Etnerging Trends in Biological Sciences
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Etnerging Trends in Biological Sciences
Editor
T. Pullaiah Department of Botany Sri Krishnadevaraya University Anantapur - 515 003
2009
DAYA PUBLISHING HOUSE Delhi - 110 035
© 2009 T. PULLAIAH (b. 1951ISBN 81-7035-606-7 ISBN 978-81-7035-606-6
)
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Preface
Sri Krishnadevaraya University organized a National Symposium on Science in '21 si century' and Annual convention of Andhra Pradesh Akademi of Sciences during February, 2008. About 100 delegates participated in the symposium. The present book is a collection of some of the papers presented during the symposium. Only some authors have responded to our invitation. Apart from the symposium articles a few invited articles have also been included in the book. We thank Andhra Pradesh Akademi of Sciences for the support. Prof. T. Pullaiah
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Contents
1.
Preface
v
Characterization of the Multifundional Nature of the Chicken M6pnGF-IIR (Mr 300 kDa) Protein Sivaramakrishna Yadavalli and Siva Kumar Nadimpalli
1
2. Field Studies of AM Fungi in Cereals and Millets K.Ammani 3.
4.
Survey of Arbuscular Mycorrhizae in Plants of Nandyal and Anantapur Environments G. Mary Sandeepa, K. Ammani and A. Subramanyam Antimicrobial Spectrum of Digera muricata (L.) Mart. and Moringa pterigosperma Gaertn. S.H.K.R. Prasad, D.Rajasekhar, K. Venkateswara Rao, M. Vijayalakshmi and G. Rosaiah
5. In vitro Antimicrobial Efficacy of Solvent Extracts of Seeds of Albizzia lebbeck (L.) Benth. S.H.K.R. Prasad, B. Rajasekhar, M. Sunny James, N.L Swapna and Madan Prasad
18
27
34
39
viii
6.
7.
8.
Production of Toxic Metabolites by Alternaria ricini Pathogenic to Castor M. Vijayalakshmi, P. Krishna Kumari, B. Rajesh and J. S. V. Anjaneyulu Fungal Infection of Chilli Pods at the Time of Harvest and Post-harvest Drying M. Vijayalakshmi, M. Prasanth, Y. Usha, M.R. Hima Bindhu and G. Rosaiah Studies on Airborne Bacteria
42
48
53
B. RamachandraMurthy andK. VMallaiah
9.
Algal (Cyanobacterial) Diversity Study in North-Eastern States with Respect to Survey and Biotechnological Inputs S.L. Gupta
10. A Study on Adoptional Behaviour of Sericulturists and their Characteristics in Anantapur District of Andhra Pradesh B. Sujatha, P. Lakshminarayana Reddy, S. Sankar Naik, A. Vijaya Bhaskar Rao and P. Sujathamma
84
90
11. Spermoderm Patterns in Some Genera of Andropogoneae D. Someswari, T. Srivalli and T.N. Mary
100
12. Phyta-Pharmacology of Plumbago zeylanica: A Review Pavankumar Bellamkondi and K. Shankaramurthy
111
13. Studies on Biosystematics of Chloris Swartz. Species (Poaceae) T.N. Man), J. Vijayamma, M. Sarada and Seshagiri Rao
122
14. Plant Diversity and Conservation in Sirumalai Hills of Eastern Ghats, Tamil Nadu, South India S. Karuppusamy, KM. Rajasekaran and T. Pullaiah 15. Taxonomy in the Service of Man P.N.Rao
133
172
16. Studies on Frerea indica Dalz.: A Critically Endangered and Endemic Species from Maharashtra, India A.B.D. Selvam, S. Bandyopadhyay and Basundhara Pillai
199
17. Conservation and Utilization of Mangrove Forests: A Case Study in Bhitarkanika Sanctuary, East Coast of India C. Pattanaik, Ch. Sudhakar Reddy, N.K. Dhal and Rashmita Dash
205
ix 18. Adventitious Shoot Bud Regeneration and Organogenesis in Hypocotyl Cultures of Acacia concinna DC.: An Important Tree Legume 211 P. Sairam Reddy, G. Prasad Babu, N. Yasodamma and G. Rama Gopal 19. Rapid In Vitro Propagation of Bixa orellana L.: An Important Dye Yielding Tree Md. ArifuUah, Ch. Kishore Kumar, D. Gayathri and G. Rama Gopal 20. Tissue Culture of Annatto Y. Venkateswara Rao, V. N. Chakravarthi, Dhavala, D. Tejeswara Rao, M. V. Subba Rao and V. Manga Subject and Plant Index
224
233
245
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Chapter 1
Characterization of the Multifunctional Nature of the Chicken M6P /IGF-IIR (Mr 300 kDa) Protein Sivaramakrishna Yadavalli and Siva Kumar Nadimpalli* Protein Biochemistry and Molecular Biology Laboratory Department of Biochemistry, University of Hyderabad Hyderabad-500 046, India
ABSTRACT Thyroglobulin (Tg) is the major secretory protein of thyroid epithelial cells. Part of thyroglobulin reaches the circulation of vertebrates by transcytosis across the epithelial wall of thyroid follicles. After its synthesis, Tg follows a complex secretion, storage and recapture pathway to lysosomes where it is completely degraded to release thyroidal hormones. In mammals some receptors (M6P / IGF-IIR) have been identified that help in metabolism of Tg, no information is available on the endocytic receptors for thyroglobulin in the non-mammalian vertebrates such as the chicken embryonic fibroblast (CEF) cell lines. In the present study we analyzed the interaction of Tg with the CEF cells to identify the pOSSible Tg binding receptors on these cells. Saturation ofTg binding on CEF cells reached at 66 nM with a Kd of 33 nM. Mannose 6-phosphate inhibits binding while glucose 6-phosphate had no effect. By affinity chromatography on Tg-Sepharose gel the
• Corresponding Author: E-mail:
[email protected];
[email protected].
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bound MPR 300 was eluted by mannose 6-phosphate. In vitro interaction between Tg and the receptor was analyzed by far-Western blot analysis. Treatment of Tg with glycosidases or alkaline phosphatase abolished the specific binding. In situ, FITC-Tg- was co-localized with MPR 300 containing endocytic compartments of CEF cells. The present study thus demonstrates the endocytosis ofTg in CEF cells and its degradation in lysosomes is mediated by the M6P /IGFIIR 300.
Keywords: Chicken embryonic fibroblast cells, M6P/lGFIIR, Thyroglobulin, Multifunctional receptor, Mannose 6-phosphate, MPR 300.
Introduction Two distinct but homologous mannose 6-phosphate receptor proteins designated as the MPR 300 (M6P /IGF-IIR, Mr 300 kDa) and MPR 46 (Mr 46 kDa) have been well characterized in mammals that mediate transport of lysosomal enzymes (Dahms and Hancock, 2002). M6P /IGF-IIR is a single polypeptide chain with an apparent molecular mass of 300 kDa consisting of 15 repetitive domains in the amino terminal region, a single transmembrane domain and a cytoplasmic tail. Each of the 15 repetitive domains of the MPR 300 are homologous to each other and to the single domain of the MPR 46 protein. Among the two receptors only mammalian MPR 300 has been shown to be a multifunctional protein and binds ligands independent of divalent metal ions. In addition to the mannose 6-phosphate (m6p) containing ligands, it also binds IGF-II. Additionalligands that the mammalian MPR 300 has been shown to bind are thyroglobulin, retinoic acid, epidermal growth factor, proliferin, rennin precursor, leukemia inhibitory factor, and precursor form of transforming growth factor (Dahms and Hancock,. 2002) and Granzymes A and B, DNase I, CD26. Some viruses have been shown to interact with the receptor for invasion (Brunett et al., 1994, Gabel CA et al., 1989 and Uta Gasanov et al., 2006). The existence of two homologous mannose 6-phosphate receptors (MPRs) with overlapping, but distinct functions and structures with ligand binding activities has raised the question of at what stage in the phylogenetic tree the two receptors have occurred for the first time, what are their functions in different species and what is their evolutionary significance? Some answers for these can only be obtained by extensive biochemical, cell biological and molecular biological comparative studies of the proteins among the non-mammalian vertebrates and the invertebrates. We identified mammalian homologues of the receptors among a number of nonmammalian vertebrate species and studies on the fish MPR protein sequences revealed that these proteins are conserved throughout the vertebrates (Suresh et al., 2006). Homologous receptors have also been identified by us in the invertebrates, [echinodermates (unpublished information) and molluscs (Siva Kumar and von Figura, 2002). Though the two putative MPR proteins from the chicken embryonic fibroblast (CEF) cells have been characterized, and shown to bind the lysosomal enzymes that contain m6p, only recently we showed that it also binds IGF-II (Suresh et al. 2006). It is not known whether it binds other ligands which also contain m6p
Characterization of the Multifunctional Nature of the Chicken M6P/IGF-IIR
3
such as the Tg and non-M6P ligands like retinoic acid etc. In view of the conserved amino acid sequences of the MPR 300 proteins among the vertebrates (fish to mammals), it would be interesting to analyze whether the multifunctional nature of the receptor is also conserved. Therefore, to gain further insight into the multifunctional nature of the non-mammalian vertebrate MPR 300 protein, CEF cells were chosen and the present study was undertaken with the following objectives: (i) To analyze the binding of purified and radio iodinated M6P /IGFIIR 300 from CEF cells and chicken liver on Tg-Sepharose matrix, (ii) binding and internalization of Tg on CEF cells in vitro and in vivo, (iii) co-localization of M6P /IGFIIR 300 and FITC-Tg in CEF cells.
Materials O-phosphonomannan was a generous gift from Dr.M.E.Slodki, USDA, Peoria, IL, USA. Affinity purified antibody to the goat MPR 300 protein was as described (Suresh et al., 2002). Mannose 6-phosphate (M6P), human IGF-II, bovine thyroglobulin (Tg), Tgmonoclonalantibody, DMEM, trypsin-EDTA, penicillin-streptomycin and FITC were purchased from Sigma. TRITC coupled anti-mouse IgG was purchased from Bangalore Genei, India. FBS was purchased from JRH Bioscience and radioactive iodine Na l25 I from MP Biomedical USA. Fresh chicken liver tissue was purchased from the local slaughter house and carried in ice box to the laboratory and used to purify the MPR 300 protein.
Methods Cell Culture Chicken embryonic fibroblast cells (CEF cells) were cultured as described (Suresh
et al., 2006).
Extraction of Membrane Proteins from CEF Cells Confluent monolayers grown in 90 cm petri plates were scraped with the help of a cell scraper, and the cell pellet was collected by centrifugation in a Biofuge stratos centrifuge, at 2991 x g for 10 min. The cells were suspended in 0.1 M sodium acetate buffer pH 6.0, containing 0.2 M NaCl, 1 mM PMSF, 5 mM iodoacetic acid, 1 mM EDTA, sonicated thrice for 35 sec each time with an interval of 1 min, incubated for 20 min on ice, and centrifuged in a Beckman ultracentrifuge, using a fixed angle 80Ti rotor at 161,280 x g for 35 min. The pellet obtained at this step was dissolved in 50 mM imidazole-HCl buffer pH 7.0 containing 0.5 per cent Triton X-lOO, sonicated thrice for 35 sec each time with an interval of 1 min, incubated for 20 min on ice, and recentrifuged as described above. The MPR300 protein from this membrane extract as well as from the chicken liver membrane extract processed as above, was purified to homogeneity by phosphomannan affinity chromatography (PM gel) as described (Suresh et al., 2006).
Protein Assay Protein concentrations were determined using BCA reagent following manufacturer's instructions. BSA was used as the standard.
4
Emerging Trends in Biological Sciences
In vitro Iodination Thyroglobulin (50 llg), purified CEF cell MPR 300 protein (10-20 llg) were radioiodinated using 150 llci of 11251] NaI as described (Suresh et al., 2006) to a specific activity of 1200-2500 cpm per ng protein. 1125llTg was used for quantitation of Tgbinding and internalization studies on CEF cells and radiolabeled MPR 300 was used for affinity chromatography on Tg-Sepharose gel.
Affinity Chromatography on Tg-Sepharose Tg-Sepharose gel and BSA coupled to affigel-10 were equilibrated at 4°C with 50 mM Tris-HCl, 0.9 per cent NaCl, 0.1 per cent BSA, 5 mM CaC~ pH 7.4 containing 0.05 per cent Triton X-lOO (buffer A) as described (Lemansky and Herzog,1992). In brief, iodinated CEF M6P jIGFIIR300 (10, 00,000 cpm) in buffer A (100111), was loaded on to the Tg affinity gel (200 111) or onto the BSA gel (200 111) at a flow rate of 2.5 ml/min. Unbound proteins were removed by washing with 10 volumes of wash buffer (buffer A). Bound proteins were eluted with buffer A containing 5 mM glucose 6phosphate followed by 5 mM mannose 6-phosphate. Column fractions were TCA precipitated, separated by SD5-PAGE and the bands visualized by autoradiography.
Quantitation of Tg Binding and Internalization [Binding Ofl125 I] Tg to CEF Cells] Radio iodinated Tg was diluted in buffer A to give final concentrations ranging from 8 to 86 nM. Cells were grown in 12 well culture plates, to 80-85 per cent confluency and incubated with 10 different concentrations (8 to 86 nM) of 1251_Tg for 90 min at 4°C in binding buffer (buffer A without TritonX100) in the presence of 2 mM mannose [to avoid interference with the possible binding of Tg by its mannose residues]. In a separate experiment, non-specific binding was determined in the presence of 2 mg/ml nonradioactive Tg. Analysis was performed in duplicates and the average values presented. After the incubation, the cells were washed in PBS containing 1 per cent BSA five times, and lysed with lysis buffer (1 per cent Triton X100,50 mM Tris-HCl buffer (pH 8.0),150 mM NaCl, 0.02 per cent NaN3 supplemented with 1 mM PMSF, and 1 mM EDTA as protease inhibitors on ice for 30 min. The cell suspension was centrifuged at 100 x g for 2 min. The supernatant was discarded and pellets were counted in a gamma counter, and the amount of bound Tg was calculated and normalized to the concentration of the membrane protein. Saturation and scatchard plot analysis were carried out by non-linear regression using graph pad prism software with standard computer (www.graphpad.com) .
Binding and Internalization Ofl125 I] Tg by CEF cells The binding and internalization of 125I_Tg in CEF cells was compared by preincubating the cells with mannose 6-phosphate (5 mM), glucose 6-phosphate (5 mM), goat MPR 300 IgG (10 llg), unlabeled Tg (200 nM), rabbit IgG (51lg) and human IGF-II (21lg). Binding analysis was carried out as described above. For internalization, cells were grown to 80-85 per cent confluency in 6 well culture plates. The cells were rinsed five times with DMEM containing 20 mM HEPES to remove the residual serum. 11251] Tg (5, 00,000 cpm) was then added to the cells in 1 ml of serum-free medium and
Characterization of the Multiftmctional Nature of the Chicken M6PIIGF-IIR
5
incubated for 30 min at 37'C. At the end of incubation the cells were washed six times with serum-free medium, three times with medium containing 1 mg/ ml bovine serum albumin and five times with phosphate-buffered saline. The volume of each wash was 1 ml. After washing the cells, they were incubated with 0.5 ml of lysis buffer as above. Radioactivity in the lysate was measured in a gamma counter.
Treatment of Thyroglobulin with Endo H, PNGase F, Alkaline Phosphatase and its Effect on Binding and Internalization For glycosidase (Endo H, PNGase F) treatment, (1251] thyroglobulin (5,00,000 cpm) was denatured by boiling with SDS and incubated for 12 hr at 37°C in incubation buffer containing 10 microunits/pl Endo Hand 20 micro unitS/lll PNGase F (Sigma) (pH 7.0). For phosphatase treatment (125IJ Tg was incubated in 0.15 M NaCl and 0.01 M Tris-HCl buffer pH 8.0, 50 milliunits/lll alkaline phosphatase (Sigma), for 1 hr at 37°C, and then diluted to 1 ml with ice-cold minimal essential medium with 3 mM glutamine or in binding buffer. Effect of these treatments on the binding and internalization of Tg to CEF cells was studied as described above.
Ligand Blotting The membrane proteins extracted from the CEF cells were separated on a 7.5 per cent SD5-PAGE under reducing conditions and transferred onto a polyvinylidene difluoride membrane. After blocking with 5 per cent BSA in PBS the membrane was probed with radio iodinated (l25IJ thyroglobulin (5, OO,OOOcpm) in PBS containing 1 per cent BSA for 1hr at room temperature. The membrane was extensively washed with 0.05 per cent Tween 20 in PBS, dried and the bands visualized by autoradiography.
Far Western Blotting Analysis of Enzyme Treated and Native Thyroglobulin Thyroglobulin (unlabeled) was treated with EndoH, PNGase F and alkaline phosphatase as described above. The enzymes treated as well as the native Tg were electrophoresed and transferred to a polyvinylidene difluoride membrane. After blocking, the membrane was incubated with 50 llg of purified CEF cell M6P /IGFIIR, followed by incubation with goat anti-MPR 300 antibodies (total IgG prepared from the antiserum, 1:1000 dilution) followed by horseradish peroxidase-conjugated antirabbit IgG. Detection was done using ECL reagent.
Endocytosis precipitation
of(125IJ
Thyroglobulin by CEF Cells and Immuno-
CEF cells were grown in two 6 cm culture plates one without and one with 5 mM M6P. The plates were rinsed as described above. Iodinated (125IJ Tg (5,00,OOOcpm) was then added to both the plates in 1 ml of semrn-free medium and the cells were incubated for 6 hr at 37°C (Lemansky and Herzog, 1992). After removal of the medium, cells were incubated for 3 hr with a chase medium containing 4 mg/ml unlabeled thyroglobulin. At the end of incubation the cells were washed as described above. The cells were lysed by treating with 0.5 ml of 0.05 per cent Triton X-lOO containing 0.02 per cent EDTA at 25°C for 5 min and the lysate was transferred into 1.5 ml tubes.
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The radioactivity in the lysate was measured as above. Thyroglobulin was iI];ununoprecipitated from both the lysates by incubating with mouse monoclonal antibody against Tg (1:200) as described (Siva Kumar and von Figura, 2002). The immunoprecipitates were finally separated on a 7.5 per .:ent SDS-PAGE under nonreducing conditions and the bands visualized by autoradiography. Table 1.1: Comparison of Ligand Binding Ability of M6PIIGFIIR 300 of Mammalian and Non-mammalian Species M6P-containing ligands
M6PIIGFII-R 300 Mammalian Species
Non-mammalian Species fchicken)
Yes
Yes
Yes
NO
Lysosomal enzymes Transforming growth
factor-~
precursor
(TGF-~)
Leukemia inhibitory factor
Yes
Proliferin
Yes
NO NO
Thyroglobulin
Yes
Yes (present stCldy)
NO NO NO NO
Renin precursor
Yes
Dnase I
Yes
C026
Yes
Epidermal growth factor receptor
Yes M6PIIGFII-R 300
Non M6P-containing ligands
Mammalian Species
Non-mammalian Species (chicken)
Insulin-like growth factor 11 (IGF-II)
Yes
Yes/R
Retinoic acid
Yes
NO
Urokinase-type plasminogen activator receptor (uPAR)
Yes
Yes
Plasminogen
Yes
Yes
R: Recent study in our laboratory revealed that the reptilian MPR 300 protein binds human IGF-II and has the critical isoleucine residue as in mammalian protein in the 11th domain.
In vitro Fluorochromation of Tg Bovine Tg, 5 mg was incubated with 550 ].11 borate buffer (50 mM, pH 9.0) and 100 ].11 fluorochrome solution of fluoresceinisothiocyanate [5 mg FITC dissolved in one ml of DMSO] overnight at 4°C. Free fluorochrome was removed by desalting using a Sephadex G-25 gel as described above. Immunofl uorescence Microscopy Cells were grown on cover glass slides. For Tg binding, the CEF cells were fixed in 4 per cent formaldehyde followed by blocking with 5 per cent BSA in PBS before incubation with 200 nM Tg in binding buffer (buffer A without TritonX100) for 90
Characterization of the Multifunctional Nature of the Chicken M6PIIGF-IlR
7
min at 4°C. After incubation, cells were immunolabeled with monoclonal mouse antiTg (1:200) for 1hr. Washing was done as described above, and TRITC coupled antimouse IgG (1:1000) was used as the secondary antibody. For internalization of Tg, cells were incubated for 30 min with serum free DMEM supplemented with 200 nM FITC-Tg. After incubation, cells were washed with PBS containing 1 per cent BSA (five times, for 2 min). Then cells were fixed with 4 per cent formaldehyde, and observed under confocal microscopy. For detection of the Tg and arylsuHatase A in the endosomal compartments, FITC-Tg (200 nM) was internalized on CEF cells at 37"C for 30 min and then cells were fixed with 4 per cent paraformaldehyde, permiablized with 0.2 per cent Triton X-lOO, after blocking, cells were incubated with the Human arylsuHatase A antibody and detected using Alexa fluor-594 labeled secondary antibody. For the colocalization of Tg with M6P /IGFIIR, cells were incubated with FITC-Tg at 37"C for 30 min to allow internalization and permeabilized as described above, washed and incubated with goat MPR 300 IgG (10 pg), followed by TRITC labeled secondary antibody (1:1000).
Results Tg-Sepharose Affinity Chromatography The MPR 300 protein from CEF cells and chicken liver were purified to homogeneity (data not shown) and radioiodinated as described under methods. These proteins were separately applied on to Tg-Sepharose gel. (BSA-affigel-lO matrix prepared in the laboratory was used as a control gel). The gels were processed as described under methods and the column fractions and eluates were analyzed by SOS-PAGE. The CEF cell M6P /IGFIIR and the chicken liver MPR 300 were both bound on Tg-Sepharose gel and could be specifically eluted using 5 mM M6P (Figures 1.1A and B). [Under similar conditions the goat purified and radioiodinated MPR 300 was bound to the gel, data not shown]. When the iodinated receptors were applied on BSA-affigel, all the radioactivity could be recovered in the unbound fraction suggesting no binding of the receptors to BSA (data not shown). Upon increasing the incubation time with fixed concentration of the radiolabeled thyroglobulin with the CEF cells, there was an increase in the number of counts bound to the cell surface or internalized until about 90 min beyond which there is no change in the number of counts bound to the cell surface. Similar results were obtained with respect to the internalized counts suggesting that the Tg was bound and internalized into the cells (Figure 1.2).
Characterization of Tg Binding to Chicken Embryonic Cells To characterize the Tg binding to the CEF cells, these were incubated with radioiodinated Tg with or withOut 3 pM nonradioactive Tg at 4°C. In the absence of non-radioactive Tg, the amount of cell-bound [l25I] Tg increased with increasing amounts of free radiolabled Tg exhibiting saturation kinetics (Figure l.3A open circles). In the presence of non-radioactive Tg, the cell-bound radioactivity was lower, and increased in a linear fashion (Figure 1.3A, triangles). The specific binding of [12511 Tg to the surface of CEF cells was calculated (Figure 1.3B) by subtracting nonspecific binding
Emerging Trends in Biological Sciences
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1
4
3
2
(A)
1
2
3
4
(B) Figure 1.1: Affinity Chromatography on Tg-Sepharose Gel A purified and radio-iodinated CEF MPR 300. 7.5 per cent SOS-PAGE analysis. Lane 1 unbound fraction, lane 2 Wash, lane 3, glucose 6-phosphate eluate and lane 4 mannose 6-phosphate eluted (specific elution). B Purified and radio iodinated chicken liver MPR 300. 7.5 per cent SOS-PAGE analysis. Lane 1 unbound fraction [It is seen from the figure that some the radioactivity of the applied was found which could be due to overloading on the affinity matrix] lane 2 wash, lane 3 glucose 6-phosphate eluted and lane 4 mannose 6-phosphate eluted.
(Figure 1.3A, triangles) from total binding (Figure 1.3A, open circles). Saturation of specific Tg binding to CEF cells was reached at 66 nM of Tg. Scatchard analysis revealed a dissociation constant of 33 nM (Figure 1.3C).
Characterization of the Multifunctional Nature of the Chicken M6P/IGF-IIR
9
05~--------------------~======================~ ___ Time (min) vs Surface-Bound ···0·· Time (min) vs Internalized
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Time in (mn) Figure 1.2: Time Course for Binding and Internalization of (1 25 1-Tg] by CEF Cells CEF cells were grown in 12 well culture plates and incubated with 1, 00,000 cpm (1 25 1-Tg] in binding buffer at different time intervals at 4°C as well as 37°C, washings and lysis was done as described in methods. Solid line and closed circles are surface-associated, dotted line and open circles are Internalized (125 I-Tg] respectively.
Binding and Internalization of Tg in Presence of Various Ligands, and Effect of Glycosidases and Alkaline Phosphatase Treatment of Tg on Binding and Internalization When radiolabeled Tg was incubated with the CEF cells at 4°C, the binding of Tg to the cell surface was observed. This binding was inhibited by mannose 6-phosphate, and MPR 300 IgG, while glucose 6-phosphate, rabbit IgG and human IGF-II had no effect. Similar observations were seen for the internalization of the radiolabeled Tg. Additionally, deglycosylation and dephosphorylation of the bovine Tg was done as described under methods and the effect of these treatments on binding and internalization in CEF cells was also studied. The data from these experiments reveals that treatment with glycosidase enzymes abolished almost 90 per cent of the binding and internalization of radiolabeled Tg while treatment with alkaline phosphatase suggested that the uptake was inhibited to 95 per cent. These results are presented in Figure 1.4 A and B.
M6PIIGFII-R Interacts with Thyroglobulin In vitro The binding of the radiolabeled Tg to the CEF cell MPR 300 was analyzed in a ligand blot experiment. From Figure L5A, it is apparent that a band could be visualized
Emerging Trends in Biological Sciences
10 0.'
~0.7
1
0••
11::0 0.5
{! 0 .•
r
0.3
1!
0.2
~
0.1 10 20 30 40 50 10 70 10 10 100
Fr. .'21 I·Tg (nM)
(A)
0.02's-.---------... ~
0.01
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Fre. ' .....Tg (nM)
B
O.ooO-t---or---'I""""----t 0.00
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Figure 1.3: Saturation Binding Assay of Tg Binding on Chicken Embryonic Flbroblast Cells Cells were Incubated with Increasing amounts of radiolodlnated Tg at 4°C. A In the presence (triangles) or absence (circles) of2 mg/ml nonradlolodlnated Tg. Non-specific binding (A, triangles) was subtracted from total binding (A, circles) to give specific binding ofTg to chicken embryonic fibroblast cells (B, filled circles). B The amount of bound Tg increased with Increasing amounts of freeTg, and was saturable at 66 nM Tg. C Scatchard plot analysis demonstrated a dissociation constant of 33 k d •
in the region of the MPR 300 protein suggesting binding of the iodinated Tg to the CEF cell MPR 300 protein. The binding capacity of Endo H, PNGase F and alkaline phosphatase treated Tg to MPR 300 protein was demonstrated in vitro using the purified M6P /IGFIIR as a probe in"a far Western blot analysis. Tg untreated with enzymes, showed clear recognition by the receptor (Figure 1.5B, lane 1). Treatment of the Tg with Endo H, PNGase F or with phosphatase completely abolished the binding (Figure 1.5B, lanes 2, 3 and 4) suggesting the importance of m6p moieties for specific binding.
Degradation of Tg in Chicken Embryonic Fibroblast Cells Intracellular degradation of Tg was analyzed by incubation of CEF cells with radioiodinated Tg at 37"C with or without 5 mM m6p as described under methods. The celllysates were irnrnuno-precipitated using monoclonal antibody to Tg and
100
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Figure 1.4: Binding and Internalization of [125ITg] by CEF Cells Cells were incubated with [125I-Tg] without any ligand and with different ligands shown in as described under methods. The per cent of radioactivity bound (panel A) and internalized (panel B) was analyzed.
Emerging Trends in Biological Sciences
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., -. (A)
2
1
-
.•
2
(8)
3
4
(C)
Figure 1.5: In vitro binding of Tg to CEF MPR 300 A. CEF membrane proteins (25 IJg) were separated on 7.5 per cent SOS-PAGE, transferred to PVOF membrane and incubated with 5, 00,000 cpm of radioiodinated Tg, washed and exposed to X-ray film. Protein band detected by autoradiography. B. Binding of native and enzymes treated thyroglobulin with purified CEF cell M6R! IGFIIR was analyzed by far Western blotting using M6P/IGFIIR as a probe Thyroglobulin (20 IJg) was incubated with buffer alone (lane 1), Endo H (lane 2), PNGase F (lane 3) and alkaline phosphatase treated (lane 4). C. Mannose 6-phosphate dependent endocytosis of Thyroglobulin by CEF cells. Thyroglobulin was incubated with CEF cells in the presence and absence of 5 mM mannose 6-phosphate. From these celllysates, Tg was immunoprecipitated, analyzed by 7.5 per cent SOS-PAGE and protein bands detected by fluorography. Lane 1, proteolysis of Tg can be seen in the absence of 5 mM man nose 6-phosphate while no such effect was seen in presence of 5 mM mannose 6-phosphate (lane 2).
analyzed by SDS-PAGE and autoradiography. From the results (Figure l.SC, lane 1) it is suggestive that the radiolabeled Tg was internalized and fragmented in the cells possibly by proteolysis. However the presence of m6p abolished its binding to the receptor and could not be internalized for further proteolytic breakdown (Figure l.SC, lane 2).
Fluorescence Microscopy Analysis CEF cells were incubated with Tg to study binding as well as internalization using fluorescence microscopy. Incubation of CEF cells with Tg at 41lC followed by immunolabeling, revealed a patched staining pattern along the entire cell surface (Figure 1.6A). Incubation with FITC-Iabeled Tg for 30 min at 3f'1lC, showed fluorescence of Tg in endocytic vesicles. Tg containing vesicles were distributed throughout the cytoplasm of CEF cells. For identification of those vesicles as early or late endocytic compartments, CEF cells were immunolabeled with antibodies against the lysosomal
13
Characterization of the Multifunctional Nature of the Chicken M6P/IGF-IlR Transmission Image
(a) Bind"'g of Tg at 40C to CEF cds
FIle-Tg
AryIsuIfatase A
Merge
Transmossoon lmage
(b ) Endocytosis of FITe-Tg and localization of Aryfsultatase A at 370C in CEF cells
D
FITC·Tg
M6PI IGFIIR
Me rge
TransmisstOn Image
(c ) Endocytosts of FITe-T9 and colocaliza!lon of M6pIIGFIIR at 37')C ,n CEF cells
Figure 1.6: (a) Binding of Tg by CEF Cells Cells were incubated with Tg at 4 ~ C , and were immunolabeled with monoclonal antibodies against Tg or mouse IgG (Isotopic control). Tg binding was seen along the cell surface (arrowheads) . (A-D , see details in figure , Bar 11.7 IJm). b) Endocytosis of FITC-Tg and localization of Arylsulafatase A in the endocytic compartments (A) FITC Tg, (B) localization of arylsulfatase A and (C) merge of (A and B) (see details in figure A-D, Bars, 11.32IJm) c) Colocalization of M6PIIGF-IIR and FITC-Tg within endocytic compartments of CEF cells. (A) Internalization of FITC labeled Tg at 37l!C (B) After fixation, cells were immunolabeled with polyclonal antiGoat MPR 300 antibody and TRITC-Iabeled secondary antibody. (C) Co-localization of M6PIIGF-IIR (A) and FITC-Tg (B) within early or late endosomal compartments of CEF cells. (C) merge of A and B. (See details figure A-D) Bars, 11.5IJm.
enzyme arylsulfatase. A (Figure 1.6B). In mammals it is well established that the arylsulfatase. A enzyme is a lysosomal marker. In our experiments with CEF cell lines, using the arlysulfatase A as a marker, the Tg-containing vesicles were identified as prelysosomal or early endosomes. The results indicated that Tg was bound to the plasma membrane of CEF cells at 4~, and was internalized when incubations were done at 37~. To confirm that M6P / IGFIIR and endocytosed Tg are colocalized and present in the early or late endosomal compartments, cells were immunolabeled with antibodies against M6P / IGFIlR and examined by immunofluorescence microscopy. As shown in Figure 1.6C, colocalization is distinctly observed and M6P / IGFIIR is located together w ith Tg in vesicles surrounding the nucleus corresponding to late endosomes.
14
Emerging TreruIs in Biological Sciences
Discussion The mammalian M6P /IGF-IIR has been characterized as one of the type-1 transmembrane glycoprotein belonging to P-type lectins that is present on the cell surface as well as intracellularly. The bulk of the receptor is localized intracellularly where it binds the newly synthesized lysosomal enzymes via their m6p containing N-linked oligosaccharides for subsequent sorting to endosomes and lysosomes. On the other hand the receptor at the plasma membrane endocytoses secreted lysosomal enzymes. The receptor thus recycles between the trans-golgi, plasma membrane and the endosomal compartments. The mammalian receptor has also been characterized as a multifunctional protein, binding to diverse array of ligands (Dahms et al., 2002, Bnmett et al., 1994, Gabel et al., 1989 and Uta Gasanov et al., 2006). However, the physiological significance of the binding to some ligands is still unclear. The physiological importance of multiple ligand binding to MPR 300 is evident from the fact that MPR 300-deficient mice are not viable. Lethality is apparently due to an impaired regulation of the response to IGF-ll, as viability of the MPR 300 knock-out mice is rescued by a simultaneous knock-out of IGF-ll. The mammalian MPR 300 protein was shown to play a role in the clearance and activation of hormones and growth factors, and is able to bind and internalize IGF-ll for its delivery to lysosomes in contrast to the second P-type lectin, MPR 46 which is involved in sorting ofIysosomal enzymes only. ThecDNA clones and partial sequence analysis of the putative receptors have been described in the chicken (Matzner et al. 1996) and the levels of the receptor proteins in different tissues of mammals and chicken vary (Suresh et al., 2006). Further structural analysis of the fish, chicken and the mammalian MPR 300 receptors revealed that the MPR 300 protein exhibits a conserved cassette structure in the amino terminal region throughout the vertebrates (Udaya Lakshmiet al., 2(00). The focus of research in our laboratory is to understand the structure and function of the putative MPR proteins in the animal kingdom. Although it has been well established that the mammalian MPR 300 protein is multifunctional and is capable of binding IGF-ll and other ligands, it was not clear Until recently whether this property is conserved in non-mammalian vertebrates. In a recent study we have shown that purified goat and CEF cell MPR300 protein are able to bind IGF-ll suggesting the possible multifunctional nature of these receptors (Suresh et al., 2006). Since we characterized that the CEF cell MPR 300 protein binding to IGF-ll, we wanted to analyze if this receptor can also bind other ligands that are known to interact with the II\aIIlInalian receptors and whether the multifunctional nature of the MPR 300 is also conserved among the non-mammalian vertebrates. Bovine Tg has been well characterized (Allenet al., 1993 and Eduardoet al., 1987) and has been used by earlier workers to study MPR 300 binding. Therefore, in the present study, we also used bovine Tg [Due to the non availability of chicken Tg, this was not used in the study] and the first evidence that the purified chicken M6P /IGFllR 300 protein (purified from CEF cells and chicken liver) binds to bovine-Tg in a m6p dependent manner was obtained by passing the radiolabeled receptors on TgSepharose gel. The specificity of the binding has been further substantiated by its elution from the gel by mannose6-phosphate but not by glucose 6-phosphate. These
Characterization of the Multifunctional Nature of the Chicken M6P/IGF-IIR
15
data suggest that the receptors from both CEF cells as well as from the chicken liver can bind on Tg-gel suggesting Tg as an additional ligand to this receptor protein in CEF cells. [Since we have been using the goat receptors as a mammalian model for our studies, we have also checked if the purified goat MPR 300 protein can bind Tg, and we found that this mammalian receptor also binds Tg, like the human receptor in a M6P dependent manner, data not shown1. Further studies on the binding of Tg to CEF cells, revealed that it binds M6P / IGFIIR 300 with high affinity and reaches saturation at 66 nM with Kd of 33 nM. Preincubation of CEF cells with 5 mM mannose 6-phosphate, anti-goat MPR 300 antibody showed about 80 per cent inhibition where as glucose 6-phosphate, human IGF-II did not show any remarkable inhibition on Tg binding and internalization. In a competitive binding assay with unlabeled Tg there is decline in surface bound radiolabeled Tg showing the competition between labeled and unlabeled Tg to bind M6P /IGFIIR (data not shown). Prior treatment of Tg with Endo H, PNGase F and alkaline phosphatase showed about 90 per cent inhibition. In far-Western blot analysis deglycosylated, dephosphorylated Tg did not show binding with M6P /IGFIIR compared to the untreated Tg. These data suggest that glycosylation and phosphorylation of Tg is important for binding to M6P /IGF-IIR. Additionally, immunofluorescence microscopy studies reveal that the Tg can bind to the M6P /IGFIIR on the CEF cell surface as detected using a monoclonal antibody to the Tg. Furthermore, experiments with fluorescent labeled Tg also reveals the clear distribution and colocalization of the Tg with the M6P /IGFIIR in the cytoplasm and perinuclear region. The detection of Arylsulfatase A enzyme in CEF cells in endocytic compartments suggest that these vesicles are late endosomes or prelysosomes because lysosomal enzymes distribution was consistent with the fact that the late endosome and prelysosomal network is the compartment where newly synthesized acid hydrolases containing the m6p marker are sorted from proteins that will be secreted and also it is well established in mammals that the M6P /IGFIIR localized in living cells to different endocytic compartments and the detection of the Arylsulfatase-A enzyme in these endocytic compartments along with FITC-Tg strongly supports the role of CEF M6P /IGFIIR in internalization of this specific ligand. Our experimental data shown in Figure 1.6, clearly support the idea that Tg taken up by the CEF cells via the M6P /IGFIIR is routed to the lysosomes through the intracellular compartments of the endocytic pathway and tra~ported from the early and late endosomes to the pre-lysosomal vesicles like the arylsulfatase A. It has been shown that in mammals, in cultured hepatocytes, most of the glycoproteins particularly Tg are internalized either by galactose or asialoglycoprotein receptors (Ashwell et al., 1982). However such receptors are absent in the chicken liver (Regoeczi et al., 1986 and Lunney et al., 1976). In view of this and the results we obtained described above for Tg and arylsulfatase A, it could be speculated that possibly the M6P /IGF-IIR might be a candidate for binding and clearance of circulating glycoproteins in the chicken. However, to support this, additional detailed experimentation using a wide variety of glycoproteins needs to be done.
Emerging Trends in Biological Sciences
16
Our studies carried out so far, clearly demonstrate that the CEF cells endocytose· bovine Tg through the M6P /IGF-IIR present on the cell surface which is then targeted to the lysosomes for degradation. However it remains to be established whether the . results found with the CEF cells, can be extrapolated to the chicken liver hepatocytes. Also it is not known the levels of the MPR 300 in chicken thyroidal tissues and its physiological significance. Further studies are necessary to gain new information on these which might give the necessary inputs to study this receptor and its interaction with Tg. In summary, this is the first report to provide evidence that the CEF MPR 300 protein can also bind Tg in addition to m6p containing hydrolases and IGF-II. The study thus provides new information on the multifunctional nature of the chicken MPR 300 protein and provides a strong functional significance for the conserved cassette structure of the MPR 300 protein in the vertebrates.
Acknowledgements Prof. NSK thanks DST for a research project on MPR proteins SR/SO/BB/602003. The authors thank Prof. Dr. Volkmar Gieselmann, University of Bonn, Germany, for generously providing the arylsulfatase A antibody. YSRK thanks CSIR, India for a Senior Research Fellowship.
References AlIen, B., Rawitch, H., Pollock, G. and Shi-Xin Yang, 1993. Thyroglobulin Glycosylation: Location and Nature of the N-linked Oligosaccharide units in Bovine thyroglobulin. Arch. Bioche. Biophy., 300(1): 271- 279. Ashwell, G. and Harford, J., 1982. Carbohydrate-specific receptors of the liver. Ann. Rev. Biochem., 51: 531-554. Brunett, CR, Burke, RL., Kornfeld, S., Gregory, W., Masiarz, P.R, Dingwell K.S. and Johnson, D.C, 1994. Herpes simplex virus glycoprotein D acquires mannose 6Phosphate residues and binds to mannose 6-phosphate receptors. J. Bioi. Chem., 269: 17067-17074. Dahms, N.M., Michael, K. and Hancock, A., 2002. P-Type lectins. Biochem. Biophys. Acta, 1572: 317-340. Eduardo, C, Angela, M., Acquaviva, S., Domenico, S.P., Liguoro, S., Adriana, G.S.,Tassi, V.S., Stebanijll, P .S., Lucaij, MD., Sidney, S., Herman, J., Yehij and C, Leonard, D.K., 1987. Characterization of phosphate residues on thyroglobulin. J. Bioi.
Chem.,262: 10304-10314. Gabel, CA., Dubey, L., Steinberg, S.P., Sherman, D., Gershon, M.D. and Gershon, A.A., 1989. Varicella-zoster virus glycoprotein oligosaccharides are phosphorylated during posttranslational maturation. J. Virol., 63: 4264-4276. Lemansky, P. and Herzog, V., 1992. Endocytosis of Thyroglobulin is not mediated by mannose 6-phosphate receptors in thyrocytes. Eur. J. Biochem., 209: 114-119. Lunney, J.K. and Ashwell, G.A., 1976. Hepatic receptor of avian origin capable of binding specifically modified glycoproteins. Proc. Natl. Acad. Sci., USA, 73: 341343.
Characterization of the Multifunctional Nature of the Chicken M6PIIGF-IIR
17
Matzner, U., Hille-Rehfeld; A., Von Figura, K. and Pohlmann, R., 1996. Expression of mannose 6-phosphate receptors in chicken. Dev. Dyn., 207: 11-24. Regoeczi, E. and Hatton, M.W., 1986. Asialoglycoprotein clearance in the chicken: evidence for the lack of a hepatic asialoglycoprotein receptor. Can. J. Physiol. Pharmacol., 54(1): 27-34. Siva Kumar, N. and von Figura, K., 2002. Identification of the putative mannose 6-phosphate receptor (MPR 46) protein from the invertebrate Mollusc. Biosci. Rep., 22: 513-521. Suresh, K., Raju, V.S.N., Siva Kumar, N., von Figura, K., Pohlmann, R. and Dennes, A., 2006. The early Vertebrate Danio reria Mr 46000 mannose 6-phophate Receptor: biochemical and functional characterization. Dev. Genes. Evol., 216(3): 133-134. Suresh, K., Siva Ramakrishna, Y. and Siva Kumar, N., 2006. Mannose 6-phosphate receptor (MPR 300) from goat and chicken bind Human IGF-II. Biosci. Rep., 26: 101-112. Suresh, K., Ramanadham, M. and Siva Kumar, N., 2002. An ELISA method to quantify the mannose 6-phosphate receptors. J. Biochem. Biophys. Meth., 52: 11-119 Uta Gasanov, Craig Koina, Kenneth, W., Beagley, R., John Aitken and Philip, M.H., 2006. Identification of the insulin-like growth factor 11 receptor as a novel receptor for binding and invasion by Listeria. Monocytogenes. Infec. and Immune., 74: 566577. Udaya Lakshmi, Y., Siva Kumar, N., Schu, P, von Figura, K.and Hille-Rehfeld, A., 2000. Conserved cassette structure of vertebrate Mr 300 kDa mannose 6-phosphate receptors: Partial cDNA sequence of fish MPR 300. Comp. Biochem. Physiol., 127B: 433-441.
Chapter 2
Field Studies of AM Fungi in Cereals and Millets K. Ammani Department of Microbiology, Acharya Nagarjuna University, Nagarjunanagar-522 510, Guntur (Dt.), A.P.
ABSTRACT The optimal development of crop plants demands a high input of minerals and fertilizers. The use of these substances is not only costly and energy demanding but also pollute soils and ground water. Hence, plant development may be improved by using biofertilizers. Arbuscular Mycorrhiza (AM), is one such biofertilizer which play a vital role in enriching soil fertility and plant growth. These fungi are most abundant in all agricultural soils and simultaneously colonizing the root and rhizosphere thereby absorbing greater quantities of water, minerals and fertilizers. There is little work on how the mycorrhizae help the agriculturally important crops under field conditions. Hence field experiments were conducted to evaluate the potential of AM fungi as biofertilizer. All the crop plants studied were found to be mycorrhizal. The AM fungi showed three phases of development in rice varieties. Also the plants with highest colonization showed more yield. Altogether 20 different AM fungi were reported in the present study. Among them Glomus is the most dominant followed by Sclerocystis, Scuteliospora, Gigaspora and Acaulospora.
Introduction Arbuscular mycorrhizal (AM) fungi are the most abundant fungi in agricultural soils found under all climates and in all ecosystems. These fungi simultaneousiy colonize the root and their rhizosphere and spread on several centimeters in the form
Field Studies of AM Fungi in Cereals and Millets
19
of filament. The filamentous network present inside and outside the root helps in absorbing greater qUaI'ltities of water and mineral salts. As a result of the improved nutrition mycorrhizal plant has better growth. The majority of cultivars are colonized by mycorrhizae. AM fungi were reported so far on grasses (Ammani et al., 1994), rice varieties, (Ammani et al., 1985), sugarcane varieties (Ammani, 1988), medicinal plants (Swapna et al., 2008, Sudha et al., 2008) in our area. The beneficial effects of indigenous VAM fungi on the nutrition of plants depends on both the abundance and type of fungi present in the soil (Ammani, 1989) but the contributions made by naturally occurring VAM fungi to plant nutrition has not been appreciated (Abbott and Robson, 1982). However, there is little work on how mycorrhizae helps the agriculturally important crops under field conditions. Hence, experiments were conducted to study the development of indigenous VAM fungi in cereals and millets in field plots in the Botanical garden and at Regional Agricultural Research Station, Lam.
Materials and Methods Upland Rice Seeds of five upland rice varieties viz., lET 7579, lET 7566, lET 7265, lET 7564, lET 7254 were obtained from the Directorate of Rice Research, Rajendranagar, Hyderabad and raised in field plots in the botanical garden. Five plants were removed at random from 20 days onwards up to harvest at regular intervals and their roots were examined for the presence of AM fungi. Percent external and internal colonization and average number of arbuscules and vesicles / spores were recorded. The number of tillers per plant, number of vesicles, grain yield and weight of 100 seeds were also recorded.
Proso Millet Fourteen varieties of proso millet viz., Br 2, Br 8, Br 379, IS 1575, L Ill, L 1512, L 232, L 4863, GS 4056, IS 363, GPUP 2, LV 13, Br 44 and L 5224 were raised in experimental plots at RARS, Lam. Five plants at random were removed for each variety at suitable interval and the following characters were recorded: days to 50 per cent flowering, days to maturity, plant height, number of basal tillers, grain yield (t/ h). The percent external and internal colonization of roots, the number of arbuscules and vesicles were determined.
Fodder Sorghum Seeds of 14 cultivars of fodder sorghum viz., UP chari 2, SRS 3, S 431, Madhira local, UPFS 23, UPFS 21, UPFS 22, PC 65, PC 66, PC 67, HC 260, S 411, S 184 and IAR! 13 obtained for RARS, Lam were raised in the Botanical garden in a randomized block design. Sampling was done in both vegetative and flowering stages and the data was recorded in the same manner as mentioned above.
Italian Millet Seeds of 9 varieties of Italian millet viz., SIA 2579, SIA 2593, SIA 2601, SIA 2640, ATPS 83, ATP 84, SIA 326 (Prasad), Arjuna and Nandyal were raised in the experimental plot in the botanical garden. Sampling was done both at the vegetative
Emerging Trends in Biological Sciences
20
and harvest stages and the roots were examined for the fungus. At least 50 root segments were observed for each cultivar and the percentage colonization was estimated as described earlier. At the last sampling plant height and yield data was recorded.
Results and Discussion AM Fungi in Upland Rice Varieties A study of development of VAM fungi in the roots of five upland varieties of rice
viz., lET 7579, lET 7566, lET 7265, lET 7254, lET 7564 was taken up in the experimental plot of the botanical garden in the kharif season.The progress of colonization on the roots of these varieties was assessed from 20 days after sowing up to harvest (100 days) at 20-day intervals. Colonization was assessed as: per cent external colonization, percent internal colonization, percent root segments with arbuscles and number of vesicles/spores per one cm root length. At the harvest stage plant height, shoot dry weight, number of tillers per plant, number of spikelet/panicle, grain yield and weight of 100 seeds were recorded. In the first sampling, at 20 days of plant growth there was no colonization. Both external and internal colonization was observed from 40 days onwards. The fungus entered the host root mainly through epidermal cells forming an appressorium and rarely through root hairs.
The AM fungus showed three phases of development in all the varieties, viz., a phase of slow development up to 60 days, by the end of which a few roots became mycorrhizal, a phase of gradual increase in colonization up to 80 days and finally a constant phase, where there was no further increase in root colonization. Arbuscles were noticed in the third sampling i.e., at 60 days. The colonization of roots was highest in the variety lET 7579 and least in lET 7566 when compared with the three varieties (Table 2.1). The shootlength, dry weight and grain yields were also highest in lET 7579 and lowest in lET 7566 (Table 2.2). The numbers of vesicles were also more inlET 7579 when compared with the other varieties. Table 2.1: Mycorrhlzal Colonization of Five Upland Rice Varieties 40DAS
Variety
a
b
lET 7579
16
12
lET 7566
4
4
lET 7265
12
8
lET 7254
12
12
lET 7564
4
8
c
60DAS
d
- - -
SODAS
a
b
c
d
36
44
20
16
20
4
24
36
28 20
100DAS
a
b
c
d
18
48
56
12
3
24
30
6
16
9
30
40
4
32
12
17
40
56
10
28
12
14
42
48
-
a
b
c
d
58
60 64 40
44
34
48
56
40
48
60
39
52
56
-
89
23
58 67 72 72
All the values are means of three replicates. a: Per cent extemal colonization; b: Per cent intemal colonization; c: Per cent roots with arbuscules; d: Average no. of vesicles/spores per one cm root bit; DAS: Days after sowing.
21
Field Studies of AM Fungi in Cereals and Millets Table 2.2: Yield Attributes In Five Upland Rice Varieties Variety
Plant Height (cm)
Number of Tillers/Plant
Spikelet Number/ Panicle
Grain Yield tonnes/ha
100 Seed Weight (g)
lET 7579
108
6
35
2.2
2.4
lET 7566
88
3
21
1.8
1.3
lET 7265
93
5
33
1.9
2.1
lET 7254
90
7
24
2.1
1.8
lET 7564
98
4
30
2.0
2.2
All the values are means of three replicates.
AM Fungi in Proso Millet (Panicum miliaceum Linn.) Fourteen varieties of Proso millet viz., Br 2, Br 8, Br 379, IS 1575, L 111, L 11575, L 111, L 1512, L 232, L 4863, GS 4956, IS 363, GPUP 2, LV 13, Br 44, and L 5224 raised in experimental plots in a randomized block design at RARS, Lam were chosen for the present study. The percent external colonization and internal colonization of roots, the number of arbuscules and vesicles/spores were determined (Table 2.3) by the technique of Phillips and Hayman (1959). Colonization of roots by the fungus at harvest time (80 days from sowing) was assessed as: 1. Percent external colonization 2. Percent internal colonization Table 2.3: Plant Growth Parameters of Proso Millet SI. No.
Entry
Days to 50% FlOWering
Days to Maturity
Plant Height (cm)
No. of Basal Tillers
No. of Nodal Tillers
Grain Yield (a/ha)
Straw Yield (a/ha)
1.
BR8
43
82
75.4
3.13
7.13
17.7
24.8
2.
BR 379
43
77
66.0
2.53
5.07
12.3
18.4
3.
IS 1575
37
81
84.3
2.67
6.27
17.1
27.2
4.
L 111
39
69
61.0
2.13
4.07
25.1
37.7
5.
L 1512
43
72
69.4
2.67
4.73
17.3
31.5
6.
L232
45
77
74.2
2.73
4.73
13.1
20.9
7.
L4863
44
83
68.3
3.33
6.87
22.1
47.7
8.
GS 4956
42
73
66.7
3.47
6.47
12.9
20.3
9.
IS 363
38
70
65.9
2.27
4.13
11.1
17.2
10.
GPUP2
54
89
92.0
2.57
4.67
22
45.8
11.
BR2
40
72
78.9
2.93
5.93
10.2
15.2
12.
LV 13
45
78
SO.1
2.87
5.40
14.3
24.5
13.
BR 44
42
76
71.5
2.87
6.SO
16.2
28.3
14.
L5224
48
85
67.8
3.73
6.27
21.7
43.5
Emerging Trends in Biological Sciences
22
3. Percent roots with arbuscules 4. Number of vesicles / spores per 25 root segments ofl cm length. Of all the varieties, L 111 and GPUP 2 showed higher level ot colonization (80 percent and 70 percent of root segments respectively) (Table 2.4). The number of vesicles was also higher (255) in L 111 and (185) in GPUP 2. Incidentally, these two varieties gave maximum grain yield and straw yield of all the varieties tested. The grain yieid of IS 363 showed least colonization (30 per cent) and also minimum yield (11.1Q/ha) when compared with other varieties. However Br 2, a variety which gave low yield showed moderate colonization (65 per cent). IS 1575, L 232, L 5224 which gave average yields also showed moderate level of colonization. Only Glomus spp. was found to occur in all the 14 varieties. PopulationofVAM fungi inRARS soil was much lower, both qualitatively and quantitatively than in the University Botanical garden soil. Only 25 spores per 100g soil were recorded in December 1988. Table 2.4: Mycorrhizal Colonization of Different Varieties of Proso Millet at Harvest SI. No.
Variety
Per cent External Colonization
Per cent Internal Colonization
Per cent Roots with Arbuscules
Average No. of Internal Vesicles/ Spores
1.
BR8
5
45
15
29
2.
BR 379
40
10
101
3.
IS 1575
70
30
151
4.
L 111
10
80
30
255
5.
L 1512
5
65
10
117
6.
L232
70
20
40
7.
L4863
5
50
5
4
8.
GS 4956
25
50
5
4
9.
IS 363
45
30
10.
GPUP 2
45
75
20
185
13
11.
BR2
40
65
20
55
12.
LV 13
20
55
10
48
13.
BR 44
55
30
10
65
14.
L5224
70
20
65
All the varieties are means of three replicates.
Study of V AM Colonization and Plant Growth in Fodder Sorghum In order to study VAM colonization in fodder Sorghum, 14 varieties viz., UP chari 2, SRS 3, S 431, Madhira local, UPFS 23, UPFS 21, UPFS 22, PC 65, PC 67, HC 260, S 411, S 184 and IAR! 13 obtained from RARS, Lam were sown in a randomized block design in both kharif and rabi seasons (1988), in the University Botanical garden. Five plants were uprooted for each variety at the vegetative and harvest stages and VAM colonization of roots and plant height, shoot dry weight and fodder yield were recorded at harvest in both kharif and rabi seasons (Table 2.5).
23
Field Studies of AM Fungi in Cereals and Millets Table 2.5: Mycorrhizal Colonization of 14 Varieties of Fodder Jowar in Kharifand RabiSeasons Khari!
Variety
Rabi
Vegetative
2
UP Chari 2 SR53 S 411 S 431 S 184 Madhira Local UPFS 23 UPFS 21 UPFS 22 PC 65 PC 66 PC 67 HC 260 IARI13
Harvest
2
3
32 30 20 22 40 42 50 52 30 69 70 72 46 46 15 51 54 54
-
56 58 46
-
72 74 76
-
78 64 66 48 48
-
38 34 38 54 54 60 62 48 44 40 40
42 46 42 56 54 56 64 46 44 38 46
3
15 15 20 20 28 32 38 30 26 16 30
4
42 47 39 68 77 83 91 63 68 29 59
56 52 40 72 70 70 72 68 68 46 58
-
-
Vegetative
2
3
4
53 20 24 10 12 28 28 160 44 46 20 43 62 66 89 42 38 5 42 40 50 108 34 36 5 39 48" 48
-
39 151 71
98 32 40 72 36 40 167 52 50 168 50 52 179 56 54 194 58 62 149 40 48 157 40 32 59 38 36 122 36 38
-
4
2
Harvest
3
15 5 18 20 20 30 25 20 5 20
4
38 27 59 68 71 82 52 58 17
42 38 60 68 68 70 58 46 40
46 42 58 64 66 72 54 52 42
32
42 40
-
-
-
89 61 61 144 156 172 181 132 128 47 101
All the values are means of three replicates. 1: Per cent external colonization; 2: Per cent internal colonization; 3: Per cent of roots with arbuscules; 4: Average no. of vesicles/spores.
All the varieties were found to be mycorrhizal and there was a progress in percent root colonization and in the number of vesicles, from vegetative to harvest stages. Of all the varieties, PC 65 showed highest colonization and maximum fodder yield whereas UP Chari -2 showed least colonization and very low yield in both lazrij and rabi seasons. The remaining varieties showed moderated colonization (Table 2.6) Both mycorrhizal colonization of roots and also the plant growth as well as fodder yield were greater in kharif than in the rabi season in all the varieties.
Colonization of V AM on foxtail millet (Setaria italica) With a view to study the colonization pattern in foxtail millet, nine varieties viz., SIA 2579, SIA 2593, SIA 2601, SIA 2610, ATPS 83, ATPS 84, SIA 326 (Prasad), Arjuna and Nandyal were raised in the Botanical garden in a randomized block design. Five plants were removed from each replicate at 40 and 80 days of plant growth and VAM colonization as well as the following plant growth parameters: days to maturity, plant height, straw yield and grain yield were recorded at harvest (Table 2.7).
Emerging Trends in Biological Sciences
24
Table 2.6. Plant parameter of 14 varieties of fodder Sorghum SI.No.
Kharif
Variety
Plant Height Shoot Dry Weightg/ (cm) Plant
Rabi Fodder YieldOlha
Plant Height (cm)
Shoot Dry Weightg/ Plant
Fodder YieldQ/ha
70.4
3.2
280
80.9
4.8
300
SR53
102.7
6.8
352
104.1
5.1
342
S411
130.8
7.1
400
122.0
6.2
382
1.
UP Chari 2
2. 3.
.
4.
S431
114.2
7.2
420
110.1
6.4
400
5.
S 184
102.9
6.8
390
109.4
6.1
382
6.
Madhira Local
124.8
7.2
440
99.3
6.6
420
7.
UPFS 23
119.9
7.4
460
122.4
6.8
446
8.
UPFS 21
129.2
7.8
488
110.2
7.4
475
9.
UPFS 22
139.9
7.8
490
119.5
7.6
488
10.
PC 65
148.4
8.2
510
121.8
7.8
492
11.
PC 66
122.2
8.1
504
100.9
7.7
482
12.
PC 67
102.6
6.4
348
112.7
4.9
332
13.
HC 260
99.9
6.5
350
89.9
5.0
340
14.
IARI13
97.2
5.4
302
91.2
3.4
292
Table 2.7: Progress of Colonization in 9 Varieties of Foxtail Millet SI. No.
40DAS
Name of the Variety
SODAS
2
3
4
2
3
4
1.
SIA2579
58
62
24
74
70
78
134
2.
SIA2599
54
60
14
61
62
68
121
3.
SIA2593
50
54
20
43
60
60
109
4.
SIA2601
52
40
14
28
56
54
93
5.
SIA2610
40
42
5
31
54
54
89
6.
SIA326
44
58
12
32
58
60
161
7.
A~una
34
38
10
21
50
52
74
8.
ATPS83
36
36
10
21
50
48
61
9.
ATPS 84
32
34
10
18
48
45
57
DAS: Days after sowing; 1: Per cent external colonization; 2: Per cent internal colonization; 3: Per cent of roots with arbuscules; 4: Average no. of vesicles/spores.
In both the samples, all the varieties were found to be mycorrhizal. SIA 2579 showed highest colonization and maximum yield when compared to the remaining eight varieties. Of the other varieties, SIA 2599, SIA 2593, SIA 2601, SIA 2610 and
25
Field Studies of AM Fungi in Cereals and Millets
Prasad showed moderate infection whereas Arjuna and ATPS- 84 showed least colonization and low yields in both the samplings (Table 2.8). Table 2.8: Plant Parameters of Nine Varieties of Fox Tail Millet at Harvest SI. No
Name of the Variety
Source/Origin
Days to Maturity
Plant Height
Grain Yield O/ha
Straw Yield O/ha
1.
SIA 2579
Selection from SIA 1063
81
91.9
28.2
30.2
2.
SIA 2599
81
89.3
25 .2
25.8
3.
SIA 2593
83
83.7
23.2
23.5
4.
SIA 2601
Selection from
93
84
74.6
20.0
20.9
5.
SIA 2610
Selection from P. 313
87
75.9
20.0
22.2 20.8
Selection from
I.se
25
Selection from (326X342)
I.se
6.
SIA 326 (Prasad)
Nandyal
78
80.9
20.4
7.
Arjuna
Guntur
80
69.9
12.8
16.2
8.
ATPS 83
Anantapur
83
71.4
14.3
15.8
9.
ATPS 84
Anantapur
85
64.5
12.2
13.2
The present study reports the occurrence of 20 AM fungi in the different crop plants surveyed. The fungi isolated were found to belong to species of Acaulospora (Figures 2.1 and 2.2), Glomus (Figures 2.3 and 2.6), Gigaspora (Figure 2.5), Sclerocystis (Figure 2.4) and Scutellospora. Of these Acaulospora was represented by two species, Sclerocystis by four species, Glomus by 12 species and Scutellospora by two species.
Figure 2.1 : Spore of Acaulospora
Figure 2.2: Pitted Spore Surface of Acaulospora
Figure 2.4: Part of Figure 2.6: Broken Spore of Sporocarp of Sclerocystis Glomus Showing Wall Layers
26
Emerging Trends in Biological Sciences
A cluster of spores that were about to release into the soils was captured. The dimorphic nature of fungi and angular projections that are the characteristic features of the fungi are clearly revealed in this photograph. This clearly suggests that a wide variety of AM fungi occur in our soils and they play a crucial role in the growth and yield of these crop plants Most of the AM fungi identified closely resembled the type species with minor deviations from the original descriptions. For example, Acaulospora scrobiculata closely resembled the type species (Trappe, 1977) in all respects except for slightly larger spore size. Sclerocystis clavispora differed from the original by the presence of a prominent and well developed peridium while all other features agree with those of the type form. The peridial form of S. clavispora is probably new to the literature. G. fasciculatum also differed slightly in size, shape and morphology of chlamydospores but it did not significantly depart from that of the original description. These minor variations of the reported AM fungi from the original descriptions might be due to the different physico-chemical factors of soil and climate and also due to the bio-chemical factors of the host plant.
References Abott, L.K and Robson, AD., 1982. The role of vesicular arbuscular mycorrhizal fungi in agriculture and the selection of fungi for inoculation. Aust. J. Agr. Res., 33: 389. Ammani, K, Venkateswarulu, K and Rao, AS., 1985. Developmental of vesicular arbuscular mycorrhizal fungi on upland rice variety. Curr. Sci., 54: 1120-1122. Ammani, K, Venkateswarulu, K and Rao, A.s., 1994. Vesicular arbuscular mycorrhizae in grasses: Their occurrence, identity and development. Phytomorphology, 44: 159-168. Ammani, K, Rao, Y.V. and Rao, AS., 1988. Incidence of vesicular arbuscular mycorrhizae in sugarcane. In: First Asian Conference on Mycorrhizae, Madras, India, pp. 29-31. Ammani, K, 1989. A study of vesicular arbuscular mycorrhizal fungi in soils of Prakasam and Guntur districts and their association with cerals and grasses. PhD. Thesis, Acharya Nagarjuna University. Phillips, J.M. and Hayman, D.S., 1970. Improved procedures for clearing and staining parasitic and vesicular arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycl. Soc.,55: 158-161. Swapna, V.L., Sunil Babu, K and Ammani, K, 2008. Survey of AM fungi on medicinal plants in and around Acharya Nagarjuna University. Asian Journal of Microbiology, Biotechnology, Env. Sci., 4: 1-9. Sudha, K and Ammani, K, 2008. Survey of AM fungi in medicinal plants in Thrissur district, Kerala. (Communicated). Research Journal of Biotechnology. Trappe, J.M., 1977. Three new Endogonaceae: Glomus constrictus, Sclerocystis clavispora and Acaulospora scrobiculata. Mycotaxon, 6: 359-366.
Chapter 3
Survey of Arbuscular Mycorrhizae in Plants of Nandyal and Anantapur Environments G. Mary Sandeepa1, K. Ammani2 and A. Subramanyam1 1
Department of Biotechnology; 2D'epartment of Microbiology, Acharya Nagarjuna University, Guntur-522 510, A.P.
ABSTRACT The growth of the plant depends on the availability of mineral elements present in the soil. Among these elements phosphorous is one of the important macroelement. About 98 per cent of Indian soils have inadequate supply of available phosphorous. The phosphorous from soils was supplied to plants by symbiotic mycorrhizae. At least 7 different types of mycorrhizal associations have been recognized. Among these arbuscular mycorrhizae is the most valuable and beneficial plant symbiont. It was found as a symbiont in 80 per cent of the terrestrial plant species. A survey was made for arbuscular mycorrhizae colonized in the roots of terrestrial plants distributed in soils of Nandyal and Anantapur. Out of 69 species collected in the present study, 74 per cent of plants found to contain VA mycorrhizae. In Nandyal region plants were collected from 4 sites. The percentage of plants showing VAM in these sites is 77 per cent, 100 per cent, 75 per cent, 100 per cent respectively. In Anantapur, plants were collected from two sites. The percentage of plants showing VAM colonization here was 63 per cent and 57 per cent. The spore types and number in the soil was assessed. Glomus mosseae, G. fasciculatum were found to be the predomina ting species and Gigaspora species occurred very less frequently.
Keywords: VAM, Nandyal, Anantapur.
28
Emerging Trends in Biological Sciences
Introduction The proper growth of the plant depends on the availability of mineral elements in soil and among such elements phosphorous is an important macro element. About 98 per cent of Indian soils have inadequate supply of available phosphorous. The phosphorous from soils was supplied to plants by symbiotic mycorrhiza. At least 7 different types of mycorrhizae associations have been recognized. Among them Vesicular and arbuscular mycorrhizae is the most valuable beneficial plant symbiont. About 80 per cent of all terrestrial plant species formed this type of symbiosis (Smith and Read 1997). They are found in wide range of habitats usually in roots of the angiosperms, gymnosperms and pteridophytes. They also occur in gametophytes and in some mosses,lycopods and Psilolates which are all root less (Pocock and Duckett, 1985). Recently they are also found in aquatic plants (Beck- Nielson and Madsen, 2001). The importance of VA mycorrhizae in the growth and nutrition of plants has recently been reviewed (Gianinazzi and Schhepp, 1994, Harrison, 1999) on soils low in available phosphate, the improved growth observed in vesicular arbuscular mycorrhizal plants compared with uninoculated controls is thought to be due largely to improved supply of phosphate. Despite these data the functioning of arbuscular mycorrhizae in the fields is attracting increasing attention (Ammani 1989; AlIen, 1991). Information on the occurrence, distribution and identification on VAM in plants growing in and around Nandyal'is still meager. Hence the present investigation is aimed in determining in nature, occurrence, distribution and identification of VAM fungi in the different soil types selected and their association with the plants in those soils.
Materials and Methods The survey was taken up in order to determine the occurrence and abundance of VAM fungi present in different plants distributed in different locations occurring in Nandyal, Kumool and Anantapur sites of Andhra Pradesh. Soil samples were collected in Nandyal, from Sri Ramakrishna degree college hostel surrounding (SRDC), from fields of Chabole village, and fields between N andyal and Mahanandi (FNM); In Kumool from C camp colony(KCC), in Anantapur from surrounding fields of Anantapuram(SFA) and Gooty Anantapuram village (GATP). Soil types selected were sandy, a silty loam, day loam, red and black soils. Sampling was carried out every month from October 2005 to October 2006. At the time of collection soil sample was picked up from 10 cm depth at five random sites by digging with a trowel after scraping the soil surface. In this manner five samples were collected mixed thoroughly, transferred to polythene bags, labeled, brought to the laboratory and stored in refrigerator.
Estimation and Identification of V A Mycorrhizal Spore Population The soil samples were passed through 2mm sieve and the spore population of each soil samples was estimated by the wet sieving and decanting method (Gerdamann and Nicolson, 1963). 100 gms of soil was taken from each site to estimate the number of spores. The root sample consists of roots of five plants of same species. Roots were
Survey of Arbuscular Mycorrhizae in Plants of Nandyal and Anantapur Environments
29
carefully dug out, washed gently, cut in to one cm segments and stained by the method developed-by Phillips and Hayman (1970). The stained root bits were placed on a clean slide covered with cover slip and pressed to observe VAM fungi. Fifty root bits were taken randomly and observed under microscope and percentage of VAM was calculated.
Results and Discussion VAM was found mmany plants collected from both Nandyal and Anantapur. Some species showed abundant infection of VAM while some species showed less colonization. The tendency of different species to have different VAM levels has been reported by Bever et al. (1996). Among 69 species selected in the present study 51 species showed the VAM colonization i.e., 74 per cent. Table 3.1 shows the mean percentage colonization in different sites. Table 3.1 : Plants Collected in and Around Sri Ramakrishna Degree College Hostel SI. No.
Name of Plant
%of External Hyphae
% of Arbuscules No. of No. of Spores Vesicles per 100 grams Internal Hyphae ofSoi/
1.
Amaranthus virdidis L.
22
50
2.
Blumea virens DC.
58
76
3.
Capsicum frutescens L.
46
82
4.
Catharanthus roseus L.
5.
Chrysanthemum indicum DC.
o
78
66
68
6
25
58
30 9
32
6.
Cleome viscosa L
7.
Crossandra infundibuliformis L.
8.
Eclipta alba L.
8
Eleusine coracana Gaertn.
11
44 62
48
9.
19
206
18
66
10
296
12. Lycopersicum esculentum Mill.
8
18
20
10
13. Oryza sativa L.
2
14
14. Pathenium hysterophorus L.
12
15. Phaseolus trilobatus L.
27
30 96
78
16. Phyllanthus amarus
38
68
22
17. Phyla nodiflora (L.) Greene
27
26
22
18. Physalis minima L.
56
33 74
21
95
38 90
56
19. Poinsenia cyathophora
24
44
34
13
22
10. Fimbristylis bisumbe/lata (Forssk.) Bubani
11. Hibiscus cannabinnus L.
4
8
3
2344
73 10
Schum. & Thonn.
23 20
21
Klotzch & Garcke
20. Ruellia tuberosa L. 21. Sida cordifolia L.
4
60 Contd...
Emerging Trends in Biological Sciences
30 Table3.1-Contd... SI.No.
Name of Plant
%of Extemal Hyphae
%of Arbuscules No. of No. of Spores Intemal Vesicles per 100 grams Hyphae of Soil
22. Tamarindus indica L. 23. Tecoma stans (L.) Kunth
72
76
4
2
24. Tagetus patula L.
72
76
4
2
54
70
32
25. Tridax procumbens L. 26. Vemonia cinerea (L.) Less
For each plant type 5 replicates were taken. All the values are means of five replicates.
Table 3.2: Plants Collected In Fields of Chabole Village (Nearer to Nandyal) SI. No.
Name of Plant
%of Extemal Hyphae
1.
Ammania baccifera L.
14
%of Arbuscules No. of .No. of Spores Intemal Vesicles per 100 grams Hyphae of Soil
44
26
2.
Arachis hypogaea Willd.
13
48
3.
Brassica juncea (L.) Czern & Coss
52
77
65
12
53
4.
Cajanus cajan (L) Millsp.
32
28
24
48
29
5.
Cicer arietinum L.
45
65
25
32
34
6.
Coldenia procumbens L.
3
66
54
4
129
7.
Croton bonplandianum Bail.
33
82
68
873
55
8.
Leucas aspera (Willd.) Link
28
79
28
250
60
9.
Ricinus cummunis L.
10. Sorghum vulgare Pers.
20 10
44
33 4
71
45
For each plant type 5 replicates were taken. All the values are means of five replicates.
Table 5.3: Plants Collected in Fields Between Nandyal and Mahanandl SI. No.
Name of Plant
1.
Acalypha indica L.
2.
Alternanthera sessilis L.
3.
Azadirachta indica Juss.
%of External Hyphae
%of Arbuscules No. of No. of Spores Intemal Vesicles per 100 grams Hyphae of Soil
11
4
5
10
4.
Cassia tora L.
5.
Corchorus aestuans L.
6.
Ficus carica L.
14
34
7.
Portulaca oleracea L.
34
36
5
4
7
8
13 3
For each plant type 5 replicates were taken. All the values are means of five replicates.
12
31
Survey of Arbuscular Mycorrhizae in Plants ofNandyal and Anantapur Environments Table 3.4: Plants Collected in Kurnool C Camp (Kurnool District) SI. No.
Name of Plant
1.
Artemesia vulgaris
%of Extemal Hyphae
%of Arbuscules No. of No. of Spores Intemal Vesicles per 100 grams Hyphae of Soil
24
78
27
139
25 8
2.
Carica papaya L.
9
52
40
16
3.
Euphorbia indica Lam.
33
97
28
18
4.
Ocimum tenuiflorum L.
27
78
54
870
27
5.
Rumex vesicarious L.
40
82
42
152
30
For each plant type 5 replicates were taken. All the values are means of five replicates.
Table 3.5: The Plants Collected in Surrounding Fields of Anantapuram SI. No.
Name of Plant
%of External Hyphae
%of Arbuscules No. of No. of Spores Internal Vesicles per 100 grams Hyphae of Soil
1.
Acanthospemum hispidum DC.
2.
Achyranthes aspera L.
11
62
56
79
3.
Albizia lebbeck L.
7
20
7
20
4.
Boerhavia erecta L.
5.
Catharanthus pusillus (Murr) G.Don
14
42
11
24
36
66
16
145
4
62
3
47
• 6.
2
Celosia argentea L.
7.
Cyamopsis tetragonaloba (L.) Taub.
8.
Chenopodium album L.
9.
Comrnelina benghalensis L.
25 12 14
10. Indoneesiel/a echioides (L.) Sreemadhavan 11. Leucaena leucocephala (Lam) Dewit 12. Sida acuta Burm.f.
5
38
13. Solanum nigrum L.
3
26
8
6
For each plant type 5 replicates were taken. All the values are means of five replicates.
Of the 6 sites studied SRKD and KCC showed 100 per cent mean percentage of colonization. Intact, viable spores were relatively infrequent while nonviable, broken spores were much more common. Glomus mosseae, Glomus fasciculatum and Gigaspora species were observed in the collected plant species. Among all the three, Glomus mosseae is the dominating species. 38 plants showed Glomus mosseae next predominating species is Glomus fasciculatum, 11 plants showed this species. And only 2 plants showed the Gigaspora species. When tested all the soil samples showed the pH between 6.5 and 7.1 i.e., neutral pH. According to Abott and Robson (1979)
Emerging Trends in Biological Sciences
32
neutral to slightly alkaline soils are known to favour the predominance of Glomus species. Our present study is also coinciding with above statement. Table 3.6: Plants Surveyed in Surrounding Fields of Gooty Anantapuram Village SI. No.
1.
Name of Plant
Corchorus capsularis L.
2.
Digera muricata CL.) Mart.
3.
Gisekia phamaceoides L.
4.
Lablab purpureus CL.) Sweet
5.
Ipomoea pentaphylla Jacq.
6.
Peristrophe paniculata CForssk.) Brummit
7.
Stemodia viscosa Roxb.
8.
Vigna unguiculata CL.) Verdc.
%of External Hyphae
%of Arbuscules No. of No. of Spores Intemal Vesicles per 100 grams Hyphae of Soil
15
50
1
8 8
15
15
8
52
40
For each plant type 5 replicates were taken. All the values are means of five replicates.
References Abott, L.K. and Robson, A.D., 1979. A quantitave study of the spores and anatomy of mycorrhizas formed by a species of Glomus with reference to its taxonomy. Aust. J. Bot., 27: 363. AlIen, M.F.,1991. The Ecology ofMycorrhizae. Cambridge University Press, Cambridge. Ammani, K., 1989. A Study of vesicular-arbuscular mycorrhizal fungi in soils of Prakasam and Guntur districts and their association with cereals and grasses. Ph.D. Thesis, Acharya Nagarjuna University. Beck-Nielsen, D. and Madsen, T.V., 2001. Occurrence of vesicular arbuscular mycorrhiza in aquatic macrophytes from lakes and streams. Aquatic Bot., 71: 141-148. Bever, J.D., Morton, J.B., Antonovics, J. and Schultz, P.A., 1996. Host-dependent sporulation and species diversity of arbuscular mycorrhizal fungi in a mown grassland. J. Ecol., 84: 71-82. Gerdemann, J.W. and Nicolson, T.H., 1963. Spores of mycorrhizal Endogone species extracted from soil by Wet sieving and decanting. Trans. British Mycol. Soc., 46: 235-244. Gianinazzi, S. and Schhepp, H., 1994. Impact ofArbuscular Mycorrhizas on Sustainable Agriculture and· Natural Ecosystems. Birkhauser Verlag, Basel, Switzerland. Harrison, M., 1999. Molecular and cellular aspects of the arbuscular mycorrhizal symbiosis. Annu. Rev. Plant Physiol. Plant Mol. Bioi., 50: 361-389.
Survey of Arbuscular Mycorrhizae in Plants of Nandyal and Anantapur Environments
33
Phillips, J.M. and Hayman, D.S., 1970. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. British Myc. Soc., 55: 158-16l. Pocock, K. and Duckett, J.G., 1985. On the occurrence of the branched and swollen rhizoids in British hepatics: Their relationships with the substsratum and association with fungi. New Phytol., 99: 281-304. Smith, S.E. and Read, D.J., 1997. Mycorrhizal Symbiosis, 2nd Edition. Academic Press, London.
Chapter 4
Antimicrobial Spectrum of Digera muricata (L.) Mart. and Moringa pterigosperma Gaertn. S.H.K.R. Prasad1, D.RajasekharI, K. Venkateswara Ra02, M. Vijayalakshmi2 and G. Rosaiah2 1St. Ann's College for Women, Gorantla, Guntur-522 035, A.P., India 2Department of Botany and Microbiology, Acharya Nagarjuna University, Guntur-522 510, A.P., India
ABSTRACT Leaf extracts of Digera muricata and Moringa pterigosperma made in acetone, benzene, chloroform, hexane, methanol and petroleum ether were tested for their antimicrobial activity against seven bacteria and three fungi. Chloroform and benzene extracts of D. muricata and acetone extracts of M. pterigosperma were highly effective against most of the test organisms.
Keywords: Digera muricata, Moringa pterigosperma, Antimicrobial activity.
Introduction The human being appears to be afflicted with more diseases than any other animal species. Plants are man's only chemists for ages. Man uses the plants around him for curing many dreadful diseases. Though several plants are tested for their medicinal value in curing diseases, the potentiality of many plants is not yet explored against many pathogenic bacteria, fungi, protozoa and viruses (Kokate, 2003). Digera
Antimicrobial Spectrum of Digera mUrlcata (L.) Mart. and NIoringa pterigosperma Gaertn.
35
muricata is an annual herb, traditionally used as cooling astringent and laxative in large doses. Flowers and seeds are used to treat urinary disorders (Kirtikar, 1981). Moringa pterigosperma, is a small tree and its parts are used to treat different diseases. Root is used to treat asthma, gout, rheumatism, epilepsy, intermittent fever and also used to cure scurvy and wounds. Fruit is used to treat liver and spleen disorders and tetanus and seeds are antipyretic (Maheshwari and Singh, 1965; Rastogi, 1993). Although the traditional medicinal information of these plants is available, their extensive antimicrobial activities are not reported. Hence, an attempt is made in the present study to test the effect of leaf extracts of D. muricata and M. pterigosperma on the growth of several bacteria and fungi.
Materials and Methods Plant Material Digera muricata and Moringa pterigosperma were collected from Gorantla, Guntur district of Andhra Pradesh and the voucher specimens were deposited in the Department of Botany, Acharya Nagarjuna University, Guntur. The sample specimens were collected in bulk quantities for analysis.
Preparation of Extracts The collected samples were washed with water and shade dried. The dried materials were powdered and passed through no.lD mesh sieve. The powdered material was subjected to extraction with different organic solvents such as acetone, benzene, chloroform and methanol by using a soxhlet apparatus for 6 hours as per the standard scheme for preparation of plant extracts for biological screening. The extracts were filtered and concentrated under reduced pressure to dryness.
Bacterial and Fungal Strains Tests were performed on seven bacteria (Bacillus megaterium-ATCC 23564, B. subtilis-ATCC 6633, Escherichia coli-ATCC 25922, Enterobacter faecalis-ATCC 35550, Proteus vulgaris-ATCC 6380, Pseudomonas aeruginosa-ATCC 27853 and Staphylococcus aureus-ATCC 25923) and three fungal strains (Aspergillus niger- NCIM 596, A. fumigatus-NCIM 291 and Candida albicans-NCIM 670). The test organisms were sub cultured at 37°C and maintained on nutrient agar media for bacteria and Sabourad Dextrose Agar (SDA) media for fungi.
Antibacterial Screening Bacterial suspensions were obtained from 18 h old cultures on nutrient broth at 3~C. A 5 ml volume of the bacterial suspension was evenly mixed with sterile nutrient
agar medium and poured into the sterile Petriplates. The sterile Whatmann no.1 filter paper discs (6 mm diam.) fed with solvent extracts of plant products were placed on the surface of freshly inoculated medium. The plates were incubated at 3rc for 24 h in the incubator. The diameter of inhibition zone was measured and the drugs such as chloramphenicol (30J.lg/ml), gentamycin (10J.lg/ml), kanamycin (10J.lg/ml), rifampicin (10J.lg/ml), streptomycin (10J.lg/ml) and vancomycin (10J.l g/ml) were used as the reference standards in each plate (Benson, 1990).
Emerging Trends in Biological Sciences
36
Antifungal Screening Fungal suspension (5 ml) was mixed evenly in SDA medium and poured into the sterile petriplates and allowed to solidify at the room temperature. Whatmann no.1 filter paper discs of 6mm diam fed with the solvent extracts of plant materials were placed on the surface of freshly inoculated medium. The plates were incubated at 37"C for 3 days. A sample of Nystatin (10J.lg/ml) was used as reference standard. Subsequently zone of inhibition was measured to determine the antifungal activity of the extracts.
Results and Discussion The benzene extracts of leaves of D. muricata were effective against all test organisms except E. Jaecalis (Table 4.1). The chloroform extracts controlled all the test organisms except E. faecalis and P. aeruginosa. The methanol extracts inhibited the growth ofB. megaterium, B. subtilis, E. coli, P. aeruginosa, C. albicans and S. aureus. But acetone extracts were effective only against E. coli and Proteus vulgaris. Table 4.1 : Antimicrobial Spectrum of Leaf Extracts of DIgera murlcats At9B of Inhibition Zone (mmi)
Microorganisms Acetone Extract 750·
1000'
Benzene Extract 750
Chloroform Extract
1000
750
StandardS" Methanol Extract
1000
750
1000
Bacillus megaterium
43.21
56.57
31.43
21.21
43.21
632
B. subtilis
31.43
56.57
56.57 106.07 31.43
43.21
172
21.21
43.21
43.21
31.43
21.21
Escherichia coli
88.00
21.21
Proteus vulgaris
21.21
31.43
21.21
21.21
71.50
-
43.21
71.50
31.43
71.50
Aspergillus niger
21.21
A. fumigatus
21.21
31.43
21.21
43.21
31.43
56.57
Candida albicans
148
31.43
Pseudomonas aeruginosa Staphylococcus aureus
148 286
Enterobacter faecslis
31.43
21.21
226
21.21
286
31.43
164 31.43
-184 21.21
276
*: Concentration-750 Ilg/ml and 1000 Ilg/ml;-: No inhibition . • Vancomycin (10 Ilg/ml) for Bacillus megaterium; kanamycin (10 Ilg/ml) forB. subtilis; streptomycin (10 Ilg/ml) for E. colt, rifampicin (51lg/ml) for E. faecslis gentayicin (101lg/ml) for Proteus vulgaris and Pseudomonas aeruginosa; chloramphenicol (30 Ilg/ml) for Staphylococcus aureus; nystatin (101lg/ ml) for Aspergillus niger, A. fumigatus and Candida albicans.
Acetone extracts of leaves of M. pterigosperma inhibited all the test microorganisms except B. megaterium. Chloroform extracts were effective against all bacteria and fungi except B. megaterium and E. coli. Methanol extracts could suppress the growth of all microorganisms tested but not B. megaterium and P. aeruginosa. Benzene extracts inhibited the growth of B. subtilis, E. coli, E. faecalis, P. vulgaris, S. aureus and C. albicans.
Antimicrobial Spectrum of Digera muricata (L.) Mart. and Moringa pterigosperma Gaertn.
37
Table 4.2: Antimicrobial Spectrum of Leaf Extracts of Morlnga pterigosperma Area of Inhibition Zone (mm")
Microorganisms Acetone Extract
Benzene Extract
Chloroform Extract
Standards" Methanol Extract
750'
1000'
750
1000
750
1000
750
1000
B. subtilis
56.57
125.7
31.43
88.00
56.57
125.7
31.43
88.00
172
Escherichia coli
106.07 125.7
31.43
88.00
31.43
88.00
148
Enterobacter faecalis
88.00
Bacillus megaterium
632
Proteus vulgaris
125.7
56.57
125.7
56.57
125.7
56.57
125.7
286
88.00
31.43
56.57
31.43
88.00
31.43
56.57
148
Pseudomonas aeruginosa
31.43
56.57
Staphylococcus aureus
88.00
169.7
Aspergillus niger
21.21
31.43
A. tumigatus
21.21
31.43
Candida albicans
31.43
47.99
31.43 31.43
21.21
226
88.00
169.7
31.43
286
21.21
31.43
21.21
164
21.21
31.43
21.21
184
31.43
43.21
21.21
276
*: Concentration-750 Ilg/ml and 1000 Ilg/ml;-: No inhibition. • Vancomycin (10 Ilg/ml) for Bacillus megaterium; kanamycin (10 Ilg/ml) forB. subtilis; streptomycin (10 Ilg/ml) for E colt, rifampicin (51lg/ml) for E faecalis gentayicin (101lg/ml) for Proteus vulgaris and Pseudomonas aeruginosa; chloramphenicol (30 Ilg/ml) for Staphylococcus aureus; nystatin (101lg/ ml) for Aspergillus niger, A. fumigatus and Candida albicans.
Antibacterial activity of Achyranthes bidentata was studied by Sangameswaran (2003). The aqueous extracts of the seeds of M. pterigosperma showed activity against E. coli, P. aeruginosa, S. aureus and Klebsiella spp.(Rajendran et al. 1998). Medicinal nature of M. oleifera was attributed to some phytochemicals such as sitosterol, caffeoyl quinic acid and kaempferol (Farooq Anwar, 2007). In the present study, the results clearly indicate that the chloroform and benzene extracts of Digera muricata and acetone extracts of Moringa pterigosperma showed good antimicrobial activity than other solvent extracts. The detailed investigation of these plants may yield promising antimicrobial agents.
References Benson, H.}., 1990. Microbiological Applications. WMC Brown, USA. Farooq Anwar, Saijid Latif and Muhammad Ashraf, 2007 Moringa oleifera: A food plant with multiple medicinal uses. Phytotherapy Research, 21: 17-25. Kokate, c.K., Purohit, A.P. and Gokhale, S.B., 2003. Pharmacognosy. Nirali Prakasan,
Pune. Kirtikar, K.R. and Basu, B.D., 1981. Indian Medicinal Plants, Vol. 1. Periodical Expert Book Agency, New Delhi.
38
Emerging Trends in Biological Sciences
Maheshwari, P. and Singh, U., 1965. Dictionary of Medicinal Plants of India, ICAR, Delhi. Rajendran, J., Arunmani, M. and Navaneetha Kannan, K., 1998. Antibacterial activity of some selected medicinal plants. Geobios, 25: 280-282. Rastogi, R.P. and Mehrota, B.N., 1993. Compendium ofIndian Medicinal Plants, Vol. 2, New Delhi. Sangameswaran, B., 2003. Antibacterial activity of aerial parts of Achyranthes bidentata. The Indian Pharmacist, 2: 67-69.
Chapter 5
In vitro Antimicrobial Efficacy of Solvent Extracts of Seeds of Albizzia lebbeck (L.) Benth. S.H.K.R. Prasad1 *, B. Rajasekhar, M. Sunny JameSZ, N.L. Swapna2 and Madan Prasad! 1St. Ann's College for Women, Gorantla, Guntur, A.P. 2Hindu College of Pharmacy, Guntur, A.P. 3Department of Biotechnology, R.P.S. College, Magadh University, Patna, Bihar
ABSTRACT The methanol and chloroform extracts of seeds of Albizzia lebbeck (L.) Benth. were tested for their antimicrobial activity. Both solvent extracts were found to be effective against all the tested organisms.
Keywords: Albizzia lebbeck; Antimicrobial activity.
Plant Albizzia lebbeck (L.) Benth. (Mimosaceae) seeds were collected from the Acharya Nagarjuna University in Nagarjuna Nagar, Guntur district of Andhra Pradesh, India in April 2007. Authenticated voucher specimen has been deposited in the department of pharmacognosy, Hindu College of Pharmacy, Guntur. "E-mail: [email protected](S.H.K.R.Prasad)[email protected]
Emerging Trends in Biological Sciences
40
Uses in Traditional Medicine Seeds are useful in inflammation, skin diseases, leprosy, leucoderma, chronic catarrh, seminal weakness opthalmopathy, and poisoning (Kirtikar and Basu, 1933).
Previously Isolated Constituents Alkaloids, Flavanoids (Rastogi and Mehrotra, 1993).
Tested Material Methanol and chloroform extract residues of seeds of Albizzia lebbeck (L.) Benth.
Studied Activity Antimicrobial activity by disc diffusion method (Bonsignor et al., 1990; Barry, 1976; Benson, 1990; Baver et al., 1996).
Used Microorganisms Escherichia coli (ATCC 25922), Bacillus subtilis (ATCC 6633), Pseudomonas aeruginosa (ATCC 27853), Aspergillus niger (NCIM 596), Aspergillus fumigatus (NCIM 291) and Candida albicans (NCIM 670) obtained from the department of pharmaceutical microbiology and biotechnology, Hindu College of Pharmacy, Guntur, India.
Results Reported in Table 5.1 (Antibacterial activity). Table 5.1: Antibacterial Activity of Seeds of Alblzzla lebbeck (L) Benth. Seed Extracts· Microorganisms
ME
M
CE
C
SteP
Escherichia coli
41.21
44.21
532
Bacillus subti/is
131.17
260.17
640
Pseudomonas aeruginosa
71.21
73.21
148
Aspergillus niger
32.17
33.71
126
Aspergillus fumigatus
33.13
39.13
148
Candida albicans
35.75
34.72
226
a: Values (zone of inhibition in mnr') are the mean of three replicates; Me: Methanol extract; M: Methanol solvent control (1001lg/ml); Ce: Chloroform extract; C: Chloroform solvent control (100llg/ml); Stdb : Streptomycin (101lg/ml) for bacteria, Nystatin (10Ilg/ml) for fungi.
Conclusion The methanol and chloroform extracts found to inhibit the growth of all the tested organisms. Bacillus subtilis and Aspergillus fumigatus were found to be highly sensitive towards chloroform extract and methanol extract showed good activity against Bacillus subtilis and Candida albicans. Chloroform extract was found to be more effective against Bacillus subtilis than methanol extract residue. The obtained result may also provide a support to the uses of the plant in the traditional medicine.
In vitro Antimicrobial Efficacy of Solvent Extracts of Seeds of Albizzia lebbeck (L.) Benth.
41
Acknowledgement The authors are grateful to the management, Department of Pharmaceutical Microbiology and Biotechnology, Hindu College of Pharmacy, Guntur for providing all the facilities and support for this research work.
References Barry, A.c., 1976. Standard Diffusion Methods for Antibiotic Susceptibility of Common Rapid Growing Bacterial Pathogens. Park Press, Baltimore, USA. Baver, A.W., Kirby, W.M.M., Sherlies, J.c. and Truck, M., 1996. Am. Journ. Pathol.,45: 493. Benson, H.J., 1990. Microbiological Applications, 5 th Edition. Wm.c. Brown Pub!., USA,
p.34. Bonsignor, L., De logn, A., Loy, G., Palmieri, G. and Seed D.ll, 1990. Farmaco, 45: 2323. Kiritikar, K.R. and Basu, B.o., 1933. Indian Medicinal Plants, Vol. 1. L.M. Basu, Allahabad. Rastogi, R.P. and Mehrota, B.N., 1993. Compendium of Indian Medicinal Plants, Vol. 2. CSIR, New Delhi.
Chapter 6
Production of Toxic Metabolites by Alternaria ricini Pathogenic to Castor M. Vijayalakshmi, P. Krishna Kumari, B. Rajesh and]. S. V. Anjaneyulu Department of Microbiology, Acharya Nagarjuna University, Guntur- 522 510, A.P.
ABSTRACT The castor crop suffers from several diseases, among which leaf spot caused by Alternaria ricini is considered to be important. Leaf spots are brown, irregular in shape and surrounded by yellow halos. In severe cases, the spots coalesce to form large patches resulting in premature defoliation. The fungus was cultured on Czapek-Dox broth for 30 days and the culture filtrates were tested for phytotoxicity against seed germination and root elongation of castor. Reduction in seed germination and root elongation over control was recorded as 80 per cent and 66 per cent respectively. For assessing antibacterial activity, cell free extracts of the pathogen grown on yeast extract-sucrose broth for 30 days extracted with diethyl ether were tested against Bacillus megaterium, B. subtilis and Escherichia coli. Basing on spectral data, the compound is identified as tetramethyl altenusin.
Introduction Castor (Ricinus communis L.) is an important oil yielding crop grown in countries like India, Brazil, China and Thailand. The crop suffers from several diseases caused
Production of Toxic Metabolites by Alternaria ricini Pathogenic to Castor
43
by fungi (Mukerji and Bhasin, 1986; Bringham, 1993). Among them, leaf blight by Alternaria ricini is considered to be one of the important diseases. Besides leaves, the fungus also attacks stems, inflorescences, capsules and seeds of castor (Tosi and Zazzerini, 1997). Alternaria species produce secondary metabolites which are toxic to plants, animals and human beings (Harvan and Pero, 1976; King and Schade, 1984; Vijayalakshmi, 1996). In view of the importance of this pathogen, an attempt has been made to screen A. ricini for the production of toxic metabolites.
Materials and Methods The fungus was isolated from the infected leaves of castor collected from the cultivated fields and its pathogenicity was proved by Koch's postulates. The pathogen was identified as Alternaria ricini (Yoshii) Hansford basing on the characteristics reported by Ellis (1971). To know the effect of several nutrients on the growth of A. ricini, Czapek-Dox (CD) broth substituted separately with different carbon, nitrogen, phosphorus and sulfur sources which provide the same quantity of carbon present in sucrose, nitrogen as in sodium nitrate, phosphorus as in potassium dihydrogen orthophosphate and sulfur as in magnesium sulfate of CD medium. The media adjusted to pH 5.0 were inoculated with actively growing culture of A. ricini and incubated for 20 days at 28 ± 2°C as stationary cultures. Mycelial mats collected at the end of incubation period were dried to record the biomass. To test phytotoxic activity, the fungus was cultured on CD broth for 30 days at 28 ± 2°C. The culture filtrates collected at the end of incubation were tested for phytotoxicity against castor and tomato. Inhibition of seed germination and reduction in root elongation were taken as criteria for testing phytotoxicity (Vijayalakshmi and Rao,1985). Antibacterial activity of the fungal metabolites was tested by culturing the fungus on yeast extract-sucrose (YES) broth for 30 days at 28 ± 2°C. The culture filtrates extracted with diethyl ether were employed for testing antibacterial activity against Bacillus megaterium MTCC 428, B. subtilis MTCC 40 and Escherichia coli MTCC 441 using Kirby-Bauer m~thod (Benson, 1994). To identify the toxic metabolites, j\. ricini was cultured on YES broth for 30 days at 28± 2°C. A sample (10 ml) of culture filtrate collected at the end of incubation was treated with ferric chloride to note the color changes as suggested by Rosett et al. (1957). Culture filtrates extracted with diethyl ether were used for IR-spectrum.
Results and Discussion Data on the influence of carbon sources on growth of A. ricini are presented in Table 6.1. The fungus exhibited marked variation in the utilization of different carbon sources. Out of eight carbon sources tested, sucrose supported good growth followed by galactose. Among the nitrogen sources tested, the fungus efficiently utilized sodium nitrate followed by magnesium nitrate (Table 6.2). Dipotassium hydrogen orthophosphate supported good fungal growth followed by potassium dihydrogen phosphate among the eight phosphorus sources tested (Table 6.3). Magnesium sulfate
Emerging Trends in Biological Sciences
44
was found to be the best sulfur source for fungal growth followed by ammonium sulfate (Table 6.4). Table 6.1 : Growth of Alternaria rlclnlon CD Broth Supplemented with Different Carbon Sources Nutrient Source
Dry Weight of Mycelium (mg)
Dextrose
1070 950 1428 1090 1200 1220 1500 1290
Fructose Galactose Lactose Maltose Raffinose Sucrose Xylulose
Table 6.2: Growth of Alternaria ricinlon CD Broth Supplemented with Different Nitrogen Sources Nutrient Source
Dry Weight of Mycelium (mg)
Aluminium nitrate
145
Ammonium nitrate
99 165 223 143 1100 692 1341
Barium nitrate Cobalt nitrate Cupric nitrate Magnesium nitrate Potassium nitrate Sodium nitrate
Table 6.3: Growth of Alternaria ricini on CD Broth Supplemented with Different Phosphorus Sources Nutrient Source
Dry Weight of Mycelium (mg)
Ammonium dihydrogen phosphate
1253 1122 1103 1490 1090 1290 987 1267
Ammonium hydrogen orthophosphate Calcium hydrogen orthophosphate Dipotassium hydrogen orthophosphate Disodium hydrogen orthophosphate Potassium dihydrogen orthophosphate Sodium dihydrogen orthophosphate Zinc phosphate
45
Production of Toxic Metabolites by Alternaria ricini Pathogenic to Castor Table 6.4: Growth of Alternaria ricini on CD Broth supplemented with Different Sulfur Sources Nutrient Source
Dry Weight of Mycelium (mg)
Aluminium sulfate
Magnesium sulfate
744 1119 711 1520
Manganous sulfate
813
Potassium sulfate
907
Sodium sulfate
1050 1070
Ammonium sulfate Calcium sulfate
Zinc sulfate
Variations in Alternaria species with regard to nutritional requirements have been reported. A. triticina exhibited good growth with galactose as carbon source followed by maltose (Vijaykumar and Rao, 1980). Rajpurohit and Prasad (1985) suggested glucose and sodium nitrate as the best carbon and nitrogen sources respectively for A. sesami. Disodium hydrogen phosphate supported good mycelial growth of A. sesami among the phosphorus tested by Ramamohana Rao and Vijayalakshmi (2000). The culture filtrates collected from 30-day old culture were toxic to castor and tomato (Table 6.5). Reduction in seed germination in treated samples was 80 per cent and 82 per cent in castor and tomato respectively as compared to control. Per cent of reduction in root length in germinated seedlings was 66 for castor and 75 for tomato. Phytotoxic activity of culture filtrates of A. brassicae, A. brassicicola and A. raphani on root and shoot growth of mustard was reported by Gangwar (1983). Wasnikar et al. (1991) recorded the phytotoxic effects of seed-borne A. sesami. Toxicity of seed mycoflora associated with foxtail millet was reported by Kavitha and Vijayalakshmi (2007). Table 6.5: Phytotoxicity of Culture Filtrates of Alternaria ricini Type
Reduction in Seed Germination (%)
Castor 80
66
Tomato
82
Reduction in Root Elongation (%)
75
Diethyl ether extracts of A. ricini were inhibitory to the growth of test bacteria (Table 6.6). B. megaterium was highly sensitive to the solvent extracts followed by B. subtilis and E. coli. Fungal metabolites have been reported to be toxic to Bacillus spp. (Vijayalakshmi and Rao, 1985; Ramamohana Rao and Vijayalakshmi, 2000). Culture filtrates of the fungus turned to brown when treated with ferric chloride indicating that they may contain altenusin or derivatives of altenusin (Rosett et al. 1957). The IR spectral data obtained for the toxic metabolites of A. ricini appears to be
Emerging Trends in Biological Sciences
46
similar to that of tetramethyl altenusin (Harvin and Pero, 1976). Attempts are in progress to characterize the toxic compound produced by A. ricini. Table 6.6: Antimicrobial Activity of Culture Filtrates of Alternaria rlclnl Bacteria
Area of Inhibition Zone (mrr1)
Gram-positive Bacillus megaterium
301.4
B. subti/is
240.5
Gram-negative Escherichia coli
235.5
References Benson, H.J., 1994. Microbiological Applications. WCB Publications, England. Bringham, RD., 1993. Castor: Return of an old crop. In: New Crops, (Eds.) J. Janick and J.E. Simon. WHey, New York, p. 380-383. Ellis, M.B., 1971. Dematmceous Hyphomyctes. CMI, Kew, Surrey, England. Gangwar, SK, 1983. Inhibition of root and shoot growth in mustard by culture filtrates of three species of Alternarm. Indmn I. Mycology and Plant Pathology, 13: 366-368. Harvan, D.J. and Pero, RW., 1976. The structure and toxicity of Alternarm metabolites. In. Mycotoxins and other fungal related food problems. In: Advances in Chemistry Series, (Ed.) J.V. Rodricks. American Chemical Society, Washington D.C., 149: 344-355. Kavitha, A. and Vijayalakshmi, M., 2007. Phytotoxic effect of seed mycoflora associated with the genotypes of foxtail millet. Seed Research (In Press). King, A.D. and Schade, J.E., 1984. Alternaria toxins and their importance in food. I. Food Protection, 47: 886-906. Mukerji, K.G. and Bhasin, 1986. Plant Diseases oflndm. Tata-McGraw Hill, New Delhi. Rajpurohit, T.S. and Prasad, N., 1985. Effect of carbon and nitrogen on growth and sporulation of Alternarm sesami. Indmn I. Mycology and Plant Pathology, 13: 234-236. Ramamohana Rao, N. and Vijayalakshmi, M., 2000. Studies on Alternarm sesami pathogenic to sesame. Microbiological Research, 155: 129-131. Rosett, T., Sankhala, RH., Stickings, C.E., Taylor, M.E.V. and Thomas, R, 1957. Studies on the biochemistry of microorganisms 103. Metabolites of Alternarm tenuis Auct: culture filtrate products. Biochemistry 1.,67: 390-400. Tosi, L. and Zazzerini, A., 1997. Alternaria ricini and Fusarium incarnatum, two new parasites of castor bean in informatore. Fitopatoiogico, 47: 37-41. Vijayalakshmi, M., 1996. Toxigenic potential of Alternarm macrospora pathogenic to cotton. Microbiological Research, 151: 239-241.
Production o/Toxic Metabolites by Alternaria ricini Pathogenic to Castor
47
Vijayalakshmi, M and Rao, A.S. 1985. Toxigenic fungi associated with sunflower seeds during development. In: Trichothecenes and other Mycotoxins, (Ed.) J. Lacey. John WHey, New York, p. 33-45. Vijaykumar, C.S.K. and Rao, A.S., 1980. Physiological changes in Alternaria infected wheat leaves. Phytopathologische Zeitschrift, 98: 76--83. Wasnikar, A.R., Deshmukh, M.R. and Sharma, S.M., 1991. Toxicity of fungal metabolites on germination and growth of sesame. Current Research, 23: 185-186.
Chapter 7
Fungal Infection of Chilli Pods at the Time of Harvest and Post-harvest Drying M. Vijayalakshmi*, M. Prasanth, Y. Usha, M.R. Hima Bindhu and G. Rosaiah Department of Botany and Microbiology, Acharya Nagarjuna University, Guntur-522 510, A. P .
• ABSTRACT Fungal invasion of red ripened as well as dried chillies of three varieties viz. G4, LCA 960 and LCA 334 was studied. Samples of red ripened chillies at the time of harvest as well as chilli pods exposed to open yard drying were collected to test the incidence of molds. Two sets of samples were taken in all the cases. One set was surface sterilized with sodium hypochlorite, while the other set was washed with sterile distilled water prior to plating on Czapek-Dox agar medium. Fungi such as Alternaria alternata, Cladosporium herbarum, Curvularia lunata, Fusarium moniliforme and F. oxysporum were abundant on red ripened chillies, while Aspergillus species like A. flavus and A. niger became prevalent on dried chillies of the three varieties.
Introduction Chilli (Capsicum annuum L.) is a lucrative spice and vegetable crop of temperate and tropical regions. Among chilli producing states in India, Andhra Pradesh, * Corresponding Author: E-mail: [email protected]
Fungal Infection of Chilli Pods at the Time of Harvest and Post-harvest Drying
49
Maharashtra, Karnataka and Tamil Nadu accounted for 75 per cent of the total area under cultivation. Andhra Pradesh ranked first in cultivation with an annual production of 3.8lakhlonnes. The use of chilli varies with its pungency, color and vitamins content. The fruits are harvested when they are fully ripened and exposed to open yard sun-drying from which the chilli powder is made. Though India ranked first in chilli production, exports from India are only about 5-6 per cent of the total production (Satyanarayana and Sukumaran, 2002). It is mainly due to poor quality of the final products in terms of high microbial count, presence of pesticides and aflatoxins. Many agricultural commodities such as cereals, oil seeds, dry fruits and spices have been reported to be contaminated with toxigenic molds (Reddy et al., 2001; Fazekas et al., 2005; Russel and Paterson, 2007). As the moulds often contaminate the agricultural products at various stages, an attempt has been made to study the fungal invasion of chilli pods of three varieties during harvest and subsequent post-harvest drying.
Materials and Methods Red ripened pods were collec~ed from three cultivated varieties of chilli viz. LCA 960, LCA 934 and G4 at the time of harvest. The chilli pods (200) collected from each variety was divided into two sets. One set was washed with sterile distilled water, blotted to dry and plated on Czapek-Dox (CD) agar medium to observe fungal colonization (unsterilized samples). The other set of samples was surface sterilized with 4 per cent sodium hypochlorite for four minutes, rinsed thoroughly with sterilized distilled water and blotted to dry prior to plating on CD agar medium to record the fungal invasion of samples (surface sterilized ones). The plates were incubated at 28±2°C for seven days. Per cent of chilli pods infected by fungi was recorded and the colonies were counted and identified. After harvesting, the chilli pods were sun-dried in open yards till their moisture content reduced to 8 per cent. Fungal incidence on dried chillies was studied for surface sterilized as well as unsterilized samples of three varieties.
Results and Discussion Data on the per cent of ripened chilli pods infected with various moulds in three varieties are presented in Tables 7.1-7.3. Mould incidence was high on unsterilized pods as compared to surface sterilized ones in all the three varieties. Fungal genera dominant on chilli pods of three varieties include Alternaria alternata, Aspergillus flavus, A. niger, Curvularia lunata, Fusarium moniliforme and F. oxysporum. Among the three varieties tested, pod invasion by fungi was found to be less in LCA 334 when compared to G4 and LCA 960. Hashmi and Thrane (1990) reported that Fusarium spp. associated with chilli seeds were toxigenic. Adebanjo and Shopeju (1993) recorded the infection of fresh and sun dried chillies with A. alternata and Fusarium species. Basak et al. (1996) noticed that Fusarium spp. associated with chilli seeds were pathogenic to chilli causing fruit rot disease.
50
Emerging Trends in Biological Sciences Table 7.1: Fungal Incidence on Chilli Pods of LCA 960 Variety During Harvest Fungus
Per cent of Infection Unsterilized Samples
Surface Sterilized Samples
Altemaria altemata
45
22
Aspergillus flavus
18
15
A. niger
40
19
Cladosporium herbarum
8
10
F. oxysporum
22 20 41
Penicillium sp.
20
17
Curvularia lunata Fusarium moniliforme
16 19
Table 7.2: Fungal Incidence on Chilli Pods of LCA 334 Variety During Harvest Fungus
Altemaria altemata Aspergillus flavus
A. niger Curvularia lunata Fusarium moniliforme
F. oxysporum Penicillium sp.
Per cent of Infection Unsterilized Samples
Surface Sterilized Samples
18 24 20 10 20
14 13 20 8 14 19 10
30 16
Table 7.3: Fungal Incidence on Chilli Pods of G4 Variety During Harvest Fungus
Altemaria altemata
Per cent of Infection Unsterilized Samples
Surface Sterilized Samples
46
26 10 12 18 10 21 14
Aspergillus flavus
17
A. niger
37
Curvularia lunata
20 18 42 20
Fusarium moniliforme
F. oxysporum Penicillium sp.
Fungal infection of dried chilli pods of three varieties is presented in Tables 7.47.6. Species of Aspergillus like A. flavus and A. niger were prevalent on dried chilli pods when compared to red ripened ones. But incidence of field fungi such as
51
Fungal Infection of Chilli Pods at the Time of Harvest and Post-harvest Drying
A. alternata, C. lunata and Fusarium spp. got reduced on dried pods which might be
due to low moisture content of the pods. Table 7.4: Fungal Invasion of Dried Chilli Pods of LCA 960 Variety Per cent of Infection
Fungus
Unsterilized Samples
Surface Sterilized Samples
Altemaria altemata
20
8
Aspergillus flavus
66
42
A. niger
75
56
A. terreus
20 10 20 19 10
10
Curvularia lunata Fusarium moniliforme
F. oxysporum Penicillium sp.
6
12 6
Table 7.5: Fungal Invasion of Dried Chilli Pods of LCA 334 Variety Fungus
Per cent of Infection Unsterilized Samples
Surface Sterilized Samples
Altemaria altemata
10
8
Aspergillus flavus
54
38
A. niger
66
45
CUNularia lunata
3
F. oxysporum
10 10
Penicillium sp.
9
2
4
Table 7.6: Fungal Invasion of Dried Chilli Pods of G4 Variety Fungus
Per cent of Infection Unsterilized Samples
Surface Sterilized Samples
Aspergillus flavus
20 42
36
A. niger
58
45
A. terreus
24
CUNularia lunata
18
12 10
Altemaria altemata
9
Fusarium moniliforme
6
4
F. oxysporum
10 10
6
Penicillium sp.
5
52
Emerging Trends in Biological Sciences
Aspergillus spp. invading the chilli pods during harvest and post-harvest drying may continue to survive and multiply during storage and produce aflatoxins. Giridhar and Reddy (1999) reported the prevalence of fungi such as A. flavus and A. niger on stored chillies. Occurrence of aflatoxin Bl in chilli powder made from dried chilli fruits was recorded by Reddy et al. (2001). Mould incidence on chillies kept in cold stores and extent of aflatoxin Bl in stored samples were studied by Ravikiran et al. (2005). Due to alarming increase in aflatoxin content in stored chillies, attempts should be made to prevent the invasion of chilli pods by Aspergillus species during harvest and post-harvest drying.
Acknowledgement This work was supported by the financial grant from CSIR, New Delhi. One of the authors (KJPN) is thankful to CSIR for providing fellowship.
References Adebanjo, A. and Shopeju, E., 1993. Sources of mycoflora associated with sun-dried vegetables in storage. International Biodeterioration and Biodegradation, 31: 255263. Basak, AB., Fakir, G.A and Mridha, M.AV., 1996. Relation of seed borne infection to different infection grades in fruit rot diseases of chilli. Seed Research, 24: 69-70. Fazekas, B., Tar, A and Kovacs, M., 2005. Aflatoxin and ochratoxin A content in spices in Hungary. Food Additives and Contaminants, 22: 856--863. Giridhar, P. and Reddy, S., 1999. Mycoflora in relation to mycotoxin incidence in red pepper. Advances in Plant Sciences, 12: 85-88. Hashmi, M.H. and Thrane, U., 1990. Mycotoxins and other secondary metabolites in species of Fusarium isolated from seeds of capsicum, coriander and fenugreek. Pakistan Journal of Botany, 22: 106-116. Ravikiran, D., Narayana, KJ.P. and Vijayalakshroi, M., 2005. Aflatoxin Bl production in chillies (Capsicum annuum L.) kept in cold stores. African J. Biotechnology, 4: 791-795. Reddy, S.V., Kiranmayi, D., Umareddy, M., Thirumala Devi, K and Reddy, D.V.R., 2001. Aflatoxin Bl in different grades of chillies (Capsicum annuum) in India as detected by indirect competitive ELISA Food Additives and Contaminants, 18: 553-558. Russel, R. and Paterson, M., 2007. Aflatoxins contamination in chilli samples from Pakistan. Food Control., 18: 817-820. Satyanarayana,. M.N. and Sukumaran, 2002 Postharvest Profile of Chillies. ANGR Agricultural University, Bapatla, AP., India.
Chapter 8
Studies on Airborne Bacteria B. Ramachandra Murthy and K. V.Mallaiah Department of Botany, Acharya Nagarjuna University, Nagarjuna Nagar-522 510, Guntur (Dt.), A.P.
ABSTRACT A volumetric study of airborne bacteria was carried out in the Nagarjuna University campus during July 1994 to June '95, using an Anderson 6-stage air sampler. Air sampling was done at 12 m above the ground level on the roof of the Botany Department building. Nutrient agar medium was used as exposure medium. The sampler was operated for a period of 10 minutes at weekly intervals and the plates were incubated at room temperature for two days before counting the colonies. A total of 10,150 colonies were recorded from 98 samples. They belonged to 32 different colony types. With respect to staining property, 77.07 per cent colonies belonged to Gram positive group of bacteria and 22.93 per cent to Gram negative group. With respect to cell morphology, 74.27 per cent belonged to rod shaped bacteria while 25.73 per cent belonged to cocci group. All coccoid bacterial colonies are Gram positive. Among the individual genera, Bacillus is the most predominant one with 35.21 per cent of total colonies and the species isolated include B. subtilis, B. megaterium.,B. sphaericus, B. circulans, B. brevis, B. cereus, B. firm us and B. licheniformis. The genus Micrococcus is the second prevalent genus contributing 22.96 per cent colonies to the total and the species include M.luteus, M.lyale, M. varians, M. roseus, M. kristinae and M. agilis. The other bacterial genera isolated include Pseudomonas, Arthrobacter, Acinetobacter, Alcaligenes, Staphylococcus, Corynebacterium, Flavobacterium and Serratia. The total bacterial colonies showed peak occurrence in summer and minimum during winter, and the two major bacterial genera viz. Bacillus and Micrococcus followed the same seasonal periodicity pattern. Staphylococcus mainly occurred during wet season while Flavobacterium, Corynebacteirum and Serratia occurred mainly during dry season.
Keywords: Airborne Bacteria, Air sampling, Seasonal periodicity.
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Introduction The earth's atmosphere contains an array of bioparticles of plant and animal origin, which become airborne as a result of several natural processes and human activities. The presence of these bioparticles in air, though suspected by Spallanzani in 1765, was first conclusively proved by Louis Pasteur in 1860 (Pasteur, 1861) during his epoch making studies to disprove the theory of spontaneous generation. Since then, various bioparticles like pollen grains, fungal spores, bacteria, actinomycetes, lichen fragments, algal filaments, protozoan cysts, insect parts like scales and hairs, plant parts like epidermal hairs, peels, xylem fibres etc. were reported as the components of air and are collectively described as Air spora. The air spora merits study "both in its own right as a natural phenomenon and also because it is a component of air we breathe" (Gregory, 1973). Hence, aerobiological studies were conducted all over the world, and abundance of air spora, especially of fungal spores and pollen grains, was reported by various workers. In addition to fungal spores and pollen grains, bacteria also occur in air, but there are very few studies on the incidence of airborne bacteria in the out door environment. An outstanding work on airborne bacteria was that of Pierre Miquel who studied airborne bacteria in France continuously for period of over 25 years in the last quarter of 19th Century. As Gregory (1973) pointed out the "Knowledge of the broad features of the bacterial population of the outdoor air near the ground remains to this day substantially as Miquelleft it at the beginning of this century." Hence, the present work on airborne bacteria was taken up for study.
Review of Literature Studies on airborne bacteria are relatively fewer compared to those of fungal spores and pollen grains. The first and perhaps the only sustained work over a long period was, that carried out by Pierre Miquel, during the last quarter of 19th century at Parc Montsouris in Paris. Basing on his daily observations in the Parc Montsouris, Miquel classified the bacteria of outdoor air as Micrococcus-66, Bacillus-25, Bacterium6 and Vibrio-1- 2 per cent. Miquel (1878-99) reported that at Parc Montsouris, airborne bacteria were nearly three times more as numerous in summer as in winter, but in the centre of Paris, bacteria showed little seasonal variation. The number of bacteria in summer averaged several hundred per cubic metre and were reduced in a few hours by rain to a mere 20 to 30 but they increased again as the ground dried. He concluded that the source of most of the outdoor airbome bacteria is the surface of the ground. He reported that airborne bacteria showed double peak pattern at Parc Montsouris (rural area), while a single peak was observed in the centre of Paris (Urban area). At Parc Montsouris, peaks were observed at 8.00 h and 20.00h, while in the centre of Paris, peak was recorded at 14.00 h. Before 1960, much of the work on airborne bacteria was mainly concerned with their occurrence at higher altitudes. Measurements of bacterial concentrations in the upper air were reported from different places like Geneva (Cristiani, 1893), Southern Bovaria (Harz, 1904), Southern Germany (Hahn, 1909), Moscow (Mischustin, 1926), Tennesse (Wolfe, 1943), over acetic and atlantic ocean (Pady and Kelly, 1954; Kelly and Layne, 1957a,b), Lav (Klyuzko et al., 1960) and Minneapolis (Wright et al., 1969).
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These studies clearly showed the incidence of bacteria in the upper air. Generally, it was reported that the concentrations decreased with height, more rapidly in winter than in summer. Further, the proportion of spore forming bacteria increased with increasing height. Different genera of bacteria isolated from upper air included grampositive coccoid genera like Micrococcus, Staphylococcus and Sarcina, gram-positive rod shaped bacteria belonging to the genera, Bacillus and Corynebacterium, gramnegative rod shaped bacteria like Achromobacter and Flavobacterium. Studies on airborne bacteria in extramural environments at ground level were reported from U .S.A., Sweden, India and Kuwait. In U.s.A., studies on airborne bacteria were reported from Kansas (Pady and Kramer, 1967), Cincinnati (Lee et al., 1973), Colorado (Mancinelli and Shulls, 1978) and Washington Oones and Cookson, 1983). Pady and Kramer (1967) exposed nutrient agar and RBS plates regularly from 1959 to 1964, using Pady-Rittis slit sampler on the roof of a Kansas State University building. They recorded the presence of bacteria throughout the growing season, usually between 20-40 per cubic foot but occasionally over 100 per cubic foot. They also found a diurnal rhythm with twin peaks, a major peak at about 10 P .M. and a minor peak around 10.00 A.M. In downtown Cincinnati, Lee et al. (1973) collected airborne bacteria at approximately 5 feet above the ground level. The concentration of viable bacteria varied from 375 to 2490 per m 3 of air. At Colorado, Mancinelli and Shulls (1978) collected air samples over a two year period and recorded bacteria in the range of 0.031-1.88 per litre of air sampled. Colonies of 19 different bacteria were identified with Micrococcus (41 per cent) dominating, followed by Staphylococcus (11 per cent) and Aerococcus (8 per cent). The natural atmospheric microbial population in the air of Washington D.e. suburban area was studied by Jones and Cookson (1983). They recorded aerobic bacteria (4.2 to 1640 per m 3) and faecal cocci (0-5.7 per m 3 0f air). In Sweden, the composition of natural airborne bacterial population was studied at different places in and around Stockholm. Andersen et al. (1973) recorded seasonal variations in airborne bacterial population with higher concentrations recorded during summer and autumn. A very low frequency of zero was recorded during winter when the ground was covered with snow. Roffey et al. (1977) reported that the concentration of airborne bacteria was high (0-500 per m 3 of air) in an inland area and was low (0-250 per m 3 of air) in coastal land. The increased concentrations during day time were attributed to increased activity, especially traffic in a city. Bovallius et al. (1978) studied the concentration of airborne bacteria during 19691973 at four different localities: Nartuna (country district), a coastal station (Kapellskar), city park in Stockholm and a city street in the centre of Stockholm. The bacterial concentrations varied with season, with the highest average concentrations for each year during summer Oune-August) and autumn (September-November), while the lowest concentration was found during winter (December-February). The genus Bacillus was reported to be the dominant one. From India, incidence of bacteria in outdoor air was reported from Visakhapatnam, Nanded and Jabalpur. Reddi and Reddi (1982) studied airborne bacteria at Visakhapatnam for a period of one year from July 1977 to June 1978. A
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total of 3804 colonies were isolated which belonged to 10 genera and 31 species. Gram-negative rod shaped bacteria belonging to the genera Acetobacter, Flavobacterium and Lucibacterium contributed 26.44 per cent followed by gram-positive cocci (Micrococcus, Streptococcus, Staphylococcus, Sarcina) with a contribution of 26.12 per cent. The spore forming Bacillus (with 16 species) contributed 24.19 per cent, nonspore forming rod shaped bacteria (Arthrobater, Corynebacterium) contributed 18.79 per cent. In addition to these, gram-variable bacteria accounted for 4.47 per cent. With respect to individual species, Flavobacterium indoltheticum is the most dominant with a tofal bf 585 colonies followed by Staphylococcus saprophyticus (179), S. aureus (157), Arthrobacter globiformis (129), Micrococcus roseus (108), Bacillus marcerans (95) and B. sphaericus (93). Highest number of colonies was isolated during December-February period and least during October-November period. The atmospheric bacterial flora of Nanded was studied in May, 1984 by Shete (1984). A total of 1244 colonies were isolated which belonged to 6 genera. Bacillus subtilis was the most dominant one (35 per cent) followed by colonies of Staphylococcus (22.8 per cent), Sarcina luteus (13.7 per cent) and Micrococcus roseus (6.9 per cent). Verma and Srivastava (1991) studied the bacterial flora in the atmosphere of Jabalpur and observed Aeromonas, Chromobacterium, Klebsiella and Proteus in the air. Gramnegative fermentative rods were found in large numbers (50.94 per cent) than grampositive aerobic cocci (33.96 per cent), gram-negative aerobic non-fermenters (7.54 per cent) and Vibrio (1.88 per cent). From Kuwait, few studies were reported on airborne bacteria. Diab et al. (1976) studied airborne bacteria and found that Staphylococcus and Micrococcus were the main components and had relatively high frequency during May and June. Marafie and Ashkanani (1991) studied the viable and culturable population of airborne bacteria by using a Casella Airborne Sampler at a height of 7m in the Allergy centre of Kuwait city between 1986-88. Bacillus was detected in 85.3 per cent of the samples followed by Micrococcus (71.3 percent) and Staphylococcusalbus (65.4 percent). Grampositive rods (53.4 per cent) were more prevalent than gram-negative rods (23.7 per cent). Peak incidence of bacteria was recorded in August, September and a minor peak in February and March. Higher average counts were observed in the days with sand storms and with raising sand compared to clear weather. From the foregoing account, it becomes clear that the concentration and composition of airborne viable (culturable) bacteria varies within broad limits for different localities, with changes in geographical, meteorological and seasonal factors. Pigmented saprophytic bacteria (Micrococcus, Sarcina), spore forming Bacillus are more frequently reported from the air by different workers.
Materials and Methods The Andersen 6-stage sampler (supplied by Andersen samplers Inc., Atlanta, Georgia) was used to study the viable and culturable fungi and bacteria present in the air over Nagarjunanagar. The sampling was carried out for a period of one year from July, 1994 to June, 1995 at weekly intervals. On the day of sampling, the trap was operated twice, at 10.30 h and at 15.30 h. The rate of suction is 28.3 litres per minute.
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The duration of sampling was fixed for 10 minutes, since preliminary studies showed that this period will give optimum number of colonies. To determine th~ diurnal rhythm of the airborne fungi and bacteria, sampling was also done at 4-hourly intervals (04.00 h, 08.00h, 12.00 h, 16.00 h, 20.00 h and 24.00h I.S.T.) during the months of March, April, May and June, 1995.
The Sampler The Andersen 6-stage sampler (Andersen, 1958) consists of six round aluminium stages clamped together to form a compact sealed cylinder, with a vacuum connection below the bottom stage. The stages are numbered 1,2,3,4,5 and 6 from the top to base. Each aluminium stage contains 400 holes in circles and these holes act as orifices for the entry of air into next chamber. The diameter of the holes range from 1.81 mm on stage #1 to a diameter of 0.25 mm on the stage # 6. These orifices function to progressively increase the speed of air-jets, when air is drawn through the sampler and force the suspended particles of progressively smaller size to impact on to the agar plates placed at each stage (~7 mm) at the top stage and 0.65 to 1.1 mm at the bottom stage.
Assembling the Sampler for Operation The orifice stages of the sampler were cleaned and disinfected, each time the instrument was used. Just before use, all the six stages were wiped with 70 per cent isopropyl alcohol using a guaze pad. One collection agar plate was inserted on each stage of the sampler and the cover lid was removed aseptically. The petriplates with nutrient agar medium were placed on stages #6 and #5 for isolation of bacteria. The plates with Czapek-Dox agar medium were placed on stages #4, #3, #2, and #1 for isolation of fungi. After placing the petriplates in all the six stages, the inlet nose cone was placed on the top of stage #1 and all the stages were clamped together with the help of springs provided at the base plate. Each stage was fitted with an o-ring (neoprene) for air-tight sealing.
Operation After the sampler has been assembled, it was taken to the site of air sampling. The outlet nipple present at the base plate was connected to the vacuum motor pump (110 volts A.C.). The plastic cover on the inlet nose cone was removed and the motor pump was switched-on to draw the air into the sampler. It was operated for a period of ten minutes and was brought back to the laboratory.
Incubation The exposed agar plates were removed from the sampler and the covers were replaced under aseptic conditions. The exposed agar plates were incubated in an inverted position at a room temperature of 25 ± 2°C. The plates were observed on 3rd, 5th and 7'" day to record the fungal and bacterial colonies. Each colony type was subcultured on agar slants and maintained for further study.
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Estimation of Colonies per m 3 of Air The number of bacterial and fungal colony forming units (du) on each sampler plate were counted. The mean number of cfu per cubic foot of air sampled was calculated using the formula: Total number of colonies from all plates Mean number of du per ft3 of air = Total sampling time in minutes The resultant values were converted into dui m3 of air using a conversion factor, 35.31. The conversion factor was calculated in the following manner. 1 cubic foot of air =0.02832 cubic metre of air 1 cubic metre of air = 1/0.02832 = 35.31 cubic feet. So, the conversion factor for estimating the number of cfu/m3 of air =35.31.
Observations Airborne Bacteria at Nagarjunanagar The bacterial population present in the air over N agarjunanagar was studied by using a 6-stage Andersen Volumetric Sampler, for a period of one year during July, 1994 to June, 1995. A total of 10,150 colonies (cfu-Colony Forming Units) were isolated from 98 cubic metres of air, with an average concentration of 103.6 du/m3 of air. The photomicrographs of the exposed agar plates showing different colony types are shown in Plate 8.1. (a)
Composition of Bacterial Flora
Basing on morphological characteristics, 32 different types of bacterial colonies were picked up from the exposed nutrient agar plates. They were brought into pure culture and maintained on nutrient agar slants at 4°C for further studies. Of the 32 colony types, 27 types were identified up to genus and I or species level. They belonged to ten genera, of which Bacillus is represented by 10 colony types, Micrococcus with 8 colony types. Pseudomonas with 2 colony types and the other genera were represented by single colony type each. Five colony types could not be identified up to genus level and all of them were gram-positive, aerobic, non-spore forming bacilli. They were counted as separate colony types basing on the colony morphology. The photomicrographs of the individual colony types major airborne bacteria isolated in this study are presented in the Plate 8.2.
Description of Bacteria Isolated from Air The isolated bacteria were studied in detail with respect to their morphological and biochemical characteristics following the recent edition of Bergey's Manual of Determinative Bacteriology (Holt et al., 1994). Basing on the above observations, the bacterial isolates were identified up to genus and I or species level. The chief characters of the isolated bacteria are described below and the bacteria are arranged in the order of their prevalence in air.
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Plate 8.1
1: Exposed Agar plate showing different Bacterial colonies at stage # 5.
2: Exposed Agar plate showing different Bacterial columns at stage # 6.
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Plate 8.2(a): Photographs of Bacterial Colonies Isolated in the Present Study Using Andersen Sampler (x 100)
1: Bacillus subtilis; 2: Bacillus megaterium; 3: Bacillus sphaericus; 4: Bacillus circulans; 5: Bacillus brevis; 6: Bacillus cereus; 7: Bacillus firmus; 8: Bacillus licheniformis
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Plate 8.2(b) 9: Micrococus luteus; 10: Micrococus Iyale; 11: Micrococus varians; 12: Micrococus rose US; 13: Micrococus kristinae; 14: Micrococus agi/is (i) Bacillus Cohn, 1872
The genus Bacillus comprises of the gram-positive rods, that grow aerobically and form endospores. Spores are central, sub-terminal or terminal in position and oval or ellipsoidal in shape. The vegetative cells are rod shaped, 0.5-2.2 mm X 1.2-70 mm in size, straight and are arranged in short or long chains. Cells are motile. Bacillus forms catalase and produces acid, but no gas from glucose, maltose and sucrose. Nitrates are reduced and gelatin is liquefied. Endospore formation is the dominant feature in the characterisation of Bacillus. This genus can be differentiated from other genera which also produce endospores
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Plate 8.2(c)
15: Pseudomonas putida; 16: Arthobacter; 17: Acinetobacter; 18: Alcaligenes; 19: Staphylococus saprophyticus; 20: Corynebacterium; 21: Flavobacterium; 22: Serratia marcescens
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(Clostridium, Sporosarcina and Sporolactobacillus) by the following differences. The genus Clostridium is anaerobic and do not produce catalase. Sporosarcina has its vegetative cells in spherical form and Sporolactobacillus closely resembles the genus lActobacillus in its physiological functions including its inability to produce catalase. In the present study, eight species of Bacillus were recognised and two colony types remained unclassified. Bacillus subtilis (Ehrenberg) Cohn, 1872 Colonies on nutrient agar medium are round or irregular, white, becoming cream coloured and opaque with age. Vegetative cells are long rods, 0.7-0.8 mm X 2-3 mm in size, usually in singles, rarely in chains. Endospores ellipsoidal and central in position. Acid was produced from glucose, sucrose and mannitol. Starch and casein were not hydrolysed. VP test positive, MR test negative and Indole not produced. Bacillus megaterium de Bary, 1884 Colonies on agar surface are large, circular or lobed, spreading, wrinkled surface, moderately dull in colour. Vegetative cells cylindrical, 1.2-1.5 mm X 2.0-5.0 mm in size and in short twisted chains. Spores ellipsoidal and central. Acid production from glucose, sucrose and fructose was negative. Starch was hydrolysed and gelatin is rapidly liquefied. Casein was actively digested. VP test was variable and Nitrates were not reduced. Bacillus sphaericus Meyer & Neide, 1904 Colonies on nutrient agar medium are white, irregular and actively spreading, smooth and translucent. Vegetative cells cylindrical, 0.6-1.0 mm X 1.5-5.0 mm in size, singles or in short chains. Spores spherical or oval in shape and terminal. Acid was not produced from carbohydrates. Starch was not hydrolysed. VP test was positive. Indole and nitrate tests were negative. Casein was decomposed. Bacillus circulans Jordan, 1890 Colonies on nutrient agar are thin, spreading actively, white and opaque. Vegetative cells cylindrical, 0.5-0.7 mm X 2.0-5.0 mm in size, spores ellipsoidal and sub-terminal. Acid was produced without gas from glucose. Casein and gelatin were weakly liquefied. Litmus milk reaction was weak. Starch was hydrolysed. VP test was positive and indole was not produced. Bacillus brevis Migula, 1900 Colonies on nutrient agar surface are spreading, thin, smooth with rhizoidal margin. Vegetative cells cylindrical, 0.6-0.9 mm X 1.5-4.0 mm in size, singles, often in short chains. Spores ellipsoidal, terminal or subterminal in position, Acid was produced from glucose. Starch was not hydrolysed. Casein was hydrolysed. VP test was positive, Indole not produced. Nitrate test variable. Bacillus cereus Frankland & Frankland, 1887 Colonies on nutrient agar are large, spreading, pinkish white, with an uneven surface and undulate margin. Vegetative cells short rods, 1.0-1.2 mm X 2.0-3.5 mm in size, and were in short chains. Spores ellipsoidal and central. Acid was produced
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from glucose and sucrose but not from lactose. Gas was not formed. Catalase was produced. Nitrate test, VP test and citrate utilization tests were positive.
Bacillus firmus Bredemann & Wemer, 1933 Colonies on nutrient agar surface are large, round, smooth, spreading, white, flat and opaque. Vegetative cells in short chains, 0.6-0.9 mm X 1.2-4.0 mm in size. Spores ellipsoidal and central. Acid was produced from glucose. Gelatin was liquefied. Starch and casein were hydrolysed. VP test and Indole test were positive. Bacillus licheniformis (Weigmann) Chester, 1901 Colonies on nutrient agar are large, spreading, with rhizoidal outgrowth, orange red in colour. Vegetative cells small rods, 0.7-0.8 mm X 2.0-3.0 mm in size, tend to occur in short chains. Spores ellipsoidal and central. Acid was produced from glucose, sucrose and maltose and gas was not liberated. Catalase and oxidase were produced. Starch was not hydrolysed. VP test was positive and Indole not produced. Unclassified Bacillus spp.
White Coloured Colonies (Type-V Colonies on nutrient agar are small, circular, glossy, opaque, white to cream coloured. Vegetative cells short rods, 1.0-1.5 mm X 3.0-4.0 mm in size, in short chains. Spores oval and central. Acid and gas were produced from glucose and mannitol. Starch was hydrolysed. Casein and gelatin not digested. Urea hydrolysed. MR test negative, VP test and Nitrate test positive. Indole not produced.
White Coloured Colonies (Type-2) Colonies are circular, milky-white, wavy, margin, opaque and smooth. Vegetative cells rod shaped, 1.5-2.0 mm X 3-4 mm in size, and in short chains. Spores oval and terminal. Acid was produced from glucose, sucrose and lactose, but without gas. Oxidase negative, catalase positive. MR test positive, VP test negative. Urease not produced. Nitrate test, Gelatin liquefaction test negative. Indole was produced.
(ii) Micrococcus Cohn, 1872 Micrococci are gram-positive, spheres, occurring singly, in pairs and in irregular clusters, but not in chains. Vegetative cells 0.5-3.5"mm in diameter, cells non-motile and non-spore forming. Colonies usually pigmented in shades of yellow or red. No acid was produced from carbohydrates. Catalase and oxidase produced. Indole was not produced. Gelatin was not liquefied. Micrococci closely resemble three other genera viz., Staphylococcus, Planococcus and Aerococcus. The genus Micrococcus includes 9 species in the 9th edition of 'The Manual' (Holt et al., 1994). The type species described was Micrococcus luteus (Schroeter) Cohn, 1872. In the present study, the following six colonies were isolated from the air and two colony types remained unclassified.
Micrococcus luteus (Schroeter) Cohn, 1872 Colonies on nutrient agar are smooth, convex, circular, golden yellow in colour. Vegetative cells spheres, 1.0-2.0 mm in diametre, occurring singly, in pairs or in irregular clusters. Non-motile. Acid was not produced from glucose. Nitrates not reduced. Casein was hydrolysed.
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Micrococcus lyale Colonies on nutrient agar are small, circular, smooth, convex with regular edge and cream coloured. Vegetative cells spheres, 1.0-2.0 mm in diameter, non-motile. Gelatin was rapidly liquefied. Nitrates were not reduced to nitrites. VP test was negative. H 2S and Indole were not produced. Micrococcus varians Migula, 1900 Colonies on nutrient agar are circular, smooth, yellow coloured. Vegetative cells spheres, 1.0-1.5 mm in diametre, non-motile. Acid, but no gas was produced from carbohydrates. Casein was weakly hydrolysed. Gelatin was hydrolysed. Citrate was utilized. H 2S was produced. Micrococcus roseus Flugge, 1886 Colonies are small, circular, pink or red or rose in colour, slightly convex, with wavy margin. Vegetative cells spheres, 1.0-2.5 mm in diametre, motile. Acid was produced from Carbohydrates. Nitrate test negative. Casein not digested. Gelatin was weekly liquefied. Citrate was not utilized. H 2S was produced. Micrococcus kristinae Colonies are small, circular, pale red to orange in colour, slightly convex with lobed margin. Vegetative cells spheres, 0.8-2.0 mm in diametre, non-motile. Gelatin was liquefied. Acid was produced from glucose, maltose and sucrose. Oxidase and catalase were produced. VP test was positive. Nitrate test positive. Citrate was not utilized. Micrococcus agilis Colonies on nutrient agar are small, circular, convex with smooth margin, red colour. Vegetative cells spherical, 0.5-1.5 mm in diametre, non-motile. Acid was not produced. Gelatin was moderately liquefied. Nitrate test positive. Oxidase test negative, catalase test positive. VP test negative and MR test positive. Citrate was not utilized. Casein digested. Unclassified Micrococcus Spp.
White Coloured Colonies (Type-l) Colonies on nutrient agar are white, circular, slightly convex with smooth margin. Vegetative cells spheres, 0.7-1.8 mm in diametre, non-motile. Acid was not produced from carbohydrates. Gelatin was weakly hydrolysed. Casein was not digested. Tndole test and VP test positive. Nitrates were not reduced.
Grey Coloured Colonies (Type-2) Colonies are circular, slightly convex with wavy margin, grey in colour. Vegetative cells spheres, 0.5-2.0 mm in diametre, non-motile. Catalase positive, oxidase negative. Starch, casein and gelatin were not hydrolysed. Acid was produced from glucose, VP test positive. Indole negative, citrate was not utilized.
(iii) Pseudomonas Migula, 1894 The genus Pseudomonas comprises of aerobic, gram-negative, non-fermentative, oxidase positive, motile rods. Vegetative cells straight or slightly curved rods, but not
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helical, 0.5-1.0 mm in size. Endospores are not produced. Old cultures produce a characteristic, grape-wine like smell. A total of 75 species were listed in the 9th edition of 'The Manual' (Holt et al., 1994), but the number of species listed was 172 in the 8 th edition of 'The Bergery's Manual of Systematic Bacteriology'. The type species described was P. aeruginosa (Schroeter) Migula, 1900. In the present study, one colony type was identified as belonging to P. putida and the other remained unclassified.
Pseudomonas putida (Trevisan) Migula, 1875 Colonies on nutrient agar medium are large, white, spreading with undulate margin. Vegetative cells cylindrical, slightly oval, 0.7-1.1 mm X 2.0-4.0 mm in size, motile, singles or in short chains. Acid was not produced from carbohydrates. Gelatin was hydrolysed. Starch was not hydrolysed. Indole, MR and VP tests negative. Gelatin liquefied. Unclassified Pseudomonas sp. Colonies on nutrient agar are small, circular, low convex and yellow. Vegetative cells short rods, non-motile, singles, in pairs or in short chains, 0.7-0.8 mm X 1.5-3.6 mm in size. Nitrates reduced to nitrites. Lactose and maltose were not oxidized. MR test positive and VP test negative. Indole not produced. Nitrates reduced. Catalase and Oxidase positive. Casein not hydrolysed. Gelatin not liquefied. Starch hydrolysed.
(iv) Acinetobacter Brison and Prevot, 1954 Colonies are white or cream coloured, smooth, circular with hairy margin, flat and opaque. Vegetative cells coccobacilli, 1.0-1.5 mm X 1.5-2.5 mm in size, gramnegative and catalase positive. Acid was produced from glucose. Indole not produced. MR and VP tests negative. Gelatin readily liquefied. Only one species was listed in the 8 th edition of Bergey's Manual of Determinative Bacteriology and the number was increased to twelve in the 9 th edition of the 'The Manual' (Holt et al., 1994). The type species described was A. calcoaceticus Beijerinck. In the present study, this particular colony type could not be assigned to any known species. (v) Arthrobacter Cohn and Dimmick, 1947 Colonies are small, yellow, smooth, convex with smooth margin. Cells in young cultures are irregular rods, cell arrangement in V or Y-shape. Coccoidal cells in old cultures. Rods are 0.5-0.8 m$ X 1.0-4.0 mm and coccoidal cells are 0.8 mm in diametre. Vegetative cells weakly gram-positive or variable, non-acid fast, non-motile. No acid was produced from carbohydrates. CatalasQ-and oxidase positive. Gelatin and starch hydrolysed. VP test negative. Citrate not utilized. Nitrate test negative. The present colony was identified as belonging to A. globiformis (Cohn) Cohnand Dimmick, 1947. (vi) Staphylococcus Rosenbach, 1884 Colonies are smooth, regular or slightly irregular margin, convex, pin-head size, white and opaque. Vegetative cells spheres, 0.5-1.5 mm in diametre, occuring in tetrads or clusters. Gram-positive, non-motile and non-sporing. Oxidase negative and catalase positive. Nitrates reduced to nitrites. Acid was produced from glucose,lactose and sucrose. Casein, gelatin, urea hydrolysed. Starch was not hydrolysed. VP and MR tests positive. Indole not produced.
Studies on Airborne Bacteria
67
Staphylococcus differs from Micrococcus, by its ability to grow anaerobically and to ferment glucose under anaerobic conditions. Holt et al. (1994) recognised 30 defined species, in the 9th edition of 'The Manual'. In the present study, the colony was identified as S. saprophyticus. (vii) Alcaligenes Castellani & Chalmers, 1919 Colonies are glistening and slimy, yellow pigment, circular. Gram-negative, cocci or coccobacilli, 0.5-1.2 mm, singles or in pairs. Motile, catalase and oxidase positive. Casein, gelatin and starch not hydrolysed. Nitrate test positive. Acid was produced from glucose, fructose and lactose. Indole not produced.
Alcaligenes can be separated from the closely related genus Acinetobacter by its inability to grow at pH 4-5, presence of motility and oxidative type of respiration. Seven species were listed in the 9th edition of 'The Manual' and the present colony type was identified as belonging to A. paradoxus .. (viii) Corynebacterium Lehmann & Neumann, 1896 Aerobic or facultatively anaerobic, gram-positive rods. Cells slender rods, with club shaped swellings, non-motile, catalase positive. Colonies white, circular, with elevated margin. Starch and gelatin hydrolysed. Caseinase and phosphatase produced. MR test negative. Acid was produced from lactose. Urease test negative. Nitrate test positive. The genus includes 46 species in the 8 th edition of Bergey's Manual of Determinative Bacteriology and the number has been reduced to 21 in the recent edition (Holt et al., 1994). The present colony could not be assigned to any known species. (ix) Flavobacterium Bergey et al., 1923
Colonies are circular, entire or undulate, moderate size. Pigmentation of the colony was yellow. Cells rod shaped, motile, gram-negative, spores not produced. Catalase, oxidase and phosphatase tests negative. Acid, but no gas was produced. Nitrate test negative. Indole test positive. Litmus milk unchanged. The genus included 12 species in the 8th edition and the number was reduced to 11 in the 9th edition of 'The Manual' (Holt et al., 1994). The type species described is F. aquatile (Frankland and Frankland) Bergey et al., 1923. The present colony was identified as belonging to F. regense Bergey et al., 1923.
(x) Serratia Bizio, 1823 Colonies of this genus are circular, convex, red or scarlet in colour, opaque. Vegetative cells motile, rods, 0.8-1.0 mm X 3.0-4.0 mm in size, gram-negative. Spores not produced. Acid and gas were produced. VP and MR tests positive. Indole not produced. Nitrate test positive. Urea not decomposed. Gelatin was liquefied. The genus included 11 species in the 9th edition of The Manual (Holt et al., 1994). The present colony was identified as belonging to S. marcescens Bizio, 1823. (d) Yearly Mean Concentrations and per cent Contributions
The yearly mean concentrations of the different bacterial taxa for which individual counts were taken are given in the Table 8.1, together with the number of times they
Emerging Trends in Biological Sciences
68
appeared and their per cent contribution to the year's total bacterial flora isolated from air. Table 8.1: Yearly Mean Concentration (cfu/m3) of Different Bacterial Colony Types and their Per cent Contributions to the Total Bacterial Colonies Isolated from Air SI. No.
Colony Type
At 10: 30h
At 15: 30 h
No. of Yearly No. of Yearly Weeks Mean Weeks Mean Appeared Concen- Appeared Concentration Concentration (cfu/rn3) tration (cfu/rn3)
Day's Percent Average Contribution ConCen- to Total tration Bacterial (cfu/rn3) Colonies
1.
Bacillus subtilis
33
8.37
23
4.28
6.33
6.11
2.
B. megaterium
29
7.88
18
4.45
6.16
5.95
3.
B. sphaericus
24
6.32
21
3.26
4.79
4.63
4.
B. circulans
28
6.25
15
2.47
4.36
4.21
5.
B. brevis
6.25
14
2.45
4.13
4.00
6. 7.
B. cereus
26 20 20
3.84
15
2.37
3.36
15
2.00
3.10 2.68
2.59
8. B. licheniformis
13
2.04
2
0.45
1.25
1.20
9.
19
4.84
8
1.43
3.13
2.02
10
1.45
4
0.86
1.15
1.11
B. firmus Bacillus white colonies
3.00
(Type-1) 10. Bacillus white colonies (Type-2) 11. Micrococcus luteus
25
6.22
16
2.53
4.38
4.23
12. M.lyale
23
5.16
24
2.96
4.06
13. M. varians
31
5.73
14
1.61
3.67
3.92 3.55
14. M. roseus 15. M. kristinae
28
4.78
18
2.37
3.57
3.45
22
4.24
11
1.20
2.72
2.63
16. M.agilis
10
1.53
8
0.84
1.18
1.14
17. Micrococcus white colonies
19
3.94
8
0.98
2.46
2.37
18. Micrococcus Grey colonies
11
1.88
9
1.59
1.73
1.67
19. Pseudomonas putida 20. Pseudomonas sp.
27 27
7.47 7.16
21 21
3.92 3.70
5.69 5.43
5.50 5.24
21. Arthrobacter globiformis 22. Acinetobacter sp.
31 25
8.29
18 22
3.12
5.70
5.50
7.12
3.08
5.10
4.93
23. Alcaligenes paradoxus
26
5.45
16
2.22
3.84
3.70
24. Staphylococcus
25
4.14
19
1.59
2.87
2.77
25 Corynebacterium sp.
16
2.39
11
2.16
2.27
2.18
26 Flavobacterium regense
16
3.41
9
1.10
2.26
2.18
27 Serratia marcescens
14
1.89
8
0.96
1.43
1.40
28 Unclassified rods
25
11.79
22
4.41
8.16
7.83
64.37
103.57
100.00
saprophyticus
Total bacteria
142.77
Studies on Airborne Bacteria
69
A total number of 10,150 colonies were isolated from 98 cubic metres of air sampled, with an average concentration of 103.58 cfu/m3 of air. From them, a total of 9,356 colonies were identified up to genus and or species level and the remaining (794 cfu/m3) were unclassified gram-positive rods. The colony counts represent the total number of colonies observed on the two agar plates exposed every time. The genus Bacillus, represented by 10 colony types was the major type with an yearly mean of 37.0 cfu per cu. metre of air, contributing 35.81 per cent to the total bacterial colonies. Among the different species of Bacillus, colonies of B. subtilis (6.33 cfu per m 3) contributed 6.11 per cent to the total colony count, followed by those of B. megaterium (6.16 cfu per m 3) which contributed 5.95 per cent to the total colony count. Colonies of B. sphaericus, B. circulans and B. brevis contributed between 4 to 5 per cent to the total number of colonies recorded. The contribution of other Bacillus spp. was between one to four per cent to the total bacterial colonies. The genus Micrococcus with eight colony types was the second major type, with an yearly mean concentration of 23.78 cfu per m 3 of air, contributing 22.96 per cent to the total number of bacterial colonies. Among the Micrococci, colonies of M. luteus (4.38 cfu per m 3) contributed 4.23 per cent to the total count, followed by colonies of M. lyale (3.92 per cent), M. varians (3.55 per cent) and M. roseus (3.45 per cent). The contribution of other Micrococcus spp. to the total number of colonies was less than 3 percent. The genus Pseudomonas represented by two colony types, with an yearly mean concentration of 11.12 cfu per m 3 of air, contributed 10.74 per cent to the total bacterial colony count. Colonies of P. putida (5.50 per cent) occurred slightly in higher concentration than unclassified Pseudomonas sp. (5.24 per cent). Colonies of Arthrobacter globiformis with an yearly mean concentration of 5.70 cfu per m 3 of air, contributed 5.50 per cent to the total colony count, and the contribution of other genera was less than 5 per cent to total bacterial spora. Colonies of Acinetobacter (4.93 per cent), Alcaligenes paradoxus (3.70 per cent), Staphylococcus saprophyticus (2.77 per cent), Corynebacterium (2.18 per cent), Flavobacterium regense (2.18 per cent) and Serratia marcescens (1.40 per cent) contributed between 1-4 per cent to the total bacterial count. The unclassified gram-negative, nonsporulating bacilli group with an yearly mean concentration of 8.16 cfu/m3 of air, contributed 7.83 per cent to the total number of colonies. The incidence of bacterial colonies varied at both times of the day. At 10.30 h, a total of 6996 colonies were recorded with an yearly mean concentration of142.77 cfu per m 3 of air. At 15.30 h, a total of 3154 colonies were recorded with an yearly mean concentration of 64.37 cfu per m 3 of air. This clearly indicates the predominance of bacterial colonies in the air during morning time, than during evening time. The morning to evening catch ratio is almost equal (1:1) in the case of Corynebacterium and unclassified white colonies of Micrococcus. The incidence of bacterial colonies in the morning hours is almost double than in the evening hours, in the case of B. cereus, B. sphaericus, B. subtilis, B. jirmus, B. megaterium, M.lyale, M. roseus, M. agilis, Pseudomonas spp. and Serratia marcescens. No single colony type was noticed as dominant in the evening time.
70
Emerging Trends in Biological Sciences
(e)
Contribution of Different Groups of Bacteria to Total Bacterial Colonies
The yearly mean concentration of bacterial colonies, belonging to different major morphological groups of bacteria and their occurrence as percentages of total bacterial colonies is given in the Table 8.2 to bring out their relative importance as contributors to total airborne bacterial colonies. Table 8.2: Contribution of Different Groups of Bacteria to Total Bacterial Colonies Isolated from Air No. of Colony Types Isolated
10: 30 h Yearly Mean Cone. (efu/rrfJ)
15 :30h Yearly Mean Cone. (efu/rrfJ)
Day's Average Cone. (efu/rrfJ)
Per eent Contribution
Gram-positive, spore forming rods
10
50.16
24.02
37.09
35.81
Gram-positive, non-spore forming rods
7
22.47
9.69
16.08
15.53
Gram-negative rods
6
32.52
14.98
23.75
22.93
Gram·posltive cocci
9
37.63
15.67
26.65
25.73
32
142.77
64.37
103.57
100.00
Group
Gram·negative cocci Total
The colonies belonging to gram-positive spore forming bacilli, were the most predominant, with an yearly mean concentration of 37.09 cfu per m 3 of air. The colonies of this type contributed 35 per cent at 10.30 hand 37 per cent at 15.30 h of the day. The contribution of colony types belonging to gram-positive, non-spore forming cocci was 26.35 per cent at 10.30 h and 24.35 per cent at 15.30 h. Gram-negative rods, represented by 5 genera and 6 colony types contributed 22.78 per cent at 10.30 hand 23.28 per cent at 15.30 h to total colony counts. Gram-positive, non-spore forming rods represented by 2 genera and 5 unclassified colony types, with an average concentration of 16.08 cfu/m3 contributed 15 per cent to the total bacterial count. Gram-negative cocci were not observed in the present study. The bacilli together contributed 74.27 per cent, followed by gram-positive cocci, whose contribution was 25.73 per cent to the total number of bacterial colonies.
(j) Highest Daily Concentrations The highest daily concentrations of different colony types observed per m 3of air and their contribution to the days total colony count are given in the Table 8.3 to bring out their relative dominance on the day of their peak incidence. The highest concentration of bacterial colonies observed during this study was 260 cfu/m3 of air on 16 April, 1995. On this day, higher concentration of Bacillus mageterium (30 per cent), B. circulans (14 per cent), B. subtilis (6.14 per cent) and Micrococcus roseus (6.7 per cent) were recorded. The other individual colony types
Studies on Airborne Bacteria
71
contributed less than 5 per cent to the total number of colonies. The second highest concentration of bacterial colonies (227 cfu/rn3 of air) observed during this study was on 19 March, 1995. Colonies of 25 types were recorded on this day, of which Pseudomonas putida (12.36 per cent), Bacillus sp. White colonies (7.7 per cent), Staphylococcus saprophyticus (6.18 per cent), Alcaligenes paradoxus (6.18 per cent) were recorded in higher concentrations. Table 8.3: The Highest Daily Concentrations of Different Bacterial Colony Types and their Contribution to the Day's Total Bacterial Colonies SI.No.
Colony Type
Date
cfulm'
Total Bacterial Per cent Count of the Contribution to Day (cfU/m'J the Day's Total
1.
Bacillus subtilis
8-5-95
36
208
17.30
2.
B. megaterium
16-4-95
79
260
30.38
3.
B. sphaericus
1-5-95
21
170
12.36
4.
B. circulans
16-4-95
37
260
14.18
5.
B. brevis
29-8-94
16
144
11.11
6.
B. cereus
6-2-95
14
128
10.93
7.
B. firmus
19-3-95
18
227
3.70
8.
Micrococcus luteus
19-9-94
20
127
15.75
9.
M.lyale
12-3-95
44
213
20.66
23-4-95
14
156
8.97
11. M. roseus
1-5-95
28
170
15.64
12. M. kristinae
10-9-94
13
113
11.50
13. Pseudomonas puntida
8-5-95
30
213
14.08
14. Pseudomonas sp.
18-6-94
24
102
23.53
15. A. globiformis
11-6-94
30
171
17.54
16. Acinetobacter sp.
17-10-94
28
90
31.11
17. A. paradoxus
9-4-95
30
185
16.22
18. S. saprophyticus
29-8-94
11
144
7.64
19. Corynebacterium sp.
4-6-94
19
141
13.48
20. F. regense
8-5-95
16
208
7.69
21. S. marcescens
9-4-95
11
135
8.15
10. M. varians
Colonies of B. megaterium (30 per cent), Acinetobacter sp. (31 per cent), M. lyale (21 per cent) and Pseudomonas sp. (24 per cent) contributed between 20-30 per cent to total colonies on the day of their peak incidence. Colonies of Arthrobacter globiformis (17 per cent), B. subtilis (17 per cent), Alcaligenes paradoxus (16 per cent), M. roseus (15 per cent), M. luteus (15 per cent), B. circulans (14 per cent), P. putida (14 per cent), Carynebacterium sp. (13 per cent), B. brevis (11 per cent), B. cereus (11 per cent), and M. kristinae (11 per cent) contributed between 10-20 per cent to the total colony count on the day of their peak incidence. The other bacterial colonies contributed less than
Emerging Trends in Biological Sciences
72
10 per cent to the total number of colonies even on the day of their peak incidence, indicating the heterogenity of bacterial flora in the air of Nagarjunanagar.
(g) Seasonal Variations Exhibited by Different Bacterial Colonies The seasonal variations observed in the concentrations of airborne bacteria observed in this study are presented in the Figure 8.1a-e as weekly totals. The periodicity curves of the different bacterial genera were arranged in the order of their prevalence. (i) Total Bacterial Flora
The incidence of airborne bacteria was relatively higher during hot summer season (March to June) followed by rainy season Guly to October) and winter season (November to February). The bacteria appeared in very low concentration from July onwards, till the end of January except for a brief period in September. From February onwards, the bacterial concentration increased gradually to record year's highest concentration the last week of April and relatively higher concentrations were maintained up to the end of June. The lowest bacterial incidence was noticed in December during 1994. (ii) Bacillus
The seasonal variations in the incidence of Bacillus colonies are almost similar to that of the variations exhibited by total bacterial colonies. The concentrations were very low from July to March and then increased to record peak in the third week of April. After the peak, the concentrations showed sharp decrease in incidence. Different species of Bacillus, showed difference in their seasonal occurrence. Colonies of B. subtilis occurred throughout the year except for a brief period in December-January period. The concentrations remained low up to March and the peak was recorded in the second week of May. Colonies of B. megaterium were observed during September to June period through December. The peak incidence was noticed in the middle of April.
--
Total Bacterial Colonies
200 100
A
S
0
N
0
F
III
A
III
Figure 8.1 (a): Weekly Concentrations of Total Bacterial Colonies Isolated from Air During July, 1994-June, 1995
73
Studies on Airborne Bacteria
300 ~E
200
I
Total Bacillus Colonies
m i'Q
~ '~ ~........ ll,Jl"llAlln..... U...... h1.......I.I.........IIl,a.,a. .........
I
I
1,I,Jl,I.I..II,I,I. ........ 1.bl.l.l.h.IU,lI•.wI.lU.ll.whII.UJ.d1w..J
80
B. subtilis
so
':I
~
B. megaterium
I1
~ 3:~~__~__~.~.n~'~'~'.I~.~.•~I~.•~.~.~.~.~'A'~.~I.•~.~.~I~.A~I.~J~I~.n~.BA.IAa.~IB~ Q
B. sphaerlcus
40 ~E 30
--5
20 10 O~~~~~~~~~~~~__~__~~~~~UU~~ 10
B. circulans
20
O+-______~~~~~~~~~~~~~Al~~~__~ 3tI
B.brevis
30 "
!
25
20 .i! 15 u
10
5
O~~~A~~~~O~~H~-O--~~~~~-U---A----M~UUUU~
Figure 8.1 (b): Weekly Concentrations of Bacterial Colonies Isolated from Air During July, 1994-June, 1995
Emerging Trends in Biological Sciences
74
..E
150
Total Micrococcus Colonies
1011
.; u
50 0
10
..E
M.luteus
40 SO
-.. ~
ZO
,. 0
40
.
E
M.lyate
so
oi! u
20
10 0
30 25 E 20
M. varians
..
-,. .. . oi!u
10
I 0
E
~
M. rose us
10 40
act 20 10 0
30
M. kristJnae
..
--5
E 20 10 0
A
o
N
.
A
..
Figure 8.1 (c): Weekly Concentrations of Micrococcus Colonies Isolated from Air During July, 1994-June, 1995
Studies on Airborne Bacteria
70
75
Pseudomonas punt/da
60
..e
60 40
~ 30 u 20 10 0
60
Acinetobacter sp.
110 40
pt
E 30
~ u 20 10
0
70
Alcaligenes paradoxus
60
..e
...
60 40
~ 30
u
20 10 0
70
Arthrobacter globiformis
80 50 pt
...
E
40
~ 30 u 20
10 0
Figure 8.1 (d): Weekly Concentrations of Bacterial Colonies Isolated from Air During July, 1994-.1une, 1995
76
Emerging Trends in Biological Sciences
Staphylococcus saprophyticus
26
.E ....
20 16
~ 10 IJ 6
I1
I1
0
,I I
11
It I 1111 Corynebacterium sp.
40 36
30
.e
25
.2u
15
... 20
I, .1 JI II II I1 •
10 6
a
Flavobacterium regense
35 30
..E .... .2u
25 20
15 10
a
I
I
0
ill
I
Serratia marcescens
25 20
.,
e
16
-IJ2
10
6
:
i
0
J
A
S
0
N
D
}
F
M
I A
M
I1
Figure 8.1 (e): Weekly Concentrations of Bacterial Colonies Isolated from Air During July, 1994-June, 1995
Studies on Airborne Bacteria
77
Colony counts of B. sphaericus showed two periods of peak incidence, one during July to October and the other during April to June. The peak was recorded in the second week of May. Colonies of B. circulans began to appear in the air from the first week of September and gradually increased in number from the last week of April. After the peak incidence, the colony counts fell sharply and almost no colonies were observed in May to June period. Colonies of B. brevis were observed in the air from the beginning of July till the end of February. After a period of no incidence during March to May, once again the colonies started to appear from July. Colonies of B. cereus were observed during July to September and again during January to June period. No distinct seasonal peak could be traced out. However, relatively higher number of colonies were observed during February to April period. B. firmus was observed in the air mainly during July to March period with a major peak in March. No colonies were recorded during April to June, the time when other Bacillus spp. showed their peak incidence. (iii) Micrococcus The genus Micrococcus was the second major bacterium found in the air and it was represented by six species and two unclassified colony types. The genus (all species combined) was observed in almost all the exposure with peak incidence in summer. Higher number of colonies was observed during September and then decreased through October to reach low in November. Once again the colonies started to appear in high numbers from December onwards till the end of June.
M. luteus appeared in the air from July onwards up to March through December. Relatively higher number of colonies were noticed in September and February and no colonies were recorded in April to June period. Colonies of M. lyale were observed through out the year but discontinuously and in very low numbers. Relatively higher numbers were observed during December to March period. The year's highest concentration was in the second week of March. Colonies of M. varians were observed during August to June through December in low concentrations. Relatively more number of these colonies were recorded in April to May period. M. roseus was observed through out the year except in July, in very low concentrations. Relatively more number of colonies were observed during April to May period. M. kristinae was recorded almost through out the year with no distinct seasonal variations. Colonies of M. agilis were observed during April to June period and in very low concentrations. (iv) Pseudomonas Colonies of P. putida occurred through out the year with relatively higher concentration during April to May period with peak in the second week of May.
78
Emerging Trends in Biological Sciences
Unclassified colonies of Pseudomonas were observed almost through out the year with no incidence in November to December period. Relatively higher concentrations were observed in February to March and June to August periods. (v) Other Bacterial Genera
Colonies of Acinetobacter sp. appeared almost through out the year with seasonal peak during September to October period.
Alcaligenes paradoxus appeared in the air through out the year except during Jury to September period. Higher concentrations were recorded in March-May period with peak in the second week of April. Arthrobacter globifonnis showed peak incidence in June to July period. The colonies were observed almost throughout the year except in January to February period. Colonies of Staphylococus saprophyticus were observed almost throughout the year in very low concentrations and without any distinct peak. Colonies of Corynebacterium sp. occurred in the air during February to June period. The seasonal peak was recorded in the first week of June, with a subsidiary peak in March. Colonies of Flavobacterium regense and Serratia marcescens were noticed in low concentrations during summer period. (h) Circadian Periodicity Patterns
The circadian rhythms exhibited by airborne bacteria, was determined from the counts taken at 4 hourly intervals during the period March-June, 1995. The curves were constructed as percentages of the peak arithmatic mean concentration of the _ component concerned and are presented in the Figure 8.2. The total airborne bacteria showed peak incidence in the early morning hours at 8.00 A.M. The concentration gradually reduced to a minimum at 16.00 h and then the concentration once again increased to record a subsidiary peak at midnight. The gram-positive rods and gramnegative rods also showed their peak incidence at.8.00 h. The rise and fall in concentration was gradual in the case of gram-negative rods, whereas the grampositive rods showed a subsidiary peak at 16.00 h. Gram-positive cocci showed gradual increase from mid-night and reached the circadian peak by 4.00 h at night. After the peak, the concentrations decreased gradually through out the day time till 20.00 h at night. In general, the incidence of bacteria was more between 8.00 h and 12.00 h during day time.
Discussion The presence and prevalence of bacteria in air is known since the classical work of Pierre Miquel, but very few studies have been carried out on the ecology of airborne bacteria in outdoor environments. The microbial concentration is reported to be varying with geography, locality, season, vegetation and human density. Hence, the present work was carried out to investigate the periodicity of viable population of bacteria in the atmosphere of Nagarjunanagar.
79
Studies on Airborne Bacteria
100
Total Colonies
ID
CO)
E
80
::J
40
't;
20
•
OHr ••
4Hr ••
IHu.
12Hr.
lIHr••
100
20Ilr ••
24Itr •.
Gram (+) rods
la
--6
!'lE so 40 20
•
OH, ••
4HrI.
IHr ••
UHr.
lIH".
20H, ••
24Hr •.
100
Gram (-) rods
10 <'I
E
I.
-6
40
2. 0 OHrl.
4Hr..
tHfI.
12Hr.
18Hr•.
100
20Hr •.
24Hr •.
Gram (-) cocci
ID
!'l
--5 E
60
40 20 0 OHrl.
4H".
IHrs.
12Hr.
18Hr•.
ZOH".
24Hn.
Figure 8.2: Circadian Rhythms Exhibited by Bacteria Isolated from Air
80
Emerging Trends in Biological Sciences
In the present study, a total of 10,150 colonies were recorded with an average concentration of 103.57 cfu/m3 of air. The result of this study was in agreement with the results of other workers. The microbial concentration was reported to be 10-100 bacteria per m 3 of air at Kansas, Canada (Kelly and Pady, 1953a, b, Pady and Kramer, 1967), Minneapolis (Wright et al., 1969), Sweden (Bovallius et al., 1978), Kuwait (Marafie and Ashkanani, 1991), Washington IX Gones and Cookson, 1983). However, a lower concentration of 0.013-1.88 bacteria per litre of air was observed in an urban environment of Colorado city by Mancinelli and Shulls (1978).
In the present study, 32 colony types belonging to 10 genera were observed in the air over Nagarjunanagar. They include Bacillus, Micrococcus, Pseudomonas, Acinetobacter,
Arthrobacter, Staphylococcus, Alcaligenes, Corynebacterium, Flavobacterium and Serratia. Pierre Miquel (1878-99) made daily counts of bacteria in the air of Paris and recognised four types of bacteria namely Micrococcus, Bacillus, Bacterium and Vibrio. Diab et al. (1976) and Maraffie and Ashkanani (1991) recognized Staphylococcus, Micrococcus, Bacillus and unclassified gram positive rods and gram negative rods in the air over Kuwait. At Colorado, 19 different bacterial genera were collected by Mancinelli and Shulls (1978). Four genera viz., Micrococcus, Bacillus, Neisseria and Corynebacterium were reported in the air of an industrial conurbation frOnt Prague by Hysek et al. (1991). In India, a total of 31 species belonging to 10 genera were reported from Visakhapatnam, AP (Reddi and Reddi, 1982). Shete (1984) identified 4 genera viz., Bacillus, Micrococcus, Sarcina, Staphylococcus and unclassified gram negative rods in the air over Nanded, Maharashtra. At Jabalpur, Verma and Srivastava (1991) identified 15 species belonging to 9 genera from the exposed agar plates. This clearly shows that the incidence of different taxa in the air of Nagarjunanagar is similar to that reported from other places. An analysis of the range of bacteria isolated in the present study reveals that
Bacillus (35.81 per cent), Micrococcus (22.96 per cent) and Pseudomonas (10.74 per cent) are the predominant genera and the individual contribution of other genera was less than 10 per cent to the total bacterial colonies. Bacillus was likewise reported to be the dominant genus of airborne bacterial flora from Nashville (Wolfe, 1943), Sweeden (Bovallius et al., 1978) and Nanded (Shete, 1984). However, Micrococcus and Staphylococcus were reported to be the predominant genera in Colorado (Mancinelli and Shulls, 1978), Kuwait (Diab et al., 1976; Marafie and Ashkanani, 1991) and Jabalpur (Verma and Srivastava, 1991). Flavobacterium was reported as the dominant genus at Visakhapatnam (Reddi and Reddi, 1982). Hysek et al. (1991) recorded Micrococcus, Bacillus, Niesseria and Corynebacterium as the predominant genera in the air of Prague. The present study indicated a regular variation in the bacterial content according to season, with highest average concentration during summer (March to June) followed by rainy season Guly to October) and winter season (December to February). The average concentrations found were 160 cfu/m3 during summer, 111 cfu/m3 during rainy season and 89 cfu/m3 during winter season. This type of seasonal variation with summer maxima and winter minima has also been reported by other workers at
Studies on Airborne Bacteria
81
Paris (Miquel, 1878-99), Kansas (Kellyand Pady, 1953a, b). Kuwait (Diab et al., 1976), Sweden (Andersen et al., 1973; Bovallius et al., 1978), Washington DC Uones and Cookson, 1983} and Prague (Hysek et al., 1991). In Jabalpur, highest concentration was recorded during October, followed by July, August and June (Verma and Srivastava, 1991). Reddi and Reddi (1982) reported that the higher number of colonies was trapped during December to June period when hot weather prevails at Visakhapatnam. In this study, colonies of Bacillus spp. and Micrococcus spp. showed peak incidence in March-June period. Summer peak of these organisms were also observed at Kuwait (Diab et al., 1976) and at Prague (Hysek et al., 1991). Hysek et al., opined that the carotene pigments present in Micrococci enables them to survive exposure to Sun's radiation better than the other organisms. This may be one of the reasons for finding a greater abundance of pigmented bacteria in the air than the colourless types. In the present study, the airborne bacteria showed a major peak at 8.00 h and a minor peak at 24.00 h. Bacillus spp. showed a diurnal peak at 8.00 h and lowest at 24.00 h. Gram-positive cocci showed a major peak at 4.00 h and minimum at 20.00 h. In general, all the four groups of bacteria were high during day time and low during night time. This is in conformity with the earlier reports. Miquel (1878-99) observed two major peaks for airborne bacteria, one at 8.00 h and the other at 20.00 h and two minima at 2.00 hand 14.00 h at Parc Montsouris. However, in the centre of Paris only one maximum at 14.00 h and one minimum at 2.00 h was observed. Pady and Kramer (1967) reported twin peaks with a major peak at about 10 P.M. and a minor peak around 10 A.M. at Kansas.
References Andersen, AA, 1958. A new sampler for the collection, sizing and enumeration of viable airborne particles. J. Bacteriol., 76: 471-484. Andersen, R., Bergstrom, B. and Buck, B., 1973. Air dispersal of bacteria from a sewage treatment plant. Vattern,2: 117-123. Blackley, CH., 1873. Experimental Researches on the Causes and Nature of Catterhus aestivus (Hay Fever). Balliere, Tindall and Cox, London, p. 202. Bovallius, A, Bush, B., Roffey, R. and Anna, P., 1978. Long range air transmission of Bacteria. Appl. Environ. Microbiol., 35: 1231-1232. Cristiani, H., 1893. Analyse bacteriologique de lair des hauteurs, puise pendant un voyage en ballon. Anns. Inst. Pasteur, Paris, 7: 665-671. Diab, A, Batran, F.G. and AI-Zaidan, A., 1976. Airborne bacteria in the atmosphere of Kuwait. Zentralbl. Bakteriol. Parastitenk. Infesktionskr Hyg. II., 131: 535-544. Gregory, P.H., 1973. The Microbiology of the Atmosphere, 2nd Edn. Leonard Hill Publications, London, p. 377. Hahn, M., 1909. Dieb estimmung und meterologische ver wertung der keimzahl in den hoheren Luftschichten. Nach Vom Luftballon aus angestellten Beobachtungen. Zbl. Bakt. Abt. 1,51: 97-114 (cited from Gregory, 1973).
Emerging Trends in Biological Sciences
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Harz, CO., 1904. Bacteriologische unterschungen der freien atmosphere mittels. Luftballons, nebst Bemerkungen uber den atmosphearischen Stau. Jb. Dtsch. Luftsch. Verb., 147-170 (cited from Gregory, 1973). Holt, J.G., Krieg, N.R, Sneath, P.H., Stanley, J.T. and Williams, S.T., 1994. Bergey's Manual of Determinative Bacteriology, 9th Edn. Williams and Wilkins, Maryland, USA. Hysek, Y_, Fisar, Z. and Binek, K., 1991. Long run monitoring of bacteria, yeasts and other micromycetes in the air of industrial conurbation. Grana, 29: 450-453. Jones, B.L. and Cookson, J.T., 1983. Natural atmospheric microbial condition in a typical suburban area. Appl. Env. Microbiol., 45: 919-934. Kelly, CO. and Layne, 5., 1957a. Bacteria found in the air over Canada and the American arctic. Can. J. Microbiol., 3: 447-455. Kelly, CD. and Layne,S., 1957b. Bacteria found in the air over the atlantic Ocean. Can. J. Microbiol., 3: 457-460. Kelly, CD. and Pady, S.M., 1953a. Microbiological studies of air over some nonarctic regions of Canada. Can. J. Bot., 31: 90-106. Kelly, CD. and Pady, S.M., 1953B. Microbial masses over Montreal during 1950 and 1951. Can. J. Bot., 32: 591--600. Klyuzko, 5.0., Kishko, Y.G. and Verschchanskiy, Y.T., 1960. Bacterial aeroplankton of the upper layers of the atmosphere during the winter. Kiev. Vrachebnoye Delo, 1: 75-76. Lee, RE. Harris, K. Jr. and Akland, G., 1973. Relationship between viable bacteria and air pollution in an urban atmosphere. J. Am. Ind. Hyg. Assoc., 34: 164-170. Mancinelli, RL. and Shulls, W.A., 1978. Airborne bacteria in an urban environment.
Appl. Environ. Microbiol.,35: 1095-1101. Marafie, S.M.RH. and Ashkanani, L., 1991. Airborne bacteria in Kuwait (1986-88). Grana, 30: 472-476. Mischustin, E., 1926. Zur Untersuchung der mikroflora der hoheren Luftschichten. Zentnbl. Bakt. Parasitkde. Abt. 11 67: 347-351 (cited from Gregory, 1973). Miquel, P., 1878-99. Annual reports in Annu. Abs. Montsouris (cited from Gregory, 1973). Pady, S.M. and Kelly, CD., 1954. Aerobiologicalstudies of fungi and bacteria over the Atlantic Ocean. Can. J. Bot., 32: 202-212. Pady, S.M. and Kramer, CL., 1967. Diurnal periodicity in airborne bacteria. Mycologia, 59: 714-716. Pasteur, L., 1861. Memoire Sur les corpuscles organizes Qui existent dans l'atmosphere. Ann. Sci. Nat., (Zool). 4. Ser., 16: 5-98. Reddi, C.S. and Reddi, J.R, 1982. A one year study of airborne bacteria at Visakhapatnam. Proc. Ind. Natn. Sci. Acad., B. 48: 819-823.
Studies on Airborne Bacteria
83
Roffey, B. Bovallius, A., Anas, P. and Konerg, E., 1977. Semi continuous registration of airborne bacteria at an inland and coastal stations in Sweden. Grana, 16: 171177. Shete, H.G. 1984. The atmospheric bacterial flora of Nanded in May 1984. Indian Botanical Reptr., 3: 158. Verma, K.S. and Srivastava, K.1991. Bacteria and actinomycetes in the air of]abalpur. Ind. J. Aerobiol., 4: 13-18. . Wolfe,F.T.1943. The microbiology of the upper air. Bull. TorreyBot. Cl., 70: 1-14 (cited from Gregory, 1973). Wright,J.J.,Greene, V.W. andPaulus,H.J., 1969. Viable micro-organisms in an urban atmosphere. J. Air. Pollut. Cont. Assoc., 19: 337-341.
Chapter 9
Algal (Cyanobacterial) Diversity Study in North-Eastern States with Respect to Survey and Biotechnological Inputs S.L. Gupta Botanical Survey of India, CGO Complex, 3rd MSO Building, Block-F, 5th Floor, DF Block, Sector-l, Salt Lake City, Kolkata-700 064
Introduction Biodiversity, i.e., biological diversity is defined as the variety and variability of plants, animals and microorganisms. Plants are the only natural productive resource of the nature and its loss is an irreversible process hence it is imperative to conserve this unique resource. India possesses about 11 per cent of total world's plants diversity and has been identified as one of the 12 'Mega Diversity' centres of the world. Due to this a national institute of biodiversity has beeI),.set up at Arunachal Pradesh Algae (Cyanobacteria in particular) is the major part of microbial diversity which is recognised as a main component of the food chain and on which no major exploration has been conducted in the past as for as Arunachal Pradesh, in particular and other north eastern states, in general, are concerned. Represented by over 6500 species (ca 40,000 known in the world), they are representing over 14 per cent of the known Indian flora. They contribute significantly to the floral diversity of India and grow in a variety of habitats ranging from fresh water habitat (ca 5800 species) to marine habitats (ca 700 species) (Rao and Gupta, 1997).
Algal (Cyanobacterial) Diversity Study in North-Eastern States
85
Algae are of source of many natural products, eco-friendly biofertilizers (Gupta, 1997) and fine chemicals etc. Biotechnology has the potential to develop modified plants, animals and microorganisms. In situ protection of ecosystem and ex situ conservation of biological and genetic resources can help in using biological resources sustainable (Tewari, 1993). Likewise algal biotechnology is such area which despite many problems, also has many prospects in solving many environmental problems. At least three research institutes in India are engaged in Biotechnology researches in addition to special advanced study centres at Central University like Banaras Hindu University, Varanasi. Further, Botanical Survey of India has given due priority on the survey and exploration of algal flora of India during 10th five year plan.
Prospects of Advance Researches on Algal Biotechnology Food The chief component of algal biomass is crude protein. Many unicellular forms of algae like ChloreIla is being exploited as a source of single protein (SCP) mainly in western countries. Large scale production of algal food is being carried out in pacific countries like Japan, Philippines etc. from marine algal species. Biofertilizers Algae, particularly cyanobacteria, is a rich source of pollution free eco-friendly biofertilizers being utilized commercially for enhancing paddy production. At least 15 genera including Tolypothrix, Nostoc, Anabaena and Cylindrospermum are used for better crop production. 'Jhum' cultivation in Arunachal Pradesh is a quite common which resulted in loss of nutrient soils. Application of nitrogen fixing cyanobacteria may maintain nutritional status of the soil. Hydrogen Production Hydrogen is considered as a cheap and pollution free energy source. Many algal species like Chlorella, Microcystis, OsciIlatoria, Scenedesmus, and Synechococcus may be exploited for hydrogen production which may become energy of the future. In addition, photoproduction of ammonia from some cyanobacteria like Anacystis nidulans is a remarkable process for conversion of solar energy into chemical form. It is to be mentioned here that ammonia is also valuable as a fuel as well as a fertilizer. Pollution Indicator Many algal species acts as an efficient pollution indicator. Microalgae may accumulate many thousand folds of heavy metal from polluted aquatic environment. Medicinal Value Algae is a potent source of many kinds of medicines including vitamins. Chlorellin from Chlorella, acrylic acid from Phacocystis, kainic acid from Digenea, tarpene from Lyngbya spp. and polysaccharides from Asterionella are some common examples. Recently a rich vitamin source is being marketed under the brand name 'Sunova Spirulina' extracted from cyanobacterium Spirulina.
86
Emerging Trends in Biological Sciences
Algal Diversity in India: A General View As cited above, about 700 marine species and over 5800 fresh water species (out of total 6500 species) of algae are represented in India under 666 genera (out of total number of world genera 2500) which responds a proportion of 27 per cent of global generic representation and surprisingly 16 percent of world species. It clearly indicates that great variety in the algal flora exist at the generic level than at the level of species in India. While comparing families, it has been found that the families Cyanophyceae, Dinophyceae, Chlorophyceae and Bacillariophyceae are better represented in India than in the world as a whole in comparison to the families Xanthophyceae, Chrysophyceae, Phaeophyceae and Rhodophyceae which are poorly represented in the Indian flora. One of the reason may be poor exploration in the country as a whole and in the northeastern region, in particular; climate may be another reason. Similar case also exist at the species level while comparing the world algal flora with the Indian algal flora. Thus at the species level Chlorophyceaee, Cyanophyceaee, Bacillariophyceae and Dinophyceae show a better representation than Rhodophyceae, Chrysophyceae, Xanthophyceae and Phaeophyceae.
Cyanobacterial Diversity in India with Emphasis to North-eastern States The cyanobacteria, commonly known as blue-green algae, are most primitive and simplest form of algae among photosynthetic plants and are characterized by procaryotic nature. They are marked by their widespread occurrence, abundance and morphological diversity within the group. It led the botanists to mention about 2500 species under 150 genera found in India, which includes about 200 species reported for the first time in India representing about 10 per cent of the Indian flora (Gupta 2002, 2003). The Cyanobacteria are characterised by the presence of chlorophyll-a, high amount of blue-coloured c-phycocyanin and low amount of red coloured r-phycoerythrin. The cell wall is made up of mucopeptide and the food reserve is myxophycean starch and proteinaceous cyanophycin. They are phylogenetically coherent group as evident by nucleotide base sequence data of 16 S and SS r-RNA. On this basis several bacteriologists advocated them to known as blue-green bacteria or cyanobacteria (Rippka et al., 1979). According to them, cyanobacteria should be classified according to the principles of the Bacteriological Code of Nomenclature rather than the Botanical Code of Nomenclature. Table 9.1 depicts some common Indian cyanobacterial species found in India and in the world. Oscillatoriales is the most dominant order in cyanobacterial groups followed by Nostocales and Chrococcales. In Oscillatoriales, Oscillatoria, Lyngbya and Phormidum are dominant genera in India. It exhibit that filamentous forms are more dominant than unicellular forms. However unicellular forms tend to have more rapid growth rates (lowest mean generation time 2.1 h at 420 C) than filamentous ones (mean generation time 3.6h at 41 0 C).
87
Algal (Cyanobacterial) Diversity Study in North-Eastern States
Table 9.1 : Some Common Indian Cyanobacterlal Species Diversity Found In India and in the World (Gupta, 2002) No. of Species in
Name of Genus India Anabaena Anabaenopis Aphanocapsa Aphanotheca Arthospira Calothrix Cylindrospermum Chrococcus Gleocapsa Gleotheca Gleotrichia Lyngbya Merismopedia Microco/eus Microcystis Nostoc Oscillatoria Phormidium Plectonema Schizothrix Scytonema Spirulina Synechococcus Tolypothrix
21 03 08 08 03 22 09 15 20 04 06 60 06 04 15 19 65 38 08 16 25 06 02 12
% Found in India
World
25 03 13 11 07 28 12 19 23 05 10 10 08 07 18 23 76 45 11 20 40 08 03 18
84 100 61 72 43 86 75 78 86 80 60 60
75 57 83 82 85 84 72 80 62 75 66 66
Filamentous and non-nitrogen fixing forms Oscillatoria is represented by 65 species in India out of which about 10 per cent are endemic to eastern part of the country. They commonly occur on damp earth surfaces, stones sides of flower ponds, muddy bank of streams and ponds. They can thrive in water where other algae cannot survive, viz., O. brevis is found in the cold region of Sikkim Himalaya. O. annae and O.luridum thrives in the mangrove ecosystem whereas O. kutzingiana ie; found on the barks of Tectona grandis in West Bengal. Likewise one species of Phormidium is dominant in eastern part of the country (P. purpurascens). It has been observed that about 15 species of Lyngbya (ca 60) shows affinity with species found in Myanmar. Table 9.2 shows comparative occurrence of cyanobacteria in different lakes of eastern and north-eastern parts of country.
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Table 9.2: Cyanobactrial Diversity In Some Indian Lakes Loktak Lake, Manipur
Salt Lake, Kolkata
Chilka Lake, Orissa
Oscil/atoria chlorina,
Oscil/atoria bra vis,
Oscil/atoria chilkensis
O. tenuis,
O. amphibia,
Phormidium coriuim
Microchaete loktakensis
O. laete-virens
P. fragile
O. limosa
Microco/eus chthonopalstis
O. salina
M. palasoscus
O. minnesotensis
Ababaena torulosa
O. simplicissima
Lyngbya aestuarii
Anabaena spherica
L. aestuarii var. tenuis
Lyngbya cinerescens Mlcroco/eus chthonoplastis
Conclusion Thus it is clear that study on algal biodiversity and advance researches on algal biotechnology may lead to proper environmental conservation programmes for achieving sustainable development for the benefit of local population. Ecologically both may act as a supplement to each other. On one hand, biotechnology can alter the fundamental processes, viz., energy-flow and biogeochemical cycles and on the other hand, it depends on the availability of a sustainable gene pool (Gupta, 1996, 1997 a, b) by keeping priorities of biodiversity in mind (Gupta 1998). Effort should be taken for their conservation and large scale survey taking in consideration all those steps being taken for the study of higher plants. Botanical Survey of India has taken steps in this direction by initiating a project on the preparation of checklist of Indian algal flora firstly on cyanobacteria.
Acknowledgement The author is thankful to Dr. M. Sanjappa, Director, Botanical Survey of India, Ministry of Environment and Forests, Kolkata for encouragement and facilities.
References Gupta, S.L., 1996. Biotechnology and sustainable development. In: Sustainable Development Strategy: Indian Context, (Eds.) S.P. Shukla and N. Sharma. Mittal Publications, New Delhi, pp. 147-152. Gupta, S.L., 1997 a. Cyanobacteria as an ecofriendly biofertilizers. Arunachal Times, 9(91): 2. Gupta, S.L., 1997 b. Role of biotechnology and biodiversity in achieving sustainable development (microbial context). Arunachal Forest News, 15: 37-39. Gupta, S.L., 1998. Algal diversity in eastern India with reference to cyanobacteria. In: Mountain Meet 1998, Intern. Symp. Environmental Management in Mountaineous Regions, Rishikesh, Oct. 4-7.
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Gupta, S.L., 2002. Cyanobacterial diversity survey in India with reference to conservation. In: Proc. Science and Ethics of Environmental Care and Sustainability, (Ed.) A. Kumar, Allahabad, 1: 38-47. Gupta, S.L., 2003. Concept ofbiodiversity with respect to algal flora: Past and Present. In: Proc. Science and Ethics ofEnvironmental Care and Sustainability, (Ed.) A. Kumar, Allahabad, 2: 145-159. Rao, P.S.N. and Gupta, S.L., 1997. Freshwater algae. In: Floristic Diversity and Conservation Strategies in India, Vol. 1: Cryptograms and Gymnosperms, (Eds.) V. Mudgal and P.K. Hajra. Botanical Survey of India, Kolkata, pp. 47-62. Rippka, R., Druelles, J.B., Waterbury J.B., Herdman, M. and Stanier, R.Y., 1979. Generic assignments, strain histories and properties of pure culture of cyanobacteria. J. Gen. Microbiol., 111: 1-6L Tewari, D.N., 1993. Conservation of biodiversity. In: Indo-British Workshop on Biodiversity, Kolkata, pp. 9-15.
Chapter 10
A Study on Adoptional Behaviour of Sericulturists and their Characteristics in Anantapur District of Andhra Pradesh B. Sujatha1, P. Lakshminarayana Reddy, s. Sankar Naikl, A. Vijaya Bhaskar Raol and P. Sujathamma2 IDepartment of Sericulture, Sri Krishnadevaraya University, Anantapur-515 003 2Department of Sericulture, Sri Padmavati Mahila Visvavidhyalayam, Tirupati -517 502
ABSTRACT The present study was undertaken in SO villages in Anantapur district of Andhra Pradesh state with an objective to study the adoptional behaviour Ilevel of improved sericultural technologies of sericulturists (among big, small and marginal farmers), to find out the relationship between socio-economic characters of farmer and their adoption and to identify the constraints for non-adoption of improved sericulture practices by sericulturists. The results based on interview of 150 sericulturists (SO big, 50 small and 50 marginal) revealed that the level of adoption was higher for the practices of spacing given while planting, FYM, disinfection, temperature and hUmidity maintenance during rearing and spacing at different instars whereas the adoption was very low Inil for practices of biofertilizers, vermiculture and mulching and remaining practices was partially adopted. The adoption level among different categories of farmers was in order
A Study on Adoptiona/ Behaviour of Sericulturists and their Characteristics in Anantapur
91
of big farmers> small farmers> marginal farmers. The factors/characteristics such as experience in sericulture, mass media participation and net income were having a positive and significant association with adoption level of all categories of sericulturists. Lack of knowledge, lack of technical guidance, lack of finance, traditional practice, strong belief on their own ideas and over confidence, non availability of cuttings in time and high cost of fertilizers were the main reasons identified for non-adoption of improved sericulture practices by sericulturists. Keywords: Adoption, Sericulture practices, Soda-economic characters, Constraints.
Introduction Sericulture is being a rural agro based labour intensive industry is ideally suited for improving the social and economic status of the rural people. With the development of new technologies in mulberry cultivation and silkworm rearing, sericulture has now emerged as a main profession and a major cash crop for the rural folk of the country. In India now the sericulture development is rapidly increasing due to the introduction of new technologies through R and D support. The introduction and adoption of Sericultural technology will not only result in increasing the silk production in the country but also help in improving the quality, standard of living, socio-economic conditions of rural population and gives high returns to the farmers. Sodo-economic conditions of the farmers influence not only the knowledge and adoption level but also the cocoon productivity (Munikrishnappa et al., 2002). However, no such study was conducted on the above aspects in Andhra Pradesh in recent years, even though the cocoon production in Hindupur region has increased tremendously after the introduction of IICA Oapan International Co-operation Agency). Hence, the present study was carried out with the following specific objectives: 1. To study the adoptional behaviour/level of improved sericultural technologies by sericulturists 2. To find out the relationship between socio-economic characters of farmers and their adoption. 3. To identify the constraints for non- adoption of improved sericultural practices by sericulturists.
Materials and Methods The present study was conducted in Andhra Pradesh state, as it is contributing 40 per cent to the country's raw silk production. In this state, Anantapur district was purposively selected for the study, as it is the first largest producer of cocoons in Andhra Pradesh. Out of forty nine mandals in Anantapur district, ten mandals were selected randomly. From each mandal five villages were selected randomly. From each village three farmers i.e., 1 big, 1 small and 1 marginal farmers were selected purposively based on their mulberry acreage. Total sample size was 150. Pre-c1assification of respondents was done based on the total land holding as big farmer (>2.5 acres of wet land), small farmer (1.25 to 2.5 acres of wet land) and
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92
marginal farmer, « 1.25 acres of wet land). Primary data were collected by personal interview method using structured schedule. The data collected include the adoption pattern of various recommended sericultural practices and the socio-economic characteristics of farmers namely age, education, family size, experience in sericulture, mass media participation, social participation, extension contact, extension participation, material possession, credit facilities, net income, etc.
Analytical Tools Used for the Study Adoption coefficient was worked out to study the impact of socio-economic characteristics on the adoption of different sericultural practices. For this purpose, full adoption was scored as 2, partial adoption as 1 and non-adoption as 0 for each technology and the total score (actual score) was worked out for each farmer, based on the level of adoption of the technologies or practices recommended for sericulture. Then the adoption coefficient was computed for each farmer by using the following formula: Adoption coefficient =
Actual score obtained x 100
Total score obtained Percentage and frequencies were worked out and simple tabular analysis was employed to analyse the data. Regression analysis was utilized in the study to find out the extent of relationship between adoption level of sericultural technologies and independent variable. The general form of the regression equation was
Y=a + blx l + b2 x2 + b3 x3 +.................. + b. x. + 11
where, Y: Dependent variable (Adoption coefficient) a: Intercept Xl to X.: Independent variables bI to b.: Coefficient to be estimated 11: Error terms In the present study, age, education, family size, experience in sericulture, mass media participation, social participation, extension contact, extension participation, material possession, credit facilities, net income, were considered as the independent variables and regressed with the dependent variables, adoption coefficient". Based on the score obtained, the independent variables were classified into high, medium and low category, for comparison of adoption levels of all categories of farmers, 11
Results and Discussion Adoption of Recommended Sericultural Practices The adoption pattern of recommended seicultural practices is presented in Table 10.1.
A Study on Adoptional Behaviour of Sericulturists and their Characteristics in Anantapur
93
Table 10.1: Extent of Adoption of Specific Recommended Practices by Serlculturlsts SI. No.
Adoption of Specific Recommended Practices of Mulberry Cultivation
Big Farmers (n=50)
FA (%)
PA
NA
(%)
(%)
1. Variety of mulberry
60.0
40.0
2. Spacing
94.0
6.0
3. Vermiculture
10.0
4. Farm yard manure application
94.0
6.0
6.0
14.0
38.0
62.0
5. Biofertilizer application 6. Fertilizer application
90.0
SO.O
Small Farmers (n=50)
FA (%)
PA
NA
(%)
(%)
40.0
60.0
90.0
10.0
6.0
FA (%)
PA
NA
(%)
(%)
30.0
70.0
84
16.0
94.0
94.0
6.0
8.0
12.0
40.0
60.0
100.0
7. Mulching
Marginal Farmers (n=50)
80.0
100.0 92.0
8.0
8.0
18.0
34.0
62.0
100.0
74.0 4.0 100.0
8. Plant Protection
30.0
30.0
40.0
20.0
36.0
44.0
10.0
40.0
50.0
9. Incubation
30.0
40.0
30.0
30.0
30.0
40.0
28.0
40.0
32.0
10. Disinfection
98.0
2.0
98.0
2.0
98.0
2.0
11. Silkworm spacing
SO.O
20.0
92.0
8.0
98.0
2.0
12. Temperate and Humidity at different instars
96.0
4.0
80.0
20.0
86.0
14.0
13. Diseases and Control
40.0
44.0
36.0
44.0
30.0
46.0
16.0
20.0
24.0
FA: Full adoption; PA: Partial adoption; NA: Non-adoption.
Big Farmers The big farmers were full adopters for practices of spacing given while planted, FYM (94 per cent), variety planted (60 per cent), disinfection (98 per cent), temperature and humidity maintenance during rearing (96 per cent) and spacing at different instars (80 per cent), whereas, partial adoption was recorded for practices of fertilizer application (62 per cent), plant protection (30 per cent), identification and control of diseases (44 per cent) and incubation (40 per cent) and non- adoption was high for the crucial practices of vermiculture (90 per cent) and biofertilizer (80 per cent) and mulching (100 per cent). Small Farmers The small farmers were full adopters for the recommended practices of FYM (94 per cent), spacing given while planting (90 per cent), disinfection (98 per cent), spacing at different instars (92 per cent), and temperature maintenance during rearing (80 per cent) whereas, partial adoption was recorded for practices of variety planted (60 per cent), fertilizer application (60 per cent), identification and control of diseases (44 per cent) and incubation (30 per cent). Non-adoption was high for the practices of biofertilizer (80 per cent), vermiculture (94 per cent) and mulching (100 per cent).
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Marginal Farmers
The marginal farmers were full adopters for practices of spacing given while planting (84 per cent), FYM (92 per cent), disinfection (98 per cent), spacing at different instars (98 per cent), and temperature and humidity maintenance during rearing (86 per cent) and whereas, partial adoption was recorded for practices of variety planted (70 per cent), fertilizer application (62 per cent) and plant protection (40 per cent), identification and control of diseases (46) and incubation (40 per cent). Nonadoption was high for the practices of biofertilizer (74 per cent), mulching and vermiculture (100 per cent). Results of the above investigation indicated that level of adoption in all categories of farmers was full for practices of spacing during planting, application of FYM, disinfection, spacing at different instars, maintenance of temperature and relative humidity. The possible reasons of the practices are simplicity of practices, experience, frequent visit to state and central sericulture departments and no cost involvement for disinfection, since this was supplied by Departmental persons. With regards to variety planted, fertilizer application, plant protection, incubation care, identification and control of silkworm diseases the adoption was partial. This may be due to traditional practice, lack of knowledge, high cost of fertilizers, observing neighbour farmers, strong belief on their own ideas, negligence of rearers at the time of disease attack. With regards to application of biofertilizers, vermiculture and mulching the adoption was low I nil. This may be due to lack of knowledge,lack of finance, lack of credit facilities, and lack of technical guidance and complexity of practices. The above findings are in conformity with that of Satheesh (1990), Dolli (1991), Sreenivasulu (1991), Dolli et al. (1993), Geetha (1993), Singhvi et al., 1994), Zeaul Ahsan (1994), Sreedhara (1996), Geetha et al. (2001), Munikrishnappa et al. (2002), Vijaya Prakash and Dandin (2005).
Relationship Between Sodo-economic Characteristics of Farmer with Adoption of Technology in the Study Area In order to study the effect of socio-economic characters on the adoption of recommended Sericultural technologies, a regression model was fitted by considering age, education, family size, experience in sericu1ture, mass media participation, social participation, extension contact, extension participation, material possession, credit facilities, net income as independent variables and adoption coefficient as the dependent variable.The regression coefficients obtained from the model are presented in Table 10.2. The value of co-efficient of multiple determination (R2) was 0.65, 0.58 and 0.63, respectively for the models fitted for big, small and marginal farmers, which implies that the variables included in the regression model put together explain 0.65, 0.58 and 0.63 percent of the variation in the data with respect to adoption.
Big Farmers It could be observed that the socio-economic variables like education, experience in sericulture, mass media participation, net income and credit facilities were positive relation with adoption behaviour Ilevel of sericulture practices with respect to big farmers. However, education, experience in sericulture and net income was significantly associated with adoption of sericultural technologies. The coefficients
A Study on Adoptional Behaviour of Sericulturists and their Characteristics in Anantapur
95
of age, social participation, extension contact, extension participation, and material possession, were negative for the model fitted for big farmers but statistically not significant. Though the number of family members involved in sericulture was found to be negative but significantly relationship with adoption of sericultural technologies. This may be due to involvement of family labour in non-farm or other activities and thus contributing negatively. Table 10.2: Relationship Between Socio-economic Characters of Farmers and their Adoption of Sericultural Technologies Anantapur District (n=150)
SI. No. • Characteristics
Correlation Coefficient Big Farmers (n=50)
Small Farmers (n=50)
Marginal Farmers (n=50)
Coefficient 't'Value Coefficient ,('Value Coefficient 't'Value
1.
Age
2.
Education
3.
Family Size
4.
Experience in Sericulture
5.
Social Participation
-{).015
-{).097
-{).036
-{).202
-{).086
-{).657
1.304
3.825*-
-2.386
-{).965
-2.790
-{).691
-{).248
-{).612-
1.037
2.172--
1.121
2.058--
1.360
2.691--
1.393
.2.456*-
1.386
2.576**
-{).21 0
-{).874
-{).194
-1.097
-{).244
-{).673
6.
Mass media Participation
0.985
1.313-
1.128
2.024*-
1.066
2.138--
7.
Extension Participation
-{).078
-{).168
0.293
1.358--
0.252
1.480--
8.
Extension Contact
-{).160
-{).502
-{).466
-1.245
-{).370
-{).156
9.
Material possession
-{).358
-{).440
0.253
0.058
0.212
2.223
1.128
1.995--
0.214
0.852-
0.282
0.633-
11. Credit facility
0.342
0.785
-{).103
-{).874
-{).253
-{).921
12. Intercept
94.463
92.689
93.373
0.65
0.58
0.63
10. Net income
13. R2
-: Significant at 5 per cent level; **: Significant at 1 per cent level.
The adoption behaviour/level of big farmers was positive and significant associated with some of the independent variables except age. From these results, it can be concluded that adoption level of big farmer's increases with education, experience in sericulture, mass media participation, and net income. The findings of the present study are in conformity with the studies reported by Shivaraja (1985), Vijaya Prakash and Dandin (2005), Lakshmannan and Geethadevi (2007) and Meenal and Rajan (2007). Small and Marginal Farmers The regression coefficient of experience in sericulture, family size, mass media participation, extension participation, material possession and net income were positive and significantly related to the adoption behaviour/level of small and marginal farmers.
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Though the regression coefficients of social participation, extension contact and credit facilities were negative, they were not statistically significant. This may be because of the fact that, the farmers have not participated in any social activities, they were not utilizing contacp; for the betterment of sericulture and credit facilities because of lack of proper information regarding credit facilities provided by government/ private sectors. The age was contributing negatively for all categories of farmers, because of the fact that, sericulture is an inherited occupation in most of the households and the experience gained over years influence adoption. Education level of the respondents was also found to be contributing negatively towards the knowledge in sericulture practices. In the case of small and marginal farmers experience in sericulture, family size, mass media participation, extension participation, material possession and net income are the independent variables were positive and significant relationship with adoption behaviour. The findings of the present study are partially in agreement with the studies reported by Prakash Kumar (1986), Geetha et al. (2001, Vijaya Prakash and Dandin (2005).
Comparison of Adoption Level Among Different Categories of Farmers Perusal of Table 10.3 indicate that adoption level among different categories of farmers was in order of big farmers> small farmers> marginal farmers. It also indicate that 52 per cent big, 54 per cent small and marginal farmers were in high adoption category, whereas 28 per cent big, 30 per cent small and 24 per cent marginal farmers were medium adoption category and 20 per cent big, 16 per cent small and 22 per cent marginal farmers were low adoption category. Table 10.3: Distribution of Different Categories of Farmers According to their Adoption BehavlourlLevel Category of Farmers
Adoption level High
Medium
Low
Total
No. %
No. %
No. %
No. %
Big farmers
26 52
14 28
10 20
80 53.34
Small farmers
27 54
8 16
41 27.33
Marginal farmers
27 54
15 30 12 24
11 22
29 19.33
Constraints for Non-adoption of the Recommended Sericultural Technologies The major constraints for partial/non-adoption of technology (Table 10.4) were low price of cocoons (75 per cent), lack of quality of dfls (60 per cent), traditional practice (40 per cent) and lack of technical guidance(training) (20 per cent) for big farmers.
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Table 10.4: Constraints for Non-adoption ofthe Recommended Sericultural Technologies in the Study Area Big Fanners
Small Fanners
Marginal Fanners
(%)
(%)
(%)
Traditional practice
60 15 40
Low price of cocoons
75
40 40 50 60
30 35 45 65
5. Lack of finance
10
60
6. 7. Lack of biofertilizers in time
5 15
10
50 15
8. High cost of fertilizer
10 15
30 50 5
40 50
8
40
30
11. Lack of credit facilities
15 20
50 60
50
12. Lack of technical guidance (Training)
SI. No.
1. 2. 3. 4.
Constraints
Lack of quality DFLs Lack of knowledge
Strong belief on their own ideas
9. Negligence of rearer 10. Lack of separate rearing house
3
60
The constraints expressed by small farmers for the adoption of recommended sericultural technologies include lack of finance, lack of technical guidance(training), low price of cocoons (60 per cent), traditional practice, high cost of fertilizers, lack of credit facilities(50 per cent), lack of quality dfls, lack of knowledge, lack of separate rearing house (40 per cent) and lack ofbiofertilizer in time (30 per cent). The constraints expressed by marginal farmers for the adoption of recommended sericultural technologies include low price of cocoons (65 per cent), lack of technical guidance (training) (60 per cent), high cost of fertilizers, lack of credit facilities, lack of finance (50 per cent), traditional practice (45 per cent), lack of biofertilizers in time (40 per cent) and lack of knowledge (35 per cent).
Conclusion Based on the findings of the study, it could be concluded that the adoption level among different categories of farmers was in order of big farmers> small farmers> marginal farmers. This is due to lack of finance and insufficient credit facilities provided to small and marginal famers.The present study reveals that farmers can achieve higher rate of cocoon production with scrupulous adoption of advanced technologies. Farmers should be provided more practical training programmes, apart from educating them on the need of their dedicated practice of advanced farming technologies. There must be a mechanism to ensure stable price for BV /CB cocoons in the form of Minimum Support Price (MSP) as in the other agriculture crops to ensure farmers to get higher profit. Further, when farmers are given credit support an assured cocoon price, they will follow the technologies recommend without much hesitation. The study also revealed that the socio-economic variables of farmers were found important in deciding about adoption of recommended sericultural
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technologies. Hence, for the introduction of new technologies in sericulture, the fanner's characters and conditions must also be considered apart from the technical factors.
References Dolli, S.5., 1991. Bench mark Survey report for the study of effectiveness of different extension communication systems. Dissertation Submitted to CSR and TI, Mysore. Dolli, S.5., Kalappa, H.K., Subramanian, RK, Chikkanna Singh, N.R, Sen, A.K, Iyengar, N. and Datta, RK., 1993. Extent of adoption of improved sericultural practices by the sericulturists. Indian Silk, 31(10): 35-40. Geetha, G.5. 1993. Socio-economic status and its implication on sericulture technology adoption in Channarayapatna Taluk, Hassan District, Kamataka. Dissertation submitted to CSR and TI, Mysore. Geetha, G.5., Srinivasa, G., Jayaram, H., Iyengar, M.N.S. and Vijaya Prakash, N.B., 2001. Socio-economic determinants of farmers oriented technology packages for sericulture: A field study. Indian. J. Serie., 40: 96-99. Lakshmannan, S. and Geethadevi, RG., 2007. Knowledge and adoption level of farmers of bivoltine and cross breeds sericultural technologies. Indian J. Serie., 46: 72-75. Meenal, R and Rajan, R: K 2007. Impact of socio-economic characters of sericulturists on knowledge, adoption and cocoon production in Tamil Nadu. Indian J. Serie., 46: 49-51. Munikrishnappa, H.M., Jagadisha, K and Srinivasa, 2002. Association of socioeconomic characters with knowledge and adoption of improved sericulture practices by sericulturists in Mysore district. Indian J. Serie., 41: 89-91. Prakash Kumar, R, 1986. A study on 1doption of improved Sericultural practices and labour utilisation among big, small and tenant farmers of Ramanagara taluk, Bangalore district. M.Se. (Agril.) Thesis, Univ. Agril. SeL, Bangalore, India. Satheesh, D., 1990. A study on the knowledge and adoption of chawki rearing practices by silk worm rearers of Kanakapura taluk, Bangalore district. M. Se. (Agril.) Thesis, Univ. Agril. Sci., Bangalore. Shivaraja, K, 1985. A study on adoption behaviour, net income and employment potential of bivoltine seed cocoon producers. M.Se. (Agril.) Thesis, Univ. Agril. Sci., Bangalore. Singhvi, N.R, Sethu Rao, Y.R, Madhava Rao, Y.R, Iyengar, M.N.S. and Datta, RK, 1994. Knowledge level and adoption of new sericulture technology by farmers in Hunsur Taluk, Mysore district, Kamataka State: An Evaluation. Indian J. Serie., 33: 48-55. Sreedhara, V., 1996. A study on knowledge and adoption of recommended practices of sericulture among farmers of Pavagada Taluk, Tumkur District. M. Se. (Agril) Thesis, GKVK, Bangalore.
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Srinivasulu, V.M., 1991. Socio-economic conditions of sericulturists in relations to adoption of new Sericultural technologies in Karnataka State. STS Dissertation submitted to CSR and TI (ICTRETS), Mysore. Vijaya Prakash, N.B. and Dandin, S.B., 2005. Factors influencing the adoption of bivoltine sericultural practice in Mandya district of Karnataka. Indian. J. Serie., 44: 55-58. Zeaul Ahsan, M.D., 1994. A study on knowledge adoption and perception of improved sericulture technology by the sericulturists of Malda district in West Bengal. M.Se. Thesis, CSR and TI, Mysore.
Chapter 11
Spermoderm Patterns in Some Genera of Andropogoneae D. Someswari, T. Srivalli1 and T.N. Marf IDepartment of Botany, T./.P.S. College, Guntur 2Department of Botany, Acharya Nagarjuna University, Guntur-522 510
ABSTRACT India includes mostly tropical and subtropical areas except the mountains with temperate climate. The members of the tribe Andropogoneae, grow under wide ecological conditions and are found important for use as forages and commercial grasses. Andropogoneae is considered to be the most highly specialized and is found in an active state of evolution. Though extensive studies were conducted on grasses all over the world, there is practically little work on the grasses of Andropogoneae from India, that too limited only to cytological aspects. Taxonomically, some of the genera in Andropogoneae are still in a confused state. Revisionary work on these taxa is the most immediate requirement for a better understanding of the tribe. Thus, there is a valid reflection of the weakness of discontinuities within genera. Seeds of the six fodder grass species belonging to three genera viz., Iseilema, Dichanthium and Eremopogon were chosen for the study. I. laxum, I. antherophoroides, I. venkateswarlui, D. annulatum, D. pertusum and E. foveolatus are the different species selected in the present investigation. Seed coat morphology was studied by observing SEM photographs of these species. The seed characters are considered fairly conservative among the species and the differences among them would be mainly due to genetic differentiation among the species. Among the present grasses, E. foveolatus showed similar seed coat structure like that of D. annulatum, indicating the genetic similarity
Spermoderm Patterns in Some Genera of Andropogoneae
101
between the two genera Eremopogon and Dichanthium. In Iseilema, the process of speciation seems to be at a higher rate. Interrelationships among the species of lseilema are evident by certain common spermoderm characters.
Keywords: Andropogoneae, lseilema, Dichanthium, Eremopogon, Spermoderm characters.
Introduction India includes mostly tropical and subtropical areas except the mountains with temperate climate. The members of the tribe Andropogoneae, grow under wide ecological conditions and are found important for use as forages and commercial grasses. Andropogoneae is considered to be the most highly specialized and is found in an active state of evolution. Though extensive studies were conducted on grasses all over the world, there is practically little work on the grasses of Andropogoneae from India, that too limited only to cytological aspects. Taxonomically, some of the genera in Andropogoneae are still in a confused state. ReviSionary work on these taxa is the most immediate requirement for a better understanding of the tribe (Claytone and Renovoize, 1986). Thus, there is a valid reflection of the weakness of discontinuities within genera. A resolution of phylogenetic trends in Andropogoneae would be very much understood by comparative investigation of SEM studies of seeds from six grass species of three genera i.e., Eremopogon, Dihcanthium, and lseilema.
Materials and Methods Seeds of the six fodder grass species belonging to three genera viz., Iseilema, Dichanthium, and Eremopogon were chosen for the study. The different species selected in the present investigation include three species of Iselima i.e., I. laxum Hack, I.antherophoroide Hack., I. venkateswarlui Satyavathi; two species of Dicanthium i.e., D. annulatum Forsk., D. pertusum L., and one species of Eremopogon i.e., E. foveolatus Del. A minimum of 30 seeds were used for measurements of seed size. SEM photographs were taken from Indian Institute of Chemical Technology, Hyderabad. Topography of the seed coat was described by following the terminology given by Lersten (1981).
Results and Discussion The seed coat morphology provides valuable information with regard to taxonomical identification of seeds. In the species of Dichanthium, Eremopogan and lseilema, seeds are lanceolate with rounded ends except in D. pertusum having smaller oval seeds (89.2/42.8) (Figure 11.4). The hilum is lanceolate, black or deep brown with light brown rim in all six species. It is evident that the seeds were largest in D. annulatum (97.70) and smallest in D. pertusum (89.2/42.8) (Table 11.1). Among the species of Iseilema, I. venkateswarlui showed the largest seeds. The spermoderm pattern found in the six species of three genera of Andropogoneae is shown in Figures 11.1-11.17. The spermoderm has complex network of interwoven rugae. The elongation of reticulate is more in the same direction. Thickness of rugae is nearly uniform but at places giving beaded appearance. It is characterized by penta to hexagonal epidermal cells, showing simple reticulate with
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distinct cell boundaries in I. laxum; with distinct to indistinct cell boundaries in D. annulatum (Figures 11.1-11.3). Cell length is usually equal to breadth in 1. laxum (Figure 9 to 11); almost double the breadth in other two species of Iseilema and Dichanthium (Figures 11.1-11.17). Cell walls in all the species are almost smooth with frequent pits and sutures, impregnated with low mounds of wax. 1. venkateswarlui (Figures 11.15-11.17) was found distinct by having single thick layer, but I. laxum (Figures 11.9-11.11) and 1. anthephoroides (Figures 11.12-11.14) were similar in having double layers. Table 11.1: Seed Size in Fodder Grass Species SI. No.
Name of the Species
Length (pm)
Breadth (pm)
1.
Iseilema laxum
90.8
30.9
2.
Iseilema anthephoroides
94.12
43.2
3.
Iseilema venkateswartui
97.64
41
4.
Dichanthium annulatum
97.7
54
5.
D. pertusum
89.2
42.8
6.
Eremopogon foveolatus
91.5
31
According to Skvortsov and Rusanovitch (1974), the spermoderm is genetically affected and is important in the studies of intra or interspecific variations. Lersten (1981) has stated that seed surface pattern reflects epidermal configuration and cuticular deposition as influenced by seed expansion. Thus, the views expressed on the study of seed coat patterns during recent times justify the utility of such investigations in solving a variety of problems concerning with systematics, ecology, genetics and evolution (Brisson and Peterson, 1976). Recently in gramineae, Korbes-Gornhe (1981) studied seed surface patterns to distinguish prehistoric cereal grains of Triticum and Secale. Buth (1982) utilized SEM studies as an aid in identification of caryopsis of Triticum. Jordan et al. (1983) discussed prominent spermoderm patterns of poaceae. SEM studies in the grasses of Andropogoneae were made in the present study, may help in systematic inferences. The spermoderm characters of different species within the same genus vary and show totally different type of ornamentation (Sharma et al., 1977; Trivedi et al., 1978a, b). Attempts have been made to understand interrelationships among the species of Chenopodium on the basis of seed surface features (Bera and Mukherjee, 1922). The work of Chuang and Heckard (1972, 1983, 1991) on the Scrophulariaceae and Bacon (1997, 1989) on Hydrophyllaceae are the two classic examples justifying the applicability of seed surface characteristics using SEM for taxonomic studies. The seed characters are considered fairly conservative and the differences among them would be mainly due to genetic differentiation among the species. Among the present grasses, E. foveolatus showed similar seed coa t structure (Figure 11.3) like that of D. annulatum, indicating the genetic similarity between the two genera Eremopogon and Dichanthium. This feature also corroborate with the opinion of Stapf (1919) for placing Eremopogon along with Dichanthium in the section Amphilopiastrae. This is
Spermoderm Patterns in Some Genera of Andropogoneae
103
104
Emerging Trends in Biological Sciences
.,... .,...
Spermoderm Patterns in Some Genera of Andropogoneae
'C C 111
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": ,.. ,.. :::l Cl
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u:::
105
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Emerging Trends in Biological Sciences
Spermoderm Patterns in Some Genera of Andropogoneae
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E
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108
Emerging Trends in Biological Sciences
Spermoderm Patterns in Some Genera of Andropogoneae
109
also supported by the observation of higher similarity values in the protein contents profiles of Eremopogon and Dichanthium (Mary and Someswari, 1999). In Iseilema, the process of speciation seems to be at a higher rate. Interrelationships among the species of Iseilema are evident by certain common spermoderm characters but the species were found different in certain features such as cells having equal length and breadth in I. laxum but double the breadth in other two species of Iseilema. I. venkateswarlui was different by showing thick layered single cell wall, where as I. anthephoroides and I. laxum were similar for double cell layers. Hence, based on SEM studies, 1. venkateswarlui could be considered a distinct species among these grasses. The SEM results of the present study are in agreement with the morphological and cytogenetical inferences regarding the species relationships of Andropogoneae.
Acknowledgements The authors D. Someswari and T. Srivalli are thankful to the Management of T.J.P.5. College for providing necessary facilities to carry out this work.
References Bacon, J.0.,1987. Systematics of Nama (Hydrophyllaceae); Seed coat morphology of Lemmonia californica and Nama species allied with Nama demissum. Aliso, 11: 441-450. Bacon, JD., 1989. Seed coat morphology of Draperia systala (Hydrophyllaceae) and its importance to the systematics of Nama. Sida, 13: 461-466. Bera and Mukherjee, K.K., 1992. Characterization of seed of three cytotypes of Chenopodium album through analysis of phenolics and proteins. Seed Sci. Technol., 20: 57-67. Brisson, J.0. and Peterson, RL., 1976. A critical review of the use of Scanning Electron Microscopy in the study of the seed coat. SEM (Part VII) Vol II. Proceedings of the worksJwp on plant science application ofSEM. lIT Research Institute, Chikago. Illinois, USA. Buth, G.M., 1982. SEM study as an aid in identification of caryopsis of Triticum (wheat). Jour. Econ. Taxon. Bot., 3(2): 537-540. Chuang, T.!. and Heckard, L.R, 1972. Seed coat morphology in Cordylanthus and its taxonomic significance. Amer. J. Bot., 59: 258-265. Chuang, T.!. and Heckard, L.R, 1983. Systematic significance of seed surface features in Orthocarpus (Scrophulariaceae-subtribe Castillejinae). Amer. J. Bot., 70: 877890. Chuang, T.!. and Heckard, L.R, 1991. Generic realignment and synopsis of subtribe Castillejinae (Scrophulariaceae-subtribe Pedicularaceae). Systematic Botany, 16: 644-666. Clayton, W.D. and Renovoize, S.A., 1986. Genera Graminum. Kew Bulletin Additional Series, 13: 23-27.
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Jordan, J.L., Jordan, L.S. and Jordan, CM., 1983. Prominent spermoderm patterns of Poaceae. Botanical Magazine Tokyo, 96: 269-272. Korbes-Grohne, U., 1981. Distinguishing prehistoric cereal grains of Triticum and Secale on the basis of their surface patterns using SEM. Jr. Agric. Sci., 8: 197-204. Lersten, N.R., 1981. Testa topography in Leguminosae, sub family Papilionoideae.
Proc.lowaAcad. Sci.,88: 180-191. Mary, T.N. and Someswari, D., 1999. Seed protein profile of a few fodder grasses of Andropogoneae (Poaceae). Plant Tissue Culture and Biotechnology: Emerging Trends, 309-313. Sharma, S.K., Babu, CR., Johri, B.M. and Hepworth, A., 1977. SEM studies on seed coat patterns in Phaseolus mungo, P. radiatus-sublobatus. Phytomorphology,27: 106111. Skvorlsov, A. K. and Rusanovitch, 1.1., 1974. SEM of the seed coat surface on Epilobium species. Bot. Not., 127: 292-401. Stapf,O., 1919. Gramineae. In: Prain D. FI. Tropical Africa, 9: London. Trivedi, B.S., Bagchi, G.D. and Bajpai Usha, 1978a. SEM studies on spermoderm of Sesbania Seop. (Leguminosae). Curr. SCi., 47: 599-500. Trivedi, B.S., Bagchi, G.D. and Bajpai Usha, 1978b. Spermoderm patterns in some taxa of Vicieae (Papilionatae: Leguminosae). Phytomorphology, 28: 405-410.
Chapter 12
Phyto-Pharmacology of Plumbago zeylanica: A Review Pavankumar Bellamkondi and K. Shankaramurthy * Department of Biotechnology, Kuvempu University, Shankaraghatta-577 451, Karnataka
ABSTRACT Plants have been one of the important sources of medicines since the beginning of human civilization. There is a growing demand for plant based medicines, health products, pharmaceuticals, food supplements, cosmetics etc. Plumbago zeylanica Linn. is used as an antimalarial, antifungal, cardiotonic, antifertility action, antibacterial, anti-inflammatory, hypolipidaemic activity, antiatheroslerotic activity and antiviral agent. It is reported to contain alkaloids, flavonoids, saponins, steroids and terpenoids. A review of chemical constituents present in various parts of P.zeylanica and their pharmacological actions is given in the present article.
Keywords: Plumbago zeylanica, Phyto-chemical constituents, Pharmacological actions, Toxicity.
Introduction There exists a plethora of knowledge and information and benefits of herbal drugs in our ancient literature of Ayurvedic and Unani medicine. One of the earliest treaties of Indian medicine, the Charaka Samhita (1000 RC.) mentions the use of over 2000 herbs for medicinal purpose. According to the WHO survey 80 per cent of the • Corresponding Author: E-mail: [email protected].
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Emerging Trends in Biological Sciences
populations living in the developing countries rely almost exclusively on traditional medicine for their primary health care needs. Exploration of the chemical constituents of the plants and pharmacological screening may provide us the basis for developing the leads for development of novel agents. In addition, herbs have provided us some of the very important life saving drugs used in the armamentarium of modem medicine. However, among the estimated 250,000-400,000 plant species, only 6 per cent have been studied for biological activity, and about 15 per cent have been investigated phytochemically (Balandrin et al., 1985; Cragg et al., 1997). This shows a need for planned activity guided phyto-pharmacological evaluation of herbal drugs. Plumbago zeylanica Linn. is an annual, stiff erect herb, about 0.6 to 1.5m high and found commonly as a weed throughout India. This article intends to provide an overview of the chemical constituents present in various parts of P.zeylanica and their pharmacological actions.
General Information Plumbago zeylanica Linn. belongs to family Plumbaginaceae. It is found through out India much-cultivated in gardens, widely in various parts of country, Western peninsula, Bengal, Uttar Pradesh and southern India. It is known as Chitramoola (Kannada), Lead wort (English), Chita (Hindi), Citraka (Sanskrit). The flowering and fruiting time is during the month of November and January. Leaves are thin, 3.8 to 7.5 mm by 2.3 to 3.8 mm, stems 0.6 to 1.5 m long, somewhat woody (Seetharam et al., 1999).
Therapeutic Uses Mentioned in Ayurvedic Pharmacopoeia The plant material is mentioned in Charaka Samitha in the preparation of Rasayana drug. The dried plant is also used in dantaroga (disease of tooth), yakrtroga (disorders of the liver), apaci (lymphadenitis), granthi (tumor), medroga (obesity). The dried plant material (Yograj Gug~lu) is used for vata related conditions (joints and muscles), Saptavimastika Guggulu (vata and pitta), Purnavadi guggulu for Vata-rakta (vitilated air in the blood). P. zeylanica has been used in Indian medicine for centuries as "Deepaniya", meaning stimulating the process of digestion and metabolism.
Therapeutic Uses Mentioned in Ethnobotanical Studies The dried powdered root of P. zeylanica mixed with goat milk, is taken to relieve body pain and arrest frequent urination. The powdered seeds were applied on boils and carbuncles which were practiced by Gonds of Ghat Parasia. A decoction of the root combined with black pepper, ginger and salt, is used to treat fever, which is practiced in the Gaddi Tribe, Himachal Pradesh. In Nigeria, the leaves are used in soap as remedy against intestinal worms and fever. In Ghana the root is administrated as an enema to treat piles. In Ivory Coast and Upper Volta, root is used to treat leprosy. The root (5 g) is kept in the mouth and chewed for cure of aphthae (Siddique et al., 2004). The dri~d powdered root mixed with goat milk to relieve body pain and arrest frequent urination (Sandhya et al., 2004). A decoction of the root is combined with black pepper, ginger and salt, and used to treat fever (5-10 ml twice a day for four days) (Singh and Kumar,1999). The plant is used as an abortefacient by introducing
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it into the vagina to produce an irritant action (Burkill, 1935). The leaves and roots have a vesicant and caustic effect on the skin. (Watt and Breyer-Brandwijk, 1962; Irvine, 1961; Chopra, 1933; Quisumbing, 1951). Table 12.1: Different Parts of Plumbago zeylanica with the Pharmacological Activity Part Used/Extract/Compound
Pharmacological Activity
Reference
Phosphate buffered saline
Anti-inflammatory
(Oyedapo, 1996)
Ethanol root extract
Anti-inflammatory
(Aparanji et al., 2005)
Suberosin
Anti-inflammatory
(Chen et al., 2007)
Aqueous extract
Antimicrobial
(Desta, 1993)
Alcoholic extracts c: roots
Antimicrobial
(Beg and Ahamad, 2000) .
80 per cent ethanolic extract
Antiviral
(Gebre-mariam et al., 2006)
Acetone, ethyl acetate extracts
Helicobacter pylori
(Wang and Huang, 2005)
50 per cent ethanolic extract + vit k Antihyperlipidemic
(Ram, 1996)
Ethanolic extract
Antimalarial
(Simosen et al. , 2004)
Aqueous/alcoholic extracts of roots
Antioxidant
(Tilak et al. , 2004)
Ethanol extract of root
Hyperglycaemia
(Olagunju et al., 1999)
Root extract
Abortificative
(Premakumari et al., 1977)
Plumbagin from organic extract
Anticarcinogenic
(Nguyen et al., 2004)
Flowers
Digestive stimulatory
(Poul et al., 1999)
50 per cent ethanolic extract
Central nervous system stimulatory
(Bopaiah and Pradhan, 2001)
Plumbagin
Antimutagenicity
(Edenharder and Tang, 1997)
Plumbagin
Antifertility
(Kini et al., 1997)
Plumbagin
Cardiotonic
(Itoigawa et al., 1991)
Pharmacognostical Studies The transverse section of root is having following components, outer single layer of epidermis, which covers a thick cuticle; below the cuticle there is a primary cortex, which is composed of parenchymatous cells. Beneath the primary cortex there is an endodermis, which is made up of lignified cell wall containing parenchyma. The vascular bundle is concentric type i.e., phloem is surrounded by xylem tissue. At the center of the section there is loosely bounded pith. In between the xylem tissue medullary ray cells are present and xylem is scattered. Starch grains were observed in primary cortical region, endodermis, and medullary rays and it is absent in phloem region as well as in pith. The starch grains measuring 7 to 9 microns in diameter are observed and it is a simple starch. The parenchymatous cells of the cortical region contain calcium oxalate in the form of needles measuring about 12 to 14 microns in length
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Figure 12.1 A: Habitat of Plumbago zeylan;ca L.; B: Flowering Twig of Plumbago zeylan;ca L.; C: An Enlarged View of a Flower
Phytochemistry There are various reports available about P.zeylanica. The main phytochemical present in this plant is 'Plumbagin', an yellow orange coloured napthoquinone compound that is in secondary cortex and medullary ray cells of roots (Mallavadhani et al,.2002). The crude extract of P.zeylanica revealed the presence of flavonoids, saponins, napthoquinones, free glucose and fructose (Beg and Ahamad, 2000). Five coumarins, seselin, 5-methoxyseselin, suberosin (Uchiyama et al., 2002), xanthyletin and xanthoxyletin were isolated from the roots of Plumbago zeylanica. The root also contains several bioactive chemical constituents which include plumbagin, 3-chloroplumbagin, droserone, chitranone, zeylanone, isozeylonone, plumbazeylone, coumarin, elliptinone, triterpenoids, ~-sitosterol, maritinone, 2-methylnaphthazarin and anthroquinones (Gunaherath et al., 1988; Gupta et al., 1993; Dinda et al., 1997). Plumbagin is a naphthoquinone and is a major component constituting about 0.03 per cent of dry weight of the roots and is considered as the
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Phyto-Pharmacology of Plumbago zeylanica: A Review
active ingredient responsible for therapeutic effects (Gupta et al., 1993). The plant is reported to contain provitamin (Bhaskarachary et al., 1995). The protein is also reported to contain in pollen grains and volatile oil from leaves and stem; 1,2-dihydro-l (S),5,5-trihydroxy-2-3(R)-dimethyl-2(R)-propyl-3, 8-bisnaphthalene-l, 4,4trione (chitranone also in root), a-and b-amyrins, lupeol, taraxasterol, j-taraxasterol (aerial parts); fructose, glucose invertase and protease (root-bark); 3,3bisplumbagin,(binaphthoquinone), elliptinone, isozeylinone, isozey lanone, and zeylinone,maritone,methy lene-3 ,3-dip lumbagin,2 methylnaphthazarin, plumbazeylanone; 5b, lla, 12, 12a-tetrahydro-l, 7-dihydroxy5b- (8-hydroxy-3-methyl-1, 4-naphthoquinon-2-yl) 5a, 12a-dimethyl-5aH-dibenzo fluorene-5,13:6,11-diquinone (a trimer of plumbagin); catechol tannin (root); amino acids; b-(2,3-dihydroxybenzoyl)-butyric acid (plumbagic acid), vanillic acid; 1,2(3)tetrahydro-33-bisplumbagin,isoshianolone, dihydrosterone and b-sitosterol also isolated from the plant (Rai et al., 2000).
o CH,
OH
0
(5 hydroxy-2 methyl, 1,4-Napthoquinone) Structure of Plumbagin
Pharmacology 1. Anti-inflammatory Activity A systemic study of anti-inflammatory effects of Indian medicinal plants began by Gujral and his associates in 1956 and they screened a number of plants for their anti-arthritic effects. Subsequently, various workers from different laboratories in India have made Significant contribution. The anti inflammatory action was reported from the phosphate buffered saline extract (Oyedapo, 1996). Extracts of Plumbago zeylanica containing suberosin exhibit anti-inflammatory activity. Suberosin is purified from such extracts and studied its effects on a set of key regulatory events in the proliferation of human peripheral blood mononuclear cells (PBMC) stimulated by phytohemagglutinin (PHA) (Chen et al., 2007). The ethanol root extract of Plumbago zeylanica (PZE) was investigated for anti-inflammatory activity in adjuvant-induced arthritic (AlA) rats. The results demonstrated that PZE inhibited the development of cutaneous Delayed type hypersensity (DTH) response in AlA rats. The T-cell proliferation induced by concanavalin A (Con A), a mitogen, was depressed in three different strains of AlA rats (Wistar, Lewis, and Fischer) when compared with that of
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normal; however, the PZE treatment of AlA rats reversed the decrease of T-cell proliferation to the normal levels (Aparanji et al., 2005).
2. Anti-microbial Activity Extracts from roots of Plumbago zeylanica showed microbiological properties. The aqueous extract and its partition (petroleum ether, dichloromethane, methanol, aqueous residue) were effective against Salmonella gallinarum, Escherichia coli, Proteus vulgaris and Klebsiella pneumoniae (Desta, 1993). Aqueous and1alcoholic extracts from roots of Plumbago zeylanica exhibited activity against Bacillus subtilis, Escherichia coli, Proteus vulgaris, Salmonella typhimurium, Pseudomonas aeruginosa and Staphyloccocus aureus. The alcoholic extract from roots of Plumbago zeylanica was tested against multidrug resistant of clinical origin (Salmonella paratyphi, Staphyloccocus aureus, Escherichia coli and Shigella dysenteriae). The extract exhibited strong antibacterial activity against all tested bacteria (Beg and Ahamad, 2000). A very dilute solution (i.e., a concentration of 1:50,000) of Plumbagin from various parts of plant is lethal to a wide spectrum of bacteria and to pathogenic fungi, i.e., Coccidioides imminites, Histoplasma capsulatum, Trichophyton spp., Candida albicans, Aspergillus niger and A. flavus (Skinner). 80 per cent of ethanolic extract have shown antiviral activities against Cox sackie virus B3 (CVB3), influenza A virus and herpes simplex virus type 1 kuoka (HSV-l) (Gebre-mariam et al., 2006). It has been shown that the presence of infection by Helicobacter pylori is strongly associated with gastric cancer and peptic ulceration. Plumbago zeylanica 1. had the highest inhibitory effects against H. pylori. Water and the organic solvents ethanol, ethyl acetate and acetone were used for P. zeylanica extraction, excluding the water extract, higher anti-H. pylori activity was demonstrated for all the extracts, both using the agar diffusion and dilution methods. The ethyl acetate extract exhibited the lowest minimum inhibitory concentrations against five H. pylori strains (Wang and Huang, 2005).
3. Anti-hyperlipidemic Activity The administration of ethanolic extract (50 per cent W IV) of P. zeylanica root, alone and in combination with vitamin E, significantly reduced serum total cholesterol, LDL cholesterol and triglyceride levels in experimentally induced hyperlipidaemic rabbits (Wang and Huang, 2005). Plumbagin, napthaquinone derivative, isolated from Plumbago zeylanica has been reported to significantly reduce serum cholesterol together with LDL-C by 53 to 86 percent and 61 to 91 per cent respectively in hyperlipidaemic rabbits and also regress atheromatous plaques in the arteries (Sharma et al., 1990). It is also reported that the ethanolic extract of Plumbago zeylanica root alone and with vitamin E lowered HDL cholesterol levels as well (Dwivedi, 1997).
4. Anti-carcinogenic Activity The antitumour activity of plumbagin (2-methyI5-hydroxy, 1,4 napthoquinone) has been evaluated against Dalton's lymphoma (DAL) in Swiss albino mice. A significant enhancement of mean survival time of Plumbagin treated tumour bearing mice was found with respect to control group. Plumbagin treatment was found to
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117
enhance peritoneal cell counts. Plumbagin treated groups were able to reverse the changes in the haemotological parameters, protein and PVC consequent to tumour inoculation. In the other reports, Plumbagin a quinonoid constituent isolated from the root of Plumbago zeylanica L., has been shown to exert anticarcinogenic, it exhibits an inhibitory effect on carcinogenesis in the intestines, causes cytogenetic and cell cycle changes in mouse Ehrlich ascites carcinoma, and possesses antiproliferation activity in human cervical cancer cells (Srinivas et al., 2004). Plumbago zeylanica causes a regression of tumor growth in methylcholanthrene-induced fibrosarcomas in Wistar rats. It is also active against P388 lymphocytic leukemia in vivo at a dose of 4 mg/kg of body weight but was not active against an L-1210 lymphoid leukemia (Krishnaswamy and Purushothaman, 1980).
5. Antimalarial Activity The treatment and prevention of human malaria reached a lot of difficulties due to the high endemicity of the disease in Amazonia, the new focuses in response to the intense migration and the resistence of Plasmodium falciparum to chloroquin as well as to other usual drugs. Plumbago zeylanica has been used in the traditional medicine against malaria and its ethanolic extract has shown high in vitro activity, being of special interest for further investigations (Simosen et al., 2004) Plumbagin shows also antimalarial effects Plasmodium falciparum enzyme, the succinate dehydrogenase (SDH) isolated from which the activity has been 50 per cent inhibited by the naphthoquinone plumbagin at an inhibitory concentration of 5mM.1t also inhibited the in vitro growth of the parasite with a 50 per cent inhibitory concentration of 0.27mM (Suraveratum et al., 2000).
6. Antioxidant Properties To examine possible mechanisms of action of P. zeylanica in relation to its reported beneficial properties, antioxidant effects of the aqueous/alcoholic extracts of root, corresponding to medicinal preparations, and the active ingredient, plumbagin, were studied. Lipid peroxidation in rat liver mitochondria induced by different agents phenolic and flavonoid content were estimated. In FRAP /DPPH [ferric reducing/ antioxidant power (FRAP), radical scavenging of 1,1-diphenyl-2-picryl hydrazyl (DPPH) I assays, boiled ethanolic extracts were the most effective, while in the ABTS (2,2'-azobis-3-ethylbenzthiazoline-6-sulfonic acid) assay boiled aqueous extracts were the most efficient. These extracts also significantly inhibited lipid peroxidation induced by cumene hydroperoxide, ascorbate-Fe (2+) and peroxynitrite and contained high amounts of polyphenols and flavonoids. This study reveals that extracts of P. zeylanica and its active ingredient plumbagin have significant antioxidant abilities that may possibly explain some of the reported therapeutic effects (Tilak et al., 2004).
7. Hyperglycemic Activity The effects of the ethanol extract of the root of Plumbago zeylanica on key enzymes of glycolysis and other biochemical parameters were studied in the rat. The results show that thigh muscle hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase activities were significantly reduced (p < 0.05) by 12.07 per cent, 51.02 per cent, 24.32 per cent and 25.16 per cent respectively in rats treated with the
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ethanol extract of Plumbago zeylanica when compared with the controls. Serum pyruvate and lactate were significantly lowered in the experimental rats by 23.64 per cent and 46.29 per cent, respectively. The reduction in the activities of the key enzymes of glycolysis and its end products suggests a reduction in flux across the glycolytic pathway in the extract-treated rats. This may be a result of impaired delivery to, and utilization of, glucose by the peripheral tissue, thus substantiating the reported hyperglycaemia in the extract-treated rats (Olagunju et al., 1999).
8. Miscellaneous Extracts of the root have been reported to be a powerful poison which, when given internally or applied to the ostium uteri, causes abortion (Bhargava, 1984; Premakumari et al., 1977). Plumbago zeylanica causes a regression of tumor growth in methylcholanthrene-induced fibrosarcomas in Wistar rats. It is also active against P388 lymphocytic leukemia in vivo at a dose of 4 mg/kg of body weight but was not active against an L-1210 lymphoid leukemia (Krishnaswamy, and Purushothaman, 1980). Plumbago zeylanica is extremely popular throughout Africa and Asia as a remedy for parasitic skin diseases, especially leprosy, scabies, acne vulgaris and surface sores and leg ulcers (Dalziel, 1956; Kokwaro, 1976). The P. zeylanica flowers showed greater effect on digestive stimulus activity than the other Plumbago species (Poul et al., 1999). Plumbagin was isolated from the organic extract of P. zeylanica and in vitro cytotoxicity against melanoma and breast cancer cell lines was demonstrated (Nguyen et al., 2004). The effects of a 50 per cent ethanol extract of the root of Plumbago zeylanica were investigated on central nervous system in rats. The extract showed enhancement of the spontaneous ambulatory activity without inducing stereotypic behavior. The neurochemical estimations revealed elevated levels of dopamine and homovanillic acid in striatum compared with the control rats. The results indicated stimulatory properties of the extract, which may be mediated by dopaminergic mechanisms in the rat brain (Bopaiah and Pradhan, 2001).
Conclusions P. zeylanica is commonly found as a weed on way side and at waste places throughout India. The plant is used in dropsy, piles, skin eruptions, colic, as a diuretic, astringent and purgative, as an antidote to snake bite, whooping cough, fever, abortion. It is reported to contain alkaloids, flavonoids, saponins, steroids and terpenoids. The pharmacological and clinical studies reported in the present review confirm the therapeutic values of P. zeylanica. Presence of wide range of chemical compounds indicates that plants could serve as "lead" for the development of novel agents having good efficacy in various pathological disorders in the coming years. Exploration of the chemical constituents of the plants and pharmacological screening will thus provide us the basis for developing such leads. However, less information is available regarding the chemical constituents of this plant. There are not many phyto-chemical and phyto-analytical studies of this plant. With the availability of primary information, further studies can be carried out like phyto-pharmacology of different extracts, standardization of the extracts, identification and isolation of active principles and pharmacological studies of isolated compound. These may be followed by development of lead molecules as
Phyto-PharmacologyofPlumbagozeylanica: A Review
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well as it may serve for the purpose of use of specific extract in specific herbal formulation.
References Aparanji Poosaria, Veerendra Kumar, B., Prasanna Kumar, S. Sreedevi, K, Rao, N. and Rama Rao Athota,2005. Induction of anti-inflammatory and altered TCell proliferative responses by the ethanol root extract of Plumbago zeylanica in adjuvant-induced arthritic rats. Pharmaceutical Bioi., 43: 784-789. Balandrin, M.F., Klocke, J.A., Wrtele, E.S. and Boilinger, W.H., 1985. Content and purity of extract solasodine in some available species of Solanum. Science and Culture, 56: 214-216. Beg, A.Z. and Ahamad, DJ., 2000. Effect of Plumbago zeylanica extract and certain curing agents on multidrug resistant bacteria of clinical origin. World Journ. Microbiol. Biotechnol., 16: 841-844. Bopaiah,CP. and Pradhan, N., 2001. Central nervous system stimulatory action from the root extract of Plumbago zeylanica in rats. Phytotherapy Research, 15: 153-156. Bhaskarachary, K, Sankar Rao, D.5., Deosthale, Y.G. and Reddy, V., 1995. Carotene content of some common and less familiar foods of plant origin. Food Chem., 54: 189-193. Bhargava, S.K, 1984. Effects of plumbagin on reproductive function of male dog. Ind. J. Exp. Bioi., 22: 153-156. Burkill, I.H., 1935. A Dictionary of the Economic Products of the Malay Peninsula, 2 Vols. Crown Agents for the Colonies, London. Chen, Y-C, Tsai, W-J., Wu, M-H., Lin, L-C and Kuo, Y-C, 2007. Suberosin inhibits proliferation of human peripheral blood mononuclear cells through the modulation of the transcription factors NF-AT and NF-KB. British Journal of Pharmacology, 150: 298-312. Chopra, R.N., 1933. Indigenous Plants of India: Their Medical and Economic Aspects. The Art Press, Calcutta. Cragg, G.M., Newman, D.J. and Sander, KM., 1997. Natural products in drug discovery and development. J. Nat. Prod., 60: 52-60. Dalziel, J.M., 1956. Useful Plants of West Tropical Africa. Crown Agents for Overseas Governments, London. Desta, B., 1993. Ethiopian traditional herbal drugs. Part 11: Antimicrobial activity of 63 medicinal plants. J. Ethnopharmacol., 39: 129-139. Dinda, B., Hajra, A.K and Chel, G., 1997. Napthoquinones of Plumbago species: A Review. J. Indian Chem. Soc., 74: 974-979. Dwivedi, S., 1997. Effect of Plumbago zeylanica in hyperlipidemic rabbits and its modification by Vitamin E. (Letter to the Editor). Indian J. Pharmacol., 29: 138. Edenharder, R. and Tang, X., 1997. Inhibition of the mutagenicity of 2-nitrofluorene, 3-nitrofluoranthene and l-nitropyrene by flavonoids, coumarins, quinones and other phenolic compounds. Food Chem. Toxicol., 35: 357-372.
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Gebre-mariam, T.,Wutzler, P., Neubert, R, 2006. Antiviral activities of some Ethiopian medicinal plants used for the treatment of dermatological disorders. J. Ethnopharmacol., 104: 182-187. Gunaherath, G.M.K.B., Gunatilaka, AAL. and Thomson, RH., 1988. Studies on medicinal and related plants of Sri Lanka: Part 18. Structure of a new naphthoquinone from Plumbago zeylanica. J. Chem. Soc. Perkin Transac., 10: 407410. Gupta, M.M., Verma, RK., Uniyal, G.C and Jain, S.P., 1993. Determination of plumbagin by normal phase high performance liquid chromatography. J. Chromat., 637: 209-212. Irvine, F.R, 1961. Woody Plants of Ghana with Special Reference to their Uses. Oxford University Press, London. Itoigawa, M., Takeya, K. and Furukawa, H., 1991. Cardiotonic action of Plumbagin on guinea-pig papillary muscle. Planta Med., 57: 317-319. Kini, D.P., Pandey, S., Shenoy, B.O., Singh, U.V., Udupa, N., Umadevi, P., Kamath, R, N agarajkumari and Ramanarayan, K., 1997. Antitumor and antifertility activities of Plumbagin controlled release formulations. Ind. J. Exp. Bioi., 35: 374-379. Kofinas, C, Chinou, I., Loukis, A., Harvala, C, Roussakis, C, Maillard, M., Hostettmann, K. 1998. Planta Med., 64: 174. Kokwaro, J.O., 1976. Medicinal Plants of East Africa. East Africa Literature Bureau, Nairobi, Kenya. Krishnaswamy, M. and Purushothaman, K.K., 1980. Plumbagin: a study of its anticancer, antibacterial and antifungal properties. Indian J. Exp. Bioi., 18: 876877. Mallavadhani, U.V., Gayatri Sahu and Muralidhar, J., 2002. Screening of Plumbago species for the bio active marker Plumbagin. Pharmaceutical Bioi., 40: 508-511. Nguyen, AT., Malonne, H., Duez, P., 2004. Cytotoxic constituents from Plumbago zeylanica. Fitoterpia, 75: 500-504. Olagunju, J.A, Jobi, A.A and Oyedapo, 0.0., 1999. An investigation into the biochemical basis of the observed hyperglycemia in rats treated with ethanol root extract of Plumbago zeylanica. Phytother Res., 13: 346-348. Oyedapo, 0.0.,1996. Studies on bioactivity of the root extract of Plumbago zeylanica. International Journal of Pharmacognosy, 34: 365-369. Poul, B.N., Mudakam, D.S., Dama, L.B. and Jadhav, B.V., 1999. Enzymatic spectrum of herbal plants Plumbago Linn. Asian Journal of Chemistry, 11: 273-275. Premakumari, P., Rathinam, K. and Santhakumari, G., 1977. Antifertility activity of plumbagin. Ind. J. Med. Res., 65: 829-838. Quisumbing, E., 1951. Medicinal.Plants of the Philippines. Tech. Bull. 16. Manila, Philippine Islands. Manila Bureau of Printing.
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Rai, M.K., Pandey, AK. and Acharya, D., 2000. Ethno-medicinal plants used by Gond tribe of Bhanadehi, District Chhindwara, Madhya Pradesh.Journal ofNon- Timber Forest, 7: 237- 241. Ram, A., 1996. Effect of Plumbago zeylanica in hyperlipidaemic rabbits and its modification by-vitamin E.Indian J. Pharmacol., 28: 161-166. Sandhya, B., Thomas,S., Isabel, W. and Shenbagarathai, R, 2006. Ethnomedicinal plants used by the valaiyan community of piranamalai hills (reserved forest), Tamil Nadu, India: A pilot study. Afr.J. Trad. CAM.,3: 101-114. Seetharam, Y.N., Gururaj Chalageri, Haleshi, e. anp Vijay, 1999. Folk medicine and ethnomedicine of North-Eastern Karnataka,. Ethnobotany, 11: 32-37. Sharma Indu, Gusain, D. and Dixit, V.P., 1990. Hypolipidaemic and antiatheroscelerotic effect of plumbagin in rabbits. Indian Physiol. Pharmacol., 35: 10-14. Siddique, N.A, Bari, M.A, Naderuzzaman, A.T.M., Khatun, N, Rahman, M.H., Sultana RS., Matin, M.N., Sharmin Shahnewaz and Rahman, M.M., 2004. Collection of indigenous knowledge and identification of endangered medicinal plants by questionnaire survey in Barind Tract of Bangladesh. Journal ofBiological Sciences, 4: 72-80. Singh, K.K. and Kaushal Kumar, 1999. Ethno-medico phytotherapy among the Gaddi tribe, Kangra Valley, Himachal Pradesh, India. Indigenous Knowledge and
Development Monitor. Simosen, H.T, Joshi, B. and Varughese, G., 2004. In vitro screening of Indian medicinal plants for Anti plasmodial activity.J. Ethanopharmacol., 74: 195. Skinner, F.A The antibiotics. In: Modern Methods ofPlant Analysis, (Eds.) K. Peach and H.V. Tracy. Springer-Vedag, West Germany, 3: 626-725. Srinivas, P., Gopinath, G., Banerji, A, Dinakar, A, and Srinivas, G., 2004. Plumbagin induces reactive oxygen species, which mediate apoptosis in human cervical cancer cells. Mol. Carcinog., 40: 201-211. Suraveratum, N., Krungkrai, S.R, Leangararamgul, P., Prapunwattana, P. and Krungkai, J., 2000. Purification and characterization of Plasmodiumfalciparum succinate dehydrogenase. Molecular Biochemistry and Parasitology, 105: 215-222. Tilak, J.e., Adhikari, S. and Devasgayam, T.P., 2004. Antioxidant properties of Plumbago zeylanica, an Indian medicinal plant and its active ingredient, Plumbagin. Redox Rep., 9: 219-227. Watt, J.M. and Breyer-Brandwijk, M.G., 1962. The Medicinal and Poisonous Plants of
Southern and Eastern Africa: Being an Account of their Medicinal and Other Uses, Chemical Composition, Pharmacological Effects and Toxicology in Man and Animal, 2nd
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Wang, Y.e. and Huang, T.L., 2005. Screening of anti-Helicobacter pylori herbs deriving from Taiwanese folk medicinal plants. FEMS Immunology and Medical Microbiology, 43: 407.
Chapter 13
Studies on Biosystematics of Chloris Swartz. Species (Poaceae) T.N. MaryI, J. Vijayamma1, M. Sarada1 and Seshagiri Ra02 lDepartment of Botany, Acharya Nagarjuna University, Nagarjunanagar-522 510, Guntur (Dt.), A.P., India 2Department of Plant Sciences, University of Hyderabad, Hyderabad-500 046
ABSTRACT About 20 morphological traits of six species of Chloris including a hybrid population were studied. The important taxonomic traits for consideration as key characters were found such as number and nature of awns and colour and shape of the seed etc. in addition to the already provided keys in the literature. The results were discussed based on features of spike and seed characters etc.
Keywords: Chloris species, Key characters.
Introduction The university campus grassland occupies about 294 acres of land, with many forage grasses belonging to various tribes of the family Poaceae, viz., Eragrostideae, Chlorideae, Panicoideae and Andropogoneae etc. Among these, the species of Chloris are a few of the dominant on the campus. In the subfamily Chlorideae, Chloris is the largest genus with approximately 55 species and appears polyphyletic (Hilu and Alice, 2001). The chIorideae had a history of unsettled taxonomic problems at the tribal and generic levels. The number of
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recognized tribes varies from two to eight (Table 13.1). The boundary between two major tribes, Chlorideae and Eragrostideae has been considered capricious (Hilu and Wright, 1982; Campbell, 1985; Van den Borre and Watson, 1997,2000). There are several important character trends of evolutionary significance in Chloridoid grasses. These include: 1) reduction in number of fertile bisexual florets per spikelet; 2) reduction of inflorescence from open paniculate to one sided digitate arrangement of spikes and 3) from perennial to annual habit. Several states along these grades may be present in anyone genus but different character states along the grades are used to delimit the genera. Further, the grass species of Chloris from this area are still unexplored (Mary and Anthoniamma, 1985; Mary and Koti Rao, 1988; Mary et al., 1990). Therefore regional biosystematic analysis of six Chloris species is initiated in the present study to judge and draw importance of other morphological traits, if any, that stand as key characters in the differentiation of species of Chloris.
Key to the Species of Chloris (Bor, 1960) 1. Spikelets 1.5 mm long, close-packed on the rhachis; spikes .............c. roxburghiana closely appressed; awns 3, up to 1.5 cm long 1. Spikelets more than 2.5 mm long: 2. Perfect florets one: 3. Empty lemmas above the fertile floret solitary, .............c. virgata well-developed or rudimentary 3. Empty lemmas above the lowest two to four: 4. A perennial, creeping below and rooting at the nodes; spikelets cuneate, with four or five awns; second .............c. montana lemma very similar to the lowest but smaller 4. An annual grass; spikelets plump, with three, rarely four awns; second lemma of thinner texture than the lowest, much smaller, truncate, obovate, eventually globose .............c. barbata 2. Perfect florets two; spikelets with four awns; culms minutely puberulous below the inflorescence .............c. bournei
Key Characters of Chloris Species (Pullaiah and Gayathri, 1997) 1. Spikes racemosely arranged .............c. roxburghaiana 1. Spikes umbelled: 2. Perfect florets 2, culms puberulous below the inflorescence .............C.bournei 2. Perfect florests 1, culms glabrous below the inflorescence: 3. Empty lemma above, the fertile floret rudimentary to well developed .............C.virgata 3. Empty lemma above, fertile lemma well developed:
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4. Sterile lemmas 2, 3-awned, keels of the fertile lemma densely ciliate on the back .............Cbarbata 4. Sterile lemmas 3, 4-awned, keels of the fertile lemma glabrous on the back .............C montana
Materials and Methods Six populations of Chloris including a hybrid derivative were studied, i.e., C. barbata Sw., C. montana Roxb., C. bournei Rang. & Tad., C virgata Sw., C roxburghaina Schult. and a hybrid. Thirty plants were picked up at random from each population at reproductive stage for the analysis of morphological characters and the data was analysed by standard statistical methods given by Schefler (1969) and Kershaw (1973). About 20 morphological characters were taken into consideration, i.e., length of culm, leaf, flag leaf, inflorescence, spikelet, number of perfect florets, number, nature and length of awn and seed characters etc. The mean, S.D. and S.E. were calculated to assess the variability between these species.
Results and Discussion About 20 morphological characters were taken to analyse the variability existing among six species of Chloris including hybrid (Tables 13.1 and 13.2). The morphological variability noticed in the Chloris species of the present study in terms of quantitative characters, length of culm, leaf, leafsheaths, flag leaf, inflorescence (Figure 13.1) might be ascertained due to climatic differences like soil pH, moisture content and availability of essential nutrients in the habitats of their occurrence. Such variations observed in these populations can be compared to annual weeds growing in fertile and less fertile soils (Salisbury, 1942; Okusanya et al., 1981; Okusanya, 1983). Bor (1960) laid emphasis on length of spike, spikelets, number of perfect florets, awns, culms pubescent below the inflorescence and plants either annuals or perennials. In Flora of Andhra Pradesh Pullaiah and Gayathri (1997) draw key characters based on type of spikes, number of florets, lemmas and awns etc. From the present observations of these species it is evident that the importance should be given to a few of the following characters, i.e., type of inflorescence, number of perfect florets, awns, nature, length and colour of awns and seed characters etc. Regarding the inflorescence C. barbata, C. bournei and C. virgata form one group being umbelled (Figures 13.1 C,D and E), while C. montana and hybrid show connate inflorescence (Figures 13.1 A, B) and C. roxburghaina is different from them showing racemose inflorescence (Figure 13.1 F). Except C. bournei (Figure 13.2 D) all species showed only single perfect floret whereas the number (Figure 13.2), nature and colour of the awns is much variable but found to be unique with a particular species (Figures 13.1 and 13.2). The colour and nature of awns are variable among them, being purple in C. barbata, C. montana and C roxburghaina but white in C virgata. The nature of awns, also differ either short and thin or long and brittle as seen in C. roxburghaina (Figure 13.1).
Table 13.1: Tribes of Chloroideae Recognized in Various Taxonomic Treatments (Tutin, 1980; Campbell, 1985 treatments are regional) Prat
Tutin
Hilu and Wright
Gould and Shaw
Wheeler et al.
Campbell
Clayton and Renvoize
(1980)
(1980)
(1982)
(1983)
(1984)
(1985)
(1986)
Chlorideae
Chlorideae
Eragrosteae
Aleuropodeae
Aristideae
Cynodonteae
Eragrostideae
Zoysieae
Eragrostideae
Pappophoreae
Aristideae
Chlorideae
Unioleae
Cynodonteae
Spartineae
Spartineae
Chlorideae
Eragrostideae
Zoysieae
Pappophoreae
Zoys.
Zoysieae
Eragrosteae
Pappophoreae
Orcuttieae
Orcuttieae
Sproboleae
Leptureae
Pappophoreae
Trioideae
Unioleae
Zoysieae
Zoysieae
.....
Table 13.2: Morphological Characters of Ch/oris Species SI No.
1. Culm length
C. hybrid
C.montana
C.barbata
5.0. Mean S.E.
5.0.
11.9 81.92 1.48
4.83
46.8
1.45
4.52 90.11
1.9
13.37
3.49 11.75
1.2
2.3
19.53 1.20
2.08
5.0. Mean S.E.
5.0. Mean S.E.
49.50 2.21
23.9 62.68
13.64 49.24
1.8
15.0
1.66
7.59 13.15 1.43
4.25
13.2
1.37
3.60 24.68 1.36
1.12
1.5
0.95
0.84
3.8
0.72
0.27
2.5
0.82
0.46
1.84 0.84
O.SO
4
0
0
6.4
0.96
0.85
8.7
0.89
0.6
5.7
0.9
0.7
1.5
5.30
67.7
1.4
4.6
39.99 1.13
1.6
SO.8
1.5
5.10
0.4
0.04
0.2
0.5
0.06
0.3
0.57
0.11
0.2
0
0.63 11.76 1.06
1.26
3. Flag leaf length
1.33
0.83 0.483 3.91
1.02
4. Inflorescence! Number of spikes
4.8
0.79
0.99 0.981
5. Number of spikelets!spike
51.5
1.86 11.97 66.93 1.11
6. Spikelet length
0.32
0.48
0.40
0.06
7.1
0.27
C.roxburghiana
5.0. Mean S.E.
5.0. Mean S.E.
2. Leaf length
C.virgata
C.boumei
Mean S.E.
1.9
N CJ'I
1.53 23.5
0.61 0.144
0.3
0
1.06
9.59
1.0
1.39
4.3
0.89
8. Length between 4.43 Inflorescence base to flag leaf
1.37 3.57 7.96
1.5
5.5
3.67
1.48 4.88
2.55
0.55
0.25
5.3
0.91
0.69
4.58
1.04
1.1
9. Length of internode leaf sheath length
9.13
0.83
0.47 14.02
1.4
4.02
10.8
1.32
3.06
17.5
1.0
1.32
9.8
1.2
2.46 17.64
1.4
4.2
10. Leaf sheath length
3.7
0.89
0.64
1.2
2.18
2.90
0.87
0.57
6.9
0.96
0.86
4.74
0.89
1.06
1.2
7. Inflorescence length
7.27
1.04
1.19
5.86 0.971 0.891 5.03
6.40
1.01
0.65
6.51
t11 ;:!
~. ~
~
~ ~
;;. ~
o· ~
[
...,en
§. ..., ~
CJ')
Table 13.3: Floret and Seed Characters of Ch/oris Species
i:
~
SI.No.
G. barbata
C. boumei
C.montana
C.virgata
C.hybrid
C. roxburghiana
~.
<::>
Nature of culms below the inflorescence
Smooth
2
Type of the inflorescence/ spikelets
Umbelled
3
Number of perfect florets
4
Number of awns
3-4
5
Nature of awns
Profuse short and thin
Smooth
Hairy
Smooth
Hairy
Smooth
;:t
tIl
~. Connate
umbelled not spreading
Connate
Racemose connate
t
2
4
2
4
4 long
~
Sparse, short and thin
Medium brittle
Brittle and long
Profuse, short and thin
Very long, brittle
Umbelled 2
~.
()
;:r-
e
~. CJ')
6
length of awns
4mm
5mm
1cm
0.9mm
0.8mm
10mm
7
Colour of awns
purple
Brown,
purple
white
white/purple
purple
8
Shape of the seed
ovate
ovate
ovate
ovate
lanceolate
lanceolate
9
length and breadth of the seed
65 x 14 mm
60 x 27 mm
60 x20 mm
50 x 13 mm
70x16mm
72 x 17 mm
,..., "'
10
Colour of the seed
brown
brown
brown
golden yellow
light brown
golden yellow
.......
E ... ~
CJ') ~
~.
'\l
2,..., Iil
~
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Emerging Trends in Biological Sciences
Figure 13.1: Inflorescence in Ch/oris Species A: C. montana; B: C. hybrid; C: C. bournei; 0: C. barbata; E: C. vlrgata; F: C. roxburghiana
Studies on Biosystematics of Ch/oris Swartz. Species (Poaceae)
129
Figure 13.2: Florets in Ch/oris Species A: C. roxburghiana; B: C.virgata; C: C. montana; 0: C. bournei;
E: C. barbata; F: C. hybrid
Regarding seed (Figure 13.3) they are ovate in C. barbata, C. bournei and C. montana and C. virgata but lanceolate in hybrid and C. roxburghiana. Increased seed size (60/ 22mm) in C. bournei may be due to its hexaploid nature (2n=60) (Mary and Koti Rao, 1988) since other species are only tetraploids (2n=40). With regard to the colour of the seed, all species showed brown colour except C. virgata and C. roxburghiana showing golden yellow.
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Emerging Trends in Biological Sciences
Figure 13.3: Seeds of Ch/oris Species
A. C. bourne/, B. C.hybrld, C. C. montana, D. C.barbata, E. C.virgata
One of the important taxonomic characters noticed in Chloris grass is the reduction in floret number, evident in all the species except C. bournei. Some of these perennial species when flourishing in their ecologicl niches, single perfect floret was associated morphological parameter. When such species have to move to the crop fields, their occupation in new habitats, where competition increased with the crops, increase in the number of florets per spikelet is an added advantage for its survival. Hence species like C. bournei, an annual with two florets/ spikelet, might be resultant of such adaptation. According to Stebbins (1982), evolution towards increased number of florets is an added advantage that must have been stabilized during the course of selection. From the example of a single character study it becomes evident that evolutionary trends are bidirectional in grasses either towards reduction or elaboration (see Soderstrometal., 1986). From the present observations, type of inflorescence, number and nature of awns and seed characters also deserve due consideration in addition to the key given by
Studies on Biosystematics o/Chloris Swartz. Species (Poaceae)
131
Bor (1960) and Pullaiah and Gayathri (1997). Another important character of microhairs of Chloridoideae find important place in taxonomic revisions of the species among the tribes (see Soderstrom et al., 1986). The SEM studies on microhairs of these species are under progress to provide framework in biosystematic approaches of ,Chloris species.
References Bor, N.L., 1960. The Grasses of Burma, Ceylon, India and Pakistan. Pergamon Press, London. Campbell, C.S., 1985. The subfamilies and tribes of Gramineae (Poaceae) in the southern United States. J. Arnod Arbor., 66: 123-199. Hilu, K.H. and Alice, L.A., 2001. A phylogeny of Chloridoideae (Poaceae) based on mat K sequences. Systemic Bot., 26: 386-405. Hilu, K.H. and Wright, K., 1982. Systematics of Gramineae: A cluster analysis study. Taxon., 31: 9-36. Kershaw, A.K., 1973. Quantitative and Dynamic Plant Ecology. 2nd edn. The English Language Book Society and Edward Arnold (Pub.) Ltd., London. Mary, T.N. and Anthoniamma, S., 1985. Phenoplasticity in Chloris montana Roxb. Geobios New Reports, 4: 20-24. Mary, T.N. and Rao, P. Koti, 1988. A new chromosome number report in Chloris bournei Rang. & Tad. Curr. Sci.,57: 617-618. Mary, T.N., Rao, P. Koti, Antonia, Sr. Charlote, Kumari, V. and Rajasekhar, T.N., 1990. Seed germination ecology of some species of Chloris (Poaceae). Proceedings of Seed Symposium, Jodhpur, pp. 75-78. Okusanya, O.T., 1983. Experimental studies on some observed variations in Luffa aegyptiaca. Can. J. Bot., 61: 202-210. Okusanya, O.T., B.A. 01 a-Adams and Banidele, J.F., 1981. Variations in size, leaf morphology and fruit characters among 25 populations of Luffa aegyptiaca. Can. J. Bot., 59: 2618-2627. Pullaiah, T. and Gayathri, M.S., 1997. In: Flora ofAndhra Pradesh. Scientific Publishers, Jodhpur, pp. 1147-1151. Salisbury, E.J., 1942. The Reproduction Capacity ofPlants. G. Bel. and Sons Ltd., London. Schefier, S.c., 1969. Statistics for Biological Sciences. Addison Wesley Publ. Co., London. Soderstrom, T.R, Campbell, S. and Barkworth, M.E., 1986. Grass Systematics and Evolution. Smithsonian Institution Press. Stebbins, G.L., 1982. Major trends of evolution in the Poaceae and their possible Significance. In: Systematics and Ecology, (Eds.) J.R Estes, RJ. Tyrl and J.N. Breenken. University of Oklahoma Press, Norman, pp. 3-36. .
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Tutin, T.G., 1980. Gramineae. In: Flora Europaea, Vol. 5. Cambridge Univ. Press, pp. 187-267. Van den Borre, A. and Watson, L., 1997. On the classification of the Chloridoideae. Australian Syst. Bot., 10: 491-531. Van den Borre, A. and Watson, L.,2000. On the classification of Chloridoideae; results from morphological and leaf anatomical data analysis. In: Grass Systematics and Evolution, pp. 180-183.
Chapter 14
Plant Diversity and Conservation in Sirumalai Hills of Eastern Ghats, Tamil N adu, South India s.
Karuppusamy, K.M. RajasekaranI and T. Pullaiah2
IDepartment of Botany, The Madura College, Madurai-655 011, Tamil Nadu 2Department of Botany, Sri Krishnadevaraya University, Anantapur-515 003, A.P.
Introduction Biodiversity provides to the humankind enormous direct economic benefits in the form of timbers, food, fibre, medicines, industrial enzymes, food flavours, fragrances, cosmetics, emulsifiers, dyes and pesticides. The indirect ecological benefits from biodiversity include regulation of the gaseous composition of the atmosphere, soil formation, processing and acquisition of nutriens, trophic dynamic regulation of populations. It is essential for the ecosystem function and stability. Biodiversity at the global scale is a balance between the rate of speciation and the rate of extinction. At a regional level it is a balance between the rates of immigration/ invasion and that of local/regional extinction. Today we seem to be losing two to five species per hour from tropical forests alone. Biodiversity is not uniformly distributed on the earth. There are broad global patterns, and regional and local concentration of diversified species. About 44 per cent of vascular plants and 35 per cent of vertebrates are concentrated as endemic species in 25 hotspots, which account for only 1.4 per cent of the global land area (Singh and Khurana, 2002). Biodiversity can be assessed at
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Emerging Trends in Biological Sciences
genetic, population, species, ecosystem and landscape levels, at the spatial scales,
viz. local, regional, and global. Measurement ofbiodiversity at a larger spatial scale is often a difficult task. However, an understanding of the structural and functional attributes of an ecosystem and associated ecological processes is essential for the assessment and management of biodiversity. In India, species richness is complemeted by enormous genetic diversity found within individual species. India is one among the 12 magagene centres of the world (Zeven and De Wet, 1982). In Indian gene centres (Vavilovian 'Hindustani' Centre) is considered the centre of origin and domestication of as many as 160 major and minor crop plants and 320 wild relatives of crop plant species (ICR, 1995). Some of the important species that have originated in India include rice, sugarcane, banana, tea, mango, citrus, jute, minor millets, Asiatic vignas, jack fruit, cardamom, black pepper, turmeric, etc. (Arora and Nayar, 1984; Paroda, 1996). There is an icreasing realization throughout the world, about the state of our environment, caused by over-exploitation of natural resources for economic development. In the context of developing versus developed nations, over-exploitation of resources has contrasting connections. Resource exploitation in the developing world has largely geared to meet the very basic needs of food, fodder, fuel wood and shelter of a large section of deprived societies. On the other hand, developed parts of the world have largely concerned themselves in trying to maintain and indeed accelerate the already very high levels of resource consumption that they have achieved for a much smaller segment of the world's population, based on an already initiative taken and advantage gained through industrialization. In this scheme of things, natural resource base has been often viewed as limitless, by the industrialized world, either due to a myopic view of the future or because of either immense faith in technology being able to substitute for the natural resource capital (Ramakrishnan, 2002). India is having rich biodiversity, strong traditional knowledge base, good network of R and D systems, and relevant national and international policy and legal frameworks, but has not advanced much in harnessing biolOgical and cultural heritage for the best economic advantage and biological security of her one billion people. The present study was aimed to examine strength of plant biodiversity and associated traditional knowledge systems in Sirumalai hills of Tamil Nadu, and to analyse the options and opportunities ahead for harnessing biocultural diversity for generation, protection and maintanence of plant resource wealth and related knowledge-based commercial and industrial ventures.
Study Area-Sirumalai Hills Sirumalai hills is lying between 10° 07'-10° 18' N latitude and 7]0 55'-78° 12' E longitude in the tropical area of Western offshoot of the Easten Ghats (Map 14.1). The hills are located 6.5 km south of Dindigul town and 22.5 km north of Madurai city. Sirumalai hills are about 19.3 km long and 12.8 km broad with an area of 247 km2 • Starting from Nadukandamalai in the northeast, one ridge slopes down in the northwest to Reddiapatti. The only motorable ghat road is on this slope. Another ridge runs northwest for 4.8 km, and abruptly descends to Gandhigram. Two ridges diverge from Sirumalai pudur on the southern slope of Nadukandamali, one is running
Plant Diversity and Conservation in Sirumalai Hills of Eastern Ghats, Tamil Nadu
Map 14.1 : Location of Slrumalal Hills
135
136
Emerging Trends in Biological Sciences
west, other is south. The southern ridge of Sirumalai hills slopes to Sattiyar valley at the eastern end. The main peaks are Mullupanrimalai (1380 m) in the north-east, Vellimalai (1350 m) in the north, Kaluguparai (1355 m) in the south and Madhagamalai (1250 m) in the north.
Geography Geographically, Sirumalai hills are an archaean formation several hundred years old, without stratification and devoid of fossils. The rocks are gneissic reffered to as charnokite, mica, felspar and quartz (Mani, 1974). The soil is sandy loam, low in bases, high in sesquioxides, with signs of leaching. The soil pH range is between 6.8 and 7.2. In high altitudes, soil becomes dark with high humus contents depending on the vegetation.
Climate The maximum and minimum temperatures are in the months of May (29.2°) and January (18.3°) respectively. The annual rainfall is around 1200 mm per year, with approximately 75 rainy days in two seasons. The maximum rainfall is received from the norh-eastmonsoon (October-November). April-June is noted as the hot summer season. The humidity is maximum in the rainy months (91 per cent) and minimum in the summer months (68 per cent).
Forests and Vegetation The variations in altitude, temperature, humidity and rainfall naturally result in a wide range of vegetation types. Accordingly the forests types of Sirumalai hills are classified based on Champion and Seth (1968) as follows:
1. 2. 3. 4.
Open scrub forest Dry deciduous forest Mixed deciduous and Savannah woodland Semi-evergreen forest
Open Scrub Forest The vegetation of the lower tracts (Alt. 380-670 m) of the open scrub forests are characterized by three layered forest, isolated tree canopy, undergrowth and ground cover. The tree canopy consists mostly of deciduous trees such as Acacia leucophloea (Roxb.) Willd.,Albizia amara (Roxb.) Boivin., Chloroxylon swietenia IX. and Catunaregam spinosa (Thunb.) Trivengadum. The undergrowth is made up of mostly thorny shrubs like Canthium parviflorum Lam., Cassia auriculata L., Dodonaea viscosa Jacq., Erythroxylum monogynum Roxb., Pterolobium hexapetalum (Roth) Sant. & Wagh, Rhus mysurensis Hyene ex Wight & Am., Tarenna asiatica (L.) Kuntz. ex Schum., Carissa spina rum L., Acacia planifrons Wight & Am., Azima tetracantha Lam., Capparis sepiaria L., Commiphora berryi Engl., Gmelina asiatica L., Randia malabarica Lam., Scutia myrtina Kurz., Toddalia asiatica Lam. var. gracilis Gamble, Ziziphus mauritiana Lam., Benkara malabarica (Lam.) Trivengadum and Hugonia mystax L. The common climbers of this forest type are Aganosma cymosum (Roxb.) G.Don, Canavalia cathartica Thouars, Cardiospermum halicacabum L., Coccinea grandis (L.) Voigt,
Plant Diversity and Conservation in Sirumalai Hills o/Eastern Ghats, Tamil Nadu
137
Cocculus hirsutus (L.) Diels, Reissantia indica (Willd.) Halle and Riven hypocrateriformis (Desr.) Choisy. The succulents such as Caralluma adscendens (Roxb.) Haw., Ceropegia juncea Roxb., Euphorbia antiquorum L., Sarcostemma acidum (Roxb.) Voigt, S. intermedium Decne. are admirably adapted to the xerophytic conditions prevailing here. The soil is sparsely covered by a few prostrate herbs with well developed rootstocks, i.e. Barleria buxifolia L., Lepidagathis cristata Willd. and Tephrosia spinosa (L.f.) Pers. Herbaceous vegetation is dominant only during the monsoon season and the post monsoon months (October- February). Grasses are poorly represented in the open scrub forest except along its upper border where it merges into the savannah. The stunted, prickly vegetation of this area is a direct response to the dry climate, poor soil conditions and hot sunshine.
Dry Deciduous Forest As climatic and soil conditions improve, a number of trees, mostly deciduous types, established themselves at the altitude of 820-950 m. the vegetation is characterized by a top canopy of tall trees usually 7.6-10.6 m high, a lower storey of smaller trees of 3-6 m high and an undergrowth of smaller shrubs, grasses and a few herbs. Thorny vegetations are the early colonizers and are soon replaced by dry deciduous flora. The top canopy is made up of Anogeissus latifolia (DC.) Bedd., Boswellia serrata Roxb., Buchanania lanzan Spreng., Butea monosperma (Lam.) Taub., Cochlospermum religiosum (L.) Alston, Pterospermum canescens Roxb., Lannea coromandeliaca (Houtt.) Merr., Dalbergia lanceolaria L.f., D. paniculata Roxb., Semecarpus anacardium L. and Vitex altissima L.f.
Anogeissus latifolia (OC.) Bedd. and Pterocarpus marsupium Roxb. are the dominant species. Albizzia odoratissima (L.f.) Benth. and Buchanania lanzan Spreng. are found only above 900 m. The second storey is sparsely represented by common trees such as Buchanania axillaris (Desr.) Ramamoorthy, Gardenia resinifera Roth, Polyalthia cerasoides (Roxb.) Bedd., Schefflera steUata (Gaertn.) Harms. and Terminalia chebula Retz. Among the climbing and scandent plants Anamirta cocculus (L.) Wight and Am., Hiptage benghalensis (L.) Kurz., Reissantia grahamii (Wight) Ding., Salacia chinensis L., Cayratia pedata (Lam.) Juss., Mucuna atropurpurea DC., Gymnema elegans Wight & Am., and Secamone emetica R.Br. are the species observed in this forest. Interesting specimens of Cycas circinalis L. are also found in some of the ravines in the drier regions. The undergrowth is a continuous stretch of grasses together with a few shrubs and undershrubs. The dominant grasses are Cymbopogon coloratus (Nees) Stapf and Themeda triandra Forssk. In places where the grass is some what discontinuous, the floor has sparse herbaceous vegetation, i.e., Desmodium triflorum (L.) DC., Euphorbia hirta L., Evolvulus alsinoides L., Hedyotis umbellata L., Oxalis corniculata L. and Polycarpaea corymbosa (L.) Lam. The vascular cryptogams are relatively a few and are represented by Actiniopteris radiata (Sw.) Link., Cheilanthes mysurensis Hook. and Selaginella longipila Hieron.
138
Emerging Trends in Biologica~ Sciences
Mixed Deciduous and Savannah Woodland Forest A further transition towards a richer flora occurs at an altitude of 950-1100 m., with the advent of several species of Terminalia, especially T. bellirica (Gaertn.) Roxb. and T. chebula Retz. They are characterized by three layered vegetation interspersed with savannah woodland. The savannah woodland correspond in general to the savannah forest. They occur on all slopes either as horizontal belts or entirely dividing deciduous forest into separate sections. Tectona gradis L.f. is rather limited in distribution but Pterocarpus marsupium Roxb. and Dalbergia lanceolaria Roxb. are common and they are of considerable economic value. The trees are deciduous, often with twisted stems and sparse, irregular branches, and occur 12-30 m apart from one another. The predominant trees are Buchanania axillaris (Desr.) Ramamoorthy, Dalbergia paniculata Roxb., Givotia moluccana (L.) Sreem., Lannea coromandelica (Houtt.) Merr., Phyllanthus emblica L., Pterocarpus marsupium Roxb., Terminalia chebula Retz. and Wendlandia thyrsoidea (Roemer & Schult.) Steud. The grassland hillocks are frequently distributed at higher elevations of about 900m. They are commonly represented by Cymbopogon coloratus (Nees) Stapf, Heteropogon contortus (L.) Beauv. and Themeda triandra Forssk. and Phoenix loureirii Kunth. an economically important species. As the rainfall begins, the ground is covered by several grasses interspersed with tuberous plants, viz. Chlorophytum malabaricum L., Habenaria elwesii Hook.f., H. digitata Lindl., H. rariflora A.Rich. and H. longicornu Lindl. In restricted areas, chiefly along small gullies, trees are located very close along with small trees,large shrubs and climbers, rocky and usually without any herbaceous growth. Undershrubs and herbs occurring in the dry deciduous forest are also found here. In addition, the following are the commonly found herbs: Crotalaria mysorensis Roth, Knoxia sumatrensis (Retz.) DC., Leucas aspera (Willd.) Link., Rynchosia rufescens (Willd.) DC. and Trichodesma zeylanicum (Burm.f.) R.Br. The aquatic flora of Sirumalai hills is represented by a few species such as Cyperus sp., Typha angustata Bory and Chaub. and Polygonum sp. The grassy meadows around water stagnation are interlaced with Centella asiatica Urban and Phyla nodiflora (L.) Green. Semi-evergreen Forest In the high altitudes of Sirumalai hills, i.e. above 1300 m, patches of undisturbed semi-evergreen forests are present and rtley are locally called shola forests. The canopy trees in these forests are over 40 meters tall and are festooned with innumerable climbers and epiphytes. Due probably to regular lumbering operations, the canopy tends to be open. Common woody species include AIseodaphne semecarpifolia Nees, Amoora canarana Hiem., Bridelia crenulata Roxb., Bischofia javanica Blume, Canarium strictum Roxb., Dalbergia latifolia Roxb., Dimocarpus longan Lour., Garuga pinnata Roxb., Gmelina arborea Roxb., Ficus beddomei King, Elaeocarpus tuberculatus Roxb., Litsea glutinosa (Lour.) Robinson, Meliosma pinnata (Roxb.) Maxim., Michelia champaca L., Sterculia urens Roxb., S. guttata Roxb. ex DC., Symplocos cochinchinensis (Lour.) Moore and Walsura trifoliata Harms. Among these trees woody climbers such as Entada pursaetha DC., Gnetum ula Brong. and Uvaria narum (Dunal) Wight & Am. are frequent. Natural forest glades are
Plant Diversity and Conservation in Sirumalai Hills o/Eastern Ghats, Tamil Nadu
139
usually dotted with Careya arborea Roxb., Phyllanthus emblica L., Cassine paniculata Lobr.-Callen., Aglaia roxburghiana Hiem. and Chrysophyllum roxburghii G.Don. The epiphytic Diplocentrum recuroum Lind1., Luisia birchea B1. and an interesting fern species Drynaria quercifolia (L.) Smith clothe many tree trunks. The peculiar species Tylophora mollissima Wight & Am. with its zig-zag inflorescence is a characteristic climber found among the large trees. Large specimens of Angiopteris evecta (Forst.) Haffm. and Cyathea gigantea (Wall. ex Hook.) Holt. are the interesting species found in some of the ravines in the high altitude forests. The rheophytic vegetation along the streams and rivulets in the southern section constitutes another interesting ecosystem. A number of trees grow along the banks of Meenparai and Kudladampatti streams. At higher altitudes are found Nothopegia beddomei Gamble, Bridelia crenulata Roxb. and Chrysophyllum roxburghii G.Don. At low levels along the streams Diospyros montana Roxb., Schleichera oleosa Oken., Lepisanthes senegalensis Leenh. and Flacourtia indica Merr. are found. Schumannianthus virgatus (Roxb.) Rolfe is a common reed that exists with Hedychium coronarium Koen. along the streams at high altitudes. The boulder-stream beds of these hills are associated with several shrubs like Homonoia riparia Lour. and Actiphila excelsa (Dalz.) Muel1. The vegetation map of Sirumalai hills shows large area of this hill covered by the scrub forest and savannah woodland (Map 14.2). Seasonal Changes in Vegetation March to August are the dry months and April to June are the very hot period. There is only one marked seasonal change that affects the vegetation on the hills, i.e., the change in the temperature. The few summer showers in April to May do not appreciably reduce the heat. The dry season is followed by a few showers in September and the setting in of the North-East monsoon rains and cold winds bring down the temperature considerably and keep the soil moist till January- February. The effect of this seasonal change is quite evident on the vegetation. As the soil becomes more and more dry from February onward, the heat increases and the herbaceous annuals disappear in the course of March-April, and leave the ground bare. Shrubs and undergrowth of forests become dormant. The sporadic rains during the South-West monsoon merely help the perennials to just survive. However, several of the large shrubs and trees in scrub, deciduous and evergreen forests flower during this hot season. The dark coriaceous leaves and large flowering branches of Schefflera wallichiana (Wight & Am.) Harms. drape the large trees in summer. The few showers in September moisten the soil and the annuals begin to appear. By the middle of the October the floor of the scrub forest and the open hills is a green carpet of seedlings, a striking and pleasing contrast to the bare look of previous months. Undershrubs and twiners produce numerous branches and leaves and the trees develop more foliage. The white fragrance of innumerable jasmines, like Jasminum cuspidatum Rott1. and J. flexile Vahl, stand out against the green of the seasonal foliage and emenate strong fragrance. The herbaceous monsoon flora of this area is rich and interesting. Besides liverworts, lichens, mosses and ferns, innumerable flowering plants thrive during the rainy seasons.
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140
•
'--_ _ _ _...;;'pklt
«'ALE
IUID Scrub forest
lmm Savannah ~Woodland
~~1M.:DeCiduous forest •
Map 14.2: Vegetation Map of Sirumalai Hills
.semi-evergreen forest
Plant Diversity and Conservation in Sirumalai Hills of Eastern Ghats, Tamil Nadu
141
Where the soil is rich and the humidity is high, the semi-evergreen trees form a compact association, especially along the streams and on the steep slopes at the higher altitude peaks. These wooden patches have often been cleaned and planted with coffee and -the accompanying shade trees like Grevillea robusta A. Cunn., Acacia holosericea Willd. and Artocarpus heterophyllus Lam. But, when the rainfall is higher and water assured, the forest canopy has been left standing, the undergrowth has been cleaned and planted with economc crops like Elettaria cardamomum (L.) Mant. and Musa paradisiaca L. Common among the native trees are Careya arborea Roxb., Gmelina arborea Roxb., Mallotus tetracoccus (Roxb.) Kurz.,M. philippensis (Lam.) Muell. and Trichilia connaroides (Wight & Am.) Benth.
Floristic Diversity One fourth of the South Indian flora occurs in this region. The maximum floristic diversity in Acanthaceae, Asteraceae, Asclepiadaceae, Caesalpiniaceae, Cyperaceae, Euphorbiaceae, Fabaceae, Rubiaceae and Poaceae are noted on Sirumalai hills. Over 120 species of legumes and 90 species of grasses mainly occur in the forest belts. The total number of plant species enumerated in this hill is shown in Table 14.1. Pallithanam (1956) has enumerated only 895 species of angiosperms in Sirumalai hills. Recently Pallithanam's work has been published in the title of Pocket flora of Sirumalai hills (Matthew, 2001). More than 14 species of Orchids and 62 species of Ferns have been reported in these hills (Karuppusamy et al., 1999 and 2001a). The present study reported that more than 100 species of plants are addition to the flora of Sirumalai hills (Table 14.2). Table 14.1: Total Number of Plant Species Recorded In Slrumalal Hills Plant Groups
Number of Families
Number of Genera
Number of Species
502 72 3 31
921
Pteridophytes
117 20 3 16
Total
156
608
1,133
Angiosperms Dicotyledons Monocotyledons Gymnosperms
163 3 46
Agriculture and forestry are basic to the economy of the region. Above 37 per cent of the land is cropped and the remaining part of the land is covered with revenue and reserve forests. The list of cultivated crops and economic plants are shown in Table 14.3. The major crops in this region are coffee, banana, pepper and arecanut. Sirumalai hills is having a rich repository of medicinal plant resources. It has been estimated that, out of the 1100 species of higher plants observed in the present study, 95 are of medicinal value. They belong to 81 genera and 49 families (Table 14.4). These medicinal plants are used in different Indian medicinal systems. Some important plants species are shown in Plates 14.1 to 14.7.
142
Emerging Trends in Biological Sciences Table 14.2: List of Additional Plant Species to the Flora of Sirumalai Hills
Annonaceae:
Miliusa tomentosa, Polyalthia korintii, Orophea indica
Brassicaceae:
Cardamine hirsuta, Rorippa indica
Capparaceae:
Capparis floribunda, C. grandis, C. divaricata, C. shervaroyensis
Polygalaceae:
Polygala erioptera, P. telephioides
Caryophyllaceae:
Cerastium glomeratum, Polycarpon tetraphyllum
Portulacaceae:
Portulaca grandiflora, P. wightiana
Sterculiaceae:
Melhania incana, Melochia corchorifolia, Pentapetes phoenicia
Linaceae:
Unum mysorense
Burseraceae:
Boswellia serrata
Meliaceae:
Aphanamixis polystachya, Aglaia tamilnadensis
Celastraceae:
Celastrus paniculatus ssp. floribunda
Hippocrateaceae:
Loesneriella pauciflora
Rhamnaceae:
Zizyphus nummularia
Vitaceae:
Ampe/ocissus araneosa, Cayratia carnosa
Fabaceae:
Tephrosia setosa, Mundulea sericea
Caesalpiniaceae:
Bauhinia purpurea, Caesalpinia bonduc, Cassia absus, Delonix alata
Myrtaceae:
Syzygium jambos
Melastomataceae:
Memecylon randerianum, Melastoma malabathricum
Cactaceae:
Opuntia ramosissima
Apiaceae:
Hydrocotyle conferta, Heracleum rigens
Araliaceae:
Schefflera wallichiana, S. alata
Rubiaceae:
Canthium parviflorum, Coffea robusta, Gardenia latifolia, Ixora arborea, Hedyotis swertioides
Asteraceae:
Dichrocephala chrysanthemifolia, Anaphalis beddomel, Bidens bispinosa
Sapotaceae:
Achras zapota
Ebenaceae:
Diospyros nigrescens
Primulaceae:
Anagallis arvensis
Salvadoraceae:
Salvadora persica
Asclepiadaceae:
Caralluma lasiantha, Cryptolepis buchananii, Secamone esculentum, Tylophora fasciculata, T. indica
Loganiaceae:
Gardneria ovata
Gentianaceae:
Canscora heteroclita, C. diffusa, Hoppea dichotoma
Solanaceae:
Datura stramonium, Solanum elaeagnifolium, S. anguivi
Gesneriaceae:
Didymocarpus gambleanus
Bignoniaceae:
Kigelia pinnata
Pedaliaceae:
Sesamum orientale
Acanthaceae:
Andrographis alata, Asystasia malabarica, A. dalzelli, Barleria grandiflora, Lepidagathis prostrata, Thunbergia tomentosa Contd...
Plant Diversity and Conservation in Sirumalai Hills of Eastern Ghats, Tarni1 Nadu
143
Plate 14.1 (a) Andrographis lineata; (b) Angiopteris evecta; (c) Hedychium coronarium; (d) Peperomia dindygu/ensis; (e) Schefflera wallichiana; (f) Ty/ophora macrantha
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144
Plate 14.2
(a) Ardisia solanacea; (b) Capparis wightii; (c) Clerodendrum serratum; (d) Gardenia gummifera; (e) Careya abrorea; (t) Morina umbel/ata
Plant Diversity and Conservation in Sirumalai Hills of Eastern Ghats, Tamil Nadu
145
Plate 14.3 (a) Arisaema leschenaultii; (b) Begonia malabarica; (c) Gloriosa superba; (d) Gymnema sylvestre; (e) Memecylon umbellatum; (f) Symplocos cochinchinensis
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146
Plate 14.4 (a) Didymocarpus gambleanus; (b) Lobelia nicotianifolia; (c) Sarcostemma intermedium; (d) Solanum tridatum; (e) Terminalia chebula; (f) Withania somnifera
Plant Diversity and Conservation in 5irumalai Hills of Eastern Ghats, Tamil Nadu
Plate 14.5 (a) Alpinia calcarata; (b) Bixa orellana; (c) Coffea arabica; (d) Coffea wightiana ; (e) Helicteres isora; (f) Stevia sp.
147
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148
Plate 14.6 (a) Buchanania axil/aris; (b) Bridelia crenulata; (c) Casearia el/iptica; (d) Dalbergia paniculata; (e) Ixora arborea; (f) Miliusa tomentosa
Pla nt Diversity and Conservation in Sirurnalai Hills of Eastern Ghats, Tarnil Nadu
Plate 14.7 (a) Cymbidium aloifolum; (b) Diplocentrum recurvum; (c) Eulophia graminea; (d) Habenaria longicornu; (e) Luisia birchea; (f) Vanda testacea
149
E~~ngTr~mBww~l~
150
Table
14.2~d ...
Verbenaceae:
Clerodendrum phlomides, C. viscosum, Vitex negundo
Lamiaceae:
Scutellaria violacea
Amaranthaceae:
AelVa zeylanica
Piperaceae:
Piper nigrum
Lauraceae:
Utsea glutinosa
Loranthaceae:
Scurrula parasitica
Santalaceae:
Osyris wightiana
Euphorbiaceae:
Bridelia velutina, B. retusa, Chrozophora baskarani Karuppusamy et al.
Podostemaceae:
Tristicha ramosissima
Orchidaceae:
Habenaria longicomu, H. elwesH, H. digitata, H. plantaginifolia, Cymbidium aloifolium, Eulophia graminea, Papilionanthe subulata
Zingiberaceae:
Hedychium coranarium
Dioscoreaceae:
Dioscorea bulbifera
Hypoxidaceae:
Molineria indica
Anthericaceae:
Chlorophytum malabaricum
Amaryllidaceae:
Crinum trifforum
Commelinaceae:
Murdannia zeylanica
Arecaceae:
Phoenix sylvestre
Araceae:
Theriophonum fischeri
Table 14.3: List of Cultivated Crops and Economic Plants In Sirumalal Hills Crop group Cereals
Pulses
Vegetable fruits
Name of the Species Echlnochloa frumentacea (Roxb) Links
Family Poaceae
lEleuslne coracana (L.) Gaertn.
Poaceae
Oryza sativa L.
Poaceae
Panicum miliare Lam.
Poaceae
Setaria Itallca (L.) Beauv.
Poaceae
Sorghum halepense L.
Poaceae
Cajanus cajan (L.) Millsp.
Fabaceae
Clcer arietlnum L.
Fabaceae
Glycine max (L.) Merr.
Fabaceae
Vigna mungo (L.) Hepper
Fabaceae
V. radlata (L.) Wllc.
Fabaceae
V. unguiculata (L.) Walp.
Fabaceae
Benlnoasa hlsplda (Thunb.) Cogn.
Cucurbitaceae
Cucurbita pepo L.
Cucurbltaceae
Lagenaria siceraria Stanley
Cucurbitaceae
Contd...
Plant Diversity and Conservation in Sirumalai Hills of Eastern Ghats, Tamil Nadu
151
Table 14.2-Contd... Crop group
Fruits and nuts
Name of the Species Luffa acutangufa (L.) Roxb.
Cucurbitaceae
Momordica charantia L.
Cucurbitaceae
Sechium edule (Jacq.) Sw.
Cucurbitaceae
Trichosanthes lobata Roxb.
Cucurbitaceae
Anacardium occidentale L.
Anacardiaceae
Ananas comosus Beauv.
Bromeliaceae
Areca catechu L.
Arecaceae
Artocarpus heterophyllus Lam.
Moraceae
Citrus aurantifolia (Christin.) Sw.
Rutaceae
C. aurantium L.
Rutaceae
C. limeta Risso.
Rutaceae
Mangifera indica L. Musa paradisiaca L.
Economic crops
Family
Anacardiaceae Musaceae
Psidium guajava L.
Myrtaceae
Punica granatum L.
Punicaceae
Ceiba pentandra (L.) Gaertn. Coffea arabica L. C. robusta Linden.
Bombacaceae Rubiaceae Rubiaceae
Elettaria cardamomum (L.) Mat.
Zingiberaceae
Hevea brasiliensis Muell.
Euphorbiaceae
Piper betel L.
Piperaceae
Piper nigrum L.
Piperaceae
Endemism Identification, documentation and assessment of rare, threatened and endemic species are important for the conservation of natural biodiversity. The Indian region presents an immense variety of climatic and altitudinal zones and is floristically rich with 34 per cent of endemism (Chatterjee, 1940). Among 25 hot spots of the world, two major hot spots are in India, i.e., Western Ghats and Eastern Himalayas. Out of 5500 flowering plants in Southern Peninsular India, 2000 flowering plants have been estimated as endemic species (Subramanyam and Nayar, 1974; Nair and Daniel, 1986; Nayar, 1980). The high degree of endemism in this region has conferred on them the hot spot status (Nayar, 1996). The riches of country's evergreen forests with endemic species are located in the Western Ghats and Eastern Ghats particularly Palnis and Nilgiris. Presently these forests cover only five per cent of the land mass where more than one fourth of the country's endemic plants (Bir, 1997) occur. Some endemic species are restricted to very small areas or with in very limited range of distribution. For example, the pitcher plant in Meghalaya and Cycas beddomei confined only to Cuddapah hills in South India Gain, 1983). Extremely restricted areas that
Emerging Trends in Biological Sciences
152
have been reported as type localities for a number of species, many of which are rare and endangered. Only little attention is given on the study of narrow habitat range of plants and the conservation of their genetic resources. The study of eco-distribution and spatial distribution of rare species and their location, degree of ecological gradients, climatic tolerance, adaptation, pollination biology, phenology, seed dispersal strategies and viability for rare and endangered species should also be established. Table 14.4: List of Common Medicinal Plants Enumerated in Sirumalai Hills SI. No.
1.
Name of the Species Abrus precatorius L.
Family
Local Name
Fabaceae
Kundumani
2.
Acacia polyantha Willd.
Mimosaceae
Silaiyunjai
3.
A. pennata Roxb.
Mimosaceae
Mulindu
Actiniopteridaceae
Mayilsigai
Amaranthaceae
Sirupulai Pumpullu
4.
Actiniopteris radiata Bedd.
5.
Aerva lanata (L.) Juss.
6.
Ageratum conyzoides L.
Asteraceae
7.
Albizzia lebbeck (L.) Benth.
Mimosaceae
Vagai
8.
A. odoratissima (Lt.) Benth.
Mimosaceae
Karuvagai
9.
Allophylus se"atus (Roxb.) Kurz.
Sapindaceae
Amali
10.
Altemanthera sessilis (L.) R. Br.
Amaranthaceae
Ponnankanni
11.
Amaranthus spinosus L.
Amaranthaceae
Mullukeerai
12.
Anacardium occidentale L.
Anacardiaceae
Munthiri
Menispermaceae
Kamakari
Combretaceae
Vekkali
13.
Anamirta cocculus (L.) Wt. & Am.
14.
Anogeissus latifolia Wall.
15.
Bacopa monnieri (L.) Pennel
16.
Boswel/ia se"ata Roxb.
17.
Butea monosperma (Lam.) Taub.
18.
Caesalpinia bonduc (L.) Roxb.
19.
Calophyllum inophyllum L.
20.
Canscora decussata (Roxb.) Schult.
21.
Canthium parvif/orum Lam.
22. 23.
Scrophulariaceae
Bhirami
Burseraceae
Kurangusambrani
Fabaceae
Purasu
Caesalpiniaceae
Kalarchikay
Clusiaceae
Punnai
Gentianaceae
Mosipudu
Rubiaceae
Mullukarai
Caryota urens L.
Arecaceae
Kunthal panal
Cassia absus L.
Caesalpiniaceae
Mulaipalvirai
24.
C. fistula L.
Caesalpiniaceae
Konnai
25.
C. occidentalis L.
Caesalpiniaceae
Paeavirai
26.
Catunaregum spinosa Trivengadum
Rubiaceae
Perumkarai
27.
Cayratia camosa Gagnep.
28.
Clitoria tematea L.
Vitaceae
Kattupirandai
Fabaceae
Sangupu
29.
Clerodendrum viscosum Vent.
Verbenaceae
Pedungunari
30.
Clerodendrum inerme Roxb.
Verbenaceae
Aathusangu
31.
Cocculus hirsutus (L.) Diels
Menispermaceae
Kattukodi Contd...
153
Plant Diversity and Conservation in Sirumalai Hills a/Eastern Ghats, Tamil Nadu Table 14.4-Contd ... SI. No.
Name of the Species
Family
Local Name
Cochlospermaceae
Malamparuthi
32.
Cochlospermum religiosum (L.) Alst.
33.
Colocasia esculenta (L.) Schott.
34. 35.
Cordia dichotoma Forst.f.
36.
Crotalaria retusa L.
37.
Cymbopogon citratus (DC.) Stapf
Poaceae
Pothaipullu
38.
Cynodon dactylon (L.) Pers.
Poaceae
Arukampullu
39.
Dalbergia sissoo Roxb.
Fabaceae
Thavasimaram
40.
Dendropthoe falcata (Lf.) Etting
Loranthaceae
Pulluruvi
41.
Desmodium gangeticum (L.) DC.
Fabaceae
Oorilai
42.
Diplocyclos palmatus (L.) Jeff.
Cucurbitaceae
Iviralkovai
43.
Drynaria quercifo/ia (L.) Smith
Polypodiaceae
Pannukizhangu
Crataeva magna (Lour.) DC.
Araceae
Saembu
Cordiaceae
Narivizhi
Capparaceae
Mavalingam
Fabaceae
Kilukilupai
44.
Elephantopus scaber L.
Asteraceae
Yanaichedi
45.
Emilia sonchifolia (L.) DC.
Asteraceae
Muyasevi
46.
Erythrina variegata L.
Fabaceae
Mulmurungai
47.
Ficus amottiana (Miq.) Miq.
Moraceae
Kodiarasu
48.
F. benghalensis L.
Moraceae
Aal
49.
F. hispida Lf.
Moraceae
Paeyatthi
50.
F. microcarpa Lf.
Moraceae
Kallichi
Moraceae
Arasu
Flacourtiaceae
Chothaikala
51.
F. religiosa L.
52.
Flacourtia indica (Burm.f.) Merr.
53.
Gardenia gummifera Lf.
Rubiaceae
Kambilipicin
54.
Gmelina arborea Roxb.
Verbenaceae
Kumalamaram
55.
Grewia tilifolia Vahl
56.
Hedyotis corymbosa (L.) Lam.
57. 58. 59.
HoIoptelea integrifolia (Roxb.) Plan.
60.
Holarrhena antidysenterica L.
61.
Ipomoea nil (L.) Roth
62.
I. pes-caprae (L.) R.Br.
Convolvulaceae
Adappankodi
63.
I. sepiaria Roxb.
Convolvulaceae
Thanikkodi
64.
Justicia beddomei Bennet
Acanthaceae
Chithadathodai
65.
Lannea coramandelica (Houtt.) Merr.
Anacardiaceae
Uthian
66.
Melia azedarach L.
67.
Merremia emarginata Hall.f.
Tiliaceae
Chadachi
Rubiaceae
Oocikothu
Heliotropium indicum L.
Boraginaceae
Thaelkodukku
Hiptage benghalensis (L.) Kurz.
Malpighiaceae
Madhavikodi
Ulmaceae
Aavimaram
Apocynaceae
Kodapalai
Convolvulaceae
Kakkatan
Meliaceae
Malaivaembu
Convolvulaceae
Elikathukeerai Contd...
Emerging Trends in Biological Sciences
154 Table 14.4-Contd... SI. No.
Name of the Species
Family
Local Name
68.
M. tridentata (L.) Hall.f.
Convolvulaceae
Muthiyarkunthal
69.
Mimosa pudica L.
Mimosaceae
Thottalsuringi
70.
Mimusops elengi L.
Sapotaceae
Makizham
71.
Naravelia zeylanica (L.) DC.
Ranunculaceae
Vathamkolli
72.
Nerium oleander L.
Apocynaceae
Sivapparali
73.
Nilgirianthus ciliatus Bremek.
Acanthaceae
Chinnakuringi
74.
Oroxylum indicum (L.) Benth.
Bignoniaceae
Palayuthachi
75.
Orthosiphon thymiflorus (Roth) Slea.
76.
Phyllanthus reticulatus Poir.
Lamiaceae
Chilanthipadam
Euphorbiaceae
Pula
Solanaceae
Chodakkuthakkali
77.
Physalis minima L.
78.
Pseudarthria viscida (L.) Wt. & Am.
Fabaceae
Muvilai
79.
Pterocarpus marsupium Roxb.
Fabaceae
Vaengai
Salvadoraceae
Kunni
Sapindaceae
Pumaruthu
80.
Salvadora persica L.
81.
Schleichera oleosa (Lour.) Oken.
82.
Solanum anguivi Lam.
Solanaceae
Karimulli
83.
S. surattense Burm.f.
Solanaceae
Kandankathri
84.
Solena amplexicaulis (Lam.) Gandhi
Cucurbitaceae
Pulivanchi
85.
Spondias pinnata (U.) Kurz.
Anacardiaceae
Mampulichai
86.
Sterculia foetida L.
Sterculiaceae
Kuthiraipudukku
87.
Strebulus asper Lour.
Moraceae
Pirayamaram
88.
Symplocos cochinchinensis Moore
Symplocaceae
Kamblivatti
89.
Terminalia arjuna Wt. & Am.
Combretaceae
Vellamaruthu
90.
Toddalia asiatica (L.) Lam.
Rutaceae
Milakaranai
91.
Tragia involucrata L.
Euphorbiaceae
Senthotti
92.
Trichoderma cindicum (L.) R. Br.
Boraginaceae
Kallidaithumbai
93.
Tylophora indica (Burm.f.) Merr.
Asclepiadaceae
Neypalai
94.
Zizyphus mauritiana Lam.
Rhamnaceae
Elanthai
95.
Z. rugosa L.
Rhamnaceae
Mullukottankay
The vegetation analyses of the Sirumalai hills in different forest sites were made based on stratified random sampling method (Champion and Seth, 1968) for the study of endemic plant diversity. Of the 66 endemic plant species spread over 23 families, 51 species showed seven per cent of endemism. A high degree of endemism was noted in the scrub forest (7.4 per cent) and was followed by the mixed deciduous forest (6.1 per cent). The number of endemic species is greater in the climax forests than in the other forests, thereby accounting for the high number (20) of endemic species in mixed deciduous forests (Table 14.5). The ecological niches could attribute the low degree of endemism (0.7 per cent) in the semi-evergreen forests. Most of the endemic taxa of Eastern Ghats are palaeoendemics and survival of some rare species
155
Plant Diversity and Conservation in Sirumalai Hills of Eastern Ghats, Tami! Nadu
which has narrow distributional range and adapted to specialized conditions, depended on the biotic, edaphic and climatic factors, genetic structure in totality and past history of their populations in accordance with gene pool niche interaction (Stebbins,1980). Table 14.5: Total Number of Endemic Species and Percentage of Endemism in Different Forests of Sirumalai Hills SI. No.
Forest Type
Total Number of Species
Number of Endemic Species
Percentage of Endemism
1.
Scrub forest
163
11
7.4
2.
Dry deciduous forest
179
11
3.9
3.
Mixed deciduous forest
227
20
6.1
4.
Semi-evergreen forest
286
14
0.7
Among the 66 species of endemic plants of Sirumalai hills, herbs have shown highest number of endemism (49) followed by shrubs (10) and trees (7). The high degree of endemic herbs has shown to prevail in drier climatic conditions in most of the months that was brought out by rainfall. Most of the endemic taxa recorded in Sirumalai hills, their distribution, narrow habitat range and the mean diversity are given (Table 14.6). The data indicated that the endemic species has high ecological amplitudes and niches. Only few species such as Andrographis linea ta, Entada pursaetha, Gymnema elegans and Knoxia sumatrensis exhibit their presence in two or more type of forests. It is prudent to consider these areas of narrow endemics as micro centers of endemism for preserving the interesting flora. The distribution pattern of endemics in Sirumalai hills is somewhat different from that of other part of Eastern Ghats. This region is isolated geographically from Eastern Ghats and very close to Palni hills in Western side and Alagar hills in Eastern side (Mathur, 1984). The discontinuous land pattern of this region might have contributed the accumulation of different types of peninsular endemic plants. The Western Ghat endemic species, Lobelia nicotianifolia is frequently distributed in high altitude valleys of Sirumalai hills. Many narrow endemic species like Ampelocissus araneosa, Andrographis linea ta, Decaschistia crotonifolia, Osbeckia stellata, Tylophora mollissima and Peperomia dindigulensis were noted'in the southern side of mixed deciduous forests. The results showed that the endemic category of Sirumalais is a diverse assemblage of herbs. Tree genera are poorly represented in the endemic category in this region. It is generally considered that woody 'life-forms' are of relictual nature. The conservation of endemic plants is important for the preservation of natural biodiversity. The reduced population of many rare plant species causes concern and their distribution is so limited that in a single catastrophe, whether environmental or anthropogenic attention of habitat, would wipe out the species. Extinction of plant species may be due to environmental factors, ecological substitutions, biological causes and human interference like overexploitation for medicinal, fibre, fuel and other purposes (MacBride, 1979). Some of the endemic species of the hills have been used by the local people for medicinal purposes. The species like Decalepis hamiltonii,
Emerging Trends in Biological Sciences
156
Entada pursaetha, Mucuna pruriens and Andrographis lineata have also been collected largely by tribals for additional income and local use. Sirumalais has wild rice, wild bean and banana varieties that are not reported elsewhere and are extremely significant genetic resources whose habitats deserve protection. Table 14.6: List of South Indian Endemic Plant Species Distributed In Different Forests of Sirumalal Hills S/.No
Name of the Endemic Species
Mean /UCN Category Diversity
Occurrence of Species in the Forest
2
3
1. Ampe/ocissus araneosa Planch. 2. Anapha/is law;; Gamble
0.2
+
0.2
+
3. Andrographis lineata Wall.
1.0
4. Anisochi/us argenteus Gamble 5. Asystasia crispata Benth. 6. 7.
A. travancorica Bedd.
8.
Bar/eria acuminata Nees
9.
B. cuspidata Heyne ex Nees
Ba/anophora fungosa Hansen
R
0.1
R
0.1
R
+ +
0.01
+
1.2
+
0.01
+
0.2 1.2
E
1.0
14. Canscora diffusa R.Br.
1.2
15. Cayratia pedata (Lam.) Juss.
0.2
16. Ceropegia juncea Roxb.
0.04 0.2
17. Crota/aria /ongipes Wight & Am. 18. C. madurensis Wight & Am.
+ +
0.01
13. Bride/ia crenu/ata Roxb.
+ + + + + +
0.01
+
19. C. priest/eyoldes Benth.
0.1
20. C. shevaroyensis Gamble
0.11
+
21. C. willdenowiana DC. 22. Cymbidium a/oifolium (L.) Sw.
1.20 0.01
+
R
+
+
23. Cymbopogon martini Watson
4.02
+
+
24. Da/bergia rubiginosa Roxb.
1.20
+
+
25. Deca/epis hamiltonii Wight & Am. 26. Decaschistia crotonifo/ia
R
0.02
+
+
0.2
10. B. courta/lica Nees 11. B. /ongiflora L.f. 12. Brachyste/ma boumeae Gamble
+
4
+
1.11
+
Wight&Arn.
27. Deccania pubescens Trivengadum
0.02
+
28. Desmodium ferrugineum Wall.
0.11
+
29. E/aeocarpus tubercu/atus Roxb.
0.01
30. Entada pursaetha DC.
E
0.02
+ +
+ Contd...
157
Plant Diversity and Conservation in Sirumalai Hills of Eastern Ghats, Tamil Nadu Table 14.6-Contd... SI.No
Name of the Endemic Species
/uCN Mean Category Diversity
Occurrence of Species in the Forest
2 31. Ficus beddomei King 32. Gymnema elegans Wight & Am.
1.00 0.20
34. j-Iabenaria elwesii Hook.f. 35. H. longicomu Lindl.
0.18
36. H. longicomiculata Graham
0.02 0.12
38. Hardwickia binata Roxb.
0.40
39. Hedychium coronarium Koen.
0.02 R
0.22
42. I. mysorensis Rottler ex DC.
0.02
43. I. trita L.
0.10
44. Jatropha tanjorensis Ellis & Saroja
0.22 R
2.30
47. Leucas hirta Spreng.
0.11
48. L. mollissima Wall.
1.02
49. L. prostrata Gamble
0.02
50. L. pubescens Benth.
0.13 E
52. Mal/otus stenanthus Muell.-Arg.
+ + + + + + + + + + + + + + +
1.40 R
1.20
54. Osbeckia stel/ata var. rostrata Hansen
R
0.04
55. Peperomia dindigulensis Miq.
0.01
56. pogostemon mollis Benth.
1.22
57. Pouzolzia wightii Benn.
2.26
58. Pterocarpus santalinus L.
0.02
59. Rhinacanthus nasutus (L.) Kurz.
1.01
60. Rhynchosla heynei Wight and Am.
+ +
+
62. Smithia setulosa Dalz.
0.01 1.02
63. Stenosiphonium parviflorum T.And.
0.02
65. Thubergia tomentosa Wall.
0.01
66. Vigna trilobata (L.) Verdc.
2.10
+ + + + +
+
+
+
+
+ +
1.11 R
+ +
+
0.02 R
+
+
0.02
53. Mucuna pruriens (L.) DC.
64. Theriophonum fischeri Sivadas.
+
0.01
46. Lepidagathis pungens Nees
61. Sarcostemma intermedium Decne.
+ +
0.02
41. I. glutinosa Roxb. ex Willd.
51. Lobelia nicotianifolia Roth.
+
+ +
1.20
37. H. rariflora A. Rich.
45. Knoxia sumatrensis (Retz.) DC.
4
0.01 R
33. Gynura nitida DC.
40. Indigofera barberi Gamble
3
+ + +
1: Scrub forest; 2: Dry deciduous forest; 3: Mixed deciduous forest; 4: Semi-evergreen forest.
Emerging Trends in Biological Sciences
158
Ethnobotany Sirumalai hills is inhabited by people of different religions, i.e., Christians, Muslims and Hindu~ belonging to various castes such as Vellalars, Nidus, Konars, Goundars, Chettiyars, Chakkiliyars and Scheduled tribes including the Paliyans. Paliyans are living in high altitude of Sirumalai hills (Altitude 900-1300 m) in caves and on rocks and two types of nomadic and semi-settled lives. They are short in stature, doichocephalic and generally black or dark brown in skin complexion like other primitive tribes. Some of them have flat nose and thick lips. The males have a tuft or curly hairs, goatee beards and very little hair on their chest. The Paliyans are capable of doing hard manual work and they collect their food such as tubers, fruits, nuts and leaves from the forests. Their main income is by the collection of non-timber forest produces (Table 14.7). They sell their products in the weekly market every Friday in Sirumalai Pudur, Kodairoad and Vadipatti. The Paliyans consumed wild edibles from the forest and local grains (Table 14.8). These wild edibles belonged to 15 families, 17 genera and 21 species. Table 14.7: Non-timber Forest Produces Collected by the Paliyan Tribes of Sirumalai Hills Grouping of Produce
Name of the Species
Family
Plant Parts Collected
Edibles
Dioscorea bulbifera
Dioscoreaceae
Root tuber
Dioscorea oppositifolia
Dioscoreacace
Root tuber
Andrographis Iineata
Acanthaceae
Whole plant
Helicteres isora
Sterculiaceae
Fruits
Gymnema sylvestre
Asclepiadaceae
Leaves
Entada pursaetha
Mimosaceae
Seeds
Caesalpinia bonduc
Caesalpiniaceae
Seeds
Mucuna pruriens
Fabaceae
Seeds
Myristica tactyloides
Myristicaceae
Seeds
Phyllanthus emblica
Euphorbaceae
Fruits
Terminalia chebula
Combretaceae
Fruits
Terminalia bellirica
Combretaceae
Fruits
Medicinals
Urginia Indica
Liliaceae
Bulb
Zizyphus xylopyrus
Rhamnaceae
Fruits
Fibre
Furcraea foetida
Agavaceae
Leaves
Gnidia glauca
Thymeliaceae
Stem bark
Basketry materials
Bambusa arundinacea
Poaceae
Stem
Brooms
Lantana camera
Verbenaceae
Stem
Phyllanthus reticulatus
Euphorbiaceae
Stem
Phoenix loureirii
Arecaceae
Leaves
Plant Diversity and Conservation in 5irumalai Hills of Eastern Ghats, Tamil Nadu
159
Table 14.8: Wild Edibles Consumed by the Paliyans of Sirumalai Hills Grouping of Edibles
Root tubers
Leafy vegetables
Fruits
Seeds and nuts
Name of the Species
Family
Dioscorea bulbifera
Dioscoreaceae
Dioscorea oppositifolia
Dioscoreaceae
Asparagus racemosus
Liliaceae
Basellaalba
Basellaceae
Marsilea quadrifolia
Marsileaceae
Solanum nigrum
Solanaceae
Talinum portulacifolium
Portulacaceae
Artocarpus heterophyllus
Moraceae
Carissa carandas
Apocynaceae
Ficus hispida
Moraceae
F. glomerata
Moraceae
Phyllanthus emblica
Euphorbiaceae
Syzygium densiflorum
Myrtaceae
S. cumini
Myrtaceae
Securinega leucopyrus
Euphorbiaceae
Zizyphus rugosa
Rhamnaceae
Artocarpus heterophyllus
Moraceae
Entada pursaetha
Mimosaceae
Mucuna pruriens
Fabaceae
Punica granatum
Punicaceae
The Paliyans are depending on the forest for their health and economy. Most of their diseases treated with plants and their own remedy. Some of them have made a name for the plants based on their uses and properties by their knowledge of the medicinal value of the herbs and roots. Among the total ethnobotanical utilities, medicinal plants represented a high percentage (54.8 per cent) (Table 14.9). Other useful plants have the following representation: edibles (9.5 per cent), fibre (8.6 per cent), socio-cultural (7.1 per cent), timbers (7.6 per cent), thatching (2.9 per cent) and miscellaneous (9.5 per cent). In recent times, continuous and indiscriminate collection of selective species and deforestation activities from diverse ecosystems, coupled with p.estruction of natural habitats has resulted in irreplaceable loss of valuable genetic diversity. As a result, a number of medicinal plants have also become vulnerable to extinction. The problem is further aggravated because the bulk of plant raw materials are still collected from the wild much before the onset of their seed setting, through untrained and unskilled labourers to earn their livelihood in response to the ever-increasing demand and export potential. Thus the problem is posing a great challenge to the survival of this traditional health care system.
160
Emerging Trends in Biological Sciences Table 14.9: Ethnomedicinal Plants Used by the Pallyans of Sirumalai Hills (Utility based classification followed for the enumeration) I.
Plants used for wound healing Allium cepa L. (Liliaceae) 'Vengayam' BUlb. Areca catechu L. (Arecaceae) 'Pakku' Seeds. Argyreia cymosa Sweet
(Convolvulaceae) 'Kattukodi' Leaves
Angiopteris evecta (Forst.) Hoffm. (Angiopteridaceae) 'Yanaivanangi' Leaves. Buchanania lanzan Spreng. (Anacardiaceae) 'Kolamavu' Stem bark. Careya arborea Roxb. (Lecythidaceae) 'Payithandri' Stem bark. Centella aSiatica (L.) Urban (Apiaceae) 'Vallarai' Leaves. Coffea arabica L. (Rubiaceae) 'Kappi' Seeds. Didymocarpus gambleanus Fischer (Gesneriaceae) 'Paraiotti' Leaves. Scutellaria violacea Heyne ex Benth. (Lamiaceae) 'Malainangai' Leaves. 11.
Plants for cold, cough and fever Cissampelos pareira L. (Menispermaceae) 'Appata' Leaves. Buddleja asiatica Lour. (Buddlejaceae) 'Karukkattan' Leaves. Exacum pedunculatum L. (Gentianaceae) 'Chakkalathlvaembu' Leaves. Lobelia nicotianifolla Roth (Lobeliaeeae) 'Kattupugayeilai' Leaves. Ocimum sanctum L. (Lamlaceae) 'Thulasi' Leaves. Pteris longipes D.Don (Pteridaceae) 'Kadavalai' Leaves. Trichosanthes lobata Roxb. (Cucurbitaceae) 'Payipudal' Leaves. Vitex negundo L. (Verbenaceae) 'Nochi' Leaves.
Ill.
Plants for stomach problems Adiantum raddianum C.Pres. (Adiantaceae) 'Kajangoral' Leaves. Alangium salvifolium Wang. (Alangiaceae) 'Alingilai' Stem bark. Alpinla calcarata Rose. (Zlngiberaeeae) 'Chittaratai' Rhizome. Cardiospennum canescens Wall. (Sapindaceae) 'Kuthumaddakan' Leaves. Cissus quadrangularis L. Mant. (Vitaceae) 'Pirandai' Stem Glycosmis pentaphylla (Retz.) DC. (Rutaceae) 'Kuttlvlzha' Fruits Oxalis comlculata L. (Oxalidaceae) 'Aaralkeerai' Leaves.
IV.
Plants to treat ulcer Ceropegia juncea Roxb. (Asclepiadaceae) 'Pulichan' Stem. Cynanchum callialatum Buch.-Ham. (Asclepiadaceae) 'Veppadalkodi' Latex. Hemidesmus Indicus (L.) R.Br. (Asclepiadaceae) 'Nannari' Roots. Ichnocarpus frutescens (L.) R.Br. (Apocynaceae) 'Paravalli' Roots. Jatropha curcas L. (Euphorbiaeeae) 'Kattukottal' Latex. Sarcostemma acldum Decne. (Asclepiadaceae) 'Kodikalli Latex. Solanum nigrum L. (Solanaceae) 'Manathakkali' Leaves. Contd...
Plant Diversity and Conservation in Sirumalai Hills of Eastern Ghats, Tamil Nadu
161
Table 14.9-Contd••• V.
Plants used to treat Jaundice Andrographis lineata Wall. ex Nees (Acanthaceae) 'Periyanagai' Leaves. Cyperus rotundus L. (Cyperaceae) 'Koraikizhangu' Root tuber. Justicia adhatoda L. (Acanthaceae) 'Aadathodai' Leaves. Phyllanthus amarus Schum. & Thonn. (Euphorbiaceae) 'Keelanelli' Leaves.
VI.
Plants used to treat nervous disorder Citrullus colocynthis (L.) Schrader (Cucurbitaceae) 'Peyikkumati' Fruits. Habenaria long/cornu Lindl. (Orchidaceae) 'Pullukizhangu' Root tuber.
VII.
Plants used to treat dysentery and diarrhoea Anisomeles ma/abarica (L.) R.Br. (Lamiaceae) 'Paeimirrati' Leaves. Combretum albidum G.Oon. (Combretaceae) 'Veragay' Flowers. Dichrostachys·cinerea (L.) Wt. & Am. (Mimosaceae) 'Vedathalai' Leaves. Punica granatum L. (Punicaceae) 'Madhulai' Seeds.
VIII.
Plants used to treat diabetes Gymnema sylvestre R.Br. (Asclepiadaceae) 'Sirukurinchan' Leaves. Gardenia resinitera Roth (Rubiaceae) 'Kumbil' Resin. Helicteres isora L. (Sterculiaceae) 'Valamburi' Seeds and root bark. Syzygium cumin/ (L.) Skeels. (Myrtaceae) 'Nava' Seeds. Zizyphus xylopyrus (Retz.) Willd. (Rhamnaceae) 'Mullukottankay' Fruits.
IX.
Plants used to treat respiratory diseases Artemisia nilagirica (Clarke) Pamp. (Asteraceae) 'Masipathrl' Leaves. Lobelia nicotianitolia Roth. (Lobeliaceae) 'Kattupugayelai' Leaves. Tylophora zeylanica Oecne. (Asclepiadaceae) 'Palaikodi' Latex.
X.
Plants used to treat ear and eye diseases Cymbidum aloitolium Hook.f. (Orchidaceae) 'Panaipulluruvi' Leaves. Gymnema elegans Wt. & Am. (Asclepiadaceae) 'Venkurichan' Leaves. Eulophia graminea Lindl. (Orchidaceae) 'Kattuvengayam' Tubers. Musa paradisiaca L. (Musaceae) 'Vazhai' Leaf :;heath. Tarenna asiatica (L.) Kuntz. (Rubiaceae) 'Velichi' Fruits.
XI.
Plants used to treat skin diseases Ardisia solanacea Roxb. (Myrsinaceae) 'Kozhikottai' Stem bark. Aristolochia indica L. (Aristolochiaceae) 'Urikaykodi' Leaves. Commiphora caudata (Wt. and Am.) Engl. (Burseraceae) 'Malaikizhuvai' Fruits. Gmelina as/atica L. (Verbenaceae) 'Madhukkarai' Root bark. Memecylon umbellatum Burm.f. (Melastomataceae) 'Kaya' Leaves.
XII.
Plants used to anti-fertility and abortion Abrus precatorius L. (Fabaceae) 'Kundumani' Seeds. Aloe vera L. (Liliaceae) 'Sortukartalai' Leaves and latex. Contd...
Emerging Trends in Biological Sciences
162 Table 14.9-Contd...
Plumbago zeylanica L. (Plumbaginaceae) 'Chithiramulam' Roots. Trichosanthes cucumerina L. (Cucurbitaceae) 'Malaipudal' Fruits. Wrightia tinctoria (Roxb.) R.Br. (Apocynaceae) 'Vetpalai' Seeds.
XIII.
Plants used as aphrodisiac Hybanthus enneaspermus (L.) Muell. (Violaceae) 'Orithalthamarai' Plants. Madhuca longifolia (Koen.) Macbr. (Sapotaceae) 'lIIupai' Seeds. Mucuna pruriens (L.) DC. (Fabaceae) 'Poonaikali' Seeds.
XIV.
Plants used to treat venereal diseases Achyranthes bidentata Blume (Amaranthaceae) 'Sennayuruvi' Leaves. Aristolochia indica L. (Aristolochiaceae) 'Esvaramuli' Leaves. Aegle marmelos (L.) Correa (Rutaceae) 'Vilvam' Leaves. Asplenium aethiopicum (Burm.) Becher. (Aspleniaceae) 'Navadakki' Leaves. Cycas circinalis L. (Cycadaceae) 'Poomisarkarai' Rhizome and roots. Elaeocarpus tuberculatus Roxb. (Elaeocarpaceae) 'Vellaithani' Seeds. Evolvulus alsinoides L. (Convolvulaceae) 'Vishnukiranthi' Leaves. Kleinia grandiflora (Wall. ex DC.) Rani (Asteraceae) 'Muyalkathu' Leaves. Mucuna pruriens (L.) DC. (Fabaceae) 'Poonaikali' Seeds. Terminalia chebula Retz. (Combretaceae) 'Kadukkay' Fruits. Ventilago maderaspatana Gaertn. (Rhamnaceae) 'Vembadakam' Stem bark.
XV.
Plants used to treat urinary troubles Asparagus racemosus A.Juss. (Liliaceae) 'Vannathikizhangu' Root tubers. Caralluma adscendens (Roxb.) Haw. (Asclepiadaceae) 'Kallimulayan' Stem.
XVI.
Plants used as antlhelmlntlc Azadirachta indica A.Juss. (Meliaceae) 'Vaembu' Leaves. Chenopodium ambrosioides L. (Chenopodiaceae) 'Plukkolli' Leaves. Momordica dioica Roxb. (Cucurbitaceae) 'Kurit'ilipavai' Fruits.
XVII.
Plant used to treat toothache Spilanthes calva DC. (Asteraceae) 'Kattupundu' Leaves.
XVIII. Plants used to treat Infant diseases Andrographis lineata Nees (Acanthaceae) 'Periyanangai' Leaves. Cassia auriculata L. (Caesalpiniaceae) 'Aavarai' Flower buds. Kedrostis foetidissima (Jacq.) Cogn. (Cucurbitaceae) 'Appakkovai' Leaves. Myristica fragrans Houtt. (Myristicaceae) 'Jaathlkay' Seeds. Piper betel L. (Piperaceae) 'Vertilai' Leaves.
XIX.
Plants used to treat poisonous bites Achyranthes aspera L. (Acanthaceae) 'Nayuruvi' Roots. Alseodaphne semecarpifolia Nees (Lauraceae) 'Vandukadipattai' Stem bark. Alstonia venenata R.Br. (Apocynaceae) 'Elaipalai' Stem bark. Contd...
Plant Diversity and Conservation in Sirumalai Hills of Eastern Ghats, Tamil Nadu
163
Table 14.9-Contd... Andrographis paniculata (Burm.f.) Wall. (Acanthaceae) 'Nilavaembu' Roots.
A lineata Nees (Acanthaceae) 'Periyanangai' Plant. Barteria prionitis L. (Acanthaceae) 'Kattukanakambaram' Roots. Cryptolepis buchananii Roem. and Schult. (Asclepiadaceae) 'Nagathali' Roots. Rauvolfia tetraphylla L. (Apocynaceae) 'Pambukadithalai' Roots. Rhinacanthus nasutus (L.) Kurz. (Acanthaceae) 'NagamaIli' Leaves. Strychnos potatorum U. (Loganiaceae) 'Theathamkottai' Seeds.
xx.
Plants used to commit suicides Breynia vitis-idaea (Burm.f.) Fischer (Euphorbiaceae) 'Karunelli' Leaves. Calotropis gigantea (L.) R.Br. (Asclepiadaceae) 'Erukku' Latex. Cascabela thevetla (L.) Lippold (Apocynaceae) 'Arali' Seeds. Cleistanthus collinus (Roxb.) (Euphorbiaceae) 'Oduvan' Leaves. Gloriosa superba L. (Liliaceae) 'Kalapaikizhangu' Rhizome.
XXI.
Plants used as miscellaneous medicines Atalantia monophylla (L.) Correa. (Rutaceae) 'Kattuelumichai' Leaves. Curculigo orchioide~ Gaertn. (AmaryIlidaceae) 'Nilappanai' Rhizome. Clematis gouriana Roxb. (Ranunculaceae) 'Kattukodi' Leaves. Dioscorea bulbifera L. (Dioscoreaceae) 'KattuvaIli' Root tubers. Clerodendrum se"atum (L.) Moon. (Verbenaceae) 'Punaitheekku' Leaves. Heracleum ringens Wall. ex DC. (Apiaceae) 'Yanaicheerakam' Seeds. Pentatropis capensis (U.) Bull. (Asclepiadaceae) 'Uppukkolli' Leaves. Theriophonum fischeri Sivadas. (Araceae) 'Narikarunaikkizhangu, Rhizome.
High value species are naturally the most threatened of the lot. For instance, in Sirumalai hills, Gymnema sylvestre which grows mostly in the foot hill tracts is now in great demand globally due to its anti-diabetic properties. This species collected by the local people in the large quantity every year for trade purposes. The commercially potential medicinal plants collected from the hills are given in the Table 14.10. If the seeds are the medicinal part, and the collectio!} of the seeds in large quantity results in the dwindling of the plant species fastly (Karuppusamy et al.,2001b). International trade of medicinal plants and plant products are rapidly increasing. Presently medicinal plants worth Rs. 2000 crores are being traded from Tamil Nadu alone. A total of 26 raw drug samples have been collected from the present study, of these three drugs such as Gymnema sylvestrte, Gloriosa superba and Lobelia nicotianifolia have high export value. Sirumalais are being served as the main habitat sources of medicinal plants in this region. Madurai and Didigul act as pooling regional centres for local plant traders. Ultimately these trading drugs are exported to various countries through Tuticorine port (Karuppusamy et al., 2002).
Emerging Trends in Biological Sciences
164
Table 14.10: Commercially Potent Medicinal Plants Collected for the Trade by Local People of Sirumalal Hills Name of the Species Andrographis paniculata
Family
Category of Availability
Useful Part
Market Rate (Rs.lkg.)
Acanthaceae
NH
Plant
35 60 130 3 6 17 15 75 10 120 850 60 30 75 150 40 25 13 24 42 60 63 25 40
Liliaceae
NH
Roots
Boswellia se"ata
BUfseraceae
NH
Resin
Cardiospennum halicacabum
Sapindaceae
NH
Leaves
Apiaceae
NH
Leaves Rhizome
Asparagus racemosus
Centella asiatica Curculigo orchioides
Hypoxidaceae
NH
Emblica officinalis
Euphorbiaceae
NH
Fruits
Entada pursaetha
Mimosaceae
NH
Seeds
Ficus glomerata
Moraceae
NH
Fruits
Gloriosa superba
Liliaceae
NH/C
Rhizome
Asclepiadaceae
NH
Leaves
Seeds Gymnema sylvestre
Sterculiaceae
NH
Fruits
Hemidesmus indicus
Asclepiadaceae
NH
Roots
Lobelia nicotianifolia
Lobeliaceae
NH
Leaves
Fabaceae
NH
Seeds
Plumbaginaceae
NH
Roots
Helicteres isora
Mucuna pruriens Plumbago zeylanica
Rubiaceae
NH
Roots
Sapindaceae
NH
Fruits
Solanum surattense
Solanaceae
NH
Fruits
Strychnos nux-vomica
Loganiac~ae
NH
Seeds
Strychnos potatorum
Loganiaceae
NH
Seeds
Tenninalia bellirica
Combretaceae
NH/C
Fruits
Tenninalia chebula
Combretaceae
NH/C
Fruits
Ventilago maderaspatana
Rhamnaceae
NH
Bark
Zizyphus rugosa
Rhamnaceae
NH
Fruits
Rubia cordifolia Sapindus emarginatus
32 17
NH: Natural habitat; C: Under cultivation.
Conservation ofbiodiversity and of the germplasm of important medicinal plant species has assumed global significance from the point of view of ecological security and for ensuring a secure livelihood. This is particularly true in the developing countries like India located in the tropical or sub-tropical belt and which have much of the biodiversity relevant to humankind. It is estimated that 50-75 percent of the popUlation still relies on traditional medicine than the modern medicine. Easy availabity at low cost and comparative safety of the traditional medicines increase people's faith in such remedies. A dictionary of Indian folk medicine (Jain, 1991) also enumerates some 2,500 Indian plants and 15,000 folk uses. A survey revealed that
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nearly 550 plant species are being used in different formulations by various pharmaceutical concerns in India (Agarwal, 1997). The bioresources of medicinal plants are increasingly being threatened due to continued degradation of their habitats and over-exploitation of the natural resources. The collection, conservation, documentation and scientific management of these invaluable resources have now emerged as priority areas. Sirumalai hills also needed for a comprehensive strategy for conservation of wild medicinal plants. This will enable the conservation status of each taxon with reasonable confidence and will provide a basis for further management. The only way of ensuring the survival of wild medicinal plants is by habitat preservation, proper management and cultivation of species. Habitat preservation must be given highest priority particularly in this region to provide suitable protection to the wild species. Sirumalai hills have a rich diversity of vegetation with much local heritage. Still a lot of plants are being used for many other purposes by the local people (Tables 14.10 and 114.1). But the impact of recent anthropogenic pressure and forest encroachment activities on the vegetation was intense. Several areas of the forest still withstand as remnants of relictual vegetation in Sirumalai hills. The protection and management of these forests from the present population pressure is urgently needed to conserve important bioresources for our posterity. Table 14.11: Non-timber Forest Produce Used by the Local People of Slrumalai Hills 5/. No.
I.
Name of the Plant Species
Family
Useful partes)
Poaceae
Stem
Borassus flabel/ifer
Arecaceae
Leaves
Cocos nucifera
Arecaceae
Leaves
Poaceae
Stem and leaves
Plants for thatching Bambusa arundinacea
Cymbopogon maritnii Schumanniathus virgatus
Marantaceae
Leaves
Poaceae
Stem and leaves
Agave americana
Agavaceae
Leaves
Themeda cymbaria
11.
Plants for fibre
Borassus flabellifer
Arecaceae
Petiole
Cocos nucifera
Arecaceae
Pericarp
Furcraea foetida
Agavaceae
Leaves
Hibiscus cannabinus
Malvaceae
Stem bark
Thymeliaceae
Stem bark
Gnidia glauca
Ill.
Plants for basket making Bambusa arundinacea Cocculus hirsutus Lantana camara Acacia p/anifrons Phyllanthus reticulatus
Poaceae
Stem
Menispermaceae
Stem
Verbenaceae
Stem
Mimosaceae
Stem
Euphorbiaceae
Stem Contd...
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Table 14.11-Contd ••• SI. No.
Name of the Plant Species
IV.
Plants for garden implements Acacia leucophloea
Mimosaceae
S\am
Alangiaceae
Stem
Fabaceae
Stem
Dalbergia paniculata
Fabaceae
Stem
Diospyros ovalifo/ia
Ebenaceae
Stem
Gmelina arborea
Verbenaceae
Stem
Shorea roxburghii
Dipterocarpaceae
Stem
Verbenaceae
Stem
Dalbergia latifo/ia
Fabaceae
Stem
Dalbergia lanceo/aria
Fabaceae
Stem
Da/bergia paniculata
Fabaceae
Stem
Tectona grandis
Plants for house construction
Fabaceae
Stem
Mangifera indica
Anacardiaceae
Stem
Tectona grandis
Pterocarpus marsupium
VI.
Useful partes)
A/angium sa/vifolium Da/bergia /atifolia
V.
Family
Verbenaceae
Stem
Diospyros ebenum
Ebenaceae
Stem
Diospyros ma/abarica
Ebenaceae
Stem
Diospyros ovalifolia
Ebenaceae
Stem
Ixora arborea
Rubiaceae
Stem
Terminalia arjuna
Combretaceae
Stem
Canarium strictum
Burseraceae
Stem
Rubiaceae
Stem
Sapindaceae
Stem
Plants for fire wood Ixora arborea Dodonaea viscosa
Ebenaceae
Stem
Clerodendrum serratum
Verbenaceae
Stem
Cassine paniculata
Celastraceae
Stem
Chukrasia tabularis
Meliaceae
Stem
Verbenaceae
Stem
Tiliaceae
Stem
Diospyros ferrea
Gmelina arborea Grewia tiliifolia
Cordiaceae
Stem
Nilgirianthus kunthianus
Acanthaceae
Stem
Wendlandia thyrsoidea
Rubiaceae
Stem
Cordia dichotoma
Contd...
Plant Diversity and Conservation in Sirumalai Hills a/Eastern Ghats, Tamil Nadu
167
Table 14.11-Contd... SI. No.
VII.
Name of the Plant Species
Family
Useful part(s)
Plants for fodder Ficus microcarpa Commiphora wightii Grewia tiliifo/ia Reissantia indica Leucaena latisiliqua Syzygium cumini Tamarindus indica
Moraceae
Leaves
Burseraceae
Leaves
Tiliaceae
Leaves
Hippocrateaceae
Leaves
Mimosaceae
Leaves
Myrtaceae
Leaves
Mimosaceae
Leaves
Biodiversity Conservation Tropical forests cover about 7 per cent of the land surface and continue to be one of the Earth's important natural resources. This resource has always been used on a sustainable basis by the local communities of the developing world, obtaining shelter, food, fodder, fuelwood and a variety of other services. Rich in biodiversity and endemism, these forests are thought to harbour about two-thirds of all living species. While they do sustain the local environment through soil and water conservation, they also influence the regional and global climate. More importantly, forests determine the social environment of the forest dwelling traditional societies, who form part of the forest ecosystem functioning. The socio-ecological interconnection between societies and their forested environment, and the social distruptions that degraded forested ecosystems still remains to be fully understood (Ramakrishnan, 1998). Successful actions to conserve biodiversity must address the full range of causes of its current loss and embrace the opportunities that genes, species and ecosystems provide for sustainable development (IUCN, 1994). There are four basic, often complementary strategies for biodiversity conservation and restoration i.e., (a) in-situ, (b) ex-situ, (c) reduction of anthropogenic pressure and d) rehabilitation of endangered species. The in-situ strategy emphasizes the protection of ecosystems for the conservation of overall diversity of genes, populations, species, communities and ecological processes. World Conservation Monitoring Centre recorded 37,000 protected areas or sites as of 1994 (Miller et al., 1995). In India, there are 448 wildlife sanctuaries, 85 national parks and 10 biosphere reserves and together with world heritage sites and RAMSAR sites, they comprise 7.3 per cent of the geographical area of country (Rodgers et al., 2000). Areas to be protected, however, need to be chosen carefully so as to maximize the conservation interest and minimize the cost of the protection. Areas rich in species, rare species or threatened species or some combination of these attributes can be delineated to help set priorities for conservation (Reid, 1998). India possesses a long tradition of conservation practices rooted in religious sentiments. Frequently species selected by people for social significance turn out to
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be of ecological significance. For example, trees of Ficus, Neem which are frequently protected by Indian communities owing to religious beliefs, are also considered keystone species by ecologists (Gadgil, 1991). Apart from individual species, whole plant communities have often been protected as 'sacred groves'. These groves are important repositories of biodiversity. In Sirumalai hills also several forest sites are conserved such local beliefs and faiths as forest temples. However, the religious beliefs and rituals which led to the conservation of sacred groves are now fast dwindling, and this calls for urgent external intervention. Large proportions of natural forests removed for cultivation of commercial crops have been conserved world wide. In these areas forest represents a matrix of modified ecosystems within which small remnants (fragments) of natural forest remain. Many fragments are too small to support populations of many native species, and begin to lose them. Turner and Corlett (1996) agreed that fragments of tropical forest do decline in species richness one time when they become isolated from continuous forest, but also found that the small fragments can retain a relatively large proportion of their biodiversity decades after isolation. Such fragments can act as last refuges for the plant and animal species and may provide an opportunity for conservation to launch last-chance attempt to rescue species from extinction. The ex-situ strategies rely on botanical and zoological gardens, conservation stands, seed and seedling banks, pollen banks, germplasm banks, tissue culture banks, gene and DNA banks etc, to help conserve species and population diverSity out side the natural habitat. In India, conservation of genetic variability of cultivated plants and their wild relatives is mandated to the National Bureau of Plant Genetic Resources (NBPGR), which has already established India's first national gene bank. Also, the National Facility for Plant Tissue Culture Repository (NFPTCR) has planned programme on cryopreservation of seeds, pollens, embryos as well as in-vitro culture of species of e<;onomic and scientific values (Chandel, 1996). Over use of natural populations poses remarkably increasing threat to taxa important to humans. For example, the degree of threat to natural populations of Indian medicinal plants has increased because more than 90 per cent of raw material for native herbal industries and for export is drawn from natural habitats (Ved et al., 1998). A reduction in the anthropogenic pressure on natural populations by cultivating them elsewhere would sustainably contribute to their conservation (Karuppusamy et al., 2000). But the systematic study on the impact of non-native species on the ecosystem structure and ecological process in all the sites of India is lacking. Restoration and rehabilitation of endangered species is an extremely difficult and expensive process. This requires species specific plans and a knowledge on ecology and reproductive biology on the seed germination, and seedling growth and establishment is vital, not only for understanding the community processes of plant recruitment and conservation, but also for developing strategies for the conservation and restoration of biodiversity (Khurana and Singh, 2001). The species restoration plans should include diagnosis of factors responsible for the decline of species, habitat conservation, captive breeding and restriction of harvesting also.
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Conclusion Biological diversity and associated traditional knowledge systems are capital assets, which need to be conserved and sustainably utilized for the socio-economic development of the regional and country level. Local natural as well as human resource endowments can lead the country to become a leader in the global market of herbal drugs and other natural products. And, these are the promising areas where forests like Sirumalai hills can contribute substantially to generate economic property of local people through sustainable utilization and conservation of natural biodiversity. This study provides a glance of plant biodiversity of Sirumalai hills and their utility for the purpose of conserving wild diversity and germplasm of natural plant resources, steps should be taken for the protection and for the conservation of potential species effectively in this region.
References Agarwal, B.K.C., 1997. Plant based health system: Revitalization needs and conservation efforts in India. Kurukshetra, 46: 5-8. Arora, R.K. and Nayar, E.R., 1984. Wild Relatives of Crop Plants in India. NBPGR Sei. Monograph 7, Lucknow. Bir, 5.5.,1997. Plant Wealth of India. Proc. Ind. Natn. Acad. Sci., 68: 211-228. Champion, H.G. and Seth, S.K., 1968. A Revised Survey of Forest Types of India. Delhi. Chatterjee, D., 1940. Studies on the endemic flora of India and Burma. J. Asiat. Soc., Bengal, 5: 19-67. Chandel, K.P.S., 1996. Biodiversity and strategies for plant germplasm conservation in India. Trop. Ecol., 37: 21-29. Gadgil, M., 1991. Conserving India's biodiversity: The social context. Evol. Treds Plants, 5: 3-8. ICR, 1995. Country report on status of plant genetic resources of India; submitted to FAO in the preparatory process for the International technical conference on plant genetic resources. IUCN, 1994. IUCN Red List Categories. Gland: IUCN. Jain, S.K., 1983. An Assessment ofThreatened Plants of India. Botanical Survey of India, Howrah. Jain, S.K., 1991. Dictionary ofIndian Folk Medicine and Ethnobotany, Deep Publication, New Delhi. Karuppusamy, 5., Rajasekaran, K.M. and Kumuthakalavalli, R., 1999. Orchids of Sirumalai hills. J. Swamy Bot. Cl., 16: 73-74. Karuppusamy, 5., Rajasekaran, K.M. and Kumuthakalavalli, R., 2000. Needs for diversity conservation of traditional medicinal plant resources of Dindigul district, Tamil Nadu. Ecol. Env. and Cons., 6: 273-278.
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Karuppusamy, 5., Karmegam, N. and Rajasekaran, KM.,2001a. Enumeration, ecology and ethnobotany of ferns of Sirumctlai hills,South India. J. Econ. Tax. Bot., 25: 631-634. Karuppusamy, 5., Karmegam, N. and Rajasekaran, KM., 2001b. Eco-biology of a medicinal plant, Lobelia nicotianaefolia in Palani hills. Geobios,28: 165-166. Karuppusamy, 5., Rajasekaran, KM. and Karmegam, N., 2002. Medicinal plant resources of Dindigul district of Tamil Nadu (Diversity, domestication trend, cultivation and conservation). J. Nature Conservation, 14: 151-158. Khurana, E. and Singh, J.S.,2oo1. Ecology of seed and seedling growth for conservation and restoration of tropical dry forest: A review. Env. Conserv., 28: 39-52. MacBride, B., 1979. Information needed to use endangered species act for plant conservation. In: Geographical Data Organization for Rare Plant Conservation, (Eds.) J.L. Morse and M.S. Henefin. New York Botanical Garden, Bronx, New York. Mani, M.s., 1974. Ecology and Biogeography in India. W. Junk Publishers, The Hage. Matthew, K.M., 2001. Pocket Flora of Sirumalai Hills. Rapinat Herbarium, Thiruchirappalli. Mathur, S.M., 1984. Physical Geology of India. National Book Trust, New Delhi. Miller, K., Allegretti, M.H., Johnson, N. and Jonsson, B., 1995. Measures for conservation of biodiversity and sustainable use of its components. In: Global Biodiversity Assessment, (Ed.) V.H. Heywood. UNEP, Cambridge University Press, Cambridge, pp. 915-1061. Nair, N.C. and Daniel, P., 1986. A floristic diversity of the Western Ghats and its conservation. Proc. Ind. Sri. Acad., 58: 127-163. Nayar, M.P., 1980. Endemism and pattern of distribution of endemic genera (Angiosperms). J. Econ. Tax. Bot., 1: 99-110. Nayar, M.P., 1996. Hotspots of Endemic Plants of India, Nepal and Bhutan. TBGRI, Thiruvananthapuram. Paroda, R.S., 1996. India's National Bureau of Piant Genetic Resources (NBPGR) leads nationwide system. Diversity, 12: 43-47. Ramakrishnan, P.S., 1998. Sustainable development, climate change and tropical rain forest landscape. Climate Change, 39: 583-600. Ramakrishnan, P.S., 2002. Coping with uncertainties: Role of traditional ecological knowledge in socio-ecologicallandscape management. Proc. Ind. Nat. Sci. Acad., 68: 235-254. Reid, W.V., 1998. Biodiversityhotspots. Tree, 13: 275-280. Rodgers, W.A., Panwar, H.S. and Mathur, V. B., 2000. Wildlife Protected Area Network in India: A Review. WII, Dehradun. Singh, J.S. and Khurana, E., 2002. Paradigms ofbiodiversity: An overview. Proc. Ind. Natn. Sri. Acad., 68: 273-296.
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Stebbins, G.L., 1980. Rarity of plant species: A synthetic view point. Rhodora, 82: 7786. Subramanyam, K. and Nayar, M.P., 1974. Ecological Biogeography of India, (Ed.) M.S. Mani. W.Junk.Publ., The Hague, pp. 178-179. Tumer,I.M. and Corlett, R.T., 1996. The conservation value of small, isolated fragments of low land tropical rain forest. Tree, 11: 330-333. Ved, D.K., Muddappa, A. and Shankar, 0.,1998. Regulating export of endangered medicinal plant species-need for scientific rigour. Curr. Sci. 75: 341-344. Zeven, A.c. and De Wet, J.M.,1982. Dictionary ofCultivated Plants and their Regions of Diversity. Wageningen.
Chapter 15
Taxonomy in the Service of Man P.N. Rao Retired Professor of Botany, Nagarjuna University, Guest Faculty, Centre for Environment, IST JNT University, Kukatpally, Hyderabad - 500 072
ABSTRACT Taxonomy is to be correctly understood if its contemporary relevance is to be appreciated. It is a multidisciplinary subject with multi- utility aspects in regard to service to man. Though it is one of the oldest branches of Botany, it showed resurgence as the most modem science and it is virtually limitless and border less when viewed objectively. Apart form the classification of the given taxa, the aspects of identification, nomenclature, biological species concept, adaptive radiation of the dynamic species evolving into more than one species, the natural and artificial modification of the existing species, conservation of our natural plant resources, drug deSigning, planning gene banks- all need significant component of taxonomic knowledge at the molecular, micro and macro levels. The environmental health is in fact contributed by the health of the green treasures, the plants. Plant taxonomic knowledge is of significance in all current applied aspects concerning herbal drugs, phytore1Jlediation, sources of herbal insecticides and pesticides and knowledge of biodiversity of botanical wealth, forests, agriculture, horticulture, sylviculture and provides effective foundation for various biological revolutions. The present article is a humble effort in motivation of students and researchers in strengthening taxonomic studies realizing the importance of obtaining knowledge about the green wealth as an Index of Nations Wealth. It is also attempted to convey that taxonomists are an indispensable human resource with wide scope of career opportunities subject to developing expertise in taxonomic science.
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Introduction "Taxonomy is often thought of as a purely academic or even superficial subject of little relevance to the urgent needs of modem man. Such a view is far from truth, for plant taxonomists have an important role to play in man's betterment" (Stace 1980). The revival of taxonomy as a most relevant and current priority is at the very basis of the concept of Diversitas (de Castri and Younes 1996). The word diversitas here means Biological diversity (equals the concept of Variation pattern) which is now considered as an Index of a Nation's wealth. The exploration, inventory preparation, distinction between utility and non-utility biological diversity is a fundamental duty of a taxonomist. In 1992 Convention ofBiological Diversity it has been cited affirming that the conservation of biological diversity is a common concern of human kind. A paragraph in preamble mentions" ..... states have sovereign rights over their own biological resources. These are an integral and most valued segments of a Nation's property". It was held that land, gold, money, diamonds etc. are priority properties of a Nation. But it is clear even from history and current global understanding that Phytodiversity (plant diversity), the present concern of this chapter, is treated as a valuable property of a Nation. It is presently conveyed to the students that biodiversity study is an allotropic modification of taxonomic study. The treatment may also look like Botany in the service of man and if so, it will be doubly justified as the science of Botany had its beginnings of search for plants useful for food, clothing, shelter, drugs and Barter trade or otherwise. The evolution of trade is from barter through cash, or jewelry exchange or the proposals through CATT (general agreement in trade and tariff) and then Dunkel draft, and as on date the WTO (World trade organization), which all became a matter of all media. It is to be understood that the explanation of the virtually synonymous terms, Variation and Diversity are gene-based. The resurgence of serious taxonomic study is equal to the basic study of our phytodiversitry analysis. As is well known taxonomic study is at the molecular, micro and macro levels, the biodiversity analysis too is concerned with genes, species and ecosystems-a triology approach. Misunderstanding is responsible for referring to taxonomy by some as Tax-onme. This emphasizes the need for eliminating ta;(onomic illiteracy. If understood correctly and practiced well it becomes the basis of additional income or a profession or awards status of a consultant. Nair (2004) mentioned "Taxonomy ought to be an area of focus but neglected due to ignorance about its importance in achieving the hopes and aspirations of the people". He further commented that taxonomy is an integral part in terms of biodiversity protection, remediation, ecodevelopment, product development and quality evaluation. Taxonomy is significant in areas of forestry, social forestry, agroforestry, agrotechnology, pollution control (phytoremediation), ecotourism, landscape development, aesthetics, and several areas with relevance to human needs. Hariharan and Balaji (2002) from M S Swaminathan Research Institute stressed the need for synthesis of taxonomy, biodiversity and biotechnology and mentioned "Let us be optimistic in achieving a complete inventory of our biological wealth by eliminating our lacunae and reorienting ourselves towards the goal."
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Taxonomic analysis of species also takes place from an ecological angle in diversity and vegetation studies in varied environmental conditions to settle the most value-returning population sample of a species (Sagar et al. 2003). Enriched taxonomic literacy is the need of the hour. Taxonomy deals with mainly identification of plants among other aspects. The identification is the basic function for further exploration of the species. Identification is as important as the knowledge of alphabet a,b,c,d, etc. Without knowing alphabet it is impossible to progress in any language. Botanists are hesitant to develop expertise in the art of naming and field identification. Pursuing systematic studies on any species is only after correct identification and authentication of nomenclature. It is often said "What is in a name?" Everything starts with name only just as the name of a human being is primary requisite for his identification, certificates, rank, career, accounts, passport etc. The name should also be supported by a photograph the equivalent of which is a herbarium sheet for plants. Correct identification, nomenclature, herbarium preparation are indispensable functions of a taxonomist as important as knowing alphabet of a language. Is it feasible to claim expertise without fundamentals? The teachers and students of taxonomy in the beginning were often shocked that modem taxonomic books dealt with the biological species concept, sub specific categories, interrelationships with ecology, genetics, evolution, molecular biology and phylogeny, relevance of numerical analysis and also the use of biotechnology skills in conservation and sustainability of the utility biodiversity as well as endangered non utility biodiversity.
History of Taxonomy It is clear that the evolution of taxonomy itself has several phases.
1. 2. 3. 4.
The pioneer phase or exploratory phase The consolidation phase The biosystematic phase The encyclopaedic phase
Turril referred to 1 and 2 as Alpha taxonomy and 3 and 4 as Omega taxonomy. All are aware of the scope and service rendered by Alpha taxonomy. If the depth, truth, technology and purpose of each one of these are appreciated-the role of Omega taxonomy proves its overpowering share in services rendered by the same. For a history of taxonomy, any text book on taxonomy need to be consulted as additional reading material apart from what is given in this textbook (works of Lawrence, Heslop Harrison, Davis and Heywood, Lyman Benson, Cronquist, Takhtajan, Porter, Dahlgren, Tippo, Radford et al., Pullaiah (1998), Radhakrishnaiah, Naik, Raju (2004) to cite a few. Presently it is contended that ALPHA taxonomy (from A to M) and OMEGA taxonomy (from N to Z) indicate the holistic scope of the subject.
ALPHA ALPHA means classical taxonomy, age of herbals, identification, nomenclature, preparation of floras, monographs, revisions, distribution, phytogeography, history
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of endemism, ecological life cycles, reproductive biology, analysis of natural biodiversity, macro propagation and classical conservation technology- dealing with species as the basic unit of taxonomy and embraces study from Species and above categories.
OMEGA OMEGA deals with individual and local population as the taxonomic unit and works out evolution and differentiation from population and above, virtually treating SPECIES as the top category. This is an integral approach in biological species concept, definition of species based on the potential limits to exchange genes, genecology, gene mutations, impact of range of variation and evolution, adaptive radiation, recognition of varieties, races, cultivars, ecads, ecotypes, coenospecies, comparium or category of subspecies and finally species, analysis of principles of incidence of hybridization, sympatry, allopatry, role of chromosomes, polyplOidy, aneuploidy, haploidy, trisomics, nullisomics, experimental taxonomy and so on. Students ought to get in touch with the book "Stages in the Evolution of Plant Species" by Clausen, Keck and Hiesey who summarised experiments covering a quarter of a century. The work is also known as " The Californian Transect Experiments" a result of hard work of multidisciplinary manpower. Later to follow are the technology of instant speciation, manipulation of genes, recombinant DNA technology and other recent Biotechnological designing of " transcendental biodiversity" and anthropogenic variation. The recombinant DNA occurs in nature as a result of gametic fusion in sexual reproduction but the artificial recombinant DNA technology is named as Biotechnogical device which is an artificial one. These explain the scope of modem taxonomy imbibing all bioadvances in achieving newer and newer variation and evolution results all to the advantage of mankind in obtaining Green revolution, evergreen revolution, white revolution (milk), blue revolution (fish aquaculture), blue green revolution (of prokaryotic blue green algae, such as Spirulina, as a source of protein, and antioxidants), white gold revolution (cotton), green gold revolution (timber), gene revolution (may be called as super green revolution, of transgenics, gene banks, design of GEMs i.e., genetically engineered microorganisms and manipulation of higher plants including rice and other crops). In all these instances sound taxonomic knowledge helps WHERE TO START? This explains the need to understand that taxonomy is a synthetic science and also a highly applied science. It is relevant to recall a great work by Chris Park (1997, p. 461): Why conserve wild life? To maintain essential ecological processes and life support systems, to preserve genetic diversity which is being dangerously impoverished, to ensure sustainable use by us and our children of species and ecosystems. Saving world heritage sites evolved as a responsibility of environmental taxonomist. Students are advised to refer IUCN since 1973 in CITES, i.e., Convention on International trade of Endangered Species. Chris Park described fundamentals immediately followed by applied aspects in each chapter. It is common knowledge that fundamental Sciences are fore runners of applied sciences. For instance: Botany is the father of Agriculture, Forestry, Herbal therapy, Horticulture, Sylviculture, Floriculture, preparation of a pollen atlas of a leading tourist destination to help
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tourists to avoid allergens and so on. This is universally accepted and uncontroversial. The emerging plant biotechnology also is an offspring of Botany with applied values and it is hence started as B.Tech Biotecnology- in several Engineering colleges, not to speak of its start in recent graduate, post graduate institutions and Universities. Mathematicians, computer software specialists, statisticians, biochemists, phytochemists formed a team in strengthening this field extremely relevant to a taxonomist. Fundamental knowledge of taxonomy reinforces advances in this subject. Please think twice or more to appreciate this view point which signifies role of Taxonomy in the service of man. Bright ideas emerge in support of this convincing version which itself is taxonomic literacy.
Taxonomy, Biodiversity and Biotechnology Just as taxonomy has been classified into four phases, it is presently attempted to give taxonomy of phases of Biodiversity and their respective scope. This is an attempt made by the present author.
Taxonomy of Phases of Biodiversity ALPHA Species richness in homogeneous habitat. BETA Range of diversity across a gradient, measure of replacement or turn over of species.
GAMMA Overall diversity within a larger region- community diversity in heterogeneous habitat. OMEGA Infraspecific/interspecific and inter/intra population variation- linked with biological species concept and biosystematics, super biodiversity, artificial biodiversity, synthetic biodiversity, anthropogenic biodiversity, transgenic and other biotechnologically introduced instant biodiversity and genetically engineered biodiversity. These manmade aspects defy traditional/basic taxonomic principles of nomenclature and classification and are designated as Transcendental biodiversity. Dealing with its taxonomy is a challenging problem and their status allotment leads to a new discipline of transcendental or ultra OMEGA taxonomy. The biodiversity is also covered by law, the NBA- National Biodiversity act of 2002. This enhances the value of taxonomists. This knowledge helps general Botany students in interviews for jobs. The agrobiodiversity which has globalization potential has to be managed appropriately by ICAR to sustain and claim our research efforts by using our brains or intellect called hence as Intellectual Property Rights (lPR). Explanation of this is also an important question in interviews for all students of science especially Botany. The effective management of genetic resources, their roots, synthesis, and engineering new recombinations, inventorization, surveying,
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collection, conservation, characterization, evaluation, documentation, exchange, utilization, dealings with TRIPS and WTO are relevant to taxonomists directly. Let us recall the explanation of Pratibha et al. (2004) about biodiversity though Jutro gave 14 definitions to the same. It means the availability among living organisms from all sources and the ecological complexes of which they are a part and includes diversity within species or between species and of ecosystems. It also referred to the following:
1. NBA: National Biodiversity Act 2. SBB: State Biodiversity Boards 3. BMC: Biodiversity Management Committees Further significant topics of taxonomic relevance of NBA are also included for students reference: Benefit claimers: means the conservers of biological resources, their byproducts, creators, holders of knowledge and information relating to the use of such bioresources, innovations and practices associated with such use and application. Biological resources: means plants, animals, and microorganisms or parts thereof, the genetic material and byproducts with actual or potential use or value but does not include human genetic material. Biosurvey and bioutilization: means surveyor collection of species, subspecies, genes, components, and extracts of bioresources for any purpose and characterization inventorization and bioassay. Equitable benefit sharing: as per section 21 of NBA- sustainable use of components of biodiversity in such manner and such rate that does not lead to the term decline of biological diversity maintaining its potential to meet the needs and aspirations of present and future generations. For the general convenience and motivation of the students, for a ready reference, taxonomy of phases in Biotechnology also are given here as designed by the author. The attempt is to illustrate the interrelationship of taxonomy, biodiversity and biotechnolgy especially at the genetic and molecular level.
Taxonomy of the Phases in Biotechnology (on a par with biodiversity) ALPHA Fermentation, formation of curd and cheese, vinegar BETA Conventional agricultural practices for food, clothing, shelter, medicine. Musical instruments, play goods, manipulation by breeding better cultivars, and cellulose products. GAMMA All above manipulated by human advancement in Science and Technology, urge for betterment; advanced practices of hybridization, improved agronomic targets, vaccination, artificial cultures, seedless fruits, antibiotics, plant and animal protection technologies, cloning of desired cultivars, vegetative propagation etc.
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OMEGA Plant and animal transformation, transgenic plants, enzyme immobilization, organelle implantation, recombinant DNA technology, modem genetic engineering, immuno biotechnology, drug designing and so on. These lead to origin of SUPER SPECIES, ARTIFICIAL SPECIES, or in other words OMEGA BIOTECHNOLOGY implies TRANSCENDENTAL BIODNERSITY OR MOLECULAR TAXONOMY. The time consuming principles of natural evolution arc surpassed. It is instant evolution with origin of new taxa, taxonomy of which.is essential to make the biotech advances more significant leading to conservation of biodiversity as gene banks, gene pools, DNA storage process and also leading to DNA fingerprinting in identifying relationships and hence it is biotechnological taxonomy. It is already a matter of common knowledge but taxonomists are to be informed that it is ultramodern taxonomy giving phylogenetic hi-stories of genetically engineered taxa. What is their evolutionary status? What is their nomenclature? Is it sufficient to refer to them as x, y,..z? What is their biochemical status? Are they mere monsters?
Nair (2004) equated deep study of taxonomy with evolutionary biology, alternatively referred to as New systematics, New taxonomy, Biosystematics, Biotaxonomy, Evolutionary taxonomy by various modem taxonomists viz: Huxley, Heslop- Harrison, Davis and Heywood, Solbrig, Stace, Radford et al., and almost equal to biodiversity analysis e.g., Heywood et al.(biodiversity treatise), de Castri and Younes who all had as basis of newness "The Origin and evolution of species" by Charles Darwin. This is basis of predarwinian taxonomy and postdarwinian taxonomy already dealt with. This also lead to proposals of Phenetic and Phylogenetic classifications. Classification is a corollary but immediate services are obtained by species identification, nomenclature, sub specific variation, molecular basis of extractable services from the species to choose and recommend for economic purpose the most value yielding sample from either the ubiquitous or endemic or endangered species of World trade importance.
Taxonomy and Herbal Medicine The study of Botany itself started with a search for herbs for health. The science of Ethnobotany is a chapter in any recent textbook of Taxonomy. The world realized that traditional herbal medicine shows little side effects and its usage spread rapidly even in advanced American and European countries. The present turn over is more than 6200 crores of rupees. It is expected to reach Rs. 1lakh cr by 2015-2020 and Rs 5 lakhs cr by 2050 as per World Bank's estimate. Europe has $2800cr share, Asia $1080cr share, Japan $ 980crores share. India has $ 690cr share. In India the turn over is ca Rs 2500cr. An increase of 25 per cent year is envisioned. However India's export is of the share of 0.5 per cent in the world. From another angle, it has been found that China exports 70 per cent finished products and 30 per cent crude, India exports only 20-30 per cent finished prod ucts and the rest crude. India has 8000 medicinal herbs and 2500 aromatics: Of these only 100 medicinal herbs and 250 aromatics figure in International market. Even these, 90 per cent are still obtained from the forests. Central Government established National Medicinal Plants Board. It started CIMAPS (Central institutes of medicinal and aromatic plants). There is one in
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Boduppal region in Hyderabad city for giving information to students. There is COR! (Central Drug Research Institute) in Lucknow.
Guidelines for the Success of the Herbal Medicine Market Medicinal herbs are valuable natural products with potential global market already made a mark. Students are advised to motivate themselves by reading articles such as that by Sukhdev (1997), Kamboj (2000) and a recent one: Ayurveda and Natural products drug discovery by Bhushan et al. (2004). Disbelief, doubts, questions and lack of confidence in herbal medicine are mainly due to wrong identification of the medicinal herb. It is taxonomists job to provide correct identification and show the same in the field to the traders and act as their botanical consultant. This prevents adulteration when field collection is assigned to novices in the subject. For instance the valuable Phyllanthus amarus is adulterated with P. maderaspatensis, P. simplex, P. urinaria, P. pinnatus. The valuable Boerhavia diffusa (puramava) is adulterated with Trianthema portulacastrum. Bark of Wrightia tinctoria is mixed with Holarrhena antidysenterica (roots of which are however medicinal for a different purpose) and that of Saraca indica with Trema orientalis. Unless specialist taxonomist certifies the identity of the species the drug cannot be relied. This is the main reason for loss of confidence in herbal medicine. The articles by Sukhdev (1997), Bamett (2000), Natesh (2001) and book by Rao CK(2000) and several ethnobotanical works are recommended to students for further study. The problems are more with polyherbal medicines suggested to include seeds of a species, bark, root, leaves etc of several other species. You are aware that list of herbs comprising herbal medicines in the market have to be printed as a mandate. The nomenclature is impressive but it is to be believed only. The authentic identity of each of these herbs is a taxonomist's professional job and not that of a trader or manual helpers. With such a paramount role, why Botanists do not render this valuable human service? Some companies may be seeking the help of taxonomists for field identification but several may not. This may not be voluntary adulteration but due to ignorance of the species identity. It is said "where ignorance is a bliss it is folly to be wise". This dictum does not hold good in respect of herbal identification. The crude drugs are supplied mostly in dried or powdered form which increases cheating activity. The taxonomist should not only be thorough with field id as well dried crude form id of each medicinal species. It is as valuable as food adulteration and this should be started as part of taxonomy curriculum. How work on herbal medicine is becoming poly technical can be illustrated by an instance. Instance of Phyllanthus emblica: The Indian Gooseberry Umashankar and Ganesaiah (1997) virtually studied the mapping of the genetic diversity of this species from population samples from 3 states, the Kamataka, Tamilnadu and Maharashtra and conducted isoenzyme analysis. The fruits of this species contain Vitamin C. They show antioxidant, anti inflammatory and antimutagenic properties. They also contain polyphenols (ellagic acid, gallic acid, tannic acid). The plant is known since decades as remedy for common cold, scurvy,
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cancer, heart diseases. It is one third of a popular drug called "Triphalachurna" the other two being fruits of Terminal ia chebula and T. bellerica. It is a part of Brahmla Hair oil. The amla extract is water soluble and effectively scavenges free radicals responsible for initiating LPO. It is also found to be pancreatoprotective. Its antioxidant properties have been highlighted by several subsequent workers. To add to this is research work on another source of Indian gooseberry. It is Phyllanthus indofischeri (Ganesan 2003). This is endemic to Deccan plateaeu and certain drier parts of South India (herbarium with FRLHT and ATREE of Bangalore) in contrast to the widely distributed P. emblica. They are not sympatric in the same forest. Both the species are valuable for sacred religious occasions e.g., "Kshirabdi dwadasi" in AP and "Uthana dwadasi" in Karnataka. P. indifischeri is to be conserved on a war footing because of its scarcity as it is introduced by businessmen as cultivar "Krishna" in Tamilnadu and "champakad large" in Kerala. Is not the role of taxonomy in the service of man becoming clearer with this single example? Service to humanity is service to God and service to plant species is service to humanity. It is also well known 11 Vruksho Rakshati Rakshitaha"
Taxonomic Notes on Herbal Oral Insulin Mimics The range of herbal medicine for polydisastrous diabetes is cited for the benefit of student who should be an expert in the field identification and the ethnobotanical history of each one of these species. The World diabetes day is on November 14th, the birth day of Frederik Banting, the insulin discoverer. This is emphasized because India is the diabetic capital of the world according to many pharmaceutical companies with China and US prospective followers; it is estimated that in developed countries there are 51-72 million and in developing countries 84-228 million diabetics! In some places there are NGOs conducting awareness camps since 3 years: e.g., APOORVA foundation in Mysore in which activity taxonomists can help in popularising herbal insulin mimics with correct identification. The biodiversity of the disease also is to be understood as Hansen (2002) from Denmark recognised in Type 11 diabetes, multiple gene forms as well as monogenic forms. Balasubramanyam and Mohan (2001) inspire taxonomists by their article on orally active insulin mimics to enable taxonomists to be expert field identifiers of the concerned species. For ready reference a few species are listed here. (After Dahanukar et al. 2000)
Phyllanthus amarus: shows hypoglycaemic effect, reduces oxidative stress, efficient antioxidant, (free radicals and 0 reduce immunity likely to result in DNA disturbances, a sort of acquired immunodeficiency not necessarily RN caused AIDSpresent comment); Ocimum album and O. sanctum: leaf powder antihyperglycaemic; Prunus amygdalus: seeds, non oil fraction soluble in ethyl ether effective; Artemisia pallens: aerial parts extract activity similar to tolbutamide, lowers fasting blood sugar, cholesterol, triglycerides and total lipids; Aegle marmelos: leaf extraxt useful in regeneration of damaged pancreas, activation of ealate dehydrogenase enzyme; Camellia sinensis: hot water extract preventive and curative; Inula racemosa: alcohol extract potentiates insulin sensitivity, Pterocarpus marsupium: heartwood extract has pterosupin, pterostilbene, activity comparable to metformin, restored normal glucose
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levels in 70 per cent patients Capparis decidua: lowers oxidative stress; Salacia oblonga: chloroform eluted fraction works like tolbutamide; Lantana camara: leaf juice hypoglycaemic. Gita Thiagarajan et al. (2003) recorded cytoprotective antioxidant properties in Withania somnifera due to its compounds glucowithanolides and this Aswagandha" 11
is a component of several polyherbal drugs for diabetes. Sangwan (2004) stressed the multiple uses of this species. Popular critical account of the current status of the drug quality is summarised by Vibha Varshney (vide Down to Earth, March 2004 issue p.15).
The taxonomist can take up such interdisciplinary issues for the benefit of mankind. A drug Institute in New Delhi deals with a pancreatic tonic (US patent No 5,886,029). The constituent species occur in India as well which need taxonomist's
intimacy with them for further work. To cite the species, the active principle in parenthesis: Pterocarpus marsupium, (epicatechin), Gymnema sylvestre (gymnemic acid), TrigoneUa foenum-graecum (sapogenins, trigonellimum, tripothylcoumarin), Tinospora cordifolia (tinosporin), Syzigium cuminii (B-cytosterols, glucosides, jamboium), Momordica charantia(kaantin, momordicin, ascorbic acid), Cinnamomum tamala (cinamaldehyde, eugenol, cinamic acid, linol, caryophyllin); Ficus racemosa (Bcytoveriglucoside, tannins, wax, silica, phosphoric acid) and so on. Kausik Biswas et al. (2002) confirmed antihyperglycaemic properties of Azadirachta indica (with sulphur, margosin, nimbine, nimbinine, nimbidine). List of antidiabetics or insulin mimics after Parnotta (2001) all are from peninsular India, a matter of great value to taxonomists. Each one is suitable for Rand D projects for confirming the claim of ethno botanical infonna tion a fertile activity of taxonomists indeed. A herb in need is a friend indeed.
Boerhavia diffusa (roots), Biophytum sensitivum (leaves contain insulin like principle, used by tribals Bhills and Dhankals of Gujarat), Bacopa monnieri (whole plant, also in memory plus), Aegle marmelos (fruits and roots), Chloroxylon sweitenia (bark powder), Curcuma longa (rhizome infusion), Abutilon indicum (raw leaves), Alternanthera sessilis (whole plant, drug LONIC), Catharanthus roseus (petals after Yoganarasimhan in Parnotta 2001), Casearia tomentosa (root decoction, tribals of Sundergharh Dt, Orissa), Derris indica (flowers, Gujarat tribals), Enicostema axillare (whole plant decoction, Bhavnagar dt. tribals), Helicteres isora (root juice in Konkan), Holopteiea integrifolia (bark and leaves), Ichnocarpus frutescens (root powder and milk), Ficus racemosa, F. benghalensis, F. religiosa (bark, roots, and figs) Lagerstroemia speciosa (leaves, dried fruits as herbal tea in Philippines), Mangifera indica (kernel of unripe fruits) used by MP tribals, Physalis minima (whole plant, in Siddha), Psora lea corylifolia (seed and seedoil), Tinospora cordifolia (mature stem of this species is "tridoshaharam tippa teega"), Sida rhombifolia (root extract), Solanum nigrum (whole plant), Striga gesnerioides (whole plant), Syzigium cuminii (root bark, fruits infusion), Tridax procumbens (leaf powder plus Cicer arietinum as 2:1 orally by tribals of Udaipur Dt. Rajasthan), Urena lobata (root tonic tribals W. Maharashtra and those of South AP).
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To add to this exhaustive list there are several other species. A few are cited further: According to Jain (1997), Scoparia dulcis and Hibiscus tiliaceceus are folk remedies for diabetes. Apparao (2003) cited Caralluma tuberculata as a reputed cure for diabetes, reduced blood glucose levels, showed antiinflammatory and analgesic activities. 19 steroidal glucosides were isolated from this. Apart from ethnobotanical data, it is significant to know that some cited magnesium deficiency as cause for diabetes mellitus. Ehtesham (2001) established a molect.llar link between obesity and diabetes. Oral consumption of the ubiquitous aquatic weed Ipomoea aquatica reduced blood glucose levels considerably (Rao 2003, present author, and recommended for aqua culture in water bodies as additional source of income for farmers resorting to the aqua-pharming of this weed.) Further the success of herbal therapy depends also on the selection of the most efficacious sample given its wide range of distribution and biodiversity. Thus advanced biotechnological skills also now became a part of the search for drug samples. Taxonomic understanding of each drug species should be both at the alpha and omega levels to provide useful database convincing to users of the herbal medicines. This elevates the status of individual taxonomists as well as the economic status of the concerned species. The end of World War 11 ushered in the glamour of modern medical science with its spectacular discoveries. Chemical "magic bullets" produced by big pharmaceutical companies and the promise of "pill for every ill" overshadowed herbal medicaments (Natesh 2001). The existing stage points out the intensive competition to be put forward by the herbal medicine experts with the merit which is absolutely result oriented and also wipe out the psychological slavery to the chemotherapy (any chemical used as medicine is equated by me as chemotherapy, a word usually associated with cancer therapy). Herbal therapy is a biochemical synergistic therapy of more than one herb or from single herb but the biochemical in its natural home is felt to be more effective encapsulated with the natural tissue which too may have its role in action. There is general renaissance of herbal medicine in recent times due to lower side effects, greater efficacy and lower cost and an eco friendly drug therapy. Genetic engineering can be used to improve quantity of drug, more efficacious bye product and saleable to larger population to meet the market demands. Also the manufacturer can retain the engineering technical skills confidential as trade secrets. The herbal medicine is also referred to as fringe medicine, alternative medicine, or as traditional medicine (as per WHO) as complementary medicine or as orthodox medicine. Various herbs are in fact basis of several allopathic, homoeopathic, ayurvedic, Siddha or Unani medicines. Asian, African and Latin American zones mostly depend on the plant remedies, felt as immediately accessible, relatively safe, cost effective, efficacious and culturally acceptable to primary health care. A report prepared by the Export-Import Bank of India (Anonymous 1997) has estimated that the international market of medicinal plant- related trade is in the region of US$ 60 billion, and growing annually at the rate of 7 per cent. The current global market is pegged at US $ 62 billion. Europe's share is nearly half of the total. Germany dominates the Europe,m trade.
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These statistics are really staggering and indicate commercial potentialities that can better be exploited by taxonomists and help enrich economic status of India. Plants have a further a role, as already stated, in allopathic armentarium design (Table 15.1 from Natesl\, 2001) Table 15.1: Examples of Crude Drugs that have Received CllnlcallParmacologlcal Support for their Therapeutic Clalms* (From Natesh 2001) Botanical Name
EnglishlHindil Sanskrit Name
Key Constituents
Type of Activity
Acorus calamus
Vacha
?
Tranquilizer
Adhatoda zelyanlca
Vasa
Vasicine
Bronchodilator
Aesculus hippocastanum
Horse chestnut
Aescine
Astringent, antiedemic
Allium sativum
Garlic
Allin;allinase
Cholesterol-lowering, antihypertensive.
Andrographis paniculata
Bhuinimba
Andrographolide
Hepatoprotective
Arctostaphylos uva-ursl
Uva-ursi
Phenoplicheterosides
Antidote for urinary tract inflammations
Artemisia annua
Qinghao
Artemisinin
Antimalarial
A. absinthium
Wormwood
Sesquiterpene lactones
Gastrointestinal disorders
Asparagus racemosus
Shatavari
Shatavarin-I
Antiabortifacient
Azadirachta indica
Nimba
Gedunin
Antimalarial
Bacopa monnieri
Brahmi
Bacosides
Memory enhancer
Boerhavia diffusa
Punarnava
Butaa frondosa
Palasha
Palasonin
Anthelmintic
Cassia angustifolia
Senna
Sennosides
Bowel stimulator, antiabsorptive
Centella asiatica
Mandookaparni
Asiaticosides
Skin diseases, psychotropic
Chamomilla recutita
Chamomile
Chamazulene, Bisabolol, Iypophyllic flavonoids
Anti-inflammatory Antispasmodic
Crataegus monogyna, C. oxycantha
Hawthorn
Glycosyl flavonoids? Procanthocyanidins?
Positive inotropic, antiarrhythmic
Diuretic, anti-inflammatory
Curcuma longa
Turmeric/Haridra
Curcumln
Anti-inflammatory
Echinacea spp.
Cone flower
Polysaccharides
Immunomodulator
Ephedra spp.
Ephedra
Ephedrine
Bronchodilator, Vasoconstrictor
Ginkgo bilobs
Ginkgo
Bilobalide, Ginkgolides, Flavonoids
Anti-ischemic, Antihypoxidotic, PAF-antagonistic, Memory enhancer Contd...
Emerging Trends in Biological Sciences
184 Table 15.1-Contd... English/Hindil Sanskrit Name
Key Constituents
Harpagophytum procumbens
Devil's claw
Harpagoside
Anti-inflammatory and anti exudative
Holarrhena antidysenterica
Kutaja
Conessine
Antidysenteric
Hypericum perforatum
St. John's wort
Hypericins
Antideppressant
Huperzine A and other alkaloids
Active cognition enhancer, facilitates memory and motor activity in the aged
Ginsenosides
Adaptogen
Botanical Name
Hyperzia serrata
Panax ginseng, P. quinquefolius
Ginseng
Phyllanthus amarus
Bhoomyaamlaki
Type of Activity
Hepatoprotector
Picromiia kurroa
Katukaa, kutki
Picroside, kutkoside
Hepatoprotector
Piper methysticum
Kava
Methysticine and related pyrones
Anesthetic, anticonvulsant, central muscle relaxant
Plantago ovata
Psyllium
Mucilages, hemicelluloses
Laxative
Prunus africana
pygeum
Psoralea corylifolia
Balsuchi
Psoralen, bakuchiol
Antileucoderma, antibacterial
Silybum marianum
Milk thistle
Silymarin
Antihepatotoxic, promotes ribosome formation and protein synthesis
Serenoa repens
Saw Palmetto
Swertia chirayita
Kairata, Chirayta
Va/eriana officinalis
Valerian
Benign prostratic hyperplasia
Benign prostratic hyperplasia Febrifuge Valepotriates
Hypnotic
Anti-hepatitis herbs are cited in Table 15.2. Table 15.2: Anti-hepatitis Herbal Taxa Allium sativum, Aloe indica (Ghikanvar), Andrographis paniculata (Kalmegh) , Boerhaavia diffusa (Punamava), Buplerum falcatum, Berberis aristata (Daru haridra), Cichorium endivia (kasan~, Curcuma longa (Haldl), Chelidonium majus, Dictamnus, Duchesni, Emblica officinalis (Amalaka), Embelia ribes (Vidang), Eclipta alba (Bhringraja), Fumaris officinalis (Pitpapra), Glycerrhiza glabra (Yashtimadhukamu), Luffa echinata (Bindaa~, Phyllanthus niruri non. L. (=P. fratemus (Wight) Webster), Plcrormiza kurroa (Kutakl), Piper nigrum (Kalimirich), Piper communis (Erandj, Silymarin (Milk thistle), Tephrosia purpurea, Tinospora cOrdifolia (Galo), Withania somnifera (Aswagandh,9-11 granules).
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There are thus several species but Phyllanthus amarus of Euphorbiaceae, a seasonal small herb with dwarf branches, is known as an effective remedy for jaundice as per my own experience during 1970s in NU campus, Kaza and Nambur villages. There were efforts to patent it in USA which were effectively snubbed by us as it is our traditional knowledge and intellectual property of our rural folk (vide Mashelkar 2002 exhorting to the need of designing a TKDL (Traditional knowledge digital hbrary), and also his contribution: Chitrakoot declaration, 2003 in a convention of the National Botanical Research Institute, Lucknow, conveying Ayurveda as a new discovery engine.) Lot of biological diversity is also known in the hepatitis virus labeled as Hepatitis A, B, C, D, E etc. Ac:ording to WHO 170 million people are effected by Hepatitis C. From the root clones of P. amarus are now extracted interferon-a and ribavirin to control C, and the turnover is of the US $ 26000 per year. A general traditional medicine strategy 2002 to 2005 has been put forward by WHO, Geneva in 2002, very relevant to emphasize renaissance of herbal medicine which could shape into a privilege of a taxonomist, in fact. The watercress Nasturtium officinale is a multivitaqlin source with Vitamins A,B,B2, C, and E, minerals I, Fe, P- a detoxifying herb, medicine for brain disorders since 5 BC, blood cleanser, diuretic and cure for bronchitis. There is Euphorbiaceous multivitamin species Sauropus androgynus, live plant specimens of which can be seen in Forest sylvicu1ture park at Rajahmundry EG Dt. Identification of this and distinction from the weed species S. quandrangularis can be done by taxonomists alone. To inspire students of taxonomy, a few antitumour herbs are also cited:
Catharanthus roseus: roots source of vincristine, vinblastine etc, administerd as injection in Leukaemia, chewed flowers also recorded as antidiabetic; Taxus baccata: source of taxol known as specific for uterine cancer; Podophyllum emodi: source of anticancerous podophyllotaxine, etoposide, tenoposide and the camptothecine from wood extract of Nothopodytes nimmoniana breaks single stranded DNA in mammals and recorded as anticancerous; The vegetables cauliflower and cabbage are considered as preventive; there are several common herbal sources (please refer Annadata periodical in Telugu of November 2002). The enumeration will be unending and hence a comma is kept here for this aspect of herbal taxonomy.
Taxonomic Knowledge About the Phytoremediation Icipact (2001) contained several research papers on aquatic plants absorbing metal pollutants in pollution control. The transfer of metals to water bodies and biota occurs due to phenomena: bioconcentration, bioaccumulation and biomagnification and so on. There are several flowering plants to be identified first and then studied in detail for their potentiality in reducing heavy metal pollution in aquatic media. A few examples are cited here. Lead and Copper are absorbed in different ranges by Elodea canadensis, Littorella uniflora, Myriophyllum alternifolium, Potamogeton crispus, P. perfoliatus. Bioindicators of metals are Ceratophyllum demersum, Myriophyllum spicatum, Potamogeton pectinatus,
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P. perfoliatus, Zanichellia palustris, Hydrilla verticillata (cadmium uptake), Vallisneria spiralis. Trace metal and heavy metal absorbers and up takers are Eichhornia crassipes, and duck weeds. Cadmium uptake by tomato cultivars has also been on record. Removal of Arsenic by Garcinia gambosa also was reported (Chandrasekhar et al. from nCT: In ICIPACT,2oo1). Margaret Greenway (in Icipact 2001) presented a review of constructed wetlands for water pollution control. Phalaris arundinacea, Baumea articulata, Carex fascicularis,
Phylidrum lanuginosum, Schoenoplectus mucronata, Arabidopsis thaliana, Arundo donax, Typha angustifolia are a few that received trials. Further work is pending in enriching this list. What are the species and from where to obtain them? are the job requirements of a taxonomists. Several Brassicaceae have been enlisted by Palmer et al. (2001) as useful in phytoremediation. Keen art of identification, introduction, propagation and ecophysiology and persistence of the various genera and species demands taxonomic expertise. The heavy metal pollution remedying species are cited for ready reference.
Cadmium: Thlaspi caervulescens; Lead: Alyssum wolfanianum, Thlaspi spp.; Nickel: nearly 50 species of Alyssum, 4 species of Bornmuellaria, Cardamine, Naccera, Peltaria, Pseudosempervivum; Selenium: Species of Stanleya Strontium: Ambis stricta; Zinc: Arabidopsis thaliana, Cardaminopsis, Cochlearia, Noccaea, and Thlaspi.
Identification of Botanical Pesticide Source Species Nearly 2000 species are known to contain insecticidal toxic substances (Varma and Dubey 1999). Most well known is Chrysanthemum cinerariefolium =Pyrethrum. Consumption of pyrethroids is more than 1000 tons in India. Combination of Blumea lacera increases pesticidal action. It is taxonomists that can identify, show and collect these. Otherwise there is chance of adulteration with other species of Blumea and even Chrysanthemum. Unawares, the pesticide source collectors are resorting to taxonomic work. For the benefit of the reader a few more species are enlisted to point out taxonomic value in this regard. Table 15.3 shows important families having number of species with anti-insect properties (Dhaliwal and KouI2001).
Azadirachta indica: seeds source of 100 tetranortriterpenoids, non-isoprenoids, azadirachtin (atleast 30 commercial brands in market), shows antifeedent properties. Artemisia capillaris: fungicide source; Ocimum suave: grain protectant; Eugenia aromatica: flower buds grain protectant; Pogostemon heyanus, Ocimum basilicum: essential oils insecticidal; Ocimum canum, Citrus medica, Cymbopogon citratus: sources of fumigant essential oils against post harvest fungal diseases; Carum carvi: insecticide in Netherlands, trade name TALENT; Chenopodium ambrosioides: essential oil against damping disease; Acacia nilotica: aqueous bark controls blue mould rot. Murugesan (2001) cited the seed powder of Leucaena leucocephala with the chemicalleucemol, Mimosa pudica with mimosine, and Annona squamosa with Annonin to be insecticidal.
187
Taxonomy in the Service of Man Table 15.3: Important Plant Families having Number of Species with Anti-Insect Properties Plant family
Number of Plant Species
Annonaceae
12
Apiaceae
23
Apocynaceae
39
Asteraceae
47
Cryptogams
58
Cupressaceae
22
Euphorbiaceae
63
Fabaceae
157
Lamiaceae
52
Leguminosae other than Fabaceae
60
Liliaceae
24
Meliaceae
>500
Moraceae
26
Myrtaceae
Poaceae
72 52 27
Ranunculaceae
55
Rosaceae
34
Rubiaceae
Pinaceae
Rutaceae
38 42
Solanaceae
52
Verbenaceae
60
Only 5-15 per cent of the existing species have been studied from this angle. Field exploration for investigation on other potential sources for authentic determination is a valuable job requirement of a taxonomist.
Taxonomy and Horticulture All botanists and taxonomists need knowledge of identification, nomenclature, origin and propagation technology of horticultural species which are eye feasting, provide landscape luxury, enrich botanical paradises, and tourism, source of job and considerable income. Few realize that it is not the job of an illiterate gardener and horticultural traders who may have picked up some knowledge just as a compounder dominates a qualified physician or a laymen running a computer institute while they are more recognizable with the help of qualified technical personnel. Origin, evolution, designing new cultivars, their ecophysiological requirements need taxonomic knowledge. After Bailey's Manual of cultivated plants (several diagrams in this are from Lawrence's Taxonomy of vascular plants). Sales men are not science experts.
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Taxonomists can be leaders and pleaders for salesmen. There are lot of economic resources and high export potential. This area needs synthesis of knowledge of morphology, ecology, ecophysiology, diversity, preservation and conservation recognised of late as limbs of taxonomic studies. Work of Barbier et al. (1994) on Paradise Lost? The ecological economics of biodiversity motivates studies in this area.
Taxonomy and Phytogeography The relevance of taxonomy to phytogeography and vegetation studies is a part of the present syllabus. Continuing the omnipresence of taxonomy, it is knowledge of phytogeography that helps in assessing phytobiodiversity and phytotaxonomy. A taxonomist is the qualified person utilizing all technology available including GIS (Geographical Information System); remote sensing to map and account the biodiversity hotspots in the world. The global biodiversity hotspots are:
* The Andes (Venezuela, Colombia, Ecuador, Peru, Bolivia) * Madagascar * Brazil's Atlantic forest region * The Phillippines * Meso American forests * Wallacea (eastern Indonesia) * Western Sunda (in Indonesia, Malayasia, Brunei) * South Africa's cape floristic region * The Antilles * Brazil's Cerado * The Darien and Choco of Panama, Colombia and Ecuador * Polynesia and Micronesian island complex including Hawaii * South West Australia * The Eastern Meditarranean region * Eastern Himalayas * The Guinean Forests of West Africa * The Western Ghats of India and ,islands of Sri Lanka * New Caledonia * South Eastern Australia and Tanzania tro~ical
Hotspots are areas rich in species, locales of endemic species. India is a major megadiversity nation and the biogeographic zones are useful for taxonomists to account for the biodiversity of species.
* Trans Himalayan Ladakh mountains * Himalayas, NW, W, central and eastern * Thar desert
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"* Semiarid Punjab plains, Gujarat and Rajputana "* Western ghats, Malabar plains "* central Deccan peninsula, central highlands, Chota Nagpur, Eastern highlands, plateau and south deccan "* Upper and lower Gangetic plains "* West and east coast and Lakhdweep "* North east hills, Brahmaputra valley "* Andaman and Nicobar islands Taxonomy, Forest Biodiversity and Afforestation The identification, nomenclature and economic yield level classification of forest flora is very important. The forests are renewable natural resource because of their potential regenerative capacities of the plants. Forests are sources of food, clothing, shelter, fuel, various drugs, and varied minor forest produce often called as the NonTimber forest produce (NTFP). What species are to be saved, regenerated, micropropagated: can be enlisted by a taxonomist correctly. Data on tree value needs to be prepared. Relative economic losses can be drafted and provided to mankind and media for wide publicity. The fact that a valuable drug source tree can yield $600 m/year considering the world market. Exports are well justified with such basic information and appeals to the world communities and the commercial WTO. This richness is superior in tropical rain forests compared to the generally less biodiversity of temperate forests. The reasons for deforestation are summarized as follows (Anjaneyulu 2004)
"* Destruction of forests and habitat loss "* Overexploitation of forest wealth "* Overgrazing in forest area "* Shifting cultivation in forests "* Urbanization. of forest area "* Industrialization in forest area "* Unchecked illegal trade of forest produce "* Smuggling and biopiracy of forest resources "* Diminishing green cover "* Mining for ores "* Intrusion of river valley and hydroelectric projects "* Making approach roads "* Exploitation of NTFP "* Loss of lan? fertility due to soil erosion "* Incidence of droughts and famines "* Increased indiscriminate devegetation
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"* Desertification and wasteland expansion "* III planned penetration of tourism business "* Unequal globalization "* GREED rather than NEED. A taxonomist can study and prepare inventory of susceptible species, publish and permeate knowledge of the threatened species following the dicta of International Union for Conservation of Nature (IUCN 2000). The following is the taxonomy of the eroding biodiversity taxa.
"* Threatened: T "* Near threatened: NT "* Vulnerable: VU "* Endangered: EN "* Critically endangered: CEN "* Extinct: EX Species classification with annotated risk factors is the applied duty of a taxonomist. (for further reading Jadhav 2003 is recommended)
Social Forestry Species To reduce pressure on the general forests and maintain their sustainability, cultivation of certain species in the core of social life areas and close by in residential areas in rural as well as urban locales can be better suggested, planned, monitored by a trained taxonomist who alone can distinguish one from the other and also analyse the biodiversity range of each species to choose the best genotype that adapts to the ambient environmental fluctuations and yield high biomass (Phyto-energy) for fuel, fodder, or green shield that prevents soil erosion. The species should be fast growing, drought resistant and relatively disease free. Several have been experimented with all over the country lead by the BESI (Bioenergy Society of India) and DNES (Department of Non-conventional Energy Sources). Few examples are here:
Acacia auriculaeformis, Casuarina equisetifolia, Caesalpinia pulcherrima, Dalbergia sissoo, Eriodendron pentandrum, Eucalyptus hybrida, Gliricidia maculata, Leucaena leucocephala, Pongamia pinnata. These species were raised by the Dept of Botany, Nagarjuna University as an implementation of a HDEP (High Density Energy Plantation) project sanctioned by DNES in nearly 70 hactares during 1986-92. The energy species are recurring source of income. Energy returns of each species and the biodiversity therein need detailed study and each species can offer a potential research problem for doctoral thesis work. The species are also taxa which are most suited in regreening of degreened wasted lands. Thousands of hactares in various parts of AP and India are supported by DNES in enriching cultivation of energy species. Severai taxonomists are associated with this activity.
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Windshields and Cyclone Shelters Along Beaches Casuarina equisetifolia (The Casuarina) and certain avenue trees near beach approach roads provide green remedies for the above unforeseen disasters. A taxonomist can study further and recommend more species suitable for mitigating these disasters.
Green Belt Species The industries have been ordered to grow certain trees which minimise pollution, act as CO2 sinks and other pollution absorbers and also to create an ecofriendly environment in that area. Taxonomists are well suited to participate in such green belt programs and apart from recommending the species, they can help by adding data on ecological efficiency of the species in their potential pollution control.: Examples are cited here for student's ready reference: All HDEP species listed above and Albizziaamara, Alstonia scholaris, Azadirachta indica,Bushes of Bougainvillaea,Caesalpinia coriaria,Cassia montana, C. renigera, c.siamea, C. sophora,Calophyllum inophyllum, Delonix regia, Enterolobium saman, Mimusops elengii and M. hexandra, Peltophorum pterocarpum, Syzigium alternijolium, Laymen and Scientists usually ask Botanists about the species and want details and if Botanists reply that they do not have touch with these, it is ridiculous. At least they should refer to a taxonomist for consultancy.
Help of Taxonomic Knowledge in Harnessing Wastelands Wastelands are in fact wasted lands with potentiality for cultivation but abandoned due to economic and other practical issues. Table 15.4 summarizes an estimate of wastelands in India which can be harnessed with suitable green wealth sources- petrocrops and also appropriate herbs of medicinal and other miscellaneous values. The contribution is most fitting when done by a taxonomist with field identification expertise, growth and survival patterns of the various species concerned. A few deserving species already with national and international demand are cited for ready reference and taxonomists to pick up field identification talent.
Parthenium argentatum, Calotropis procera, l.imtana camara, Simmondsia chinensis, Jatropha curcas, J. gbssypijolia, J. glandulifera, J. panduraefolia, J. podagrica, seven species of the genus Chamaesyce, several species of Euphorhia (leafless, glabrous with cylindrical stems; leafy, herbaceous with rich latex source; leafy, prostrate or spreading erect annuals), Azadirachta indica, Pongamia pinnata, Calophyllum inophyllum, Schleichera oleosa, Pittosporum resiniferum and P. eriocarpum. A case in point is Eichhornia crassipes from which an aviation fuel butanediol has been extracted as per a report from NEERI (National environmental engineering research Institute, Nagpur). The lignocelluloses, latex, oils, edible or nonedible, the fats and lipids, and several ingredients of phytomass is modified as green fuel or biodiesel sources. The solar energy fixing potential of green plants is harvested as utilizable energy. The MOE and F, Bio energy Society of India, The DNES (department of non conventional energy sources), various national laboratories and Universities are looking for more phytomass yielding energy species.
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Emerging Trends in Biological Sciences Table 15.4: Estimate of Wastelands in India to be Greened with Petrocrop Taxa and Drug Species State/Union
Non-forest Area
Extent of Waste in '000 of ha Degraded Forest Area
Total
Andhra Pradesh
3734
11416
Assam
7682 935
Bihar
3896
795 1562
5458
1730
Gujarat
7153
Haryana
2401
683 74
Himachal Pradesh
1424
534
1958
Jammu and Kashmir
531
1034
Karnataka Kerala
7122 1053
2043 226
1565 9165 1279
Madhya Pradesh
12947
7195
20142
Maharashtra
11560
2841
14402
Manipur
14
1424
1438
Meghalaya
815
1103
1918
Nagaland
508
1386
Orissa
3157
878 3227
Punjab
1151
79
1230
Rajasthan Sikkim
18010 131
1933 150
19943 281
Tamil Nadu
3392
1009
7836 2475
6384
Tripura
108
865
4401 973
Uttar Pradesh
1426
8061
West Bengal
6635 2177
359
2526
Uts
889
2715
3604
Total
93691
35889
129580
Endemics The Botanical Survey of India published various accounts on endemism and and endemic species restricted to certain areas which afford a congenial environment for these species. Ex situ efforts of cultivation and conservation of these species do not succeed unless the exact habitat is artificially fabricated. A critical search and research for these species is a taxonomist's job and rearing them in situ or ex situ is a real service to preserve segments ofbiodiversity.
Conservation Procedures Singh (2002) summarized the conservation strategies. In situ: cultivate at the native habitat; ex situ: cultivate elsewhere from the original habitat recreating their
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ambient environment such as in green houses, phytotrons in botanical gardens, or utilize biotechnology skills in micro propagation of the endangered species or develop their germ plasm banks, pollen banks, seed banks, gene banks or DNA banks. The conservation efforts can be integrated from molecular to macro levels to save the endangered biodiversity at any cost. Conservation of biodiversity and sustainabiIity have been recommended for a radical reassessment by Adams (1996). Kellert (1996) pointed out the effects of loss of biodiversity on physical, emotional and intellectual well being of the people. Barbier et al. (1994) even earlier stressed the ecological economics of biodiversity and referred to the loss as "Paradise Lost". A classic work on the causes and consequences of extinction of species by Ehrlich and Ehrlich (1982) is useful to all taxonomists. The use of taxonomy in the service man is thus showing border less dimensions. The works of Radford et al., Heywood et al., de Castri and Younes (1996) have already been cited. The planning responsibilities of a taxonomist reached a stage of "perform or perish". The taxonomist should be aware of the global concern shown by World Bank, World Watch Institute, UNDP, UNESCO, IUCN, IUBS, CITES, WWF, CGIAR, UNEP, CIFOR, MOE and F, BSI, DNES and so on concerned with conservation and enrichment ofbiodiversity as an Index of a Nation's wealth.
Identification of Plant Indicators Knowledge of identity of plant indicators of environmental conditions can be better done by a trained taxonomist. There are several instances of cursory indicators of climate: Temperature, Light, Rainfall, Relative humidity of representatives of evergreen, semi evergreen, deciduous, scrub jungles, mangroves, sclerophylls, grass lands etc. The terminology itself is derived by the qualities of the indicator species and there are several floras of each of these climatic situations. The recognition of plants as mesophytes, xerophytes, lithophytes, halophytes, etc. is based on indicator species. If soil is considered: sandy soils, acidic or alkaline soils, grasslands, iron rich soils, low lying lands, uplands, waste lands, salty soils, marshes etc each one of these are explained by respective examples of species, correctly enlisted by a taxonomist better than others. There are also illustrative examples of plant indicators of mines, gems and so on. Prasad (1986) enlisted plant indicators of ground water, of soil diversity, of environmental pollution and agricultural climatology. More data is found in Sharma (2001) and several books on Ecology and Environment.
Role of Taxonomist in Ecosystem Studies The main role of a phytotaxonomist is to prepare a full statement of the primary producers (photosynthetic taxa) in any given ecosystem, a foundation data for picturising the trophic chains, trophic webs, energy flow pattern, ecological pyramids, and information on gross and net productivity, an estimation of photosynthetic activity, an indication of efficiency of solar energy fixation. The comparative statement of relative efficiencies helps in preparation of a compendium of highly productive species or ecosystems. Writing a flora with productivity data is an unending realm of work fetching research and employment opportunities to taxonomists.
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The same series of events prevail in studying a cropland ecosystem as well. The hybrid crop, transgenic crop, clonal crop, male sterile crop-such data comprise the taxonomists diary of the biodata of the cultivar and helps in indexing the molecular levels responsible for the higher productivity of a crop cultivar as well as its early maturation and disease resistance and general" curriculum vitae" of a commercial crop. Singh SP (2002) cited the combination of ecosystem services and biodiversity in environmental conservation.
How a Taxonomist can be of Help to Mankind Basic qualification: capacity to identify phytobiodiversity of species in the field, certify its nomenclature and maintenance of herbarium. Professional opportunities for taxonomists (also indicates their job requirements). Field collection of authentic utility species from wilderness and supply to the concerned company trading them. General crude drug supply to private pharmaceutical companies. Search and research on specific individual species for supplying to the drug dealers or synthetic drug manufacturers recommending effective population sample.
Self maintenance of a medicinal plant nursery. Create awareness and conduct courses to workers who require this sort of taxonomic literacy. Maintenance of herbaria in schools, colleges, universities, private botanical companies, local government administrative offices supervising landscape, horticulture and parks as nodal reference points. There are serious omissions in most educational institutions. Training students in field exploration, collection, identification, preservation, herbarium development, and keys for identification and storage of data on floppies or CDs for retrieval. Maintenance of nursery of horticultural species. Publication of annotated notes on each ornamental species with eco-physiological details Enrich floras of cities, towns, rural areas,-wild, cultivated, introduced ornamentals, drugs and others of economic value. A big omission of several floras is they enlist only wild flora and not bother about cultivated species, a feature of demand by most of the public, privates, students, laymen, even botanists and surprisingly including taxonomists. Develop instructive model plant propagation units for education, also provide most needed species or their propagules Provide botanical consultancy for botanical gardens, parks, landscape, nurseries, avenue trees, seasonals, annuals, lawns and so on. Botanical consultancy for social forestry, hedges, fuels, crop rotations, green manures and other sources of green wealth.
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Consultancy about taxonomic status, parentage, technology about transgenic crops, somatic hybrids and so on. Professional taxonomic consultancy about identification of species, medicinal, or other economic utility for starting research work on biology, cytology, cytogenetics, ecology, or biotechnology of the species. Botanical consultants and suppliers of all specimens for class work, musea mounts, herbarium and life cycle charts and demonstration samples (several companies are already in the field, few making 11se of trained taxonomists. (This provE!s that Taxonomy is not tax-on-me but income- taxonomy). Professional practical leaders of field exploration to help colleges and University students where this expertise is getting endangered. The taxonomist is most suitable to lead people as guides of ecotourism, prepare inventories of pristine botanical paradises and suggest remedies for ecodegradation of tourism spots. They can strengthen this role by publishing botanical wealth brochures and also seek employment as caretaker ClOs of endangered biodiversity and prevent biopiracy. There is no exaggeration in the versatile roles prescribed for a taxonomist in the service of man. Th~re is, however, obvious need for interactive synthesis of the basics and ethnic knowledge with such applied fields like horticulture, biotechnology, green crop gene revolution, agroforestry, and various new interdisciplines. The syllabus hence is to be revised in future to foresee professional taxonomists as botanical consultants.
Conclusion Support taxonomy, conserve taxonomy, master taxonomy, practice taxonomy, generate taxonomic consultants. Let not taxonoIJIists be "an endangered species". Wipe out all misunderstanding about taxonomy. Make genuine effort to spread the foundation service which taxonomist alone can provide. A mastery of a subject alone keeps an expert in demand whether medicine, engineering, science, teaching or preaching. The same is true of taxonomy. Masters of taxonomy are a very useful human resource for developing as well as developed nations. This HRD should get top priority. Taxonomists have to learn more and more about more and more whereas other specialists learn more and more about less and less. Consummate taxonomists are the friends of the society. This branch deserves to be started as a compulsory course in all bioscience courses emphasizing the multiple applications of the subject from microbes to macrobes, from genes to species and communities. Nurturing of efficient taxonomists is sure to metamorphose as an index of a Nation's academic wealth as well as economic wealth.
Acknowledgements I am highly thankful to Prof. V. Chandrasekhara Rao, Director, Centre for Distance Education, Acharya Nagarjuna University, Nagarjunanagar - 522 510 and to Professor T.N. Mary, Dept. of Botany of the same University for their encouragement in preparation of this article. I express my thanks to Prof T. Pullaiah and to Prof. V.S. Raju for several personal discussions in this regard during the last several years with
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emphasis on revival of taxonomy. I am also thankful to Prof. Y. Anjaneyulu, Director, 1ST, Jawaharlal N~hru Technological University, for providing several opportunities to widen my horizons that resulted in proposing a taxonomic revolution in keeping with the efforts to explore sources of green wealth and health.
References Adams, W.M., 1996. Future nature: a vision for conservation. In: Kellert 1996: A Radical Reassessment of Conservation of Biodiversity and Sustainability. Island Press, New York. Apparao, AV.N., 2003. Chemical and biological investigations of Caralluma of India. In: Int. Seminar on Global Scenario ofHerbal Medicine, nCT, Hyderabad, Diamond Jubilee Celebrations, p. 31-32. Anjaneyulu, Y., 2004. An Introduction to Environmental Science (Ed.) P.N. Rao. BS Publications, Hyderabad. Balasubramanyam, M. and Mohan, V., 2001. Orally active insulin mimics. J. Bioscience, 26: 383-390. Barbier, E.B., Burgess, J.c. and Folk, c., 1994. Paradise Lost? The ecological economics of Biodiversity. Earthscan, London. Barnett, R., 2000. Traditional medicinal practitioners in Kenya-putting theory into practice. Traffic Bull., 18: 87-89. Bhushan Patwardhan, Ashok, D.B., Vaidya and Mukund Chorghade, 2004. Ayurveda and natural product drug discovery. Curr. Sci., 88: 789-800. Chris Park, 1997. The Environment: Principles and Applications. Routledge, London. Dahanukar, S.A, Kulkarni, R.A and Rege, N.N.,2000. Pharmacology of medicinal plants and natural products. Ind. J. Pharmacology, 32: 581-5118. De Castri, F. and Younes, T., 1996. Biodiversity, Science and Development: Towards a New Partnership. CAB International. Dhaliwal, G.S. and Koul, 0., 2001. Biopesticides: A global perspective. In: Biopesticides: Emerging Trends, Souvenir, Chandigarh, p. 7-16. Ehrlich, P. and Ehrlich, A, 1982. Extinction. The causes and consequences of disappearance of species. Victor Collancz, tondon. Ehteshan, N.Z., 2001. Molecular link between diabetes and obesity. Curr. Sci., 80: 1369-1370. Ganesan, R., 2003. Identification, distribution and conservation of Phyllanthus indofischerii, another source of Indian gooseberry. Curr. Sci., 84: 1515-1518. Geetha Thyagarajan, Talla Venu, and Dorairajan Balasubramanian, 2003. Approaches to relative burden of cataract blindness through natural antioxidants: Use of Aswagandha (Withania somnifera). Curr. Sci., 85: 1065-107l. Hans, P.c., Silay, R. and Devi D. Bansal, 2002. Magnesium deficiency in diabetes mellitus. Curr. Sci.,84: 1456-1463.
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Hansen, T., 2002. Genetics of Type 11 diabetes. In: Diabetes special section. Curr. Sci., 83: 1477-1483. Hariharan, G.N. and Balaji, P., 2002. Taxonomic research in India: Further prospects.
Curr. Sci.,83: 1068-70. ICIPACT, 2001. In: Proceedings ofthe International Conference on Pollution and its Control Technologies, Jawaharlal Nehru Technological University, Hyderabad. Jadhav, N.S., 2003. In situ conservation and sustainable utilization of medicinal plants in AP. UNDP and MOE and F project report. Jain, S.K., 1997. Cultural dimensions of biodiversity. Proc. Ind. Natn. Acad. Sci., B63: 449-466. Kamboj, V.P., 2000. Herbal medicine. Curr. Sci., 78: 35-39. Kausik Biswas, Chattopadhaya, I., Banerjee, RK. and Bandopadhyay, 2002. Biological activities and medicinal properties of neem. Curr. Sci., 82: 1336-1345. Kellert, S.R, 1996. The Value of Life: Biological Diversity and Human Society. Island Press, New York. Mashelkar, RA., 2001. Intellectual property rights and the third world. In special section Science in the third world. Curr. Sci., 81: 955-965. Mashelkar, RA., 2003. Chitrakoot Declaration. NBRI, Convention Lucknow, Murugesan, K., 2001. Recent perspectives in plant disease management. In: 21 st Century Perspectives in Plant Science, National Symp. UGC-SAP-CSIR July 2931. Andhra University, Abstract page No. 70. Nair, P.K.K., 2004. Plant taxonomy. Curr. Sci., 86: 665-667. Natesh, S., 2001. The changing scenario of herbal drugs: Role of Botanists. Phytomorphology, Golden Jubilee Issue, p. 75-96. Palmer, C.E., Suzanne Warwick and Wilf Keller, 2001. Brassicaceae (Cruciferae) family, Plant biotechnology and Phytoremediation. Int. J. of Phytoremediation, 3: 245287. Parnotta, J.A., 2001. Healing Plants of Peninsular India. CAB International. Prasad, E.A.V., 1986. Plant Indicators. Indian Review of Life Sciences, 6: 253-271. Pratibha Brahmi, Dua, RP., and Dhillon, B.S., 2004. The biological diversity Act of India and agrobiodiversity management. Curr. Sci., 86: 659-665. Pullaiah, T., 1998. Taxonomy of Angiosperms. Regency Publications, New Delhi. Raju, V.S., 2004. Systematics of Angiosperms. Nagarjuna University text for Centre for Distance Education, Nagarjunanagar. Rao, c.K., 2000. Database on Medicinal Plants. Kamataka state Council for Science and Technology, Malleswaram, Bangalore. Rao, P.N., 2003. Health and wealth supplements from wetland weeds with prospects of aqua-pharming. In: National Con! Recent trends in Aquatic Biology, IAAB and Department of Zoology, Nagarjuna University.
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Sagar, R, Raghubanshi, AS. and Singh, J.5.,2003. Asymptomatic models of speciesarea curve for measuring diversity of dry tropical forest tree species. Curr. Sci., 84: 1555-1560. Sangwan, RS., Chaurasiya, N.D., Misra, L.N .. Lal, P. Uniyal, G.c., Sharma, R Sangwan, N.S., Suri, K.A, Qazi, G.N.and Tuli, R,2004. Phytochemical variability in commercial herbal products and preparation of Withania somnifera (Aswagandha). Curr. Sci., 86: 461-470. Sharma, P.D., 2001. Ecology and Environment. Rastogi Publications, India. Singh, J.5.,2002. The biodiversity crisis: A multifaceted review. Curr. Sci., 82:638-647. Singh, S.P., 2002. Balancing the approaches of environmental conservation by considering ecosystem services as well as biodiversity. Curr. Sci., 82: 1331-1335. Stace, C.A, 1980. Plant Taxonomy and Biosystematics. Edward Arnold, London. Sukhdev, 1997. Ethnotherapeutics and modem drug development: The potential of A yurveda. Curr. Sci., 73: 909-928. Umashankar, R and Ganesaiah, K.N., 1997. Mapping genetic diversity of Phyllanthus emblica. Curr. Sci., 73: 560-565. Varma, J. and Dubey, N.K., 1999. Prospective botanical and microbial products as pesticides of tomorrow. Curr. Sci., 75: 172-179.
Chapter 16
Studies on Frerea indica Dalz.: A Critically Endatlgered and Endemic Species from Maharashtra, India A.B.D. Selvam,
s.
Bandyopadhyay and Basundhara Pillai
Pharmacognosy Unit, Botanical Survey of India, Howrah - 711 103
ABSTRACT Frerea indica Dalz. (Asclepiadaceae) is endemic to Maharashtra State. The International Union for Conservation of Nature and Natural Resources (IUCN) has identified this species as one of the twelve most endangered plant species on earth. In this paper, we provide information on the current status of this species viz., distribution, phenology and unreported medicinal uses. This study might help in effective methods of conserving the species and saving it from extinction in its natural habitat.
Introduction Frerea indica Dalz. is a monotypic genus belonging to the family Asclepiadaceae. This plant species was first described by N.A. Dalzell in 1864 from Cancan region, Maharashtra. The generic name 'Frerea' was named after Sir H.B.E. Frere, the then Governor of Bombay Presidency, as a mark of esteem and respect and also for encouraging and promoting scientific research in India. The specific epithet 'indica' indicates that this plant species is a native of India.
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This species is known to occur only in Maharashtra State and it is known as
'Shindal-makudi' in Marathi. The pendulous nature and succulent stem and a star shaped corolla are the distinguishing characters of this species. Several workers have studied various aspects of F. indica, such as taxonomy (Hooker, 1883; Cooke, 1958; Almeida, 2001); revisionary studies Gagtap, 1992); status Gain and Sash'y, 1980; Nayar and Sastry, 1987; Tetali et. al., 1997a); associated plants and habitat conservation (Tetali et al., 1997b).
Methodology The present paper includes survey on the available literature of F. indica and examination of voucher specimens deposited in 'CAL' and 'BSI' Herbaria of Botanical Survey of India located at Howrah and Pune respectively. A field survey was undertaken to Sajjangad, Satara District, Maharashtra, one of the natural habitats of the said plant to assess the wild population and the factors responsible for its rapid depletion in the wild. Further, personal observations and interview with the local people of the above said area were also carried out to know the usefulness of this plant.
Results and Discussion Distribution A survey of the available literature shows that F. indica is reported from seven different localities /habitats in four neighbouring districts of Maharashtra State viz., Randha falls from Ahmednagar District; Avre/Hewra Gunnar), Shivneri Fort Gunnar) and Vazirgarh hills (Purandhar) from Pune District; Shivthalgarh (Mahad) from Raigad District; Mahabaleswar and Sajjangad from Satara District. Each of these habitats shelters a few patches of individuals (Tetali et al., 1997a; Mishra and Singh, 2001). The typical habitat of F. indica consists of rocky crevices on steep hill slopes/hill cliffs covered with a thin layer of soil at an altitude between 750 to 1347 m. It is reported that rainfall greatly varies in all the six localities. This plant is a fleshy, glabrous perennial. Branches spread on barren rocks and form patches more than 1 metre across or may droop while clinging to rocks. The plant prefers slightly acidic soil (pH 5.5-7.0). The plant cannot withstand water logging conditions (Kothari and Moorthy, 1993; Tetali et. al., 1997a). During our field survey to Sajjangad locality, the following plants were found associated with F. indica in its natural habitat viz., Balsam spp., Notonia spp., umtana camara L. and Grasses. Some of the patches were inaccessible and are free from human disturbance in the above said locality. To certain extent, habitat itself is conserving this plant in the wild.
Phenology The phenological data such as flowering and fruiting period recorded from various localities have been reviewed and are reported hereunder along with our observations in the field.
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It is reported that the flowering of F. indica occurs during September-December and fruiting during December-January Gagtap and Singh, 1992; Almeida,2001), but we have observed flowering in the month of June also both in the wild in Sajjangad locality and also under cultivation in the nursery of N aoroji Godrej Centre for Plant Research, Shindewadi. This is an additional information/report for this study. From this study, it is concluded that the flowering starts in the month of June and ends in the month of December. The flowering may be peak during the months of SeptemberDecember as observed by earlier workers. Further, we have not observed any fruiting during our field survey.
Various Uses F. indica is pretty succulent, with star shaped flowers, when domesticated, it can make a good indoor plant. When leafless, the plant is eaten (Tetali et. al., 1997a). According to the local people of Sajjangad temple area, Satara District of Maharashtra, this plant is a good cattle feed. The leaves and green portion of young shoot of the plant are consumed by the local people either raw or cooked. Further, it was learnt from the local people that consumption of this plant reduces 'pitham or pitta', one of the 'tridosa' factors in Indian Systems of Medicine such as Siddha and Ayurveda. Therefore, the plant can be useful in treating 'pitham or pitta' related disorders like biliousness, nausea, acidity, indigestion and also reducing body heat. The above medicinal uses have not been previously reported elsewhere and therefore it is a new information/report for this study. The pharmacognostic study of this species is underway in our lab. It is suggested that the plant should be subjected to pharmacological and other related studies so as to ascertain the therapeutic efficacy of this plant.
Current Status The literature survey shows that there is a continuous change in the status of F.
indica in the wild from time to time. The species is classified as a Rare Species and included in the list of Threatened plants of India Gain and Sas try, 1980). Later, Ahmedullah and Nayar (1986) have placed this species under Rare and Threatened category, while Nayar and Sastry (1987) have placed this species under Endangered category. The International Union for Conservation of Nature and Natural Resources (IUCN) has enlisted this species as one of the twelve Most Endangered Species on earth. In 1988, the Government of India has included this species under Negative list of Export, which bans all its trade and export from wild collections. This species is likely to move from most endangered category to extinction in the wild if the casual factors are not checked forthwith.
Major Threats and Conservation Measures The major threats and conservation measures to be taken up immediately to save the plant in its natural habitat are discussed hereunder.
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Major Threats The major threats to this plant species in its natural habitats are pests, animal grazing, bush or ground fire, landslides and exploitation by local people for food and medicinal values. There are two types of insects that infest this plant viz., caterpillars and aphids. The former is a serious pest, which feeds on the leaves and pods of this plant and defoliates the leaves. The aphids feed on slender shoots. The domestic animals also eat the leaves and tender shoots of this plant and disturb/destroy the habitat. The bush or ground fire destroys the natural habitats during summer season. The natural calamities like landslides are unavoidable during rainy season (Tetali et al., 1977a). Apart from the above threats, the local people who live near the habitat of the plant exploit this species for food and medicine. All these threats need to be checked immediately so as to save this plant species in the wild. Conservation Measures There are two major approaches in practice for the conservation of threatened plant species viz., in situ and ex situ conservation. The conservation measures taken so far and steps to be taken in respect of this plant are discussed below.
Ongoing Ex situ Conserva'tion Considering the serious threats to this plant in the wild, it is conserved ex situ in Maharashtra State where this plant grows as an endemic. The Botanical Survey of India, Western Circle, Pune is maintaining the germplasm of this species in its Experimental Garden. Apart from this, Naoroji Godrej Centre for Plant Research (NGCPR), Shindewadi (near to Pune) is cultivating this species in its garden. It has a good collection of this plant, about 500 individuals multiplied through seeds and vegetative parts. Outside Maharashtra State, the germplasm of this species is also conserved in the Indian Botanic Garden, Howrah. It is likely that the plant is also cultivated in leading botanical gardens in the world. It has certainly been found in cultivation between 1971-1980 in the greenhouse of the Matthaei Botanical Garden of the University of Michigan, Ann Arbor, USA (personal communication from Prof. P. Dayanandan).
Ongoing In aitu Conservation The International Union for Conservation of Nature and Natural Resources (IUCN) has enlisted this species as one of the twelve most endangered plant species on earth which draws an immediate urgency for the conservation of this plant species in its natural habitat. Further, the Government of India has placed this plant species in the Negative list of Export, which bans all its trades and exports from wild collections. However, there is no effective in situ conservation measure taken so far to conserve this plant species in its natural habitat. Hence, we are suggesting the following conservation measures based on our studies. 1. Since this species is endemic to Maharashtra State especially to four neighboring Districts of Maharashtra, the responsibility of conserving this species in wild should be given to the concerned District Forest Officers under whose jurisdiction the habitat falls.
Studies on Frerea indica Dalz.: A Critically Endangered and Endemic Species
203
2. Local people are the custodians of the natural resources, without their help and co-operation, it is impossible to conserve any plant in the wild. Therefore, an awareness among the local people who live near the natural habitat of F. indica to be created about the rarity of this species and its danger of extinction in the wild, thereby stressing a vital need for conserving this species in the wild by distributing pamphlets or notices in their vernacular language. 3. Maharashtra State Forest Department should take necessary steps to cultivate this plant in a large scale and supply sapling or seeds of this plant to the local people at free of cost for cultivation in their home gardens/ agricultural lands to meet out their food and medicinal uses, thereby wild population could be conserved. 4. The Maharashtra State Forest Department should also take necessary steps to supply sapling/seeds of this plant to the traders/exporters etc. at a subsidized rate thereby encouraging them to cultivate and multiply them on a large scale for commercial exploitation.
Conclusion The plant Frerea indica has moved from Rare category to Most Endangered category in the last two decades, if the casual factors/major threats are not checked forthwith, it will lead to the extinction in the wild. The major threats that need to be checked immediately are animal grazing and exploitation by local people for food and medicine, apart from natural threats like pests, ground fire and landslides. The effective implementation of the conservation measures suggested in this paper would have this species from becoming extinct in the wild. The pharmacognostic study of this species is under investigation in our lab. Further, it is suggested that the plant should be subjected to pharmacological and other related studies so as to ascertain the therapeutic efficacy of this plant.
Acknowledgements The authors wish to record their sincere thanks to Dr. M. Sanjappa, Director, Botanical Survey of India, Kolkata for providing necessary facilities and encouragement. The authors are grateful to Dr. P. Dayanandan, Former Professor and Head, Department of Botany, Madras Christian College, Chennai for his critical comments, valuable suggestions and necessary corrections.
References Ahmedullah, M. and Nayar, M.P., 1986. Endemic Plants of the Indian Region. Botanical Survey of India, Calcutta, Vol. I, pp. 23-24 and 127. Almeida, M.R., 2001. Flora ofMaharashtra. Blatter Herbarium, Mumbai, pp. 241-242. Cooke, T., 1958 (Reprinted). The Flora of the Presidency of Bombay,. Botanical Survey of India, Calcutta, Vol. 11, pp. 243. Dalzell, N .A., 1864. A New Genus of Asdepiadaceae. J. Proc. Linn. Soc. Bot.,8: 10-11.
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Hooker, JD., 1883. The Flora of British India. L. Reeve and Co., London, Vol. IV, pp. 76. Jagtap, AP., 1992. Revision of Family Asclepiadaceae of India. Ph.D. Thesis, University ofPoona. Jagtap, AP. and Singh, N.P., 1999. Fasicles ofFlora of India. Botanical Survey of India, Kolkata, Vol. 24, pp. 243-244. Jain, S.K. and Sas try, AR.K., 1980. Threatened Plants ofIndia: A State of the Art Report. Botanical Survey of India, Calcutta and Man and Biosphere Committee, D.S.T., New Delhi, pp. 41. Kothari, M.J. and Moorthy, S., 1993. Flora ofRaigad District, Maharashtra State. Botanical Survey of India, Kolkata, pp. 232-233. Mishra, D.K. and Singh, N.P., 2001. Endemic and Threatened Flowering Plants of Maharashtra. Botanical Survey of India, Calcutta, pp. 154-157. Nayar, M.P. and Sastry, AR.K., 1987. Red Data Book ofIndian Plants. Botanical Survey of India, Calcutta, Vol. I, pp. 72-73. Tetali, P., Tetali, S., Kulkarni, D.K. and Kumbojkar, M.S., 1997a. Studies on the Status and conservation of Frerea indica Dalz. J. Bombay Nat. Hist. Soc., 94: 115-121. Tetali, P., Tetali, S., Kulkami, D.K., Kumbojkar, M.S. and Sujatha Tetali, 1997b. Association of Frerea indica Dalz., an endangered plant species with Euphorbia neriifolia L. and its importance in habitat conservation. Curr. Sci., 73(7): 563-65.
Chapter 17
Conservation and Utilization of Mangrove Forests: A Case Study in Bhitarkanika Sanctuary, East Coast of India C. Pattanaikl, Ch. Sudhakar Reddyl, N.K. DhaP and Rashmita Dash} IForestry and Ecology Division, NRSA, Hyderabad 2Regional Research Laboratory (CSIR), Bhubaneswar 3Botany Department, Berhampur University, Berhampur
ABSTRACT Bhitarkanika in the east coast of India has got a special significance as it harbours an important manghal formation in the Indian sub-continent. From the floristic studies, it is evident that, this region was very rich in plant diverSity in the remote past. Nearly 75 mangrove species and its associates are found in this region. Among these, many plants are of certain medicinal properties and socioeconomic values. One lakh inhabitants of 300 villages are living close to this mangrove ecosystem. In course of time, the flora of this region has been depleting at an alarming rate due to adverse impacts of human development activities tied with natural processes such as industrial sewerage pollution, reclamation, fresh water diversion for agriculture, extraction of timber, other forest products and conversion of mangrove forests for agricultural purposes. Most of the mangrove forests are in the verge of denudation. People in surrounding areas utilized these plants for various purposes. In this region, plants having medicinal and economic
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values are at a great degree of threat. As a result, there is an urgent need for the conservation of these mangrove ecosystems. This can only be achieved by the combined efforts of individuals, community participation and effective government policies. The present paper deals with the distribution and utilization of 45 mangrove plants and its associates from Bhitarkanika Sanctuary area, their present status, strategies for conservation and steps for sustainable management.
Introduction Bhitarakanika is a potential site of various plants of medicinal and other socioeconomic importance. The vegetation of this region has got a special significance as it constitutes an important manghal formation on the Indian sub-continent. Bhitarkanika is located in Kendrapara district of coastal state of Orissa on eastern India. It is one of tht: largest and vibrant mangrove ecosystems in the country. It is situated between 20°04' to 20° 08' N latitudes and 86° 45' to 87" 50' E longitudes in the east coast of India covering an area of 625 sq km. It is bounded on the east by the Bay of Bengal, on the west by villages of Kendrapara district of Orissa, on the north by the Baitarani and the Dhamra rivers and on the south by the Mahanadi estuary. The Government of Orissa declared this area as a sanctuary in 1975 for better protection of the habitat. The temperature ranges from 21°C-40°C. The annual rainfall is 141.15 cm. The soil types are sandy on the fringes of the coast and alluvial silty along the river mouth. This congenial ecological factor provides ample scope for an important manghal formation in this region. Among the mangroves many plants have medicinal and other socio-economic importance.
Past Floristic studies From the past floristic studies conducted by number of workers such as Haines (1921-25), Mooney (1925), Raoand Banarjee (1967, 1982),Panigrahi(1964),Choudhury et al. (1991), Subudhi et al (1992), Saxena and Brahmam (1996) etc., it is evident that the flora of this region was much rich in remote past. The floral diversity of Bhitarkanika includes a total of more than 300 plant species (Banerjee, 1984). It includes both mangroves and non-mangroves belonging to 80 families (209 plants belonging to 62 dicotyledon families and 93 plants belonging to 18 monocotyledon families). However, in course of time; the floristic elements including those of the mangroves have been depleted at an alarming rate'due to operation of various biotic factors which include a lot of prawn cultures, settlements of immigrants, conversion of mangrove forests into cultivated fields, fresh water diversion for agriculture etc. As a result of this, the present vegetation is extinct in most denuded condition on different areas of the Bhitarakanika sanctuary.
Ongoing Project Keeping in view of the various socio-economic importance of the mangroves and other floristic elements, intensive floristic studies have been conducted in different areas of the Bhitarkanika sanctuary in order to identify the plants of medicinal, ecological and other socio-economic importance and to devise possible strategies of their conservation. This study is in connection with a project entitled "Biodiversity
bl
Table 17.1: Enumeration of Mangrove Plants with Medicinal and Economic Uses SI. No.
Species Name
1.
Acanthus ilicifolius L.
2.
Acrostichum aureum L.
Family
Local Name
Locality
Acanthaceae
Harakancha
All over sanctuary
Acrostichaceae
Kharkhari
Dangmal PF
;:t
MedicinaVEconomic Uses
~
~....
C·
Dyspepsia, Rheumatism
;:t
Applied on wounds and boils
~
;:t
l:l..
3.
Aegialites rotundifolia Roxb.
Myrsinaceae
Kharsi
4.
Aegiceras comiculatus (L.) Blanco
Myrsinaceae
Banarua
Bhltarkanika RF
5.
Aglaia cucullata (Roxb.) Pellagrin
Meliaceae
Ooanra
Mahisamada creek
Construction purpose
c:: .... ::::.: §. .... C·
6.
Avicennia alba Blume
Avicenniaceae
Kalabani
along Maipura river
Fodder, Fuel wood
~
Bhitarkanika RF
Wood for construction Firewood, honey collection
;:t
7.
Avicennia marina (Forssk.) Vierh
Avicenniaceae
Singlabani
along Maipura river
8.
Avicennia officinalis L.
Avicenniaceae
Dhalabani
Bhitarkanika RF
Aphrodisiac, Astringent
9.
Bruguiera cylindrica (L.) Blume
Rhizophoraceae
Kaliachua
Hetamundia FB
Timber, Fuel, Tannin
10. Bruguiera gymnorhiza (L.) Savigny
Rhizophoraceae
Bandari
Kansaridiha
~ ~
Fodder, Fuel wood
;:t
11. Bruguiera parviffora (Roxb.) W. & A.
Rhizophoraceae
Dot
Kalibhanjadia RF
Timber, Fuel
12. Bruguiera sexangula (Lour.) Poir.
Rhizophoraceae
Bandari
Bhltarkanika RF
Timber, Fuel, Tannin
13. Caesalpinia bonduc (L.) Roxb.
Caesalpiniaceae
Nentei
Common in sanry
Stone bladder, Eye disease
14. Caesalpinia crista L.
Caesalpiniaceae
Garani
Hukitola
Tannin extracted from bark
Apocynaceae
Garani
Satabhaya PF
15. Cerbera odoIlam Gaertn.
~
2;l
....
Fodder,Building fishing boat
~
Yr
~
~
en .... ~
Haemorrhage, Ulcer
~ 3' ~
16. Ceriops decandra (Griff.) Ding Hou
Rhizophoraceae
Chiani
Dangmal RF
17. Ceriops tagal (Perr.) Robins
Rhizophoraceae
Singda
Bhltarkanika RF
Piles, Purgative
18. Clerodendrum inerme (L.) Gaertn.
Verbenaceae
Kalakatira nai
Bhitarkanika RF
Fibre extracted from stem
t
19. Cynometra iripa Kostel
Leguminosae
Guan
Sunirupi RF
Epilepsy, Ulcer
~
20.
Darris trifoliata Lour.
Papilionaceae
Khasai lata
Dangmal PF
Asthma
;:t
21.
Excoecaria agallocha L.
Euphorbiaceae
Badasundari
Kalibhanjadia RF
Asclepiadaceae
Dhalasundari
Dangmal PF
22. Finalaysonia obovata Wall.
Rheumatic pain, Veneral infection
B= ;:t
~
Timber, For boat building
'...."'
Timber, House constuction
«
~
Contd...
N 0 'I
N 0
Table 17.1-C ontd...
00
Medicinal/Economic Uses
Family
Local Name
Locality
Sterculiaceae
Bania
Bhitarkanika RF
Sterculiaceae
Maasitha
Dangmal PF
Timber, House constuction
Malvace ae
Sinduka
Bhitarkanika RF
Diabetes, Fodder, Firewood
Leguminosae
Churund a
Bhitarkanika RF
Herpes, Itches
Rhizophoraceae
Nalia ghasa
All over sanctuary
Combre taceae
Nypa palm
Dangmal PF
Thatching, Fruit edible
Poaceae
Hental
Sunirupi RF
Fruit edible
Arecace ae
Karanja
All over sanctuary
Arecace ae
Rai
Kansaridiha
Papilionaceae
Rai
Bhitarkanika RF
Rhizophora apiculata Blume 34. Rhizophora mucronata Poir. 35. Rhizophora stylosa Griff.
Rhizophoraceae
Rai
Kansaridiha
Firewood, Fodder, Tannin
Rhizophoraceae
Batula
KhoIa creek
Itch, Emmenogogue
Rhizophoraceae
Orua
Ekakula island
Fodder, Timber
36. salicomia brachiata Roxb. 37. sonneratia alba J.Smith
Chenopodiaceae
Koruan
Bhitarkanika RF
Fodder, Timber
Orua
Khola creek
SI. No.
Species Name
23. Herietiera tomes Buch. Ham 24. Herietiera fittoralis Dryand 25.
Hibiscus tifiaceus L.
26.
Instia bquga (Colebr.) O.Kuntze
27. Kandelia candel (L.) Druce 28. Lumintzera racemosa Willd. 29. Myriostachya wightiana Nees ex Steud
30. Nypa truticans (Thunb.) Wurmb. 31.
Phoenix paludosa Roxb.
32.
Pongamia pinnata (L.) Pierre
33.
Sonneratiaceae
Earache, Wash ulcer and wound
Fodder
Bronchitis, Whooping cough Fodder, Firewood Astingent, Diabetes
t"T1
;!
'"
Fodder, Timber, Vegetable
~
Skin disease, Dysentery
Oq
5'
Sonneratiaceae
Jagula
Satabhaya RF
Sonneratiaceae
Kathabadam
Fringe areas
Habali
Scabies, Gonorrhoea
I:l...
Tamaric aceae
Kalibhanjadia RF
Combre taceae
Sisumar
Bhitarkanika RF
Timber
t:x:I
Malvaceae
Pitamari
Bhitarkanika RF
Tannin, Furniture making
43. Xylocarpus granatum Koeing. 44. Xylocarpus mekO[lgensis Pierre
Meliaceae
Pitakorua
Dangmal PF
Cholera, Diarrhoea, Fever
Meliaceae
Gilo
Gokhani creek
Jaundice, Anthelmintic prop
(.f)
Meliaceae
;:$
45. Xylocarpus mofuccensis (Lamk.)
Bark is Purgative
38. sonneratia spatala Buch. Ham 39. sonneratia caseolaris (L.) Engler 40. 41. 42.
Tamarix troupii Hole Terminalia catappa L. Thespesia populnea (L.) Sol.ex.Corr.
Pani amba
Khola creek
Scabies, Rheumatism, Skin diseases
~
;:$
'"5'
I
;:;. la.
.., .., '"'"
;;;-
Conservation and Utilization of Mangrove Forests: A Case Study in Bhitarkanika Sanctuary
209
Characterization at landscape level of east coast of India using RS and GIS" sponsored by Department of Biotechnology and Department of Space (DBT-DOS). During the present investigation many plants were collected. Out of these, some valuable and interesting plants are presented. In the present paper, an exhaustive idea has been given about the distribution and utilization of 45 mangrove speci~s which have various medicinal and socioeconomic importance. A comprehensive idea about the species name, family, local name, locality where it found and various uses of the species are given in Table 17.1.
Strategies for Conservation Conservation and judicious utilization of this valuable wealth is an urgent need of the hour. Many economic plants belonging to either mangroves or their associates have become threatened due to over exploitation and clearing of forests for industrialization, urbanization, pisciculture, human settlements etc. Conservation of the present vegetation and natural regeneration of the species having medicine and other socio-economic importance should be done on top priority. Some of the species are Rhizophora apiculata, R. mucronata, Excoecaria agallocha, Kandelia candel should be planted extensively in the estuarine belt to restore the past vegetation. Steps should be taken by the state government as well as central government for the in-situ conservation of the existing vegetation. Extensive plantations of mangrove plants which have economic importance should also be done by the active participation of the voluntary agencies, medicinal plant growers and the local people. Mass propagation of threatened and other species which are better adopted in this region should be done extensive plantations and for sustainable utilization of vegetable wealth of this region. The conservation of mangrove habitats and species in the sense of direct science based management is increasingly described as protection. Conservation now means more besides protection, sustainable use and sharing the benefits of use. All the measures suggested above should help the state to protect whatever mangroves we still have and restock degraded mangroves, besides helping to regenerate the potential mangrove areas. This will enable to protect the human population from natural calamities, besides improving the economic conditions of the people depending on mangroves.
Acknowledgement The authors are grateful to Dr. R. R. Navalgund, Director, National Remote Sensing Agency and Dr. P. S. Roy, Deputy Director (RS and GIS, Application Area), NRSA for their valuable suggestions and encouragement. We are also thankful to Regional Research Laboratory (C.S.I.R.) for verifying herbarium specimens. The help received from colleagues of Forestry and Ecology Division, NRSA are thankfully acknowledged.
References Banerjee, L.K., 1984. Vegetation of the Bhitarkanika Sanctuary in Cuttack district, Orissa, India, J. Econ. Tax. Bot., 5(5): 1065-1079.
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Emerging Trends in Biological Sciences
Banerjee, L.K and Rao, T.A., 1990. Mangroves OfOriS5a Coast and their Ecology. Bishen Singh and Mahendrapal Singh, Dehradun. Biswal, A.K and Choudhury, B.P., 1993. Bhitarkanika wildlife sanctuary: Exploitation and management. Neo Botanica, (1 and 2): 17-22. Chadha, S.K and Kar, e.S., 1999. Bhitarkanika: Myth and Reality. Natraj Publishers, Dehradun. Champion, H.G. and Seth, S.K, 1968. Revised Survey ofForest Types in India. Government of India, Press Publication, New Delhi. Chop ra, RN., Nayar, S.L. and Chop ra, I.e., 1956. Glossary of Indian Medicinal Plants, e.S.I.R, New Delhi. Choudhury, B.P., 1990. Bhitarkanika: Mangroveswamps.J. Env. Sci.,3(1}: 1-16. Choudhury, B.P., Bisawl, A.K and Rath, S.P., 1995. Biodiversity in the Bhitarkanika wildlife sanctuary in the state of Orissa. In: Environment Change and Management, (Ed.) Re. Mohanty. Kamal Raj Publication, New Delhi. Choudhury, B.P., 1987. Socio-economic aspect of Bhitarkanika Wildlife Sanctuary in the state of Orissa. Orissa Review, 43(9}: 45-47. Choudhury, B.P., Biswal, A.K and Subudhi, H.N., 1991. Mangroves of Orissa and aspects of their conservation. Rheedea, 1(1 and 2}: 62-67. Das, H.S., Panda, P.e. and Patnaik, S.N., 1996. Traditional uses of wetland plants of eastern Orissa. J. Econ. Tax. Bot. Additional Series, 12: 306-313. Gamble, J.5. and Fischer, e.E.e., 1967. Flora of Presidency ofMadras, Vols. I-Ill (Repr. Edn.). Botanical Survey of India, Kolkata. Haines, H.H., 1926. The Botany ofBihar and Orissa. London. Kartikar, KR and Basu, B.D., 1935. Indian Medicinal Plants, Vols. 1-4. Dheradun (Rep. Edn. 1975). Mishra, M.K., Dash, 5.5. and Das, P.K, 1999. Additions to the flora of Orissa. Rheedia, 9: 163-172. Mooney, H.F., 1950. Supplement to the Botany ofBihar and Orissa, Ranchi. Patnaik, S.K, Kar, e.S. and Kar, S.K, 2000. Biodiversity in tidal swamps with special references to Bhitarkanika. In: Mahanadi Delta Geology: Resources and Biodiversity, (Ed.) N.K Mahalik, pp. 139-153. Saxena, H.O. and Brahmam, M., 1996. The Flora of Orissa, Vols. I-IV. Orissa Forest Development Corporation, Bhubaneswar. Subudhi, H.N., Choudhury, B.P. and Acharya, B.e., 1992. Potential medicinal plants from Mahanadi delta in the state of Orissa. J. Econ. Tax. Bot., 16: 479-487
Chapter 18
Adventitious Shoot Bud Regeneration and Organogenesis in Hypocotyl Cultures of Acacia concinna DC.: An Important Tree Legume P. Sairam Reddy,2, G. Prasad Babu1, N. Yasodamma and G. Rama Gopal1 IDepartment of Botany, S. V. University, Tirupati - 517 502 4th Floor, Varun Towers, Begumpet, Hyderabad - 16
2J.K. Agrigenetics Ltd.,
ABSTRACT Efficient protocols have been developed to induce callus-mediated organogenesis and adventitious shoot regeneration in hypocotyl cultures of Acacia concinna. Hypocotyl segments cultured on MS (Murashige and Skoog, 1962) basal medium containing 26.85 mM a-naphthalene acetic acid (NAA) and 2.22 mM 6benzylaminopurine (BA) induced green friable callus, which further showed efficient shoot bud induction (76 per cent) on MS basal medium containing 22.2 mM BA and 0.57 mM indole-3-acetic acid (IAA). Addition of 0.05 per cent activated charcoal (AC) to the medium promoted the shoot bud regeneration (88 per cent) from callus. Adventitious shoot induction was achieved when hypocotyl segments of one-week old seedlings were cultured on MS basal medium containing 8.9 mM BA and 0.54 mM NAA. Addition of 10 per cent tender coconut milk (TCM) improved the regeneration efficiency. Agar concentration in the media showed a
212
Emerging Trends in Biological Sciences significant effect on adventitious shoot regeneration. Regenerated shoots rooted profusely on 1h strength MS basal medium devoid of growth regula tors. Plantlets were successfully acclimatized to the green house conditions.
Keywords: Acacia concinna, Hypocotyl cultures, Shoot bud regeneration. Abbreviations: 2, 4-D-2, 4-dichlorophenoxy acetic acid; IAA-Indole-3-acetic acid; NAA-anaphthalene acetic acid; B-6-benzylaminopurine; TCM- Tender Coconut Milk
Introduction Acacia concinna DC., commonly known as Soap acacia or 'Shikai' belongs to the family Leguminosae. It occurs in tropical forests of India and Myanmar. Pods of this plant are known to promote the growth of the hair and the anti-dandruff effect has also been well-documented (Sastri, 1956; George, 1972). The pods are being extensively used in various commercial herbal shampoo and detergents. It is used for skin diseases in the form of ointment. Bark contains lupeol, hexacosamol, a-spinasterol, a-soinpsterone and an amorphous saponin. Insecticidal activity was reported in bark (Pandey et al., 1982) and the bark extract is used as a remedy for leprosy (Asholkar et ai., 1992). The increasing demand for A. concinna as a valuable forest product made the forest authorities to choose it as an important plant for afforestation programme. However, pest problem like inflorescence galls (Reddy and Rama Gopal, 1998); inadequate conservation steps and natural calamities like forest fires contribute greatly to the depletion of this economically useful forest legume. Although, vegetative propagation through stem cuttings has been reported (Reddy et al., 1998a), the limitations in the protocol may aggravate the mass production of propagules for plantation. Hence, the desire to seek better methods for the mass multiplication of A. concinna has been intensified. Tissue culture has been suggested as an effective surrogate method for the rapid propagation of the tree species (Anand 1984; Bajaj et al., 1988). The recalcitrant behaviour is found as a limitation for the in vitro regeneration of tree legumes. However, the success has been obtained in inducing organogenesis and somatic embryogenesis in tree legumes (Ahee and Duhox, 1994; Garg et al., 1996; Babbar et al., 1996; Xie and Hong,2001a; Xie and Hong,2001b). For the first time, we report here an efficient protocol for the induction of direct and indirect shoot organogenesis, and whole plant regeneration from the hypocotyl segments of A. concinna seedlings.
Materials and Methods Plant Material Seeds collected from mature dry pods were treated with 98 per cent (v Iv) H 2S04 for 2-3 min, washed with distilled water for five times and then rinsed with 2 per cent (v Iv) detergent (Teepol) for 5 min. The scarified seeds were surface sterilized with 0.1 per cent (w Iv) aqueous solution of HgC~ for 8 min followed by 4-5 rinses in sterilized
Adventitious Shoot Bud Regeneration and Organogenesis in Hypocotyl Cultures
213
DDHp. The treated seeds were aseptically germinated in conical flasks (250 ml) containing 50 ml of water agar medium (2 per cent sucrose + 0.7 per cent agar). Hypocotyl segments, cotyledons and leaflets were collected from one-week old aseptic seedlings and used in further studies after excising them into small pieces.
Culture Media and Incubation Conditions The basal medium used in the present study consisted of the salts and vitamins of MS medium (Murashige and Skoog, 1962). Unless specified, all media were fortified with 3 per cent sucrose (w Iv) and 0.8 per cent agar (w Iv; Qualigen, India). The pH of the medium was adjusted to 5.7 prior to the addition of agar. Molten medium (15 ml) was dispensed into 25x150 mm glass tubes (Borosil, India), plugged with nonabsorbent cotton wrapped in a layer of cheese-cloth and autoclaved at 121°C for 15 min. All cultures were incubated at 25°C ± 2 °C and under 16 h light/8 h dark photoperiod and with light intensity of 50 mE m-2 S-2 provided by cool-white fluorescent lamps.
Callus Induction Hypocotyl and root segments 5 mm long, and cotyledons cut into 4 x 4 mm were used as explants. The pinnate leaves were excised and minute incisions were made on leaflets using a sharp scalpel. Leaflets were cultured facing the abaxial side towards the medium. They were inoculated on MS medium containing auxins like 2,4-0 (9.05,22.62 mM) or NAA (10.74,26.85 mM) alone, or in combination with cytokinins like kinetin (2.23, 4.62, 9.3, 23.2 mM) or BA (2.22, 4.45, 8.9 mM). The callus was subcultured in fresh medium after every 30 days of interval. In each experiment 60 explants were cultured and all the experiments were repeated thrice.
Organogenesis Two sets of experiments were conducted on shoot differentiation from callus. In the first set of experiments, the green nodular callus was cut into small pieces and cultured on MS medium containing different concentrations of various BA (8.9, 22.2 mM) or TDZ (2.27,4.55 mM) or kinetin (9.3, 23.2 mM) in combination with NAA (0.54 mM) or !AA (0.57 mM). In the second set of experiments, the effect of growth adjuvants like TCM (10 per cent, v Iv), CH (0.05 per cent, w Iv) and AC (0.05 per cent, w Iv) was tested, respectively, o~ shoot differentiation during optimal growth regulator treatment. MS basal medium lacking growth adjuvants served as control for both the experiments. In each experiment 60 calli (0.3 x 0.3 cm) were cultured and all tests repeated thrice. Data like the percentage of calli inducing shoots, the mean number of the shoots per callus and the shoot length were recorded after 8 weeks of culture.
Direct Shoot Regeneration Hypocotyl segments measuring 5-10 mm length were dissected from one week old aseptic seedlings and inoculated horizontally as well as vertically on MS medium supplemented with BA (2.22,4.4,8.9,22.2 mM) and NAA (0.54, 1.07 mM). In the next stage of experiments growth adjuvants like AC (0.05 per cent, w Iv) and TCM (10 per cent, v Iv) were added to the MS basal medium containing optimal concentrations of growth regulators for shoot bud regeneration. The concentration of
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agar (O.S per cent, 0.4 per cent and no agar) in the medium was also altered to study its role on shoot organogenesis. In each experiment 60 explants were cultured and all the experiments repeated thrice.
Rooting and Hardening The regenerated shoots containing 2-4 pinnate leaves with 3-5cm height were separated from the callus by cutting them with a sharp razor. Single shoots were transferred to full-strength, half-strength and quarter-strength MS basal medium with and without the addition of auxins (IAA 0.57-2S.54 mM, NAA 0.54-26.S5 mM). In each experiment 30 explants were cultured and all the experiments were repeated thrice. The percentage of rooted shoots, number of roots per shoot and the root length were recorded after 4 weeks of culture. In order to acclimatize, rooted plants were removed from the rooting medium and transferred to cultured tubes containing sterile distilled water. After 10 days, plantlets were transferred to plastic pots containing sterile vermiculite covered with plastic bags and kept under the culture room conditions for 15 days. Plants with newly formed leaves were then transferred to the field and kept under shade for about a week before planting in the soil.
Histological Studies Organogenic callus and regenerating hypocotyl segments were cut into small pieces (0.5cm2) and fixed in FAA (formaldehyde: acetic acid: ethanol; 5:5:90; v Iv Iv) for 24h. The tissue was dehydrated by passing through the ethanol series (20, 30, 50, 70 and 100 per cent) for 5 min at each stage. The tissue was embedded in paraplast (mp. 5S°C) and sectioned at 6-Sm thickness on a rotary microtome. The sections were placed on clean microscopic glass slides coated with egg albumin and stained with safranin after bringing them to the aqueous media by passing through the ethanol series in a reverse direction. Sections were mounted with DPX (BDH) and viewed under inverted light microscope (Nicon).
Results and Discussion Callus Formation Hypocotyl, cotyledon, leaf and root explants produced callus after 4 weeks of inoculation on MS basal medium containing all combinations of growth regulators tested. However, the frequency of callus formation varied among explants and media tested (Data not shown). The highest frequency of callus formation (SS.4 per cent) was noticed from hypocotyl explants when cultured on media containing BA and NAA. The combination ofNAA (10.74 mM) and BA (2.22 mM) induced the clusters of compact green nodular structures in callus after 6 weeks of inoculation (Figure lS.lA). The same medium did not induce any shoot formation. However, callus produced on 2,4-D (9.05-22.62 mM) alone or in combination with BA (2.22-S.9 mM) or kinetin (2.23-23.2 mM) and also the callus formed on either NAA (10.74-26.S5 mM) alone or in combination with kinetin (2.23-23.2 mM) induced neither nodular structures nor shoot formation. Formation of regenerative callus from various explants on MS basal medium containing higher concentrations of NAA (10.74 mM) and lower concentrations of BA (O.S9mM) has been reported in the same family member Prosopis
215
Adventitious Shoot Bud Regeneration and O rganogenesis in Hypocotyl Cultures
(A)
(B)
.
.. "
V
...
).
........ • T.....
I
(C)
(D)
Figure 18.1 : Acacia concinna Regeneration through Organogenesis from Callus (A) Formation of green nodular callus cluster from hypocotyl derived callus on MS medium containing 10.74 mM NAA and 2.22 mM BA (bar 0.38cm); (B-C) Leafy shoot formation from green callus cluster in presence of 8.9 mM BA and 0.54 mM NAA (B. bar 0.37cm; C. bar 0.9cm); (D) Histological section of shoot bud formed from green nodule derived from hypocotyl callus (xl 00) (LP = Leaf primordia, AM = apical meristem); (E) Histological section of adventitious shoot bud regeneration from hypocotyl segments (x20) (SB shoot bud, VB vascular bundle, C callus).
=
=
=
=
=
=
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tamarugo (Nandwani and Ramawat, 1992). The efficiency of nodule formation from the calli obtained from hypocotyl explants were significantly higher (p<0.05) than in calli produced from cotyledon, leaf and root (Table 18.1). Similar variations in regeneration efficiency, among different explants were reported in A. mangium (Xie and Hong, 2001a) and Eucalyptus grandis x E. urophylla (Cid et al., 1999). Table 18.1: Nodule Formation from Callus Derived from Various Explants of A. concinna on MS Medium Supplemented with NAA (10.74 mM) and BA (2.22 mM). Observations were taken after 6 weeks of subculture. Source of Cal/us
Frequency of Shoot Regeneration (per cent)
Mean No. of Nodules/Explant
Hypocotyl
62.1 ± 3.S"
4.2 ± 0.3"
Cotyledon
2.1 ±0.1b
Root
14.3 ± O.Sb 12.3 ± 0.6b
Leaf
12.1 ± 2.Sb
1.0 ± 0.3e
1.2 ± 0.3e
Values represented above are the average of 3 independent experiments (each with 60 hypocotyl segments). Mean values followed by the same letter are not significantly different from each other at p:=O.OS.
Shoot-bud Induction from Callus The green nodular structures further showed shoot morphogenesis (Figures 18.1 B, C) upon transferring onto MS medium containing lower concentration of NAA (0.54 mM) or !AA (0.57 mM) and higher concentrations of BA (8.9 mM) or kinetin (9.3-23.2 mM) or TDZ (2.27-4.55 mM). However, the significant variation in shoot morphogenesis was noticed, depending on the type of cytokinin present in the medium. Among all the concentrations and combinations of growth regulators tested, BA (8.9 mM) in combination with IAA (0.57 mM) showed the highest frequency (76.3 per cent) of shoot morphogenesis (Table 19.2). Presence of lAA in medium promoted the frequency of shoot bud formation, whereas, NAA produced elongated shoots. The synergistic effect of BA with IAA or NAA on shoot morphogenesis has been well documented (Rout et al., 1999, Caboniet al., 2000). The organogenic efficiency of BA has been reported in callus cultures of A. auriculiformis (Rao and Prasad, 1991). Kinetin (9.3-23.2 mM) in combination with either NAA (O.57mM) or !AA (0.54 mM) showed comparatively poor morphogenic efficiency. Histological studies showed the evidence of shoot bud and leaf primordial development from callus (Figure 18.10). Rout et al. (1995) reported the callus mediated shoot regeneration via somatic embryogenesis inA. catechu on MS medium containing kinetin and NAA. However, in the present study kinetin in combination with NAA did not induce somatic embryogenesis in A. concinna. Callus cultured on TDZ (2.27-4.55 mM) alone or in combination with NAA or IAA produced malformed shoots and they never elongated more than 2cm length. Similar effect of TDZ on shoot regeneration was reported in Petasites hybridus (Wildi et al., 1998). In contrast with the recommendations of using lower concentrations of TDZ (0.01-0.1 mM) for woody plant regeneration (Lu, 1993), in the present study, lower concentrations of TDZ
217
Adventitious Shoot Bud Regeneration and Organogenesis in Hypocotyl Cultures
(0.01-1.48 mM) failed to produce either nodules or shoot buds (Data not shown). Xie and Hong (2001a) showed efficient shoot regeneration in callus cultures of A. mangium on MS medium containing higher concentrations of TDZ (4.55 mM). However, BA showed 2 folds higher shoot morphogenesis than TDZ or kinetin in the present study. The differential response of the different species in the same genus to various growth regulators may be due to the variations in their genetic background. Table 19.2: Effect of Various Combinations of PGR in MS Medium on Indirect Shoot Regeneration from Hypocotyl Callus of A. concinna. Observations were documented after 8 weeks of culture. Percentage of Shoot Regeneration (per cent)
Mean No. of Shoots/ explant
62.1 ± 3.5b
4.2 ±0.3°
4.7±0.1"
0.57
76.3 ± 1.0" 52.6 ± 2.6d
8.7 ±0.1" 3.5 ±0.5d
3.4 ±0.3b
0.57
59.5 ± 2.5e
7.9 ±0.2b
2.8 ± 0.2e
0
0
0
PGR (pM)
BA
Kin
TDZ
NAA
IAA
0.54
8.9 8.9
0.54
22.2 22.2 9.3
0.54
9.3 23.2 2.27
11.3 ± 2.2'
1.0 ±O.og
1.0 ± 0.0'
0.57
23.8 ± 2.40 30.7± 1.90
3.4 ±0.8° 3.6 ±0.3d
3.2 ± 0.5e 2.9 ±0.3e
0 28.5 ±2.6e 28.2 ± 3.80
0
0
0.57
2.8 ± 1.2'
1.5 ±0.5e
2.6 ±0.4'
1.3 ± 0.5'
0.57
57.5 ± 3.3e
3.7 ± 1.3d
1.8±0.1d
0.54
2.27 4.55 4.55
2.2 ±0.2d
0.57 0.54
23.2
Mean Length of the Shoot (cm)
0.54
Values represented above are the average of 3 independent experiments (each with 60 hypocotyl segments). Mean values followed by the same letter are not significantly different from each other at
.0=0.05.
Various growth adjuvants were incorporated in regeneration media to improve the morphogenic efficiency of the callus derived from hypocotyl explants. Addition of AC (0.05 per cent) to the medium containing 8.9 mM BA and 0.57 mM IAA hiked the regeneration frequency (88.4 per cent), whereas TCM and CH could not improve the shoot regeneration (Figure 18.2). However, presence of TCM in medium showed a significant (p<0.05) enhancement in shoot elongation rather than the regeneration frequency. Quoirin et al. (2001) showed the enhancement of shoot multiplication in nodal cultures of A. mearnsii, when 0.2 per cent AC was supplemented to MS basal medium. In contrast with the present study, CH and TCM showed positive effect on shoot regeneration in other species like Carthamus tinctorius (Nikam and Shitole, 1999) and Acacia auriculiformis (Mittal et al., 1989). However, Barrettet al. (1997) tested the various nitrogen sources including CH in regeneration media, and found no improvement on somatic embryo differentiation in white spruce.
Emerging Trends in Biological Sciences
218
100~----------------------~==============~
ID ControlllllAcmTCM EaCH
80
11
I
60 40
20
% Shoot Regeneration
No. ofShoots
Shoot Length (cm)
Figure 18.2: Effect of Growth Adjuvants Activated Charcoal 0.05 per cent (AC), Tender Coconut Milk 10 per cent (TCM), Casein Hydrolysate 0.025 per cent (CH) in MS Medium with 8.9 mM BA + 0.57 mM IAA on Shoot Morphogenesis from Hypocotyl Derived Callus of A. conclnna. Observations were taken after 8 weeks of culture. Table 18.3: Effect of Orientation of the Explant on Direct Shoot Regeneration from Hypocotyl Segments of A. conclnna, on Agar Gelled MS Medium Supplemented with 8.9 mM BA + 0.54 mM NAA. Observations were documented after 8 weeks of culture. Orientation of the Explant
Percentage of Shoot Regeneration (%)
No. of Shoots per Explant
Length of the Shoot (cm)
Vertical
73.6 ± 1.5
1.4 ± 0.2
2.4 ±0.2
Horizontal
56.5 ± 1.8
3.6 ± 0.1
4.8 ± 0.1
Values represented above are the mean of 60 replicates ± standard error.
Table 18.4: Effect of Agar Concentration on Direct Shoot Regeneration from Hypocotyl Segments of A. concinna, Inoculated Horizontally on MS Medium Supplemented with 8.9 mM BA + 0.54 mM NAA. Observations were documented after 8 weeks of culture. Agar Strength
Percentage of Shoot Regeneration (%)
Mean No. of ShootlExplant
Mean length of the Shoot (cm)
56.5 ± 1.8"
3.6 ± 0.1"
4.8 ±0.1"
Semi-solid (0.4 per cent)
43.8 ±
3.6 b
1.6 ± 0.8"
1.8 ±0.8b
Liquid (without agar)
68.6 ±2.9"
2.5±0.~
4.5 ±0.3a
Solid. (Q;8 per cent)
Values' represented above are the average of 3 independent experiments (each with 60 hypocotyl segments). Mean values followed by the same letter are not significantly different from each other at
p:0.05.
Adventitious Shoot Bud Regeneration and Organogenesis in Hypocotyl Cultures
219
Direct Shoot Regeneration Direct shoot regeneration was achieved from hypocotyl explants when cultured horizontally as well as vertically on MS medium supplemented with BA (8.9 mM) and NAA (1.07 mM). The first visible sign of adventitious shoot bud formation was the appearance of a dark green, ring like lump of tissue around the circumference of the cut ends. Thereafter, tiny leaf-like shoot clusters started to emerge from the green ring region, which further developed into shootlets (Figure 18.3A). Similar pattern of shoot formation was reported in hypocotyl cultures of Capsicum annuum (Carl and David, 1996). However, hypocotyl explants showed significant variation in organogenesis based on its orientation on culture medium. The highest frequency of shoot regeneration was achieved in hypocotyl explants, when cultured vertically on medium, but the significant (p>0.05) increase in shoot number and the length was observed in horizontally placed explants. Proximal and distal parts of the explant also showed differential regeneration behaviour. Most of the shoots (98.8 per cent) regenerated only from the proximal end. Adventitious shoot buds regenerated from hypocotyl segments elongated on the same medium within 6 weeks of culture. In the present study it was noticed that the orientation of the explant is a major factor contributing to shoot regeneration. It was found that 73 per cent of the explants were organogenic in vertical orientation and only 56 per cent in the horizontal position. In contradiction to these results, Cerdas et al. (1997) reported the reverse effect of hypocotyl orientation on shoot regeneration in Pithecellobium saman. Azmi et al. (1997) noticed the differential response of shoot regeneration to the explant orientation on regeneration medium. Interestingly hypocotyl explants cultured on either solid medium (0.8 per cent agar) or liquid medium (without agar) showed no significant (p>0.05) variation in shoot regeneration. However, suppression of adventitious bud formation was noticed on semi-solid (0.4 per cent agar) medium. It might be due to the excess in diffusion of the media ingredients and growth regulators into the explant, which may have caused the callus proliferation rather than differentiation. Histological studies showed the formation of multiple shoot buds directly from the hypocotyl segments with a little interference of callus (Figure 18.lE) Leafy shoots derived from hypocotyl callus and adventitious shoots regenerated from hypocotyl segments were transferred to MS basal medium for rooting. One week after inoculation, root formation was noticed from basal cut portion of the shoot (Figure 18.3B). Reduction of MS salts to half-strength, enhanced the root formation from the shootlets. The maximum frequency of root formation (98.4 per cent) and the maximum number (13.7) of roots were achieved in half-strength MS medium devoid of growth regulators (Figure 18.4), whereas quarter-strength MS nutrient medium showed poor response of root formation with low frequency and low root number, but yielded very lengthy roots (9.6 cm). However, addition ofIAA (0.57,2.85, 5.7 ~M) and IBA (2.46, 4.9 ~M) induced the formation of basal callus which in turn inhibited the root formation. Success of auxin-free basal medium in the present study for efficient root induction is also reported by Reddy et al. (2001), Reddy et al. (1998b). Xie and Hong (2001a) reported the root formation on Vz MS medium for A. mangium, however, the incorporation of NAA and kinetin is needed for the induction of roots in it. The incidence of highly efficient root formation on auxin-free medium may be due to the
220
Emerging Trends in Biological Sciences
(B)
(A)
(D)
(C) Figure 18.3
(A) Adventitious shoot formation from hypocotyl segments of Acacia concinna (bar = O.5cm); (8) Root formation from In vitro derived shoots on 112 strength MS basal medium devoid of growth regulators (bar = O.78cm); (C) Plantlets during transplantation (bar = O.83cm); (0) A plant growing in pot (bar = 3.7cm).
availability of required quantities of endogenous auxin in in vitro raised shootlets (Minocha, 1987). To acclimatize these in vitro developed plantiets, they were transferred to fresh test tubes containing autoclaved tap water. One week later, plants were removed from the tubes and transferred to plastic bags containing autoclaved vermiculite (Figure 18.3C). Plants were covered with polythene bags to maintain humidity and watered with autoclaved tap water. After one month they were transferred to greenhouse and found that all plants grew normally (Figure 18.30 ). In vitro produced plants grew well under greenhouse conditions and showed no morphological variations. In conclusion, the present communication presents an efficient protocol
221
Adventitious Shoot Bud Regeneration and Organogenesis in Hypocotyl Cultures
DFS
% Root funnatbn
No. ofroots
fiilHS
IbIQS
Root length (cm)
Figure 18.4: Rooting Efficiency of In vitro Raised Shoots of A. concinna on Full-strength (FS), Half-strength (HS) and Quarter-strength (QS) MS Basal Medium. Observations were taken after 8 weeks of culture.
for the large-scale mass propagation of A. concinna through tissue culture, which should be suitable for conservation and large-scale commercial cultivation of the threatened A. concinna.
References Ahee, J. and Duhoux, E. 1994. Root culturing of Faidherbia =Acacia albida as a source of explants for shoot regeneration. Plant Cell Tiss Org Cult. 36: 219-225. Anand, M. and Bir, 5.5. 1984. Organogenic differentiation in tissue cultures of Dalbergia lanceolaria. Curr Sci. 53: 1305-1307. Asholkar, L.V., Kakkar, K.K. and Chakre, O.J. 1992. Second Supplement to Glossary of Medicinal Plants with Active Principles. Part-I. Acacia concinna, CSIR Publications, New Delhi, pp 11. AzIni, A., Noin, M., Iandre, P., Prouteau, M., Boudet, A.M. and Chriqui, 0.1997. High frequency plant regeneration from Eucalyptus globulus Labill. hypocotyles: Ontogenesis and ploidy level of the regenerants. Plant Cell Tiss Org Cult., 51: 9-16.
Babber, K., Vinod, S.and Vargese, T.M. 1996. Micropropagation of Cassia roxburghii DC through in vitro technique. J. Ind. Bot. Soc. 75: 263-266. Bajaj, Y.P.S., Furmanowa, M. and Olszowskao. O. 1988. Biotechnology of the micropropagation of medicinal and aromatic plants. In: Bajaj, Y.P.S. (ed.) Biotechnology in Agriculture and Forestry. Springer and Verlag, New York, Vol. 4, pp 60-103.
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Barrett, J.D., Park, Y.S. and Bonga, J.M. 1997. The effectiveness of various nitrogen sources in white spruce [Picea glauca (Moench) Voss] somatic embryogenesis. Plant Cell Rep. 16: 411~15. Caboni, E., Lauri, P. and Angeli, D. 2000. In vitro plant regeneration from callus of shoot apices in apple shoot culture. Plant Cell Rep. 19: 755-760. Carl, M.R. and David, W.M.L. 1996. Influence of BA and sucrose on the competence and determination of pepper (Capsicum annuum L. var. Sweet banana) hypocotyl cultures during shoot formation. Plant Cell Rep. 15: 974-979. Cerdas, L.V., Dufour, M. and Villalobos, V.M. 1997 In vitro propagation of Pithecellobium saman (Rain-tree). In Vitro Cell Dev. BioI. 12: 33-38. Cid, L.P.B., Machado, A.C.M.G., Cavalheira, S.B.R.C. and Brasileiro, A.C.M. 1999 Plant regeneration from seedling explants of Eucalyptus grandis x E. urophylla. Plant Cell Tiss. Org. Cult. 56: 17-23. Garg, L., Bandari, NN., Vijay Rani, Bojwani, 5.5.1996. Somatic embryogenesis and regeneration of triploid plants in endosperm cultures of Acacia nilotica. Plant Cell Rep. 15: 855-858. George, W. 1972. Dictionary of Economic Plants of India. Vo!. I, CSIR Publications, New Delhi, pp 4~5. Lu, c.Y. 1993. The use of thidiazuron in tissue culture. In vitro Cell Dev. BioI. 29: 9296. Minocha, S.c. 1987. Plant growth regulators and morphogenesis in cell and tissue culture of forest trees. In: Bonga, J.M. and Durjan, D.J. (eds.) Cell and Tisssue Culture in Forestry, Martinus Nijhoff Pub. Dordrecht. Vo!. I, pp 50-66. Mittal, A., Agarwal, R. and Gupta, S.c. 1989. In vitro development of plantlets from axillary buds of Acacia auriculiformis-a leguminous tree. Plant Cell Tiss. Org. Cult. 19: 65-70. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant. 15: 473~97. Nandwani, D. and Ramawath, K. 1992. High frequency plantlets regeneration from seedling explants of Prosopis tamarugo. Plant Cell Tiss. Org. Cult. 29: 173-178. Nikam, T.D. andShitole, M.G.1999. In vitrocultureofSafflowerL. cv. Bhima: initiation, growth optimization and organogenesis. Plant Cell, Tiss. Org. Cult. 55: 15-22. Pandey, U.K., Verma, G.S., Lekha, C. and Singh, A.K. 1982. Note on the use of some insecticides against Bagrada cruciferarum, Painted bug, extracts of Acacia concinna, Nyctanthes arbor-tristis. Indian J. Agri. Sci. 52: 205-206. Quoirin, M., da Silva, M.C., Martins, K.G. and de OHveira, D.E. 2001. Multiplication of juvenile black wattle by microcuttings. Plant Cell Tiss. Org. Cult. 66: 199-205. Rao, G.V.R. and Prasad, M.N .V. 1991. Plant regeneration from the hypocotyl callus of Acacia auriculiformis-multipurpose tree Legume. J. Plant Physiol. 137: 625-627.
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Reddy, P.S. and Rama Gopal, G. 1998. Inhibition of inflorescence in Acacia concinna by gall formation. The Indian Forester 124: 167-168. Reddy, p.s., Rajasekhar, A. and Rama Gopal, G. 1998a. Vegetative propagation of Acacia concinna by stem cuttings. The Indian Forester 124: 164-166. Reddy, P .5., Rama Gopal, G. and Sita, G.L. 1998b. In vitro multiplication of Gymnema sylvestre an important medicinal plant. Curr. Sci., 75: 843-845. Reddy, P.S., Rosaline, R. and Rajasekharan, R. 2001. Shoot organogenesis and mass propagation of Coleus forskohlii from leaf derived callus. Plant Cell, Tiss. Org. Cult. 66: 183-188. Rout, G.R., Samantaray, 5., Das, P. 1995. Somatic embryogenesis and plant regeneration from callus cultures of Acacia catechu-a multipurpose leguminous tree. Plant Cell Tiss. Org. Cult. 42: 183-185. Rout,G.R., Saxena, c., Samantaray, S. and Das, P. 1999. Rapid plant regeneration from callus cultures of Plumbago zeylanica. Plant Cell Tiss. Org. Cult. 56: 47-51. Sastri, BN. 1956. The Wealth of India, Raw Materials, Vol. I, Acacia concinna. CSIR Publications, New Delhi, pp 13. Wldi, E., Schaffner, W. and Berger, K.B. 1998. In vitro propagation of Petasites hybridus (Asteraceae) from leaf and petiole explants and from inflorescence buds. Plant Cell Rep. 18: 336-340. Xie, D.Y. and Hong, Y. 2001a. In vitro regeneration of Acacia mangium via organogenesis. Plant Cell Tiss. Org. Cult. 66: 167-173. Xie, D.Y. and Hong, Y. 2001b. Cell biology and morphogenesis: Regeneration of Acacia mangium through somatic embryogenesis. Plant Cell Rep. 20: 34-40.
Chapter 19
Rapid In Vitro Propagation of Bixa orellana L.: An Important Dye Yielding Tree Md. Arifullah, Ch. Kishore Kumar, D. Gayathri and G. Rama Gopal* Department of Botany, Sri Venkateswara University, Tirupati - 517 502
ABSTRACT A protocol is presented for the micropropagation of Bixa orellana, a dyeyielding tree. Results indicate that different explant types require different growth regulator regimes for regeneration. The best organogenic response, including adventitious shoot number and elongation was obtained when 14 day old hypocotyl segments (0.5 cm) were cultured on to MS medium supplemented with 2.0 mg/l 2-isopentenyl adenine (2-iP) along with 2.0 mg/l I-naphthalene acetic acid (NAA). Basal media, size and orientation of the explant also influenced the efficiency of shoot organogenesis. Regenerated shoots rooted on MS medium fortified with 2 mg/12-iP along with 2.0-mg/1 indole-3- acetic acid (IAA). Rooted plants were transferred to soil medium and acclimatized successfully. Histological studies revealed the clear regeneration pattern of shoot from hypocotyl.
Keywords: Bixa, Hypocotyl, Adventitious shoot, 2-ip, NAA, [AA.
• Corresponding Author: E-mail: [email protected]
Rapid In Vitro Propagation ofBixa orellana L.: An Important Dye Yielding Tree
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Introduct~on
Bixa orellana L. is a small tree belonging to family Bixaceae locally called as Japhra (commonly known as Annatto) is native to tropical America, West Indies and naturalized in India. The plant is an erect branched tree, leaves are simple and much dissected, bluish green. Annatto, a major nutritive orange-red colour dye used for flavouring cakes, tablets etc. is highly priced and the yield is 4.8-6 per cent by weight of the seeds. Bixin (C 2SH300J, the principal coloUling matter present in the seeds is accompanied by small amounts of orellin, a water soluble yellow substance fat resin used for colouring foods stuffs. About 200 tons of seeds per annum were exported from South India (Wealth of India, 1976; Kirtikar and Basu, 1975; Kochhar, 1982; Madhuri Sharon et al., 2000). All parts of Bixa possess medicinal value and are widely employed in Ayurvedic medicine (Solkar et al., 1992; Caius, 1986; Irobi et al., 1996). Leaves are used against jaundice and snake bite (Nadkarni, 1976). The plant possess antigonorrhoeal, antimicrobial, antidysentric, antitumour, antihepatic, antiperiodic, antipyretic, hypoglycemic properties (Caceres-Armando et al., 1995; Srivastava Anil et al., 1999; Irobi et al., 1996; Ganesan, 1994; Ontengeo Delia et al., 1995; Suhaila Mohamed et al., 1996). Since annatto is one of the 13 basic pigments derived from natural sources that are currently permitted for food colouring by the US-FDA, there is an ever-increasing demand (Collins and Hughes, 1991; Srinivasulu, 1996). The plants require long period (4 years) for the production of seeds and show seed dormancy due to impermeability of tegument to water (Amaral-Lourdes et al., 1995). Its propagation is slowed down due to low percentage of flower setting (30-40 per cent), long time of seed germination (40 days) and low percentage of seed germination (5-7 per cent). It requires a specific soil, rich in Mn and extremely favourable climatic conditions for growth. More over its conventional propagation via cuttings has limitations because of the intense leaching of a gummy substance and phenolics from the cut ends, which obscure rooting (Madhuri Sharon et al., 2000). To overcome the hindrance and for rapid commercial propagation, plant tissue culture technology was initiated making significant contributions to agriculture and industry throughout the year. Many reports have been covered on Bixa shoot proliferation either directly from shoot apex or nodal explants (Madhuri Sharon et al., 2000 and Souza marie Claire et al., 2001) or indirectly from callus or somatic embryos (Almeida et al., 1996; Debata and Pank, 1996; Ramamurthy et al., 1999; Souza et al., 2001; Khan et al., 2002; Vieira, 2000). The present investigation is aimed at the direct organogenesis through hypocotyl segments of in vitro germinated seedlings of Bixa orellana.
Materials and Methods Fully ripe fruits were collected during January- February from 7-year-old Bixa orellana plants,growing in the Botanical garden, Sri Venkateswara University, Tirupati, Chitoor District, Andhra Pradesh. India. The seeds were removed from the dried fruits and treated with different concentrations of sulphuric acid (5-60 per cent) at different time intervals (5-20 min.). After acid treatment the excess sulphuric acid was removed by repeated washing with double distilled water. Then seeds were surface sterilized with 70 per cent ethanol for 30 seconds followed by 0.1 per cent HgCl2 for 5 min. Finally, the seeds were washed thrice with sterile double distilled
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water. Seeds were sown aseptically on MS basal medium (Murashige and Skoog, 1962) supplemented with 30 g/l sucrose and solidified with 0.8 g/l agar-agar. After 6-8 days of incubation in the dark (at 28 DC), cultures were moved to light conditions (16-h photoperiod daily, illuminated with cool-white fluorescent tubes at 25±2 DC). Seed germination percent differed based on the concentration of H 2S04 and duration of the treatment. Highest percentage of germination (96 per cent) was observed at 10 per cent H 2S04 for 5 min. Hypocotyl segments (0.5-2.5 crns) were excised from vigorously- growing 14- day-old seedlings and inoculated on MS medium containing various concentrations and combinations of plant growth regulators (Table 19.1). Explants were also cultured on four kind of basal media, i.e., MS and ~ MS (half strength concentrations of the major and minor salts of the MS medium), Bs and ~ Bs (half strength concentrations of the Bs medium, Gamborg et al., 1968) to compare and detect the basal media suitable for shoot organogenesis (Table 19.2). All cultures were maintained at 25±2 DC under a 16-h photoperiod daily, illuminated with fluorescent light (15 m E m·2 s·1) and sub cultured onto the same fresh medium at 2-week intervals. After 4- weeks of culture, the number of explants forming adventitious shoots was counted. Explants with more than four adventitious shoots (defined as multiple shoots) were distinguished from those with fewer than three adventitious shoots. Each experimental treatment consisted of 20 replicates. This experiment was repeated at least twice. Students' t test was used to examine differences between means. Table 19.1: Effect of Cytokinins and Auxins on Shoot Organogenesis from Hypocotyl Explants of Bixa orellana Medium
Hormone Concentration (mg//)
BAP MS
2-ip
NAA
IAA
% of Mean Numbflr Response of Shoots per Explant
1.0
22
1.5
26
3.4 ±0.8
2.0
40
3.5 ±0.4
1.0
36
3.1 ±0.2
1.5
46
3.0 ±0.7
2.0
56
2.5 ±0.3
3.2 ±0.6
1.0
2.0
24
3.4±0.2
1.5
2.0
34
3.6 ±0.9
2.0
2.0
48
3.8±0.2
2.0
12.0 ± 0.5 16.0 ± 0.7
0.5 1.0
2.0
52 62
1.5
2.0
64
20.0 ±0.6
2.0
2.0
80
26.0±0.2
0.5
0.5
38
4.4 ± 0.8
1.0
1.0
41
4.2 ±0.5
1.5
1.5
43
4.6±0.8
2.0
2.0
47
Data represent mean ± SE, based on 10 replicates.
Rnpid In Vitro Propagation ofBixa orellana L.: An Important Dye Yielding Tree
227
Table 19.2: Influence of Basal Media on Bixa orellana Shoot Organogenesis Medium
Hormone Concentration (mg/l)
BAP MS half
85
85 half
2-ip
NAA
% of Mean Number Response of Shoots per Explant
1.0
16
2.4 ± 0.1
15
1.5
21
2.6 ±0.7
20
2.0
30
2.7 ±0.3
10
10
2.0
31
4.8 ±0.6
15
2.0
36
8.0 ±0.6
20
2.0
42
12.0 ± 0.5
1.0
35
8.0 ±0.7
10 15
1.5
38
10.0 ± 0.3
20
2.0
43
14.0 ± 0.2
10
1.0
52
16.0 ± 0.6
15
1.5
68
18.0 ± 0.3
20
2.0
75
22.0 ±0.5
10
1.0
18
4.0 ±0.6
15
1.5
21
4.3 ±0.5
20
2.0
23
4.6±0.7
1.0
16
4.0 ±0.4
1.5
22
4.4 ±0.5
2.0
24
4.8 ±0.6
Data represent mean ± SE, based on 10 replicates.
Adventitious shoots induced from the explants were transferred to 1h MS medium without phytohormones to promote elongation of the adventitious shoots. Elongated shoots (2.5-45 cm long) were excised from the explants and transferred to full strength and half strength MS basal medium with and without supplemented various auxins (Table 19.3) for rooting and further elongation. After 3- weeks, the number of rooted shoots and the number of roots per shoot were recorded. For histological studies, hypocotyl explants cultured on regeneration medium for 0, 4, 8 and 16 days respectively were fix1?d in FAA (Formaldehyde: Acetic acid: 50 per cent Ethyl alcohol, 5: 5: 90 v Iv Iv) before dehydration through a graded ethanol series (20 per cent, 30 per cent, 50 per cent, 70 per cent, and 90 per cent) sequentially for 20 min. at each step, each step being repeated three times, and finally in 100 per cent ethanol for 3 times for 30 min. All ti~sues were embedded in paraffin wax. Sections of 10 l1m were cut and then stained with safranin and light green.
Results and Discussion Madhuri Sharon and Marie Claire D' Souza (2001) reported the germination after 20 days when the seeds of Bixa were treated in hot water. But in our study, the acid treated seeds germinated (96.3 per cent) within 6-8 days in agreement with the
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Emerging Trends in Biological Sciences
reports of Amaral Lourdes et al. (1995) that the chemical scarification caused rupture in the dense palisade, which occurs in the tegument, creating an efficient way of water entrance into the seeds. Thermal scarification was ineffective in promoting seed germination and in some cases it caused loss of viability of the seeds. Table 19.3: Effect of auxin treatments on In vitro rooting of cultured Shoots of Blxa orellana on MS Medium
.
Medium
Hormone Concentration (mgl/) BAP
MS
2-ip
NAA
0.1
1.0
0.5
2.0±O.6
0.2
2.0
3.0
4.5±O.8
0.3
3.0
3.5
4.8±O.4
0.1
1.0
73.0
5.6±O.5
0.2
2.0
78.0
6.8±O.3
0.3
MS half
IAA
% of Mean Number Response of Shoots per Explant
3.0
75.0
7.2±O.2
0.1
1.0
1.0
2.5±O.7
0.2
2.0
0.9
2.6±O.8
0.3
3.0
4.0
2.6±O.5
1.0
0.8
1.0±O.6
0.2
1.5
0.9
1.0±O.2
0.3
2.0
2.5
2.8±O.9
0.1
0.1
1.0
20.0
2.9±O.7
0.2
1.5
32.0
4.8±O.5
2.0
38.0
5.2±O.6
1.0
0.7
1.0±O.9
0.2
1.5
0.4
2.0±O.8
0.3
2.0
2.0
1.6±O.6
0.3 0.1
Data represent mean ± SE, based on 10 replicates.
Adventitious shoot buds appeared on hypocotyls within 3 weeks after culture, but the final shoot count was performed at 4 weeks. Bud initials formed along the cut ends of explants. Preliminary studies revealed that 2-iP was more effective plant growth regulator (PGR) for inducing adventitious shoots from hypocotyl fragments. Of all the combinations used, 2-iP + NAA induced adventitious shoots most efficiently from hypocotyls, although others have reported only 2-iP (Madhuri Sharon et al., 2000), or BAP+ IBA (Ramurthy et al., 1999) for other explants. Explants of different origin react to growth regulators in distinct ways and, as with choice of explant, this is particularly obvious in the formation of adventitious shoots. In some species, one type of explant may respond to a particular growth regulator treatment while the other does not. In some instances it may even be advantageous to vary the regulatory compounds used for different kinds of explants. Adventitious shoots were obtained in greatest numbers from petiole sections of Oxalis tuberosa using 3 mg/l BAP and
Rapid In Vitro Propagation of Bixa orellana L.: An Important Dye Yielding Tree
229
3 mg/l NAA, but the best regeneration from internode sections occurred when they were grown on a medium with 3 mg/l Zeatin plus 3 mg/l NAA (Khan et al., 1988). Among all the concentrations tested in the present study (Table 19.1), the best response (80 per cent) was noticed with 2.0 mg/12-iP + 2.0 mg/l NAA (Figure 19.1 A, B). In the present study, kinetin did not promote shoot multiplication in any of the combinations tested, which was also reported by Madhuri Sharon et al. (2000), Ramamurthy et al. (999) and Sairam Reddy et al. (998). In the present study, the frequency of shoot formation was influenced by both size of hypocotyl fragments and the kind of basal media. The highest frequency of shoot formation (80 per cent) was obtained using MS medium with 0.5 cm fragment oriented horizontally (Figure 19.1C) and could initiate more multiples than the longer (1.5 cm) hypocotyl segments. Better response (75 per cent) was observed with B5 medium followed by MS medium. The superiority of MS medium over other salt formulations has been demonstrated for many plant species such as Hybanthus enneaspermus (Prakash et al., 1999), Guizotia abyssinica (Nikam and Shitole, 1997) and Cunila galioides (Fracaro and Echegavirray, 2001), and it has been frequently and successfully used in tissue culture studies of tree species (Duns tan and Thorpe, 1986). These results indicate that salt concentrations in media influence shoot organogenesis in Bixa. Half MS and half B5 media proved ineffective in promoting shoot organogenesis as reported by Ramamurthy et al. (999). Wright et al. (986) reported that lower salt concentrations in MS basal medium supplemented with BA 0.15 mg/D had minimal effects on shoot formation and they also indicated that half MS medium did not induce multiple shoots effectively, when compared to MS medium. All the shoots continued to elongate on the same medium in which shoot initiation had taken place (Figure 19.1E). In all cases where the shoots were inoculated on MS half and full strength media with auxins, roots were found formed with basal callus. But when a cytokinin (2-iP) is added, roots developed (after 4 weeks) without interference of basal callus. No root was found to develop on half and full strength MS media free from hormone (Madhuri Sharon et al., 2000). Among the different concentrations and combinations of auxins tested, 0.2 mg/l IAA with 2.0 mg/l 2-iP was more effective than the other growth regulators (Table 19.3). This treatment not only induced more number of roots (7.2±O.2) but also promoted root length (Figure 19.1F, G). Healthy plants with 3-5 cm roots were individually removed from the culture tubes. After washing the roots carefully with sterile distilled water, plantlets were transferred to plastic pots containing autoclaved vermiculite and pelrite (3:1). The plants were watered and covered with polythene bags for one week. After hardening (Figure 19.1 I), plants were gradually transferred to the field for developing into mature plants. Thus rooted plants were successfully acclimatized in vermi~ulite with gradual decrease in air humidity and 95 per cent of transferred plantlets survived without any morphological variations. Histological studies revealed that, at the time of inoculation seedling had no preexisting meristems. However after the incubation period, clusters of actively dividing cells were present. Cell divisions were most apparent in the subepidermal and cortical regions of hypocotyl segments. The development of shoot organization could clearly be observed from these clusters (Figure 19.1H).
Emerging Trends in Biological Sciences
230
Figure 19.11 (A-C) Multiple shoot regeneration (0) rooted hypocotyls (E) shoot elongation (F,G) rooting (H) histology, and (I) hardened plants of Bixa orellana regenerated from hypocotyl segments.
Rapid In Vitro Propagation ofBixa orellana L.: An Important Dye Yielding Tree ~
231
Since annatto is one of the 13 basic pigments derived from natural sources that are currently permitted for food colouring by the U5-FDA, there is an ever-increasing demand (Collins and Hughes, 1991; Srinivasulu, 1996). The protocols established in the present study to achieve rapid shoot organogenesis for Bixa orellana can be utilized in commercial micropropagation as well as in genetic transformation studies and other fields such as mutation breeding.
References Almeida, J. L., Almeida, F. C. G., Nunes, R De. P. and Almeida, F.A.G. 1996. Induction of shoot buds in leaf explants of annatto seedlings in different cytokinins. Ciencia Rural (Brazil) 26(1): 54-49. Amaral Lourdes, LV., Pereira Maria De Fatima, A. and Cortelazzo Angelo, L. 1995. Dormancy break in seeds of Bixa orellana. Revista Brasileira De Fisiologia Vegetal. 7 (2): 151-157. Caius, J. F. 1986. In: The Medicinal and Poisonous Plants of India, Scientific Publishers, Jodhpur, India pp.141-142. Caceres Armando, Menindez Herlinda, Mendez Emilia, Cohobon Erickar, Samayoa Blanca E, Jauregul EIsa, Peralto Eduardo, Carrillo Guillermo. 1995. Journal of Ethanopharmacol. 48(2): 85-88. Collins and Hughes. 1991. Report in prospective in Natural Food Symposium Overseas food Ltd, England. Debata, B. K and Pank, F.1996. Micropropagation of Bixa orellana. Proceedings, International symposium, Breeding research on medicinal and aromatic plants, Quedinburg, Germany. Souza, D, and Madhuri Sharon M.C. 2001. In vitro clonal propagation of annatto (Bixa orellana L.). In Vitro cell. Dev. Biol.-plant 37(2): 168-172. Dunstan DJ. and Thorpe T.A. 1986. Regeneration of forest trees In: Vasil,l..K (ed.) cell culture and somatic cell genetics of plants. Academic press, New York. 3: 223-341. Fracaro F and Echeverrigary. 2001. Micropropagation of Cunilaga lioides, a popular medicinal plant of South Brazil. Plant Cell Tiss. Org. Cult. 64: 1-4. Gamborg, O. L., Miller, RA, and Ojima, K 1968. Nutrient requirements of suspension cultures of soyabean root cell. Exp. Cell. Res. 50: 148-151. Ganesan, T. 1994. Antifungal properties of wild plants. Adv. Plant Sci.. 7 (1): 185-187. Irobi O.N., Moo Young M and Anderson W. A. 1996. Antimicrobial activity of Annatto (Bixa orellana) extract. Int. J. Pharmacog. 34: 87-90. Khan, P.S.S.V., Prakash, E. and Rao, K. R. 2002. Callus indution and plantlets regeneration in Bixa orellana L. An annatto yielding tree. In Vitro cell. Dev. Bioi.
plant 38(2):186-190. Kirtikar, KR and Basu, B.D.1975. In Indian Medicinal Plants, Bishen Singh Publishers, India, Vol. 1,200 edn. pp. 216-218.
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Kochhar S.L. 1981. In Economic Botany in the Tropics, Macmillan PbI. India, pp. 377378. Madhuri Sharon and 0' Souza M.C. 2000. In vitro clonal propapagation of annatto (Bixa orellana L.) Curr. Sci. 78(12): 1532-1535. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497. Nadkami KM. 1976. Indian Materia Medica (ed.) KM Nadkarni Popular Prakashan Pvt Ltd., Mumbai, 1: 199-200. Nikam T.D. and Shitole, M.G. 1997. In vitro plant regeneration from callus of niger (Guizotia abyssinica Cass. C.V. Sahyadri). Plant Cell Rep.17: 155-158. Ontengeo Delia, c., Dayap Lourdes, A. and Capal Terisita, V. 1995. Screening for the antibacterial activity of essential oils from some Phillipine plants. Acta manilana. 43: 19-23. Prakash, E., Shavallikhan, p.s., Sairam Reddy, P. and Rao KR.1999. Regeneration of plants from seed derived callus of Hybanthus enneaspermum, a rare ethnobotanical herb. Plant Cell Rep.18: 873-878. Ramamurthy N, Savithramma, N., Usha, R. and Swamy, P.M. 1999. Multiple shoot induction and regeneration of Japhra (Bixa orellana L.) through axillary bud derived callus cultures. J. Plant Bioi. 26 (3): 231-235. Sairam Reddy, P., Rama Gopal, G. and Lakshmi Sita, G. 1998. In vitro multiplication of Gymnema sylvestre R. Br. -An important medicinal plant. Curr. Sci. 75(8): 843845. Solkar, L.V., Kakkar, K.K and Chakre, O.J. 1992. In Glossary of Indian Medicinal Plants with Active Principles (part 1) Orient Longman, p.126. Srinivasulu, C. 1996. Annatto-The Natural Colour. In the suppliment to cultivation and Utilization of Medicinal Plants (eds. Handa S.S. and Kaul M.K), National Institute of Science Communication, New Delhi, pp. 537-543. Srivastava Anil, Shukla Y.N, Jain S.P. and Kumar Sushi!. 1999. Chemistry, pharmacology and uses of Bixa orellana: A review. Journal ofMed. Arom. Plant Sci. 21(4): 1145-1154. Suhaila Mohamed, Saka Suzana, El-Sharkawy saleh H, Ali Abdul Manaf and Muid Sepiah. 1996. Antimycotic screening of 58 Malaysian plants against plant pathogens. Pesticide Science. 47(3): 259-264. The Wealth of India. 1959. Raw Material Vol. (5), CSIR Publications, New Delhi. pp. 106. Vieira, I.M.S., Barbosa, AS. A, Serra, AG.P., Silva, S.P.G., Mota, M.G.C. and Botelho, MN. 2000. Embryogenese sorruitica de urucu (Bixa orellana), cultivar EMBRAPA37. Proceedings of 51°Congresso Brasileiro de Botanica. Brasllia pp. 23. Wright M. S., Koehler S.M., Hinchee, M.A and Cames, M.G. 1986. Plant regeneration by organogenesis in Glycine max. Plant Cell Rep. 5: 150-154.
Chapter 20
Tissue Culture of Annatto Y. Venkateswara Rao, V. N. Chakravarthi, Dhavala, D. Tejeswara Rao, M. V. Subba Rao and V. Manga Department of Botany, Andhra University, Visakhapatnam - 530 041, Andhra Pradesh, India E-mail: [email protected]
ABSTRACT Bixa orellana L., commonly known as Annatto is one of the important medicinal and commercial shrubs belonging to the family Bixaceae. It is native to America and also grows in various tropical and subtropical regions of the world. The development of Annatto through conventional breeding had limited success due to its long generation time and low viability of seeds. Since the beginning of this decade work has been going on in this plant, and most of this is focused on in vitro aspects. The present review summarises work on the effect of factors like explant type, its position, type of growth regulators used, secondary metabolites produced etc., and on in vitro propagation of Bixa orellana.
Introduction Bixa orellana (2n =14) belonging to the family Bixaceae, is an economically important cultivated plant grown in tropical and subtropical regions of the world (Figure 20.1A). It is a good source of two important natural carotenoid dyes bixin and norbixin present in the seed coat as well as in some aerial parts of the plant system (Yabiku and Yamayaki, 1992; Mercadante et al., 1997; Scotter et al., 1998; Bouvier et al., 2003; Castello et al., 2004) (Figure 20.IB). Annatto has also been used in traditional medicines as well as in new generation medicines to cure several diseases
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Emerging Trends in Biological Sciences
(Morrisone et al., 1991; Irobi et al., 1996; Castello et al., 2002; Sabitha Rani et al., 2003, Russell et al., 2005). Although Annatto is propagated by seeds as well as by stem cuttings, only limited success has been achieved through seeds since they exhibit very low viability (Belfort et al., 1992; Eira and Mello, 1997) and delayed germination. Even though scarification of seeds yields good germination it can be achieved only in fresh seeds (Amaral et al., 1995). Plant tissue culture has progressed immensely from its inception to its present state as a variable tool for improvement of crop plants and includes somaclonal variations, somatic hybridization and genetic transformation. With the help of these techniques novel variability for biotic and abiotic stress can be created or transferred from unrelated sources. However efficient and high regeneration protocols are fundamental for utilizing the power and potential of these techniques. Apart from its use for rapid micropropagation, PIC technique has a number of other major applications of commercial value like (I) production of virus free plants. (il) Germplasm preservation through cryopreservation (iil) Genetic transformation (iv) Generation of breeding material of agricultural importance and for secondary metabolite production (George and Sherrington, 1984). Although annatto does not lend itself readily to in vitro propagation due to the gummy substance and phenolic compounds that ooze out from the cut surfaces and obscure the entry of the nutrients into the tissues and result in necrosis of the explant, successful attempts have been made by several workers to standardize protocols for regeneration of this plant system and considerable progress has also been made in understanding the physical and morphological mechanisms underlying the morphogenesis. In this review, an attempt is made to sum up the information on various aspects of in vitro regener~tion in annatto, evaluate recent progress and its future utilization in the product improvement.
Factors Affecting In vitro Propagation Use of practical approaches for in vitro propagation of plants showed that success largely depends on factors that are related to donor plant characteristics, composition of culture medium and control of the physical elements. Systematic screening of one or more of these factors with the plant of interest has been the most common experimental approach for achieving in vitro regeneration.
Effect of Age, Size and Orientation of Explant In annatto, variety of explants i.e., nodal explants, shoot apex (Madhuri and D'Souza, 2000; D'Souza and Sharon, 2001), cotyledonary nodes and stem nodes (Nassar-Abla et al., 2001), seeds (Shavalli khan et al., 2002), zygotic embryos (PaivaNeto et al., 2003), hypocotyl (Paiva- Neto et al., 2003), hypocotyls, cotyledonarynodes and inverted hypocotyls (Carvalho et al., 2005a,b) have been used for induction of somatic embryogenesis as well as direct organogenesis. The choice of explant is considered to be an important factor in the induction of somatic embryogenesis as well as organogenesis. Madhuri and D'Souza, (2000) used shoot apex and nodal segments of which, the former showed better response than the later ones. They also
Tissue Culture of Annatto
235
noticed that the size of the explant was an important factor in producing multiple shoots. Small sized segments (O.5cm) produced maximum multiple shoots than the larger ones. Nassar-Abla et al. (2001) reported the positional effect on in vitro response of the explants. According to them the nodal segments obtained from different sources revealed differential response i.e., the cotyledonary nodes responded better compared to the stem nodes. Similar position effects on in vitro response were observed in Solanum (Sharma and Rajam 1995), pepper (Kintzios et al., 2000), Lilium davidii (Long Chun Lin et al., 2004). According to Carvalho et al. (2005a) the explant type and the different combinational concentration of hormones used revealed variable in vitro responses found to be significant. Hypocotyls and cotyledonary nodes in this order had significantly better embryogenic as well as regeneration potential compared to the inverted position of hypocotyls. Paiva- Neto et al. (2003), observed response of explants at different stages of development. According to them juvenile explants (viz., immature embryos) produce optimum response while mature ones did not show any response (mature embryos). They also found that the embryogenic response was significantly affected by the genotype, of the source plant. Profound effects of explant type and genotype on in vitro response were noticed in other species such as alfa alfa (Hammad Picconi and Standardi, 1993), pea (Doome et al., 1995) Solanum (Sharma and Rajam, 1995) and tomato (Moghaieb et al., 1999).
Physical Factors Light is an important factor, for the growth and differentiation of explants and it depends on the length of exposure and quality of light. Some growth processes show enhanced response while in other processes growth is delayed or inhibited with changing environmental conditions. Light affects in vitro response of annatto at different stages, i.e., the callus induction frequency is found to be greater under 24h dark period as compared to 16h light/Bh dark photoperiod or 24h light period, while 16h light is required for regeneration (Shavalli khan et al., 2002). Madhuri and D'Souza (2000); D'Souza, and Sharon (2001), reported that 16h photoperiod (using 3000 lux) resulted in highest frequency of regeneration while 16h photoperiod using 36 J.l mol mlg-1 intensity produced the same result (Carvalho et al., 2005a).
Temperature Warmer temperature (27°C± 2) was found to be optimum for somatic embryogenesis as well as organogenesis in Bixa orellana (Madhuri and D'Souza, 2000; D'Souza and Sharon 2001; Nassar-Abla et al., 2001; Shavalli khan et al., 2002; Paiva- Neto et al., 2003; Paiva- Neto et al., 2003; Paiva- Neto et al., 2003; Carvalho et al., 2005a,b)
Chemical Factors Carbon source: Sucrose has been commonly used as carbon source in tissue culture media (Petersen et al., 1999; Fuentes et al., 2000; Vespasiano et al., 2003). Sucrose is the sugar of choice for in vitro annatto culture, and other sugars failed to give promising results. Paivo-Neto et al. (2003) reported effect of carbon source in hypocotyl derived explants of annatto. Sucrose and glucose induced the maximum organogeneic response. The affect of carbon source on shoot regeneration was found to be more
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prominent in rooted hypocotyls than in cut hypocotyJ segments. This difference could not be directly linked to the carbohydrate nutritional aspects, but to carbohydrate osmotic contribution. Carbohydrates control morphogenesis by acting as energy source and also by altering the somatic potential of the culture medium. They also observed browning of explants in medium supplemented with fructose as a carbon source which was also supported by the Buter et al. (1993) according to whom high temperatures produced during autoclaving of culture media can result in toxic compounds, such as 5-(hydroxypentyl)-2- furaldehyde and these compounds are primarily derived from the breakdown of fructose. Paivo-Neto et al. (2003) suggested that sucrose supply was highly essential for inducing morphogenesis in annatto. They also evaluated the feasibility of mannose as a selection system for the future genetic transformation studies on annatto. Several other studies have also demonstrated the influence of carbon source on in vitro morphogenesis of different plant species like carrot (Devi and Dougall, 1977), Lavandula vera (Nakajima et al., 1989), Solanum eleagnifolium (Nigra et al., 1990), Cherry (Borkowska and Szczerba, 1991), Pinus sylvestris (Sui and Korbon, 1998), Euonymus europaeus (Biahoua and Bonneau, 1999), Miscanthus (Petersen et al., 1999) Coffea canephora (Fuentes et al., 2000), oil palm (AsIan and Sompong, 2005) and in banana (Madhulatha et al., 2006).
Gelling Agents Agar-agar was the common gelling agent used in tissue culture of Bixa. According to Paiva- Neto et al. (2003) phytagel was the best gelling agent for regeneration, and Gelrite containing media produce low frequency of shoots while agar-agar has shown intermediary response.
Nutrient Media Most researchers have been using MS or modified MS medium (Madhuri and D'Souza, 2QOO; D'Souza, and Sharon 2001; Nassar-Abla et al., 2001; Shavalli Khan et al., 2002; Paiva- Neto et al., 2003, Paiva- Neto et al., 2003a,c; Carvalho et ai., 2005a,b), and media additionally contain myo-inositol at a concentration of 100mg to 200mg/l (Shavalli khan et al., 2002). Bixa orellana L. explants respond differently to varying nutrient concentrations. Nassar-Abla et al. (2001) used WPM (Woody Plant Medium), MS and Gamborg's B5 media. Of those, MS and WPM were found to be good for the growth and proliferation of high quality shoots. B5 vitamins along with M.5 basal major and minor nutrients were successfully used by Paiva- Neto et al., 2003b. Regeneration of plant lets from shoot apex and nodal explants could be achieved by Madhuri and D'Souza, (2000) and by D'Souza, and Sharon (2001) on B5 medium. The type and effect of gelling agents in other plant systems has been reported by earlier workers (Scholten and Pierik 1998; Nestakova et aI., 2000; Klimaszewska et al., 2000 Kalatejari et ai., 2006).
Effect of Charcoal Paiva Neto et al. (2003) observed accumulation of the secondary metabolites during somatic embryogenesis in annatto. They also found that use of activated charcoal suppressed the effect of these compounds and enhances the number of somatic embryos per explant.
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Plant Growth Regulators (PGRs) Plant growth regulators are small organic molecules that are produced in specific organs or tissues, and which elicit defined responses either directly at the site where they are synthesized or after transfer to other organs or tissues. Plant development is believed to be controlled by a tight network of interactions among several different classes of such PGRs. Each class of PGR affects a large number of developmental processes, by more than one class of hormones. It is accepted that without plant growth regulators, in vitro culture of plants is almost impossible (George, 1996). Cytokinins appear to play an important role in somatic embryogenesis as well as direct organogenesis of annatto. Earlier, Cytokinins in combination with auxin have been used for somatic embryogenesis in Bixa. The following combinations have been used by Nassar-Abla et al. (2001)-BAP 1.0 mg/l + 0.2 mg/l NAA, KN 1.0 mg/ 1+ 0.2 mg/l NAA; Shavalli khan et al. (2002)-BAP 2.5~ + 5.0 llM NAA; Paiva- Neto et al. (2003)-2.2611M 2,4-D + 4.52 llM kinetin; Carvalho et al. (2005a,b)-O.3011M IAA + 2.28 llM/4.5611M zeatin, 0.3011M IAA + 4.5611M BAP. Direct organogenesis was reported by Madhuri and D'Souza, (2000) and by D'Souza and Sharon (2001) on 1mg/12iP, Paiva- Neto et al. (2003) on 4.5611m Zeatin, TDZ and BAP supplemented medium respectively.
Organogenesis Organogenesis of annatto has been achieved first by Madhuri and D'Souza, (2000) from shoot apex and nodal explants on B5 medium supplemented with 1.0mg/ I 2-isopentinyl adenine (2-iP). Paiva- Neto et al. (2003c) reported that the best organogenic response, including high adventitious shoot number and shoot elongation, was obtained from hypocotyl segments and rooted hypocotyls cultured on MS medium supplemented with 4.56J.lID zeatin + 87.6mM sucrose, and 2.8gm/1 phytagel. A comparative analysis on the effect of different cytokinins viz., Zeatin, TDZ, BAP on regeneration revealed both Zeatin and TDZ resulted in higher frequency of organogenesis than BAP treatment respectively. Paiva- Neto et al. (2003b) inoculated hypocotyls, hypocotyls in inverted position and immature zygotic embryos, on MS medium supplemented with Bs vitamins, 87.6 mM sucrose and mannose in different combinations, 2.8g/1 phytagel and 4.56 llm zeatin. Annatto explants responded better in sucrose containing medium and did not regenerate on medium having mannose as the carbon source.
Somatic Embryogenesis Somatic embryogenesis (SE) in annatto has been reported from hypocotyls, cotyledonary nodes, stem nodes, seeds, immature embryos, nodal explants and shoot apices. The first report of somatic embryogenesis in Bixa orellana was by NassarAbla et al. (2001) on MS medium supplemented with BAP or kinetin (KN) + NAA 100mg/1 tyrosine and 40 mg/l Adenine sulphate. Where as Shavalli Khan et al. (2002), obtained somatic embryos from seed explants inoculated on M.S medium supplemented with 5.0 J.lID N AA and 2.511m BAP.
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The study on somatic embryogenesis carried out so far reveal that auxin in combination with cytokinin enhances somatic embryo differentiation. However, its optimal concentrations as well as response in different hormonal combinations varied with the explant used. Immature embryos required 2.26 pm 2, 4-D + 4.5pm kinetin (Paiva- Neto et al., 2003a). While hypocotyls, cotyledonary nodes and inverted hypocotyls require 0.30 pm IAA + 2.28 pm zeatin (ZEA) (Carvalho et al., 2005 a,b). Paiva- Neto et al. (2003) studied the effect of genotype, explant type and age of the explant on somatic embryogenesis. Studies on different stages of embryos revealed that the juvenile explants responded better and produced somatic embryos, while mature ones did not show any in vitro response. They also found that the embryogenenic response was significantly affected by the genotype of the source plant.
Histological Analysis Histological investigation of Paiva- Neto et al. (2003a) in Bixa revealed that primary direct somatic embryos differentiated exclusively from the protodermis or together with the outer ground meristem cell layers of the zygotic embryo axis, and from the protodermis of zygotic cotyledons. They also observed diverse morphological structures such as malformed embryos among somatic embryos. Although they found high frequencies of histodifferentation of all embryo stages, a very low conversion frequency to normal plants from somatic embryos was observed in their study. Histological studies of Paiva- Neto et al. (2003c) on hypocotyls derived explants of annatto showed that, TDZ induced high frequency of mitotic divisions resulting in proliferation of several meristemoid zones nearby epidermis and outer cortical tisssues, but without individual bud formation and shoot elongation. On the other hand, zeatin based medium promoted low mitotic activity, but allowed bud differentiation and further shoot elongation. In both these cases, cortical and epidermal cells formed on additional tissue to heal the wounding surface of the explant and in some regions, the epidermis appeared to form a periderm like structure mainly in explants inoculated on TDZ supplemented medium. Similar results were also reported earlier by Karkoken (2000), Compton and Veilleux (1991), Pugliesi et al. (1999) Warren (1993), Ovecka et al. (2000).
Cytological Analysis According to the cytological observationpf Carvalho et al. (2005a), there is no genetic variation among the regenerations produced from various explants viz., hypocotyls, cotyledonary nodes and inverted hypocotyls inoculated on to MS supplemented with Zeatin (2.28 pM) + IAA 0.30 pM and 0.30 pM IAA + 4.56 Zeatin. Carvalho et al. (2005b), induced in vitro polyploidy in hypocotyl and cotyledonary node cultures. The culture medium used to induce polyploidisation was supplemented with MS salts, B5 vitamin complex, 100 mgll myoinositol, 3 per cent w Iv sucrose, 2.28 um ZEA (hypocotyl segments) or 4.56pM ZEA (cotyledonary nodes), 0.8w Iv agar and different concentrations of colchicine (0, 25, 250 and 1250pM) and oryzalin (0, 5,15 and 30 pM). They also determined the optimum duration of either colchicines or oryzalin treatment for the induction of tetraploids by placing the
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explants on regeneration medium for 15-30 days. High frequency of ployploids in regenerated shoots from cotyledonary nodes were obtained in culture medium supplemented with 15 pM oryzalin for 15 days. Among the characteristics evaluated in their studies, the measurements based on stomata length, width, area and frequency enabled greater discrimination between diploid and polyploid regenerated shoots.
Rooting and Acclimatization of Plantlets Based on the classification by Marks and Simpson (2000), annatto can be considered as a difficult to root woody species. But exogenous supply of auxins considerably enhances the rhizogenesis efficiency (rooting frequency and root number), Madhuri and 0' Souza (2000), 0' Souza and Sharon (2001) reported best rooting on MS medium supplemented with 0.5 mg/l NAA. They obtained 80 per cent survival rate on transfer of seedlings in to the feild. Nassar-Abla et al. (2001) achieved best rooting response on half strength MS medium supplemented with 3mg/l IAA and 2 per cent sucrose and 80 per cent and 67 per cent survival rate observed in cotyledonary node and stem node explants respectively. Mature shoots were rooted in half strength MS medium containing IBA at 50 J.1M concentration and about 85 per cent of these plants were acclimatized successfully by Shavalli Khan et al. (2002). The best rhizogenesis efficiency (rooting frequency and root number) was noticed by Paiva- Neto et al. (2003 a,b,c) in MS medium supplemented with 5.0J.1M of IBA. Rooting of elongated shoots derived form juvenile hypocotyls, cotyledonary leaves and hypcotyls was achieved on half strength MS medium supplemented with 5 J.1M of IBA by Carvalho et al. (2005 a).
Genetic Engineering The first report on the transient gus gene expression in two annatto varieties through Agrobacterium tumefaciens was reported by Zaldivar et al. (2003). Hypocotyls from annatto seedlings were inoculated with Agrobacterium tumefaciens strain LBA 440A harboring a binary vector PBI121 and PCAMBIA 2301, containing the Bglucuronidase (gus) gene. Histochemical GUS assay of infected hypocotyls from two annatto varieties showed transient gus gene expression between 3-12 days after inoculation. Paiva- Neto et al. (2003) evaluated the feasibility of mannose as a selection system for future genetic transformation of annatto. They also observed that this system is useful for manipulation of genes involved in pigment biosynthesis and accumulation.
Conclusion Remarkable progress has been made in the induction of somatic embryogenesis as well as direct organogenesis in annatto during last decade, much of the work being focused on the physical and chemical aspects of in vitro production. The prospects of annatto improvement appear brighter with the advent of biotechnology tools. The areas that need to be strengthened in annatto research are genetic manipulations, germplasm conservation through cryopreservation, generation of breeding material of agricultural importance and for secondary metabolites production. Further, Bixa orellana is an important plant extensively used in the food
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industry as a source of coloring agent and the dye has been proved to be non-carcinogenic and nontoxic. Work should be strengthened for in vitro production of the dye, its commercial extraction and long term storage. Work on this aspect is in progress in our laboratory.
Acknowledgements The first authors are very much thankful to University Grants Commission, New Delhi, for financial assistance (F30-155/2004(SR Dated 10.11.2004» and UGCSAP, Department of Botany for providing facilities.
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Madhulatha, P., Kirubakaran, S.1. and Sakthivel, N. 2006. Effects of carbon sources and auxins on in vitro propagation of banana. Biologia Plantarum 50 (4): 782-784. Madhuri Sharon and D'Souza, M.C.2000. In vitro clonal propagation of annatto (Bixa orellana L.). Curr. Sci. 78(12}: 1532-1535. Marks T. M. and Simpson, S.E. 2000. Interaction of explant type and indole-3- butyric acid during rooting in vitro in a range of difficulty and easy-ta-root woody plants. Plant cell tiss. Org. Cult. 62: 65-74. Mercadente, A.Z., Steck, A. and Fander, P.H. 1997. Isolation and structure elucidation of minor carotenoids from annatto (Bixa orellana L.) seeds. Phytochemistry 46(8}: 1379-1383. Moghaieb, RE.A., Saneoka,H. and Fujita, K 1999. Plant regeneration from hypocotyls and cotyledon explant of tomato (Lycopersicon esculantum Mill.). Soil Sci.- Plant. Nutr. 45: 639-646. Morrisone, Y.S.A., Thompson, H., Hompson, H., Pascoe, K, West, M. and Fletcher, e. 1991. Extraction of an hyperglycemic principle from the annatto (Bixa-orellana) a medicinal plant in the West Indies. Tropi. Geograp. Med. 43(1-2}: 184-188 Nakajima, H., Sonomoto, K, Sato, F., Ichimura, K, Yamada, Y. and Tanaka, A. 1989. Influence of carbon source on pigment production by immobilized cultured cells of Lavandula vera. J. Ferm. and bioeng. 68(5}: 330-333. Nassar Abla, H., Khalifa, S. F., Hammouda, F.M. and Shams, KA .. 2001. In vitro propagation of Bixa orellana L. (annatto). Egyptian J. Bot. 41(2}: 241-253. Nestakova M., Havrlentova, M. and Farago, J. 2000. Effect of gelling agent on in vitro manipulation of two ornamental plants. Biolugia, Bratislava 55: 409-411. Nigra, H. M., Alvarez, M. A. and Giulietti, A. M. 1990. Effect of carbon and nitrogen sources on growth and solasodine production in batch suspension cultures of Solanum eleagnifolium Cav. Plant Cell Tiss. Org. Cult.21 (1): 55-60. Ovecka, M., Bobak, M. and Samaj, J. 2000. A comparative structural analysis of direct and indirect shoot regeneration of Papaver somniferum.L In vitro J. plant physiol. 157:281-289. Paiva-Neto, V.B., Botelho, M.N., Aguiar, R, Silva, E. A. M and Otoni, W. e. 2003a. Somatic embryogenesis from immature zygotic embryos of annatto (Bixa orellana L.). In Vitro Cell Dev. Bio. Plant. 39(6): 629-634. Paiva-Neto, V.B. Carvalhi, e.Rand O.J;oni, W.e.2oo3b. Mannose a potential selection system for genetic transformation of annatto (Bixa orellana L.). Bioi. Plant. 47(3): 441-444. Paiva-Neto, V.B., Mota, T.R and Otoni, W.e. 2003c. Direct organogenesis from hypocotyl-derived explants of annatto (Bixa orellana). Plant Cell Tiss. Organ Cult. 75(2): 159-167. Petersen, K,1<., Hansen, J. and Krogstup, P. 1999. Significance of different carbon sources and sterilization methods on callus induction and plant regeneration of Miscanthus x ogiformis Honda 'Giganteus'. Plant cell Tissue org. cult. 58: 189-197.
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Pugliesi C, Cionini, G., Bertram, L. and Lercari, B. 1999. A histological study of light dependent shoot regeneration in hypocotyl explants of tomato cultured in vitro. Adv. Hort. Sci. 13: 168-172. Russell, K.R.M., Morrison, E.Y.A and Ragoobirsingh, D. 2005.The effect of annatto on insulin binding properties in the dog. Phytotherapy Res. 19(5): 433-436. Sabitha Rani, A, Satyakala, M. and Suryanarayana murthy, U. 2003. Bixa orellanaan important source of natural colorant: a review. Journal ofMed. Aromat. Plant Sci. 25: 733-742. Scotter, M. J., Wilson, L.A Appleton, G.P. and Castle, L. 1998. Analysis of annatto (Bixa orellana) Food coloring formulations.l. Determination of coloring components and colored thermal degradation products by high-performance liquid chromatography with photodiode array detection. J. Agric. Food Chem. 46: 1031-1038. Scholten H. J. and Pierik, RL.M. 1998. Agar as a gelling agent: differential biological effects in vitro. Sci. Hort. 77: 109-116. Sharma, P. and Rajam, M.V. 1995. Genotype, explant and position effects on organogenesis and somatic embryogenesis in eggplant (Solanum melongena L.). J. Expt. Bot. 46:135-14l. Shavalli Khan, P.S., Prakash, E. and Rao, K.R 2002. Callus induction and plantlet regeneration in Bixa orellana L., an Annatto-yielding tree. In Vitro Cell. Dev. BiolPlant, 38(2): 186-190. Sui, I. and Korbon, 5.5.1998. Effect of media, carbon sources and cytokinins on shoot organogenesis in the Christmus tree Scots pine (Pin us sylvestris L.). J. Hort. Sci. Biotech. 73: 822-827. Vespasiano B. D., Paiva Neto, V.B. and Otoni, W.C 2003. Carbon sources and their osmotic potential in plant tissue culture: does it matter? Scien. Horti. 97 (3-4): 193202 Warren, G. 1993. The regeneration of plants from cultured cells and tissues. In: Stafford A, and Warren G (eds.). Plant cell and tissue culture. John Wiley and sons. Chichester. Yabiku, H.Y. and Yamazaki, T.M.1992. Determination ox bixin in annatto seeds: Collaborative study. Revista-do-Instituto-Adolfo-Lutz. 52(1-2): 31-36. Zaldivar, CJ.M., Ballina, G.H., Guerro, RC, Aviles, B.E. and Hernandez, G.CG. 2003. Agrobacterium-mediated transient transformation of annatto (Bixa orellana) hypocotyls with the gus reporter gene. Plant Cell Tiss. Org. Cult. 73(3): 281-284.
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Subject and Plant Index
Abrus precatorius 152, 161 Abutilon indicum 181 Acacia auriculaeformis 190 Acacia concinna 211 A holosericea 141 A leucophloea 136, 166 A pennata 152 Aplanifrons 136, 165 Acacia polyantha 152
Aegiceras corniculatus 207 Aegilites rotundifolia 207 Aegle marmelos 162, 180 Aeromonas 56 Aerva lanata 152 A zeylanica 150 Aesculus hippocaslnum 183
Acanthaceae 142
Afforestation 189
Acanthus ilicifolius 207 Acaulospora 18,25 Achromobacter 55 Achyranthes aspera 162 A bidentata 162 Acinetobacter 53, 62, 66 Acorus calamus 183 Acrostichum aureum 207 Actiniopteris radiata 137 Adhatoda zeylanica 183 Adiantum raddianum 160
Aganosma cymosum 136 Ageratum conyzoides 152 Aglaia cucullata 207 A roxburghiana 139
Adoption 91 Adventitious shoot 224
Airborne bacteria 53 Air sampling 53
Alangium salvifolium 160, 166 Albizia amara 136, 191 A lebbeck 39, 152 Aodorotissima 137, 152 Alcaligenes 53, 62, 67
246
Aleuropodeae 125 Algae 84
Allium cepa 160 A. sativum 183
Allophylus serratus 152 Aloe vera 161 Alpha taxonomy 174
Alpinia calcarata 147 AIseodaphne semecarpinifolia 138 Alstonia schplaris 191 A. venenata 162 Alternanthera sessilis 152, 181 Alternaria alternata 48, 50, 51 A. ricini 42 Alyssum wolfanianum 186 Amaranthaceae 150
Amaranthus spinosus 152 Amaryllidaceae 150 AM Fungi 18
Amoora canarana 138 Ampelocissus araneosa 156 Anabaena 87 A. sphaerica 88 A. torulosa 88 Anacardium occidentale 152 Anabenopsis 87 Anamirta cocculus 137, 152 Anaphalis lawii 156 Andrographis lineata 143, 156, 161, 162 A. paniculata 163, 164, 183 Andropogoneae100
Angiopteris evecta 143 Anisochilus argenteus 156 Anisomeles malabarica 161 Annatto 233 Annonaceae 142
Annona squamosa 186
Emerging Trends in Biological Sciences
Anogeissus latifolia 137, 152 Anthericaceae 150 Antibody 13 Anti-carcinogenic activity 116 Anti-hepatitis 184 Anti-hyperlipidemic activity 116 Anti-inflammatory activity 115 Anti-insect properties 187 Anti-malarial activity 117 Anti-microbial activity 34, 39, 46,116 Antioxidant properties 117
Aphanocapsa 87 Apiaceae 142
Arabidopsis thaliana 186 Arabis stricta 186 Araceae 150 Araliaceae 142 Arbuscular mycorhizae 18, 27
Arctostaphylos uva-ursi 183 Ardisia solanacea 144, 161 Areca catechu 160 Arecaceae 150
Arisaema leschenaultii 145 Aristideae 125
Aristolochia indica 161, 162 Artemisia absinthium 183 A. annua 183 A. capillaris 186 A. nilagirica 161 Arthospira 87 Arthrobacter 53, 62, 66 Artocarpus heterophyllus 141, 159 Asc1epiadaceae 142
Asparagus racemosus 159, 162, 164,183 Aspergillus flavus 48, 50, 51 A. fumigatus 36, 40 A. niger 35, 40, 48, 49
Subject and Plant Index
Asplenium aethiopicum 162 Asteraceae 142
Asystasia crispata 156 A. travancorica 156 Atalantia monophylla 163 Avicennia marina 207 A.officinalis 207 Auxins 226
Azadirachta indica 162-; 181, 183, 191 Azima tetracantha 136 Bacillus 61 B. brevis 53, 60, 63 B. cereus 53, 60, 63 B. circulans 53, 60, 63 B. firm us 53, 60, 63 B. lioheniformis 53, 60, 64 B. marcerans 56 B. megaterium 35, 42, 53, 60, 63 B. sphaericus 53, 60, 63 B. subtilis 35, 40, 42, 53, 60, 63 Bacopa monnieri 152, 183 Balanophora fungosa 156 Bambusa arun143acea 165 Barleria acuminata 156 B. courtallica 156 B. cuspidata 156 B. longiflora 156 B. prionitis 163 Basella alba 159 Benincasa hispida 150 Bhitarkanika 205 Bignoniaceae 142 Biodiversity 176
Biophytum sensitivum 181 Biotechnology 176 Boerhavia diffusa 179, 181, 183
247
Borassus flabellifer 165 Boswellia serrata 152, 164 Bougainvillea 191 Brassicaceae 142
Brachystelma bournaeae 156 Breynia vitis-idea 163 Bridelia crenulata 148, 156 Bruguiera cylindrica 207 B. gymnorhiza 207 B. parviflora 207 B. sexangula 207 Buchanania axillaris 148 B.lanzan 160 Budleja asiatica 160 Burseraceae 142
Butea frondosa 183 B. monosperma 152 Cactaceae 142
Caesalpinia bonduc 152, 158, 207 C. crista 207 C. pulcherrima 190 Callus induction 213
Calophyllum inophyllum 152, 191 Calothrix 87 Calotropis gigantea 163 Camellia sinensis 180 Canarium strictum 138, 166 Candida albicans 35, 40 Canscora decussata 152 C. diffusa 156 Canthium parviflorum 152 Capparaceae 142
Capparis decidua 181 C. sepiaria 136 C. wightii 144 Capsicum annuum 48
248
Caralluma adscendens 137, 162 Caralluma tuberculata 182 Cardiospermum canescens 160 C. halicacabum 164 Careya arborea 141, 144, 160 Carissa carandas 159 C. spinarum 136 Carum carvi 186 Caryophyllaceae 142
Caryota urens 152 Cascabela thevetia 163 Casearia elliptica 148 C. tomentosa 181 Cassia absus 152 C. angustifolia 183 C. fistula 152 C. auriculata 136 C. montana 191 C. occidentalis 152 C. renigera 191 C. siamea 191 C. sophora 191 Cassine paniculata 138 Castor 42
Casuarina equisetifolia 190 Catharanthus roseus 181 Catunaregam spinosa 136, 152 Cayratia carnosa 152 C. pedata 137 Centella asiatica 138, 160, 164, 183 Ceratophyllum demersum 185 Cerbera odollum 207 Cereals 18
Ceriops decandra 207 C. taga1207 Ceropegia juncea 137, 156 Chamomilla recutita 183
Emerging Trends in Biological Sciences Cheilanthus mysurensis 137 Chenopodium ambrosioides 162, 186 Chicken embryonic cells 7, 10 Chilli 48 Chlorideae 125
Chloris 122 C. barbata 123, 128 C.bourneil23,128 C. montana 123, 128 C. roxburghiana 123, 128 C. virgata 123, 128 Chloroxylon swetenia 136, 181 Chroococcus 87 Chrysanthemum cinerariefolium 186 Chrysophyllum roxburghii 138 Chukrasia tabularis 166 Cinnamomum tamala 181 Circadian rhythms 79
Cissampelos pereira 160 Cissus quadrangularis 160 Cladosporium herbarum 48, 50, 51 Cleistanthus collinus 163 Clerodendron inerme 152, 207 C. phlomides 150 C. serratum 144, 163, 166 C. viscosum 150, 152 Clitoria ternatea 152 Cochlospermum religiosum 137, 153 Cocculus hirsutus 137, 152, 165 Cocos nucifera 165 Coffea arabica 147, 166 C. wightiana 147 Colocasia esculenta 153 Combretum albidum 161 Cotnmelinaceae 150
Commiphora wightii 167 Conservation 192
Subject and Plant Index
249
Crotalaria longipes 156 C. madurensis 156 C. priestleyoides 156 C. retusa 153 C. shevaroyensis 156 C. willdenowiana 156 Cryptolepis buchanani 163 Curculigo orchioides 164 Curcuma longa 183 Curvularia lunata 48, 50, 51
Deccania pubescens 156 Delonix regia 191 Dendrophthoe falcata 153 Derris indica 181 D. trifoliata 207 Desmodium ferrugineum 156 D. gangeticum 153 D. triflorum 137 Dichanthium 101 D. annulatum 102, 103 D. pertusum 102, 104 Dichrostachys cinerea 161 Didymocarpus gambleanus 160 Digera muricata 34 Dimocarpus longan 138 Dioscorea bulbifera 159, 163 D. oppositifolia 159
Cyanobacteria 84
Dioscoreaceae 150
Cycas circinalis 162 Cylindrospermum 87 Cymbidium aloifolium 149, 156 Cymbopogon citratus 153, 186 C. coloratus 137 C. martini 156, 165 Cynanchum callialatum 160 Cynodon dactylon 153 Cynometra iripia 207 Cyperus rotundus 161
Diospyros ebenum 166 D. malabarica 166 D. ovalifolia 166 Diplocentrum recurvum 149 Diplocyclos palmatus 153 Dodonaea viscosa 166 Drynaria quercifolia 139, 153
Cytokinins 226
Ecosystem studies 193
Cordia dichotoma 153, 166 Corynebacterium 53, 62, 67 Crataeva magna 153 Crategus monogyna 183 C. oxycantha 183 Crinum triflorum 150 Critically endangered 199
Dalbergia lanceolaria 166 D. latifolia 138, 166 D. paniculata 148, 166 D. rubiginosa 156 D. sissoo 153, 190 Decalepis hamiltonii 156 Decaschistia crotonifolia 156
Ebenaceae 142
Echinacea 183 Elaeocarpus tuberculatus 138 Elephantopus scaber 153 Elettaria cardamomum 141 Elodea canadensis 185 Emblica officinalis 164 Emilia sonchifolia 153 Endangered 190 Endemic 192, 199
250 Endocytosis 5,13
Enicostemma axillare 181 Entada pursaetha 138,156, 159, 164 Enterlobium saman 191 Enterobacter faecalis 36 Ephedra 183 Eragrostideae 125
Emerging Trends in Biological Sciences
Furcraea foetida 158, 165 Fusarium moniliforme 48,50,51 F. oxysporum 50, 51 Gardenia gummifera 144, 153 G. resinifera 137 Garuga pinnata 138 Gigaspora 18, 25
Eremopogon 101 E. foveolatus 102, 105 Eriodendron pentandrum 190 Erythrina variegata 153 Escherichia coli 36, 40, 42 Eucalyptus hybrida 190 Eulophia graminea 149 Euphorbia atttiquorum 137 E. hirta 137
Ginkgo biloba 183 Givotia moluccana 138 Gleocapsa 87 Gleotheca 87 Gliricidia maculata 190 Glomus 18, 25 Gloriosa superba 145, 163, 164
Euphorbiaceae 150
Glycoproteins 15
Evolvulus alsinioides 137, 162
Glycosmis pentaphylla 160 Gmelina arborea 138, 166 G. asiatica 161 Gnetum ula 138 Gnidia glauca 158, 165
Extinct 190
Excoecaria agallocha 207 Fibroblast cells 2, 10
Fiats arnottiana 153 F. beddomei 157 F. benghalensis 153, 181 F. glomerata 159, 164 F. hispida 153, 159 F. microcarpa 153, 167 F. racemosa 181 F. religiosa 153, 181 Finlaysonia obovata 207 Flacourtia indica 139, 153 Flavobacterium 53, 62, 67 F. regense 69
Gesneriaceae 142
Gram-positive 70 Gram negative 70 Green belt species 191 Grewia tiliifolia 166, 167
Gymnema elegans 157, 161 G. sylvestre 145, 161, 164, 181 Gynura nitida 157 Habenaria digitata 150 H. elwesii 150, 157
H. longicorniculata 157 H. longicornu 149, 150, 157
Fodder sorghum 19
H. plantaginifolia 150
Foxtail millet 23
H. rariflora 157
Freeria indica 199
Hardwickia binata 157
Subject and Plant Index
Harpagophyton procumbens 184 Hedyotis cor.ymbosa 153 Hedychium coronarium 139, 143, 150, 157 Hedyotis umbellata 137 Helicteres isora 158, 161, 164, 181 H. wightiana 147 Heliotropium indicum 153 Hemidesmus indicus 164 Heracleum ringens 163 Herbal medicine 178
Heritiera fomes 208 H.littoralis
Hibiscus cannabinus 165 H. tiliaceus 182, 208 Hiptage benghalensis 137, 153 Holarrhena antidysenterica 153, 184 Holoptelia integrifolia 153, 181 Hugonia mystax 136 Hybanthus enneaspermus 162 Hyperglycemic activity 117
Hypericum perforatum 184 Hyperzia serrata 184 Hypocotyl cultures 212
Ichnocarpus frutescens 181 Immunofluorescence microscopy 6 Indian gooseberry 179
Indigofera barberi 157 1. glutinosa 157 1. mysorensis 157 1. trita 157 Instia bijuga 208 Inula racemosa 180 Ipomoea aquatica 182 I. nil 153 1. pes-caprae 153 1. sepiaria 153
251
Iseilema anthephoroides 102, 107 1. laxum 102, 106 1. venkateswarluii 102, 108 Italian millet 19
Ixora arborea 148 Jasminum cuspidatum 139 J. flexile 139 Jatropha curcas 191 J. glandulifera 191 J. gossypifolia 191 J. panduraefolia 191 J. podagrica 191 Jatropha tanjorensis 157 Justicia adhatoda 161 J. beddomei 153 Kandelia candel 208 Kedrostis foetidissima 162 Kleinia grandiflora 162 Knoxia sumatrensis 157 Lagenaria siceraria 150 Lannea coromandelica 137, 153 Lantana camara 181 Lepidagathis pungens 157 Leucaena latisiliqua 167 L. leucocephala 186, 190 Leucas hirta 157 L. mollissima 157 L. prostrata 157 L. pubescens 157 Linaceae 142
Litsea glutinosa 138, 150 Littorella uniflora 185 Lobelia nicotianifolia 164, 161 Loganiaceae 142
252
Luisia birchea 139, 142 Lumnitzera racemosa 208 Lyngbya 87 L. aestuarii 88 L. cinerescens 88
Emerging Trends in Biological Sciences O. canum 186 O. sanctum 180 O. suave 186 Omega taxonomy 175 Orcuttieae 125 Organogenesis 211,237
Mad/lUea longifolia 162 Mallotus stenanthus 157 Mangrove 205 Melastomataceae 142
Melia azedarach 153 Meliaceae 142
Memecylon umbellatum 161 Merremia emarginata 153 M. tridentata 154 Miliusa tomentosa 148 Mimosa pudica 154, 186 Mimusops elengi 154, 191 M. hexandra 191 Momordic.z dioica 162 Mucuna pruriens 157, 162, 164 Murdannia zeylanica 150 Musa paradisiaca 161 Myriophyllum alternifolium 185 Myriostachya wightiana 208 Myristica fragrans 162 M. tactylodes 158 Myrtaceae 142
Naravelia zeylanica 154 Nerium oleander 154 Nilgirianthus kunthianus 166 Nostoc87 Nypa fruticans 208 Ocimum album 180 O. basilicum 186
Oroxylum indicum 154 Orthosiphon thymiflorus 154 Oryza sativa 150 Osbeckia stellata 157 Oscillatoria 87 O. amphibia 88 O. bravis 88 O. chilkensis O. chlorina 88
O.limosa 88 O. minnesotensis 88 O. salina 88 O. simplicissima 88 O. tenuis88 Oxalis corniculata 160 Panicum miliaceum 21 P. miliare 150 Pappophoreae 125
Papilionanthe subulata 150 Parthenium argentatum 191 Peltophorum pterocarpum 191 Pentatropis capensis 163 Peperomia 143dygulensis 143, 157 Pharmacological actions 111
Phoenix loureirii 158 P. paludosa 208 P. sylvestre 150 Phormidium 87 Phyla nodiflora 138 Phyllanthus amarus 161, 179, 184
Subject and Plant Index
P. emblica 138, 158, 165, 179 P. maderaspatensis 179 P. pin natus 179 P. reticulatu.s 154, 158, 165 P. simplex 179 P. urinaria 179 Physalis minima 154, 181 Phytochemical constituents 111 Phytochemistry 114 Phytogeography 188 Phytoremediation 185
253
Pseudomonas 65 P. aeruginosa 35, 40 P. putida 62, 66 Psoralea corylifolia 181, 184 Pteris longipes 160 Pterocarpus marsupium 137, 154 P. santalinus 157 Pterospermum canescens 137 Punicagranatum 159, 161
Picrorhiza kurroa 184 Piper betel 162 P. methystichum 184 P. nigrum 150 Pittosporum eriocarpum 191 P. resiniferum 191 Plantago ovata 184
Randia malabarica 136 Reissantia grahamii 137 R. indica 167 Rhinacanthus nasutus 157 Rhizophora apiculata 208 R. mucronata 208 R. stylosa 208 Rhynchosia heynei 157
Plant indicators 193
Rice 19
Plectonema 87
Ricinus communis 42
Plumbagin 113, 115
Rubiaceae 142
Plumbago zeylanica 111, 162, 164
Rubia cordifolia 164
Poaceae 150 Polygalaceae 142
Salacia oblonga 181 Salicornia brachiata 208
Pongamia pinnata 190,208
Salvadoraceae 142
Portulacaceae 142
Salvadora persica 154 Sapindus emarginatus 164
Pogostemon mollis 157
Potamogeton crispus 185 P. pectinatus 185 P. perfoliatus 185 Pouzolzia wightii 157 Proso millet 19 Protein 1
Proteus vulgaris 35 Prunus africana 184 P. amygdalus 180 Pseudarthria viscida 154
Sapotaceae 142
Saraca indica 179 Sarcostemma acidum 137 S. intermedium 146, 157 Schefflera wallichiana 139, 143 Schizothrix 87 Schleichera oleosa 154, 191 Schummannianthus virgatus 165 Sclerocystis 18
254
Scoparia dulcis 182 Scutellaria violacea 150 Scutellospora 18 Scytonema 87 SOS-PAGE 8
Emerging Trends in Biological Sciences
Staphylococcus 53,66 S. aureus37 S. fumigatus 35 S. saprophyticus 62 Stenosiphonium parviflorum 157
Seasonal periodicity 53
Sterculiaceae 142
Secamone emetica 137 Securinega leucopyrus 159 Serenoa repens 184
Sterculia foetida 154
Sericulture practices 91 Sericulturists 90
Serratia 53, 67 S. marcescens 62 Setaria italica 23 Shoot bud regeneration 212
Shorea roxburghii 166 Simmondsia chinensis 191 Sida rhombifolia 181 Silybum marianum 184 Sirumalai hills 133
S. guttata 138 S. urens 138 Stevia 147 Strebulus asper 154 Striga gesnerioides 181 Strychnos nux-vomica 164 S. potatorum 164 Swertia chirayita 184 Symplocos cochinchinensis 138, 145, 154 Synechococcus 87 Syzygium alternifolium 191 S. cumini 159,181 S. densiflorum 159
Smithia setulosa 157 Socio-economic characters 91
Talinum portulacifolium 159 Tamarix troupii 208
Solanaceae 142
Taxonomy 172
Solanum anguivi 154
Somatic embryogenesis 237
Tectona grandis 138, 166 Tephrosia spinosa 137 Terminalia arjuna 154, 166 T. bellirica 158, 164 T. catappa 208 T. chebula 138, 146, 158, 162
Sonneratia alba 208 S. apetala 208
Themeda cymbaria 165 T. triandra 137, 138
Spartineae 125
Therapeutic uses 11
Spermoderm 100
Theriophonum fischeri ISO, 157, 163 Thespesia populnea 208
Social forestry 190
S. nigrum 160, 181 S. surattense 154, 164
S. tridatum 146 Solena amplexicaulis 154
Spilanthes calva 162 Spirulina 87 . Spondias pinnata 154
Threatened 190
Thunbergia tomentosa 157
Subject and Plant Index
Thyroglobulin 2, 5
Tinospora cordifolia 181 Toddalia asiatica 136, 154 Tolypothrix 87 Tragia involucrata 154 Trema orientalis 179 Trianthema portulacastrum 179 Trichodesma indicum 154 T. zeylanicum 138 Trichosanthes cucumeria11,a 162 T. lobata 160 TrigoneUa foenum-graecum 181 Tristicha ramosissima 150 Tylophora indica 154 T. macrantha 143 T. zeylanica 161 Typha angustata 138 Unioleae 125
255
Vanda testacea 149 Ventilago maderaspatana 162, 164 Vigna mungo 150 V. radiata 150 V. trilobata 157 V. unguiculata 150 Vitaceae 142
Vitex altissima 137 V. negundo 150 Vulnerable 190
Walsura trifoliata 138 Wendlandia thyrsoidea 138 Withania somnifera 146, 181 Wrightia tinctoria 162 Xylocarpus granatum 208 X. mekongensis 208 X. moluccensis 208
Uvaria narum 138 Valeriana officinalis 184 VAM22 VAMfungi28
Ziziphus mauritiana 136, 154 Z. rugosa 154, 164 Z. xylopyrus 158, 161 Zoysieae 125
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