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UNIVERSITY OF RIZAL SYSTEMAngono, Rizal :

EFFECTS OF GAMMA RADIATION IN VITRO CULTURE

ON PROTOCORM DEVELOPMENT OF DENDROBIUM ''

Pattaya Beauty

A Thesis

Presented to the Faculty of

the Institute of Science

and Education

In Partial Fulfillment of the Requirements

For the Degree of Bachelor of Science :

in Biology

FLORES, JOHN MARTIN R.i

LANIC, ALVIN D.

: MATUTO, MARK ANTHONY C.

VIDALf JOHN FAUSTUS C.

2005 - 2006

Republic of the Philippines

Angono, Ri/al

Approval Sheet

Thus thesis entitled EFFECTS OF GAMMA RADIATION IN

VITRO CULTURE ON PROTOCORM DEVELOPMENT OF DENDROBIUM

Pattaya beauty has been prepared and submitted by: JOHN

MARTIN R. FLORES, ALVIN D. LANIC, MARK ANTHONY C. MATUTO,

JOHN FAUSTUS C. VIDAL in partial fulfillment of the

requirements for the Degree BACHELOR OF SCIENCE IN BIOLOGY,

is hereby recommended for approval.

MR.

Aovi se rDATE

JOSEPHINE B. JAMERO

Chairman

Approve in partial fulfillment of the requirements for

the Degree BACHELOR OF SCIENCE IN BIOLOGY by the

examination Committee with grade of

MR

MRS. NORMPJ F. ELVINA

Member

Accepted in partial fulfillment of the requirements

for the Degree, BACHELOR OF SCIENCE IN BIOLOGY j

MR. JERRyWT-ESTERANZA MRS. F L O $ D E L 1 ? A R ! DIAZ

0TW Instructor Institute Head, iISE

—MR. RENATO M.

Date1 Date

lit


(WMiAngono, Rizal

Acknowledgment ;

We were blessed to have had people who were generous

in sharing with us their time and resources. We are forever

indebted to all of you. Thi3 thesis would not have been

possible if were to do this alone.

Dr. Olivia F. De Leon, University President

Dr. Araceli B. Bobadilla, vice President of :Academic

Affairs.

Dr. Valentina R. Catmunan, Director of Angono Campus,

for her moral support and kind consideration;

Mrs. E'lordeliza R. Diaz, Institute of Science and

Education for her generosity. |

Mr. Jerry £speranza, thesis adviser, statistician for

his patience, assistance, comments and suggestions in our

thesis;

Dr. Regina Noemi R. Abarabar, for her advice arid moral

support;

Mrs. Josephine B. Jamero, for her guidance, moral

support and share some ideas;

Mrs. Avelina G. Lapade, Head of Agriculture researchi • i ! I '

Section PNRI, who shared some knowledge about the study.

JV


WERS1TY OF RIZAL SYSTEMAngono, Ki/al

Ms. Ana Marie Veluz, Adviser, Supervisor, who were

always around to give some advice on laboratory techniques,

and for guiding the members every inch of the study, -for

her encouragement, guidance, and for lending us the use of

laboratory equipments. I

: Ms.: ; Jane, : Ma'am Suzette, and Mang Gil for the• : • i

guidance, support, encouragement, sharing us some stories,

giving some advice on the study.

To : the Library . staff of PNRI who shared ;written

materials, journals, books and the use of computer. '<• . I •

To all the Soccer team, Petroglyphs adviser jeditors

and staff for their support and philosophical iviews about

the study. i : • I | i : :

To Ms. Lord Apple Maquiling for sharing Isome stories,

advices, constructive criticism, encouragement, ; ;

Friends and classmates of the researchers from BS-

Biology, the critics of our group, who were one way or

another to serve as an;aspiration in continuing Lhe study.

To ;our Beloved parents, Mr, Romeo C. Matuto and Mrs.

Teresita C. Matuto, for my sisters and brother, land my

cousins. : • ; :

mm UNIVERepublic of the Philippines

FTY OF RIZAL SYSTEMAngono, Ri/iil

Mrs. Rebecca G. Flores, to my brother Karbie and Kaye,

and to all my Aunties and cousins for their support.!

Mr. Mansueto Lanic and Mrs. Linda Esperanza D. Lanic,

to my brothers Malvin Kelly and Mansueto,: Mommy Aida,

Masong, Ate Precy, and especially to the BS Bilogy

classmate Ronnie Cerr&ro, Carmen Zainora, Gladys Melissa,

Melissa Bayot, Ella Arrambulo, Alice Anain, Anna Grace

Francisco and others for their support and encouragement to

finish this study.

Mr. Faustino C. Vidal and Mrs. Elizabeth C. Vidal for

their moral support, love, encouragement, inspiring views

and financial support. To my brother J.R. and sister J.C.

for their laughs and catfights. To someone special for her

moral support, encouragement, laughs and cries,

inspiration, if she is not around the study will not be a

complete one. And most of all to Almighty God, for all his

plans, guidance, courage, love, and for listening

prayers. None of these would be possible without You.

A!

! M . A'

J. F

to our

. R. F .

. D. L .

. C. M.

. C. V .

VI


R1 RJZAL SYSTE1

Angono, Riw»!

Dedication ! •

This piece of work is whole heartedly dedicated to the f f:

i)hr. and Mrs. Teresita C. Matuto,

f\hr$. Rebecca G.FIores, •

Mr. and Mrs. Linda Esperanza D. Lanic,

Mr. arid Mrs. Faustina C. Vidai.

To all our friends, classmates, schoolmates,

To all the teachers who helped us.

By giving their encouragement

And Most of All to .

Sod Almighty for guiding us, •-;

For giving us strength to make it possible. i

J'.M.R. F.

: A. D. L

M:. A. C M.

J. P. <:. v.

vn


Angono, Rial I

abstract

This study sought to determine if Gamma radiation had

an effect on in-vitro cultured orchids in its regeneration

of a protocorm into plantlet within 20 days duration.. To

find the effects of gamma radiation in terms of weight,

development^ "the method used was experimental- procedure.

The design used in this study was Random Complete Block

Design.

The researchers' used Dendrobium Pattaya Beauty as a

subject of the study. Applying gamma radiation to change

the maturing time of the protocorm.

The researchers' findings about the study shows that

the average weights increase of each treatment/s in the

three (3) data observation after inoculation vary from

sample to sample. As can be seen in the table that the

average weight increase of dendrobium pattaya beauty

i i

protocorm does not have an obvious trend considering the

increase of gamma radiation on the different treatments.

It can be seen that the converted f average of 1.5 is

lower than the critical f value of 4.07 at 0.'05 level of

significance with the degree s of freedom of 3^8. The null

viiiRepublic of the Philippines

UNIVERSITY OF RIZAL SYSTEM .'Aiigono, Riza!

hypothesis stating that gamma radiation has no effects on

the weight of dendrobium pattaya beauty protocorm is

accepted. :

Therefore the researchers concluded, that out: of the

eight {8} samples the general average weight increase for

the control (1.01 grams), 30 Gy (1.22 grams), 50 Gy (1.06

grams), 70 Gy (0.44 grams). Hence, it can be concluded that

the radiation does not show positive effect on the weight

of Dendrobium pattaya beauty protocorm. One-way ANOVA shows

that there is no significant effect on the weight of

dendrobium protocorrn.

The researchers recommend that Dendrobiuni pattaya

beauty is a good example; of plant, to have undergone this

kind of experiment; i :

The researchers also -recommend that gamma radiation

would not affect the groWth of the protocorm upon the

: !

result of one-way ANOVA that there is no significance using

gamma radiation. The researchers would like to recommend

continuing this study;. :from embryo to complete piantlet.


Rizai

Table of Contents ;

TITLE PAGE i

APPROVAL SHEET ii

ACKNOWLEDGEMENT iii

DEDICATION vl

ABSTRACT vii

TABLE OF CONTENTS Ix

LIST OF TABLES xi

LIST OF FIGURES xiii

CHAPTER 1 THE PROBLEM AND ITS BACKGROUND 1

Introduction 1

Setting of the Study 5

Statement of the Problem 6

Objective of the Study 6

Hypothesis .--, 7

Significance of the study i 7

Scope and Delimitation of the Study ; 8

• Definition of Terms . i 8

CHAPTER 2 REVIEW OF RELATED LITERATURE AND STUDIES 14

Foreign Literatures : 14

Foreign Studies 16


OF RDAugcmo. Rizal

Local Studies 20

Local Literature 22

CHAPTER 3 METHODOLOGY OF RESEARCH .AND SOURCES OF DATA 26

Research Method and Design 26

Experimental Design 29

; Experimental Layout 30

: Research Materials and Equipment 31

Procedure of Study 32

Statistical Treatment 34

CHAPTER 4 PRESENTATION, ANALYSIS AND INTERPRETATION OF

DATA 47

CHAPTER 5 SUMMARY OF FINDINGS, CONCLUSION AND

; RECOMMENDATIONS 59

Summary of Findings 59

Conclusions 59

Recommendations . £;Q

Appendices . -61

: A. Letter of Endorsement • ! 66

B. ' Letter of Acceptance.: : 67

Refarenc^ List . 53i :

Routing Slip ! 71

Curriculum Vitae : . ; 72

XI


SYSTEMAugono, liv/x-A

List of Tables

TABLE

1. Experimental Design of Dendrobium

Pattaya Beauty

2. Chemicals used for Knudson C

Page

30

32

3. a.)Difference in weight of Dendrobium

Pattaya Beauty Protocorm after six {6}

interval days in three(3) data observation

after irradiation and Inoculation (Control; 47

b.) Difference in weight of Dendrobiurn

Pattaya Beauty Protocorm after six (6)

interval days in three (3) data observation

after irradiation and Inoculation (30 Gy) 49

c.)Difference in weight of Dendrobium

Pattaya Beauty Protocorm after six (6) :

interval days in three (3) data observation

after irradiation and Inoculation (50 Gy)

\.)Difference in weight of Dendrobium

Pattaya Beauty Protocorm after six {6);

interval days in-three (3) data observation

after irradiation and Inoculation (70 Gy) 53

4. Composite table of the weight increase in the

51

XII


Angono, Riza!

different treatment after the three (3)

data observation Period upon irradiation

and inoculation. 55

5. One-way ANOVA on the effects of gamma radiation

On dendrobium pattaya beauty protocorm under

laboratory condition 57


LIST OF FIGURES

xni

Figures : Page

1. Map of Phil. Nuclear Research

Institute (PNRI) 5

2. Entrance gate of PNRI 37

3. Infrastructure of PNRI 37

4. Greenhouse of agriculture research

Section 38

5. Dendrobium Pattaya beauty plant 38

6. Building of Agriculture Research

Section 3 9

7. Building of Irradiation of gamma radiation 39

8- Dendrobium Pattaya beauty culture bottles 4 0

9. Dendrobium Pattaya beauty control and

Treated bottles 4 0

10. control bottle of Dendrobium Pattaya beauty : 41

11. 30Gy bottle of Dendrobium Pattaya beauty ; 41

12. 50Gy bottle of Dendrobium Pattaya beauty : 42

' 13- 70Gy bottle of Dendrobium Pattaya beauty I 42

14. Preferred bottles for the media(Knudson C) ; 43

15. Hotplate stirrer with Knudson C media 43

xsv


Angono, Rizal

16. Actual preparation for inoculation 4 4

17. Actual presentation of inoculating 4 4

18.: plugging of Catsup Bottles 4 5

19. Air conditioned culture room 45

20. Actual Plugging of Knudson C media 4 6

21. Actual autoclave preparation 46

Republic of the Philippines; OF RIAngono, Riza

• • \ Chapter X : •- '•

; INTRODUCTION

Orchid a member of the Orchidaceae, order orchidales

belonging to the family of perennial monocotyledonous

terrestrial plants. Some are epiphytes or saprophytes; they

are cosmopolitan and very abundant in the tropics.; Within

the genus of orchidaceae there exist a number of va-riet-*s

of species, which have different characteristic^ with

respect to the growth habits and general relativity.

Orchids are specially describe as houseplants or home based

plants that; are known for their adaptability, elase of

drowing,: availability and production of beautiful flowers.:

i,t is important to know that orchids have different types

according to their cultural requirements; Plalaenopsis,

Cattleya, and Dendrohium. These beautiful orchids produce

beautiful flowers but will take up to 5 years to bloom.

i :

i The general culturingof orchids require the following

t:o survive: temperature,, light, fertilizer, humidity,

proper containers and watering. This study shows that in

vitro culture is the aseptic (free from microorganism.)

culture of any plant parts in. vitro. Tissue culture is• i

utilized' in the field of ;Biotechnology. Micro-propagation


' OF RIZAL SYSTEMAngono, Rial

is the rapid vegetative propagation of plants; via; tissue

culture .'techniques. ; i

Micro-propagation permits the manipulation of physical

and chemicals conditions in the production in a; large

numbers of high quality plant materials within a short

period of time. In vitro culturing, also known as micro

propagation was developed or produced slow growing

temperamental orchids, which will create an impact in

agricultural research and production. Tissue culture is a

process in which micro-propagating the species though it

gives a vexing problem that people look at tissue culturing

as part of cloning, hence it gives more further interest to

the researchers to understand a tinge of apprehension to

extend their mind or broaden their IQ' s to understand ..ie

meaning of tissue culturing as micro-propagation of plant

or a certain species of plant like Cattleya. \ Tissue

cuituring is not just done in sterile laboratories.:

: )

Essentially, this is what: must be done. These; containers

irmst be sterilized; everything you see must be sterile to

reduce the possibility of pathogens being introduced into

the medium. There are different mediums (usually a gel) to

produce undifferentiated callus tissue, multiply; the: number


I UNIVERSITY OF REAL SYSTEPAugono, Riza!

of plantlets, grow roots or multiply embryos for the

"artificial seed". The medium is dosed with a chemical to

encourage root growth. You insert one leaf (or other type

plant material) into the baby food jar that has the

appropriate medium. Although In. Vitro culture requires a

lot of materials and mediums to be used, it shows that

plants are the most usable materials for experiments.

Dendrobium contains a very large number of

heterogeneous species. Members of this group have an erect,

cane-type pseudobuib and have flowers with an relatively

long vase like. With a wide range of brilliant colors

possessed by their flowers, dendrobium species and hybrids

have always occupied a dominant position in the minds of

floriculturist and amateur orchidists. In the Philippines

the dendrobium group (which include Den, Pnaiaenopsis

Fitzg., and Den. Bigibbum, Lindl. Among other:3) is

generally prepared. The dendrobiuitt group is naturally

: i :

distributed in the warm, humid areas, near the: equatorialjI

zone of the Australian - Asian region. Thus, the resulting

hybrids require warm and humid environment with abundant!

sunlight:for optimum growth and development. ;


:MSITY OF RIZAL SYSTEMAngono,Rizal

i Orchids are important to some orchid hobbyist because

they thrive to explore more about the plant. It is also

important to our local agriculture in which the flowers

were used to some experiments in hybridizing the plant. In

agriculture sectors orchids strengthens the :propagation

capability through the production of disease-free plant

material via micro-propagation techniques. It will

contribute to propagate more species of orchids and will

give the interest to some researchers about the plant

itself.

As isotopes sources are relatively cheap, the

instrumentation is readily available, and the application

in practically all fields of science and industry. It is

not surprising that the importance of the us'e of these

tools, in spite of growth of other new methods, Us steadily

increasing in both developing and developed countries..

In agricultural research, isotopes and radjiatidn play;

a: part in so many fields and in so many ways that ii is so

difficult to obtain a proper picture of their enormous'1

importance. In laboratories of developed ! countries,:.. : • i i •

isotopes are used routinely with an ever-increasing

assortment of modern research aids. In emerging

Republic of the Philippines,

biotechnologies, which ' are used increasingly byi

agricultural scientists, isotopes are a basic tool (without

i iwhich research in molecular biology could not be'donej.

! ISetting of the Study | ,

i I

The study was conducted at the Tissue culture1 i

laboratory of the Agricu] ture Research Section | Atomic

Research Division of the Philippine Nuclear Research

i iInstitute located at Diliman, Quezon City, Philippine^.

Figure 1. Map of Phil. Nuclear Research Institute (PNRI)


UNIVERSITY OF RIZAL SYSTEMi Angono, Rizal

o£ the Problem ;

The study was conducted to determine the effects of

Gamma Radiation In-vitro cultured of Dendrobium jPattaya

Beauty using the KNUDSON C medium as basal medium for

regeneration within 20 days study from protocorrn. TO solve

the aforementioned problems the researcher sought answers

to the following specific questions:

1. What is the effect of Gamma radiation on protocorm

development of Dendrobium PatLaya Beauty in terms of

weight?

2. Is there any significant difference on the rate of

increase in weight of Dendrobiuin Pattaya Beauty

treated with different dosage of Gamma ray?

Objectives of the Study

The study aimed to determine the effect, of gamma rays

on in-vitro cultured orchid in the regeneration of an

undergone protocorm into plantlet within 20 days duration.

Ajlso this study, is to find the effects of gamma radiation

In; terms of weight development.


H I UNIVERSITY OF REAL SYSTEMAngono, Rizal

Hypothesis

There is no effect of Gamma radiation in propagating

the species of Dendrobium Pattaya Beauty in terms of

weight, Protocorm developed. There is no significant

difference on in-vitro cultured using Gamma radiation of

Bendrobium Pattaya Beauty and its control in terms of

weight and Protocorm developed.

Significance of the Study

The study will strengthen the propagation capability

in agricultural sectors and will contribute more to the

product! i of disease-free plant material via micro-

propagation techniques. In-vitro culture is the aseptic

culture of any plant part; it will permit the manipulation

of physical and chemical conditions in the production of

large numbers of quality plant material within a short

period of time. Also, it wills cheaper the market value of

Dendrobium because more plants produce and may result toi i

drive the price down. Other researcher may use this data toi ' :

propagate other plants:. This study will help to emphasize

the • importance of using radiation through gamma rays to

develop new and novel varieties of plant particularly!

orchid and other ornamental: crops. ; , :


ITY OF RIZAL S YS1Angono, Riza!

Scopa aad Delimitation of tSie Study i

The data that was gathered or collected was from

protocorm development within 20 days of duration. The

protocorm of the plant was grov/n in a KNUDSOM C basal

medium with 15% bungulan. Data collection was on a 6 day

interval from protocorm irradiation. The irradiation

treatments were 30 Gray (Gy), 50 Gy and 70 Gy with

unirradiated protocorm as control.

Definition of Terms

The following terms are defined here under in the

context of the study:

Aseptic culture - Culture grown under a sterile

condition, free from microorganism elements (Torres, 1991)

Auxin - A group of compounds known by their capacity

to induct elongation in shoot cells; a principal ingredient

for the creation of the cell colony (Weaver/ 1979)

Bloom - an individual flower.

Callus culture - In vitro culture growth of tissues

arising from the disorganized proliferation of cells from

segments {explants) of the plant organ. Callus culture is.

usually grown as a. mass of cells on a solid medium. Callus

grown in vitro has some similarities to tissue arising in


SITY OF RIZAL SYSTEM

vivo from injury to plants (called wound callus). There can

be a difference in the morphology, cellular structure,

growth and metabolism of callus derived through tissue

culture and natural wound callus (Torres, 1991}

Cultivars - a clone or a number of morphologically

indistinguishable clones or a line or line-hybrid

maintained in cultivation.

Culture cell - High vacuolated cell, thin cytoplast,

very large, of diverse shape and contain faintly visible

nuclei, these cells are differentiated but perhaps

organized (Weaver, 1979)

Culture Medium - A process of growing ox~ developing

microorganism or plant parts through formation of nutrient

media under artificial and controlled condition (Weaver,

1979)

: Cytokinin - Naturally occurring or; synthetic

substances that induce cell division in certain plant

tissue in the presence of auxin (Weaver, 1979) ;

: Development - The sum total events that contribute to

the' progx'essive elaboration of the body of :an organism

[Torres,; 1991) :; .;

10Republic of the Philippines

Aiigono, Rizal•u i-Jmi*oVjt^*Tt*t. mtv- ' W . ^ **»>;»•• trwnsto*"in*uaivutiM HJ«»I»H m i.«mwiwi»w»'"t*«

Dendrobivm - Their flower spikes are commonly used as

cut flowers and orchid leis.

EDTA (ethylenediaminetetraacetic acid - A chemical,

chelating agent used to keep iron from precipitating in

culture media (Torres, 1991)

Epiphyte - A plant which naturally grows upon another

plant or other means of support above earth's soil but is

not parasitic; deriving it's needed moisture from the air

Explants - part of a plant taken from natural site and

under-go inoculation in medium (Torres, 1991).

Gray ~ Unit used in gamma rays (Encyclopedia of Agri.

Science, 1979).

Gamma Radiation (y) - belongs to the class known as

electromagnetic radiation. It consists of quanta or packets

of energy transmitted in the form of wave motion. Gamma

radiation travels very large distances and loses its energy

by interacting with atomic electrons. It is difficult to

absorb completely. It can travel through the body.

In vitro- Kind of artificial environment outside the

living organism (in Glass). Outside the body;. under

laboratory conditions or in an artificial environment

(Bajaj 1984, Torres, 1991).


mm UNIVERSITY OF REAL SYSTEM

Induction - The act or progress that causes initiation

of structure (Torres, 1991).

Inoculation - act, process or an instance of

•a

inoculating; the introduction of a microorganism into a

suitable medium for its growth (Webster's Dictionary 1986).

In vitro transmission - A heritable change occurring

in cells in culture result from treatment with chemical

carcinor-ns, oncogenic viruses, irradiation, etc, and

leading to acquisition of altered morphological, antigenic,

proliferation, etc., properties (Torres, .1991)

Irradiation - a giving off or emission of radiant,

energy, an emanation, diffusion, or radiation of something

from a common center or point of origin as a result of an

activity

; •• Isotopes - are atoms of a particular element in which

contains the same number of protons but may have differing

numbers of neutrons, this means that an element can have

sjeveral [types of atoms and any given element ar& chemically•; t- ' - i l l

i d e n t i c a l . . . ' . ; • '''• '

Medium ~ The potting material or mixing materials

being used inside the orchid pot.


Angono, Riza!

Knudson C basal medium - The medium used for the in

vitro culturing of orchids.

Micro-propagation - Another name for in vitro

propagation or tissue culture (Torres, 1991)

Nodal Section - A point on a plant stern at which a

leaf or leaves are attached (Torres, 1991)

Orchid - A member of the Orchidaceae, a family in the

monocotyledons, which are a major group of flowering

plants. Presently; the orchid family comprises about 35,000

species and 60,000 registered hybrids. '• *

Pathogen - A disease producing organism.

Petiole - The stem like portion of a leaf which the

blade is connected to the stem (Torres, 1991).

ppm - Refers to part of per million, which is equal to

Img per liter (Sharp, 1979)

Regeneration - In plant tissue culture, the formation

of new parts from in-vitro derived tissues;! particular

formation of entire plantiets from cultured1 callus or

organs (Torres, 1991). ij

• Shoot tip or Apex - The apical meristem together with

primordial and developing leaves and subjacent stem tissue

(Torres, 1991). I

.,1,1

Republic of the Philippines7 OF SI2Angono, Rizal

« ^ V ^ 1

Species - A group of plants showing intergradations

among its individuals and having in common one or more

characteristics which definitely separate it from any other

group, a kind of plant distinct from other plant's. \

Terrestrial - The maintenance or growth of tissues in

vitro in a way that , may allow differentiation and

preservr'-ion of the architecture and or function. The term

plant tissue culture is often used generically to offer to

ail types of aseptic, in vitro plant culture (Torres,:; 1991)

i Protocorm - Early stage in seed germination having a

globose structure with : fine root-like projections

(rhizoids) with or without leaves; pre-seedling stage.i

Variety - a given kind; a plant which varies from the

type in one or more, important characters; see cultivdrs

\4Republic of the Philippines

UNIVERSITY OF REAL SYSTEMA»go»ot Rival

II

• REVISE OB' BELATED LITERATURE AfcJD STUDIES \

! This chapter contains the relevant literature and

study. This study like many others is•anchored ion a theory

upon which a scientific investigation is based. \

Foreign Literature

Kovacs et al (1985) a |study indicated lin the

irradiation of apples extend the shelf-life : of the fruit

stored at 2°c to 9 months. (Rigney et al 1985) according

his study gamma radiation of apples didn't alter the rate

of suffering of fruit, which could be expected if there's a

major change in calcium metabolism.

L.M. Revetti studies the Maize and kidney beans know

insect population, exposed to 60Co gamma rays. In the maize

population insect consist of coleopteran and Lepidoptera.

• In kidney beans only coleopteran. The doses radiation

applied; varying from 10 to krad. After radiation

temperature and humidity condition is under controlled. The• • « * • '

sampling carried out in order at suitable interval action

of radiation in grain components. The maize was made on the

acidity of grain have no changes in greater possibility of

rancidness of grain detected. The iodine concent ration in

35Republic ofthe Philippines

CRSITY OF RIZAL SYSTFEAuj>ouo, Rizal

maize oil did not vary with radiation dose. Direct affected

of gamma rays grain protein fraction found, analytical

methods applied in irradiated. The samples revealed no

significant changes. The doses of 20 krad and 30 krad

prevent contaminations.

According to Chalermpol (200.1) the advantages of

tissue culture's ability to dramatically speed up the

research and development, of improvement plant varieties -

process that can take months or years using traditional

propagation methods such as sowing seeds or growing plant,

cuttings in soil. Currently working with 100 tree species

to determine which grow best in the saline soil. And also

have potential to reduce genetic diversity by displacing

species with single genetically identical plant.

Gavino Rotor (1949) demonstrated that inflorescence

nodes from Phalaenopsis could be.induced to form a plantlet

if aseptically placed on seed germination media. George,

Morel (1960) reported that excised Cymbidium Sw. shoot tips:i • • i

could be' induced to f<j>rm multiple plant let si when cultured^

on 3eed germination media ^supplemented with phytohormones.I

Donald Wimber (1963) developed a successful method of using

tissue culture to commercial propagate cymbidi.urn.

16


Angono, Rizai

Radiation Preservation of Food N. Grecz, H. LO, E.J.

Kennedy,, E. Durban Gamma radiation studies of Clostridium.

botulinum Types A, B and E (November 1972) The profiles of

gamma radiation survival curves of spores of Clostridiurn

botulinum types A, B and E have been analyzed for 14

strains of high, medium and low radiation resistance. An

attempt was made to correlate radiation resistance with

electron microscopic and phase contrast cell morphology mid

with gross colonial appearance in deep agar cultures. The

general features of multihit profile of radiation survival

were obtained. The appearance of colonies in deep agar

culture was of three general types: (1) strongly lobed

solid colonies, associated as a rule with high radiation-

resistant strains; (2) lacy or small-lobed colonies usually

but not always associated with radiation-sensitive,

particularly type E strains; (3) small, fluffy, diffuse,

cottony colonies usually associated with radiation-

sensitive strains. 8

Foreign Studies

In Korean, in green house nurseries, tissue cultured

temperate cymbidiums suffer from diseases causing great

37


Angono, Rizai

loss in plant life. Two species of orchids Cyin. goeringii

and Cym. kanran showed relatively better growth and a

lesser death rate in the HKNO soil than in other culture

media, including sterilized HKNO soil.

Colombia is well known worldwide for its extremely

high indexes of biodiversity of both plants and animals.

The paper Orchids and Ecology in Colombia represents

an attempt to show the correlations between ecosystems and

main orchid species in Colombia, as a basic step towards

the establishment of protective measures for both specific

and ecological conservation of these wonderful plants that

for many are the true jewels of our jungles and savannas.z

Great variability and beauty of Dendrobium nobile

hybrids has attracted interest of biotechndlogical

laboratory for rapid clonal propagation of superior

genotypes of this species.

This study was performed to establish a protocol for:

micro propagation of Dendrobium nobile using approaches to;

reduce the undesirable somaclonal variation. In order to

get this aim, following procedures were used: low;

13


Angono, Riza!

concentration of fitoregulators, reduction and limitation

of subcultures, avoidance of callus formation and finally

stimulation of axillary's bud propagation.

Bel ore cell division takes place, the genetic

blueprint must be replicated and correctly separated

(mitosis) so that each cell has the identical blueprint, in

the life span of larger organisms, many tens of thousands

of cell divisions are accomplished every day.

Alam, M.K., Rashid, M.S., Hossain, M.S., Salam, M.A.

and Rouf, M.A. in 2002 reported that the four different

media namely - Hyponex, Murashige and Skoog (MS), OKF1, and

Knudson C (KC), were tested for large scale multiplication

of sympodial native orchid, Dendrobium transparens Wail.

Via seeds. MS medium was found best for characters studied

in the said orchid, and. it wars followed by Hyponex medium

but OKF1 gave the least performance. Days required for

germination was the minimum of {50 days) in MS medium while

maximum (59 days) was required by OKF.1 medium. Considering

other characteristics, such as days required to protocorm

formation and plantlet development, number of leaves and

roots per plantlet, plantlet height and root length and

19


Angono, Riza!

finally plant survivability percentage, MS medium showed

significantly better performance for in - vitro seed

propagation of Dendrobium transparens. It was also stated

that the different media significantly influenced the

protocox-rn formation and plantlet development (Aiam et al

2002}. The maximum duration (48 days) was required by OKF1

medium, which was significantly similar to Knudson C medium

{43 days) while MS medium took the least period (36 days) .

Similar trends were also observed in plantlet development.

Hoque (1993) stated that protocorms differentiated into

plantlets within 72 - 78 days in different media in which

is closely related to the present findings.

In 2005 Mar.kus Axelsson reported that Knudson C medium

are old formulae in which proven to be good for growth in

cultures of terrestrial species. Micronutrien'ts and

vitamins are naturally occurring in pretty much everything

arid may enhance the growth of the specimen.

; According to Simmonds, N. W. (1966) reported that

banana's composition and nutritional value is mostly

composed of water and carbohydrate and ha3 almost

negligible contents of protein and fats. It is

20


Angono, Rizal

comparatively rich in potassium, magnesium, sodium and

phosphorus; according to Loesecke (1950) copper, iodine,

manganese, zinc and cobalt have also been identified.

Standard figures for nutritional calculations {Chatfield,

1954; Watt and Merrill, 1950) are as follows: Calcium .80,

Phosphorus .290 and Iron .6 these are calculations in p.p.in

from fresh pulp.

Local Studies

Most of the habitats of the Orchid species are located

in developing least developed countries. Conservation of

nature, though advocated with all fanfare, is slow to take

root in these countries and obviously they have other

compelling priorities.

Encouragingly, there is a world-wide concern for

conservation of biological diversity in all its facets for

which this august City happened to play the most

significant role as the host in very recent years; RIO

Convention is;i now the pass-word in all world environmental

meets.

iThe former looking open then as a source of economic

gains, whether by eco~tourism or scientific growing, and

21


1 1 1 ; UNIVERSITY OF REAL SYSTEMkJM Angono, Rhiil

the later as proud possessor of a unique natural creation

which modern technology cannot create. A key-note address

on this topic followed by series of panel discussions both

within and outside the Conference Carried over a week

should help us to arrive at a possible solution or at least

a world--.,xde accepted approach . 5

In 1983 Garcia, Nardo Q. reported that; the agronomic

parameters were significantly affected by the irradiation

with different doses in Ml generation were decreased the

percentage of germination in Red Rice from 6% to 42%,

shortening of seedling height from 3% to 8% in IR 52 and in

Red Rice from 4% to 10%. Heading dates of IR 52 was delayed

from 6% to 11% and 3% to 11% for Red Rice. Number of filled

grains of IR 52 was decreased from 12% to 70% and for the

Red Rice was from 45% to 288%. Increased in the nuftiber of

unfilled: grains for IR 52 and Red Rice was from 26% to 61%

and 96% to 288% respectively. '.

i : . 2fi '1999 Dimaano, • Maritess M. and Imperial Maria

Angelica: Liza V. reported that the effects of gamma

radiation on the meotic ceils of Tradescantia bracteata

clone 02 (BNL). Showed that flower buds were exposed

: 22


„ j UNIVERSITY OF RIZAL SYSTEM!^W Angono, Riail

through dosages ranging from 1 Gy to 5 Gy using Gamma Cell

220 machines (AECL) in central axis position (c/a) and

grown in Peralta's solution for three days. In the study

shows the occurrence of micronuclei among irradiated pollen

mother cells suggested a linear relation with the quantity

of radiation dose. Experimental results show chromosomal

breaks, sticky chromosomes/ and anaphase bridges in the

pollen mother cells of irradiated buds. A significant:

numbers of cells were also found to have micronucleif which

may vary from 1 to 6 per pollen mother cell, and this

showed no relationship with radiation dose.

Local Literature

Lapade ET. Al in 2000 reported that there is induced

Mutation in In vitro Techniques for the Genetic Improvement

of Ornamentals. Phil. Nuclear Journal. In Press, Jtfutation

Breeding through the use of gamma radiation coupled with

tissue culture techniques and related biotechnology is

being undertaken for the improvement of foliage ornamentals

and cut flowers. Produce chlorophyll growing and selection

of 'desirable mutants in D. sanderiana. In there study five

chlorophyll mutants were selected in D. sanderiana with the

green leaves? 18 in D; sanderiana with yellowish-geen

23


i UNIVERSITY OF REAL SYSTEIAngono, Rizal

leaves stripes; and 16 D. sanderiana with green and white

stripes in the total M1V4 population of 164,1463 and 139,

respectively. These variants were grown up to M1V6

generat.i is. These mutants were propagated and to date,

there are 50 plants exhibiting chlorophyll mutations.

Selection of dwarf mutants in Murraya exotica was

undertaken. There are 12 dwarf mutant induced through gamma

radiation of 10 and 20 Gy. Propagation of these mutants wan

done. At present, 50 dwarf mutants of Murraya exotica are

available for distribution. During the Atomic Energy Week

dissemination of information regarding the peaceful

application of Atomic Energy through Radio and TV was

undertaken. The Chlorophyll Dracaena Mutant and the Murraya

Mutant induced through;gamma radiation were highlighted and

test marketed. A total of 4 7 Murraya Mutants and 7

Chlorophyll Dracaena Mutants were sold and 50 irradiated

Dracaena;were given to the Press. Tissue culture studies in:

orchids and chrysanthemum are being done \ as tool for

mutation induction and as means of micro propagation.

Protocorms of Dendrobium sp. and Vanda sanderiana were

irradiated with gamma rays. Profuse growth of protocorms

was observed in all doses except 50 Gy. Shoots were formed


ITY OF RIZAL SYSTEMAngoiK), Rizal

from protocorms irradiated with 10-30 Gy as well as in the

control. Protocorms of Vanda sanderiana irradiated with 10

Gy and grown in Knudson C medium resulted in the

development of plantlets that are bigger and more vigorous

than those at 20 Gy and the control. Micro propagation of

the irradiation and unirradiation chrysanthemum using (MS)

basal medium being done. Growing of chrysanthemum in-vitro

is now in the M1V2 generation. A total of 330 plantlets

were micro propagated. These in-vitro cultured planlets

(control and irradiated) were maintained in MS medium (full

or half strength) until roots are well-developed and ready

for potting out. (Lapade A.G. et al)

Dr. Evelyn Mae Tecson-Mendoza (2001) reported that

Crop biotechnology encompasses technologies such as tissue

and cell culture. These technologies have been utilized at

different stages of plant breeding such as varietals

improvement and seed multiplication. Some of the tissue

culture technologies developed at the University of the

Philippines, specifically at the Los Bafios campus are:: L

embryo culture of makapuno coconut, rapid micro propagation

of orchids, and other ornamentals. Protocols for conserving

some of these crops in vitro have been developed for

25


(ccfc/onrw

Aiigono, Rizal

banana, yam, sweet potato, white potato, garlic and

shallot.

Lapade et al. reported that genetic mutation of the

queen variety of pineapple through induced mutation and in

vitro culture techniques by using ionizing radiation

coupled with in vitro culture. By irradiating the axillary

buds and tissue culture techniques were utilized for

mutation induction with the objective of eliminating or

reducing the spines or thorns along the margin of the leaf

of pineapple. The specimens were irradiated with 5, 10, 20,

30, 40, and 50 Gy of gamma radiation. 50 samples were used

in the experiment with three replications arranged in

randomized complete block design. The percentage survival

i

of the buds that emerged were collected 65 days after

irradiation and was measured 95 days after transplanting or

250 days after irradiation.

26


Angono, Rizal

Chapter III

METHODOLOGY OF RESEARCH AM3 SOURCES OF DATA

; The chapter presents method and design, the subject of

the study, research procedure, experimental layout, and

statistical treatment to be used to be able to answer the

problems presented.

Research Method

This study will make use of Experimental Method of

Research and Randomized Complete Block Design to get

accurate findings on the problems.

This design may be used when the experimental units

can be meaningfully grouped, the number of units in the

group being equal; to the number of treatment or some

multiple of it. Such a group is called a block or

replicate. The object grouping is to have the units in a

block as uniform as possible so that observed differences

will be greater, on the average than variability among

units in the same block if no treatments were to be

applied. Clearly variability among blocks does not affect

differences among treatments means since each treatment

appears in every block.. Each block usually1 consists of

27


Angono, Rizal

compact, nearly square group of plots. Likewise, placed in

outcome group or blocks on the basis of such

characteristics as initial weight. During the course of the

experiment all units in a block must be treated as

uniformly as possible in every respect other than

treatment. Any changes in technique or other condition that

might affect results should be made on the complete block.

If the number of observation per unit equal the number of

observers then each observers should make one observation

per unit. This practice help control variation within

blocks and thus experimental error; at the same time, they

contribute nothing to differences among treatment means.

Variation among blocks is arithmetically removed from

experimental error. Notice the balance that exists in this

design. Each treatment appears an equal number of,* times,

usually once, in each block and each blocic contains all

treatments. It is sometimes stated that blocks and

treatment are orthogonal to each other. It is the property

g.

that leads to the very simple arithmetic involved in the

analysis of resulting data. This design is used more

frequently that any other design and if it gives.

28


satisfactory precision, there's no point in using an

alternative.

: The layout of RCBD experiment is very similar to that

of a pairing experiment. The only difference is that we may

have two or more treatments (or varieties) in RCBD

experiment, while there are only two treatments (varieties)

involved in paring experiments. A pairing experiment is a

specific type of a RCBD experiment. In which there are only

two treatments (varieties) included. The- randomized

complete-block design has many advantages over other

designs. It is usually possible to group experimental units

in to blocks so that more precision is obtained than with

the completely random design. There are no restrictions on

the number of treatments or varieties extra replication is

desired for certain treatments, these maybe applied £*o two

or more units per block. The statistical analysis of the

data is simple. If, as a result of mishap, the data from a

complete block or for certain treatments are unusable/ this

data may be omitted without complicating the analysis. If

data fro individual units are missing, they can be

estimated easily so that arithmetic convenience is not


J

Aiigono, Riza!

estimated easily so that : arithmetic convenience is not

lost. If the experimental is heterogeneous, unbiased

components applicable to testing specific comparison can be

obtained. The chief advantage of randomized complete-blocks

is that when the variation among the experimental units

within a block is large, a large error term results. This

frequently occurs when the number of treatments is large;

thus it may not be possible to secure a sufficiently

uniform group of units per block in such situations, other

designs to control a greater proportion of the variation

are available.

Experimental Design

This study "used the Randomized Complete Block Design

(RCBD) to determine if Gamma Radiation and In-Vitro

culturing is effective on Dendrobium Pattaya Beauty.'

30


SYSAngono, Rizal

Table 1.

Experimental Design of Dendrobiura Pattaya Beauty

TreatmentTl Control

22 30 Gy

23 50 Gy

74 70 Gy

Total

Replication 18

8

8

8

32

Experimental Layout

Tl - Control (In-vitro)

Rl R2 R3 R4 R5 R6 R7 R8

T2 - 30 Gy (In-Vitro)

Rl R2 R3 R4 R5 R6 R7 R8


Rl R2 R3 R4 R5 R6 R7 R8


Rl R2 R3 R4 R5 R6 R7 R8

Legend: . '

Tl '- Control under : Tissue Culture

(Dendrobium Pattaya Beauty)

T2 - !Dosage of 30 Gy (Irradiated Dendrobium)

Protocorm

31


Angono, Riza!

T3 - Dosage of 50 Gy {Irradiated Dendrobium)

T4 - Dosage of 70 Gy (Irradiated Dendrobiuni)

Subject of the Study

The subject of the study is about Gamma Radiation

on Protocorm Development of Dendrobiurn Dendrobiuni Pattaya

Beauty.

Materials used in the Study

Equipments and supplies used in the tissue culture are

analytical balance; (for weighing nutrients for media/

graduated cylinders and beakers {for measuring stock

solutions), pH meter (to regulate pH of media), hot plate

or stove (to heat and dissolve gelling agent), glass

containers (for heating and dissolving media), a still or

de-ionizer (water needed for media), Autoclave machine (for

sterilizing instruments and media), alcohol lamp/burner,

transfer instruments (forceps, scalpels, spatulas, blades),

refrigerator (storage; of chemicals and stock solutions),i : j ; : : . i • \\ : •

sterile ' petri plates; and Catsup bottles; that can be

sterilized with media in them and flowering orchid

{Dendrobium. Pattaya Beauty) Laminar flow hood . Reagents

needed are from Knudson C basal medium are: CaN'04, NH4SO4,

32

Republic of the Philippines7 OF RGAisgono, Rizal

MgS04, KH2PO4, FeS04, MnS04f Thiamine HC1 are the

components of KNUDSON C Basal medium, with 15% of bungulan

(banana), sucrose and agar.

; Table 2.

Chemicals used for Knudson C

Elements :; : : CaNO4

NH4SO4: MgS04

KH2PO4FeS04

' MnS04 :Thiainine HC1

: : Sucrose! Agar

• i

Weight in gramsl.OgO.SQ

0.25g0.25g0,025g0.0075gO.OOlg20g ;

9g/L

Procedure of fch© Study

: In vitro culture of orchid protocorm was done in the

study. ;

Preparation of medium. Knudson C basal medium was used

in the experiment the concentration of each mineral

element, vitamins and other components of the medium are

shown in table 2. Bungulan banana was likewise added in

the solution as additives,: after mixing all the mineral

elements ;-and additives: the solution was adjusted to pH 5

and agar: was added for solid medium. The medium was

dispensed in catsup bottles at about 75 ml. per bottle.

33


Angono, Rizal

Bottles were plug with cotton to avoid contamination of thei

prepared medium and then placed in the autoclave to be

sterilized at temperature of 121° C at 15 psi for 20

minutes. Bottles were taken out of the autoclave and were

placed in a cool and dry room before inoculation.

Irradiation of Protocorm. Protocorm was selected from

four bottles of control and three bottles was irradiated

with gamma ray3 at dosages of 30, 50, 70 Gy using Gamma

Cell-20 at the Cobalt 60° C facility of the Philippine

Nuclear Research Institute. Control protocorms was also

maintained immediately in the Knudson C medium. They were

kept in an air-conditioned room maintained at 20 - 25°

Celcius.

Transferring/Subculture of protocorm. protocorm were

observed every 6 days for changes in weight,., after

irradiation. First, sterilize the laminar flow hood, and

then wait for 30 minutes to begin the transferring of

protocorms. Prepare; the necessary materials needed such as:

sterilized spatula, forceps, petri plates, alcohol lamp,;

ligh'ter/match, and flask or catsup bottles with media, 70%

etnanol / ethyl alcohol for hand sterilization and

analytical balance. Remove the protocorm from catsup

34


Angono, Rizai

bottles and placed in a sterile petri plates, weigh the

protocorm and subculture in the newly prepared KC media

with bungulan banana. These procedures are done aseptically

to prevent contamination of cultures (protocorm). The same

procedure was done to all the treated protocorms. After all

the cultures have been weighed and sub cultured the bottles

was placed in an air-conditioned culture room maintained at

20 - 25°C until the next data collection.

Statistical Treatment

The statistical treatment was used:

To determine the significant difference on the effects

of Gamma Radiation on Protocorm development of Dendrobium

Species (Dendrobium Pattaya Beauty). One-way Analysis of

Varience v/as used:

One-way ANOVA (one way analysis of Varience)

Formula 1

C = (grand total)2

No. of Observation

Formula 2

Total SS = 2 X2 - C

Formula 3

35

Republic ofthe Philippines

Angono, Rizal

Block SS - S B2 - C

Formula 5

Error SS = Total SS - Block SS - Treatment SS

Formula 6

MS = Error SS

df Error

MS = Block SS

df Block

MS Treatment = Treatment SS

; df Treatment

Formula 7

, ' F Observed = MS Treatment

(trt. Effect) MS Error

36


' OF REAngono, Rizal

Formula 8

F Observed = MS Block

MS Error


Ango'no, Rizal

37

Figure 2 Entrance gate of PNRI

Figure 3 Infrastructure of PNRI


STY OF RIZAL SYSTEM

Figure 4 Greenhouse of agriculture research Section

Figure 5 Dendrobium ;Pattaya beauty plant


Angono, Rizal

Ejigure 6 Building of agriculture research Section

]: !Figure 7 Building"of Irradiation of gamma radiation

; I ;

4J


UNIVERSITY OF MZALAngono, Rizal

Figure 3,0 Control bottle of Dendrobium Pattaya beauty

Figure 11 30Gy bottle of Dendrobium Pattaya beauty

it lilii

tilliiiiiiiB


42

Angono, Rizal



I Republic of ti}e Philippines

IJMVERSITY OF REALAiigono, Rizal

43

Figure 3 4 Preferred bottles for the media (Kriudson C)

Figure 15 Hotplate stirrers with Knudson C media


Angono, Ri

44

Figure 16 Actual preparations for inoculation

Figure 2 7 Actual presentation of inoculating


AngonQ, Rizal

45

Figure 18 Plugging ot Catsupi Bottles

Figure 19 Air conditioned culture room

46


Angono, Riral

Figure 20 Actual Plugging of Knudson C media

Figure 21 Actual autoclave preparations

47


Angono, Rizal

Chapter 4

PB&SEHTMJIOM, ANALYSIS AND INTERPRETATION OF DATA

This Chapter presents and discusses briefly the data

analysis and the findings of the study.

Table 3a.

Difference in weight of Dendrobium Pattaya Beauty Protocorm

After six (6) interval days in three (3) data

Observation after irradiation and

Inoculation (Control)

Control

Sample 1

Sample 2

Sample 3

Sample A

Sample 5

Sample 6

Sample 7

Sample 8

Total:

First 6 Days

After

Inoculation

I? 02 g

0.90 g

0.42 g

0.43 g

0.29 g

<• 0.33 g

0.21 g

0.33 g

0.49 g

Second 6

Days after

Inoculation

1.45 g

1.61 g

1.12 g

l~~ 1.30 g

1.06 g

0.06 g

0.56 g

0.51 g

0.96 g

Third 6 Days

After

Inoculation

2.65 g

2.62 g

0.84 g

1.16 g

1.64 g

0.95 g

1.38 g

1.28 g

1.57 g

Average

1.72 g

3 .73 g

0.79 g

0.96 g

1.00 g

0.45 g

0.72 g

0.71 g

1.01 g

48


i UNIVERSITY OF RIZAL SYSTEMAngono, Rizal

The table above shows the effects of gamma radiation

on dendrobium pattaya beauty protocorm.

It can be gleaned from the table above that the

averager of weight increase in the three (3) data

observation after inoculation vary from sample to sample.

Therefore, it can be implied that out of the eight (8)

samples the general average weight increase for the control

is 1.01 grams.

49


Angono, Rizat

T a b l e 3 b .



: Observation after irradiation and

Inoculation (30 Gy)

306y

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Sample 6

Sample 7

Sample 8

Total:

First 6 Days

After

Inoculation

1.40 g

1.21 g

0 .29 g

0.15 g

1.0'4 g

0.98 g

0.32 g

0.20 g

0.70 g

Second 6

Days after

Inoculation

2 . 2 0 g

1.49 g

0.34 g

0.16 g

1.96 g

1.62 g

0 .27 g

0 .42 g

1.05 g

Third 6

Days after

Inoculation

3.28 g

3 . 9 0 g

0 .46 g

0 .62 g

2 . 9 8 g

2 . 4 9 g

0 .48 g

0.98 g

1.90 g

Average

2 . 2 9 g

2 . 2 0 g

0 . 3 6 g

0.31 g

1.99 g

1.70 g

0.36 g

0.53 g

1.22 g

The table above shows the effects of gamma radiation|

on dendrobium pattaya beauty protocorm. •

50


k.

Angono, Rizal


averages of weight increase in the three (3) data



samples the general average weight increase for the 30 Gy

is 1.22 grains.

51


UNIVERSITY OF REAL SYSTEMAngono, Rizal

Table 3c.




Inoculation (50 Gy)

50Gy

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Sample 6

Sample 7

Sample 8

Total:

First 6

Days After

Inoculation

0.93 g

0.68 g

1.16 g

1.04 g

0.35 g

0.23 g

0.29 g

0.21 g

0.61 g

Second 6 Days

after

Inoculation

1.53 g

1.33 g

1.73 g

1.54 g

0.94 g

0.66 g

0.18 g

0.41 g

1.04; g

Third 6 Days

After

Inoculation

2.48 g

1.73 g

2.33 g

2.18 g

0.82 g

1.01 g

0.75 g

0.85 g

1.52 g

Average

1.65 g

1.25 g

1.74 g

1.59 g

0.70 g

0.63 g

0.41 g

0.49 g

1.06 g

The table above shows the effects of gamma :radiation

on dendrobjium pattaya beauty protocorm.

52


UNIVERSITY OF RIZAL SYSTEMAngono, Rizal :






is 1.06 grams.

53

Republic of !he Philippines

IT

Angono, Rizal

T a b l e 3d .




Inoculation (70 Gy)

70Gy

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Sample 6

Sample 7

Sample 8

Total:i -!

i :

First 6

Days After

Inoculation

0.29 g

0.37 g

0.26 g

0.19 g

0.12- g

0.05 q

0.25 g

0.14 g

0.20 g

Second 6 Days

after

Inoculation

0.38 g

0 .46 g

0.94 g

0.42 g

0.76 g

0.10 g

0.81 g

0.14 g

0.50 g

Third 6 Days

After

Inoculation

0.73 g

0.59 g

0.20 g

0.91 g

0.15 g

0.88 g

0.65 g

0.83 g

0.61 g

Average

0.47 g

0.47 g

0.4 7 q

0.51 g

0.34 g

0.34 g

0.57 g

0.37 g

0 .44 g

The table above shows the effects of gamma radiation

on dendrobium pattaya beauty protocorm.

54


! UNIVERSITY OF MZAL SYSTEM:Angono, Riznl






is 0.4 4 grains.

55


Angono, Rizal

T a b l e 4 .

Composite table of the weight increase in the different

treatment after the three (3) data observation

Period upon irradiation and inoculation.

Treatment

Tl

(Control)

T2 (30 Gy)

T3 (50 Gy)

T4 (70 Gy)

1st 6 Days

after

Inoculation

0.49 g

0.70 g

0.61 g

0.20 g

2IK1 6 Days

after

Inoculation

0

1

1

0

.96 g

.05 g

.04 g

50 g

3ld 6 days

after

Inoculation

1

1

1

0

.57 g

90 g

52 g

61 g

Average

1.01 g

1.22 g

1.C6 g

0.44 g

Table 4 presents the effects of gamma radiation on

dendrobium pattaya beauty protocorm under laboratory

condition.

As can be seen in the table that the average weight

increase of dendrobium pattaya beauty protocorm does not

have an obvious trend considering the increase of gamma

radiation -n the different treatments. ;


56

Hence, it can be concluded that the radiation does not

show positive effect on the weight of dendrobium pattaya

beauty protocorm.


UNIVERSITY OF RIZALAngono, Rizal

Tab le 5 .

One-way ANOVA on the effects of gainma radiation

On dendrobium pattaya beauty protocorrn

Under laboratory condition

ANOVA

Source ofVariation

SS Df MS P-value F crit

BetweenGroupsWithinGroups

Total

1.042425

1.852667

2.895092

3 0.347475 1.500432 0.286786 4.06618

8 0.231583

11

Ho

Accepted

VINotSignificant

The table 5 presents the one-way anova of dendr&bium

pattaya beauty protocorm under laboratory condition which

is treated with gamma radiation:.

It can be seen that the converted f value of 1.5 is

lower than the critical f value of 4.07 at 0.05 level of

significance with the degree Si of freedom of 3/8. The null

hypothesis stating that gamma .radiation has no effects on


Angono, Rizal

the weight of dendrobium pattaya beauty protocorm is

accepted. '••

Hence, no significant effect on the weight of

dendrobium protocorm.

59


Angoito, Rizal

Chapter V

SUMMARY OF FINDINGS, CONCLUSIONS AND RECCOJ4MEJNDATIONS

Suoaaaxy of Findings

The researchers' findings about the study shows that

the average weights increase of each treatrnent/s in the

three (3) data observation after inoculation vary from

sample to sample. As can be seen in the table that the

average weight increase of dendrobium pattaya beauty

protocorm does not. have an obvious trend considering the

increase of gamma radiation on the different treatments.

It can be seen that the converted f value of 1.5 is

lower than the critical f value of 4.07 at 0.05 level of

significance with the degree s of freedom of 3/8. The null

hypothesis stating that gamma radiation has no effects on

the weight of dendrobium pattaya beauty protocor^n is

accepted.

Concilia ion

The researchers concluded, that it can be applied that

out of the eight (8) samples the general average weight

increase for the control (1.01 grams), 30 Gy (1.22 grams),

50 Gy (1.06 grams), 70 Gy (0i.44 grams). Hence, it can bei

concluded that the radiation does not show positive effect

60


Augono.Rizal

on the weight of dendrobium pattaya beauty protocorm. Hence

in one-way ANOVA shows that there is no significant effect

on the weight of dendrobium protocorm.

Recommendation

The researchers recommend that dendrobium pattaya

beauty is a good example of plant, to undergo this kind of

experiment.

The researchers also recommend that gamma radiation

would not affect the growth of the protocorm upon the

result of one-way ANOVA that there is no significance using

gamma radiation. The researchers would like to recommend

continuing this study, from embryo to complete plantlet.


Aiigono, Rizal

Appendices

Taxonomy of the Subject

Kingdom: Plantae

Division: Magnoliophyta

Class: Liliopsida

Order: Asparagales

Family: Orchidaceae

Subfamily: Epindendroideae

Tribe: Podochileae

Subtribe: Dendrobiinae

Genus: Dendrobium

Variety: Pa€taya

Beauty

62Republic ofthe Philippines

Angonq.Rizal

Table a. Shows the weight of the Protocorm in grains from 6

days of interval of control

CONTROL

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Sample 6

Sample 7

Sample 8

2-08-06

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

02-14-06

3.52

3.40

2.92

2.93

2.79

2.83

2.71

2.83

02-20-06

4.97

5.06

4.04

4.23

3.85

2.89

3.27

3.34

02-27-06

7.62

7.68

4.80

5.39

5.49

3.84

4.65

4.62


Angono, Ri/al

Table b. Shows the weight of the Protocorm in gram3 from 6

days of interval with a dose of 30 Gy

30Gy

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Sample 6

Sample 7

Sample 8

02-08-06

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

02-14-06

3.90

3.71

2.79

2.65

3.54

3.48

2.82

2.70

02-20-06

6.10

5.20

3.13

2.81

5.50

5.10

3.09

3.12

02-27-06

9.38

9.10

3.59

3.43

8.48

7.59

3.57

4.10

64


3FAngono, Rizal

Table c. Shows the weight of the Protocorm in grams from 6

clays of interval with a dose of 50 Gy

50Gy

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Sample 6

Sample 7

Sample 8

02-08-06

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

02-1406

3.43

3.18

3.66

3.54

2.85

2.73

2.79

2.71

02-20-06

4.96

4.51

5.39

5.08

3.79

3.39

2.97

3.12

02-27-06

1.44

6.24

7.72

7.26

4.61

4.40

3.72

3.97


SMSITY OF REAL SYSTEMAngono, Rizal

Table d. Shows the weight of the Protocorm in grams from 6

days of interval with a dose of 70 Gy

70Gy

Sample

Sample

Sample

Sample

Sample

Sample

Sample

Sample

1

2

3

4

b

b

7

8

02-08-06

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

02-

2

2

2

2

2

2

2

2

14-06~

.79

.87

.76

.69

.62

.55

.75

64

02-20-06

3.17

3.33

3.70

3.11

3.38

2.65

3.56

2.78

02-27-06

3.90

3.92

3.90

4.02

3.53

3.53

4.21

3.61


Angono, Riral

FIRST ENDORSEMENT

Respectfully forwarded to the Office of the Director,

Dr. Valentina R. Catmunan, the herein communications of

Flores, John Martin R., Lanic, Alvin D., Matuto, Mark

Anthony C , and Vidal, John Faustus C, presenting three

copies of there manuscript in there undergraduate thesis

entitled "EFFECTS OF GAMMA RADIATION IN VITRO CULTURE ON

PROTOCORM DEVELOPMENT OF DENDROBIUM Pattaya Beauty".

Approved by:

Dr. Valentina R. Catmunan Mr. Jerry C. Esperanza

Campus Director Thesis Adviser and Statistician


Angono, Rizal

! Bibliography

Denniem, R. A., Ahmed, E. M.

Irradiation Treatment of Fruits and Vegetables.

Symposium; Postharvest Biology and Handling of Fruits

And Vegetables. (1975) Connecticut: the Avi; Pub: 118-

129

Dimaano, Maritess M. and Imperial, Maria Angelica Liza V.

The effects of varying doses of Gamma Radiation on

Locally adapted Transdescania Clone 02 (BNL); 60 p.

Facts about Low - level Radiation (1981) Vienna:

International Atomic Energy Agency; 20 p.

Garcia, Nardo Q.

Argonornic Variability in Two Rice Varieties ,t

Induced by Gamma Radiation. (1983); 77

Grecz, N.', Lo, E.J., Kennedy,E.D. (1973)

Gamma Radiation studies j>f Clostridium botulinum types

A, B, and E; Biological aspects. Radiation

Preservation of Food, (STI/PUB 317); 177 - 191.

Knudson, L. 1946. A new nutrient solution for germination


Angono.Rizal

Of orchid seeds. Amer. Orchid Soc. Bull. 15: 214 - 217

Lakshmi Sita, G. Sagawa, V.,

In vitro propagation of pineapple, Hortic. Sci. 16

(1981) 496.

Lapade, A.G., Barrida, A.C., Veluz, A.M.S., Aurique, F.B.,

Marabella, L.J. and Rama, M.G. (2004)

The Effects of Ganuna Radiation on Cashew and

Mangosteen. (14): 1 - 11.

Lapade, A.G., Veluz, A.M.S., Santos, I.S.

Genetic Improvement of the Queen Variety of Pineapple

Through induced Mutation and In Vitro Culture

Techniques. JIAEA - SM - 340/145P)

Rangan, T.S., Handbook of Plant Cell Culture, Vol. 3,

Macmillan, New York (1984)

Revetti, L.M. 1973 Preservation of Maize and Kidney Beans

By Gamma Irradiation. Radiation Preservation of Food,

i (ST±/PUB*317): 339 - 34$

Simmonds, N.W., Bananas. 2nd Ed. (1966). Great Britain;

Longman; 252 - 27 6 •

70


Angono, Rizal

The Philippines Recommends for Orchids, 1977, Laguna:

Philippine Council for Agriculture and Resources

Research. 99p.


3F REAL SYSTEMAngono, Ri/al

Routing Slip

This is certifying the undergraduate thesis entitled

"EFFECT OF GAMMA RADIATION ON PROTOCORM DEVELOPMENT OF

DENDROBIUM Patlaya beauty IN VITRO CULTURE" by Flores, John

Martin R., Lanic, Alvin D., Matuto, Mark Anthony C , and

Vidal, John Faustus C. has been review and scrutinized and

is now ready for finalization and book binding.

MR JERRY C.ESPERANZAAdviser

Date

Mrs, NormaMember, Boar.

MR JERRY C. ESPERANZAStatistician

Date

Elvinaf Panelist

Mr. RenaMember, Boar!

Mrs. 'Joseph iiieB. JarneroChairman Board of Panelist

Date

Mr. Jerry C. EsperanzaUTW Instructor

: Date

MRS F L O J ^ E L S S K DIAZIns t i tu te 1 Head

Date


Angono, Rizal

CURRICULUM VITAE

JOHN MARTIN R. FLORES

J. Sumulong st. Lunsad Saudi Village Binangonan, Rizal

09208756321

PERSONMi PROFILE

AGE :

SEX

PLACE OF BIRTH :

DATE OF BIRTH :

RELIGION :

20

MALE

TAYTAY, RIZAL

AUGUST 11, 1985

BORN AGAIN

EDUCATION

PRIMARY LAMBAK ELEMENTARY SCHOOL

CARDONA, RIZAL

1992- 1998

SECONDARY TUNA BALIBAGO NATIONAL HIGHSCHOOL

CARDONA, RIZAL :

1998- 2000 :

TERTIARY UNIVERSITY OF RIZAL SYSTEM ANGONO

BS-Biology

2005-2006

73


UNIVERSITY OF RlZAL SYSTEMAngono, Rizn!

ORGANIZATIONS

PEP SQUAD

Member (2004 - 2005)

YOUTH ENVIRONMENTAL STUDENT (YES)

P.R.O. (2005)

JOHN MARTIN R. FLORES

Signature

74


Angono, Rizal

ALVIN D. LANIC

38C Tiffany St. Bloomingdale Subd. Brgy. San Pedro

Angono, Rizal

0927-311-7812

PERSOMM. PROFILE

AGE

SEX

PLACE OF BIRTH

DATE OF BIRTH

RELIGION

23 years old

MALE

Manila

March 29, 1982

Roman Catholic

ED0C&TIQN

PRIMARY' Roxas Central School

Roxas, Oriental Mindoro

1990 - 1996

SECONDAP.Y Angono National High School

Angono, Rizal

1996 - 1999

TERTIARY: : UNIVERSITY OF RIZAL SYSTEM ANGONO

BS-Biology

2005-2006

JOB EXPERIENCE : ;

Angono General Hospital

7 5


VAnyono, Riwil

On Job Training (OJT)

January to March 2004

Responsible for Patients Admittance and also for

Other request such as Medico - Legal.

ABS - CBN Bantay Kalikasan

On Job Training

November to December 2003

Responsible for hotline Bantay Kalikasan.

Kentucky Fried Chicken (KFC)

Service Crew

February to April 2001

Responsible for preparation of food at the

Kitchen, assigned at the fry station. Conducting

Costumer relation.

Jollibee Food Corporation

Service Crew

July to November 2000 : ,•

Responsible for the food handling, food serving,

; Implementing the l(j>0% satisfaction of the

i. Customer. Maintaining the cleanliness and

| Sanitation of the store. ; ' '•>-\

ALVIN D. LANIC

Signature


UNIVERSITY OF REALAngouo, Kizal

MARK ANTHONY C. MATUTO

Lot 5 Block 5 Rosario Village San Isidro

Angono, Rizal

PERSOUM. PROFILE

AGE

SEX

PLACE OF BIRTH

DATE OF BIRTH

RELIGION

24 years old

MALE

358 Dr. Sixto Antonio Ave. Caniogan,

Pasig City

July 28, 1981

Roman Catholic

EDUCATION

PRIMARY Caniogan Elementary School

Pasig City

1988 - 1994

SECONDARY Rizal High School Main

Rotonda, Pasig City

1994 - 19!98 ' !

TERTIARY UNIVERSITJY OF RIZAL SYSTEM ANGONO

BS-Biology

2005-2006!


'OFRJAngono, RiV;

JOB EXPERIENCE:

Philippine Nuclear Research Institute

Diliman, Quezon City

On Job Training (OJT)

January - March 2006

MARK ANTHONY C. MATUTO

Siganture

78


Angono, Rizal

JOHN FAUSTUS C. VIDAL

122B Sitio Villamayor Brgy. Pag-asa, Tayuinan

Binangonan, Rizal

PERSONAL PROFILE:

AGE

SEX

PLACE OF BIRTH

DATE OF BIRTH

RELIGION

23 years old

MALE :

MORONG, RIZAL

MARCH 8, 1983

BORN AGAIN CHRISTIAN

EDUCATION

PRIMARY "

SECONDARY

TERTIARY

NATIONAL COLLEGE BUSINESS AND ARTS

TAYTAY, RIZAL

1990 - 1994

SHALOM LEARNING CENTER

MUZON TAYTAY, RIZAL

1994 - 1996

: ANGONO PRIVATE HIGH SCHOOL

I ANGONO, RIZAL

1996 -•: 2000. •! :

SYSTEM TECHNOLOGY INSTITUTE

! MANDALUYONG, CITY

2000 -; 2001

79


Angono, Rizal

UNIVERSITY OF RIZAL SYSTEM ANGONO

BS-Biology

2005-2006

OBGRNI^ATION: j

t

The Petroglyphs • • ; i

Official Publication of University of Rizal System

Angono. i

Feature Editor (2005 -- Present)

Editorial Cartoonist {2004 - 2005)

Teatro Rizalino jI I :

Member (2002 - Present)

JOB EXPERIENCE:'' i

Philippine Nuclear Research Institute

Diliman, Quezon City : . ; * :

On Job Training !(OJT) }

January ~ March 2006 j ] ' ;

Student Assistant ;

University of Rizal System Angono (2002 - 2005)

Responsible for cleaning Faculty Room/ Registrar,

Cashier, and Library.

JOHN FAUSTUS C. VIDAL

Signature

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