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Analysis of Protoplasts and Somatic

Embryogenesis in Medicago truncatula

A thesis submitted for the degree of Doctor of Philosophy of The Australian National

University

By

Fern ke de J ong

November 2006

(Research School of Biological Sciences,

Genomic Interactions Group)

Declaration

The research in this thesis is my own work, except where acknowledgment is made,

and has not been submitted for any other degree.

,/ ~ ............. " ,t .. "... )L: ~

Femke de Jong

II

Acknowledgements

There are many people I would like to thank and acknowledge for their help and

support during the course of my PhD. I would like to start by thanking my

supervisors for giving me the opportunity to do my PhD: Barry Rolfe for his wide

knowledge about plant development and stem cells and discussions with him about

these subjects, and Ulrike Mathesius, for her knowledge about proteomics,

discussions about protoplasts and somatic embryogenesis, and for her support and

belief, in my ability to succeed.

I would like to thank my advisors Nijat Imin, for his help and his knowledge about

proteomics and molecular biology, and for the many discussions about somatic

embryogenesis and protoplast proliferation, and Jeremy Weinman for the discussions

and help with forming my ideas so I could put them onto paper.

I would like to thank Elena for her help and patience with my English writing, and

for sharing the lab with me. I also would like to thank all other members of the

Genomic Interactions Group for their support and friendship.

I would like to thank the ARC Centre of Excellence for Integrative Legume Research

for their funding and scholarship, which enabled me to do my research, and all their

members, particularly the Newcastle node, for the discussions during the annual

meetings.

For their technical support, I would like to thank the Mass Spectrometry Facility at

the Research School of Biological Sciences at the Australian National University and

the Biomolecular Resource Facility at The John Curtin School of Medical Research

at the Australian National University. I am especially grateful to Charles Hocart and

Caroline McKinlay of the Mass Spectrometry Facility, and Peter Milburn of the

Biomolecular Resource Facility for their time and for running my samples.

For their help with the setting up the in vitro hybridization I like to thank Riccardo

Natoli and Daryl Webb. I also like to thank the ANU Electron Microscopy Unit and

especially Daryl Webb for his expertise in microscopy.

I am indebted to the staff of the Controlled Environment Facility, and especially Sue

Lyons for keeping the growth chambers running and my plants alive.

Finally I want to thank my family and friends in RSBS and Fenner Hall for their

support and I especially want to thank Jasmine and Leah for keeping me sane.

iii

Abstract

This thesis examined protoplast proliferation and somatic embryogenesis, by

comparing a highly with a poorly embryogenic Medicago truncatula line through

microscopic, proteomic and in situ hybridization analysis. Proteome analysis of M.

truncatula was used to identify proteins involved in protoplast proliferation and the

initiation of somatic embryogenesis. Furthermore, an in situ hybridization study was

done to compare the expression of genes known to be involved in zygotic

embryogenesis with the expression during somatic embryogenesis. A large number

of proteins were up-, and down-regulated during the first 5 days of protoplast culture

indicating that cellular reorganization took place. An up-regUlation of PR 1 O-like

proteins and flavonoid synthesis proteins and a down-regulation of energy

metabolism proteins were observed, indicating an initiation of a stress response. The

observed stress response in protoplasts was down-regulated before the first cell

divisions at 5-7 d. A stress-inducing bioassay on protoplasts showed that the ability of

protoplasts to overcome stress and to proliferate under stress conditions depended on

the level of stress and density of the protoplast culture, whereby more stress or a

lower culture density resulted in higher levels of cell death. Proteomic analysis of the

initiation of somatic embryogenesis showed that similar metabolic pathways were

involved in the initiation of somatic embryogenesis and protoplast proliferation. By

using a highly embryogenic (2HA) line, and a poorly embryogenic (A 17) line of M.

truncatu!a, it was shown that particular proteins were specifically accumulated

during the initiation of somatic embryogenesis. A high accumulation of a peroxidase

was observed only in At7 tissue at the time of initiation of somatic embryogenesis

and might be the reason why the initiation of somatic embryogenesis is inhibited in

A 17 tissue. The specific accumulation of flavonoid synthesis proteins might also

indicate that flavonoids are involved during the initiation of somatic embryogenesis.

In situ hybridization with probes to genes known to be involved in zygotic

embryogenesis, showed that M. truncatula somatic and Arabidopsis thaliana zygotic

embryogenesis both followed similar developmental pathways. However, a few

genes showed distinct patterns of gene expression in M. truncatula somatic embryos.

iv

Abbreviations

#: number

% Vol: percentage volume

§: paragraph

°C: degrees Celsius

2,4-0: 2,4-dichlorophenoxy acetic acid

20E: 2-dimensional electrophoresis

20-LC: 2-demsionalliquid chromatography

ABA: abscisic acid

AGP: arabinogalactan proteins

ANT: AINTEGUMENTA

ANT: AINTEGUMENTA

APC: anaphase promoting complex

A. thaliana: Arabidopsis thaliana

ARF: auxin response factor

AS1: ASYMETRIC LEAVESl

ASl: ASYMETRIC LEAVESl

ATP: adenosine-5'-triphosphate

Avr: avirulence

AXR1: auxin-resistance protein 1

BAC: bacterial artificial chromosome

BAP: 6-benzyl amino purine

BBM: BABY BOOM

BBM: BABY BOOM

BCIP: 5-Bromo-4-chloro-3-indolyl phosphate, toluidine salt

BOL: BODENLOS

BDL: BODENLOS

BLAST: basic local alignment search tool

bp: base pair

BR1: BRASSINOSTEROID-INSENSITIVEI

BRl: BRASSINOSTEROID-INSENSITlVEl

BSA: bovine serum albumin

C .elegans: Caenorhabditis elegans

CAK: CDK activating kinase

v

CBB: coomassie brilliant blue

CCoAOMT: Caffeoyl-CoA O-methyltransferase

CDK: cyelin-dependent kinase

eDNA: complementary DNA

CHAPS: (3-[ (3-cholamidopropyl) dimethylammonio ]-propanesulfonate

CID: collision induced dissociation

CKI: CDK inhibitory protein

CLV:CLAVATA

CLV: CIA VATA

em: centimetre

Cond+-medium: conditioned medium supplemented with a final concentration of 10

JlM NAA and 1 JlM BAP

Cond4-medium: conditioned medium from 4 x 105 cells/ml

Conds-medium: conditioned medium from 8 x 105 cells/ml

Cond-medium: conditioned medium

cue: CUP SHAPED COTYLEDON

CUC: CUP SHAPED COTYLEDON

Cye: cyelin

CZ: central zone

d: day

Da: dalton

dA TP: deoxyadenosine 5' triphosphate

DCF: 2',7'-dichlorofluorescein

dCTP: deoxycytidine 5' triphosphate

DEPC: diethyl pyrocarbonate

dGTP: deoxyguanosine 5' triphosphate

DIG: digoxigenin

DNA: deoxyribonueleic acid

dNTP: deoxynucleoside triposphate

dpi: dots per inch

dT: 2' -deoxyribo-thymine

DTT: dithiothreitol

dTTP: deoxythymidine 5' triphosphate

E. coli: Escherichia coli

ERFl: ethylene response factor 1

vi

ERF1: ethylene response factor 1

ESI: electrospray Ionisation

EST: expressed sequence tags

ET: ethylene

eV: electronvolt

FASTA: FAST-All

g: acceleration due to gravity

g: grams

GCIMS: gas chromatography mass spectrometry

h: hour

H20CFDA: 2',T-dichlorodihydrofluorescein diacetate

HBT: HOBBIT

BBT: HOBBIT

HR: hypersensitive response

IAA: indole-3-acetic acid

id: internal diameter

inj: injection

IPTG: isopropyl-B-D-thio galactopyranoside

ISR: induced systemic resistance

JA: jasmonic acid

KAPP: kinase-associated protein phosphatase

kDa: kilodalton

KPR: p27kip-related protein

I: litre

LCIMS: liquid chromatography mass spectrometry

LEC: LEAFY COTYLEDON

LEe: LEAFY COTYLEDON

LRR: leucine rich repeat

M. truncatula: Medicago truncatula

m1z: mass-to-charge ratio

rn: metre

M: molar

rnA: milli Ampere

MALOI-TOF: matrix assisted laser desorption ionisation - time of flight

vii

MALDI-TOF -TOF: matrix assisted laser desorption ionisation - time of flight -time

of flight

MAPK: mitogen activated protein kinase

Mbp: mega base pair

MeJA: methyl jasmonic acid

mg: milligram

min: minute

miRNA: micro RNA

ml: millilitre

mm: millimetre

mM: millimolar

MP: MONOPTEROS

MP: MONOPTEROS

mRNA: messenger RNA

MS: mass spectrometry

msec: millisecond

Mudpit: multi-dimensional protein identification technology

MW: molecular weight

M!l: mega Ohm

NAA: a-napthalene acetic acid

NAC-domain: N-acetyl-cysteine-domain

NBT: nitro blue tetrazolium chloride

ng: nanogram

NO: nitric oxide

nt: nucleotide

OGA: oligosaccharide

Pl+: PI medium supplemented with a final concentration of to 11M NAA and 1 11M

BAP

PCR: polymerase chain reaction

pH: p(otential of) h(ydrogen)

pI: isoelectric point

PIN: PIN-formed

PIN: PIN-formed

PLTl: PLETHORA1

PLTl: PLETHORA]

viii

POL: POLTERGEIST

POL: POLTERGEIST

PP2C: protein phosphatase 2C

ppm: parts per million

PR: pathogen related

PRZl: PROPORZI

PRZl: PROPORZI

psi: pounds per square inch

PSK: phytosulfokine

PZ: peripheral zone

QC: quiescent center

R: resistance

RAM: root apical meristem

Rb: retinoblastoma-related protein

REML: restricted maximum likelihood

RLK: receptor like kinase

RNA: ribonucleic acid

RNAi: RNA interference

ROP: Rho-like GTPase

ROS: Reactive Oxygen Species

rpm: rotations per minute

RZ: rib zone

SA: salicylic acid

SAM: shoot apical meristem

SAR: systemic acquired resistance

SCF: Skp I-Cullin-F-box

SCR: SCARECROW

SCR: SCARECROW

SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis

sec: second

SERK: Somatic Embryo Receptor Kinase

SHD: SHEPHERD

SHD: SHEPHERD

SHR: SHORTROOT

SHR: SHORTROOT

ix

SIPK: salicylic acid-induced protein kinase

siRNA: small interference RNA

STM: SHOOT MERISTEM LESS

STM: SHOOT MERISTEM LESS

TF A: trifluoroacetic acid

Tm: melting temperature

UV: ultra violet

V: volt

VDC: vein derived cells

vol: volume

v/v: volume/volume

W: watt

WIPK: wounding-induced protein kinase

WOX: WUSCHEL HOMEOBOX

WOX: WUSCHEL HOMEOBOX

WUS: WUSCHEL

WUS: WUSCHEL

w/v: weight/volume

X-gal: 5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside

JIg: microgram

JlI: microliter

JIm: micrometer

JIM: micromolar

Jlmol: micromole

x

Table of contents

Declaration .............................................................................................................. ii

Acknowledgements ............................................................................................... iii

Abstract ................................................................................................................. iv

Abbreviations .......................................................................................................... v

Table of contents ................................................................................................... xi

Chapter 1: Introduction ........................................................................................... 1

1.1 Introduction .................................................................................................................................... 1

1.2 Medica~o truncatula as a model legume ....................................................................................... 3

1.3 Protoplasts ...................................................................................................................................... 4

1.4 Growth regulators .......................................................................................................................... 4

1.5 Stem cells ......................................................................................................................................... 5 ! .5.1 Meristem organization .............................................................................................................. 7 1.5.2 Shoot apical meristem .............................................................................................................. 7 1.5.3 Root apical meristem ............................................................................................................... 1 0

1.6 Dedifferentiation and differentiation ........................................................................................... 11 1.6.1 Wound, defence and stress responses ...................................................................................... 12

1.6.1.1 Wounding and jasmonic acid ........................................................................................... 14 1.6.1.2 Pathogen infection ........................................................................................................... 15 1.6.1.3 Interaction of different stress/defence responses ............................................................. 16 1.6. 1.4 Mitogen acti vated protein kinase ..................................................................................... 17

1.7 Cell cycle ........................................................................................................................................ 18

1.8 Embryogenesis ............................................................................................................................... 21 1.8.1 Zygotic embryogenesis ............................................................................................................ 23 1.8.2 Somatic Embryogenesis .......................................................................................................... 25 1.8.3 Genes regulating zygotic embryogenesis ................................................................................ 26

1.9 Scope of this thesis ......................................................................................................................... 30

Chapter 2: Materials and Methods ...................................................................... 32

2.1 Biological material, media, buffers and solutions ....................................................................... 32

2.1.] Biological materials .................................................................................................................... 32 2.1.1.1 Plant species and cultivars ............................................................................................... 32 2.1.1.2 Bacterial strains used in this study ................................................................................... 32 2.1.1.3 Plasmids and constructs used in this study ...................................................................... 33

2.1 .2 Protoplast culture .................................................................................................................... .35 2.1.2.1 PAC-potting mix ............................................................................................................. .35 2.1.2.2 PES-5 enzyme medium .................................................................................................... 35 2.1.2.3 PES-Y medium ............................................................................................................... .35 2.1.2.4 Perc 011/ Mannitol solution ............................................................................................... 35 2.1.2.5 Hormone stock solutions ................................................................................................. 35 2.1.2.6 PI medium ....................................................................................................................... 36 2.1.2.7 PI agarose culture medium .............................................................................................. 36

Xl

2.1.2.7 P4 culture medium ........................................................................................................... 37 2.1.3 Proteomics ............................................................................................................................... 38

2.1.3.1 Extraction solution I ....................................................................................................... .38 2. 1.3.2 Extraction solution 2 ........................................................................................................ 38 2.1.3.3 Protein sample buffer ....................................................................................................... 38 2.1.3.4 Rehydration solution ........................................................................................................ 38 2. 1.3.5 Equilibration solution A .................................................................................................. .38 2.1.3.6 Equilibration solution B ................................................................................................... 38 2.1.3.7 Fixation solution .............................................................................................................. 39 2.1.3.8 Sensitizer solution ............................................................................................................ 39 2. 1.3.9 Silver solution .................................................................................................................. 39 2.1.3.10 Developer solution ........................................................................................................ .39 2.1.3.11 Stop solution .................................................................................................................. 39 2.1.3.12 CBB staining solution .................................................................................................... 39 2.1.3.13 Sample elution solution LC-MS .................................................................................... 39 2.1.3.14 Sample elution solution MALDI-TOF-TOF .................................................................. 39

2.1.4 In situ hybridization ................................................................................................................ .40 2.1.4.1 Stock solutions for bacteria medium ............................................................................... .40 2.1.4.2 LB-medium ..................................................................................................................... .40 2.1.4.3 SOC medium .................................................................................. ................................ 040 2.1.4.4 5x TBE buffer ........................................................................ ......................................... 040 2.104.5 5x MOPS buffer ........................................................................ ...................................... 040 2.104.6 1%, agarose RNA gel ...................................................................................................... 041 2.1.4.7 RNA sample buffer ......................................................................................................... .41 2.1.4.8 RNA loading buffer ........................................................................................................ .41 2.1.4.9 Maleic acid buffer ............................................................................... ............................ 041 2.1.4.10 Blocking solution .......................................................................................................... .41 2.1.4.11 Anti-DIG antibody solution .......................................................................................... .41 2.1.4.12 Washing buffer ............................................................................................................. .41 2.1.4.13 Detection buffer ............................................................................................................. 42 2.1.4.14 Colour substrate solution ............................................................................................... 42 2.1.4.15 PBS Phosphate buffered saline ...................................................................................... 42 2.1.4.164% formaldehyde ..................................................................................... ..................... 042 2.1.4.17 Proteinase K solution ..................................................................................................... 42 2.1.4.18 20x SSC (stock) ............................................................................................................ .42 2.1.4.19 Probe mix ....................................................................................................................... 42 2.1.4.20 Hybridization solution .................................................................................................. .43 2.104.21 Hybridization mix ......................................................................................................... .43 2.1.4.22 Neutral red staining solution .......................................................................................... 43

2.2 Methods .......................................................................................................................................... 44 2.2.1 Protoplast culture ..................................................................................................................... 44

2.2.1.1 Growth conditions for plants ........................................................................................... 44 2.2.1.2 Protoplast isolation .......................................................................................................... 44 2.2.1.3 Protoplast purification ..................................................................................................... 44 2.2.1.4 Protoplast culture ............................................................................................................. 45 2.2.1.5 SPE fractionation and GC/MS analysis .......................................................................... .45

2.2.2 Proteomics .............................................................................................................................. .47 2.2.2.1 Protein extraction ............................................................................................................ .47 2.2.2.2 Bradford assay ................................................................................................................ .47 2.2.2.3 First dimension isoelectric focusing ............................................................................... .48 2.2.2.4 Second dimension SDS-P AGE ....................................................................................... .48 2.2.2.5 Silver staining .................................................................................................................. 49 2.2.2.6 Coomassie Brilliant Blue (CBB) staining ....................................................................... .49 2.2.2.7 Scanning and analysing the gels ..................................................................................... .49 2.2.2.8 CBB de-staining .............................................................................................................. .50 2.2.2.9 Silver de-staining ............................................................................................................. 50 2.2.2.10 Trypsin digestion ........................................................................................................... 50 2.2.2.11 MALDJ analysis ............................................................................................................ 51 2.2.2.12 LC-MS analysis ............................................................................................................. 51 2.2.2.13 Protein identification ..................................................................................................... 51

2.2.3 In situ hybridization ................................................................................................................. 53 2.2.3.1 Probe and primer design .................................................................................................. 53

XII

2.2.3.2 RNA isolation .................................................................................................................. 53 2.2.3.3 First strand cDNA synthesis ............................................................................................ 54 2.2.3.4 PCR .................................................................................................................................. 55 2.2.3.5 DNA-gel electrophoresis ................................................................................................. 55 2.2.3.6 Plasmid ligation ............................................................................................................... 55 2.2.3.7 E.coli transformation ....................................................................................................... 56 2.2.3.8 Plasmid isolation .............................................................................................................. 56 2.2.3.9 Plasmid restriction ........................................................................................................... 57 2.2.3.10 DIG-RNA labelling ...................................................................................................... .57 2.2.3.11 RNA-gel electrophoresis ................................................................................................ 58 2.2.3.12 Control labelling efficiency ........................................................................................... 58 2.2.3.13 Dot blot hybridization .................................................................................................... 59 2.2.3.14 Tissue sample fixation and embedding .......................................................................... 59 2.2.3.15 In situ hybridization ....................................................................................................... 60

Chapter 3: Protoplast culture optimization and analysis .................................... 62

3.1 Introduction ................................................................................................................................... 62 3.1.1 Aims of the chapter ................................................................................................................. 62 3.1.2 Protoplast culture ..................................................................................................................... 62 3.1.3 Growth regulators .................................................................................................................... 63

3.2 Results ............................................................................................................................................ 64 3.2.1 Protoplast culture ..................................................................................................................... 64 3.2.2 Optimization of protoplast isolation ........................................................................................ 66 3.2.3 Jasmonic acid influence on protoplast culture ......................................................................... 67 3.2.4 Influence of conditioned medium on protoplast culture .......................................................... 68 3.2.5 Hormone influence on protoplast culture ................................................................................ 71 3.2.6 GC/MS analysis of conditioned medium ................................................................................. 72

3.3 Discussion ....................................................................................................................................... 74 3.3.1 Optimization of protoplast isolation ........................................................................................ 74 3.3.2 Stress and Jasmonic acid ......................................................................................................... 75 3.3.3 Stress and medium conditioning .............................................................................................. 76 3.3.4 Further research ....................................................................................................................... 78 3.3.5 Concluding remarks ................................................................................................................. 79

Chapter 4: Proteomic analysis of protoplast proliferation of Medicago truncatula .............................................................................................................. 81

4.1 Introduction ................................................................................................................................... 81 4.1.1 Aim of the chapter ................................................................................................................... 81 4.1.2 Proteomics ............................................................................................................................... 81

4.2 Results ............................................................................................................................................ 85 4.2.1 2-Dimensional gel analysis ...................................................................................................... 85 4.2.2 Time point analysis .................................................................................................................. 87 4.2.3 Genotype analysis .................................................................................................................... 88 4.2.4 Interaction of time point with genotype analysis ..... , .. ,.,., ... , .................................................... 89 4.2.5 Protein identification ........................................................................... , ................................... 89 4.2.6 PRI0-like proteins ................................................................................................................... 91

4.3 Discussion ....................................................................................................................................... 94 4.3.1 Protein synthesis and folding ................................................................................................... 95 4.3.2 Energy metabolism .................................................................................................................. 95 4.3.3 PRJ O-like proteins ................................................................................................................... 96 4.3.4 Flavonoid metabolism ................................................................... , .................. , ...................... 97 4.3.5 Genotype analysis .................................................................................................................... 98 4.3.6 Genotype and time point analysis ............................................................................................ 98 4.3.7 Protein isolation and identificatioll .......................................................................................... 99 4.3.7 Concluding remarks ............................................................................................................... 100

xiii

Chapter 5: A proteomic analysis of the initiation of somatic embryogenesis of Medicago truncatula . ........................................................................................... 101

5.1 Introduction ................................................................................................................................. 101 5.1.1 Aims of this chapter ............................................................................................................... 101 5.1.2 The characteristics of embryogenic cells ............................................................................... 10 I 5.1.3 The influence of growth regulators on somatic embryogenesis ............................................ 102

5.2 Results .......................................................................................................................................... 104 5.2.1 Protoplast culture ................................................................................................................... 104 5.2.2 Proteome analysis .................................................................................................................. 105 5.2.3 Genotype differences ............................................................................................................. 107 5.2.4 40-50 d time points ................................................................................................................ 1 08 5.2.5 50-80 d lime period ............................................................................................................... 109 5.2.6 Protein spot identification ...................................................................................................... 110 5.2.7 Identified proteins whose accumulation is genotype dependent... ......................................... 112 5.2.8 Identified proteins whose accumulation was genotype and time point dependent ................ 112

5.2.8.1 Metabolic proteins ......................................................................................................... 112 5.2.8.2 Peroxidases .................................................................................................................... 114 5.2.8.3 PR 10-like proteins ......................................................................................................... 115 5.2.8.4 Cell structure .................................................................................................................. 115 5.2.8.5 Primary metabolism ....................................................................................................... 116 5.2.8.6 Protein folding and processing ...................................................................................... 117

5.3 Discussion ..................................................................................................................................... 119 5.3.1 Comparing A 17 and 2HA ...................................................................................................... 119 5.3.2 Somatic embryogenesis influences protein accumulation ..................................................... 120

5.3.2.1 Proteins involved in protein synthesis and folding ........................................................ 120 5.3.2.2 Cell structure .................................................................................................................. 120 5.3.2.3 Primary metabolism ....................................................................................................... 121 5.3.2.4 Peroxidases .................................................................................................................... 1 21 5.3.2.5 PRIO-Iike proteins and f1avonoids ................................................................................. 122 5.3.2.6 Metabolic proteins ......................................................................................................... 123

5.3.3 Comparison of the proteomes of leaf explants and protoplast cultures ................................. 123 5.3.4 Future approaches .................................................................................................................. 124 5.3.5 Concluding remarks ............................................................................................................... 124

Chapter 6: Expression analysis of embryogenic genes in Medicago truncatula somatic embryos .................................................................................................. 126

6.1 Introduction ................................................................................................................................. 126 6.1.1 Aim of this chapter ................................................................................................................ 126 6.1.2/11 situ hybridization ............................................................................................................... 127

6.2 Results .......................................................................................................................................... 129 6.2.1 Selection of candidate genes for in situ hybridization analysis of somatic embryos ............. 129 6.2.2 Cloning and labelling of the probes ....................................................................................... 1 29 6.2.1 Dot blot hybridization ............................................................................................................ 133 6.2.3 Morphology of somatic embryos ........................................................................................... 134 6.2.4 In situ hybridization ............................................................................................................... 135

6.3 Discussion ..................................................................................................................................... 141 6.3.1 Are the analysed genes homologues or orthologues to their Arabidopsis counterparts? ....... 141 6.3.2 Morphology of somatic embryos ........................................................................................... 142 6.3.3 Comparison of the expression in somatic embryos to that in zygotic embryos ..................... 143

6.3.3.1 Hobbit ............................................................................................................................ 143 6.3.3.2 CLYI. STM, and AS1 ................................................................................................... 144 6.3.3.3 WUS .............................................................................................................................. 145 6.3.3.4 BBM .............................................................................................................................. 147 6.3.3.5 SERK, LEC1, and GNOM ............................................................................................. 148

6.3.4 Dot blot hybridization analysis .............................................................................................. 148 6.3.5 In situ hybridization specifity and probe specifity ................................................................. 149

xiv

6.3.5 Concludi ng remarks ............................................................................................................... 149

Chapter 7: Final discussion/conclusion .............................................................. 151

7.1 Introduction ................................................................................................................................. 151 7.1.1 Main findings of this thesis ................................................................................................... 151 7.1.2 The scope of this chapter ....................................................................................................... 152

7.2 What are the processes controlling protoplasts proliferation? ............................................... 153 7.2.1 Dedifferentiation and chromatin reorganization .................................................................... 154 7.2.2 Re-entry into the cell cycle .................................................................................................... 155 7.2.2 Summary and a model for proliferation ................................................................................. 156 7.2.3 Implications of the model on current research ....................................................................... I 58

7.3 What are the changes in cell state during the initiation of somatic embryogenesis? ............. 159 7.3.1 Influence of asymmetric cell division .................................................................................... 160 7.3.2 Summary of the initiation of somatic embryogenesis ........................................................... 162

7.4 ProposaJ for future experiments to answer remaining questions ............................................ 162 7.4. 1 Future experiments on protoplast proliferation ..................................................................... 163 7.4.2 Future experiments on regulation of the initiation of somatic embryogenesis ...................... 165 7.4.3 Future experiments comparing the gene expression between zygotic and somatic embryos 166

7.5 Other possible factors that may influence the process of protoplast proliferation and the initiation of somatic embryogenesis ................................................................................................. 166

7.6 Summary ...................................................................................................................................... 167

References ........................................................................................................... 168

Appendix 1 .......................................................................................................... 189

Appendix 2 ............................................................................................................... 191

Appendix 3 ............................................................................................................... 200

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