Chapter I THE PROBLEM AND ITS BACKGROUND Introduction The increasing price and scarcity of fossil fuels are exposed as an environmental challenge, a vital and immediate concern of the society as a whole. Amidst this challenge, island ecology is inherently fragile due to the predictable consequences mankind has brought to the motherland, caused by their old habits of carelessness as greenhouse effect contributors. A solution to this problem is to find an alternative for fossil fuels. There are 51 of the plant species that yield higher level of substances for biofuels and one of them is Jatropha curcas. (Fernandez,et al 2007) Jatropha curcas L. (purging nut, physic nut, barbadose nut or sabudum, tubang-bakod or tubang-balayan as is commonly called in Quezon Province), a member in the family Euphorbiaceae, is a large drought-resistant shrub with multipurpose. Physic nut has been the important target in many countries for substitute energy. Various parts of the plant have been used for many aspects. The development of techniques for in vitro culture has been found to be the best procedure to develop tissue for potential mass propagation in order to produce plants in large scale. Currently, the need of plants is increasing from government institutes, private sectors, and farmers for research, development and producing stem

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cuttings, seeds, oil, and substitute energy. During the QUESCAA Convention on the launching of the Science Investigatory Manual at Gavinas Restaurant in Tayabas (Oct. 17, 2007), Governor Rafael P. Nantes of Quezon Province stated that Jatropha can be intercropped with prevailing coconut trees. He

has chosen the said specie among the rest of the plant species due to the fact that it can grow almost anywhere, even on gravelly, sandy and saline soils as well as crevices and rocks. It can also grow in cold climate because it can withstand light frost. Because of its excellent drought resistance; it is also suitable for preventing soil erosion and shifting of sand dunes underscoring its potential as a plant that can possibly grow in areas such as wastelands (Fernandez,2007). There are limited research efforts on this plant as measured by intellectual properties filed before the Phil. Development of Trade Intellectual Property Office (Biofuels from Phil. Plants); hence, this Masters Thesis has been identified for the procurement of further studies on propagating the Jatropha plant for mass production through the culture media such as potatodextrose-agar, coconut-dextrose-agar and Murashige & Skoog. This present investigation significantly plays its greatest part in initially pursuing on the village level with the scientific know-how it can provide the farmers in economic farming. It is to the success of this investigation that the community will hopefully depend on in the most economical way possible, and be part of the scientists processes for further studies in coming up with

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standard culture media to ultimately be utilized solely for the Jatropha curcas mass propagation. Background of the Study To think of the forestland depletion that has caused grave climatic changes, an unfolding crisis has haunted the earth of global warming from the past to our present situation.The earth provides every living organism with the food to eat, air to breath and water to drink; but if people produce a lot of carbon dioxide by burning fossil fuel, if they do not maintain the vehicles properly, fuel is burned incompletely, so what would these bring to the surroundings ? As what was stated by Al Gore in The Inconvenient Truth (April

27, 2008), people all over the world are contributing to the greenhouse effect of 70 million tons a day of carbon dioxide in the air. This is a big problem, nevertheless, the solution to this problem is just within mans reach for it is just a matter of individuals concern to change those old habits. It is the major concern of most developing countries specially the government at present to increase and conserve energy supplies. There is a huge unserved demand for biofuel. The potential demand for the years to come is even larger as country signatories of the Kyoto Protocol will have to comply with their commitments in the use of clean fuels. With the foregoing scenario this thesis attempted a contribution of closing the gap on the side of cultivating the biodiesel plant, Jatropha curcas

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in vitro to hasten mass propagation and production that will provide feedstock to be processed into biofuels. This present investigation could contribute much on its ability to keep up with the growing demand, the biggest challenge the country is currently facing. The key to the future of viable biodiesel is in search for inexpensive feedstocks that can be grown possibly by farmers on marginal agricultural land and become part of the standardized agronomy practices on Jatropha sustainability. Biofuel production is an emerging reality both globally and locally. Due to the fossil fuel depletion the country is now experiencing the continuing increase of fuel prices that has affected most of the basic commodities in terms of their commercial prices. And to think of these increasing prices, related literature has been procured on the utilization of available materials at its possible minimum cost for the culture media utilized for the in vitro cultivation of the biodiesel plant: Jatropha curcas, to hasten multiple production of plantlets. Shoots produced through tissue culture are generally easy to root than the same cultivar by cutting propagation. Techniques for rooting tissue culture shoots are currently receiving a great deal of attention in research studies for different plant species as are methods for establishing these shoots into the greenhouse environment, more advances in the mass production of woody plants have been made. Much research was (and is still is) needed to define the cultural conditions required by plant species.

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With the optimum use of the information, the researcher came up with the study of using potato and coconut milk extractions as substitute for other components in the culture media to define and determine the nutritional requirements and conditions necessitated as compared with the commercially utilized Murashige and Skoog culture media for commercial scale tissue culture production of Jatropha curcas; particularly on village level. Furthermore, for economical modification of these propagation systems and hopefully for contribution to the countrys industry in agronomy, specifically the nursery industry, in the community and as to the national levels. Conceptual Framework This section illustrates the variables used in the study. Independent Variables Kinds of culture media: Coconut-dextrose-agar (CDA) macronutrients:calcium, potassium,phosphorus,nitrogen, micronutrients: sodium,iron Potato-dextrose-agar (PDA) macronutrients:calcium, potassium,phosphorus,nitrogen, micronutrients: sodium,iron Murashige & Skoog (MS) macronutrients:calcium, potassium,phosphorus,nitrogen, micronutrients: sodium,iron Frame1 Figure 1: Research Paradigm Dependent Variables

In vitro cultivation of Jatropha curcas: Level of Growth Parameters: nodal explant number of leaves number of shoots apical explant number of leaves number of shoots

Frame 2

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Frame 1 presents the kinds of culture media like coconut-dextroseagar (CDA); potato-dextrose-agar PDA); and Murashige & Skoog (MS) which were characterized as to their nutritional conditions in terms of macro and micronutrients present to cultivate the Jatropha tissues showing growth parameters as in Frame 2 . Statement of the Problem This present investigation aimed to determine the growth of Jatropha curcas (Tubang- bakod) in three different culture media:, coconut- dextrose agar, potato-dextrose-agar, and Murashige and Skoog. Specifically, this answered the following questions: 1. What is the status of the nutritional condition of the kinds of culture media used for the cultivation of J. curcas plantlets in vitro cultures based on the chemical analysis in terms of: 1.1. Coconut-dextrose-agar a. macronutrients:calcium,potassium, phosphorus,nitrogen, b. micronutrients: sodium,iron 1.2. Potato-dextrose-agar a. macronutrients:calcium,potassium, number of leaves, and shoots which were presented in

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phosphorus,nitrogen, b. micronutrients: sodium,iron 1.3. Murashige & Skoog a. macronutrients:calcium,potassium, phosphorus,nitrogen, b. micronutrients: sodium,iron? 2. What is the level of growth parameters of Jatropha curcas explants in terms of: 2.1. nodal explants a. number of leaves b. number of shoots 2.2 . apical explants a. number of leaves b. number of shoots 3. Is there a significant difference in the level of growth parameters in the three kinds of culture media? Research Hypothesis This thesis forwarded the following hypothesis: There is a significant difference in the growth level parameters of

Jatropha curcas in vitro protocols of three different culture media. Significance of the Study

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This study will be found very significant to the following people, agencies, and organizations:

Business Investors Opportunities Investment on the Jatropha System and Development could lead the investors to great opportunities in the marketing business on provisions of seedlings or plantlets. The use of 11 million hectares of wastelands for J. curcas cultivation can lead to generation of minimum 12 million jobs, (as sourced from a reliable information) . There are by-products derived from the waste of the Jatropha or from the transesterification processes of the biofuel, like soap production,and fertilizers. These by-products are another opportunity business for interested investors. Local Government Organizations There will be improvements in the local government units in terms of employment status and revenues, through the mass propagation of the Jatropha plant in vitro tissue culture. There will be virus-free stocks of the specie and improvements in agronomy. A basic knowledge in the present investigation will lead to further studies on improving the hybridization of the Jatropha plant until an established propagation has reached its maximum scale for sustainable biofuel. Farmers

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Biofuels creates new market specially for many of our farmers. At the present trend farmers produce only enough to feed their families. From a reliable source almost two thirds of the people in the developing world derive their incomes from agriculture and since biofuels are derived from crops, they hold an enormous potential for farmers economic status. Researchers This study, as an eye opener, will lead the researchers to increase the yield traits of Jatropha seeds and intellectually invest on these seeds to be sown and planted on large scale of land aiding the farmers of know-how. Scope and Limitation The research included several techniques in following the protocols for the preparation of culture media and the preparation for PDA, CDA and MS. Samples used (nodal and apical explant) were randomly assigned to systematically varying sequences of conditions . The study evolved around 54 apical and nodal explants of Jatropha curcas , proportionately cut to 3 cm each which were used as cultivars in invitro investigation. The more observations the study evolved in , the more random error tended to balance out. The growth parameters of the Jatropha were measured considering the number of leaves, and number of shoots that sprouted in the explants

within one and a half weeks.

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The initial experiment was operated in one of the U.P-immunological laboratories in the National Institute of Molecular Biology and Biotechnology, UP Los Baos, Laguna for its protocol preparatory procedures, specifically on the Murashige & Skoog preparations. In-vitro cultivation of the explants in PDA, and CDA. was meticulously prepared and done in a non-laboratory setting. Utilizing an improvised biological safety-clean cabinet/box in the cool area , incubation was done in the vegetable compartment of the refrigerator with artificial light utilizing a 25 watt bulb of rechargeable emergency light (8 hours darkness and 16 hours light), under a temperature of 26C. Definition of Terms The terminologies in the study presented in alphabetical arrangement were given following operational meanings: Apical explants. These are the meristem of Jatropha curcas plant tips observed to determine its growth in different culture media such as Coconut-dextrose-agar (CDA), Potato-dextrose-agar (PDA), and Murashige & Skoog (MS). Biodiesel plant. This pertains to the locally viable plant same as tubang bakod or tubang-balayan utilized for biofuel feedstock. Coconut-dextrose-agar (CDA). It is a culture medium that contains coconut milk extract, sugar and agar in definite amounts for rapid proliferation

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of single specie cultivars (J. curcas) that can be manipulated in the laboratory for study. Culture media. They are semi-solidified base containing agar, nutrients and mineral salts in definite amounts necessary for rapid shoot proliferation of single specie cultivars (J. Curcas), used for mass propagation of plant species and for establishment and maintenance of virus-free stocks. In this study, these are the Coconut-dextrose-agar (CDA), Potato-dextroseagar (PDA), and Murashige & Skoog (MS). Cultivar. This is an explant with existing/signs of growth parameters grown in the three culture media (CDA,PDA,MS). Growth parameters. This pertains to the factors on the growth level of the plant specie under study as to number and length of shoot/plantlets and number of leaves to be measured. In-vitro Cultivation. This pertains to aseptically embedding explants into the culture media allowing a week and half gestation period. Jatropha curcas. This is a bio-diesel plant specie belonging to the tree family of Euphorbiaceae that yields 28-36% high level of oil substances. Its apical shoot tip and nodal part are taken as explants in the culture methods In vitro protocols. Macronutrients. This pertains to the large amounts of minerals essential in the proliferation/growth of plant tissues included in the three culture media: the calcium, potassium, phosphorus, and nitrogen,

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Micronutrients. This pertains to the small amounts of minerals essential in the proliferation/growth of plant tissues included in the three culture media, such as: iron and sodium. Murashige & Skoog (MS). It is a type of culture media invented by Murashige and Skoog containing definite amounts of nutrients, hormones, mineral salts, for rapid shoot proliferation of single specie cultivars that can be manipulated in the laboratory for study. Nodal Explants. These are lateral meristematic plant tissues of Jatropha curcas put in culture/extract media and observed to determine the growth parameter of the plant. Nutritional condition. This pertains to the macro and micronutrients which are needed in large and small quantities respectively for the proliferation of growth parameters, such as calcium, potassium, phosphorus, nitrogen, iron and sodium. Potato-dextrose-agar (PDA). This is another culture medium that contains potato extracts rich in cytokinin and auxin hormones, sugar and agar in definite amounts for rapid proliferation of single specie cultivars that can be manipulated in the laboratory for study

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Chapter 2 REVIEW OF RELATED LITERATURE AND STUDIES This chapter presents literature and studies which are found significant to the present research. Related Literature Jatropha curcas The genus name Jatropha derives from the Greek jatrs (doctor) and troph (food) which implies medicinal uses. Its common name is physic nut. Locally, it is known as tubang-bakod or tubang-balayan in Mauban, Quezon. Jatropha curcas, as stated by Fernandez (2007), is a shrub or tree to 6m, with spreading branches and stubby twigs, and a milky/yellowish refuscent exudates. Since Jatropha plants average height is about 3 meters, harvesting is easy and the plant can grow practically anywhere (ordinary soils sandy, gravely or rocky soil) and adapts easily to different climates. It can withstand drought of up to 2 years without rainfall. It is resistant to a high degree of

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aridity (it can be planted even in the dessert) and as such does not compete with food crops as claimed by some authors. Jatropha is well-suited for growing in arid conditions, has low moisture requirements (Sirisomboon et al. 2007), and may be used to reclaim marginal, desert, or degraded land (Wood 2005). The oil content of the seeds ranges from 30% to 50%, and the unmodified oil has been shown to perform adequately as a 50/50 blend with petroleum diesel (Pramanik 2003). The potential use of Jatropha curcas lies in its oil profile as an alternative fuel. Jatropha.curcas was considered (Fernandez, Elvira et al2007) as the second most promising prospect for energy generation, second to Cocos nucifera. The articles/studies written by Fernandez et al (2007), Sirisomboon (2007), Promanik (2003) are related to this research because all of them made mention of Jatropha curcas which was considered as subject being investigated. In 2006, the Food and Agricultural Organization studied the potential of Jatropha as biodiesel feedstock in the Philippines. In UPLB R & D program teams of scientists and researchers are currently working on the germplasm collections, varietal improvement, component technologies with respect to plant propagation, pruning, flower and fruiting management, fertility management, pests and diseases management, post-production technologies , and processing of biodiesel and by-products of Jatropha curcas Linn. Var.

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It has been reported that UPLB and CHED are working together for the provenance testing of Jatropha in 17 locations where cooperating SUCS are located.(www.relocalize.net/jatropha_what_the_public_should_know)

As stated in one study of Luis Roy I. Velasco (Chancellor of UPLB) on UPLB R & D Programs on Jatropha and Biofuels, UPLB worked on the promotion of biofuels. That during the process, the food security and general welfare of Filipinos are not jeopardized. UPLB is one with the government, inspite of the mass production of Jatropha, in ensuring that food crop areas are maintained and protected for food security and should not be neglected and deprived of much attention. In Jomalig and Panukulan, Polilio Group of Islands, small scale exploration efforts pursues the use of Jatropha nut shells to fuel stoves and the processed seed oil to other small-time village-level power generators. Depleted minerals and climatic conditions like degraded grasslands, denuded uplands, lahar-affected areas and even mine silted areas are targeted sites for Jatropha plantation, areas where food crops are not planted or cannot be planted . ( www.relocalize.net/jatropha_what_the_public_should_know ). Recently, December 30,2008, a passenger Air New Zealand Boeing 747-400s Rolls-Royce RB211 engine jet, with one of its four engines running on a biofuel blend made of 50:50 Jatropha and Jet A1 fuel completed the worlds first commercial aviation test flight. The Jatropha used on this flight

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was grown in Malawi, Mozambique and Tanzania. The criteria for sourcing the Jatropha oil required that the land was neither forest land nor virgin grassland within the previous two decades. The seeds for this test flight are rain-fed and not mechanically irrigated, growing on poor soil and in arid climate not suitable for most food crops. The test flight partners engaged Terasol Energy, a leader in sustainable Jatropha development projects, to independently source and certify that the Jatropha-based fuel for the flight met all sustainability criteria. Air New Zealand aims to meet 10% of its fuel needs through sustainable biofuel by 2013. (www.jatropha.de/news/jel-news.htm) Relatively speaking the present study selected Jatropha curcas as mother plant of explants to grow in the culture media to match up with the growing demand of biodiesel fuel, through mass cultivation in vitro. And

primarily, to eventually cope up with the communitys mass propagation. Matured leaves of Jatropha are three-five lobed with a spiral

phyllotaxis. Leaves are ovate large green to pale green basally cordate 3-5 lobed in outline, 6-40 cm long, 6-35 cm broad, the petioles 2.5-7.5 cm long which are arranged alternately . The cells of meristematic tissues are similar in structure and

have thin and elastic primary cell wall made up of cellulose. They are compactly arranged without inter molecular spaces between them. Each cell contains a dense cytoplasm and a prominent nucleus. Dense protoplasm of meristematic cells contains very few vacuoles. Normally the

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meristimatic cells are oval, polygonal or rectangular in shape. The shoot of Jatropha is a dome- shaped growth proliferating from the apical or nodal meristematic part of the plant arising within 1-2 weeks of a soil propagated plant as bserved in t his present pre-investigation. Shoots are the source of all above-ground organs. Cells at the SAM (Shoot apical meristem) summit serve as stem cells to the surrounding peripheral region, where they proliferate rapidly and are incorporated into differentiating leaf or flower primordia. Nodal meristem consists of cells which mainly divide in one plane and cause the organ to increase in diameter and girth. It is also present at the base of node, internode and on leaf base. They are also responsible for growth in length of the plant. Nodal meristem also occurs between nodes beneath the bark of the tree in the form of cork cambium and in vascular bundles of dicots in the form of vascular cambium. The activity of this cambium results in the formation of secondary growth. (http://en.wikipedia.org/wiki/Meristematic. Relatively, in the present study it was shown that growth from the nodal explants elongated laterally and no sign of growth at the top were seen. This only indicated the initial growth leading to bush growth. Nodal explants in the present study were used as subjects, where growth

parameters were observed to develop and lateral elongation of shoots and increase in girth continued. The explant from which the growth parameters were observed came from this secondary growth of mother plant. The shoot

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apical meristem is the site of most of the embryogenesis in flowering plants. Primordia of leaves, sepals, petals, stamens and ovaries are initiated there at the rate of one every time interval called a plastochron. It is where the first indications that flower development has been evoked are manifested. One of these indications might be the loss of apical dominance and the release of otherwise dormant cells to develop as axillary shoot meristems, in some species in axils of primordial as close as two or three away from the apical dome. ( www.nationmaster.com/encyclopedia/Meristem - 84k ) Single cells of leaf tissue can regenerate whole plants, as shoot tips, leaf pieces, root pieces, lateral buds, or stem sections. Not all of the methods used to regenerate the above tissues as prepared and made by various

scientists/propagators were applicable to woody plants and indeed, not all had been applied to a commercial level to any plant. But in this study, regeneration of leaf tissues from cultivars existed from the nodal and apical meristems/explants. Apical dominance is phenomenon where one meristem prevents or inhibits the growth of other meristems. As a result the plant will have one clearly defined main trunk. For example, in trees the tip of the main trunk bears the dominant meristem. Therefore the tip of the trunk grows fast and is not shadowed by branches. If the dominant meristem is cut off, one or more branch tips will assume dominance. The branch will start growing faster and the new growth will be vertical. Over the years the branch may begin to look

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more and more like an extension of the main trunk. Often several branches will exhibit this behavior after the removal of apical meristem, leading to a bush growth.

Culture Media and their Nutritional Components Culture media are growth media, solutions freed of all microorganisms by sterilization (usually in an autoclave, where it undergoes heating under pressure for a specific time) and containing the substances required for the growth of different tissues of organisms. Specific procedures are employed like isolation, cultivation, and manipulation of microorganism for the

propagation of plant cells and tissues. A relatively massive minute number of cells , the explant, is introduced into a sterilized nutrient environment, the medium. The culture medium in a suitable vessel is protected by loose-fitting covers with overlapping edges so as to allow diffusion of air, yet prevent access of contaminating organisms from air or from unsterilized surfaces. The transfer, or in vitro cultivation usually is done with an aseptic, sterilized forceps to carry the explants and embed at about 0.5cm deep gently on the surface of the culture medium . There is at present no way to predict the exact growth medium and growth protocol, to generate a particular type of callus. These characteristics are determined through a carefully designed and observed experiment for each new plant species, and frequently also for each new variety of species

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which was taken into tissue culture.

The basis of the experiment would be

media and protocols that give the desired effects in other plant species and experience.

Culture Media a. Coconut-dextrose-agar (CDA) , which was derived from a successful study made in An alternative culture medium for rapid detection of aflatoxins in agricultural commodities ( Atanda et al, 2006). This literature on the success of the effective use of a desiccated coconut agar had triggered the researchers thoughts on preparing the coconut-dextrose-agar as culture medium. It contains coconut milk rich in nutrients and sugars necessary for multiple proliferation or growth of plant tissues b. Potato-dextrose-agar (PDA) which was derived from the preparation used effectively with 20 % leaf extract of Jatropha curcas as culture medium in : Antifungal Effect of Leaf Extract of Some Medicinal Plants Against Fusarium oxysporum Causing Wilt Disease of Solanum melogena L. (Siva et al, 2008), also made the researcher a place in the choice of culture medium for this present investigation. It contains potato extract rich in cytokinin, auxin for growth of plant tissues, and in sugar and nutrients necessary for the shoot proliferation.. c. Murashige & Skoog (MS) which is routinely used for majority of invitro preparations on micropropagation of higher plants like in the study of

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orchids, contains the much-needed nutrients that the explants normally acquire in its native habitat during proliferation of shoots and develop into plantlets that are large enough to grow on their own. Similarly the above culture/extract media were re-investigated in the present study. The nutritional value of the two cultures, potato and coconut as extracts of raw materials used in the preparation of potato -dextrose-agar and coconut -dextrose-agar culture media is chemically analyzed. For every composition gram values per 100 grams the following were found, such as 1. potato had significantly more carbohydrates and water contents than coconut, fats were more in coconut than in potato ; 2. significantly more phosphorus and potassium nutrient content in potato than in coconut, but more of the salts were in coconut than in potato; 3. There are significantly more Vit. A and C in potato than in coconut; 4. there are significant amounts of lipids in coconut than in potato. Furthermore, the

minerals generally needed by plants in large quantities (macronutrients) are: calcium which forms part of the cell wall of plant cells, calcium is an ionically stable divalent cation with important beneficial and toxic properties in cell culture. It is a component of a wide range of cell culture media. Calcium is involved with a wide range of vital cell functions including enzyme activities, attachment, motility, tissue morphology, metabolic processes, signaltransduction, replication, and is stored primarily in the endoplasmic reticulum (ER); the biggest chemical issues with calcium in cell culture are its solubility

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and bioavailability. Calcium is frequently added to cell culture systems as freely soluble calcium chloride. Once in solution, calcium redistributes and associates with other ions and molecules in the medium. There are a number of ions and molecules in cell culture systems to which calcium may bind and form insoluble or slightly soluble molecules. Chelators such as citrate and ethylenediamine tetra-acetate (EDTA) are sometimes used in cell culture, like the ones used in the preparation of Murashige & Skoog. EDTA has a log affinity for calcium of approximately 10.6 and it is often used to remove calcium from cell cultures media, especially when cells are being detached from substrates or when cell clumping is a problem;

(http://www.sigmaaldrich.com/life-science/cell-culture/learning-center/mediaexpert/calcium.html) potassium, which controls opening and closing of stomata and activates enzymes, potassium also regulates osmotic potential, the principal inorganic cation; phosphorus, which forms part of ATP (adenosine triphosphate), nucleic acids, and other organic molecules for energy metabolism of the cell. It is available in tissue culture media as sodium hydrogen phosphate or potassium hydrogen phosphate, similar to the preparations of Murashige & Skoog. Its deficiency results in delayed growth and dark green colour of the leaves; nitrogen, which forms part of amino acids, proteins and other organic molecules; sulfur which forms part of some amino acids; and magnesium which forms part of chlorophyll; minerals in small quantities (micronutrients) are the following: sodium which is a component in the enzymatic action for active transport of materials; iron which

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is needed in the formation of chlorophyll; chlorine which helps in water balance; zinc which activates enzymes; manganese which regulates enzyme function and growth of pollen grains; boron which activates enzymes; copper which is a component of enzymatic actions; and molybdenum which is essential in nitrogen fixation. (http://www.oup.com/uk/orc/bin/9780199282616/ch02.pdf); Coconut milk contains substance/s that stimulated mature cells to enter and remain in cell division cycle. In past 50 years, coconut milk was a necessary addition in plant tissue culture. Scientists knew of a growth factor in coconut milk but could not separate it from coconut milk. Later the growth factor was known as

cytokinin. There were hundreds of cytokinins found in plants, the most effective is zeatin. "Plant zeatin stimulates the rapid split of cells, promotes the growth of lateral buds and the expansion of water plant leaves. (www.biologie.uni-hamburg.de/bonline/e31/31.htm) Relatively, this information had triggered the researchers choice in utilizing coconut-dextrose-agar as one of the culture medium. People trying to get tissues to grow in vitro, knew the importance of minerals and vitamins in the medium. They tried various additives to get tissues to grow optimally. Single tissues could grow for at least a limited time, but getting a whole plant to develop from the culture was not possible. One additive that seemed to help a lot was the addition of coconut water (liquid endosperm).

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The major components of the nutrient medium that influence water availability are the concentrations of agar, the carbon source as an osmoticum. Dextrose as a carbohydrate plays an important role in the regulation of the external osmotic potential. A suitable starting point for the initiation of callus from a dicot tissue plant would be a preparation of the MS basal medium since it is relatively high in potassium and nitrogen in comparison to other nutritional media, (http://books.google.com/books) hence was chosen by the researcher for this investigation. Related Studies Jatropha curcas The country is in its track of joining the competitive world in search for sustainable alternative fuel and enthusiasts alike, should not be left behind in doing their part, particularly on the village level on Jatropha propagation. The research reiterates the development track and for being true on mandating initially the deliverance of concrete results for the community in the choice of an economical home tissue culture media for mass cultivation of Jatropha, leading to the ultimate mass soil propagation. From the work of Rashida Soomro and Rabia Asma Memon in Establishment of callus and suspension culture in Jatropha curcas(2007) callus cultures were initiated from leaf and hypocotyl explants isolated from four-day old seedling of Jatropha curcas L., on Murashige & Skoog (1962)

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basal medium supplemented with different growth regulator formulations including 2,4-D, ABA, GA3 gibberellic acid,and coconut milk.(Soomro et al, 2007). The present study is related in its use of coconut milk extract to induce the growth of leaves and shoots from Jatropha curcas L. explants. Shoot regeneration from apical shoots, nodes, axillary bud-derived shoots, petioles and leaf explants were assessed on Murashige and Skoog (MS) medium supplemented with different concentrations of N6-benzyladenine (BA) alone, or in combination with indole-3-butyl-butyric acid. Sujatha and Mukta (1996) have reported a method for the differentiation of adventitious shoots through callus derived from hypocotyl, petiole, and leaf explants of J. curcas. In vitro clonal propagation of seven-month old Jatropha curcas L. variety was achieved employing nodal explants (Mukul Manjari et al, 2007). They found in their study that axillary shoot bud proliferation was best initiated on Murashige and Skoogs basal medium supplemented with 22.2 M N6-benzyladenine (BA) and 55.6 M adenine sulphate, in which culture produced 6.20.56 shoots per nodal explant with 2.00.18 cm average length after 4-6 weeks. The present study, likewise used a seven-month old mother plant similar to the selected mother plant in the experiment of Manjari, et al. In the works of Kalimuthu, et. al. (2007) in : In vitro Propagation of the Biodiesel Plant Jatropha curcas L.The biodiesel plant, Jatropha curcas L. was

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micropropagated using nodal explants on MS supplemented with BAP (1.5 mg/l), Kn (0.5 mg/l) and IAA (0.1 mg/l). Somatic embryos were induced directly from green cotyledon explants on MS fortified with 2 mg/l of BAP. Culture Media In the work of Kalimuthu et. al. (2007) In vitro propagation of the biodiesel plant Jatropha curcas L. Basal medium comprised MS basal salts, 3% sucrose and 0.8 % agar supplemented with different concentrations of auxins - IAA (0.05 - 0.1mg/l), cytokinins BAP (0.5 - 3.0 mg/l), Kn (0.5 mg/l) before autoclaving. In this aspect the present study had utilized the same plant: Jatropha curcas L. variety for the explant cultured in the three culture media, leading to similarities of subjects. Other aspects of the micropropagation process can be performed using methods known to those of skill in the art in plant tissue culture. Micropropagation typically involves the following steps:1) culturing explants in initiation media to generate multiple shoots; proliferation and elongation media; 2) transferring shoots to

3) transferring t he elongated shoots to

rooting media; 4) hardening the plantlets; and 5) transferring the hardened plantlets to fields. But in this present study cultivation of the explants with

existing or signs of growth parameters are cultured in the three culture media to investigate the efficiency or effectiveness of the components in the three prepared culture media: coconut-dextrose-agar, potato-dextrose-agar and the

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Murashige

&

Skoog

which

would

eventually lead made by

to further other

the

micropropagation process to be investigators/scientists alike. through for farmers too.

yet be

interested

This present study could be starters, a break-

The basal media used to culture Jatropha can be any of those already known in the field of the art for plant tissue culture, such as Murashige & Skoog, Gamborg's, Vacin & Went, White's, Schenk & Hildebrandt or the like. Basal media can also be supplemented with various carbon sources . The carbon source may be sucrose or glucose, typically, at a concentration of about 2-5%. The carbon source may also be sugar alcohol like myoinositol, typically, at a concentration of about 50-500 mg per liter. In relation to the concept on the field of art for plant tissue culture, it has been gleaned from the experiment that basic components of nutrients most especially macronutrients of calcium, potassium, phosphorus and nitrogen; micronutrients of iron and sodium; furthermore, like sugars, lipids, vitamins, and minerals are essential in the induction of leaf and shoot from apical and nodal meristematic explants.

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Chapter 3 RESEARCH METHODOLOGY This section, reveals the procedures and process of the investigation to determine the growth of Jatropha curcas (tubang- bakod/tubang-balayan) in three different culture media: coconut- dextrose agar, potato-dextroseagar, and the Murashige & Skoog. Research Design The intent of this study was to describe briefly the contemporary techniques of tissue culture of woody plants as an initial propagation, that is, in vitro cultivation, a method for Jatropha curcas, utilizing experimental research method. According to Sevilla (1997) experimental research is a method of manipulating certain stimuli, treatment or condition observed to determine the change or effect it brings to the phenomena under

investigation. This experimental design allows the results of researches in comparing them with each other. A completely randomized design was used because it is the easiest design to employ and analyze, even when there were missing data or when treatments were unevenly spaced, with the only recognizable differences between explants with the treatments being applied

29

to them. All treatments consisted of three replicates and each replicate contained three explants. The method was appropriate because when the treatment was administered, all groups represented a random sample of the population consisting of every cultivar (explants with signs of growth). The variety of techniques that were used to get plant development in vitro was considerable and completely dependent upon the species in question. Subjects of the Study This study focused on the growth of Jatropha in 27 culture vessels

involving matured seven - month old plant of the Jatropha curcas from which green soft tissues of meristematic in nature were selected. The shoot apical and the nodal portion of the plant were cut into 3 cm each as explants. Data Gathering Procedures Supplies, Materials and Equipment for Media Preparation 1. Preparing the materials for Murashige and Skoog tissue Culture medium and for sterilization in laboratory setting: The following materials used for the protocol procedures came from the Institute of Molecular Biology, UPLB, Los Baos:. 500 ml Erlenmeyer flask for a 250 ml preparation; magnetic stirrer; one liter of triple distilled water; mayonnaise jars as culture vessels, with lids which can withstand the heat inside a pressure cooker; agar (2 g); aluminum foil to wrap scalpels and forceps for sterilization; 500 ml graduated cylinder for measuring liquids;

30

Closed type electronic analytical balance for weighing solids accurately. Pressure cooker for sterilization; microwave oven for cooking the media and drying other instruments; pH meter calibrated to pH 5.7; paper toweling/A4 white paper used for sterile cutting surface, cut to size about as big as a cup saucer; scalpel and forceps; pH adjusters: 6N HCl and 2N NaOH (for home tissue culture alternative, kalamansi juice and sodium bicarbonate or baking soda). 2. Other items used in the home culture were as follows: A sterile fish tank or an improvised clean box made of plastic cover as safety biological transfer cabinet was utilized to transfer explants with dimensions of 50 cm (length), x 40 cm (height) , x 40 cm (width) which laid on its side on

a flat table where most of the operations in the manipulations of the explants took place; 2. Pressure cooker for sterilization of media, instruments, water, paper toweling, etc; 3. Glass jars (baby food jars) with lids/microwaveable plastics that withstood the heat of the pressure cooker.These were used for culturing ( as culture vessels); 4. Scalpel and forceps; 5. Paper toweling / A4 white copier paper used for sterile cutting surface, cut to size about as big as a cup saucer; 6. A spirit lamp containing 70% ethanol for flaming instruments; 7. Hand held spray bottle containing 70% ethanol to spray the transfer/cabinet and other surfaces; 8. Skin disinfectant (hand sanitizer) was used for disinfecting hands before and after explants; 9. Media preparations ( see research procedures). any manipulations of the

31

3. Equipment for media preparation and materials for use in the transfer chamber/cabinet were: forceps (2 or more), tweezers, kitchen paring knife , scalpel, rubber gloves, spray bottle with 70% ethyl alcohol (flammable), household bleach, plastic dish to hold 10% bleach solution (1 part bleach and 9 parts sterile water) for sterilizing instruments and gloved hands. Plastic dish to hold 1% rinse solution (1 part bleach and 99 parts sterile water) for rinsing instruments and gloved hands . Device to hold sterilized instruments as they are cooled or dried: Metal rack or wooden rack; Sterile surface on which to cut cultures; 12 inch square of plate glass (spray with alcohol); and single sheet paper towels which were wrapped in foil and sterilized in pressure cooker for one hour at 15 lbs pressure. The tissue culture media: coconut-dextrose-agar; potato-dextrose-agar

were homely prepared in the kitchen while the Murashige & Skoog utilized the standard preparation in the UP laboratory. Construction Procedures A. Preparation of tissue culture- media 1. Coconut-dextrose-agar culture medium: Medium sized coconut about the size of a small kids basketball was selected; shells cracked, and the white meat grated. A measured 50 ml of coconut milk was poured into the 500 ml beaker. A weighed dextrose of 5 grams and 1.625 grams of agar (Mr. Gulaman) was measured and mixed into the prepared solution, stirring constantly. The agar did not dissolve. The pH

32

was checked and adjusted to 5.7 by adding kalamansi juice to a lower level or a base: baking soda to a higher level. 250 ml line on the flask and stirred. Distilled water was added to the The mixture was poured into the

sterilized culture vessels so that a depth of 2cm media was in each of the 9 vessels. The culture vessels were covered and sealed with microwaveable plastics and rubber band.. The sealed vessels were boiled in the pressure cooker for 15 minutes at 15 psi. They were cooled and allowed to set at about one hour. After the medium was completely cooled, the vessels were

wrapped in plastic baggies to keep moisture in and stored in the refrigerator as needed. 2 Potato-dextrose-agar tissue culture medium Potatoes free from any brownish spots were selected, weighed, peeled and soaked in water to prevent browning of the starch during preparation. A weighed 62.5 grams of healthy potatoes were peeled, boiled in a casserole of 500 ml water and drained and mashed in cheesecloth. The drained water The mashed potato was

was set aside for later use as stock solution.

extracted of its juice. The stock solution of 200 ml was measured in 500 ml graduated cylinder and poured into the 500 ml beaker of the extracted juice. . A weighed dextrose of 5 grams and 1.625 grams of agar (Mr. Gulaman) was measured and mixed into the prepared solution, stirring constantly. The agar did not dissolve.The pH was checked and adjusted to 5.7 by adding kalamansi juice to a lower level or a base: baking soda to a higher level. Distilled water was added to the 250ml line on the flask and stirred. The

33

mixture was poured into the sterilized culture vessels so that a depth of 2cm media was in each of the 9 vessels. The culture vessels were covered and sealed with microwaveable plastics and rubber band. The sealed vessels were put in the pressure cooker and submerged in water and boiled for 15 minutes at 15 psi. The vessels were cooled and allowed to set at about one hour. After the medium was completely cooled, the vessels were wrapped in plastic baggies to keep moisture in and stored in the refrigerator until needed. 3. Murashige & Skoog basic tissue culture medium The Murashige & Skoog utilized the standard preparation in the UP laboratory. A 500 ml flask for 250 ml of medium was used to contain boil- over that occured during the sterilization process. Two hundred ml of Weighed chemical substances for MS

distilled water was poured into it.

preparations was added slowly into the first flask stirring constantly using the magnetic stirrer. Using squirt bottles with 25 ml distilled water, left

over powder was flushed out from their weighing containers. In preparation for the culture medium to be poured into culture vessels the following amount of each component of the Murashige & Skoog was prepared: MS I = 10 ml b. MS II = 2.5ml c. MS III = 2.5ml . Iron Chelate = 2.5 ml a.

The above amounts were mixed together in a 500 ml beaker, with 5 grams of dextrose and 1.625 grams of agar stirring constantly. The agar did not dissolve. The pH was checked and adjusted to 5.7 by adding 6N HCl to a lower level or a base: 2N NaOH to a higher level. Distilled water was added

34

to the 250 ml line on the flask and stirred. The mixture was poured into the sterilized culture vessels so that a depth of 2cm medium was in each of the 9 vessels. The culture vessels were covered and sealed with microwaveable plastics and rubber band. The sealed vessels were sterilized in the pressure cooker for 15 minutes at 15 psi. The vessels were cooled and allowed to set at about one hour. The medium was completely cooled, the vessels were wrapped in plastic baggies to keep moisture in and stored in the refrigerator until needed. B. Preparation of Jatropha curcas explant 1. Sterilizing the explants The explants were initially washed with running tap water to remove dust. Forceps were used to hold the tip of the explants as were kept in a systemic fungicide for 3 hours. The explants were immersed and soaked in a jar of 70% ethanol for 30 seconds. The sterilized explants were then

transferred to a jar of 10% sodium hypochlorite/chlorox bleach solution for 5 minutes. To free the explants from the bleach solutions, they were then

washed by soaking in distilled water in four successive beakers. They were soaked for 2 minutes in each of the beakers. 2. Culturing of the explants into the media In every manipulation, bottom part (base) of explants were trimmed to about O.5cm on the sterile paper toweling. Used paper toweling was discarded after every manipulation to minimize contamination. Using the

35

sterilized forceps, explants were placed on the growth medium by gently embedding the trimmed bottom part of the explants at a depth of 0.5cm. Three pieces per culture vessel were cultured. In every manipulation of the explants embedded, the forceps were flamed over the spirit lamp; immersed in 70% ethanol; then washed into distilled water before picking up the explant. Before sealing the inoculated medium, the top surface was flamed over a spirit lamp to prevent/reduce any contamination. The culture vessels were then incubated at 26o C in 16 hours of light (artificial light of 25 watts: Fluorescent bulb/emergency light) and 8 hours of darkness in the vegetable compartment of the refrigerator. Try-out and Revision Data were recorded for the visible growth parameters. A. For the number of leaves: The same cultivar selected for the viable healthy shoots were utilized for the counting of the number of leaves, provided they were free of contamination of any sort of organisms. Bright green, smooth growing leaves were an indication of a healthy cultivar. B. For the Number of Shoots: Through the transparency of the culture vessel, observation on viable shoots were carefully examined with the naked eye. All the cultivars with

36

viable growth of shoots were selected. The number of shoots grown from the base of the same cultivar were counted and recorded.

Research Instrument Instruments used in data gathering for measurements using an investigators notebook. Data were recorded on a checklist for the visible growth parameters, please refer to each tables as presented in this investigation in Chapter 4 from table numbers 5 to 10. The following were done systematically: Each culture vessel was labeled during the examination for growth using gum labels to indicate the values for growth parameters and to minimize confusion on which vessel was already examined. Using forceps the explant were gently and cautiously retracted out from the culture vessel , placed on the sterilized A4 paper towel and carefully observed for of the cultivar, the

contamination and counted. With every manipulation

forceps were flamed and sterilized prior to each handling. As each cultivar in the culture vessel was examined , values were also prepared Format of Data table. Statistical Treatment of Data The mean for each treatment was calculated. All treatments of three recorded in the

triplicates and each triplicate contained three explants. The number of shoots and leaves were recorded after 14 days in culture. At 0.05 alpha level of

37

significance was used to test if there were significant differences in the growth level parameters of J. curcas explants in the three types of culture media .The following statistical tools were used to treat the data gathered in the study: ANALYSIS 1. Status of the kinds of the culture media in terms of nutritional conditions . STATISTICAL TOOL Based on the chemical analysis of DOST, in appendices are results of analysis, page 96 2. Level of Growth parameters of J. curcas explants in different media. 3 .Difference in the growth level parameters in three culture media. One way- ANOVA Frequency

38

Chapter 4 PRESENTATION, ANALYSIS, AND INTERPRETATION OF DATA This chapter presents the results of the statistical treatment that led to the following discussions. Status of the Kind of Culture Media in terms of Nutritional Conditions based on chemical analysis: 1. Coconut-dextrose-agar (CDA) The following list summarizes the nutritional components per 100 grams of prepared media: Table 1 Nutritional Components in the Preparation of Coconut-dextrose-agar Nutrtional Components Macronutrients calcium Potassium phosphorus nitrogen Micronutrients Iron sodium CDA mg/ 100 Grams 2.01 270 39.24 0.22 0.71 19.77 TOTAL

311. 47 mg

20.48 mg

39

It is gleaned in the table that there is dominant potassium content in the coconut-dextrose-agar (CDA) culture medium by a milligram component of 270 per 100 grams. Among the nutritional components present in the coconut-dextrose-agar, nitrogen of 0.22 mg per 100 grams is found to be

the least. Although, coconut is found to be high in lipid content, refer to any nutritional status (RDA allowances) of nutritional values of its raw material. 2. Potato-dextrose-agar media The following table shows the summarized potato nutritional components of the media: Table 2 Nutritional Components in Preparation of Potato-Dextrose-Agar (PDA) Nutrtional Components Macronutrients calcium Potassium phosphorus nitrogen Micronutrients Iron sodium PDA mg/ 100 Grams 3.38 470 22.38 0.12 0.80 17.02 TOTAL

495.88 mg

17.82 mg

Table 2 evidently shows the nutritional components of potato-dextrose agar. It can be gleaned that potassium of 470 mg is in the highest nutrients of potato. Nitrogen at 0.12 is the

composition among the other

least but that the least amount does not necessarily mean it is insufficient to favor growth of tissues but the amounts are suffice to cell proliferation.

40

The culture potato-dextrose-agar (PDA) has a total of 495.88 mg macronutrients comparably of higher amounts, hence produced/continued growth/elongation of more shoots. As to the effects of dextrose in potato-dextrose-agar: absence of sugars inhibited shoot production. Shoot production was also limited on the low dextrose concentration, but comparable with the control at high dextrose. 3. Murashige and Skoog Table 3 Nutritional Components of Murashige & Skoog (MS) Nutrtional Components Macronutrients calcium Potassium phosphorus nitrogen Micronutrients Iron sodium M&S mg/ 100 Grams .044 .77 0.0164 0.164 0.278 0.3724 TOTAL

0.9944 mg

0.6504 mg

Table 3 shows the raw materials utilized for the laboratory culture media as follows; for every composition gram values per 100 grams, macronutrients is found to be the greatest components at 0.9944 mg. Murashige & Skoog is a highly balanced salt mineral culture media hence expected more number of leaves and shoot tissues. MS media had higher organic fractions than the two media types, hence a longer shoot

41

formation was also observed. Murashige & Skoog was regarded as a highsalt medium because of its macroelement composition,obviously growth was observed for having an optimized regeneration of shoots and leaves. At low and zero levels of mineral salts, it is said that no growth will occur. Table 4 Summary on Nutritional Components of the Three Culture Media Nutritional Components Macronutrients Micronutrients Culture Media mg/100gCDA PDA MS

311.47 20.48

495.88 17.82

0.9944 0.6504

Table 4 indicates the amount of nutritional components of the three culture media used in the cell proliferation of Jatropha curcas. It appears that potato has significantly more macronutrients with content of 495.88 mg/100g. Referring to the complete components of the coconut and potato extract from reliable sources, it shows that more phosphorus and potassium nutrients are contained in potato than in coconut, but there is more of the salts in coconut than in potato. There are more Vitamins A and C in potato than in coconut but there are bigger amount of lipids in coconut than in potato.

42

Figure 2:The Nutritional Components of the Three Culture Media It is gleaned in Table 4 and in the line graph Figure 2 that nutritional components of the Murashige & Skoog is significantly the least, but more balanced ratio of nutrients than in PDA and CDA culture media as seen from the closeness of the red and blue lines against their values. Referring to standards Murashige & Skoog has balanced values or relatively close values in basal salts (micronutrients) or other minerals as compared to the culture media of CDA and PDA.

Level of Growth Parameters

43

The following data reveal

the distribution of values on growth

parameters on nodal explants and apical shoot in terms of numbers. The raw data are computed for their total and average. Table 5 Growth Parameters in Coconut-Dextrose-Agar MediumType of medium CDA-A Explants in Vessel Nodal-A1 Leaf Number Per Explant 3 4 3 4 3 3 4 3 4 31 Ave. 3.33 Shoot Number Per Explant 1 2 1 2 2 2 1 1 2 14 Ave. 1.33

CDA-A

Nodal-A2

3.33

2

CDA-A

Nodal-A3

3.67

1.33

Total

10.3 33.44

4.6 61.55

Ave.

Per shoot

Per cultivar

Table 5 presents the growth of leaves and shoots in the 9 nodal explants in Coconut-dextrose-agar medium. It appears that the total number of leaves in the 3 triplicates with 3 explants per triplicate of 3 cultures is 31, the total shoot number is 14 with an average of 3.44 leaves per shoot and an

average of 1.55 shoots per cultivar.

44

Table 6 Growth Parameters in Potato-dextrose-agar MediumT ype of media Explants in vessel Leaves number Per-explant Shoot number per-explant

Av e.

Av e.

PDAB

nodal-B1

2 4 6

4

1 2 3

1.33

nodal-B2

3 4 4

3.67

1 2 2

1.67

nodal-B3

3 4 6

4.33

2 2 2

2.33

Total Ave.

36 Per shoot

12 4

17 Per cultivar

5.3 3 1.7 8

Table 6 presents the growth of leaves and shoots in the 9 nodal explants in Potato-dextrose-agar medium. It appears that the total number of leaves in the 3 triplicates with 3 explants per triplicate of 3 cultures is 36, The total shoot number is 17 with an average of 4 leaves per shoot and an

average of 1.78 shoots per cultivar.

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Table 7 Growth Parameters in Murashige & Skoog MediumType of medium MS-A Explants in Vessel Nodal-C1 Leaf Number Per Explant 6 4 6 MS-B Nodal-C2 2 3 3 MS-C Nodal-C2 2 2 3 total Ave. 31 Per shoot 10.33 3.44 2.33 2.67 Ave. 5.33 Shoot Number Per Explant 3 2 3 1 2 2 1 1 2 17 Per cultivar 9.67 3.22 5.33 1.67 Ave. 2.67

Table 7 presents the growth of leaves and shoots in the 9 nodal explants in Murashige & Skoog Medium. It appears that the total number of leaves in the 3 triplicates with 3 explants per triplicate of 3 cultures is 31, The total shoot number is 17 with an average of 3.44 leaves per shoot and an

average of 3.22 shoots per cultivar. The high mineral salts in Murashige & Skoog also allowed the production of true-to-type, disease-free cultivars of shoots and leaves..

46

In Vitro Cultivation of Jatropha curcas in three different culture media Nodal Explants Number of shoots Number of leaves CDA 3.44 1.55 4.99 PDA 4 1.78 5.78 MS 3.44 3.22 6.66

Total Average Growth

Figure 3: Growth Parameters of the Nodal explants in the three culture media It is gleaned in Figure 3 that the values of the number of leaves and shoots grown in MS have relativity in closeness, while those grown in CDA and PDA show a farther relationship of values for the number of leaves and shoots. This farther relationship of values on CDA and PDA are most probably the effect of a high value in the potassium contents (270 mg/100g and 470 mg/100g respectively). The principal inorganic element is

47

responsible for the osmotic potential, closing and opening of stomata in the internal plant tissues for the exchange of gases while cultivated in enclosed vessel of the culture media. The more balanced ratios of nutrients present in Murashige and Skoog could also account for such factors in influencing the significant effect on the growth parameters for having the highest total average growth of 6.66 among the three culture media. And since CDA contributed to the more unbalanced ratios of nutrients as gleaned from figure 2, then this could probably be the factor that caused a significant effect on its lowest value of total average growth of 4.99. Growth Parameters of Apical Explants Table 8 Growth Parameters in Coconut-Dextrose-Agar MediumLeaf Number Type of Medium CDA-A Explants in Vessel Apical-A1 Per Explant 2 4 3 2 3 4 3 3 2 27 Per shoot Ave , 3 Shoot Number Per Explant 1 2 1 1 2 2 3 2 2 16 Per cultivar Av e. 1.33

the

CDA-A CDA-A

Apical-A2 Apical-A3

3 2.67

1.67 2.33

Total Ave.

8.6 7 2.89

5.3 3 1.78

Table 8 presents the growth of leaves and shoots in the 9 Apical Explants in Coconut-Dextrose-Agar Medium. It appears that the total number of leaves in the 3 triplicates with 3 explants per triplicate of 3 cultures is 27,

48

The total shoot number is 16.The table, likewise, shows an average of 2.89 leaves per shoot and an average of 1.78 shoots per cultivar. Table 9 Growth Parameters in Potato-dextrose-agar MediumType of Medium PDA-B Explants in Vessel Leaf Number Per Explant 3 2 2 PDA-B Av e, 2.33 1 1 1 2.67 1 1 1 2.33 1 1 1 7.33 2.44 9 Per cultivar 3 1 1 1 Shoot Number Per Explant e. 1 Av

Apical-B1

Apical-B2

3 2 3

PDA-B

Apical-B3

3 2 2

Total Ave. Per shoot

22

Table 9 presents the growth of leaves and shoots in the 9 Apical Explants in Potato-Dextrose-Agar Medium. It appears that the total number of leaves in the 3 triplicates with 3 explants per triplicate of 3 cultures is 22, The total shoot number is 9. The table, likewise, shows an average of

2.44 leaves per shoot and an average of 1 shoot per cultivar.

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Table 10 Growth Parameters in Murashige & Skoog MediumType of Medium MS-C Explants in Vessel Leaf Number Per Explant 3 2 3 4 3 4 3 2 2 26 Av e, 2.67 Shoot Number Per Explant 2 1 2 2 1 2 1 1 1 13 Per cultivar Av e. 1.67

Apical-C1

MS-C

Apical-C2

3.67

1.67

MS-C

Apical-C3

2.33

1

Total Ave.

Per shoot

8.67 2.89

4.34 1.44

Table 10 presents the growth of leaves and shoots in the nine apical explants in Murashige & Skoog medium. It appears that the total number of leaves in the 3 triplicates with 3 explants per triplicate of 3 cultures is 26, The total shoot number is 13 with an average of 2.89 leaves per shoot and an average of 1.44 shoots per cultivar. Juvenile tissues like the apical

meristematic tissue permitted shoots to arise on the tip of the explant, this is due to to the apical dominance. Stimulated by the proteolytic actions of the minerals present in the media such as: potassium, zinc and boron, this

blocked the induction of shoots to arise from the lateral side of the shoot tip. It allowed the production of true-to-type, disease-free shoots and leaves.

In Vitro Cultivation of Jatropha curcas in three different culture media

50

Apical Explants Number of shoots Number of leaves

Total Average Growth

CDA 2.89 1.78 4.67

PDA 2.44 1 3.44

MS 2.89 1.44 4.33

Figure 4: Growth Parameters of Apical Explants in the Three Culture Media It is gleaned in Figure 4 that the influence of potassium content which is the principal inorganic element is mostly effective on the growth of shoots than on leaves.The influence on the apical dominance with auxin hormone concentration and ratio of potassium present in the culture medium have a great effect on the formation and elongation of shoots than on leaves in the apical explants. Potassium also acted as a proteolytic enzyme that did not inhibit the formation of few lateral shoots at the internodal portion of the apical explants, hence this added to the total proliferation of shoots. And furthermore, there is an indication that the ratio of inorganic salts and organic

51

elements (sugars) are relatively suffice for proliferation of shoots and leaves from each culture media, since the values of growth parameters (number of leaves and number of shoots) are somehow of close relationship with each other from each media . This figure also conveyed an account that since CDA contributed to have the more unbalance ratios of nutrients among the three culture media, as gleaned from Figure 2, this could be the factor that caused no effect on the significance in growth parameters on development of meristematic tissues in the apical portion of the plant in study; nevertheless to mention also the apical dominance that has great influence against the development of lateral shoots, but caused an effect in the significance of growth in the number of leaves. Thus, it could mean that the utilization of unbalanced ratio of nutrients in the coconut-dextrose-agar would have greater benefits on the use as culture medium for both the apical portion or nodal portions of the plant in the in-vitro cultivation for the ultimate mass propagation of the biodiesel plant: Jatropha curcas, since both total average growth in the two explant variables grown in the CDA have relatively close values ( 4.99 on nodal explants and 4.66 on apical explants). Both having the extremes of highest and lowest values in terms of the total average growth in number of leaves and shoots utilizing the two explant variables. Although using the Murashige and Skoog on the nodal explants would give similar benefits.

Significant Differences in the Growth Parameters

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The following summary of calculations and tests will determine the significant differences in the growth parameters of nodal explants and apical explants in terms of the numbers of leaves and shoots grown in the three Media cultures. Table 11 Computation of F-Value based on Measured Growth Parameters of Nodal Explants in terms of Leaf NumbersSource of Variation Treatments (between columns) Residuals (within columns) Total Degrees of Freedom 2 Sum of Squares 2 .30 Mean Square 1.15 O.72 24 26 38.22 40.52 1.59 0.497 NS P-value F-value Interpretation

Table 11 reveals the computed F-value based on the measured growth parameters of nodal explant in terms of leaf number. As shown in the table, The F value of 0.72 was found to have

exceeded the P-value of 0.497 which is interpreted as not significant at .05 alpha level. Therefore, the alternative hypothesis which states that there is significant difference in the level of growth of nodal explant in terms of number of leaves in the three different culture media was accepted. There is significant difference in the level of growth parameter of nodal explant in terms of the number of leaves of Jatropha curcas . This means that the difference in nutritional contents of the raw materials of the three different

53

culture media gave significant effect/difference on the growth parameters in terms of number of leaves from the nodal explant. Table 12 Computation of F-Value based on Measured Growth Parameters of Nodal Explants in terms of Shoot NumberSource of variation (between columns) (within columns) Total 24 26 10.000 10.667 0.4167 0.8000 0.461 NS Degrees of freedom 2 Sum of squares 0.6667 Mean square 0.3333 F-value Pvalue Interpretation

Table 12 shows the computed F-value based on the measured growth parameters of nodal explant in terms of shoot number. As shown in the table, the F value of 0.8000 is greater than the probability value of 0.461 is not significant at .05 alpha level of significance

Therefore, the alternative hypothesis which states that there is significant difference in the level of growth of nodal explant in terms of number of shoots in the three different culture media was not supported. This means that the difference in nutritional contents of the raw materials of the three different culture media gave no significant effect/difference on the growth parameters in terms of number of shoots from the nodal explant. Table 13 Computation of F value based on the Measured Growth Parameters of Apical Explant in terms of Leaf Numbers

54

Source of variation between columns within columns Total

Degrees Sum of Mean of Squar Squa FFreedom es re value 2 24 26 1.185 12.000 13.185 0.5926 1.185 0.5000

Interpretation Pvalue

0.323

NS

As shown in the table, the F value of 1.185 is greater than the pvalue of 0.323 is not significant at 0.05 alpha level of significance . Therefore, the alternative hypothesis which states that there is significant difference in the level of growth of apical explant in terms of number of leaves in the three different culture media was not supported. This means that the difference in nutritional contents of the raw materials of the three different culture media gave a no significant difference on the growth parameters in terms of number of leaves from the apical explants.

Table 14

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Computation of F value based on the Measured Growth Parameters of Apical Explants in terms of Shoot Numbers

Source of variation

Degrees of freedom 2 24

Sum of squares

Mean square

Fvalue

Pvalue

Interpretation

(between columns) (within columns) Total

2.741 5.778

1.370 0.2407 5.692 0.009 S

26

8.519

Indicated in

Table 14, the F value of 5.692 is lesser than the P-

value of 0.009 is significant at 0.05 alpha level of significance. Therefore, the alternative hypothesis which states that there is significant difference in the level of growth of apical explant in terms of number of shoots in the three different culture media was supported. It means that there is no significant difference in the level of growth parameter of apical explant in terms of the number of shoots of Jatropha curcas. The difference may be due to the

varied nutritional contents of the raw materials of the three different culture media and the apical dominance of auxin at the shoot apical portion of the explant.

56

.

The following table shows different values of the F- level of

significance in the growth parameters of the apical and nodal meristematic explants on their shoot, leaves and root number. Table 15 F-test Values Computed based on the Measured Parameters in Three Different Culture Media

Growth Parameters On Explant Apical

Leaf number P-value F

Remark S

Shoot number P-value F

Remark S

0.323

1.185

0.009

5.692

nodal

0.497

0.72

S

0.461

0.8000

NS

Table 15 shows the F-test values computed based on the measured parameters in three different culture media. In terms of leaf number, the F= 1.185 was found to have P-value of 0.323, which is greater than the 0.05 alpha level of significance. It indicates that there is no significant difference in the number of leaves in three different culture media located at the apical explant. Still in terms of leaf number at the nodal explant., the F= 0.6098 was found to have the P-value of 0.497, which is greater than the 0.05 alpha level of significance. It indicates that there is no significant difference in the number

57

of leaves in three different culture media : coconutdextrose-agar (CDA), potato-dextrose-agar (PDA) and Murashige & Skoog (MS). On the other hand, in terms of shoot number at the apical explant., the F= 5.692 was found to have the P-value of 0.009 which is less than the 0.05 alpha level of significance. It means that there is significant difference in the number of shoots in three different culture media-CDA, PDA, & MS. In terms of shoot number at the nodal explant., the F= 0.8000 was found to have the P-value of 0.46 which is greater than the 0.05 alpha level of significance. It supports that there is no significant difference in the number of shoots in three different culture media. The significant difference in the number of leaves and shoots from the nodal explants can be attributed to the

composition of the basal salts and minerals present in the growth media designed to sustain the plant cells. Likewise, the significant difference in the

number of leaves from the apical explant can also be attributed to the close composition of minerals in terms of grams per culture preparation. The absence of significant difference in the number of shoots from the apical explant in the three culture media could also be attributed to the varied composition of the basal salts and the apical dominance of auxin at the shoot apical meristem.

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Chapter 5 SUMMARY, FINDINGS, CONCLUSION AND RECOMMENDATIONS The following accounts convey the acceptance of this study through its summary, findings, conclusions and to further the investigation with recommendations. Summary In this study, the experimental method was used to find out the differences in the growth rate of meristematic tissues using the three

different culture media Murashige & Skoog, Potato-dextrose-agar and Coconut-dextrose-agar. The culture media were unsupplemented, meaning there were no additional minerals vitamins or hormones to further stimulate growth. The media were generally basic in their composition. A total of fifty-four (54) apical and nodal meristematic explants were employed aseptically. Observations on results were recorded and analyzed using the statistical treatment of ANOVA with the help of the Microsoft Excel 2007 and Graph Pad Instat. Findings Based on the experimentation and observations, the following findings were derived. The status of the kinds of culture media used for the cultivation of Jatropha curcas plantlets into vitro cultures is as follows:

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1.

As in their nutritional components per 100 grams prepared media: 1.1. Coconut-dextrose-agar nutritional condition per 100 g are with

macronutrients of 311.47 mg/100g and micronutrients of 20.48 mg/100 g; 1.2. Potato-dextrose-agar nutritional condition per 100 g are highest in macronutrients of 495.88 mg/100g and micronutrients of 17.82 mg/100g ; 1.3. Murashige & Skoog nutritional condition per 100g are with macronutrients of 0.9944 mg/100g and micronutrients of 0.6504 mg/100g ; 2. Level of growth parameters 2.1. For the number of leaves in nodal explants per culture medium,

whereas in coconut-dextrose-agar there are 31 leaves,14 shoots; in potatodextrose-agar there are 36 leaves, 17 shoots; and in Murashige & Skoog there are 31 leaves, 17 shoots; 2.2. For the number of shoots in apical explants per culture medium, whereas in coconut-dextrose-agar there are 27 leaves,16 shoots; in potatodextrose-agar there are 22 leaves, 9 shoots and in Murashige & Skoog there are 26 leaves, 13 shoots ; 3. As to the difference in the growth parameters of explants in the three kinds of culture media: In terms of leaf number at the apical explants., the F= 1.185 was found to have the P-value of 0.323, which is not significant at 0.05 alpha level; in shoot number at the apical explants., the F= 5.692 was found to have the Pvalue of 0.009, which is significant at 0.05 alpha level.

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On the other hand, in terms of leaf number at the nodal explants., the F= 0.72 was found to have the P-value of 0.497, which is not significant at 0.05 alpha level; on shoot number at the nodal explants., the F= 0.8000 was found to have the P-value of 0.497, which is not significant at 0.05 alpha level. Conclusions Based on the foregoing findings, the following conclusions are made:1.In terms of nutritional status, coconut-dextrose-agar had a relatively

lower macronutrients thanpotato-dextrose-agar, and a relatively unbalanced ratios with micronutrients; potato-dextrose-agar had a high level of macronutrients but unbalanced ratio with micronutirents; Murashige & Skoog had high levels basal salts/minerals and with relatively balanced ratios of macronutirents and micronutrients.2.

The total average growth as shown in Figure 3 on growth

parameters of nodal explants in the three culture media revealed a higher value of measurement, both in terms of number of leaves and shoots grown in Murashige and Skoog and lowest in coconut-dextrose-agar (6.66 and 4.99, respectively). On the other hand, in Figure 4 on growth parameters of

apical explants in the three culture media revealed a higher value of measurement on the total average growth in terms of number of leaves and shoots grown in coconut-dextrose-agar and lowest in potato-dextrose-agar3.

There is

no significant difference in the level of growth

parameters in the three different culture media in terms of number of leaves

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and shoots in the nodal explants, as well as in the number of leaves in the apical explants, but found to be significant in the growth parameters in terms of number of shoots in the apical explants grown in the three culture media. Recommendations In view of the foregoing conclusions, the following recommendations are forwarded: 1. Considering that all three media obtained visual survivability for shoot proliferation, number of leaves or continuous growth of explants, the farmers must be oriented to use them in the mass propagation of Jatropha curcas. 2. Experts should continue the experiments on different rations of mineral salts and hormones supplemented with the basic CDA media utilized in the study for more satisfying results to precultivation of the plant. 3. Investors and farmers who can venture to propagate Jatropha

curcas may use coconut as resource material treatment in tissue culturing for immediate mass production of the plant. 4. The future researchers may conduct further experiments on

genetic diversity and yield potential of Jatropha curcas and its adaptations to different rations of environmental, hormonal and nutritional conditions.

BIBLIOGRAPHY

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Books Fernandez, Elvira C., et al. Biofuels from Philippine Plants. Quezon City: Asia- PacificBiofuels Corporation. 2007. Reyes, Rodolfo S., et al. Biology: Science and Technology Textbook for Second Year. Mandaluyong City, M.M.: Book Media Press,Inc. 2009 Sas, A.C. Plants and Health. Manila: Eastern Publishing Association. 1990. Sevilla, Consuelo G. Research Methods. Quezon City: Rex Printing Co., Inc. 1997. Periodicals Atanda O., I. Akpan and O.A. Enikuomehin . An alternative culture medium for rapid detection of aflatoxins in agricultural commodities African Journal of Biotechnology, Vol. 5, No. 10, 16 May 2006, pp. 1029-1033 Bordadora, Norman.Alternate Fuel seen to grow from 2M ha of idle land. Philippine Daily Inquirer. August 20, 2006. Braid, Floranel Rosario.Protection of the Environment Biofuels Law and Conserving Biodiversity, Manila Bulletin. Nov. 25, 2006. Faylon, Patricio S. Crop, R & D Milestones. Vol. 3, No. 3. March 2000. Grecia, Dell Hitalla.Alternative Fuels from our trees and plants. Journal. Feb. 18, 2006. Womens

K. Kalimuthu, S. Paulsamy1, R. Senthilkumar and M. Sathya . In vitro Propagation of the Biodiesel Plant Jatropha curcas L.Plant Tissue Cult. & Biotech. 17(2): 137-147(2007) Mabasa, Roy. Bioadditives for Gasoline, diesel made Mandatory, Manila Bulletin. 2006. Pramanik, K. Properties and use of Jatropha curcas oil and diesel fuel blends in compression ignition engine. Renewable Energy Journal, Bhawan Road, Patna, India, 2003.

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Sirisomboon, P., et al Physical and mechanical properties of Jatropha curcas L. fruits, nuts and kernels, Biosystems Engineering. 2007 . Siva N. , S. Ganesan , N. Banumathy and Muthuchelian Antifungal Effect of Leaf Extract of Some Medicinal Plants Against Fusarium oxysporum Causing Wilt Disease of Solanum melogena L. Ethnobotanical Leaflets 12: 156-163. 2008. Soomro, Rashida and Rabia Asma MemonEstablishment of callus and suspension culture in Jatropha curcas Pakistan Journal Botany 39(7): 2431-2441, 2007. Wood, P. Out of Africa: Could Jatropha vegetable oil be Europes biodiesel feedstock?, Refocus, 2005. Unpublished/published Materials Augustus, G.S., Jayabalan, M., & Seiler, G.J. . Evaluation and bioinduction of energy components of Jatropha curcas. Biomass and Bioenergy., 2002. Heller, J. . Physic nut Jatropha Curcas L. Promoting the conservation and use of underutilized and neglected crops. Institute of Plant Genetics and Crop Plant Research (Gartersleben) and International Plant Genetic Resources Institute: Rome Vol. 1. 1996. Sujatha and Mukta Morphogenesis and Plant regeneration from tissue cultures of Jatropha curcas, Plant Cell Tissue & Organ Culture, 1996. Weida Lu, Tang Lin, Yan Fang & Chen Fang Induction of callus from Jatropha curcas and rapid propagation, College of Life Science, Sichuan University Chengdu 610064, China. 2003.

Website/Online Sources

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Academic Journals retrieved August 27, 2009 academicjournals.org/AJB/PDF/pdf2006/16May/Adebowalean dAdedir. pdf PNOC News retrieved Sept. 20, 2009. Alternat1ve.com/ 2006//2-million-ha-up-for-jatropha-planting Biotech news retrieved Sept. org/news/2008-07-16/ 20, 2009 biofuel/

bioenergy.checkbiotech.

Blogspot retrieved Sept. 20, 2009. Lagarden.blogspot.com/2008/01/plantPropagation meristem and terms. html82kdatabase.prota. org/PROTAhtml/Jatropha curcas_En.htm Cached Datta, Mukul Manjari et al retrieved November http://www.ias.ac.in/currsci/nov252007/1438.pdf 28, 2008.

DOE retrieved June 3,2009 . www.doe.gov.ph/AF/jatropha.htm Garden tech retrieved July 25, 2009 www.gardentech.com.sg/view vendor.asp?id=105 Google Books retrieved Oct 18,2009 http://www.oup.com/uk/orc/bin/ 9780199282616/ch02.pdf GMA NEWS retrieved Sept. 20, 2009 gmanews.tv/story/58054 /LandBank-to-extend-up-to-P10B-assistance- to-jatropha Henning, Reinhard K. retrieved August 28, 2009 .www.jatropha de/news/jel-news.htm Jha, Hani, Timir Baran & Bivajit Ghosha http://books.Google.com/books retrieved Oct. 18,2009

Ummu retrieved Aug. 28, 2009. www.futureenergyevents.com/ jatropha/attend

Kalimuthu, et. Al. retrieved Nov. 28, 2008 http://www.baptcb .org/ptc/Full_article/ptc17_2_05.pdf) . News Update retrieved July 4, 2009. www.biofuels.com.ph/news.html PNOC retrieved August 25, 2009 www.pnoc-afc.com.ph

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PNOC

News retrieved Sept. 20, 2009. Alternat1ve.com/ biofuel/2006//2-million-ha-up-for-jatropha-planting www.relocalize.net/jatropha _what_the_

PCARRD August 25,2009 public _should_know

Sigma Aldrich retrieved Oct 18,2009(http://www.sigmaaldrich.com/lifescience/cell-culture/learning-center/media-expert/calcium.html) Todd, John H.retrieved Oct.18,2009 http://books.google.com/books Trigiano, Robert Nicholas & Dennis John Gray Oct. 17,2009 http://books.google.com/books Wikipedia retrieved August 20,2009 http://en. Apical_dominance Wikipedia retrieved Meristematic August 20,2009 Wikipedia. Org/ wiki/

http://en.wikipedia.org/wiki/ 1/0

www.ingentaconnect.com/content/maik/rupp/2003/00000050/0000000 046 1666?crawler=true OTHER SOURCES Gore,Al (April 27, 2008) Cable Vision HBO: The Inconvenient Truth. Nantes, Rafael P. Tayabas,Quezon (October 13, 2007) QUESCAA

Convention

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APPENDICES RESEARCH CONTACT DIRECTORIES

On potentialities and demand of Jatropha curcas as alternative biogas feedstock Reading and interview sources.Names and Contacts Topic The odds on mass propagation of curcas against vegetative crops

1. Dr. Pedro D. BalagasQEPC,DENR, Region !V-A, Jatropha Talipan, Pagbilao, Quezon Tel.# : 042-710-2815 2. Reinhard K. Henning,author of Jatropha Booklet the Bagani GBR, Rothkreuz D-88138, Neissensberg, Germany System

Downloading of Jatropha Booklet;The Value of the Jatropha

e-mail: henning@bagani.de, Jatropha website: www.Jatropha,orgTel. #: +49-8389-984129 Fax: +49-8389-984128 3.Mr. Ummu Hani, General Manager Promotions Center for Management Technology Development Downloading of the Best Practices for Long Term Jatropha

Website: www.futureenergyevents,com/jatropha4. Dr. Jessamyn D. Recuenco-Adorada, University Extension Specialist Crop Protection Center,U.P. Los Baos, 4031 College,Laguna Phone: +63(049)536-2410 Adaptive trials of Pest control on Jatropha

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Fax: +63(049)536-2409 Mobile: +63-926-874-556

Request Letter Applying for the Use of Facilities in U.P.

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Approved Request to Work and Use the facilities of BIOTECH, U.P.Los Baos

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Results of Chemical Analysis from the Department of Science and Technology

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CURRICULUM VITAE

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PERSONAL INFORMATION NAME ADDRESS DATE OF BIRTH CIVIL STATUS SPOUSE FATHER MOTHER EDUCATIONAL BACKGROUND Elementary : Keravat International Primary School, Graduated Dec. 1979 Secondary : Liceo de Pila Graduated March 1984 Special Mention in Honor Roll Miss Liceo de Pila, Talent and Journalist of the Year. College : Far Eastern University, Graduated 1994 : Laguna State Polytechnic University 18 education units, :Gracie O. Ching : Sitio Mapuputi,Brgy.Liwayway , Mauban,Quezon : August 28, 1967 : Married with four children : Arnold V. Ching : Carlos D. Olivares : Iluminada E.Isles (deceased)

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: UP Open University ,Los Baos, Laguna.DST Biology (under Teachers Devt Program) June 2009- present Graduate School : Laguna State Polytechnic University Master of Arts in Teaching major in Science and Technology Education May, 2009 ELIGIBILITY Licensure Examination for Teachers TEACHING EXPERIENCES Liwayway National High School, Mauban,Quezon Pablo D. Maningas National High School Quezon,Quezon : June 2007 March 4, 2008 : March 4, 2008 to present : August 1-2,1998 - 75.60%

Pedro Guevara Memorial National High School Sta. Cruz,Laguna San Padua College, Pila, Laguna Cometa National High School-Annex Quezon, Quezon De La Salle University, : March 4 April 4, 2003 : Sept. 25- Oct.26,2001 :May October,2005 :July 1 Aug.29,2005

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Lipa, Batangas Laguna State Polytechnic University, Sta.Cruz, Laguna Laguna Santiago Educational Foundation,Inc. Sta. Cruz, Laguna PRESENT POSITIONS AND AWARDS RECEIVED Liwayway National High School - Secondary Teacher II, teaching Biology and Chemistry subjects - DORP Coordinator and Science Club Adviser : June, 2009 to present Cluster Science Environmental Camp held at Luis Palad National High School, Tayabas - Coach Science Essay Writing : First Place TRAININGS AND SEMINARS In-service Training (INSET) for Teachers and Employees held at Liwayway Natl High School, Liwayway,Mauban,Quezon Division Training Workshop on Innovative Teaching Techniques and Strategies as a Tool for Raising Academic Excellence (for Secondary Science Teachers), Held at Lutucan National High School,Sariaya,Quezon Capacity Building on SBM BESRA Implementation and Mid-Year Adjustment of Divisional Operations Plan :May 26-30,2008 :Oct.27-29,2008 : Sept. 2008 : June 1 July 31,1998 : July 11, 2001 Sept. 2,1999 : Sept. 20-Nov.19,1999

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for Elementary and Secondary School Heads and Teachers Quezon Science Teachers Convention: Theme:Science towards Education Excellence, held at Convention Center, Lucena, Quezon Teachers Convention:Theme:Education towards Digital Age, held at Convention Center, Lucena,Quezon District Seminar-Workshop for Secondary School Teachers and Personnel held at Cometa National High School-Main Cometa, Quezon, Quezon.

:Feb.9,2008

:Jan.12,2008

:Dec.17,2007

:Oct.22-24,2007

5th Quezon Science Club Advisers Association, Inc. Convention, Theme:The Role of Science Investigations in the Conservation Of Quezons Natural Resources, held at Gavinas Restaurant, Tayabas, Quezon sponsored by QUESCAA and DepEd In-service Training Program for Private Secondary School Teachers,Theme: Multiple Intelligence held at Lyceum Institute of Technology,Calamba,Lag. OVERSEAS TRAVEL HISTORY Study on Elementary Education (Grades 3-6) Keravat International Primary School,Keravat, East New Britain, Papua New Guinea Study on Secondary Education (Year Levels 7-8) Lae International High School,Lae,Papua New Guinea : 1980-1981 : 1976-1979 : May 2-6,2005 : Sept. 17,2007

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Employment as Kitchen Supervisor at Popondetta General Hospital,Popondetta,PNG : Sept.22,1992.Mar.22,1993

Employment and Training for RAM Services, Port Moresby,Papua New Guinea : Sept.27-Oct.20,1997

_____________________________ ______

Gracie O. Ching