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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. ,/

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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.

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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 1O-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.

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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 dATP: 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

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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

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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 ............................................................................................................... 10

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

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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 ................................................................................................................ 108 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 .................................................................................................................... 121 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 ....................................................................................... 129 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

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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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