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
 2

cuttings, seeds, oil, and substitute energy. During the QUESCAA Convention

on the launching of the Science Investigatory Manual at Gavina’s 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 Master’s Thesis has

been identified for the procurement of further studies on propagating the

Jatropha plant for mass production through the culture media such as potato-

dextrose-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
 3

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 man’s 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
 4

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.

 5

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 country’s 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 Dependent Variables

Kinds of culture media:
Coconut-dextrose-agar (CDA)
macronutrients:calcium, In vitro cultivation of Jatropha
potassium,phosphorus,nitrogen, curcas:
micronutrients: sodium,iron
Level of Growth Parameters:
Potato-dextrose-agar (PDA) nodal explant
macronutrients:calcium, number of leaves
potassium,phosphorus,nitrogen, number of shoots
micronutrients: sodium,iron
apical explant
Murashige & Skoog (MS) number of leaves
macronutrients:calcium, number of shoots
potassium,phosphorus,nitrogen,
micronutrients: sodium,iron

Frame1 Frame 2

Figure 1: Research Paradigm

 6

Frame 1 presents the kinds of culture media like coconut-dextrose-

agar (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 number of leaves, and shoots which were presented 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,
 7

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

 8

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
 9

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 in-

vitro 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.

 10

The initial experiment was operated in one of the U.P-immunological

laboratories in the National Institute of Molecular Biology and Biotechnology,

UP Los Baños, 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 26°C.

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
 11

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-dextrose-

agar (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,

 12

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

 13

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 jatrós (doctor) and

trophé (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 plant’s 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

 14

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 al-

2007) 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.

 15

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

747-400’s 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

world’s first commercial aviation test flight. The Jatropha used on this flight
 16

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 community’s 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

 17

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
 18

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
 19

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
 20

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 researcher’s 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 in-

vitro preparations on micropropagation of higher plants like in the study of

 21

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, signal-

transduction, replication, and is stored primarily in the endoplasmic reticulum

(ER); the biggest chemical issues with calcium in cell culture are its solubility
 22

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/media-

expert/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
 23

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 researcher’s 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).

 24

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)
 25

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 Skoog’s basal medium supplemented with 22.2

µM N6-benzyladenine (BA) and 55.6 µM adenine sulphate, in which culture

produced 6.2±0.56 shoots per nodal explant with 2.0±0.18 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
 26

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; 2) transferring shoots to

proliferation and elongation media; 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

 27

Murashige & Skoog which would eventually lead to further the

micropropagation process to be yet be made by other interested

investigators/scientists alike. This present study could be starters, a break-

through for farmers too.

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 myo-

inositol, 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.

 28

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-dextrose-

agar, 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 Baños:. 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 any manipulations of the

explants; 9. Media preparations

( see research procedures).

 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 kid’s 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. Distilled water was added 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 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

was set aside for later use as stock solution. The mashed potato was

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

distilled water was poured into it. Weighed chemical substances for MS

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: a.

MS I = 10 ml b. MS II = 2.5ml c. MS III = 2.5ml . Iron Chelate = 2.5 ml

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

investigator’s 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

contamination and counted. With every manipulation of the cultivar, the

forceps were flamed and sterilized prior to each handling. As each cultivar in

the culture vessel was examined , values were also recorded in the

prepared Format of Data table.

Statistical Treatment of Data

The mean for each treatment was calculated. All treatments of three

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 STATISTICAL TOOL

1. Status of the kinds of the culture Based on the chemical

media in terms of nutritional conditions . analysis of DOST, in

appendices are

results of analysis,

page 96

2. Level of Growth parameters Frequency

of J. curcas explants in different media.

3 .Difference in the growth level One way- ANOVA

parameters in three culture media.

 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

CDA TOTAL
Nutrtional Components mg/ 100 Grams
Macronutrients
calcium 2.01
311. 47 mg
Potassium 270
phosphorus 39.24
nitrogen 0.22
Micronutrients
Iron 0.71 20.48 mg
sodium 19.77
 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)

PDA TOTAL
Nutrtional Components mg/ 100 Grams
Macronutrients
calcium 3.38
495.88 mg
Potassium 470
phosphorus 22.38
nitrogen 0.12
Micronutrients
Iron 0.80 17.82 mg
sodium 17.02

Table 2 evidently shows the nutritional components of potato-dextrose

agar. It can be gleaned that potassium of 470 mg is in the highest

composition among the other nutrients of potato. Nitrogen at 0.12 is the

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)

M&S TOTAL
Nutrtional Components mg/ 100 Grams
Macronutrients
calcium .044
0.9944 mg
Potassium .77
phosphorus 0.0164
nitrogen 0.164
Micronutrients
Iron 0.278 0.6504 mg
sodium 0.3724

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 high-

salt 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 Culture Media mg/100g

CDA PDA MS
Macronutrients 311.47 495.88 0.9944
Micronutrients 20.48 17.82 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 Medium

Shoot
Type of Explants in Leaf Number Ave. Number Ave.
medium Vessel Per Explant Per Explant
CDA-A Nodal-A1 3 3.33 1 1.33
4 2
3 1
CDA-A Nodal-A2 4 3.33 2 2
3 2
3 2
CDA-A Nodal-A3 4 3.67 1 1.33
3 1
4 2
Total 31 10.3 14 4.6
3 6

Ave. Per shoot 3.44 Per cultivar 1.55

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 Medium

T Leaves Shoot
ype of Explants in number Av number Av
media vessel Per-explant e. per-explant e.

PDA- nodal-B1 2 4 1 1.33

B

4 2
6 3
nodal-B2 3 3.67 1 1.67
4 2
4 2
nodal-B3 3 4.33 2 2.33
4 2
6 2
Total 36 12 17 5.3
3

Ave. Per shoot 4 Per 1.7

cultivar 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.

 45

Table 7
Growth Parameters in Murashige & Skoog Medium

Shoot
Type of Explants in Leaf Number Ave. Number Per Ave.
medium Vessel Per Explant Explant
MS-A Nodal-C1 6 5.33 3 2.67

4 2

6 3

MS-B Nodal-C2 2 2.67 1 1.67

3 2

3 2

MS-C Nodal-C2 2 2.33 1 5.33

2 1

3 2

total 31 10.33 17 9.67

Ave. Per shoot 3.44 Per cultivar 3.22

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 CDA PDA MS

Number of shoots 3.44 4 3.44
Number of leaves 1.55 1.78 3.22
Total Average Growth 4.99 5.78 6.66

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 the

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 Medium

Leaf Number Shoot Number

Type of Explants in Ave Av
Medium Vessel Per Explant , Per Explant e.
CDA-A Apical-A1 2 3 1 1.33
4 2
3 1
CDA-A Apical-A2 2 3 1 1.67
3 2
4 2
CDA-A Apical-A3 3 2.67 3 2.33
3 2
2 2
Total 27 8.6 16 5.3
7 3
Ave. Per shoot 2.89 Per cultivar 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 Medium

Shoot Number
Type of Explants in Leaf Number Av Per Explant Av
Medium Vessel Per Explant e, e.

PDA-B Apical-B1 3 2.33 1 1

2 1

2 1

PDA-B Apical-B2 3 2.67 1 1

2 1

3 1

PDA-B Apical-B3 3 2.33 1 1

2 1

2 1

Total 22 7.33 9 3

Ave. Per shoot 2.44 Per cultivar 1

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.

 49

Table 10
Growth Parameters in Murashige & Skoog Medium

Shoot
Type of Explants in Leaf Number Av Number Av
Medium Vessel Per Explant e, Per Explant e.
MS-C Apical-C1 3 2.67 2 1.67
2 1
3 2
MS-C Apical-C2 4 3.67 2 1.67
3 1
4 2
MS-C Apical-C3 3 2.33 1 1
2 1
2 1
Total 26 8.67 13 4.34
Ave. Per shoot 2.89 Per cultivar 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 CDA PDA MS

Number of shoots 2.89 2.44 2.89
Number of leaves 1.78 1 1.44
Total Average Growth 4.67 3.44 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

 52

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 Numbers
Source of Degrees Mean P-value Interpretation
Variation of Sum of Square F-value
Freedom Squares
Treatments 2 2 .30 1.15
(between
columns)
O.72 0.497 NS
Residuals 24 38.22 1.59
(within columns)

Total 26 40.52

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 Number

Source of Degrees Sum of Mean Interpre-

variation of squares square F-value P- tation
freedom value

(between 2 0.6667 0.3333

columns)
(within columns) 24 10.000 0.4167 NS
0.461
0.8000
Total 26 10.667

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

Degrees Sum of Mean Interpretation

Source of of Squar Squa F- P-
variation Freedom es re value value

between 2 1.185 0.5926

columns
1.185 0.323 NS
within 24 12.000 0.5000
columns

Total 26 13.185

As shown in the table, the F value of 1.185 is greater than the p-

value 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
 55

Computation of F value based on the Measured Growth Parameters

of Apical Explants in terms of Shoot Numbers

Source of Degrees Sum of Mean F- P- Interpretation

variation of squares square value value
freedom

(between 2 2.741 1.370

columns)

(within 24 5.778 0.2407 5.692 0.009 S

columns)

Total 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 Leaf number Shoot number

Parameters P-value F P-value F
On Explant Remark Remark

Apical 0.323 1.185 S 0.009 5.692 S

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 : coconut–dextrose-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.
 58

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:

 59

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 potato-

dextrose-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 potato-

dextrose-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 P-

value of 0.009, which is significant at 0.05 alpha level.

 60

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-agar

3. There is no significant difference in the level of growth

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

 61

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
 62

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”. Women’s
Journal. Feb. 18, 2006.

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.
 63

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 Memon“Establishment 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 Europe’s

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
 64

Academic Journals retrieved August 27, 2009

academicjournals.org/AJB/PDF/pdf2006/16May/Adebowalean dAdedir.
pdf

PNOC News retrieved Sept. 20, 2009. Alternat1ve.com/ biofuel/

2006/…/2-million-ha-up-for-jatropha-planting

Biotech news retrieved Sept. 20, 2009 bioenergy.checkbiotech.

org/news/2008-07-16/…

Blogspot retrieved Sept. 20, 2009. Lagarden.blogspot.com/2008/01/plant-

Propagation meristem and terms. html82kdatabase.prota.
org/PROTAhtml/Jatropha curcas_En.htm – Cached

Datta, Mukul Manjari et al retrieved November 28, 2008.

http://www.ias.ac.in/currsci/nov252007/1438.pdf

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, Timir Baran & Biśvajit̲ Ghosha retrieved Oct. 18,2009

http://books.Google.com/books

Hani, 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

 65

PNOC News retrieved Sept. 20, 2009. Alternat1ve.com/

biofuel/2006/…/2-million-ha-up-for-jatropha-planting

PCARRD August 25,2009 www.relocalize.net/jatropha _what_the_

public _should_know

Sigma Aldrich retrieved Oct 18,2009(http://www.sigmaaldrich.com/life-

science/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. Wikipedia. Org/ wiki/

Apical_dominance

Wikipedia retrieved August 20,2009 http://en.wikipedia.org/wiki/

Meristematic

www.ingentaconnect.com/content/maik/rupp/2003/00000050/0000000 1/0
046 1666?crawler=true –

OTHER SOURCES

Gore,Al (April 27, 2008) Cable Vision HBO: “The Inconvenient Truth”.

Nantes, Rafael P. (October 13, 2007) QUESCAA Convention

Tayabas,Quezon
 66

APPENDICES

RESEARCH CONTACT DIRECTORIES

On potentialities and demand of Jatropha curcas as alternative biogas

feedstock – Reading and interview sources.
Names and Contacts Topic

1. Dr. Pedro D. Balagas The odds on mass

QEPC,DENR, Region !V-A, propagation of
Jatropha
Talipan, Pagbilao, Quezon curcas against vegetative crops
Tel.# : 042-710-2815

2. Reinhard K. Henning,author of Jatropha Booklet Downloading of

the
Bagani GBR, Rothkreuz D-88138, Jatropha Booklet;The
Neissensberg, Germany Value of the Jatropha
System
e-mail: henning@bagani.de,
Jatropha website: www.Jatropha,org
Tel. #: +49-8389-984129
Fax: +49-8389-984128

3.Mr. Ummu Hani, General Manager Downloading of the

Promotions Center for Management “Best Practices for Long
Technology Term Jatropha
Development”
Website: www.futureenergyevents,com/jatropha

4. Dr. Jessamyn D. Recuenco-Adorada, Adaptive trials of

University Extension Specialist Pest control on Jatropha
Crop Protection Center,U.P.
Los Baños, 4031 College,Laguna
Phone: +63(049)536-2410
 67

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

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

 68

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

Baños
 69

Results of Chemical Analysis

from the Department of Science and Technology
 70

CURRICULUM VITAE
 71

PERSONAL INFORMATION

NAME :Gracie O. Ching

ADDRESS : Sitio Mapuputi,Brgy.Liwayway ,
Mauban,Quezon
DATE OF BIRTH : August 28, 1967
CIVIL STATUS : Married with four children
SPOUSE : Arnold V. Ching
FATHER : Carlos D. Olivares
MOTHER : Iluminada E.Isles (deceased)

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,
 72

: UP Open University ,Los

Baños, Laguna.DST
Biology
(under Teacher’s Dev’t 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 : August 1-2,1998 - 75.60%

TEACHING EXPERIENCES
Liwayway National High School,
Mauban,Quezon : March 4, 2008 – to present

Pablo D. Maningas National High School

Quezon,Quezon : June 2007 – March 4, 2008

Pedro Guevara Memorial National High School

Sta. Cruz,Laguna :July 1 – Aug.29,2005

San Padua College,

Pila, Laguna :May – October,2005

Cometa National High School-Annex

Quezon, Quezon : March 4 – April 4, 2003

De La Salle University, : Sept. 25- Oct.26,2001

 73

Lipa, Batangas

Laguna State Polytechnic University, : July 11, 2001– Sept. 2,1999

Sta.Cruz, Laguna : Sept. 20-Nov.19,1999
Laguna Santiago Educational Foundation,Inc.
Sta. Cruz, Laguna : June 1– July 31,1998

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 : Sept. 2008

TRAININGS AND SEMINARS

In-service Training (INSET) for Teachers and Employees
held at Liwayway Nat’l High School,
Liwayway,Mauban,Quezon :Oct.27-29,2008

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 :May 26-30,2008

Capacity Building on SBM BESRA Implementation

and Mid-Year Adjustment of Divisional Operations Plan
 74

for Elementary and Secondary School Heads and Teachers :Feb.9,2008

Quezon Science Teachers’ Convention:
Theme:”Science towards Education Excellence”,
held at Convention Center, Lucena, Quezon :Jan.12,2008

Teachers’ Convention:Theme:”Education towards Digital Age”,

held at Convention Center, Lucena,Quezon :Dec.17,2007

District Seminar-Workshop for Secondary School Teachers

and Personnel held at Cometa National High School-Main
Cometa, Quezon, Quezon. :Oct.22-24,2007

5th Quezon Science Club Advisers Association, Inc. Convention,

Theme:”The Role of Science Investigations in the Conservation
Of Quezon’s Natural Resources”, held at Gavina’s Restaurant,
Tayabas, Quezon sponsored by QUESCAA and DepEd : Sept. 17,2007

In-service Training Program for Private

Secondary School Teachers,Theme: “Multiple Intelligence”
held at Lyceum Institute of Technology,Calamba,Lag. : May 2-6,2005

OVERSEAS TRAVEL HISTORY

Study on Elementary Education (Grades 3-6)
Keravat International Primary School,Keravat,
East New Britain, Papua New Guinea : 1976-1979

Study on Secondary Education (Year Levels 7-8)

Lae International High School,Lae,Papua New Guinea : 1980-1981
 75

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