hieracium neopinnatifidum; B1-B2, Hieracium pilosella. (A1, B1, T.S. of cypsela
(diagrammatic); A2, B2, a part of cypsela in T.S.; Cv, cavity; Ep, epidermis; En-endosperm; Scl, scerenchyma; T, testa; Tu,
tubercle; VT, vascular trace)
Hieracium semigothiciforme Zahn (Fig. 5A1A2)
Cypsela was widely elliptic in transection, with near about 10 lobes. Cypselar wall was 75 m wide with 68
m thick pericarp. Pericarp was differentiated into two zones- epicarp and mesocarp. Epicarp was uniseriate,
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made up of square to rectangular, compactly arranged and tangentially oriented parenchymatous cells. Papillate
out growths were noted in the epicuticular layer. Mesocarp was heterogenous, consists of outer
sclerenchymatous and inner parenchymatous tissues. Sclerenchymatous tissue was composed of continuous,
four to five cell layers. Cells were thick-walled, angular with narrow elongated lumen. Inner parenchymatous
tissue was uniseriate, made up of rectangular, thin-walled, compactly arranged and tangentially oriented cells.
Inside of sclerenchyma tissue vascular trace was noted. Corresponding to each vascular trace, single vallecular
cavity was found to exist inside of parenchyma tissue. Testa was attached with pericarp, approximately 6.9 m
thick, unilayered and cellular. Testal cells were parenchymatous, elliptic, thin-walled, compactly arranged and
tangentially oriented. Inner cross walls of testal cells were much more thickened than outer wall. Endosperm
was persisted in mature cypsela and biseriate. The two cell layers were dissimilar with large, barrel-shaped outer
cells and small, narrow elliptic inner cells. Cells of both the endospermic layers were thick-walled, tangentially
oriented and compactly arranged. The mature embryo occupied major part of the cypsela. Cotyledons were two
in number, plano-convex in shape, anterior-posteriorly oriented (parallel to surface) with three secretory ducts in
each cotyledon of which middle one was larger than others.
Hieracium umbellatum L. (Fig. 5B1B2)
Both disc and ray cypselas were more or less anatomically similar. Cypsela was circular in transaction, with
ten broadly triangular lobes including five more pronounced lobes. Wall of the cypsela was 160 m and 93 m
wide at rib and furrow region respectively with on an average 119 m thick pericarp. Pericarp was differentiated
into epicarp and mesocarp. Epicarp was uniseriate, made up of thin-walled, elliptic, compactly arranged and
tangentially oriented parenchymatous cells. Epicuticular layer was present forming papillate outgrowths.
Mesocarp was homogenous, sclerenchymatous, made up of continuous three to four layers of cells. Mesocarpic
cells were thick-walled, angular, with small and elliptic lumen. Single vascular trace and single vallecular canal
were situated inside of sclerenchyma tissue at each lobe. Testa was represented by thin, approximately 4.13 m
wide, non-cellular pellicle layer. Endosperm was persisted in mature cypsela and biseriate. The two
endospermic cell layers were dissimilar with outer larger cells. Cells of both the layers were thick-walled,
parenchymatous, barrel-shaped, tangentially oriented and compactly arranged. The mature embryo occupied
more or less the entire part of the cypsela. Cotyledons were two in number, plano-convex in shape, anterior-
posteriorly oriented (parallel to the surface) with four more or less equally developed secretory ducts in each
cotyledon.
Figure 5. Cypselas anatomy: A1-A2, Hieracium semigothiciforme; B1-B2, Hieracium umbellatum. (A1, B1, T.S. of cypsela
(diagrammatic); A2, B2, a part of cypsela in T.S.; Cv, cavity; Ep, epidermis; En, endosperm; Ppl, papillate structure; Scl,
scerenchyma; T, testa; VT, vascular trace)
DISCUSSION
Cypselas of the studied species were categorised into heteromorphic type and homomorphic type.
Heteromorphic cypselas were noted in H. neopinnatifidum and H. umbellatum. Cypselas were black to blackish
brown in colour and oblong to narrow oblong in shape. However, ray cypsela of H. neopinnatifidum and H.
umbellatum were lorate in shape. Length of cypselas was ranged from 2.04.0 mm (excluding pappus) and
width varied between 0.1 and 0.8 mm. Cypselas were mostly cylindrical but dorsiventral differentiation was
noted in cypsela of H. semigothiciforme. In contrast to other species, two apical spiny projections were observed
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on cypsela of H. pilosella. Cypsela surface of all the studied taxa was glabrous and marked with varied striation.
Mostly cypselas were ribbed or lobed, but rib was totally absent in heteromorphic cypselas of H.
neopinnatifidum. In ribbed cypselas, number of ribs was generally 10. Similarly, 10-ribbed cypsela was also
noted in Lactuca pseudo-umbrella of the tribe Cichorieae (Maity & Maity 2001). According to Babcock and
Stebbins (1937), the longitudinal main ribs of the cypsela are generally five in this tribe. However, the
occurrence of other than five-ribbed (i.e. 10, 15 or 20 ribbed) cypsela as noted in the present observation was
well explained by Kilian (1997). Generally, main ribs were frequently differentiated and sub-divided into less-
defined, smaller secondary ribs on either side, resulting in 10, 15 or 20-ribbed cypsela (Kilian 1997).
In all of the presently studied species stylopodium and carpopodium were well developed. Carpopodium
showed a wide range of variation between the studied taxa and could be species delimiting factor. Two basic
types of carpopodium was noted in the present study, A. complete ring like as in H. semigothiciforme, in disc
cypsela of H. umbellatum and in both the cypselas of heteromorphic H. neopinnatifidu and B. interrupted ring
like as in H. pilosella and ray cypsela of H. umbellatum. Completely ring like carpopodium also varied in shapes
of the ring, which may be either circular in H. semigothiciforme and in disc cypsela of H. umbellatum or
triangular in ray cypsela of H. neopinnatifidum. A complete ring like carpopodium with marked interruption was
observed in H. jutlandicum while carpopodium was complete ring like in H. norvegicum (Mukherjee & Das
2008). Marked variations also exist in carpopodium thickness among the different studied species. While
highest magnitude of thickness (78 rowed) was exhibited in H. umbellatum carpopodium, it was moderately
thick in H. pilosella and H. semigothiciforme (56 rowed). Contrastingly, lowest extent of carpopodium
thickness (25 rowed) was noted in H. neopinnatifidum. Mostly, diameter of the carpopodium was found to be
lesser than the body of the cypsela except in ray cypsela of H. neopinnatifidum and disc cypsela of H.
umbellatum. Pappus features were found to be less variable among the studied taxa, suggesting this feature may
not be a taxonomic delimiting factor in these four taxa. Pappus was mostly persistent, represented by
unbranched, free, scabrous and uniseriate bristles of fragile nature. In H. neopinnatifidum biseriate bristles and
in H. semigothiciforme heteromorphic bristles were noted.
Distinct differences were noticed between different species in many of the micro-morphological features of
cypsela like surface characters, nature of carpopodium etc. Such type of inter-specific demarcation between H.
pilosella and H. umbellatum was also evidenced by their chromosomal and ploidy level variations (Mrz 2003).
This correlation between morphological markers and genomic variations in turn signifies the fact that visual
morphology of any parts of plant is as competent as any molecular marker in predicting interrelationship of taxa.
SEM analysis clearly revealed rectangular surface cells of cypsela with visible anticlinal and periclinal wall,
although surface cells were totally non-visible under SEM in H. umbellatum. Anticlinal and periclinal wall of
the surface cells were mostly straight, but in H. semigothiciforme wavy anticlinal wall and in H.
neopinnatifidum shallow periclinal wall was also noted.
Cypselas were circular to elliptic in cross section. Among the studied species, pericarp was the thinnest in
H. pilosella (58.5 m) but the thickest in H. umbellatum (119 m), indicating high range of variation in shapes
of cypsela. Formation of tubercle or papillate outgrowths was noted on the cuticle layer except in H.
neopinnatifidum. Pericarp was clearly differentiated into epicarp and mesocarp. Epicarp was usually uniseriate;
made up of thin walled, rectangular to elliptic, tangentially oriented, parenchymatous cells. Occurrence of dark
brown tanniniferous substance in epicarpic cells of Hieracium L. was reported (Pandey et al. 1978), but in
present observation this was totally absent. Absence of mesocarpic parenchyma as observed in the present case
was also observed in Sonchus asper of Cichorieae (Uyar & Cirli 1982), and in Mikania and Ageratum of
Eupatorieae (Talukdar & Mukherjee 2008). Only heterogenous mesocarp comprising of both sclerenchymatous
and parenchymatous cells was noted in H. semigothiciforme among all the studied taxa. Mesocarpic
sclerenchyma in all of the studied taxa was present as continuous and multiseriate zone of thick walled
compactly arranged angular cells. Mesocarpic parenchymatous layer in cypselas of H. semigothiciforme was
uniseriate.
On the basis of mesocarpic tissue distribution pattern, two types of fruit wall structures -winged type and
ribbed type was earlier proposed in Ixeris of Cichorieae tribe of Asteraceae (Pak & Lawano 1990). In more or
less similar way, Zhu et al. (2006) defined winged type cypsela, in which mesocarp was composed of
parenchymatous cells and distinct fibrous strand (i.e. thick-walled sclerotic cells). In the contrary, cypselas of
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.
the studied taxa are supposed to be of ribbed type due to bearing continuous thick-walled sclerotic cells in their
mesocarp.
Usually few vascular traces were noted in the sclerotic layer. Notably, few larger vallecular canals were
present in the same layer corresponding to each vascular trace side by side. They are generally considered as
collapsed protoxylem and might be an indicator of reduced mechanical strength. On the other way these empty
canals were also act as aerating passage in the cypsela and possibly favour their wind dispersal and
dissemination over varied eco-geographical regions and thus, could be regarded as one of the adaptive strategies
of Hieracium spp.
Testa or seed coat characters are found to be very diacritical and can be considered as potential marker in
sub-genus level of Hieracium L. Usually testa was uniseriate and cellular as also reported earlier in the tribe
Cichorieae (Borthwick & Robbins 1928; Lavialle 1912). Non-cellular pellicle like testa was noted only in H.
umbellatum among the studied species. Occurrence of non-cellular pellicle like seed coat was also noticed in
Aposeris foetida of Cichorieae by (Pandey et al. 1978). In H. neopinnatifidum testal layer was secondarily
separated from pericarp, but testa was adpressed to pericarp in heteromorphic species like H. pilosella and H.
semigothiciforme. Notably, in H. semigothiciforme differential thickening was noted on inner tangential wall of
testal cells, as reported earlier in other members of Cichorieae (Borthwick & Robbins 1928, Pandey et al. 1978).
Uniformity in endosperm layer in all the studied species was manifested as biseriate with two dissimilar cell
layers, of which outer cells were larger than inner. The embryo occupied major to entire portion of the cypsela.
Cotyledons were 2 in number, equal, plano-convex, anterio-posteriorly oriented and possess secretory ducts.
Cotyledon of H. umbellatum bears 4 equally developed secretory ducts. However, in rest of the studied species
secretory duct was 3 in number, of which central one was well developed and larger than others.
Considering all these micro features of cypselas, an artificial key to the species was constructed by which
studied species can be identified in their fruiting stage.
Key to the species

Genus - Hieracium
1a. Cypsela heteromorphic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2a.Cypsela cylindrical; ribs absent; pappus biseriate; epicarp without any papillate outgrowths; surface
cells visible in SEM study; testa cellular. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. neopinnatifidum
2b. Cypsela dorsiventrally compressed; ribs present; pappus uniseriate; epicarp with papillate
outgrowths; surface cells not visible in SEM study; testa disorganized . . . . . . . . . . . . H. umbellatum
1b. Cypsela homomorphic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3a. Apex with spiny projections; cypselar margin muricated; carpopodium asymmetric, ring-like, with
one marked interruption; mesocarp with sclerenchyma tissue only; testal cells without any wall
thickenings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. pilosella
3b. Apex without spiny projections; cypselar margin not muricate; carpopodium symmetric, ring-like,
without interruption; mesocarp with both sclerenchyma and parenchyma tissue; testal cells with inner
cross-wall thickenings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. semigothiciforme
CONCLUSION
The present study on detailed macro and micro-morphological and anatomical features of cypselas of four
different species of Hieracium L. is a strong attempt to assess the potentiality of cypsela as species delimiting
factor. The analysis clearly indicates that in comparison to size, shape and colour of cypsela; surface characters,
nature of carpopodium, mesocarpic differentiation, testal features etc. are much more reliable for inter-specific
taxonomic grouping or separation of Hieracium genus and should have a positive impact on its present status.
Talukdar (2015) 2(2): 156167
.
ACKNOWLEDGEMENTS
Author is grateful to Prof. Sobhan Kumar Mukherjee, Department of Botany, University of Kalyani for his
valuable suggestions and Dr. Hans Vilhelm Hansen, Curator, Denmark for his active assistance in despatching
the materials for studies. Kind acknowledgement is also due to the Incharge, Regional Sophisticated
Instrumentation Centre (RSIC), Bose Institute, Calcutta for permitting SEM analysis.
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ISSN (E): 2349 1183

ISSN (P): 2349 9265

In vitro antioxidant activity of selected Ganoderma species found

MATERIALS AND METHODS

Additions to lichen flora of Jammu and Kashmir, India

MATERIALS AND METHODS

RESULTS AND DISCUSSIONS

Two species of Zygnemopsis (Skuja) Transeau from West

MATERIALS AND METHODS

The effect of sodium silicate and silica nanoparticles on seed

MATERIALS AND METHODS

Stem gall of Michelia champaca L. (Magnoliaceae) induced by

MATERIALS AND METHODS

Assessment of land use land cover change in Chakrar watershed

Abstract: Rapid urbanization, anthropogenic and socioeconomic activities and environmental

MATERIALS AND METHODS

Figure 1. Location map.

RESULT AND DISCUSSION

Table 1. Land use land cover changes from 19902013.

Figure 3. Trend of land use land cover classes.

A rapid and economical method for the maceration of wood fibers

MATERIALS AND METHODS

RESULTS AND DISCUSSION

Floristic composition, life-forms and biological spectrum of

RESULTS AND DISCUSSION

Hemicryptophyte Geophyte Epiphyte

Phanerophyte Therophyte Chamaephyte

Table 3. Arrangement of taxa of Baraytha forest.

Estimation of genetic divergence, association, direct and indirect

MATERIALS AND METHODS

Figure 1. Biplot graph using mean data of sixty cotton genotypes.

Table 3. Path analysis of numerous attributes in (Gossypium hirsutum L.).

Heavy metal and phytochemical screening of anti-jaundice and

Abstract: Increasing population world-wide is depending on herbal medicine as a source of

MATERIALS AND METHODS

Table 3. Phytochemical screening of anti-malaria and anti-jaundice concoctions.

Table 4. Heavy metal analysis of anti-malaria and anti-jaundice concoctions.

Effect of fluoride contaminated irrigation water on Eco-physiology,

MATERIALS AND METHODS

Electrolyte leakage (EC)

Shoot-root length, total leaf area and number

Stem dry weight (g)

Root dry weight (g)

Average biomass production

Biocomputation assisted data base of plant stress responsive traits:

BIOCOMPUTATION IN PLANT STRESS RESPONSIVENESS: The Arabidopsis and rice model

BIOCOMPUTATION IN PLANT STRESS RESPONSIVENESS: The miRNAs

BIOCOMPUTATION IN PLANT STRESS RESPONSIVENESS: Other data bases

Recollection of Stellaria congestiflora H. Hara (Caryophyllaceae)

Comparative investigations on fruit microcharacters of four species

*Corresponding Author: talukdartulip12@gmail.com [Accepted: 23 August 2015]

MATERIALS AND METHODS

Table 1. Source of materials with collection number.

Key to the species

1b. Cypsela homomorphic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

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