IN-VITRO PLANTLET REGENERATION AND …psasir.upm.edu.my/619/1/t_fp_2006_42.pdfIN-VITRO PLANTLET REGENERATION AND AGROBACTERIUM - MEDIATED GENE TRANSFORMATION OF CARICA PAPAYA L. cv

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IN-VITRO PLANTLET REGENERATION AND AGROBACTERIUM-MEDIATED GENE TRANSFORMATION OF CARICA PAPAYA L. cv.

EKSOTIKA

By

SYAIFUL BAHRI PANJAITAN

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirement for the Degree of Master of

Agriculture Science

2006

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Specially Dedicated

To

My Father and Mother: Lokot Panjaitan and Boniyem

……….. who inspired, supported and gave me tremendous courage to

be a well educated person

All teachers

……….. who crossed their paths with mine and taught me essential

things in life

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Agriculture Science

IN-VITRO PLANTLET REGENERATION AND AGROBACTERIUM-

MEDIATED GENE TRANSFORMATION OF CARICA PAPAYA L. cv. EKSOTIKA

By


November 2006

Chairman : Associate Professor Maheran Abdul Aziz, PhD Faculty : Agriculture The study was conducted at the In Vitro Laboratory, Institut Pertanian

Persekitaran Terkawal (IPPT), Faculty of Agriculture and Molecular and Cell

Biology Laboratory, Faculty of Biotechnology and Biomolecular Sciences,

Universiti Putra Malaysia, Serdang, Selangor. The main objectives of the

study were to develop in-vitro plantlet regeneration systems and to carry out

preliminary studies of Agrobacterium-mediated gene transformation of Carica

papaya L. cv. Eksotika.

The study consisted of in-vitro plantlet regeneration through organogenesis,

and somatic embryogenesis using different types of explant and different

concentrations and combinations of plant growth regulators whereby the most

appropriate system could be applied in the genetic transformation of the crop.

The experiments were arranged in a completely randomized design (CRD).

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The study on organogenesis included shoot induction and rooting. On shoot

induction, modified MS (Murashige and Skoog, 1962) medium supplemented

with 500 mgL-1 (w/v) casein hydrolysate, 30 gL-1 (w/v) sucrose and 0.2% (w/v)

phytagel was used as the basic medium, and different concentrations of BAP

(0, 0.1, 1.0, and 2.0 mgL-1 (w/v)) in combination with 0, 0.05, 0.1 and 0.2 mgL-

1 (w/v) NAA were applied as treatments.

A modified MS medium containing 1 mgL-1 (w/v) BAP combined with 0.05

mgL-1 (w/v) NAA was the most suitable treatment combination to induce shoot

proliferation from shoot tip, leaf and petiole explants of field-grown

hermaphrodite plant. The mean number of shoots produced was 73 on shoot

tip explant, 47 on leaf explant and 10 on petiole explant. The treatment also

successfully enhanced the percentage of explant producing shoots which

were 60.24% on petiole explants, 70.07% on leaf explants and 100% on

shoot tip explants.

In the rooting study, shoots obtained above were pretreated in different

concentrations of IBA (0, 0.5, 1.0, and 2.0 mgL-1 (w/v)) for one week followed

by culture on medium with or without vermiculite. Pretreatment in 1 mgL-1

(w/v) IBA for one week stimulated root initiation on shoots cultured on MS

medium with vermiculite supplement. Pretreatment in 1 mgL-1 (w/v) IBA

followed by culture on medium with vermiculite was most suitable for root

induction on shoots derived from shoot tip, leaf and petiole. Shoots derived

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from field-grown shoot tip and petiole explants produced 90% rooting while

87.50% rooting occurred on shoots derived from leaf explant. However, the

treatments and methodology used for rooting still need to be modified and

improved in order to produce roots of good characteristics and root quality.

Plantlet regeneration through somatic embryogenesis was successfully

achieved on leaf and petiole explants derived from proliferated shoots of

hermaphrodite field grown shoot tips as well as from embryogenic cell

suspension culture. Modified MS medium with the macro nutrients reduced to

half-strength, full-strength of other elements, 500 mgL-1 (w/v) glutamine, 30

gL-1 (w/v) sucrose, 0.2% (w/v) phytagel, and 2,4-D at 1.0, 2.5, 5.0 and 7.5

mgL-1 (w/v) in combination with 0, 0.001, 0.005 and 0.010 mgL-1 (w/v) BAP as

treatments was employed for the induction of somatic embryos on leaf and

petiole explants. For the establishment of cell suspension culture, two

treatments which were liquid full strength MS and liquid half-strength MS

medium containing 2 mgL-1 (w/v) 2,4-D were assessed.

Treatment containing 5 mgL-1 (w/v) 2,4-D was the most suitable for induction

of somatic embryos from leaf explant with the highest percentage of explants

that responded to form somatic embryos at 55% and mean number of

somatic embryos produced at 76.56. Meanwhile, treatment containing 2.5

mgL-1 (w/v) 2,4-D was most suitable for production of somatic embryos on

petiole explant with the highest percentage of explants that responded to form

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somatic embryos at 20.50% and mean number of somatic embryos produced

at 11.79.

High frequency of somatic embryo formation was achieved through cell

suspension culture method. Liquid full-strength MS medium supplemented

with 2 mgL-1 (w/v) 2,4-D significantly produced the highest percentage of

somatic embryo formation in cell suspension derived from embryogenic calli

of leaf or petiole explants compared to treatment consisting of liquid half-

strength MS medium containing 2 mgL-1 (w/v) 2,4-D. 58.33 ± 5.86

cotyledonary somatic embryos were produced per mL of suspension culture

derived from embryogenic calli of leaf explant in liquid full-strength MS

medium supplemented with 2 mgL-1 (w/v) 2,4-D whereas only 35.67 ± 1.53

cotyledonary somatic embryos were obtained per mL of suspension culture

established from embryogenic calli of petiole explant in the same treatment

(liquid full-strength MS supplemented with 2 mgL-1 (w/v) 2,4-D).

Different concentrations of NAA (0, 0.01, 0.02 and 0.03 mgL-1 (w/v)) in

combination with 0, 0.1, 0.2 and 0.3 mgL-1 (w/v) BAP were used as treatments

for germination of somatic embryos derived from the leaf and petiole explants

and from the cell suspension cultures. For somatic embryos obtained from

leaf explant, treatment containing 0.02 mgL-1 (w/v) NAA combined with 0.1

mgL-1 (w/v) BAP gave significantly the highest percentage of somatic

embryos germination (92.50%) and also the highest percentage of normal

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plantlet formation (83%). Whereas for somatic embryos obtained from petiole

explant, treatment containing 0.02 mgL-1 (w/v) NAA combined with 0.2 mgL-1

(w/v) BAP produced significantly the highest percentage of somatic embryos

germination (93.25%) and also the highest percentage of normal plantlet

formation (83%). Meanwhile, treatment containing 0.02 mgL-1 (w/v) NAA

combined with 0.1 mgL-1 (w/v) BAP was the most suitable for germination of

somatic embryos derived from cell suspension culture, whereby 92% of the

somatic embryos germinated into seedlings (plantlets).

In the development of genetic transformation protocol via Agrobacterium-

mediated gene transfer, the minimal inhibitory concentration (MIC) of

hygromycin on target materials such as leaf, petiole and embryogenic calli

was evaluated. The different concentrations of hygromycin tested were 2, 4,

6, 8, 10, 12 and 15 mgL-1 (w/v). Other parameters for genetic transformation

that were evaluated, were the different bacterial concentrations (1 X 105, and

1 X 107 cfu) combined with co-cultivation periods of 5, 15 and 30 minutes.

Hygromycin at 12 mgL-1 (w/v) was chosen as the minimal inhibitory

concentration (MIC) that caused the death of leaf explant and embryogenic

calli derived from leaf and petiole explants, while 15 mgL-1 (w/v) hygromycin

was chosen as the minimal inhibitory concentration for petiole explant.

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Although plantlet regeneration via organogenesis and somatic

embryogenesis and through cell suspension culture were successfully

established in this study and applied in the genetic transformation of papaya,

no transgenic plant was obtained. This means that further work on the

establishment of an efficient genetic transformation protocol via

Agrobacterium-mediated gene transfer for papaya cv. Eksotika need to be

pursued.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains Pertanian

KAEDAH REGENERASI ANAK POKOK SECARA IN-VITRO DAN

TRANSFORMASI GEN MELALUI PERANTARA AGROBAKTERIA PADA BETIK KULTIVAR EKSOTIKA

Oleh


November 2006 Pengerusi : Profesor Madya Maheran Abdul Aziz, PhD Fakulti : Pertanian Kajian ini telah dijalankan di Makmal In Vitro, Institut Pertanian Persekitaran

Terkawal (IPPT), Fakulti Pertanian dan Makmal Biologi Sel dan Molekul,

Fakulti Bioteknologi dan Sains Biomolekul, Universiti Putra Malaysia,

Serdang, Selangor. Matlamat utama kajian ini ialah untuk mengembangkan

sistem regenerasi anak pokok secara in vitro dan melakukan kajian awal

transformasi gen melalui perantara Agrobakteria pada betik kv. Eksotika.

Kajian ini terdiri daripada regenerasi anak pokok secara in vitro melalui

organogenesis dan embriogenesis soma menggunakan jenis eksplan yang

berbeza dan kombinasi serta kepekatan pengawal atur pertumbuhan yang

berbeza dan sistem regenerasi yang sesuai akan digunakan untuk kaedah

transformasi genetik tanaman tersebut. Kajian-kajian tersebut telah disusun

dalam reka bentuk berawak penuh.

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Kajian organogenesis terdiri daripada induksi tunas dan pengakaran. Bagi

kajian induksi tunas, media MS (Murashige dan Skoog, 1962) yang telah

diubahsuai dengan menambahkan 500 mgL-1 (b/i) kasein hidrolisat, 30 gL-1

(b/i) sukrosa dan 0.2% (b/i) agar phytagel telah digunakan sebagai media

asas, dan kombinasi kepekatan BAP berbeza-beza (0, 0.1, 1 dan 2 mgL-1

(b/i)) dengan 0, 0.05, 0.1 dan 0.2 mgL-1 (b/i) NAA telah digunakan sebagai

rawatan.

Suatu media MS yang telah diubah suai yang mengandungi kombinasi 1

mgL-1 (b/i) BAP dan 0.05 mgL-1 (b/i) NAA merupakan kombinasi rawatan

yang sesuai untuk penggandaan tunas daripada mercu pucuk daripada

pokok hermafrodit yang di tanam di lapangan, daun dan tangkai daun. Purata

bilangan tunas adalah sebanyak 73 pada eksplan mercu pucuk daripada

pokok yang ditanam di lapangan, 47 pada eksplan daun dan sebanyak 10

pada eksplan tangkai daun. Rawatan ini juga berjaya meningkatkan

peratusan eksplan yang telah menghasilkan tunas iaitu 60.24% pada eksplan

tangkai daun, 70.07% pada eksplan daun dan 100% pada eksplan mercu

pucuk.

Bagi kajian pengakaran, tunas yang dihasilkan di atas telah diberikan pra

rawatan kepekatan IBA yang berbeza (0, 0.5, 1.0 dan 2.0 mgL-1 (b/i)) untuk

seminggu dan kemudian dikultur pada Medium MS dengan bahan tambahan

vermikulit atau tanpa vermikulit (MSO). Penggunaan 1 mgL-1 (b/i) IBA

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sebagai pra rawatan selama satu minggu telah merangsang pembentukan

akar pada tunas yang dipindahkan ke atas media MS dengan tambahan

vermikulit. Kombinasi pra rawatan 1 mgL-1 (b/i) IBA diikuti dengan

pengkulturan di atas medium mengandungi vermikulit adalah yang paling

sesuai untuk induksi pengakaran pada tunas yang diperoleh daripada mercu

pucuk, dari lapangan, eksplan daun dan eksplan tangkai daun. Tunas yang

diperoleh daripada mercu pucuk dan tangkai daun menghasilkan 90%

pengakaran manakala sebanyak 87.50% pengakaran diperoleh daripada

tunas yang berasal daripada eksplan daun. Walau bagaimanapun rawatan

dan kaedah yang telah diguna pakai untuk pengakaran tersebut masih perlu

diubahsuai dan diperbaiki untuk mendapatkan ciri dan kualiti akar yang baik.

Pada regenerasi anak pokok melalui kaedah embriogenesis soma telah

berjaya dicapai bagi dua jenis eksplan iaitu daun dan tangkai daun yang

diperoleh daripada proliferasi tunas mercu pucuk pokok hermafrodit yang

ditanam di lapangan dan juga daripada kultur ampaian sel. Media MS yang

telah diubah suai dengan mengurangkan kadar pemakanan makro kepada

setengah kuasa, unsur lain pada kuasa penuh, 500 mgL-1 (b/i) glutamin, 30

gL-1 (b/i) sukrosa, 0.2% (b/i) agar phytagel dan dengan mengandungi

berbagai kepekatan 2,4-D (1.0, 2.5, 5.0 dan 7.5 mgL-1 (b/i)) yang

dikombinasikan dengan berbagai kepekatan BAP (0, 0.001, 0.005 dan 0.010

mgL-1 (b/i)) telah digunakan sebagai rawatan-rawatan untuk induksi embrio

soma pada eksplan daun dan tangkai daun. Manakala untuk kajian kultur

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ampaian sel, dua rawatan iaitu medium cecair MS dengan kuasa penuh dan

medium cecair MS dengan setengah kuasa yang masing-masing

mengandungi 2 mgL-1 (b/i) 2,4-D telah diuji.

Rawatan yang mengandungi 5 mgL-1 (b/i) 2,4-D adalah paling sesuai untuk

induksi embrio soma daripada eksplan daun dengan peratus paling tinggi

eksplan membentuk embrio soma sebanyak 55% dan purata bilangan embrio

soma yang terbentuk sebanyak 76.56. Manakala rawatan yang mengandungi

2.5 mgL-1 (b/i) 2,4-D adalah yang paling sesuai untuk eksplan tangkai daun

dengan peratus paling tinggi eksplan yang membentuk embrio soma

sebanyak 20.50% dan purata bilangan embrio soma yang terhasil sebanyak

11.79.

Pencapaian yang tinggi dalam pembentukan embrio soma telah dicapai

melalui kaedah kultur ampaian sel. Rawatan medium cecair MS kuasa penuh

yang mengandungi 2 mgL-1 (b/i) 2,4-D telah memberikan pembentukan

embrio soma yang sangat bererti bagi ampaian sel yang diperoleh daripada

kalus embriogenik eksplan daun dan eksplan tangkai daun berbanding

dengan rawatan medium cecair MS setengah kuasa yang mengandungi 2

mgL-1 (b/i) 2,4-D. Sebanyak 58.33±5.86 embrio soma kotiledonari dihasilkan

per mL kultur ampaian yang terbentuk daripada kalus embriogenik eksplan

daun pada rawatan medium cecair MS kuasa penuh yang mengandungi 2

mgL-1 (b/i) 2,4-D. Manakala sebanyak 35.67±1.53 embrio soma kotiledonari

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dihasilkan per mL kultur ampaian yang terbentuk daripada kalus embriogenik

eksplan tangkai daun pada rawatan yang sama (medium cecair MS berkuasa

penuh yang mengandungi 2 mgL-1 (b/i) 2,4-D).

Berbagai kepekatan NAA (0, 0.01, 0.02 dan 0.03 mgL-1 (b/i)) yang

dikombinasikan dengan 0, 0.1, 0.2 dan 0.3 mgL-1 (b/i) BAP telah digunakan

sebagai rawatan untuk kajian percambahan embrio soma yang diperoleh

daripada eksplan daun, eksplan tangkai daun dan daripada kultur ampaian

sel. Untuk embrio soma yang diperoleh daripada eksplan daun, rawatan yang

mengandungi kombinasi 0.02 mgL-1 (b/i) NAA dengan 0.1 mgL-1 (b/i) BAP

telah memberikan peratus percambahan tertinggi yang sangat bererti

sebanyak 92.50% dan juga peratus pembentukan anak pokok normal

tertinggi sebanyak 83%. Manakala bagi bagi embrio soma yang diperoleh

daripada eksplan tangkai daun, rawatan yang mengandungi kombinasi 0.02

mgL-1 (b/i) NAA dengan 0.2 mgL-1 (b/i) BAP telah memberikan peratus

percambahan tertinggi yang sangat bererti sebanyak 93.25% dan juga

peratus pembentukan anak pokok normal tertinggi sebanyak 83%.

Sementara rawatan yang mengandungi kombinasi 0.02 mgL-1 (b/i) NAA

dengan 0.1 mgL-1 (b/i) BAP adalah yang paling sesuai untuk percambahan

embrio soma yang diperolehi daripada kultur sel ampaian dengan 92%

daripada embrio soma tersebut bercambah menjadi anak pokok.

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Bagi mengembangkan suatu protokol transfromasi genetik melalui kaedah

pemindahan gen menggunakan perantaraan Agrobakterium, kajian

kepekatan perencatan minimal (MIC) hygromycin ke atas bahan sasaran

transformasi genetik seperti eksplan daun, tangkai daun dan kalus

embriogenik dijalankan. Berbagai kepekatan hygromycin (2, 4, 6, 8, 10, 12

dan 15 mgL-1 (b/i)) telah diujikan dalam kajian MIC ini. Parameter lain yang

diuji untuk kajian transformasi genetik ialah kepadatan bakteria (1x105 dan

1x107 cfu) yang dikombinasikan dengan masa pengkulturan bersama (5, 15

dan 30 minit) rawatan kajian.

Hygromycin pada kepekatan 12 mgL-1 (b/i) telah dipilih sebagai kepekatan

perencatan minimal yang menyebabkan eksplan daun dan kalus embriogenik

yang berasal daripada eksplan daun dan tangkai daun mati, manakala

rawatan yang mengandungi 15 mgL-1 (b/i) hygromycin telah dipilih sebagai

kepekatan perencatan minimal bagi eksplan tangkai daun.

Meskipun kaedah regenerasi anak pokok melalui organogenesis dan

embriogenesis soma diikuti dengan kultur ampaian sel telah berjaya

dilaksanakan untuk menyokong transformasi genetik pada betik tetapi

tanaman transgenik tidak diperolehi. Ini bermakna kajian lanjut untuk

mendapatkan protokol transformasi genetik yang efisien melalui pemindahan

gen dengan perantaraan Agrobakterium masih diperlukan untuk betik kv.

Eksotika.

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ACKNOWLEDGEMENTS

In the name of Allah The Beneficial and The Compassionate Praise be to Allah the Most Gracious and Merciful, upon His permission I

could complete this thesis well.

I would like to express my sincere thank to my supervisor, Associate

Professor Dr. Maheran Abdul Aziz of the Department of Agriculture

Technology, Faculty of Agriculture for her supervision, kind support beyond

duties and at heart for being so patient and understanding.

I am also indebted to my committee members, Mr. Azmi Abdul Rashid,

M.Phil., Department of Agriculture Technology, Faculty of Agriculture and

Associate Professor Dr. Norihan Mohammad Saleh, Department of Molecular

and Cell Biology, Faculty of Biotechnology and Biomolecular Sciences for

their guidance and constructive suggestion during the course of this project.

I would also like to express my deepest thanks to my father Lokot Panjaitan,

my mother Boniyem, my elder sister Murniwati, S.E. (almh.), my brother

Khayamuddin Panjaitan, S.P., M. Agric. Sc., and Syahril Fitriadi Panjaitan,

S.T., my sister Mira Herawati Panjaitan, S.Pd., and Ramadhaniyah Panjaitan,

A.M.Kes., my lover Armiyanti, S.Si., my best friends in the village, Idris and

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Hetty Mirawaty for their encouragement, patience, moral support and

inspiration during the period of my study.

I would like to express my sincere gratitude to the Malaysian people for the

opportunity on behalf of the Ministry of Science Technology and Innovation

(MOSTI) through the Universiti Putra Malaysia for the financial support (GRA

scheme) given to me to accomplish this goal.

Thanks to my housemates, Ismail bin Iberahim, Ikhsan Nugroho, Hendrawan

Samodra, H. Mohammad Cholil Mahfud, Albertus Soemargono, Teguh

Prasetyo, Muhammad Zukhrufuz Zaman and Mohd. Shamzi bin Mohamed,

Finally, special thanks to my lab mates Dr. Md. Humayun Kabir (Mithu), Budi

Winarto, Suleiman Mohamed Ahmed Elhory (Abu Fatimah), Tajul Afif

Abdullah (gorilla comel), Aini Mohd. Zainol Azlin (cool lady), Beverlien

Christine Daiman (big baby), Elixon Sunian, Norwaty Bita (best gang), Dalila

Bahrun (stubborn but also best friend), Nor Azwa Salim and Roslahalila

Abdullah for their help and cooperation in making all things easier for me and

an enjoyable stay in Malaysia.

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I certify that an Examination Committee has met on 3rd November 2006 to conduct the final examination of Syaiful Bahri Panjaitan on his Master of Agriculture Science thesis entitled “In Vitro Plantlet Regeneration and Agrobacterium-Mediated Gene Transformation of Carica papaya L. cv. Eksotika” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Act 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows: Mihdzar Abdul Kadir, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Chairperson) Radzali Muse, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Datin Siti Nor Akmar Abdullah, PhD Assosciate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Abdul Rahman Milan, PhD Senior Research Officer Horticulture Research Centre MARDI, P.O. Box 12301 50774 Kuala Lumpur (External Examiner)

_____________________________ HASANAH MOHD. GHAZALI, PhD Professor/Deputy Dean School of Graduate Studies, Universiti Putra Malaysia Date : ________________

xviii

The thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirement for the degree of Master of Agriculture Science. Members of the Supervisory Committee are as follows: Maheran Abdul Aziz, PhD Associate Professor Department of Agriculture Technology, Faculty of Agriculture, Universiti Putra Malaysia. (Chairperson) Azmi Abdul Rashid, M.Phil. Lecturer Faculty of Agriculture Universiti Putra Malaysia (Member) Norihan Mohd. Saleh, Ph.D Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member)

______________________ AINI IDERIS, PhD Professor/Dean School of Graduate Studies, Universiti Putra Malaysia Date : 2006

xix

DECLARATION

I hereby declare 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.

___________________________


Date : 8th December 2006

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TABLE OF CONTENTS

Page DEDICATION ii ABSTRACT iii ACKNOWLEDGEMENTS xvi APPROVAL xviii DECLARATION xx LIST OF TABLES xxv LIST OF FIGURES xxvii LIST OF PLATES xxxi LIST OF ABBREVIATIONS xxxv CHAPTER 1. INTRODUCTION

1.1. Background 1.1 1.2. Objectives 1.4 1.3. Significance of Study 1.4

2. LITERATURE REVIEW 2.1. Botany of Papaya 2.1 2.2. In-Vitro System 2.3 2.2.1. Field-grown shoot tip culture 2.4 2.2.2. Organogenesis 2.6 2.2.3. Somatic embryogenesis 2.7 2.3. Plant Growth Regulators 2.10 2.3.1. Auxins 2.11 2.3.2. Cytokinins 2.12 2.3.3. Amino acids as supplement 2.13 2.4. Fruit Ripening and Postharvest Physiology 2.14 2.5. Plant Transformation 2.15 2.5.1. Agrobacterium-mediated gene transfer 2.18

3. IN-VITRO PLANTLET REGENERATION OF PAPAYA THROUGH ORGANOGENESIS: SHOOT INDUCTION 3.1. Preface 3.1 3.2. Materials and Methods 3.2 3.3. Results 3.8 3.3.1. Induction of multiple shoots from field-grown shoot tip

of papaya 3.8 3.3.2. Direct shoot regeneration on leaf explant of papaya

cv. Eksotika 3.13

xxi

3.3.3. Direct shoot regeneration on petiole explant of papaya cv. Eksotika 3.20

3.4. Discussion 3.26 3.4.1. Induction of multiple shoots from field-grown shoot tip

of papaya 3.26 3.4.2. Direct shoot regeneration on leaf explant of papaya

cv. Eksotika 3.29 3.4.3. Direct shoot regeneration on petiole explant of papaya

cv. Eksotika 3.31

4. IN-VITRO PLANTLET REGENERATION OF PAPAYA THROUGH ORGANOGENESIS: ROOT INDUCTION 4.1. Preface 4.1 4.2. Materials and Methods 4.2 4.3. Results 4.6 4.3.1. Induction of roots on shoots proliferated from field-

grown shoot tip explant 4.6 4.3.2. Induction of roots on shoots regenerated directly from

leaf explant 4.11 4.3.3. Induction of roots on shoots regenerated directly from

petiole explant 4.15 4.4. Discussion 4.22 4.4.1. Induction of roots on shoots proliferated from field-

grown shoot tip explant 4.22 4.4.2. Induction of roots on shoots regenerated directly from

leaf explant 4.24 4.4.3. Induction of roots on shoots regenerated directly from

petiole explant 4.26

5. IN-VITRO PLANTLET REGENERATION OF PAPAYA THROUGH SOMATIC EMBRYOGENESIS 5.1. Preface 5.1 5.2. Materials and Methods 5.3 5.2.1. Induction of somatic embryo from leaf and petiole

explants 5.3 5.2.2. Establishment of cell suspension culture and somatic

embryo formation 5.8 5.2.3. Germination of somatic embryos 5.12 5.3. Results 5.16 5.3.1. Induction of somatic embryo from leaf explant of

papaya cv. Eksotika 5.16 5.3.2. Induction of somatic embryo from petiole explant of

papaya cv. Eksotika 5.28 5.3.3. Establishment of cell suspension culture from leaf

embryogenic callus and somatic embryo formation 5.42

xxii

5.3.4. Establishment of cell suspension culture from petiole embryogenic callus and somatic embryo formation 5.56

5.3.5. Germination of somatic embryos derived from leaf explant 5.61

5.3.6. Germination of somatic embryos derived from petiole explant 5.70

5.3.7. Germination of somatic embryos derived from cell suspension culture 5.75

5.4. Discussion 5.75 5.4.1. Induction of somatic embryo from leaf explant of

papaya cv. Eksotika 5.75 5.4.2. Induction of somatic embryo from petiole explant of

papaya cv. Eksotika 5.78 5.4.3. Establishment of cell suspension culture from leaf

embryogenic callus and somatic embryo formation 5.80 5.4.4. Establishment of cell suspension culture from petiole

embryogenic callus and somatic embryo formation 5.83 5.4.5. Germination of somatic embryos derived from leaf

explant 5.85 5.4.6. Germination of somatic embryos derived from petiole

explant 5.86 5.4.7. Germination of somatic embryos derived from cell

suspension culture 5.89

6. PRELIMINARY STUDIES ON DEVELOPMENT OF GENETIC TRANSFORMATION PROTOCOL IN PAPAYA via Agrobacterium-MEDIATED GENE TRANSFER

6.1. Preface 6.1 6.2. Materials and Methods 6.2 6.2.1. Location of study 6.2 6.2.2. History and construction of ACC oxidase genes 6.2 6.2.3. Plasmid CAMBIA 1301 and gene use 6.4 6.2.4. Preparation of gene construct 6.5 6.2.5. Determination of minimal inhibitory concentration

(MIC) 6.6 6.2.6. Genetic transformation through Agrobacterium-

mediated gene transfer 6.9 6.3. Result 6.14 6.3.1. Determination of minimal inhibitory concentration


mediated gene transfer 6.19

xxiii

6.4. Discussion 6.22 6.4.1. Determination of minimal inhibitory concentration


mediated gene transfer 6.23

7. GENERAL DISCUSSION 7.1. In Vitro Plantlet Regeneration of Papaya (Carica papaya L.)

cv. Eksotika Through Organogenesis 7.1 7.2. In Vitro Plantlet Regeneration of Papaya (Carica papaya L.)

cv. Eksotika Through Somatic Embryogenesis 7.5 7.3. Preliminary Studies on Development of Genetic

Transformation System for Papaya (Carica papaya L.) cv. Eksotika 7.9

8. CONCLUSION REFERENCES APPENDICES BIODATA OF THE AUTHOR

xxiv

LIST OF TABLES

Table Page 3.1. The combination of BAP (B) and NAA (N) concentrations

for induction of shoots on shoot tip taken from field-grown trees, leaf and petiole explants of papaya cultivar Eksotika 3.6

4.1. Combination of IBA pretreatment (I) and cultured on basal

MS medium with and without vermiculite supplement (S) on root induction of shoots derived from field-grown shoot tip, leaf and petiole explant of papaya cv. Eksotika 4.6

5.1. The combination of 2,4-D (D) and BAP (B) concentrations

for induction of somatic embryogenesis on leaf and petiole explant of papaya cultivar Eksotika 5.6

5.2. The combination of NAA (N) and BAP (B) concentrations

for germination of somatic embryos 5.14 5.3. Effect of 2,4-D and BAP concentration on percentage of

leaf explants producing callus (%) from one to five month-old culture 5.20

5.4. Effect of 2,4-D and BAP concentration on percentage of

leaf explants forming somatic embryo (%) from two to five month-old culture 5.23

5.5. Effect of 2,4-D and BAP concentration on mean number of

somatic embryos produced per leaf explants from two to five month-old culture 5.26


petiole explants producing callus (%) from one to five month-old culture 5.34


petiole explants forming somatic embryo (%) from two to five month-old of culture 5.37

5.8. Effect of 2,4-D and BAP concentration on mean number of

somatic embryos produced per petiole explant from two to five month-old culture 5.40

xxv

5.9. Estimated yield of somatic embryos in cell suspension cultures established from embryogenic callus of leaf explants 5.55

5.10. The Estimated yield of somatic embryos in cell suspension

cultures established from embryogenic callus of petiole explants 5.60

6.1. The hygromycin treatments used for determination of

minimal inhibitory concentration (MIC) 6.8 6.2. The combination of bacterial concentrations and co-

cultivation periods for transformation 6.11

xxvi

LIST OF FIGURES

Figure Page 3.1. Effect of BAP in combination with NAA on mean number of

shoots produced per shoot tip explant after twelve weeks of culture 3.11

3.2. Effect of BAP in combination with NAA on mean shoot

height (cm) after twelve weeks of culture 3.11 3.3. Effect of BAP in combination with NAA on percentage of

shoot tip explant that responded to form callus (%) after twelve weeks of culture 3.12

3.4. Effect of BAP in combination with NAA on percentage of

leaf explants that producing shoots after twelve weeks of culture 3.18

3.5. Effect of BAP in combination with NAA on mean number of

shoots produced per leaf explant after twelve weeks of culture 3.18

3.6. Effect of BAP in combination with NAA on mean shoot

height (cm) attained after twelve weeks of culture 3.19 3.7. Effect of BAP in combination with NAA on percentage of

leaf explant that responded to form callus (%) 3.19 3.8. Effect of BAP in combination with NAA on percentage of

petiole explants produced shoots after twelve weeks of culture 3.24

3.9. Effect of BAP in combination with NAA on mean number of

shoots produced per petiole explant after twelve weeks of culture 3.24

3.10. Effect of BAP in combination with NAA on shoot height

(cm) attained after twelve weeks of culture 3.25 3.11. Effect of BAP in combination with NAA on percentage of

petiole explant that responded to form callus (%) after twelve weeks of culture 3.25

xxvii

4.1. Effect of IBA pretreatment followed by culture on basal MS medium with and without vermiculite supplement on percentage of root formation on shoots proliferated from field-grown shoot tip explant after eight weeks of culture 4.9

4.2. Effect of IBA pretreatment followed by culture on basal MS

medium with and without vermiculite supplement on mean number of roots produced per shoot after eight weeks of culture 4.10


medium with and without vermiculite supplement on mean root length (cm) attained after eight weeks of culture 4.11


medium with and without vermiculite supplement on percentage of root formation on shoot regenerated directly from leaf explant after eight weeks of culture 4.13






medium with and without vermiculite supplement on percentage of root formation on shoot regenerated directly from petiole explant after eight weeks of culture 4.17





5.1. Percentage of leaf explants forming callus (%) from one to

five month-old culture 5.21

xxviii

5.2. Percentage of leaf explants that formed somatic embryo formation (%) from one to five month-old of culture 5.25

5.3. Mean number of somatic embryos produced per leaf

explants from one to five months of culture 5.28 5.4. Percentage of petiole explants forming callus (%) from one

to five month-old culture 5.35 5.5. Percentage of petiole explants that formed somatic embryo

formation (%) from one to five month-old culture 5.38 5.6. Mean number of somatic embryos produced per petiole

explant from one to five month-old culture 5.41 5.7. Cell growth in full strength liquid MS medium containing 2

mgL-1 (w/v) 2,4-D 5.43 5.8. Cell growth in half-strength liquid MS medium containing 2

mgL-1 (w/v) 2,4-D 5.44 5.9. Cell growth in full strength liquid MS medium containing 2

mgL-1 (w/v) 2,4-D 5.57 5.10. Cell growth in half strength liquid MS medium containing 2

mgL-1 (w/v) 2,4-D 5.58 5.11. Percentage of germination of somatic embryos derived

from leaf explant after two months of culture 5.66 5.12. Percentage of normal plantlet production from somatic

embryos derived from leaf explant after eight weeks on germination medium 5.67

5.13. Percentage of abnormal plantlet production from somatic

embryos derived from leaf explant after eight weeks on germination medium 5.69

5.14. Percentage of germination of somatic embryos derived

from petiole explant after two months on germination medium 5.71

5.15. Percentage of normal plantlet production from somatic

embryos derived from petiole explant after two months on germination medium 5.72

xxix

5.16. Percentage of abnormal plantlet production from somatic embryos derived from petiole explant after two months on germination medium 5.74

6.1. Effect of different concentrations of hygromycin that

caused death of leaf explant after ten days of culture 6.15 6.2. Effect of different concentrations of hygromycin that

caused death of petiole explant after ten days of culture 6.16 6.3. Effect of different concentrations of hygromycin that

caused death of embryogenic callus derived from leaf explant after ten days of culture 6.17

6.4. Effect of different concentrations of hygromycin that

caused death of embryogenic callus derived from petiole explant after ten days of culture 6.18

xxx

LIST OF PLATES

Plate Page

3.1. Proliferation of minute shoots from field-grown shoot tip of Eksotika papaya after eight weeks of culture. Bar = 5mm 3.10

3.2. Further development of the shoots after 12 weeks of culture.

Bar = 5mm 3.10 3.3. Prolific emergence of shoot meristems on leaf explant after

two weeks of culture. Bar = 5mm 3.16 3.4. Shoots developing young leaves within three to four weeks

of culture. Bar = 5mm 3.16 3.5. Further development of the shoots within five to six weeks of

culture. Bar = 5mm 3.17 3.6. Mass production of shoots on leaf explant after twelfth

weeks of Culture. Bar = 5mm 3.17 3.7. Swelling of petiole explant and emergence shoot primordial

(red arrow) after two weeks of culture. Bar = 1cm 3.22 3.8. Development of shoots from the shoot primordial (red arrow)

after four weeks of culture. Bar = 1cm 3.22 3.9. More shoots produced from petiole explant within ten weeks

of culture. Bar = 1cm 3.23 3.10. Shoots produced from petiole ready to be separated from

the group and transferred into MSO medium for rooting. Bar = 1cm 3.23

4.1. Single shoots excised and cultured on MSO medium to

neutralize the residual effect of PGR from previous shoot induction experiment. Shoots from field-grown shoot tip explant (A); from leaf explant (B) and from petiole explant (C). Bars = 1 cm 4.4

4.2. Shoots cultured on modified MS medium with IBA as

pretreatment to stimulate root initiation. Bar = 1 cm 4.20

xxxi

4.3. Normal plantlets produced from shoots pretreated in 1 mgL-1 IBA and cultured on medium with vermiculite for eight weeks. Root produced from the shoots (red arrow). Callus sometimes grew at the cut end of shoot (blue arrow) Bar = 1 cm 4.20

4.4. Plantlets obtained from shoots pretreated in 1 mgL-1 IBA and

cultured on MS medium without vermiculite for eight weeks. Roots produced from shoot (red arrow). Bar = 1 cm 4.21

4.5. Abnormal root growth with roots growing upward during

rooting (red arrow). Bar = 1 cm 4.21 5.1. In vitro proliferated shoots as source material for leaf (red

arrow) and petiole (blue arrow) explants. Bar = 1 cm 5.5 5.2. Swelling of leaf explants after a few days on somatic embryo

induction medium. Bar= 1 cm 5.17 5.3. Nodular structures grew on the surface of the callus within

two to three months of culture (red arrow). Bar = 1 cm 5.17 5.4. Formation of heart-shaped somatic embryo (red arrow) from

the nodular structures by the 12th to 14th week of culture. Bar = 0.5 mm 5.18

5.5. Somatic embryos at the torpedo stage (red arrow) by the

fourth month of culture. Bar = 5 mm 5.18 5.6. Petiole explant swelled and turned yellowish and translucent

after two weeks of culture. Bar = 1 mm 5.29 5.7. Formation of callus from both cut ends of a petiole after

three to four weeks of culture. Bar = 1 mm 5.30 5.8. Growth and enlargement of callus which covered the whole

explant surface by the eighth week of culture. Bar = 1 mm 5.30 5.9. Petiole explant that responded to form somatic embryo (red

arrow) and petiole explant without somatic embryo formation (blue arrow). Bar = 1 cm 5.31

5.10. Nodular structures (red arrow) grew from the surface of

callus within two to three months of culture. Bar = 1cm 5.31

xxxii

5.11. Nodular structures turning into heart-shaped somatic embryo (red arrow) by the 12th to 14th week of culture. Bar = 1 cm 5.32

5.12. Somatic embryos at the torpedo stage (red arrows)

observed by the 16th week of culture. Bar = 1 cm 5.32 5.13. Diagram of a standard haemocytometer chamber. Bar =

1mm 5.46 5.14. The number of cells was counted on a haemocytometer. Bar

= 0.2mm 5.46 5.15. Rounded to elongated and oval shaped cells with varied cell

size and cell content (cytoplasm and vacuole) during cell suspension establishment. Bar = 100µm 5.47

5.16. Viable cells with dense cytoplasm fluoresced apple green

when assessed using FDA solution. Bar = 100µm 5.47 5.17. Turbidity of cell suspension indicated more cells becoming

embryogenic after three sub cultures. Bar = 1cm 5.49 5.18. The cells were small, rounded and with distinct nucleolus

undergoing to divide (red arrow) and then divided into two (blue arrow). Bar = 100µm (A); Cell divided into four (white arrow). Bar = 100µm (B) and cell divided into eight celled structure (green arrow). Bar = 100µm (C) 5.49

5.19. Pro embryogenic masses in liquid cell suspension culture.

Bar = 100µm 5.50 5.20. Single globular embryo formation in liquid cell suspension

culture. Bar = 500µm 5.50 5.21. Globular embryos isolated by sieving and transferred to

fresh liquid medium of the same composition to the previous treatment. Bar = 1cm 5.51

5.22. Early heart stage somatic embryo formation. Bar = 500µm 5.51 5.23. Heart shaped somatic embryo. Bar = 400µm 5.52 5.24. Torpedo shaped somatic embryo. Bar = 400µm 5.52 5.25. Maturation into cotyledonary somatic embryos in hormone

free liquid MS medium. Bar = 100µm 5.53

xxxiii

5.26. Maintenance of cell suspension culture as stock material for

continuous production of somatic embryos. Bars = 1cm 5.53 5.27. Maintenance of cell suspension culture as stock material for

continuous production of somatic embryos. Bars = 1cm 5.62 5.28. A clump of somatic embryos (consisting of 10-15 somatic

embryos) placed on germination medium containing NAA and BAP. Bar = 50µm 5.62

5.29. Somatic embryos germinating within a week on germination

medium. Bar = 1mm 5.63 5.30. Meristem tip of somatic embryo grew upward, the hypocotyl

elongated (red arrow) and plumule (blue arrow) emerged within two to three weeks on germination medium. Bar = 2mm 5.63

5.31. Plantlets produced from somatic embryos of petiole explant

(A and B) and leaf explant (C and D) after eight weeks on germination medium. Bars = 5 mm 5.64

5.32. Abnormal plantlets production. Somatic embryo developing

into an abnormal shape and callus grew from the swollen embryo tissues (A) and somatic embryo growing like a vase (B). Bars = 5mm 5.64

6.1. Nomenclature of pCAMBIA vector 6.5 6.2. Plating of bacteria E. coli containing recombinant DNA in LB

medium containing 50 mgL-1 (w/v) of Kanamycin 6.6 6.3. Plating of Agrobacterium tumefaciens onto LB medium (A);

Single colony of Agrobacterium after incubated in incubator shaker at 37 oC, 250 rpm for 18-20 hours OD 0.4 (B); Pellet was resuspended in 10 ml MSO (C) 6.13

6.4. Somatic embryos obtained on transformed leaf embryogenic

callus (red arrow); Callus formed at the base of the explant turned brown and started to die (blue arrow) and caused somatic embryos that emerged from the callus to die when transferred onto selectable medium containing 12 mgL-1 (w/v) hygromycin and 100 mgL-1 (w/v) carbenicilin (B2C3 treatment) (Bar=1mm) 21

xxxiv

6.5. Somatic embryos (red arrow) that formed on petiole embryogenic callus died following transfer onto selectable medium containing 12 mgL-1 (w/v) hygromycin and 100 mgL-

1 (w/v) carbenicilin (B2C3 treatment) (Bar=1mm) 6.21

xxxv

LIST OF ABBREVIATION

ACC oxidase 1-aminocyclopropane 1-carboxylate oxidase

ANOVA Analysis of Variance

BAP 6-Benzylaminopurine

bp Base pair

oC Degree Celsius

CaCl2 Calcium chloride

Car. Carbenicilin

cfu Colony forming unit

cm Centimeter

CoCl2 6H2O Cobalt chloride 6-water

CuSO4 5H2O Cuprum sulfate 5-water

cv. Cultivar

2,4-D 2,4-Dichlorophenoxyacetic acid

dH2O Deionized sterile distilled water or

Denatured water

DMSO Dimethyl sulfoxide

DNMRT Duncan’s New Multiple Range Test

E. coli Escherichia coli

EDTA Ethylene diamine tetraacetate

e.g. Exempli gratia (for example)

et al. Et alia

xxxvi

etc. Et cetera

FDA Fluorescein diacetate

FeSO4 7H2O Ferrous sulfate 7-water

gL-1 Gram per liter

GTE Glucose, Tris Cl, EDTA solution

H3BO3 Boric acid

HCl Hydrochloric acid

HgCl2 Mercuric chloride

Hyg. Hygromycin

2-iP γ, γ-(dimethylaly) aminopurine

IAA Indole-3-acetic acid

IBA Indole-3-butyric acid

IPPT Institut Pertanian Persekitaran Terkawal

Kan. Kanamycin

kbp Kilo base pair

KH2PO4 Potassium dihydrogen phosphate

KI Potassium iodide

KNO3 Potassium nitrate

KOH Potassium hydroxide

µL Microliter

LB medium Luria and Broth medium

lb/inch2 Pound per square inch (newton)

LAF Laminar Air Flow

xxxvii

M Molar; Molarity

MARDI Malaysia Agriculture research and Development Institute MgSO4 Magnesium sulfate

mM Millimolar

µM Micromolar

µmolm-2s-1 Micromole per square per second

mgL-1 Milligram per liter

MnSO4 4H2O Manganese sulfate 4-water

MS medium Murashige and Skoog medium

MSO medium Hormone-free Murashige and Skoog Medium NAA α-Naphtaleneacetic acid

NaCl Sodium chloride

Na2EDTA Dinatrium ethylene diamine tetraacetate

NaOH Sodium hydroxide

Na2MoO4 2H2O Natrium molybdate 2-water

NH4NO3 Ammonium nitrate

OD Optical density

p=0.05 Probability at 95%

PCR Polymerase chain reaction

PGR Plant growth regulator

pH Potential Hydrogen or –Log (H)

pPACOA1 Plasmid PACOA1

xxxviii

pPACOA2 Plasmid PACOA2

PRSV Papaya ring-spot virus

SD Standard deviation

TAE Tris-acetate/EDTA electrophoresis buffer

TFB Buffer for preparation of competent E. coli

ZnSO4 7H2O Zinc sulfate 7-water

% Percent

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