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Date
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
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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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xv