TABLE OF CONTENTS
CHAPTER 1
General Introduction
PAGE
1.1
Impatiens balsamina background
1
1.2
The importance study of I. balsamina
4
1.3
Plant regeneration system
5
1.4
Transformation system
7
1.4.1
Agrobacterium tumefaciens transformation
8
1.4.2
Biolistic-mediated transformation
10
1.5
Marker genes
15
1.5.1
Selectable marker
15
1.5.1.1 Antibiotic
16
1.5.1.2 Herbicide
17
Reporter genes
20
1.5.2
1.6
Transient gene expression and stable gene expression
22
1.7
Promoters
23
1.8
Objectives of study
25
vi
CHAPTER 2
Tissue culture system of
PAGE
Impatiens balsamina
2.1
Introduction
26
2.2
Materials and methods
30
2.2.1
Plant materials and seeds sterilization
30
2.2.2
Germination of seeds
30
2.2.3
Plant growth regulators and stock solutions
30
2.2.4
Explant preparations
31
2.2.5
Shoot induction experiment
31
2.2.6
Root induction experiment
34
2.2.7
In vitro plant regeneration
35
2.2.8
Data analysis
35
2.3
Results
2.3.1
36
The effect of explant age, types and sections
36
on shoot induction
2.3.2
Shoot induction
39
2.3.3 Root induction
44
2.3.4 In vitro plant regeneration
48
2.4
Discussion
50
2.5
Conclusion
57
vii
CHAPTER 3 Transformation of Impatiens balsamina
PAGE
through biolistics
3.1
Introduction
58
3.2
Materials and Methods
61
3.2.1
Bacterial strain and plasmid
61
3.2.2
Bacteria growth and condition
61
3.2.3
Plasmid DNA
62
3.2.4
Agarose gel electrophoresis
62
3.2.5
Measurement of DNA concentration
63
3.2.6
Gold particles preparations
63
3.2.7
Preparation of DNA coating gold particles
64
3.2.8
Explants preparation
64
3.2.9
Bombardment media
65
3.2.10 Bombardment technique
65
3.2.10.1 Physical parameters
65
3.2.10.2 Biological parameters
66
3.2.11 ß-glucuronidase (GUS) assay buffer
66
3.2.12 ß-glucuronidase (GUS) expression assay
66
3.2.13 Genomic DNA
67
3.2.14 Statistical analysis
68
3.2.15 Transformation frequency
68
3.2.16 Biolistic transformation using hph gene
69
for hygromycin resistant
viii
3.2.16.1 Determination of minimal lethal
69
dose of hygromycin on cotyledon explants
3.2.16.2 Delay selection method on hygromycin media
69
3.2.16.3 Polymerase chain reaction (PCR) analysis
70
3.2.17 Biolistic transformation using bar gene for
71
phosphinothricin (Basta) resistance
3.2.17.1 Herbicide applications on I. balsamina
71
3.2.17.2 Determination of minimal lethal dose of
71
phosphinothricin (PPT) on explants
3.2.17.3 Delay selection method on phosphinothricin
72
(PPT) media
3.3
Results
3.3.1
73
The effect of target distance and helium pressure
73
on GUS gene expression
3.3.2
The effect of number of bombardments on
75
GUS gene expression
3.3.3
The pre-culture time prior bombardment on transient
77
gene expression
3.3.4
The effect of plasmid concentration on transient gene
79
expression
3.3.5
The effect of pre-culture treatments on transient
gene expression
ix
81
3.3.6
The effect of post-bombardment incubation time on
83
transient gene expression
3.3.7
The effect of optimal bombardment conditions on
85
GUS gene expression and regeneration of I. balsamina
3.3.8
Biolistic transformation using pRQ6 (uidA+, hph+)
88
for hygromycin resistant
3.3.8.1 Minimal lethal dose of hygromycin
88
3.3.8.2 The effect of delay selection on
91
regeneration of transformed plant in hygromycin
media
3.3.8.3 Chimera expression of I. balsamina
95
3.3.8.4 The effect of hygromycin in selection system
95
on regeneration of I. balsamina
3.3.8.5 Transformation frequency
96
3.3.8.6 Polymerase chain reaction (PCR) analysis of
97
transformed plants
3.3.9
Biolistic transformation of bar gene for Basta
99
(phosphinothricin) resistant
3.3.9.1 The effect of herbicide Basta (phosphinothricin)
99
on I. balsamina
3.3.9.2 The effect of herbicide phosphinothricin (PPT)
100
on plant tissue culture of I. balsamina
3.3.9.3 The effect of selection on phosphinothricin
(PPT) media and GUS gene expression
x
102
3.4
Discussion
105
3.5
Conclusion
114
CHAPTER 4
115
General Conclusion
FUTURE WORK
117
REFERENCES
119
APPENDICES
136
xi
LIST OF TABLES
TABLE NO.
2.1
TITLE
The usage of different types and concentrations of
PAGE
33
cytokinins in shoot induction media.
2.2
The different types and concentrations of auxins on
34
root induction.
3.1
The plasmids used in this study.
61
3.2
The effect optimal bombardment conditions on
86
GUS gene expression of explants 24 h post-bombardment.
3.3
The effect of different concentrations of hygromycin in
89
various ages of explants on the mortality of explants using
the unbombarded explants for minimal lethal dose after five
weeks in culture.
3.4
The effect of selection on number of regenerating
93
plants when cultured on 75 mg/L hygromycin and 100 mg/L
hgromycin after five weeks in culture.
3.5
The effect of delay selection using 35 days shooting explants
in 75 mg/L hygromycin on regenerating of the transformed plants
after 5 weeks in culture.
xii
96
3.6
The effect of different concentrations of phosphinothricin (PPT)
101
on unbombarded explant ages for minimal lethal dose after
five weeks in culture.
3.7
The effect of 1 mg/L phosphinothricin (PPT) on bombarded
explant with co-transformation of pRQ6 (hph +, uidA+)
and pAHG11(bar+) in regenerating transformed plants.
xiii
104
LIST OF FIGURES
FIGURE NO.
TITLE
PAGE
1.1
I. balsamina with white and purple flower colours
2
1.2
Lawsone and Me-lawsone, chemical structures found in
3
roots cultures of I. balsamina.
1.3
The genetic organization of the TL-DNA of an octopine
9
–type Ti plasmid with eight open reading frames (ORFs)
(1-7).
1.4
PDS-1000/He Particle Delivery System, a device used
12
in biolistic transformation (BioRad, USA).
1.5
Structure of hygromycin.
17
1.6
The chemical structure of phosphinothricin (PPT).
19
1.7
Structure of X-Gluc
21
(5-bromo-4-chloro-3-indoxyl-ß-D-glucuronide)
1.8
Nucleotide sequence of the Cauliflower Mosaic Virus
24
35S promoter and upstream region.
2.1
The different types and sections of 7 days old seedling
32
explants.
2.2
The effect of explant age from proximal cotyledon on average
number of shoot per explant after three weeks in culture on
MS media supplemented with 1 mg/L BAP.
xiv
37
2.3
The effect of cytokinin on shoot regenerated from different
38
types of explants used after three weeks in culture.
2.4
Multiple shoots development in MS media supplemented with
40
1 mg/L BAP within two weeks in culture (A-E).
2.5
The effect of different concentrations of hormones and control
42
(without hormone treatments) on shoot induction using
7 days old cotyledon explants after three weeks in culture.
2.6
Shoot induction from proximal section of 7 days old
43
seedling cotyledons on MS media supplemented with and
without hormones after three weeks in culture.
2.7
The effect of different concentrations of auxin hormones on
46
root induction after two weeks in culture.
2.8
Comparison of root morphology from different auxins
47
treatment in half strength MS media after two weeks in culture.
2.9
In vitro plant regeneration of I. balsamina using
49
7 days old seedling cotyledons within eight weeks in culture.
3.1
The effect of target distance and helium pressure on
74
GUS gene expression after pre-culture of explants in bombardment
media (0.4 M sorbitol and mannitol) 24 h prior bombardment using
1.0µg DNA, 9 cm target distance and 1100 psi helium pressure.
3.2
The effect of number of bombardment (one and two times) on
75
explants after a week of bombardment.
3.3
The effect of number of bombardments on GUS spots per
explants after 24 h post-bombarment using 9 cm target distance
xv
76
and 1100 psi helium pressure.
3.4
The effect of pre-culture time of explants with osmotic
78
treatment of 0.4 mannitol and sorbitol on GUS gene
expression after bombarding explant at 9 cm target distance
and 1100 psi helium pressure.
3.5
The effect of DNA concentrations (µg) on GUS spots per
80
explants 24 h post-bombardment using 9 cm target
distance and 1100 psi helium pressure.
3.6
The effect of pre-culture treatments with different types and
82
concentrations of osmotic treatment on GUS gene expression
after 24 h post-bombardment using 9 cm target distance and
1100 psi helium pressure.
3.7
The effect of post-bombardment incubation time on
84
GUS expression after bombardment using
9 cm target distance and 1100 psi helium pressure.
3.8
The effect of bombardments using optimal biolistic conditions
(target distance 9 cm, 1100 psi helium pressure,one time
bombardment, 1.0 µg DNA, pre-culture with combination of
mannitol and sorbitol (0.4M) in 16 h pre-culture time,
post-bombardment incubation time 24 h before GUS) assay on
GUS gene expression of explants
xvi
87
3.9
The effect of different concentrations of hygromycin on
90
explants after five weeks in culture.
3.10
The effect of hygromycin on plantlets regeneration and
94
GUS assay of I. balsamina.
3.11
Agarose gel analysis of polymerase chain reaction (PCR)
98
product of transformed I. balsamina.
3.12
The effect of Basta on I. balsamina after 48 h of exposure
99
by spraying.
3.13
The effect of phosphinothricin (PPT) in different concentrations
on shoots for minimal lethal dose.
xvii
102
ABBREVIATIONS
BAP
-
Benzylaminopurine
PPT
-
Phosphinothricin
C
-
Celcius
psi
-
Pounds per square inch
cm
-
Centimeter
RNA
-
Ribonucleic acid
DNA
-
Deoxyribonucleic acid
SE
-
Standard error of mean
GUS
-
ß-glucuronidase
TDZ
-
Thidiazuron
h
-
Hour
µg
-
Microgram
HCl
-
Hydrochloric acid
µl
-
Microliter
IAA
-
Indole acetic acid
µm
-
Micrometer
IBA
-
Indole butyric acid
ß
-
Beta
L
-
Liter
V
-
Volt
M
-
Molar
%
-
Percentage
mg
-
Milligram
mM
-
Millimolar
ml
-
Milliliter
MS
-
Murashige and Skoog (1962)
NAA
-
α-naphthylene acetic acid
NaOH
-
Natrium hydroxide
nm
-
Nanometer
pH
-
Per hydrogen
xviii
LIST OF APPENDICES
APPENDIX NO
A
TITLE
PAGE
Murashige and Skoog (1962) media containing the
136
macro element, micro element, iron source and vitamins.
B
Plant growth regulators and the solvents used for
137
stock preparation.
C
Plant growth regulators and the chemical names
138
D
Luria- Bertani (LB) media.
138
E
The bacterial growth with different plasmid
139
in LB media with ampicilin.
F
Plasmid pRQ6 (8.25kb) with uidA ß-glucuronidase (GUS)
140
gene and hph gene encoding for hygromycin resistant.
G
Plasmid pAHG11 (7.3kb) with bar gene confers to
Basta (PPT) resistant.
xix
141