IN VITRO CULTIVATION OF Jatropha curcas (TUBANG-BAKOD: A BIODIESEL PLANT) USING THREE DIFFERENT CULTURE MEDIA S.Y. 2008-2009

A Thesis Presented to the Faculty of Graduate Studies and Applied Research Laguna State Polytechnic University Main Campus, Sta.Cruz, Laguna

In Partial Fulfillment of the Requirements for the Degree in Master of Arts in Teaching, Major in Science and Technology Education

GRACIE O. CHING

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Summer, 2009 Republic of the Philippines Laguna State Polytechnic University Santa Cruz Campus,Santa Cruz, Laguna APPROVAL SHEET This Thesis Proposal entitled IN VITRO CULTIVATION OF Jatropha curcas (TUBANG-BAKOD:A biodiesel plant) USING THREE DIFFERENT CULTURE MEDIA submitted by GRACIE O. CHING is hereby recommended for Final Oral Thesis Defense._____________________ Date ________________________ JUVY L. TOBIAS, M.A. Thesis Adviser

Accepted in partial fulfillment of the requirements for the Degree of Master of Arts in Teaching major in Science and Technology Education by the Examination Committee. _________________________LYRMA C. HIFE, Ed. D Member _______________ _________ __________________________ LUCITA G. SUBILLAGA, Ed. D REDELLA P. VISTA, M.A. Member External Panel Member Member _________________________ HON. RICARDO A. WAGAN, Ph.D., D.P.A. University President/Chairman

MILA E. ISLES, Ed. D

Approved In partial fulfillment of the requirements for the Degree of Master of Arts In Teaching, Major In Science and Technology Education.

______________________ Date

ENRIQUETA C. REYES, Ed. D.

Dean Graduate Studies and Applied Research

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DEDICATION This study is dedicated to all fellowmen behind the Jatropha Development for the alternative biofuel feedstock the world over, and most especially to whom this progress on sustainability of the biofuel will be most beneficial to my children:

Kyleen, Kyveen, Kyreen and Kydreen.

GOC

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ACKNOWLEDGMENT

Extension to all assistance where this present study had been successfully written, acknowledgement is hereby granted to the following unforgettable important persons behind the possibility of this investigation: HONORABLE RAFAEL P. NANTES, Governor of Quezon Province for his encouraging words on the Jatropha Development in the Philippines during seminars and convention of Science Teachers, that uprooted my inspiration and motivation; HONORABLE RICARDO A. WAGAN, President of LSPU for his unending support on humane relationship, courtesy and service to the country. Dr. ENRIQUETA C. REYES, Dean of the Graduate Studies and Applied Research, for all her professional support on my success of completing my requirements;

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DR. IDA F. DALMACIO, Director, Biotech, U.P. Los Baos, for allowing me to use the facilities and equipment at National Immunological Testing Laboratory; MRS. REDELLA P. VISTA, Division Science Supervisor of DepEd Laguna, as External Panel Member; Mrs. JUVY L. TOBIAS, Thesis Adviser and Dr. SEVERINA E. EXCONDE, Laboratory consultant , for their efforts in making the research study a success; Dr. MILA E. ISLES, Dean of Education, for all her encouragement, and moral support, and as panel member on language critiquing; Dr. LYRMA C. HIFE, Panel member on Technical Writing; Dr. LUCITA G. SUBILLAGA, Panel Member on analytical and statistical treatment critiquing; Dr. LUZVIMINDA C. CALARAMO, for her moral support and guidance on the thesis problem; ATE ANNIE, Laboratory Technician of Biotech for all her assistance on the Murashige & Skoog preparations and the laboratory protocols on aseptic techniques; Sir CONRADO C. GABARDA, Teacher-in-Charge, Liwayway National High School, Mauban, Quezon and the TEACHING FORCE for their support and generosity, especially on the utilization of the faculty computer;

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Mr. CARLOS D. OLIVARES, a father in all ways, for his unending generous support especially during tough times; And lastly, Mr. RODERICK V. ALVARO, for his generous support on transportation from Mauban, Quezon during the progress of the experimental research at U.P. Los Baos and around the region in search for the parent plant: Jatropha curcas; Above all, to THE ALMIGHTY CREATOR, the source of everything in particular from strength, inspiration and support. GOC ABSTRACT In this study, the experimental method was used to find out the differences in the growth parameters of meristematic tissues using the three different culture media: coconut-dextrose-agar, potato-dextrose-agar and the Murashige & Skoog. 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 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 software Graph Pad Instat. Based on the chemical analyses and observations,the status of the three kinds of culture media used for the cultivation of Jatropha curcas

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plantlets in vitro was as follows: as in their nutritional components in unit measurement per 100 grams of prepared media: coconut-dextrose agar had 2.01 mg of calcium, 270 mg of potassium, 19.77 mg of sodium, 39.24 mg of phosphorus, 0.22 g of nitrogen and 0.71 mg of iron; B) potato-dextrose-agar was highest in calcium content (3.38 mg), potassium of 470 mg, and lowest in sodium of 17.02 mg,phosphorus of 22.38 mg ,0.12 g of nitrogen and 0.80 mg of iron.;C) Murashige & Skoog had the lowest total in nutrient

parameters:1.6448 mg/100g, but with relatively balanced ratios of macro and micronutrients. As to the level of growth parameters in nodal explants per culture medium it was found out that :a) Coconut-dextrose-agar had 31 leaves,14 shoots,; b) Potato-dextrose-agar had 36 leaves, 17 shoots; and c)

Murashige & Skoog had 31 leaves, 17 shoots. While in apical explants per culture medium: a) Coconut-dextrose-agar had 27 leaves,16 shoots; b) Murashige & Skoog

Potato-dextrose-agar had 22 leaves, 9 shoots; and c)

had 26 leaves, 13 shoots. In this light of information , therefore nodal explants grown in potato-dextrose-agar and those apical explants grown in coconutdextrose-agar had the greatest number of leaves, while those nodal explants grown in potato-dextrose-agar and Murashige & Skoog, and those apical explants grown in coconut-dextrose-agar had the highest number of shoots. As to the level of growth parameters in the three kinds of culture media: in terms of leaf number at the apical explants., the F= 1.185 was found to have

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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 P-value of 0.009, which is significant at 0.05 alpha level, and; On the other hand, in terms of leaf number at the nodal explants., the F= 0.6098 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 0.46, which is not significant at 0.05 alpha level. Based on the above data, it was found out that the significant differences are due to the different amounts of macronutrients and micronutrients present in the culture media.

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TABLE OF CONTENTS Preliminaries Page

TITLE PAGE..i APPROVAL SHEET....ii DEDICATION .....iii ACKNOWLEDGMENT..........iv ABSTRACT.....vi TABLE OF CONTENTS....ix LIST OF FIGURES .. xi LIST OF TABLES.....xii CHAPTER 1 THE PROBLEM AND ITS BACKGROUND Introduction.......1 Background of the Study....3 Conceptual Framework...5

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Statement of the Problem......6 Research Hypothesis .....7 Significance of the Study..........................7 Scope and Limitation..9 Definition of Terms....10

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REVIEW OF RELATED LITERATURE AND STUDIES Related Literature ......13 Jatropha curcas.......................13 Culture media ...20 Related Studies.......24 Jatropha curcas . .........24

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Culture Media ...26

3 RESEARCH METHODOLOGY Research Design28 Subject of the Study......29 Data Gathering Procedures.29 Construction Procedures..31 Try-Out Revision35 Research Instrument.....36 Statistical Treatment of Data...36

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PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA Status of the Kind of Culture Media Coconut-dextrose-agar...38 Potato-dextrose-agar media..39 Murashige and Skoog.40 Level of Growth Parameters....43

Significant Differences in Growth parameters of Explants in three different culture media.52 5. SUMMARY, FINDINGS, CONCLUSIONS AND RECOMMENDATIONS .58 Summary58 Findings..58 Conclusion.....60 Recommendations61 BIBLIOGRAPHY.............................................................62 A. Books....62 B. Periodicals62 C. Published and Unpublished Materials.....63 D. Website/Online sources..64 E. Other Sources...65 APPENDICES....66 A. Research Contact Directories....66

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B. Request Letter Applying for the Use of Facilities in U.P67 C. Approved Request to Work and Use the Facilities of BIOTECH, U.P.Los Baos.68 D. Results of the Chemical Analysis in DOST.....69 CURRICULUM VITAE .....70

LIST OF FIGURES

Figure 1 2 3 4 Research Paradigm.5 Nutritional Components of the Three Culture Media...42 Growth Parameters of the Nodal Explants in the Three Culture Media.46 Growth Parameters of Apical Explants in the Three Culture Media.50

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LIST OF TABLES

Table 1 2 3 4 5 6 7 8 9 Nutritional Components of CDA......................38 Nutritional Components of PDA....39 Nutritional Components of Murashige & Skoog..40 Summary of Nutritional Components of the Three Media41 Growth Parameter of Nodal Explants in CDA.43 Growth Parameter of Nodal Explants in PDA.....44 Growth parameter of Nodal Explants in MS....45 Growth parameter of Apical Explants In CDA....47 Growth Parameters in Potato-dextrose-agar Medium.........48

10 Growth Parameters in Murashige & Skoog Medium.,...49 11 Computation of F-Value Based on Measured Growth Parameter of Nodal Explants in Terms of Leaf Numbers...52

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Computation of F-Value Based on Measured Growth Parameter of Nodal Explants in Terms of Shoot Numbers. ...53 13 14 ....55 15 F-test Values Computed Based on the Measured Paramete in Three Different Culture Media....56 Computation of F-Value Based on Measured Growth Parameter of Apical Explants in Terms of Leaf Number....54 Computation of F value Based on the Measured Growth Parameters of Apical Explant in Terms of Shoot Numbers..

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