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SOMATIC EMBRYOGENESIS OF CARICA PAPAYA CV. EKSOTIKA

By

BEVERLIEN CHRISTINE

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfilment of the Requirement for the Degree of Master of Agricultural Science

November 2005

� Dedicated TO:

My Parents (Daiman Lamat and Maina

Godoun)

My Sisters (Jeniffer and Gwendoline)

My Brothers (Sherwin and Reno)

�

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment

of the requirement for the degree of Master of Agriculture Science

SOMATIC EMBRYOGENESIS OF CARICA PAPAYA CV. EKSOTIKA

By

BEVERLIEN CHRISTINE

November 2005

Chairman : Associate Professor Maheran Abdul Aziz, PhD

Faculty : Agriculture

This study was carried out with the main objective of establishing somatic embryo

production in Carica papaya cv. Eksotika that can be used for further genetic

improvement of the crop. The study included induction of somatic embryogenesis

from immature zygotic embryos and anatomical study of the embryo development,

establishment of plant regeneration from the somatic embryo, and establishment of

cell suspension culture from embryogenic callus.

The specific objective of the study on induction of somatic embryogenesis from

zygotic embryos was to study the effect of growth regulators on the induction of

somatic embryos with or without monthly subculture. The combination of 2,4-

dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP) at various

concentrations were assessed. The experiment was conducted in a Randomized

Completely Block Design.

In the experiment without subculture, MS medium supplemented with 5 mg/L 2,4-D

induced the highest percentage (100 %) of somatic embryo formation on the second

and third month of culure. At higher 2,4-D concentrations (6-8 mg/L) the

percentage of callus formation increased. Monthly subculturing delayed the

maturation of somatic embryos and increased callus formation. The percentage of

somatic embryo formation was highest (97.5 %) on MS medium supplemented with

5, 6 and 8 mg/L 2,4-D by the third subculture.

The specific objectives of the study on plant regeneration from somatic embryos

were to determine suitable plant growth regulators, as well as medium formulation

on regeneration of somatic embryos. The combination of BAP and NAA at various

concentrations were assessed. Different medium formulations evaluated were MS

medium (Murashige and Skoog, 1962), LS medium (Linsmair & Skoog, 1965) and

B5 medium (Gamborg, 1968) either at full strength or half strength in

macronutrients. All experiments were conducted and arranged in a Completely

Randomised Design (CRD).

MS medium without plant growth regulator induced the highest percentage (59.3

%) of regeneration from the somatic embryos. It was observed that the higher the

concentration of BAP and NAA either alone or in combination, the lower the

percentage of regeneration and the higher the percentage of callus formation. In the

experiment on effect of different medium formulations on regeneration, MS full

strength medium produced the highest percentage of regeneration of the somatic

embryos.

The third study was the establishment of cell suspension culture from embryogenic

callus of papaya cv. Eksotika. Four weeks after the transfer of embryogenic callus

into liquid medium, single cells were released and after eight weeks pro-

embryogenic masses (PEMs) were formed. The highest mean number of cells per ml

(56.9) was obtained on liquid MS medium containing 2 mg/L 2,4-D. Maturation of

somatic embryos was achieved on transferring the globular or heart-shaped somatic

embryos to liquid MS medium without growth regulator. Germination of somatic

embryos occurred following transfer of cotyledonary embryos from liquid MS

medium onto solid hormone-free MS medium

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains Pertanian

EMBRYOGENESIS SOMA BAGI CARICA PAPAYA KULTIVAR EKSOTIKA

Oleh

BEVERLIEN CHRISTINE

November 2005

Pengerusi : Profesor Madya Maheran Abdul Aziz, PhD

Fakulti : Pertanian

Penyelidikan ini dilaksanakan dengan objektif utama untuk mewujudkan produksi

embrio soma betik kultivar Eksotika, yang boleh digunakan untuk pembaikan

genetik tanaman tersebut. Kajian ini terdiri daripada induksi embrio soma

daripada embrio zigotik yang belum matang dan kajian anatomi ke atas

pembentukan embrio soma tersebut; mewujudkan regenerasi pokok daripada

embrio soma, dan mewujudkan kultur ampaian sel daripada kalus yang

embriogenik.

Objektif khas daripada kajian induksi embrio soma daripada embrio zigotik yang

belum matang adalah untuk mengkaji kesan pengawalatur tumbesaran terhadap

induksi embrio soma sama ada dengan subkultur setiap bulan atau tanpa

subkultur. Kombinasi asid diklorofenoksi asetik (2,4-D) dan 6-benzilaminopurina

(BAP) pada beberapa kepekatan telah ditaksirkan. Kajian ini dijalankan dengan

menggunakan Rekabentuk Rawak Berblok Penuh (RCBD).

Dalam eksperimen tanpa subkultur, medium MS yang mengandungi 5 mg/L 2,4-D

menghasilkan peratus pembentukan embrio soma tertinggi (100 %) pada bulan

kedua dan ketiga. Pada kepekatan 2,4-D yang lebih tinggi (6-8 mg/L) peratus

pembentukan kalus bertambah. Subkultur setiap bulan melambatkan kematangan

embrio soma dan meningkatkan pembentukan kalus. Peratus pembentukan embrio

soma tertinggi (97.5 %) adalah pada medium MS yang mengandungi 5, 6 dan 8

mg/L 2,4-D pada subkultur ketiga.

Objektif khas daripada kajian regenerasi pokok daripada embrio soma adalah

untuk menentukan pengawalatur tumbesaran serta formulasi medium yang sesuai

untuk regenerasi embrio soma tersebut. Kombinasi NAA dan BAP pada beberapa

kepekatan telah dicuba. Pelbagai formulasi medium yang dikaji adalah medium MS

(Murashige dan Skoog, 1962), medium LS (Linsmair dan Skoog, 1965) dan medium

B5 (Gamborg, 1968) sama ada nutrien makronya berkepekatan penuh atau

separuh. Kesemua kajian ini dijalankan dengan menggunakan Rekabentuk Rawak

Lengkap (CRD).

Medium MS tanpa pengawalatur tumbesaran menghasilkan peratus regenerasi

embrio soma yang tertinggi (59.3 %). Pemerhatian menunjukkan lebih tinggi

kepekatan NAA dengan atau tanpa BAP lebih rendah peratus regenerasi dan lebih

tinggi peratus pembentukan kalus. Di dalam eksperimen ke atas kesan pelbagai

formulasi medium terhadap regenerasi juga menunjukkan medium MS pada

kepekatan penuh memberikan peratus regenerasi embrio soma yang tertinggi.

Kajian ketiga adalah mewujudkan kultur ampaian sel daripada kalus embriogenik.

Empat minggu selepas kalus embriogenik dipindahkan ke dalam media cecair, sel-

sel baru terbentuk dan selepas lapan minggu ‘pro-embryogenic masses’ (PEM)

terbentuk. Min bilangan sel per ml (56.9) tertinggi diperoleh pada medium MS

cecair yang mengandungi 2 mg/L 2,4-D. Kematangan embrio soma dicapai dengan

memindahkan embrio berbentuk globul atau hati ke dalam medium cecair MS

tanpa pengawalatur tumbesaran. Percambahan embrio soma berlaku berikutan

pemindahan embrio soma berkotiledon dari media cecair MS ke medium pepejal

MS tanpa pengawalatur tumbesaran.

ACKNOWLEDGEMENTS

I would like to express my deepest appreciation and gratitude to the chairman of my

supervisory committee, Assoc. Prof. Dr. Maheran Abdul Aziz, for her help and

guidance in making the completion of this thesis a success. I am also indebted and

very grateful to my committee members Mr. Azmi Abdul Rashid, M.Phil. and

Assoc. Prof. Dr. Norihan Mohd. Saleh for their kind advice, suggestions, invaluable

discussion and support during the preparation and completion of this thesis.

Special thanks to Mr. Agus Sutanto, Mr. Md. Humayun Kabir, Mr. Elixon Sunian

and Mr. Budi Winarto for their invaluable assistance in the laboratory work and

professional photography. I would also like to express my gratitude to Mr. Wahab

from Jabatan Pertanian Serdang for supplying the papaya fruit throughout my

study, the Head, Department of Crop Science faculty of Agriculture for allowing me

to use the Histology and Scanning Electron Microscopy facilities. Not to forget to all

my lab mates at the In Vitro Laboratory IPPT, Faculty of Agriculture, Aini, Dila,

Yea, Syaiful, Suleiman and Tajul, thanks for your help and strong support.

I wish to thank my parents for their patience, understanding, faith and strength

during my study. Lastly, but not least to my brothers and sisters, thank you for your

understanding, encouragement and love. To someone special, thanks for everything

and may you will fullfill your dreams. Above all, God the Most Gracious and

Merciful who gave me strength to complete the work and made all things well.

I certify that an Examination Committee has met on 22nd November 2005 to conduct

the final examination of Beverlien Christine on her Master of Agriculture Science

entitled “Somatic Embryogenesis of Carica papaya cv. Eksotika” in accordance with

Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian

Malaysia (Higher Degree) Regulation 1981. The Committee recommends that the

candidate be awarded the relevant degree. Member of the Examination Committee

are as follows:

Halimi Mohd Saud, PhD

Associate Professor

Faculty of Agriculture

Universiti Putra Malaysia

(Chairman)

Mihdzar Abdul Kadir, PhD

Associate Professor



(Internal Examiner)

Abdul Ghani Mohd. Yunus, PhD

Associate Professor



(Internal Examiner)

Normah Mohd. Noor, PhD

Professor

Faculty of Science and Technology

Universiti Kebangsaan Malaysia

(External Examiner)

HASANAH MOHD. GHAZALI, PhD

Professor/Deputy Dean

School of Graduate Studies


Date:

This thesis submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfilment of the requirement for the degree of Master of Agriculture

Science. The members of the Supervisory Committee are as follows:

Maheran Abdul Aziz, PhD

Associate Professor



(Chairman)

Azmi Abdul Rashid, M. Phil.



(Member)

Norihan Mohd. Saleh, PhD

Associate Professor

Faculty of Biotechnology and Biomolecular Science


(Member)

AINI IDERIS, PhD

Professor/Dean

School of Graduate Studies


Date:

DECLARATION

I hereby certify that this thesis is based on my original work except quotations and

citations which have been duly acknowledged. I also declare that it has not been

previously or concurrently submitted for any other degree at UPM or other

institutions.

BEVERLIEN CHRISTINE

Date :

TABLE OF CONTENTS

Page

DEDICATION i

ABSTRACT ii

ABSTRAK

v

ACKNOWLEDGEMENTS viii

APPROVAL ix

DECLARATION

xi

LIST OF TABLES

xv

LIST OF FIGURES xvi

LIST OF PLATES xvii

LIST OF ABBREVIATIONS/CITATIONS xxi

CHAPTER

1 GENERAL INTRODUCTION 1

Background 1

Objectives 4

2 LITERATURE REVIEW 5

History of papaya 5

Botany 5

Cultivated Papaya varieties in Malaysia 6

Principles of Plant Tissue Culture 8

Tissue Culture of Papaya 10

Somatic Embryogenesis 13

Factors Effecting Somatic Embryogenesis 15

Plant Growth Regulators 15

Genotype 17

Explant Choice 17

Culture Medium 18

Culture Environment 19

Carbohydrates 19

Crop Improvement In Papaya 20

Genetic Transformation Work on Papaya 20

Histology 22

3 SOMATIC EMBYROSGENESIS FROM IMMATURE

ZYGOTIC EMBRYOS 24

Preface 24

Materials and Methods 26

Location of Study 26

Plant Material 26

Surface Sterilization 26

Preparation and Culture of Explant 26

Basic Medium 28

Medium Preparation 28

Culture Conditions 28

Experimental Design, Statistical Analysis and Data

Evaluation 28

Experiments 30

Effect of 2,4-D and BAP on Somatic Embryo

Formation (without subculture) 30


Formation (with subculture) 30

Histological Study and Scanning Electron Microscopy of

Somatic Embryos 30

Results 35


Formation (without subculture) 35


Formation (with subculture) 47

Histology and Scanning Electron Microscopy of

Somatic Embryo 54

Discussion 58

Induction of Somatic Embryogenesis 58

Recurrent Embryogenesis 60

Effect of Subculture on the Induction of Somatic

Embryogenesis 61

Histological Study and Scanning Electron Microscopy

of the Somatic Embryo 62

4 GERMINATION OF SOMATIC EMBRYOS 63

Preface 63


Experiments 64

Effect of BAP and NAA on the Germination of Somatic

Embryos 64

Effect of Different Medium Formulations on

Germination of Somatic Embryos 64

Experimental Design, Statistical Analysis and Data

Evaluation 65

Rooting of Shoot Derived from Somatic Embryo 66

Results 66


Embryos 66

Effect of Different Medium Formulations on the


Rooting of Shoot Derived From Somatic Embryo 86

Discussion 90


Embryos 90

Effect of Different Medium Formulations on the


Effect of Induction Medium on Somatic Embryo

Germination 93

Rooting of Shoots Derived from Somatic Embryos 94

5 ESTABLISHMENT OF CELL SUSPENSION

CULTURE FROM EMBRYOGENIC CALLUS OF

PAPAYA cv. EKSOTIKA 96

Preface 96


Results 100

Discussion 112

GENERAL DISCUSSION AND CONCLUSION 116

REFERENCES 120

APPENDICES 134

BIODATA OF THE AUTHOR 150

LIST OF TABLES

Table Page

3.1 The combination of 2,4-D and BAP concentration for the

induction of somatic embryogenesis 31

3.2 Percentage (%) of explant that responded to form somatic

embryos by months (without subculture) 39

3.3 Mean number of somatic embryos formed per explant by

months (without subculture) 41

3.4 Percentage (%) of explant that responded to form callus by

months (without subculture) 45

3.5 Percentage (%) of explant that responded to form somatic

embryos following each subculture

48

3.6 Mean number of somatic embryos formed per explant following

each subculture 50

3.7 Percentage (%) of explant that responded to form callus

following each subculture 52

4.1 The combination of BAP (mg/L) and NAA (mg/L)

concentrations for the germination of somatic embryos 65

4.2 Percentage (%) of rooting of shoots derived from somatic

embryos 87

5.1 Treatments for embryogenic cell suspension 98

5.2 Averaged number of cell per ml (10 5) from the different cell

suspensions after 2 months of culture 103

5.3 Estimation of yield of embryos at various development stages on

T2CS medium. 109

LIST OF FIGURES

Figure Page

2.1 Different stages of somatic embryo development 14

4.1 The percentage of somatic embryos producing shoot 71

4.2 The percentage of somatic embryos producing normal plantlet 71

4.3 The percentage of somatic embryos producing abnormal

plantlet 73

4.4 The percentage of germination of somatic embryos (A), normal

plantlets (B), and abnormal plantlets (C) following transferred

to different medium formulation for germination. Treatment 1

(MS full strength), T2 (MS half strength macro salt), T3 (LS full

strength), T4 (LS half strength macro salt), T5 (B5 full strength)

and T6 (B5 half strength macro salt). Means followed by the

same letter were not significantly different at the 5 % level 82

LIST OF PLATES

Plate Page

3.1 Immature fruits of papaya cv. Eksotika (2 ½ - 3 month-old)

collected from Jabatan Pertanian, Serdang 27

3.2 Immature seeds from immature papaya fruit placed in petri

dish prior to zygotic embryo excision 27

3.3 Zygotic embryo cultured on induction medium 36

3.4 Opening of the cotyledons and enlargement of the zygotic embryo

(red arrow) after a week on induction medium 36

3.5 Globular somatic embryos (blue arrow) arose directly from the

swollen apical dome (red arrow) of the zygotic embryo after

three weeks of culture 37

3.6 Germination of zygotic embryo into plantlet on medium without

plant growth regulators 37

3.7 Globular somatic embryos (pink arrows) and callus (blue

arrow) growing together from the apical dome of a zygotic

embryo 38

3.8 Development of globular (blue arrows) and heart-shaped (red

arrows) somatic embryos 42

3.9 Development of torpedo shaped somatic embryos (blue arrows) 42

3.10 Development of cotyledonary somatic embryos 43

3.11 Less number of somatic embryos formed on medium containing

1 mg/L 2,4-D 43

3.12 Two type of callus formation. (A) callus which is friable and

yellowish, (B) callus with watery/sticky liquid excreted on the

surface 46

3.13 Recurrent or secondary somatic embryos (pink arrows)

emerged from the surface of primary globular embryos after

two months of culture 46

3.14 The formation of somatic embryos from the apical dome of

immature zygotic embryos. (A and B) globular somatic embryos

(pink arrows); (C and D) heart (blue arrows) and torpedo

shaped (pink arrows) somatic embryos; (E) cotyledonary

somatic embryos (purple arrows); (F) callus formation which

covered the somatic embryos (red arrows). 53

3.15 Histological observation of somatic embryo development from

a zygotic embryo of papaya.(A) zygotic embryo (apical meristem

is the site of formation of the globular somatic embryo), (B)

globular somatic embryo, (C) heart-stage somatic embryo and,

(D) cotyledonary somatic embryo. Cot=cotyledon, Am=apical

meristem, Rm=root meristem, Pd=protoderm, Pm=procambium 55

3.16 Scanning electron microscopy of somatic embryos at different

stages of development. (A) initial globular stage (pink arrows),

(B) globular stage, (C) heart stage (yellow arrow) and torpedo

stage (purple arrow), and (D) cotyledonary stage (blue arrow) 56

3.17 Secondary globular somatic embryos (blue arrows) emerging

From primary somatic embryos (pink arrows)

57

4.1 The formation of shoot only (A) and roots only (B) from the

somatic embryos 68

4.2 Germination of somatic embryos into plantlets, with normal

shoot and root 69

4.3 Hypocotyl and callus growing together on germination medium

containing BAP and NAA. Red arrow = callus, blue arrow =

new leaf 70

4.4 Abnormal germination of somatic embryos. Formation of non-

functional shoots (pink arrows) and roots (blue arrows) 70

4.5 Intensed callus formation (pink arrow) on medium containing

high concentration of BAP 74

4.6 Numerous root formation (red arrow) on medium containing

high concentration of NAA 74

4.7 Somatic embryos germinated on MS medium without PGRs

(T1) (A). Upon subculture on to the same medium the somatic

embryos formed well-developed shoot and roots (B) 76

4.8 Somatic embryos germinated on medium with higher BAP and

lower NAA concentration (T6-T10). Stunted shoots with callus

formation at the base (A). Upon subculture on to the same

medium the shoots grew but became vitrified with continued

formation of callus at the base of the shoot (B) 77

4.9 Numerous roots formation from the somatic embryos upon

subculture on to the same medium with higher concentration of

NAA alone (T13) 78

4.10 Callus formation occurred upon subculture on to the same

medium containing higher combination of BAP and NAA

concentration medium (T14-T16) 78

4.11 On medium containing BAP alone or in combination with 0.05

mg/L NAA the somatic embryos only produced shoot. Upon

subculture on to the same medium somatic embryos produced

multiple-shoots 79

4.12 The germination of somatic embryos. (A) somatic embryos, (B)

somatic embryos on maturation medium, (C) matured somatic

embryos cultured on germination medium and (D) germination

of somatic embryos 81

4.13 Shoot production from somatic embryos. (A) elongation of the

hypocotyls, (B) emergence of the first leaves, (C) emergence of

the whole shoot and (D) complete plantlet 83

4.14 Abnormal germination of somatic embryos, with no shoot

development, and roots produced were generally stout, thick

and short 84

4.15 Several patterns of abnormal germination of somatic embryo 85

4.16 Shoots on rooting medium after 2 weeks of culture 88

4.17 Adventitious root formation on MS medium with 1 mg/L IBA

after 5-6 weeks of culture 89

5.1 Cell suspension, (A) before counting using haemocytometer and

(B) cell counting on haemocytometer 99

5.2 Embryogenic callus with somatic embryos prior to transfer into

liquid MS medium for the establishment of an embryogenic cell

suspension 101

5.3 Four week-old cell suspension in medium containing 2,4-D. (A)

turbid cells suspension in flask. (B) observation under a

microscope 104

5.4 Embryogenic callus transferred to MS liquid medium after the

first subculture (four weeks). (A) T1CS medium, (B) T2CS

medium 105

5.5 (A) Cells suspended in medium containing 2,4-D after two

months of culture (before sieving) and, (B) after sieving 106

5.6 Cell suspension two weeks after sieving. (A) using 250 µm nylon

mesh, and (B) using 100 µm nylon mesh 107

5.7 The development of somatic embryos in cell suspension. (A) The

embryogenic cells with distinct nucleolus and dense cytoplasm

(blue arrow). (B) Embryogenic cell has divided into two-celled

structure. (C) Pro-embryogenic masses. (D) Globular somatic

embryo with epidermal layer (white arrow). (E) Heart-shaped

embryo. (F) Torpedo-shaped embryo. (G) Cotyledonary embryo 108

5.8 Maturation of somatic embryos (pink arrows) in liquid

hormone-free MS medium 110

5.9 Germination of somatic embryos on hormone-free MS medium 111

5.10 Addition of fresh medium to prolong culture without sieving

resulted in continuous release of cells in the suspension culture. 112

LIST OF ABBREVIATIONS/CITATIONS

2,4-D 2,4-dichlorophenoxyacetic acid

AC activated charcoal

ACC 1-aminocyclopropane-1-carboxylic

ANOVA analysis of variance

BAP 6-benzylaminopurine

BA 6-benzyladenine

B5 Gamborg (1968) basal medium

CH casein hydrolysate

CRD Completely randomised design

cv. cultivar

DNMRT Duncan new multiple range test

EC embryogenic cell

e.g. exempli gratia (for example)

et al. et alia

etc. et cetera

FAA formaldehyde acetic acid

FDA fluorescein diacetate

Fe-EDTA iron ethylene diamine tetraacetic acid

GA3 gibberellic acid

g/L gram per liter

IAA indole acetic acid

IBA indole butyric acid

i.e id est (that is)

IEDCs induced embryogenic determined cells

LS Linsmaier and Skoog (1965) basal medium

M molar

MARDI Malaysian Agriculture Research and Development Institute

mg/L milligram per liter

MS Murashige and Skoog (1962) basal medium

NAA naphthalene acetic acid

PBA 6-(benzylamino)-9-(2-tetrahydropyranyl)-9H-purine

PCR polymerase chain reaction

PEDCs pre-embryogenic determined cells

PEMs pro-embryogenic masses

PGR plant growth regulator

pH - log [H

+]

PRV papaya ringspot virus

Ri root-induction

RNA ribonucleic acid

Rpm rotation per minute

SAS statistical analysis system

SEM scanning electron microscopy

sp. species

Ti tumor-induction

v/v volume to volume

w/v weight per volume

% percent

α level of significance

UKM Universiti Kebangsaan Malaysia

µg microgram

µm micrometer

µM micromolar

UV ultra violet light

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