Emerging Trends in Biological Sciences

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Etnerging Trends in Biological Sciences

"This page is Intentionally Left Blank"

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

)

All rights reserved, including the right to translate or to reproduce this book or parts thereof except for briefquotations in critical reviews.

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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


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


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].

Emerging Trends in Biological Sciences

2

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


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.


6

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


8

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

~

U'> N

0.4

0 ........ 0 ....... 0

.o ..... ···O' ... ····O=-.··_···~··.--4""--

~-roe:

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60

80

100

120

140

160

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


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

J 10 20 30 40 50 10 70 ID 10 100

Fre. ' .....Tg (nM)

B

O.ooO-t---or---'I""""----t 0.00

0.50

0.25

0.75

Bound

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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'
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


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).

94


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


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.


96

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.


97

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

98


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.

A Study on Adoptionai Behaviour of Sericuiturists and their Characteristics in Anantapur

99

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


102

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


103

104


.,... .,...


'C C 111

1/1

": ,.. ,.. :::l Cl

e

u:::

105

106



....: c::

III

.~

~

Il.

E

"C

Q)

E

.. ..o c-

Q)

OO

~ ""': ,....

~

""': ,.... ,....

107

108



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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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

115

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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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

118


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.

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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

edn. E and 5 Livingstone Ltd., Edinburgh.

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

Studies on Biosystematics ofChloris Swartz. Species (Poaceae)

123

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:


124

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

~

128


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.

130


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

134


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


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.


140

•

'--_ _ _ _...;;'pklt

«'ALE

IUID Scrub forest

lmm Savannah ~Woodland

~~1M.:DeCiduous forest •

Map 14.2: Vegetation Map of Sirumalai Hills

.semi-evergreen forest


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

Emerging Trends in Biological ScienceI'

144

Plate 14.2

(a) Ardisia solanacea; (b) Capparis wightii; (c) Clerodendrum serratum; (d) Gardenia gummifera; (e) Careya abrorea; (t) Morina umbel/ata


145

Plate 14.3 (a) Arisaema leschenaultii; (b) Begonia malabarica; (c) Gloriosa superba; (d) Gymnema sylvestre; (e) Memecylon umbellatum; (f) Symplocos cochinchinensis

Emerging Trends in Biological Sdences

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


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...


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


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...


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,


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.


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...


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...


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...


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).


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


165

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...

166


Table 14.11-Contd ••• SI. No.


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.


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

168


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 e0.05) 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


(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

225

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


226

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


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

228


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


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).


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

234


(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

236

Emerginx Trends in Biological SCIences

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.


237

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


239

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

240


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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