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Study on the Characterization of Coelomic Fluid of Eudrilus Eugeniae and Its Effect on Plants and Animal Cells in the In-Vitro Culture System

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Study on the Characterization of Coelomic Fluid of Eudrilus Eugeniae and Its Effect on Plants and Animal Cells in the In-Vitro Culture System Study on the characterization of coelomic fluid of Eudrilus eugeniae and its effect on plants and animal cells in the in-vitro culture system Thesis submitted to Bharathidasan University, Tiruchirappalli for the award of the degree of DOCTOR OF PHILOSOPHY IN BOTANY Submitted by A. VEERAMANI Under the guidance of Dr. R. RAVIKUMAR Ph.D., Assistant Professor PG and Research Department of Botany Jamal Mohamed College (Autonomous) Tiruchirappalli - 620 020. November 2010 DECLARATION I do hereby declare that this work has been originally carried out by me under the guidance and supervision of Dr. R. Ravikumar, Assistant Professor, P.G and Research Department of Botany, Jamal Mohamed College (Autonomous), Tiruchirappalli and this work has not been submitted elsewhere for any other degree, diploma or other similar titles. Place: Tiruchirappalli Date: (A. Veeramani) POST GRADUATE AND RESEARCH DEPARTMENT OF BOTANY JAMAL MOHAMED COLLEGE (Autonomous) TIRUCHIRAPPALLI-620 020. Dr. R. RAVIKUMAR Ph.D., CERTIFICATE Study on the characterization of coelomic Thisfluid isof to Eudrilus certify thateugeniae this thesis and itsentitled effect on plants and animal cells in the In-vitro culture system submitted by Mr. A. VEERAMANI, for the degree of Doctor of Philosophy in Botany, to the Bharathidasan University is based on the results of studies carried out by him under my guidance and supervision. This thesis or any part thereof has not been submitted elsewhere for any other degree. Place: Tiruchirappalli Date: R. Ravikumar Office: 0431-2331235 Extn. 302 Mobile: 9787486255 E-Mail: [email protected] Residence: Plot No.63, Sriram Nagar, Kattur, Tiruchirappalli-620 019. Acknowledgement I owe a great many thanks to a great many people who helped and supported me during my Ph.D work. My deepest thanks to Dr. R. Ravikumar, my research supervisor and also Assistant Professor in the Department of Botany, Jamal Mohamed College (Autonomous), Tiruchirappalli, Tamil Nadu, India, for guiding and correcting various documents of mine with attention and care. He has taken pains to go through the project and make necessary correction as and when needed. I am much indebted to Dr. V. Nandhagopal, Associate Professor, Department of Botany, National College (Autonomous), Tiruchirappalli, Tamil Nadu, India, for his valuable advice as a Doctoral Committee member. I express my thanks to the Management of Jamal Mohamed College and Dr. M. Sheik Mohamed, Principal, for extending support and the facilities to carry out part of my research work in the Departments of Botany and Biotechnology. I extend my sincere thanks to Dr. S. Ahamed John, Head, PG and Research Department of Botany, Jamal Mohamed College (Autonomous), Tiruchirappalli, Tamil Nadu, India for permitting me to undertake my research programme in the Department. I would like to express my thanks to the members of the faculty of the PG and Research Department of Botany for giving me valuable suggestions and support in course of my work. My deep sense of gratitude to Dr. M.M. Shahul Hameed, Head, Department of Biotechnology, Jamal Mohamed College (Autonomous), Tiruchirappalli, Tamil Nadu, India for extending the facilities of the Department to perform in vitro studies in Plant and Animal cells that remains a vital part of my research work. I am grateful to the Principal, Head and members of the faculty of the Department of Botany, Arignar Anna Govt. Arts College, Namakkal, for their constant encouragement all through my Ph.D work. I wish to specially thank my friend and colleague Dr. M. Rajasekarapandian, Assistant professor, Department of Zoology, in every possible way for the encouragement that he showed upon me. I gratefully acknowledge Mr. S. Senthil Kumar and Mr. M.S. Mohamed Jaabir, Assistant Professors, Department of Biotechnology, Jamal Mohamed College (Autonomous), Tiruchirappalli, for their timely help and interpretation of the results. My family deserves special mention for their inseparable support and prayers. Though I need not thank them, I record my sincere gratitude for the care and support of my wife, son, daughter and my son-in-law in this occasion. It is a pleasure to pay tribute also to all those friends and fellow-scholars in the Department of Biotechnology for various helps rendered to me. I am proud to record that I had several opportunities to work with exceptionally hard working scholars like them. Finally, I would like to thank everybody who was important to the successful realization of thesis, as well as expressing my apology that I could not mention personally one by one. A. Veeramani S. No. CONTENTS Page No. 1. INTRODUCTION 01 2. REVIEW OF LITERATURE 09 CHAPTER I: Evaluation of different collection methods for coelomic fluid 3. 36 from Eudrilus eugeniae and its biochemical characterization 4. CHAPTER II: Antimicrobial activity of the coelomic fluid of E .eugeniae 64 CHAPTER III: Investigation of plant growth promoting property of the 5. coelomic fluid in plant tissue culture system 79 CHAPTER IV: Evaluation of anticancer property of coelomic fluid of 6. 104 Eudrilus eugeniae in SiHa cell line CHAPTER V: To study the impact of textile effluent on the fecundity and 7. 118 population of Eudrilus eugeniae 8. SUMMARY 133 10. REFERENCES 11. PUBLICATIONS “Earthworms-The intestines of the soil" – Aristotle. "The plow is one of the most ancient and most valuable of man's inventions; but long before he existed, the land was in fact regularly plowed and still continues to be thus plowed by earthworms. It may be doubted whether there are many other animals which have played so important a part in the history of the world, as have these lowly organized creatures. Without the work of this humble creature, who knows, nothing of the benefits he confers upon mankind, agriculture, as we know it, would be very difficult, if not wholly impossible" –Charles Darwin. These are the words, which reveal the importance of Earthworms. It is well known that the earthworms have the ability to support the growth of plants and they can increase the fertility of the soil. There are about 3920 named species of earthworm so far reported worldwide. In India, so far, 509 species, referable to 67 genera and 10 families, have been reported (Kale, 1991). Earthworms play an important role in agro-ecosystem like enhancing decomposition, humus formation, nutrient cycling and soil structural development (Kladiviko et al., 1986). The practice of vermiculture is at least a century old but it is now being revived worldwide with diverse ecological objectives such as waste management, soil detoxification and regeneration and sustainable agriculture. Earthworms act in the soil as aerators, grinders, crushers, chemical degraders and biological stimulators. They secrete enzymes, proteases, lipases, amylases, cellulases and chitinases which bring about rapid biochemical conversion of the cellulosic and 1 the proteinaceous materials in the variety of organic wastes which originate from homes, gardens, dairies and farms. Recent works have elucidated some of the mechanisms by which earthworms enhance soil aggregation. Ingested aggregates are broken up in liquid slurry that mixes soil with organic material and binding agents. The defecated casts become stable after drying and also earthworms initiate the formation of stable soil aggregates in mining soils. Ecology of earthworms Earthworms are burrowing animals and form tunnels by literally eating their way through the soil. The distribution of earthworms in soil depends on factors like soil moisture, availability of organic matter and pH of the soil. They occur in diverse habitats specially those which are dark and moist. Organic materials like humus, cattle dung and kitchen wastes are highly attractive sites for some species. Earthworms are generally absent or rare in soil with a very coarse texture, in soil and high clay content, or soil with pH < 4 (Gunathilagraj, 1996). Earthworms are very sensitive to touch, light and dryness. Water-logging in the soil can cause them to come to the surface. Worms can tolerate a temperature range between 5ºC to 29ºC. A temperature of 20ºC to 25ºC and moisture of 50–60 percent is optimum for earthworm function (Hand, 1988). Biology of earthworms Earthworms are long, narrow, cylindrical, bilaterally symmetrical, segmented animals without bones. The body is dark brown, glistening and covered with delicate cuticle. They weigh about 700–1400 mg after 10 weeks. They have a muscular gizzard which finely grinds the food (fresh and decaying plant debris, living or dead larvae and small animals, and bacteria and 2 protozoa mixed with earth) to a size of 2–4 microns. The gut of the earthworm is inhabited by millions of decomposer micro-organisms. They are bisexual animals and cross-fertilization occurs as a rule. Copulation may last for about an hour, the worms then separate. Later the clitellum of each worm eject cocoon where sperms enter to fertilize the eggs. Up to 3 cocoons per worm per week are produced. From each cocoon about 10–12 tiny worms emerge. Earthworms continue to grow throughout their life and the number of segments continuously proliferates from a „growing zone‟ just in front of the anus. Earthworms contain 70–80 percent high quality lysine rich protein on a dry weight basis. They can be useful as animal feed. Usually the life span of an earthworm is about 3 to 7 years depending upon the type of species and the ecological situation. Eudrilus eugeniae Eudrilus eugeniae is an earthworm species indigenous in Africa but it has been bred extensively in USA, Canada, Europe and Asia for the fish bait market, where it is commonly called as the African Night-crawler. E. eugeniae is a large worm appearing brown and red to dark violet like animal flesh. Their length ranges from 3.2 to 14 cm, and 5 to 8 mm in diameter. It grows faster and better than other species. Life span of this worm ranges from 1 to 3 years.
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