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.
_________________________
MILA E. ISLES, Ed. D LYRMA C. HIFE, Ed. D
Member Member
_______________
_________ __________________________
LUCITA G. SUBILLAGA, Ed. D REDELLA P. VISTA, M.A.
Member External Panel Member

_________________________
HON. RICARDO A. WAGAN, Ph.D., D.P.A.
University President/Chairman

Approved In partial fulfillment of the requirements for the Degree of

Master of Arts In Teaching, Major In Science and Technology Education.

______________________ ENRIQUETA C. REYES, Ed. D.

Date 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 Baños, 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 Baños 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)

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

had 26 leaves, 13 shoots. In this light of information , therefore nodal explants

grown in potato-dextrose-agar and those apical explants grown in coconut-

dextrose-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

2 REVIEW OF RELATED LITERATURE AND STUDIES

Related Literature ….…………….……………………………...…….13

Jatropha curcas…………………………….......................13

Culture media ……………………………….….………….20

Related Studies…………………………………………..………….....24

Jatropha curcas ………. …………………..…..……….....24

, Culture Media ……………………………………….……..26

3 RESEARCH METHODOLOGY

Research Design………………………………………………………28

Subject of the Study………………………………………………......29

Data Gathering Procedures………………………………………….29

Construction Procedures……………………………………………..31

Try-Out Revision………………………………………………………35

Research Instrument………………………………………...….…….36

Statistical Treatment of Data………………………………………...36

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4 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

Summary……………………………………………………………………58

Findings……………………………………………………………………..58

Conclusion...…………………………………………………………..……60

Recommendations…………………………………………………………61

BIBLIOGRAPHY………..................................................………….……..........62

A. Books…………………………………………………………...……….62

B. Periodicals………………………………………………………………62

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.P……………67

C. Approved Request to Work and Use the Facilities of BIOTECH,

U.P.Los Baños………………………………………………………….68

D. Results of the Chemical Analysis in DOST.………………………....69

CURRICULUM VITAE ………………………………………………………..…...70

LIST OF FIGURES

Figure

1 Research Paradigm………………………………………………….…5

2 Nutritional Components of the Three Culture Media…………..….42

3 Growth Parameters of the Nodal Explants in the Three

Culture Media………………………………………………………….46

4 Growth Parameters of Apical Explants in the Three

Culture Media………………………………………………………….50
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LIST OF TABLES

Table

1 Nutritional Components of CDA……………………………......................38

2 Nutritional Components of PDA…………………………………………....39

3 Nutritional Components of Murashige & Skoog……………………….….40

4 Summary of Nutritional Components of the Three Media………………41

5 Growth Parameter of Nodal Explants in CDA…………………………….43

6 Growth Parameter of Nodal Explants in PDA………………………….....44

7 Growth parameter of Nodal Explants in MS……………………….….…..45

8 Growth parameter of Apical Explants In CDA………………….……..….47

9 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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12Computation of F-Value Based on Measured Growth Parameter of

Nodal Explants in Terms of Shoot Numbers………………….
………...53

13 Computation of F-Value Based on Measured Growth

Parameter of Apical Explants in Terms of Leaf Number…………....…54

14 Computation of F value Based on the Measured Growth

Parameters of Apical Explant in Terms of Shoot Numbers……..
…....55

15 F-test Values Computed Based on the Measured Paramete in

Three Different Culture Media………………………………………...….56