TISSUE CULTURE AND BIOLISTIC –MEDIATED TRANSFORMATION OF
IMPATIENS BALSAMINA
AISHAH MOHD TAHA
UNIVERSITI TEKNOLOGI MALAYSIA
ACKNOWLEDGEMENTS
First of all I would like to express my sincere gratitude and appreciation to my
supervisors, Dr Fahrul Huyop, Pn Alina Wagiran, Cik Zaidah Rahmat and Pn. Hamidah
Ghazali (MARDI) for their invaluable guidance throughout the course of this project.
To all the lab members, Pn Fatimah and Kak Radiah for technical assistance
during my time in the lab.
Finally I thank my parents for continual encouragement and assistance, without
which none of this would have been possible.
iii
ABSTRACT
Biolistic- mediated transformation is an approach to transfer a gene of interest into a
plant cell using particle bombardment method. Tissue culture experiment was
conducted to optimize shoot regeneration from cotyledon explant of 7 day-old seedlings
I. balsamina. The maximum average number of shoots (6.8 ± 1.05 shoots) per explant
was obtained on full strength MS-based medium containing 1 mg/L BAP after three
weeks of culture. For rooting induction, there was no significant difference on
percentage of root regeneration on full strength (84%-94%) and half strength MS–based
medium (78%-96%) supplemented with IAA (0.1 - 1mg/L), IBA (0.5 - 2 mg/L) and
NAA (0.1 - 1 mg/L) after two weeks of culture. Plantlets were successfully produced on
full strength MS plates supplemented with 1 mg/L BAP (93%) for shooting and half
strength MS media supplemented with 0.1 mg/L IAA (92%) for rooting in eight-week
culture. Biolistic technique was used for the transformation of uidA and hph genes into I.
balsamina. The 7 day- old cotyledons explants were bombarded with pRQ6 containing
uidA gene encode for ß-glucuronidase (GUS) and hph gene conferring resistance to
hygromycin and co-transformation was carried out using pRQ6 with pAHG11 (contain
bar gene conferring resistance to herbicide Basta). The physical and biological factors
of bombardment such as target distance (6 - 12 cm), helium pressure (650 - 1100 psi),
number of bombardments (once and twice), DNA concentrations (0.5 - 1.5µg), preculture times (4 - 32h), osmotic treatments using mannitol , sorbitol and combination of
mannitol and sorbitol at 0.2 M, 0.4 M and 0.6 M and post-bombardment incubation
times (4 - 48 h) were optimized. Transformation of I. balsamina with pRQ6 and cotransformed with pRQ6 and pAHG11 at 28 mm Hg vacuum using optimized
bombardment conditions (9 cm target distance, 1100 psi helium pressure, one time
bombardment, 1.0 µg DNA, 16 h pre-culture time with osmotic treatment of 0.4 M
mannitol and sorbitol and 24 h post-bombardment incubation time) showed the highest
average number of GUS spots of 149.3 and 128.1, respectively. Delay selection method
was used to delay the timing of selection on shooted explants after bombardment to
obtain transformed plants. Out of 160 bombarded explants with pRQ6, only 84 plants
were successfully regenerated from selected 35-day old shooted explants after five
weeks grown on MS –based medium containing 75 mg/L hygromycin. All regenerated
plants (84 plants) were GUS positive. However, there was no plant regenerated on MSbased medium containing 1 mg/L phosphinothricin (PPT) after co-transformed with
pRQ6 and pAHG11. Furthermore, PCR results showed only 14 of 40 GUS positive
plants survived at 75 mg/L hygromycin were hph gene positive. In conclusion, the PCR
results also showed the possibility integration of hph gene into the genomic DNA of
transformed plants.
iv
ABSTRAK
Transformasi melalui biolistik adalah satu cara untuk pemindahan gen yang dikehendaki
ke dalam sel tumbuhan menggunakan kaedah penembakan partikel. Eksperimen kultur
tisu dilakukan untuk mengoptimumkan penumbuhan pucuk daripada kotiledon eksplan I.
balsamina selepas 7 hari. Maksimum purata pertumbuhan pucuk yang tertinggi (6.8 ±
1.05 pucuk) per eksplan didapati dalam kepekatan media MS penuh yang mengandungi
1 mg/L BAP selepas tiga minggu dikultur. Untuk pertumbuhan akar, tiada perbezaan
yang signifikan pada peratusan pertumbuhan akar yang dihasilkan menggunakan
kepekatan MS yang penuh (84%-94%) dan setengah kepekatan MS (78%-96%) dengan
menggunakan IAA (0.1 - 1 mg/L), IBA (0.5- 2 mg/L) dan NAA (0.1 - 1 mg/L) selepas
dua minggu dikultur. Anak pokok yang berjaya dihasilkan dalam kepekatan media MS
penuh dengan 1 mg/L BAP (93%) untuk pertumbuhan pucuk dan setengah kepekatan
MS dengan 0.1 mg/L IAA (92%) untuk pertumbuhan akar dikultur selama lapan minggu.
Teknik biolistik digunakan untuk transformasi gen uidA dan hph ke dalam I. balsamina.
Kotiledon eksplan yang berumur 7 hari ditembak dengan plasmid pRQ6 yang
mempunyai gen uidA untuk ß-glucuronidase (GUS) dan gen hph yang rintang kepada
hygromycin dan ko-transformasi dengan plasmid pRQ6 dan pAHG11 (mempunyai gen
bar rintang ke atas herbisid Basta). Faktor fizikal dan biologikal seperti jarak sasaran (6
- 12 cm), tekanan helium (650 - 1100 psi), bilangan penembakan (sekali dan dua kali),
kepekatan DNA (0.5 - 1.5 µg), masa kultur (4 - 32 jam), rawatan osmotik menggunakan
mannitol, sorbitol dan kombinasi mannitol dan sorbitol pada kepekatan 0.2 M, 0.4 M
dan 0.6 M dan masa pengeraman eksplan selepas penembakan (4 - 48 jam) telah
dioptimumkan. Transformasi I. balsamina dengan pRQ6 dan ko-transformasi dengan
pRQ6 dan pAHG11 pada 28 mm Hg vakum menggunakan penembakan yang optimal (9
sentimeter jarak sasaran, 1100 psi tekanan helium, 1.0 µg DNA, 16 jam masa kultur
dengan rawatan osmotik 0.4 M mannitol dan sorbitol dan 24 jam masa pengeraman
eksplan selepas penembakan) menunjukkan purata bilangan bintik GUS yang tertinggi
dengan 149.3 dan 128.1, masing-masing. Kaedah penangguhan pemilihan digunakan
untuk menangguhkan masa pemilihan pada eksplan yang mempunyai pucuk selepas
penembakan bagi mendapatkan tumbuhan yang telah ditransformasikan. Daripada 160
eksplan yang ditembak dengan pRQ6, hanya 84 tumbuhan berjaya ditumbuhkan
daripada penangguhan pemilihan 35 hari eksplan yang mempunyai pucuk selepas 5
minggu tumbuh dalam media MS mengandungi 75 mg/L hygromycin. Kesemua 84
tumbuhan adalah positif GUS. Walaubagaimanapun, tiada penumbuhan dalam media
MS yang mengandungi 1 mg/L phosphinothricin (PPT) selepas ko-transformasi dengan
pRQ6 dan pAHG11. Keputusan PCR menunjukkan hanya 14 dari 40 tumbuhan yang
ditumbuhkan dalam 75 mg/L hygromycin adalah positif gen hph. Kesimpulannya,
keputusan PCR juga menunjukkan kemungkinan integrasi gen hph ke dalam genom
DNA tumbuhan yang ditransformasi.
v
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
CHAPTER 1
General Introduction
1.1
Impatiens balsamina background
I. balsamina is a plant from Asia, North America and South Africa or known as
‘Keembung’, Garden balsam or Touch Me Not. The name of this plant refers to the
elasticity of the valves of the seedpods, which discharge the seeds when ripe. These
plants have thick stems and light green leaves with wide range of colors including rose
red, rose purple, white and pink (Figure 1.1). The plant is an annual and easily grown in
evenly moist, an organically rich, well drained soil in full sun to part shades.
The photoperiodic studies on growth and development of I. balsamina have been
reported by Nanda and Krishnamoorthy (1967). It was found that while floral bud of
this species are initiated with three short day cycles, at least eight such cycles are
required for flowering.
The number of floral bud and open flowers bear a linear
relationship with number of short day cycles.
1
The induced floral buds revert to
vegetative growth unless the plants receive a minimum number of short day cycles
needed for flowering and this reversion occurring in a basipetal direction.
This plant also has properties and action according to indigenous medical
system. For example, the flowers as cooling agent for external application to burns and
scalds and posses marked antibiotic activity against some pathogenic fungi Aspergillus
flavus and Candida albicans (Lee et al., 1999).
Lawsone-forming multi enzyme
complex from I. balsamina root cultures contain lawsone, an antimicrobial
naphthoquinone. It was found that the root cultures of I. balsamina could produce a
number of natural products, mostly belonging to the chemical groups of coumarins and
napthoquinones including lawsone and Me-lawsone (Figure 1.2) (Eknamkul, 1999).
Figure 1.1 I. balsamina with white and purple flower colors. Bar= 0.5m
2
Figure 1.2 Lawsone and Me-lawsone chemical structures, found in roots cultures of I.
balsamina (Eknamkul, 1999).
3
1.2
The importance study of I. balsamina
The inflorescence study in flower development using I. balsamina was reported
by Pouteou et al., (1998). This can be explained by the response of axillary meristem
primordial to the quantity of inductive signal, a response that is conditioned by the age
or position of the primordia and allows undifferentiation of axillary primordial initiated
before evocation to adopt different fates.
The interpretation of this study is that
vegetative and reproductive phase are not separate and antagonistic but interpenetrate
each other to varying extends depending on the quantity of inductive signal.
The research on role of the leaves in flower development using I. balsamina has
been reported by Tooke and Battey (2000). In this research, the leaf derived signal was
found to have a role in floral maintenance. The increased petal number was neither a
response to a stress affects associates with leaves removal nor a result of alternation of
petals for stamen. Rather, the petal initiation phase was prolonged when the amounts of
a leaf derived signal were limiting. Therefore, leaf derived signal has a continuous and
quantitative role in flower development. This may provide an explanation for the wide
variety and instabilities of floral form seen among certain species in nature.
The research on Impatiens was also carried out for Impatiens Necrotic Spot
Virus. Over the past decades, the virus began to develop in floriculture industry and
becoming one of the most important viral pathogens of floral crops. More than 300
species from 50 plants families were susceptible to Impatiens Necrotic Spot Virus
including Impatiens. Symptoms of virus including ring spots, brown to purple on the
4
leaves and stem and flower breaking of plants (Windham et al., 1998). To date, there
are few reports of genetically engineered disease resistance in floral crops including the
transformation using nucleocapsid gene of Tomato Spotted Wilt Topovirus (TSWT) into
chrysanthemum plants (Daughtery et al., 1997).
Baxter (2005) also reported on
development of Impatiens Necrotic Spot Virus resistant using the nucleocapsid gene to I.
wallenaria via Agrobacterium-mediated transformation.
1.3
Plant regeneration system
Plant regeneration system could be achieved via tissue culture. Tissue culture is
the culture and maintenance of the plant cell or organs in sterile and environmentally
supportive condition in vitro. It also refers as micropropagation, which is a set of
techniques for the production of whole plants from cell cultures, derived from explants
of meristem cells. Plant cells do need to multiply in vitro in certain condition and the
most important is the freedom of the cells from competition of other organ. Therefore, it
is necessary to remove the contaminants from the culture in aseptic conditions for the
plant cells to multiply and produce the whole plant. This is usually conducted by
surface sterilization of the explants with chemicals such as bleach and mercury chloride
at the certain concentration and duration that killed or removed the pathogens without
injuring the plant cells. Sterilizations of medium and apparatus used and maintenance
the sterile condition while culturing the explants also need to be done to avoid the
pathogens (Lineberger, 2003).
5
The successful of plant cell cultures is depended on the type of media
(Murashige and Skoog, (1962); Gamborg B5) containing macro element, micro element,
iron and vitamins supplemented with appropriate hormones for the growth.
By
providing the necessary chemicals in appropriate combinations and forms, it has been
possible to establish cultures from virtually every plant part, of plants ranging from
mosses to monocotyledonous angiosperms and to pursue diverse academic and
economic aims. The ingredients of plant culture media can be categorized as inorganic
salts, organic compounds, complex natural preparations and inert supportive materials
(Huang and Murashige, 1976).
Tissue culture has wide applications in research as it offers numerous benefits
over the traditional propagation methods.
For example, a single explant can be
multiplied into several thousand plants in less than one year. Once established, actively
dividing cultures are a continuous source of microcuttings, which can result in plant
production under greenhouse conditions without seasonal interruption.
The rate of
growth is much faster due to the addition of exogenous hormones in vitro compared with
traditional methods. The environment in culture provides nutrient and sterile conditions
to avoid from other organism and the competitor of nutrient uptake (Lineberger, 2003;
Slater et al., 2003).
Example of plant regeneration of Impatiens reported were I. wallenaria (Baxter,
2005), I. platypetala (Kyungkul, 1993), Impatiens L. interspesific hybrids T63-1, a Java
(J) x New Guinea (NG) (Kyungkul and Stephens, 1987) and Java, New Java and Java x
6
New Guinea (Stephens, 1985). Till now, no plant regeneration of I. balsamina is
reported.
1.4
Transformation system
Transformation is the introduction of foreign DNA into the plant cell by means
of Agrobacterium tumefaciens and biolistic method. According to Birch (1997), the first
plant transformation was reported in 1984 and has been extended to over 120 species in
about 35 families.
Plant transformation research has been widely provided as the
expectations of this approach in improving the quality, to obtain the high yield and
varieties resistant of plants (rice, orchid, soybean and pepper) against herbicides and
diseases.
The plant transformation remains as an alternative used to enhance the
productivity and quality of plants. This improvement could lead for the future prospect,
as the novel genes can be introduced for the useful production ranging in industrial,
economical and agricultural product.
The conventional breeding limited the high yield of plants, as it is a time
consuming process to develop a variety of desired plants. The major limitation of
conventional methods derive from the limitations of the sexual process itself and include
constrains on the amount of genetic variation available within the crop (the gene pool)
and the fact that all traits differing between the parents are subject to segregation and
thus of selection are required to identify rare individuals that combine the best qualities
of both parents (Manshardt, 2004).
7
The most widely successful systems used for plant transformation are biolistic and
A. tumefaciens mediated transformation system. Biolistic is one of the methods that
used high velocity microprojectiles to penetrate into the cells. This method depends on
the apparatus optimization to enhance the transformation. In contrast, Agrobacterium
technique uses the biological approach of plant pathogenic bacteria that moves the DNA
from its plasmids into the plant cells as part of its life cycle (Slater et al., 2003).
1.4.1
Agrobacterium tumefaciens transformation
The A. tumefaciens, the causative agent of crown gall disease is a Gram negative
bacterium, rod in shape, found in rhizhophere and survives on plant nutrient. This
bacterium can infect the wound site of wounded plants and cause tumors.
A.
tumefaciens attracted to the wound site via chemotaxis shows the reaction to the
phenolic compounds released from the damage cell.
The Ti plasmid region of A.
tumefaciens containing the T-DNA is defined as the presence of right (TR-DNA) and
left borders (TL-DNA). Any DNA inserted between these borders will be transferred
into the genome of the plant host and this system has been used to introduce the desired
gene into plant during transformation. However, only the TL-DNA is oncogenic and
contains eight open reading frames. The organization of the genes in the TL- DNA is
shown in Figure 1.3 (Slater et al., 2003).
The TR-DNA can be transferred and
maintained in the absence of TL-DNA. Therefore, TR-DNA is considered as a naturally
occurring T-DNA vector which might be an alternative to manipulated TL-DNAs from
which all onc genes have been deleted (Leemans et al., 1983).
8
Although the used of A. tumefaciens has been widely reported in transformation
of dicotyledonous, there were reports of the use of biolistic as an alternative method to
enhance the expression and incorporation of genes into the plant cell in dicotyledonous
species. For example, the first report describing recovery of an intact transgenic plant
soybean (Glycine max) using biolistic was published (Christou, 1994). However, there
was no report on transformation work of I. balsamina using A. tumefaciens and biolistic
methods.
LB
aux
5
7
2
cyt
1
4
tm1
6a, b
ocs
RB
3
Border repeat (LB=left border, RB=right border).
Figure 1.3 The genetic organization of the TL-DNA of an octopine – type Ti plasmid
with eight open reading frames (ORFs) (1-7) (Slater et al., 2003).
9
1.4.2
Biolistic- mediated transformation
Biolistic is one of the methods used for genetic transformation. Many
transgenic plants have been produced via this approach. The first generation of this
technique for gene transfer into plant cells was developed in 1960s by plant virologist
(Birch and Bower, 1994). Transgenic plants generated by this method have been
reported for monocotyledonous such as Kentucky bluegrass (Gao et al., 2005), jute
(Ghosh et al., 2002), rice (Jain et al., 1996; Li et al., 1993) and dicotyledonous species
including mothbean (Kamble et al., 2003), cowpea (Ikea et al., 2003), sunflower
(Molinier et al., 2002) and soybean (Moore et al., 1994).
This method involves the use of a high velocity of particles to penetrate the
cells and introduce the DNA into the cells. The introduced DNA will be expressed in
the plant cell if it is into plant chromosomal DNA (Wong, 1994). The particles that
are commonly used are gold and tungsten. For biolistic transformation, DNA is coated
onto the surface of gold or tungsten by precipitation with calcium chloride and
spermidine. Generally with the high velocity, the DNA was delivered into the cells
and once inside the cells, the DNA will elute off. If the foreign DNA reaches the
nucleus, then transient expression will likely result and the transgene may be stably
incorporated into host chromosomes (Kikkert et al., 2003; Birch and Bower, 1997).
One of the widely used device for biolistic transformation is PDS-1000/He
Particle Delivery System (Figure 1.4). This instrument used the high pressure helium
10
which carries the most power for given pressure of compressed gas without danger of
explosion associated with hydrogen compared to other gases, with the less shock wave
(Wong, 1994).
One of its components is the rupture disc. The rupture disc determines the
acceleration of shock wave to deliver the particle and available in different thicknesses
ranging from 450 psi to 2200 psi. The rupture disc sealed the gas tube and released the
shock wave when the valve is opened as the helium gas flows into the gas tube and
pressuring the cavity. The energy of shock wave will accelerate the particles to the
target cell. The distance of the target distance can be adjusted at different positions (3
cm, 6 cm, 9 cm and 12 cm) from the stopping screen. This target distance determines
the spread of the particles to the target cell. The macrocarrier disk is held by the metal
ring and the particles coated with the DNA were spread on the surface of macrocarrier.
The stopping screen and macrocarrier sheet are subjected to the violent forces during
operation. The operation is held under vacuum to help the blast of helium shock wave
(Wong, 1994).
The physical parameters depend on the apparatus settings. Generally, the wide
ranges of settings have been tested (target distance, helium pressure and number of
bombardments) and show the optimal for most of plants including banana
(Sreeramanan et al., 2005), wheat (Ingram et al., 1999) and rice (Jain et al., 1996).
The use of gold particle in range 0.7 µm to 1.0 µm, vacuum of 28 Hg, helium pressure
11
Figure 1.4 PDS-1000/He Particle Delivery System, a device used in biolistic
transformation (BioRad, USA).
12
of 1100 psi and gap distance of 6 cm to 10 cm result in high transformation rates
(Kikkert et al., 2003). For example, biolistic-mediated transformation on callus of rice
resulted on transformation frequency with 37.5% (Ramesh and Gupta, 2005) and 8.9%
(Lee et al., 2003).
The necessity to optimize the velocity using the target distance and helium
pressure is for optimal transformation rates with different tissues types, depending on the
cell wall thickness and the need to penetrate several layers (Birch and Bower, 1994).
For example, target distance of 9 cm and 1100 psi helium pressure in single bud of
banana (Sreemanan et al., 2005), immature embryos of sorghum (Tadesse et al., 2003),
embryos of cowpea (Ikea et al., 2003) and in embryogenic cells of rice (Jain et al., 1996)
was the optimal condition in biolistic-mediated transformation.
Multiple bombardments are carried out with the objective of getting better
coverage of targeted areas in banana tissues (Sreeramanan et al., 2005). This is in
consensus with Ikea et al., (2003) who reported that bombarding the embryos of cowpea
twice increased the number of GUS gene expression with 70% of shoots embryos
showed more than 20 blue spots per embryos hit and this could be due to the fact that
multiple bombardments allow better coverage of the target areas and compensate for
misfires from faulty and poorly set rupture discs.
The biological factor includes the physiological condition of cell such as cell
turgor pressure. The osmotic treatment with stabilizer sugar such as mannitol and
13
sorbitol can increased the transformation rates. The mechanism of osmotic
enhancements could include both reduced vacuole volume and reduced turgor, which
would alter penetrability by particles (Birch and Bower, 1997). It has been proposed
that osmotic treatment could prevent the damages of cell membrane and loss of
cytoplasm. In addition, the reduction of the volume of the vacuoles increased the
possibility to reach the nucleus, resulting in larger number of cells successfully
expressing the introduced gene (Santos et al., 2002). Transformation efficiency is also
affected by subjecting the bombarded tissue at the right stage to the GUS assay for the
recovery of the cells from injuries caused by bombardment (Sreeramanan et al., 2005).
Other biological parameter includes the DNA concentration, tissue type, cell
culture age and mitotic age (Wong, 1994). However, increasing the concentrations of
DNA precipitated will also increase transient expression frequencies until particle
aggregation occurs, resulting in poor dispersal and increased cell damage (Birch and
Bower, 1994). On the other hand, the exogenous DNA used in the transformation
experiments typically comprises a plant expression cassette inserted in a vector based
on the high copy number bacterial cloning plasmid. Neither of these components is
required for DNA transfer and only the expression cassette is required for transgene
expression. The expression cassette typically compromises a promoter, open reading
frame and polyadenylation site that are functional in plant cells. Once this plasmid has
been isolated from the bacterial culture, it is purified and used directly as a substrate
for transformation (Altpeter et al., 2005).
14
Therefore, the application of particle bombardment has been wide in transient
gene expression studies. The transient expression study which initiates the genetically
transformed plants enable for the study of mutations, promoter, and the effect of gene
expressions (Taylor et al., 2002). In contrast, major disadvantage of biolistic is the
tendency for complex integration patterns and multiple copy insertion could cause
gene silencing (Kikkert et al., 2003).
The application of biolistic has demonstrated transient gene expression in plant
studies, production of genetically transformed plant and tissue and inoculation with
viral pathogens (Taylor et al., 2002). The universality of application through cell
types, size, shape, presence or absence of cell wall, and direct introduction of
biological material into the cell have very high delivery efficiency to enhance the
transformation rates (Sanford et al., 1987).
1.5
Marker genes
1.5.1
Selectable marker
The used of selectable marker genes is to identify those cells that successfully
integrate and express the transferred DNA. Genes conferring resistance to various
antibiotics or herbicides such as hygromycin and phosphinothricin (PPT) respectively
are commonly used in transformation research. The genes (hph gene and bar gene)
15
encode proteins that detoxify corresponding selection agents and allow the growth of
transformed cells (Goodwin et al., 2003; Slater et al., 2003).
1.5.1.1 Antibiotic
Hygromycin B is an aminocyclitol antibiotic with broad spectrum activity
against prokaryotic and eukaryotic cells by interfering with protein synthesis (Waldron
et al., 1985; Rao et al., 1983). Hygromycin B appears to interfere with amino acyl tRNA recognition and cause misreading in cell free polypeptide synthesizing system that
could prevent the synthesis of protein needed for the growth of cells (Rao et al., 1983).
The resistance can be conferred by hph gene encoding hygromycin
phosphotransferase, which was originally found in the strain of E.coli (Waldron et al.,
1985).
Resistance
to
hygromycin
B
is
determined
by
an
aminocyclitol
phosphotransferase that modifies hygromycin B. The specific modification of
hygromycin B is a phosphorylation of the hydroxyl on the 4 position of the cyclitol ring
(hyosamine) (Rao et al., 1983) as showed in Figure 1.5.
The hygromycin B resistant plants have been developed for both monocots and
dicots such as rice with 8.9% transformation frequency (Lee et al., 2003), Kentucky
bluegrass with 77.8% transformation frequency (Gao et al., 2005) and orchids with 12%
transformation frequency (Men et al., 2003).
16
Figure 1.5 Structure of hygromycin. The arrow indicates the 4-hyroxyl group which is
the site of phosphorylation for inactivation of hygromycin (Rao et al., 1983).
1.5.1.2 Herbicide
Weeds are one of the major problems encountered in crop management because
of competition with crops for water, nutrients and as result decrease farming yields and
productivity. Herbicide resistant crops are planted and non- selective (broad spectrum)
herbicides are used for weed management. Provided that the field crops are genetically
modified the carry gene for herbicide resistance, the broad spectrum herbicides
(glufosinate and glyphosate) will not harm the resistant crops (corn, soybean, sugar beet,
17
carnation, flax, tobacco, rice and wheat). The other herbicides used are bromoxynil,
sulfonamides and sulfonylurea (Mayer et al., 2004).
Phosphinothricin (PPT) is also known as glufosinate, one of the active
ingredients of herbicide Basta. It belongs to the family of aminoalkylphosphonic acids,
structural analogues of amino acids in which the carboxyclic group is replaced by a
phosphonic. This group resembles the tetrahedral transition state of several enzymatic
reactions, particularly amide bond formation and hydrolysis. Several enzymes unable to
discriminate between carboxylic and phosphonic function for binding to active sites,
therefore results in inhibition of enzyme activity (Guiseppe et al., 2006).
Phosphinothricin (PPT) acts by inhibiting the glutamine synthetase, a key
enzyme in nitrogen metabolism in bacteria and plant cells (Berlicki et al., 2006;
Wolfgang et al., 1994). This rapidly leads to ammonium accumulation and lack of
glutamine. Glutamine synthetase catalyzes a reaction of central importance in plant
metabolism, the conversion of glutamate to glutamine (Eugene et al., 1991).
The
enzymes catalyze the conversion of glutamate to glutamine in the presence of
ammonium ion with accompanied hydrolysis of adenosine triphosphate (ATP) as an
energy source. Inhibition of this enzyme in plants causes total impairment of nitrogen
metabolism resulting in accumulation of toxic amount of ammonium, which followed by
plant death (Giuseppe et al., 2006).
18
Being nonselective herbicides, the exposure of phosphinothricin (PPT) to the
plant may also block the recycle of carbon from the photorespiratory pathway to the
Calvin cycle (Eugene et al., 1991). The genetic engineering of phosphinothricin (PPT)
resistant plants allows the enlargement of the application of the herbicide in transgenic
plant (Wolfgang et al., 1994). Figure 1.6 shows structure of phosphinothricin (PPT).
The bialaphos (bar) resistance gene of Stereptomyces hygroscopicus that encodes
for phosphinotricin acetyltransferase, which confers resistant against Basta has been
widely used in transformation research (Ghosh et al., 2002). The bar gene converts
phosphinothricin (PPT) into nontoxic acetylated form and allows growth of transformed
plant even in the presence of phosphinothricin (PPT) (Goodwin et al., 2003).
Figure 1.6 The chemical structure of phosphinothricin (PPT) (Eugene et al., 1991).
19
1.5.2
Reporter genes
The most convenient measure of efficiency for DNA delivery into intact cells is
the number of cells, which transiently express incoming reporter gene. Reporter genes
or reporter proteins have played an important role in developing and optimizing
transformation protocols for plant species (Sreeramanan et al., 2005). Reporter genes
should be easy to assay, preferably with a non-destructive assay system and there should
be little or no endogenous activity in plant to be transformed (Slater et al., 2003).
Reporter genes codes for enzymes or protein can be detected using the
biochemical assay. The efficient reporter gene allows the detection of transgenic events
after bombardment in either a transient or stable expression. The most commonly used
reporter gene is uidA gene encoding the ß-glucuronidase (GUS). Other reporter genes
are green fluorescent protein (gfp), chloramphenicol transferase (CAT), nopaline syntase
(NOS), octopine syntase (OCS) and luciferase (Datta et al., 1997).
The GUS gene, originally isolated from E.coli has been widely used in transient
expression study and plant transformation as the stability of the enzymes and the high
sensitivity of the GUS allowed qualitative and quantitative assessment of transgene
expression levels (Cervera, 2003; Taylor et al., 2002; Jefferson et al., 1987).
Various ß–glucuronide acid substrates are available for GUS expression, which
contained the D-glucopyranosiduronic acid attached glycosidic linkage to a hydroxyl
20
group of detectable molecule.
The preferred substrate for detection is 5-bromo-4-
chloro-3-indoxyl-ß-D-glucuronide (X-Gluc) (Datta et al., 1997). After bombardment,
the explants were incubated with GUS histochemical substrate, 5-bromo-4-chloro-3indoxyl-ß-D-glucuronide (X-Gluc) (Kikkert et al., 2003) (Figure 1.7). Those cells that
express GUS activity stained blue and distribution of cells received and expressed the
bombarding DNA is easily visualized (Jefferson et al., 1987). However, disadvantage of
using GUS is the blue stain is toxic and the assay therefore resulting in destruction of
GUS expressing cells (Birch and Bower, 1994).
Figure 1.7 Structure of X-Gluc (5- bromo 4-chloro-3-indoxyl-ß-D-glucuronide, a
substrate for GUS which is encoded by uidA, a widely used reporter gene (BioWorld,
USA).
21
1.6
Transient gene expression and stable gene expression
Generally, there are two types of gene expression in transformation. The
transient gene expression is temporary, occurs almost immediately after gene transfer
with the higher frequency than stable integration and does not require the regeneration of
whole plants. Therefore, transient expression is a rapid and useful method for analyzing
the function of gene of interest (Altpeter et al., 2006; Lincoln et al., 1998) and the
transient expression frequency provides the most convenient measure of the frequency
of introduction of DNA into explants during the optimization of bombardment
conditions (Birch and Bower, 1994). In contrast, although the stable expression occurs
with the lower frequency, the expression was maintained for long term as the DNA
incorporate into the chromosome of the recipient cell (Datta et al., 1997; Mitrovic, 2003).
Introduction and expression of an exogenous gene into cells does not always
involve stable integration of the gene into the genome of the recipient cell. Frequently,
plasmid DNA may be introduced into host cells and may express in a transient fashion.
This activity declines over time and eventually disappears. Even though measuring
levels of such transient activity may be useful in specific cases, when it comes to the
creation of stable transgenic phenotypes, only transformation events leading to
integration of the foreign gene into the genome of the host cell are useful (Christou,
1994).
22
1.7
Promoters
According to Odell et al., (1985), the cauliflower mosaic virus (CaMV) promoter
35S is a major promoter of cauliflower mosaic virus that infects member of Cruciferae.
The promoter is well characterized and stronger than other plant promoter expression in
dicots, but confers low level of expression in monocots. The rice actin promoter and
maize ubiquitin promoter achieved far better expression compared to 35S promoter in
most monocots tested. The cauliflower mosaic virus (CaMV) fragments containing 400
to 1000 base pairs of 35S upstream sequences have been shown to be active when
integrated into nuclear genome of transgenic tobacco. The upstream fragment -343 to 46 is responsible for the majority of the 35S promoter strength. The upstream fragments
can be subdivided into three functional regions, -343 to -208, -208 to -90, and -90 to -46.
The first two regions can potentiate the transcriptional activity and the third region itself
increasing the transcriptional activity of the first two regions (Figure 1.8) (Fang et al.,
1989).
The specific sequence (a -60 nucleotide region (S1)) downstream of the
transcription initiation site of the cauliflower mosaic virus (CaMV) 35S can enhance
gene expression. By using transient expression assays with plant protoplast, this activity
was shown to be at least partially due to the effect of transcriptional enhancers within
this region.
The sequence motif with enhancers function is identified, which are
normally masked by the powerful upstream enhancers of the 35S promoter. A repeated
CT-rich motif is involved both in enhancer function and in interaction with plant nuclear
proteins (Pauli et al., 2004).
23
-343
5’ TGAGACTTTT CAACAAAGGG TAATATCCGG AAACCTCCTC GGATTCCATT GCCCAGCTAT
CTGTCACTTT ATTGTGAAGA TAGTGGAAAA GGAAGGTGGC TCCTACAAAT GCCATCATTG
-208
CGATAAAGGA AAGGCCATCG TTGAAGATGC CTCTGCCGAC AGTGGTCCCA AAGATGGACC
CCCACCCCAC GAGGAGCATC GTGGAAAAAG AAGACGTTCC AACCACGTCT TCAAAGCAAG
-90
-46
TGGATTGATG TGATATCTCC ACTGACGTAA GGGATGACCC ACAATCCCAC TATCCTTCGC
AAGACCCTTC CTCTATATAA GGAAGTTCAT TTCATTTGGA GAGGACACGC TG 3’
Figure 1.8 Nucleotide sequence of the Cauliflower Mosaic Virus 35S promoter and upstream regions (Fang et al., 1989).
24
1.8
Objectives of study
The objectives of this study were:
1.
To establish the regeneration system of I. balsamina with high regenerating
plants using plant growth regulators for further study on biolistic
transformation.
2.
To establish the biolistic transformation technique by optimizing of
bombardment conditions (physical and biological parameters), selection
system and analysis of transformed I. balsamina.
25
CHAPTER 2
Tissue culture system for Impatiens balsamina
2.1
Introduction
A highly efficient and reproducible in vitro regeneration system is an absolute
prerequisite for producing transgenic plants. Particularly in cereals, stably transformed
plants from all major species have been generated. In routine, application of molecular
improvement independent of the chosen method of transformation is still hampered by
the lack of readily available, highly efficient and long term regenerable cell and tissue
culture systems (Sharma et al., 2004). The ability of genetically engineered floral crops
depends first on development of protocols that allow for high frequency shoot
regeneration then needs to be coupled with an effective gene transfer commonly which
use Agrobacterium vectors or biolistic transformation (Daughtery et al., 1997).
26
Plant regeneration system of Impatiens species from multiple shoots was
achieved using different types of explants for example, cotyledons of immature ovules
of I. platypetala Lindl (Kyungkul, 1993) and shoot tips from both of Impatiens hybrids
(Kyungkul and Stephens, 1987) and Java x New Guinea Impatiens (Stephens et al.,
1985). Multiple shoots were also successfully regenerated from cotyledon of explants
from other species such as from Pinus (Sul and Korban, 2004), barley (Sharma et al.,
2004), squash (Ananthakrishnan et al., 2003), Bambara groundnut (Lacroix et al., 2003),
Terminalia chebula (Shyamkumar et al., 2003) and cotton (Agrawal et al., 1997).
Cotyledons usually utilize the meristematic cells and were chosen to establish a plant
regeneration system. Based on previous reports, cotyledons proved to be responsive
explants for many species (Kyungkul, 1993).
Plants growths regulators are the media components produced in small
concentration and have an effect on growth and development of plant organs. It is used
commonly as plant hormones or the synthetic hormones. The use of auxin in high ratio
generally induces the roots whereas high cytokinin will induce the shoots and the
intermediate ratio favors on callus formation. There are five classes of plant growth
regulator including auxins, cytokinins, gibberellins, abscisic acid, and ethylene.
Generally, auxins (indole acetic acid (IAA), α-naphthalene acetic acid (NAA) and
indole butyric acid (IBA)) and cytokinins (benzylaminopurine (BAP) and Kinetin) are
the most widely used plant growth regulator in plant tissue culture. Auxin promotes
both the cell division and cell growth and cytokinins promote the cell division (Slater et
al., 2003).
27
Cytokinins has clearly play important role in various processes in the growth and
development of plants, including the promotion of cell division, the counteraction of
senescence, the regulation of apical dominance and the transmission of nutritional
signals. The natural plant cytokinins are adenines that have been substituted at the N6
terminal either an isoprene-derived chain (isoprenoid cytokinins) or an aromatic
derivative side chain (aromatic cytokinins). Another well-known type of cytokinins is
the phenylurea type, however there is no evidence that any of them occur naturally in
plant (Sakikabara, 2004).
The endogenous hormones refer to the hormones that are naturally synthesized
such as IAA and zeatin. IAA, the most important naturally synthesize is unstable to both
heat and light. However, the more stable of synthetic auxins and cytokinins have been
widely used in plant cell culture (Appendix C) (Slater et al., 2003).
Auxins are defined as organic substances that promote cell elongation growth
when applied in low concentrations to plant tissue segments in a bioassay (Jennifer et al.,
2004). There is evidence on the cell elongation growth of tobacco young shoot using
GUS assay, indicating the presence of auxin on the lower side of the stem and this side
elongates faster causing a bending. One of the native auxin is IAA, synthesized in
young apical meristem and is transported basipetally to the growing zones of the stem
and more distantly to the root via polar transport system (Davies, 2004).
The auxin transport is a polar transport, which requires energy driven by
adenosine triphosphate (ATP). The mechanism of auxin transport is one of
28
chemiosmosis system that depends on the hydrogen proton (H+) gradients generated
from the proton pump. The proton pumps located in plasma membrane play a role in
growth response of cells. The acid growth theory states that auxin stimulates the proton
pump to lower the pH in cell wall. The acidification of the wall activates enzymes that
break the hydrogen bonds between the cellulose microfibrils and loosening of the wall.
The cells are free of water uptake by osmosis and turgor pressure pushes the loosening
of the wall. The pressure enhances the elongation of cells and continuously the growth
of plant cell (Lack and Evans, 2005). This experiment was carried out to optimize shoot
and root regeneration of I. balsamina by various plant regulators concentrations.
29
2.2
2.2.1
Materials and Methods
Plant materials and seed sterilization
The seeds were obtained from wild type species. All seeds were washed under
running tap water for 1 hour. Then, surface sterilization was carried out with 70 % (v/v)
ethanol for 1 minute and 10% (v/v) solution of Clorox, Selangor, Malaysia (commercial
bleach containing 5.25% sodium hypochlorite) for 10 minutes (Kyungkul, 1993). Seeds
were rinsed with sterile distilled water three times and blotted dry on sterile filter paper.
2.2.2
Germination of seeds
Seeds were germinated on MS media (Appendix A) supplemented with 3% (w/v)
sucrose, vitamins (w/v), and 0.8 % (w/v) Bacto Difco agar. The pH was adjusted to 5.7
with 0.1 M NaOH or 0.1 M HCL and solidified with 0.8% (w/v) Bacto Difco agar
(Sigma Aldrich, Malaysia) prior to autoclaving (121° C/ 15 psi) for 20 minutes.
The
seeds were germinated at 25° C ± 2° C under 16 h photoperiod with light supplied by
cool white fluorescent lamps at an intensity of 17 µmol s-1m-2 using light meter (LICOR,
USA).
2.2.3
Plant growth regulators and stock solutions
Plant growth regulator stock solutions were prepared in 1 mg/ml by dissolving in
an appropriate solvent (Appendix B). All plant growth regulators except for IAA and
30
IBA were added into the media before sterilization. Filter sterilization was carried out
using 0.2 µm filters (Whatman, USA) for IAA (Sigma Aldrich, Malaysia) and IBA
(Sigma Aldrich, Malaysia). They were added into the media after autoclaving (121° C/
15 psi) for 20 minutes.
2.2.4
Explant preparations
Cotyledons and hypocotyls were chosen as source of explants. Cotyledons were
excised from 7 days, 14 days and 21 days old in vitro seedlings. Cotyledons were
separated into proximal and distal parts. Hypocotyls were cut into three different
sections for near base, center and near cotyledon. All explants with different ages and
sections from cotyledons and hypocotyls were transferred to shooting media containing
MS media with 1 mg/L BAP (Sigma Aldrich, Malaysia) and 3 mg/L BAP, respectively
(Ananthakrishnan et al., 2003). Each experiment contained three replicates with ten
explants per plate. Figure 2.1 shows examples from 7 days old seedling cotyledons and
hypocotyls explants.
2.2.5
Shoot induction experiment
Explants were transferred to shooting media consisting of MS media
supplemented with various concentrations of TDZ, BAP alone or combination with
NAA. Total number of explants per each hormone treatment was 90. Each replicate
contained ten explants per plate (Table 2.1).
31
Figure 2.1 The different types and sections of 7 days old seedling explants.
A. Cotyledon used in the study. Bar= 0.5cm.
B. Proximal section of cotyledon. Bar= 0.2cm.
C. Distal section of cotyledon. Bar= 0.2cm.
D. Hypocotyls with different section in H1 (near cotyledon), H2 (center), and H3 (near roots). Bar=1 cm.
32
Table 2.1 The usage of different types and concentrations of cytokinins in shoot
induction media.
Cytokinins
Concentrations (mg/L)
BAP
1.0
3.0
5.0
BAP:
1.0: 0.5 and 1.0:1.0
NAA
3.0:0.5 and 3.0: 1.0
5.0: 0.5 and 3.0: 1.0
TDZ
0.5, 1.0, and 3.0
33
2.2.6
Root induction experiment
Explants were transferred to rooting media consisting of MS media
supplemented with NAA, IAA or IBA (Table 2.2). The effect of media types on rooting
was carried out using full strength and half strength MS medium. Each experiment
contained three replicates with ten explants per plate. Experiment was repeated three
times.
Table 2.2 The different types and concentrations of auxins on root induction.
Auxins
Concentrations (mg/L)
IAA
0.1, 0.5 and 1.0
IBA
0.5, 1.0 and 2.0
NAA
0.1, 0.5 and 1.0
34
2.2.7
In vitro plant regeneration
The experiment was conducted using MS media supplemented with 1 mg/L BAP
for shoot induction and half strength MS media supplemented with 0.1 mg/L IAA for
root induction based on results obtained in previous experiment. Cotyledons of 7 days
old seedling from proximal section were cultured on MS media supplemented with 1
mg/L BAP for three weeks. The shoots were transferred to rooting medium (half
strength MS media supplemented with 0.1 mg/L IAA). After 2 weeks in rooting media,
plantlets were sub-cultured in different media for observation of development plantlets.
Half of plantlets (n= 90) were sub-cultured on half strength MS medium supplemented
with 0.1 mg/L IAA and the other half (n= 90) were transferred in MS free hormone. The
height of plantlets was measured for further growth within three weeks.
2.2.8
Data analysis
Data on the effect of hormones and explant age on shoot and root induction and
height of plantlets were analyzed using post hoc test (Bonferroni) from ANOVA (SPSS
version 12.0 : SPSS Inc., Chicago, USA).
35
2.3
2.3.1
Results
The effect of explant age, types and sections on shoot induction
The morphological and biochemical characteristics of cotyledons vary with age
and will affect on cytokinin uptake. The plant also has regenerated potential emerges
from proximal and distal section within a cotyledon and different section of hypocotyls
of I. balsamina.
Therefore, this experiment (Section 2.2.4) was carried out using
different age, types and sections of cotyledons and hypocotyls to induce shoot
regeneration.
In the present study, the highest shooting percentage (88%) was obtained from 7
days old seedling of proximal cotyledon sections followed by 14 days (86%) and 21
days old seedling (81%), respectively (Figure 2.2). There was a significance difference
(P< 0.05) on number of shoots per explant between all explant age tested using 7 days
(P=0.00), 14 days (P= 0.002) and 21 days (P=0.19) cotyledons explants. This showed
that explant age did affect on number of shoot regenerated from cotyledons of I.
balsamina.
No shoot was induced from distal of cotyledons (Figure 2.3). The callus
formation was observed in distal cotyledon section with the highest percentage (76%) of
explants produced white callus (Figure 2.2 and Figure 2.3). However, necrosis of callus
did not induce any shoot after four weeks of culture on shooting media.
36
Hypocotyls were also tested for shoot induction.
In all explants ages and
sections tested, hypocotyls only produced green to brown callus without shoots after two
weeks on MS plates supplemented with 3 mg/L BAP (Figure 2.3). The results suggested
that 7 days old seedling of proximal cotyledon section was the best explant for shoot
induction.
5
4.5
4
3.5
Average
3
number of
shoots per
explants 2.5
2
1.5
1
0.5
0
7 days
14 days
21 days
Explant age
Figure 2.2 The effect of explant age from proximal cotyledon on average number of
shoot per explant after three weeks culture on MS media supplemented with 1 mg/L
BAP. Data were analyzed using ANOVA and error bar represents standard error (SE).
37
A
B
C
Figure 2.3 The effect of cytokinins on shoot regenerated from different types of
explants used after three weeks in culture.
A. Proximal sections with shoots formation on MS media supplemented with 1 mg/L
BAP. Bar = 0.5cm.
B. Distal parts with white calli formation on MS media supplemented with 1 mg/L
BAP. Bar = 0.2cm.
C. Hypocotyls with green to brown calli on MS media supplemented with 3 mg/ L
BAP. Bar = 2 cm.
38
2.3.2
Shoot induction
Different types of cytokinins and concentrations have been used widely for shoot
induction. In the present study, the used of BAP and TDZ showed significance different
(P=0.00< 0.05). However, treatment using BAP: NAA showed no significance different
with P value equal to 1.00 (P> 0.05). Proximal section of 7 days old seedling cotyledon
showed the highest percentage of shooting (93%) with the highest average number of
shoots (6.8 ± 1.05) at 1 mg/L BAP alone after three weeks in culture (Figure 2.4).
Multiple shoot (50%-93%) were observed in all treatments BAP (1- 5 mg/L), BAP (1 - 5
mg/L): NAA (0.5 mg/L and 1 mg/L) and TDZ (0.5 - 3 mg/L) (Figure 2.4 and Figure
2.5). Development of shoots was illustrated in Figure 2.4 A- E.
Combination of BAP (1 - 5 mg/L) with NAA (0.5 mg/L and 1 mg/L) promoted
callus and roots formation (Figure 2.6 B). Even though the used of combination of
BAP( 1-5 mg/L) and NAA (0.5 mg/L and 1 mg/L) treatments resulted on lower
percentage of shooting explants (50%-70%) and lower average number of shooting per
explant (1.2 ± 0.47 shoots -1.7 ± 0.59 shoots) but there was no significant difference in
all treatments (P= 1.00).
39
Figure 2.4 Multiple shoots development in MS media supplemented with 1 mg/L BAP
within two weeks in culture (A-E). Bar=0.1 cm.
40
Shoot induction using TDZ treatments (0.5- 3 mg/L) induced both shoots and
roots with significance different (P= 0.00< 0.05). However, TDZ was not consistent in
terms of number of shoots. TDZ treatment (1 mg/L) resulted on high number of shoots
or lower number of shoots.
This may cause by intermediate actions of thidiazuron
(TDZ) as cytokinin and auxin in culture system affect on shoots or roots inhibition
(Slater et al., 2003; Wang et al., 2003) (Figure 2.6 C). The average number of shoots
per explants produced using TDZ treatments (0.5 - 3mg/L) was in range of 3.8 ± 0.9
shoots to 4.4 ± 1.09 shoots per explant (82%-87% explants produced shoots) compared
to BAP treatments (1 - 5 mg/L) with average number of shoots per explants 3.6 ± 0.63
shoots to 6.8 ± 1.05 shoots per explant (84%-93% explants produced shoots). Therefore,
in terms of higher percentage of shooting (93%) and higher average number of shoots
per explant (6.8 ± 1.05 shoots), BAP treatment at 1 mg/L was the best treatment used for
shoot induction.
High percentage of shoots produced per explant obtained from control culture
(80 %). However, the average number of shoots (1.2 ± 0.02 shoots) in control culture
was lower compared to BAP (6.8 ± 1.05 shoots) and TDZ (4.4 ± 1.09 shoots) treatments
(Figure 2.5). In addition, shoot with roots induction in control was initiated after two
weeks in culture compared to the treated one, which were within the first week in culture
(Figure 2.6 D). The shoot and roots induction was observed in control due to the
presence of endogenous cytokinin in explants (Sakikabara, 2004).
41
8
7
Average number of
shoots per explants
6
5
4
3
2
1
L
O
Z
NT
R
TD
Z
O
TD
TD
Z
1
3
C
P:
N
B
A
0.
5
(5
:0
1)
A
A
A
A
B
A
P:
N
A
A
P:
N
A
B
(5
:.5
)
(3
:0
1)
.5
)
(3
:
A
A
B
A
P:
N
A
B
P:
N
A
A
A
A
P:
N
A
B
(1
:0
1)
.5
)
(1
:
A
P
5B
B
3
1
B
A
P
A
P
0
Different concentrations of hormones ( mg/L)
Figure 2.5 The effect of different concentrations of hormones and control (without hormone treatments) on shoot induction
using 7 days old cotyledon explants after three weeks in culture. Data were analyzed with ANOVA and error bar represents
standard error of mean (± SE).
42
Figure 2.6 Shoot induction from the proximal section of 7 days old seedling cotyledons
on MS media supplemented with and without hormones after three weeks in culture.
A.
1 mg/L BAP. Bar= 1.5 cm.
B.
0.5 mg/L BAP with 0.5 mg/L NAA. Bar= 1.5 cm
C.
0.5 mg/L TDZ. Bar= 1.5 cm.
D.
Control. Bar= 0.5 cm.
43
2.3.3
Root induction
The highest percentage of rooting was obtained when explants were cultured on
half strength MS media supplemented with 0.5 mg/L IBA (96%) followed by 0.1 mg/L
IAA (92%)(Figure 2.7). There was no significant difference (P= 1.00>0.05) between
treatments of IAA (0.1 - 0.5 mg/L), IBA (0.5 - 2 mg/L) and NAA (0.1 - 1 mg/L) in half
strength MS media with percentage of rooting explants range from 78% to 96%.
The used of higher concentrations IAA at 1.0 mg/L in half strength MS media
showed the lowest percentage of rooting (78%) with significant difference (P= 0.08<
0.05) compared to the used of IAA at 0.1 mg/L (92%) and 0.5 mg/L (90%). On
contrary, the used of higher concentrations of IBA at 2mg/L and NAA at 1 mg/L
resulted on high percentage of rooting when cultured on half strength MS media.
Control culture in half strength MS media produced 92% of rooting explants.
However, control culture induced roots after 6 days in culture compared to auxin
treatments (IAA, IBA and NAA) within a week (4-6 days) culture. Therefore, 0.1 mg/L
IAA (92%) in half strength MS medium was used for rooting of I. balsamina.
In the present study, half strength MS medium induced rooting with 78% to 96%
of explants compared to full strength MS medium (85%-94%) with no significant
different (P= 0.913 > 0.05) between MS strength (Figure 2.7). Rooting in full strength
MS medium showed no significant difference for all auxin treatments, IAA (0.1 – 0.5
mg/L), IBA (0.5 - 2 mg/L) and NAA (0.1 - 1 mg/L) with P value equal to 0.75 (P>0.05)
44
on percentage of rooting explants (85%-94%). Control culture in full strength MS
media produced 90% of rooting explants. Therefore, different types and concentrations
of hormones and MS strength did not effect on percentage of rooting explants of I.
balsamina (Figure 2.8).
45
full strenght MS media
half strenght MS media
12
10
8
Average number
of rooting explants
6
4
2
0
0.1 IAA 0.5 IAA 1.0 IAA 0.5 IBA 1.0 IBA 2.0 IBA 0.1 NAA 0.5 NAA 1.0 NAA Control
Different concentrations of auxin (mg/L)
Figure 2.7 The effect of different concentrations of auxin hormones on root induction after two weeks in culture.
46
.
Figure 2.8 Comparison of root morphology from different auxins treatment in half strength MS media after two weeks in
culture.
A. Root induction in half strength MS media supplemented with 0.1 mg/L IAA. Bar=0.1cm.
B. Root induction in half strength MS media supplemented with 0.5 mg/L IBA. Bar=0.1cm.
C. Control in half strength MS media. Bar=0.1cm.
47
2.3.4
In vitro plant regeneration
After eight weeks in culture, plantlets were obtained when using MS media
supplemented with 1 mg/L BAP for shoot induction and half strength MS media
supplemented with 0.1 mg/L IAA for root induction (Figure 2.9).
After rooting,
plantlets (100%) were sub-cultured in same rooting medium (half strength MS media
supplemented with 0.1 mg/L IAA) showed lower height (5.0 ± 0.11 cm) compared when
sub-cultured in MS medium free hormone showed higher in height (9.8 ± 0.13 cm) with
significant different (P=0.00< 0.05) after three weeks in culture. Slower growth of
plantlets in half strength MS medium with 0.1 mg/L IAA possibly affect on growth of
plantlets because it was under pressure by hormones that could interferes with
elongation and development of plantlets (Davies, 2004; Herath et al., 2004).
48
A
C
B
Figure 2.9 In vitro plant regeneration of I. balsamina using 7 days old seedling cotyledons within eight weeks in culture.
A. Proximal section of 7 days old seedling cotyledon on MS supplemented with 1 mg/L BAP after three weeks in culture.
Bar=0.8 cm.
B. Roots formation on half strength MS media supplemented with 0.1 mg/L IAA after five weeks in culture. Bar= 1cm.
C. Well rooted plantlets sub-cultured in MS free hormone media after eight weeks in culture. Bar= 1cm.
49
2.4
Discussion
The highest shoot induction using proximal cotyledons has been reported from
previous reports in Terminalia chebula (Shyamkumar et al., 2003) and bottle gourd
(Han et al., 2004). In plant, the meristematic zones are exclusively constitutive to the
regions of dividing cells (Luc and Harry, 2004). The choice of proximal cotyledons
including the axillary meristem in the present study showed the highest shooting
compared to other types of explants.
The inclusion of proximal region showed
maximum shoot regeneration while removal of this region led to the reduction of direct
shoot development (Han et al., 2004; Ananthakrisnan et al., 2003).
The greater
induction of organogenic tissues was obtained from whole and proximal halves of
cotyledons than from distal halves and whole cotyledon explants. If compared with
distal half explants, whole and proximal half explants also resulted in fewer explant
death and larger diameter of organogenic tissue (Kyungkul, 1993).
The organogenic tissues were not induced from distal-half cotyledon explants at
any BAP concentrations tested (Kyungkul, 1993). Similar results were observed in the
present study with no shoots induced from all explants age of distal section on the shoot
induction media. Such differences in response among explant types suggests that an
unidirectional polarity exists in the cotyledon explants in which a certain gradient of
plant regeneration potential exists from proximal to the distal region within a cotyledon
(Kyungkul, 1993). However, callus formation was observed in the present study from
the distal sections. There were no reports on shoot induction from callus derived from
distal sections.
Therefore, although the callus was sub-cultured in shooting media
50
continuously in the present study, there were no shoots observed and all the white calli
turned into necrosis.
Similar result was also observed for hypocotyls explants in the present study
with white calli turned into brown calli after eight weeks in culture. This finding
suggested hypocotyls as non-responsive explants for regeneration system of I.
balsamina. Moreover, there was no report on shoot induction from hypocotyls of I.
balsamina. In contrast, shoot induction from hypocotyls were reported in other species
such as Arabis gunnisoniana (Taskin et al., 2003) and Niger (Murthy et al., 2003).
The explant age is one of the factors that affect shooting percentage. As in the
present study, the younger cotyledons with 7 days old cotyledon showed the highest
shooting percentage compared to other ages (14 days and 21 days) with significance
difference (P<0.05). The highest number of shoots with younger cotyledon explants has
been reported from previous studies. For example, the greater number of shoots with
less callus formation was obtained from 13 days old cotyledons from I. platypetala
Lindl (Kyungkul, 1993). For other species, cotyledon explants at various ages were also
tested for sunflower which showed higher shoot regeneration frequency using four days
old seedling cotyledon compared to six days old seedling cotyledon (Baker et al., 1999).
The younger cotyledons contained high endogenous hormone concentrations such as
cytokinin (Ruth, 2004).
The morphological and biochemical characteristics of
cotyledons change with age and these changes can affect cytokinins uptake and
competency of cells to initiate buds. The used of cotyledons of lettuce (Lactuva sativa
L.) explants 2-4 days after germination considered to be the time at which explants
51
showed maximum competence for shoot regeneration (Hunter and Burritt, 2002).
Therefore, like other plant species reports, the explant age of Impatiens cotyledon was
also an important factor affecting the induction of organogenic tissues and shoot
regeneration (Kyungkul, 1993).
Generally, the benefit of BAP over the other cytokinins on shoot regeneration of
I. balsamina has been reported such as in I. platypetala Lindl (Kyungkul, 1993) and
Impatiens L. interspesific hybrids T63-1, a Java (J) x New Guinea (NG) (Kyungkul and
Stephens, 1987). Multiple shoots of Hibiscus cannabis with the highest number of
shoots (11 shoots) per explants was produced when using 8.8 µM of BAP (Herath et al.,
2004). In the present study, the shoots were produced when using BAP (1 - 5 mg/L) and
TDZ (0.5 - 2 mg/L) with significance different (P=0.00< 0.05). However, the highest
frequency of explants producing number of shoots (6.8 ± 1.05 shoots) was obtained at 1
mg/L BAP after three weeks in culture. This finding was similar to Kyungkul and
Stephens (1987) where shoots of I. platypetala was achieved from cotyledon explants on
media supplemented with BAP (10 µM) showed the highest frequency of shoots (9
shoots per explants). However, cytokinins generally inhibited shoot elongation as the
concentration was increased. As BAP concentrations increased, lower average number
of shoots per explants was observed as reported before in I. platypetala when higher
concentration (40 µM) used delayed the induction of organogenic tissue and overall
process of regeneration (Kyungkul, 1993). The similar result was found in the present
study that higher concentration of BAP (3 mg/L and 5 mg/L) showed lower number of
shoots (3 shoots per explants) with inhibition of shoot elongation.
52
The combination of BAP (1 - 5 mg/L) with NAA (0.5 mg/L and 1 mg/L) in the
present study showed lower percentage of shooting (50%-70%) and average number of
shoots (1.2 ± 0.47 shoot to 1.7 ± 0.59 shoots) per explant , callus and root production
with no significance different (P=1.0> 0.05). In Impatiens hybrid of ‘T63-1’ and ‘Star
Fire’, NAA stimulated shoot elongation of T63-1 at the lowest concentration (2 µM),
and inhibited shoot elongation of ‘Star Fire’ at all concentrations of NAA (2 µM, 4 µM
and 6 µM) ( (Kyungkul and Stephens, 1987). Combination of BAP (3 mg/L and 5mg/L)
with NAA (0.5 mg/L and 1 mg/L) or IAA (0.01 - 1 mg/L) treatments resulted on lower
shoot regeneration frequency, suggesting that only BAP at 3 mg/L may be considered a
crucial factor for shoot induction in bottle gourd (Han et al., 2004). The combination of
BAP (1 - 5 mg/L) and NAA (0.5 mg/L and 1 mg/L) also led to increased callus
formation in the present study and continuous sub-culturing of the callus led to the
necrosis of explants. Previous studies also reported callus formation from cotyledon
explants of sunflower (Baker et al, 1999) when the cotyledon explants were cultured
with combination of BAP (1 mg/L) and NAA (0.5 mg/L).
Non-purine based chemicals, such as substituted phenylureas are also used as
cytokinins in plant cell culture media. These substituted phenylureas can substitute for
auxin in some culture system (Slater et al., 2003). Therefore, in the present study, shoot
and root formation was also found in all concentrations of TDZ at 0.5 mg/L, 1mg/L and
2 mg/L. Yang et al., (2001) reported on higher percentage of shoots (83.2%) when
culturing cotyledons of Swainsola salsula Taubert using TDZ (2 mg/L) treatment with 9
average numbers of shoots. Although TDZ treatments was produced shoots at 82% to
87% with high average number of shoots (3.8 ± 0.97 shoots to 4.4 ± 1.09 shoots) per
53
explant in this study, TDZ was not consistent on producing high induction of shoots
when shoots inductions were inhibited in certain experiments and replicates due to the
intermediate actions of TDZ as cytokinin and auxin in culture systems. This coincides
with Wang et al., (2003) finding that both BAP and TDZ were also effective in inducing
shoot regeneration of Spartina alterniflora. However, treatment with TDZ in the shoot
regeneration medium inhibited the root regeneration of Spartina alterniflora. Other than
that, there was no evidence of TDZ occurring as endogenous hormone of cytokinins
compared to BAP which was effective in uptake and led to the high shoot induction
compared to TDZ (Sakikabara, 2004). For control, lower average number of shoots (1.2
± 0.02 shoots) per explants was observed in this study compared with cytokinins
treatments (BAP and TDZ) although high percentage of explants produced shoots (80%)
was obtained. The shoots induced without any treatment was achieved due to the effect
from the endogenous cytokinin naturally present in the explants at axillary meristem
(Sakikabara, 2004).
Generally, root induction was obtained using all the auxins (IAA, IBA and NAA)
tested with the highest percentage of rooting (96%) obtained when using 0.5 mg/L IBA.
However, 0.1 mg/L IAA with high percentage of rooting (92%) was used for rooting
treatment in this study. The rapid growth of roots in the present study was observed in
four days when using IAA treatment possibly due to native auxin IAA occur in plant as
reported by Jennifer et al., (2004). Han et al., (2004) reported shoots of bottle gourd
from cotyledons explants were successfully rooted in half strength MS supplemented
with 0.1 mg/L IAA. Generally, rooting treatments using IAA (0.1 - 1 mg/L), IBA (0.5 2 mg/L) and NAA (0.1 - 1 mg/L) showed no significance difference on percentage of
54
rooting in half strength MS media (P 1.00>0.05) and full strength MS media (P=0.75
<0.05). However, IAA treatments using higher concentrations of IAA could inhibit
roots formation as reported by Swamy et al, (2004) on average number of rooting of
Albizia procera using IAA at 10µg/L (2 roots) compared to the used of IAA at lower
concentration (8 µg/L) with 4 roots. In this study when using 1 mg/L IAA affect on
lower percentage of rooting with 78% compared to lower concentrations of IAA (0.1
mg/L and 0.5 mg/L) treatments with high percentage of rooting (90%-92%) in half
strength MS media. The used of IBA was effective for rooting of Hordeum vulgare L.
at 0.5 mg/L and 1 mg/L (Sharma et al., 2004) and Swainsona salsula Taubert at 2 mg/L
(Yang et al., 2001). Although NAA treatment was not effective for rooting treatment of
Swainsona salsula Taubert (Yang et al., 2001), multiple shoots of Vigna subterranea
(Bambara groundnut) and shoots of I. platypetala hybrid responded well to NAA at 0.5
mg/L and 1 mg/L to develop roots (Lacroix et al., (2003) ; Stephens et al, (1985)). The
presence of endogenous auxin also induced roots for control culture in both types of MS
media (full and half strength) in this study with average of 90% of rooting. This finding
coincides with Kyungkul (1993) who found multiple shoots of I. platypetala Lindl were
successfully rooted in free medium.
The used of different MS strength with full and half strength showed no
significant difference (P=0.913>0.05) on percentage of rooting in this study. However,
greater root induction of Spartina alterniflora was produced when using half strength
MS medium (91% of rooting) compared to full strength MS (82% of rooting) (Wang et
al., 2003). The highest rooting percentage (100%) from cotyledons explants was
reported when using half strength MS media in bottle gourd and Terminalia chebula
55
(Han et al., 2004; Syhamkumar et al., 2003). The favorable effects of a diluted mineral
solution on rooting can be explained by the reduction of nitrogen concentration. The
reduction at the mineral concentration of medium to half normal values increased the
rooting percentage in Prunus persica using IBA treatments (Fotopoulus and
Sotiropoulus, 2005). Therefore, in this study, half strength media was used for rooting.
In this study, elongation of plantlets in MS free medium after rooting resulted on
10 cm in average of height compared when sub-culturing in the same half strength MS
rooting medium supplemented with 0.1 mg/L IAA ( 5 cm in height) after three weeks in
culture. The used of MS free medium was reported before for elongation of Hibiscus
cannabinus multiple shoots (96% shoots) as the presence of hormones could interferes
with shoot elongation and development (Herath et al., 2004).
56
2.5
Conclusion
In conclusion, after eight weeks in culture, plantlets were successfully obtained
in MS medium supplemented with 1 mg/L BAP for shooting and half strength MS
media supplemented with 0.1 mg/L IAA for rooting. This system would be used as a
fundamental study for I. balsamina for further investigation.
57
CHAPTER 3
Transformation of Impatiens balsamina through biolistics
3.1
Introduction
Plant transformation is normally carried out in order to improve crop plant
characteristics. However, the success of plant transformation depends on the stable
expression of the gene of interest into the genome of the plant. Basically, there are two
important methods that have been successfully used for transformation. They are,
utilizing Agrobacterium, a plant pathogen of dicotyledonous plants and a direct gene
transfer using particle bombardment or biolistics. The latter is the method of choice for
the transformation of many monocotyledonous plants. In this chapter technique of
particle bombardment or biolistics was used throughout the investigation.
The effort to use biotechnology techniques to the transformation of Impatiens sp.
has increased. The regeneration systems of Impatiens have been established such as I.
wallenaria (Baxter, 2005), I. platypetala (Kyungkul, 1993), Impatiens L. interspesific
58
hybrids T63-1, a Java (J) x New Guinea (NG) (Kyungkul and Stephens, 1987) and
Impatiens Java, New Java and Java x New Guinea (Stephens, 1985). However, there is
no literature to describe plant regeneration and transformation of I. balsamina.
Particle bombardment or biolistics is the most important and effective direct gene
transfer method in regular use. It is also useful in the transformation strategies involving
plant viruses (Altpeter et al., 2005). Impatiens is one of the host plants for Impatiens
Necrotic Spot Virus (INSV), the virus that could infect the crops in many species.
Therefore, efforts have been carried out on development of transformation system using
biolistics for obtaining resistant plants (Daughtery et al., 1997).
In certain study, the transformation efficiency as reported in literatures can be
very low as described by Jain et al., (1996). It could be due to several reasons to
account for low efficiency for example the optimal bombardment conditions which vary.
Other reasons would depend on the type and quality of the target cells and tissues or it
could be the media requirements for bombardments. Therefore, Kikkert et al., (2003)
suggested that improvement on the optimization of biological and physical parameters
need to be carried out for high transformation efficiency.
Biolistics system had never been tested on cotyledonary explants from I.
balsamina and there was no report on the successful of the stable transformation of I.
balsamina using biolistics. Therefore, there is the possibility of utilizing the biolistics
system to produce transformed I. balsamina using cotyledonary explants with axillary
meristem in comparison to the A. tumefaciens system. However, using the meristematic
59
tissue, high proportion of transformed plants is likely to be chimeric (Birch and Bower,
1994). A plant is said to be a chimera when cells of more than one genotype are found
growing adjacent in the tissues of that plant. The chimeral plants may originate by
grafting, spontaneous mutation, induced mutation, sorting-out from variegated seedlings,
mixed callus cultures, or protoplast fusion. Tissue culture methodology provides a
useful way to separate plant chimeras into their component genotypes. Conditions that
favor adventitious shoot formation (leaf or callus culture, suspension culture, extremely
rapid shoot proliferation rates) encourage genotypic segregation. Genotypic segregation
can confirm the chimeral nature of the cultivar in question and can allow conclusions to
be drawn about the ontogeny of in vitro adventitious shoot formation (Lineberger, 2003).
This research focused on plant transformation via biolistics.
The ß-
glucuronidase (GUS) expression system used to assess the expression of the reporter
gene. The hph gene for hygromycin resistance was used to develop the transformation
procedure using the biolistics system. Further study with bar gene was also carried for
Basta resistance.
60
3.2
Materials and Methods
3.2.1
Bacterial strain and plasmids
Bacteria strain of Escherichia coli (E.coli) and plasmids were provided by
Biotechnology Research Centre, MARDI, Serdang (Table 3.1).
Table 3.1 The plasmids used in this study.
Plasmid
pRQ6
uid
pAHG11
3.2.2
Phenotype
Others
+
GUS reporter gene
hph +
Resistant to hygromycin
bar +
Resistant to Basta
Bacteria growth and condition
Bacteria cells E.coli from frozen stock (-80 ºC) were streaked onto LB media.
Luria bertani (LB) media contained yeast extract 10.0 g/L, tryptone 5.0 g/L and NaCl
10.0 g/L (Appendix D) and ampicilin (Appendix E) was solidified with 1.5% (w/v)
Bacto Difco agar. The plate was incubated in incubator (Memmert, Malaysia) at 37 ºC
for overnight to obtain an independent single colony. A single colony of E.coli was
cultured in 10 ml of LB broth (LB media without agar) contained ampicilin and were
61
incubated in a rotary orbital shaker incubator ( Protech, Malaysia) at 200 rpm at the 37°
C temperature for overnight (Bloom et al., 1996).
3.2.3
Plasmid DNA
Two plasmids were used for bombardment. The plasmid pRQ6 (hph+) (Appendix
F) contained the GUS gene (uidA gene) and hygromycin phosphotransferase gene (hph
gene) conferring to hygromycin resistance. The plasmid was driven by Cauliflower
Mosaic Virus (CaMV) 35S promoter. The plasmid pAHG11 (bar+) (Appendix G)
contained the bar gene conferring to Basta driven by ubiquitin promoters.
Both
plasmids were provided by Biotechnology Research Centre, MARDI (Serdang) and
were isolated from E.coli using Qiaprep Spin protocol from Qiagen, USA.
3.2.4
Agarose gel electrophoresis
Complete DNA digested by endonuclease enzymes were analyzed by submarine
gel electrophoresis through agarose gels. Generally, 0.8% agarose gel (w/v) prepared in
TAE buffer (40 mM Tris-acetate pH 7.6, 1 mM EDTA + ethidium bromide at 0.5 μg/ml)
was used. Samples (between 2 µl to 5 μl) were mixed with 5 µl of gel loading buffer
[0.25% bromophenol blue (w/v), 0.25% xylene cyanol (w/v), 30% glycerol (w/v)] before
loading. Mini gels were run at a constant 80 volts for 1 hour to 2 hours.
62
DNA fragments on the gel were visualised using an ultraviolet (UV)
transilluminator (Superbright, Vilbert Loumart, Germany). The sizes were estimated by
comparison with a 1 kb ladder (Promega, USA) standard DNA marker (1μg of DNA
marker was used each time). The DNA ladder could also be used as a means of
estimating the amount of DNA present in a sample as the band makes up 10 % (100 ng)
of the total DNA present in the marker used (Bloom et al., 1996).
3.2.5
Measurement of DNA concentration
DNA concentration was estimated by ultraviolet spectrophotometry (Cary 100
Bio, Siber Hegner, Selangor, Malaysia). An A260nm of 1.0 corresponds to 50 μg of
double stranded DNA per ml. The DNA purity was estimated from the ratio of A260nm /
A280nm. Ratio of less than 1.8 indicates that the preparation is contaminated with protein
(Maniatis et al., 1982).
3.2.6
Gold particle preparations
Gold particles (6 mg) (BioRad, USA) with 1.0 µm in diameter were sterilized in
100 µl absolute ethanol with vortexing.
The particles were washed twice and
resuspended in 100 µl sterile water (Lee et al., 2003).
63
3.2.7
Preparation of DNA coating gold particles
Sterile gold particles in sterile water (100 µl) (Section 3.2.6) (BioRad, USA)
were mixed with 20 µl plasmid DNA (1.0 µg/ µl). Then the gold particles and DNA
mixture were mixed in 100 µl CaCl2 (2.5 M) and 40 µl spermidine (0.1 M). The gold
particle/DNA suspension was quick vortex (Grant, Malaysia) and left for 10 min at room
temperature.
The DNA coated particles were pelleted by centrifugation at 10,000 g for 10 sec
at room temperature. The supernatant was completely removed. The pellet was
resuspended in 100 µl absolute ethanol. Then, 12 µl of particle/ DNA mixture was
placed in the center of microcarrier. The bombardment was carried out using the PDS 1000/He Biolistic Particle Delivery (BioRad, USA) (Lee et al., 2003).
3.2.8
Explants preparation
Proximal cotyledons (20 units) of 7 days old were placed at the center of 9 cm
petri disc containing bombardment media prior to bombardment. Total number of
explants used depends on experiments. Optimization of biolistic parameters used 80
explants per treatment while bombardment using optimal concentrations using 960
explants for both transformation (pRQ6) and co-transformation (pRQ6 and pAHG11).
All explants were cultured for 16 h before bombardment on bombardment media
(Section 3.2.9) at 25° C with light supplied by cool white fluorescent lamps at an
intensity of 17µmol m2 s-1 using light meter (LICOR, USA).
64
3.2.9
Bombardment media
Bombardment media (NBO media) consist of MS media supplemented with
vitamins (w/v), sucrose 3% (w/v), benzylaminopurine (BAP) (1 mg/L) and mannitol (0.2
M) / sorbitol (0.2 M). The pH was adjusted to 5.7 with 0.1 M NaOH or 0.1 M HCL and
solidified with Bacto Difco agar (0.8% (w/v) (Lee et al., 2003).
3.2.10 Bombardment technique
Physical and biological parameters for DNA delivery into I. balsamina explants
were optimized. Vacuum pressure was maintained at 28 Hg. Each experiment had two
replicates and was conducted twice. Control was using the unbombarded plant tissues.
Bombarded explants depends on experiments were histochemically GUS tested for the
optimal parameter (physical parameters and biological parameters) of biolistic condition.
For further study on selection system, bombarded explants (n=280 explants) were
subjected to GUS assay while other explants (n= 680) were sub-cultured in selection
media consisting of 75 mg/L hygromycin and 1 mg/L phosphinothricin (PPT) after
bombardment or used as control culture.
3.2.10.1 Physical parameters
Optimization of the physical factors was carried out under the following
conditions, combination of helium pressure (650 psi, 900 psi, 1100 psi) and distance
65
from stopping plate to the target distance (6 cm, 9 cm, and 12 cm) and the number of
bombardments.
3.2.10.2 Biological parameters
The biological parameters included the pre-culture time prior bombardment (4 h,
16 h and 32 h), DNA concentrations (0.2 µg, 0.5 µg and 1.0 µg), post-bombardment
incubation time (4 h, 24 h, and 48 h) and osmotic treatments (bombardment media)
containing mannitol, sorbitol and combination of both mannitol and sorbitol in different
concentrations (0.2 M, 0.4 M and 0.6 M) appropriately.
3.2.11 ß-glucuronidase (GUS) assay buffer
Histochemical GUS assay was carried out as described by Jefferson et al., (1987).
GUS assay buffer consist of 1 mM 5-bromo-4 –chloro-3-indoxyl-ß-D-glucuronide (XGluc), 0.2 M sodium phosphate (pH 7.0), and 0.1% (v/v) Triton X-100. The presence
of GUS spots on explants was examined under the dissecting microscope.
3.2.12 ß-glucuronidase (GUS) expression assay
Histochemical GUS assay of explants for transient gene expression was tested
after 24 h of bombardment in 1 mM 5-bromo-4 –chloro-3-indoxyl-ß-D-glucuronide (XGluc) reaction buffer. All explants were incubated at 37°C for 24 h in the reaction
66
buffer (GUS buffer). After incubation, all explants were soaked in 70 % (v/v) ethanol
for 15 min to remove the chlorophyll.
The GUS gene expression was recorded in terms of percentage GUS positive
explants and average number of GUS spots per explants.
In order to obtain the
transformed plants, half of the explants were regenerated in selection media. The GUS
assay was tested again after the plants were successfully regenerated in selection media.
3.2.13 Genomic DNA
In this study, the genomic DNA was carried out using cetyl metyl ammonium
bromide (CTAB) modified method from Zidani et al (2005). An extraction buffer
consisted of 2% cetyl metyl ammonium bromide (CTAB) (w/v) (Sigma Aldrich,
Malaysia), 100 mM Tris (pH 8.0), 20 mM EDTA (pH 8.0), 1.4 M natrium chloride
(NaCl), 1% (w/v) polyvinylprolidone (PVP) (Sigma Aldrich, Malaysia) and 3 M sodium
acetate (pH 5.2). In addition, chloroform: isoamylalcohol (24:1), 75% and 100% (v/v)
ethanol and TE buffer consisting of 10 mM Tris (pH 8.0) and 1 mM EDTA (pH 8.0)
were also prepared.
A 1.0 g of leaf sample (10 weeks) from transformed and non transformed plants
was homogenized in liquid nitrogen using mortar and pestle. The homogenized tissues
were transferred into microcentrifuge tube and 1 ml of 65°C cetyl metyl ammonium
bromide (CTAB) buffer was added. The tube was incubated at 65°C for 5 min. The
mixture of chloroform: isoamylalcohol (24:1) with 1.0 ml was added and centrifuged by
67
microcentrifuger (Micro 200, Zentrifugen, Malaysia) at 5000 g for 10 min to separate
the phases. The supernatant (top layer) was carefully decanted and transferred to a new
microcentrifuge tube and 200 µl of 5 % (w/v) cetyl metyl ammonium bromide (CTAB)
solution was added. Then, chloroform: isoamylalcohol (24:1) (1 ml) was added again,
centrifuged at 5000 g for 10 min to separate the phases and ending with decanting of
supernatant into a new microcentrifuge tube. The supernatant was precipitated with 95
% (w/v) ethanol and 100 µl of sodium acetate (pH 5.2) was added. The DNA pellet was
obtained by centrifuging the supernatant at 10,000 g for 10 min at room temperature.
The DNA pellet were washed using 100 % ethanol, dried and resuspended in 100 µl TE
buffer.
3.2.14 Statistical analysis
Data from optimization of biolistic conditions (biological and physical
parameters) and selection system were subjected to post hoc test (Bonferroni) in one
way ANOVA using SPSS 12.0 (SPSS Inc., Chicago, USA) with significance level at
0.05.
3.2.15 Transformation frequency
Transformation frequency (%) was calculated as described by Lee et al., (2003)
on rice callus transformation using biolistic method. The assumption of transformation
frequency was mentioned as below.
68
Transformation frequency (%) =
(Number of GUS positive plants/ total plants analyzed) x regenerated plants x 100%
Number of explants bombarded.
3.2.16 Biolistic transformation using hph gene for hygromycin resistant
3.2.16.1 Determination of minimal lethal dose of hygromycin on cotyledon explants
The optimal concentration of hygromycin for the selection of transformants
were determined by placing the unbombarded cotyledons on MS media supplemented
with 25 mg/L, 50 mg/L, 75 mg/L and 100 mg/L hygromycin. The minimum
concentration for mortality of explants was determined as minimal lethal dose. This
result was observed within five weeks in culture with sub-culturing in the same media
every two weeks.
3.2.16.2 Delay selection method on hygromycin media
Delay selection method was used to delay the exposure of selective agents
(hygromycin; phosphinothricin) at the later regeneration phase (Jones et al., 2005). The
bombarded explants using pRQ6 (hph+) were cultured in MS media supplemented with
1 mg/L BAP (without hygromycin ) in 1 day, 7 days, 21 days and 35 days before
transferred on hygromycin media. Selection method was conducted when culturing
69
cotyledons explants (1 day and 7 days) post-bombardment on MS media supplemented
with 1 mg/L BAP and 75 mg/L hygromycin. The effect of delay selection using shooted
explants (21 days and 35 days) post-bombardment on MS media containing 75 mg/L
hygromycin was observed. The GUS gene expression assay as described in section
3.2.12 and percentages of the regenerated plants were observed after five weeks in the
selection media (75 mg/L hygromycin).
3.2.16.3 Polymerase chain reaction (PCR) analysis
Plants regenerated following bombardment and antibiotic resistance selection
were analyzed by polymerase chain reaction (PCR). The DNA derived from selected
transformed plants was tested for the presence of hph gene using the specific primers.
The expected size of polymerase chain reaction (PCR) product is 0.8 kb. Polymerase
chain reaction (PCR) was performed with Gene Amp PCR System 2400 (Perkin Elmer,
USA). The primers used for amplification of hph gene were 5’ –GGGGGGTCGGTTT
CCACTA-3’ (forward primer) and 5’-ATCGTTATGTTTATCGGCACTTTG-3’
(reverse primer). The primers sequences were provided by Biotechnology Research
Centre, MARDI, Serdang.
Polymerase chain reaction (PCR) was carried out for initial denaturation of 2 min
at 94 °C, followed by 29 cycles of 45 sec at 94° C, 30 sec at 55° C annealing,
temperature, 30 sec at 72° C of 30 sec and final elongation of 5 min at 72 °C.
Polymerase chain reaction (PCR) products were analyzed in 1.2% (w/v) agarose gel
70
electrophoresis. The primers condition was optimized by Biotechnology Research
Centre, MARDI, Serdang.
3.2.17 Biolistic transformation using bar gene for phosphinothricin (Basta)
resistance
3.2.17.1 Herbicide applications on I. balsamina
The effect of herbicide on I. balsamina was observed using commercial Basta
(Bayer Crop Science, Malaysia), an active ingredient containing 200 g/L glufosinate
ammonium with 0.25 % (v/v) and 0.10 % (v/v) of Basta aqueous solution.
3.2.17.2 Determination of minimal lethal dose of phosphinothricin (PPT) on
explants
Minimal lethal dose is minimum concentration for mortality of explants
(necrosis). The unbombarded 7 days old cotyledon explants were cultured on MS media
supplemented with different concentrations of phosphinothricin (PPT) (0.5 mg/L, 1
mg/L, 5 mg/L and 10 mg/L). This result was observed within five weeks in culture by
subculture the cotyledon explants every two weeks.
71
3.2.17.3 Delay selection method on phosphinothricin (PPT) media
Cotyledon explants (7 days old) were bombarded using co-transformation
pRQ6 (hph+) and pAHG11 (bar+) which confers phosphinothricin (PPT) resistance.
Using the optimal concentration of phosphinothricin (PPT) (1 mg/L), the bombarded
cotyledons explants were transferred to selection MS media supplemented with 1 mg/L
phosphinothricin (PPT) and 1 mg/L BAP after 1 day and 7 days post-bombardment. The
delay selection method was conducted on 21 days and 35 days shooted explants and
plantlets on MS media containing 75 mg/L hygromycin. The GUS expression assay was
conducted as described in section 3.2.12 and percentages of the regenerated plants were
observed after five weeks in the selection media.
72
3.3
3.3.1
Results
The effect of target distance and helium pressure on GUS gene expression
This experiment was carried out to determine the best correlation between the
target distance and helium pressure during transformation procedure. Correlation test
between helium pressure and target distance on average number of GUS expression
showed significant at 0.01 level. In the present study, the DNA concentration was fixed
at 1.0 µg and 16 h pre-culture explants in bombardment media contained 0.4 M sorbitol
and mannitol (Lee et al., 2003), the target distance at 9 cm combined with 1100 psi
helium pressure showed the highest average number of blue spots with significance
different (P< 0.05) to other target distance (6 cm and 12 cm) and helium pressure (650
psi and 900 psi). The highest average number of 137.4 spots was achieved using this
optimal target distance and helium pressure (9 cm: 1100 psi). The use of helium
pressure less than 1100 psi resulted in lower GUS gene expression (Figure 3.1).
73
160
140
120
Average
100
number
GUS spots per
80
explants
650psi
900psi
1100psi
60
40
20
0
6cm
9cm
12 cm
Target distance
Figure 3.1 The effect of target distance and helium pressure on GUS gene expression
after pre-culture of explants in bombardment media (0.4 M mannitol and sorbitol) 24 h
prior bombardment using 1.0 µg DNA ,9 cm target distance and 1100 psi helium
pressure. Data were analyzed using One-way ANOVA and error bar represents standard
error of mean (+ SE).
74
3.3.2
The effect of number of bombardments on GUS gene expression
The second physical parameter was to study the effect of number of
bombardments on GUS gene expression. The bombardment was carried out using
cotyledons (7 days old) showed high frequencies of transiently expressing cell in both
numbers of bombardments when bombarded at 9 cm target distance and 1100 psi helium
pressure.
The average number of GUS spots was high when using two times bombardment
(104.5 spots) compared to one time bombardment (98.9 spots). However, there was no
significant difference (P= 0.1>0.05) on multiple bombardment and one time
bombardment. Besides, two times bombardment caused physical injury of explants
(Figure 3.2).
Therefore, one time bombardment was chosen to enhance the high
recovery after bombardment and minimize the cell damage (Figure 3.3).
A
B
C
Figure 3.2 The effect of number of bombardments (one and two times) on explants after
a week of bombardment.
A-B. The physical injury on meristem and explants. Bar= 0.3 cm
C.
The non-injury cotyledon explants. Bar= 0.3 cm
75
105
104
103
102
Average number 101
of GUS spots
per explants 100
99
98
97
96
One time
Two times
Number of bombardments
Figure 3.3 The effect of number of bombardments on GUS spots per explants after 24 h
post-bombardment using 9 cm target distance and 1100 psi helium pressure. Data were
analyzed using One-way ANOVA and error bar represents standard error of mean (+
SE).
76
3.3.3
The pre-culture time prior bombardment on transient gene expression
One of the biological factors determined was pre-culture time prior to
bombardment.
The result suggested that high transient gene expression (average
number of GUS spots per explant with 110.7 spots was obtained after 16 h pre-culture
before bombardment on bombardment media contained 0.4 M sorbitol and mannitol.
There was a significance difference (P< 0.05) between pre-culture times on average
number of GUS spots per explant. The shorter pre-culture time (4 h) resulted in less
average number of GUS spots per explants (5.8 spots) whereas prolonged pre-culture
time (32 h) reduced the ability of explants for high GUS gene expression (average
number of GUS spots per explant with 31.4 spots) (Figure 3.4). Pre-culture at 4 h (P=
0.02) resulted lower results because the cells need more time to acclimatize in the
osmotic media whereas 32 h (P=0.02) inhibited shoot induction.
77
120
100
Average
number of
GUS spots per
explants
80
60
40
20
0
4h
16 h
32 h
Preculture times
Figure 3.4 The effect of pre-culture time of explants with osmotic treatment of 0.4 M
mannitol and sorbitol on GUS gene expression after bombarding explants at 9 cm target
distance and 1100 psi helium pressure. Data were analyzed using One-way ANOVA
and error bar represents standard error of mean (+ SE).
78
3.3.4
The effect of plasmid concentration on transient gene expression
The various plasmid concentrations were tested on GUS gene expression ranged
from 0.5 µg to 1.5 µg. The result suggested high GUS gene expression using 1.0 µg and
1.5 µg plasmid DNA concentrations with average GUS spots per explants 106.7 spots
and 135.8 spots, respectively. There was significant difference at the 0.05 level between
concentrations used in this study (P< 0.05). The average number of GUS spots per
explant using different concentration of plasmid was shown in Figure 3.5. The plasmid
concentration at 1.5 µg per bombardment resulted in highest GUS gene expression
(Figure 3.5). Although the used of 1.5 µg DNA showed the best result, high GUS gene
expression was not consistent at each bombardment. At certain time, poor dispersal
could be observed under microscope. The particle aggregation occurs due to high
amount of DNA used affect on poor dispersal and cell damage (Birch and Bower, 1994).
This observation may interfere with gene expression at the later stage of the experiment.
As a result 1.0 µg plasmid DNA was used in this research.
79
160
140
120
Average
100
number of
GUS spots per
80
explants
60
40
20
0
0.5
1
1.5
DNA concentrations
Figure 3.5 The effect of DNA concentrations (µg) on GUS spots per explants 24 h postbombardment using 9 cm target distance and 1100 psi helium pressure. Data were
analyzed using One-way ANOVA and error bar represents standard error of mean (+
SE).
80
3.3.5
The effect of pre-culture treatments on transient gene expression
This experiment was carried out to reduce cell injury during bombardment
procedure. In the present study, the osmoticum treatments used consisted of mixture of
sorbitol and mannitol showed 7.2-fold higher frequency of GUS gene expression
compared to the sample without treatment. All explants were pre-culture (16 h) with
different types and concentrations of osmotic treatment.
Then, explants were
bombarded using constant 1.0 µg DNA, 9 cm target distance and 1100 psi of helium
pressure resulted on average number of GUS spots per explant in range of 45.2% to
110.2 %.
Bombarded explants with 16 h pre-culture time in 0.4 M mannitol and sorbitol
resulted on the highest average number of GUS spots (110.2 spots) compared without
osmotic treatment (sucrose) 15.3 GUS spots. The significance level at 0.05 was shown
between using the osmotic treatment and without using osmotic treatment (P< 0.05).
Therefore, osmotic treatment did affect on transformation efficiency (average number of
GUS spots per explant). Different types and concentrations of osmoticum (mannitol,
sorbitol and mannitol: sorbitol) showed significance different between treatments
(P<0.01). Osmoticum treatments using mannitol alone showed higher of GUS gene
expression compared to sorbitol treatments alone (Figure 3.6). From this observation,
treatment using both osmotica gave better results.
81
120
100
0.2M
0.4M
0.6M
Average
80
number
GUS spots per
60
explants
40
20
l
or
bi
to
ito
l
M
an
ni
to
l+
S
So
rb
M
an
n
ito
l
0
Pre-culture treatments
Figure 3.6 The effect of pre-culture treatments with different types and concentrations
of osmotic treatment on GUS gene expression after 24 h post-bombardment using 9 cm
target distance and 1100 psi helium pressure. Data were analyzed using One-way
ANOVA and error bar represents standard error of mean (+ SE).
82
3.3.6
The effect of post-bombardment incubation time on transient gene
expression
The bombarded explants were tested in GUS assay after the incubation time for
the recovery and high expression of the cells. The post-bombardment incubation times
tested were 4 h, 24 h, and 48 h after bombardment using optimal condition (9 cm target
distance, 1100 psi helium pressure, 1.0 µg DNA, 16 h pre-culture time of explants on
bombardment media using 0.4 mannitol and sorbitol, one time bombardment).
The post-bombardment incubation time affect on the recovery cells for high
expression of cells. The results showed that the highest transient expression (110.7 spots)
was obtained after 24 h of post-bombardment showed significant different (P < 0.05)
between other post-bombardment time (4 h and 48 h). The incubation time for 4 h (P=
0.00< 0.05) showed less expression (19.0 spots) compared to 24 h, whereas 48 h (P=
0.007 <0.05) showed higher expression (99.3 spots) compared to 4 h post-incubation
time (Figure 3.7).
83
120
100
Average
number of GUS
spots per
explants
80
60
40
20
0
4h
24 h
48 h
Post-bombardment incubation time
Figure 3.7 The effect of post-bombardment incubation time on GUS expression after
bombardment using 9 cm target distance and 1100 psi helium pressure. Data were
analyzed using One-way ANOVA and error bar represents standard error of mean (+
SE).
84
3.3.7
The effect of optimal bombardment conditions on GUS gene expression and
regeneration of I. balsamina
Total number of 960 explants bombarded using pRQ6 (hph+) and cotransformation with pRQ6 (hph+) and pAHG11 (bar+) in the optimal bombardment
+
conditions. Bombarded explants (n=140 explants) for each transformation pRQ6 (hph )
and co-transformation pRQ6 (hph+) and pAHG11 (bar+) were subjected to GUS assay
resulted on high average number of GUS spots per explants with 149.3 spots (95%) and
128.1 spots (90%), respectively (Table 3.2 and Figure 3.8).
The other 180 bombarded explants out of 340 bombarded explants used for each
+
+
of pRQ6 (hph ) and co-transformation of pRQ6 (hph
, uidA+)
+
and pAHG11 (bar )
were cultured in MS media supplemented with 1 mg/L BAP or 0.1 mg/L IAA resulted
on 100% of plantlets. The other 160 bombarded explants were further tested in selection
media. Analysis of bombardment condition (target distance: helium pressure, osmotic
treatment, DNA concentration, pre-culture time and post-incubation time) showed
significant different between parameters tested (P< 0.05). In this study, only number of
bombardments (physical parameter) showed no significant difference between
bombardment for once and twice (P > 0.05). However, in terms of less injury of cells
that effect on shoot regeneration (number of shoots), one time bombardment was the
optimal parameter for biolistic condition.
85
Table 3.2 The effect optimal bombardment conditions on GUS gene expression of
explants 24 h post-bombardment.
Plasmid
Number of
Number of
Average
Percentage of
bombarded
explants for
number of
GUS positive
explants
GUS assay
GUS spots
explants (%)
± SE
+
480
140
149.3+ 2.14
95
pRQ6 (hph )
480
140
128.1+ 1.54
90
pRQ6 (hph )
+
and
+
pAHG11(bar )
86
A
Figure 3.8
B
C
D
The effect of bombardment using optimal biolistic conditions (target distance 9 cm: helium pressure 1100 psi,
one time bombardment, 1.0 µg DNA, pre-culture explants with combination of mannitol and sorbitol (0.4 M) in 16 h preculture time and post-bombardment incubation time 24 h before GUS assay) on GUS gene expression of explants.
A. GUS gene expression on explants after 24 h bombardment. Bar= 0.3 cm
B. GUS gene expression on meristematic region of explants after 72 h bombardment. Bar= 0.1cm
C. Lower GUS gene expression on explants after 24 h bombardment. Bar= 0.2cm.
D. Control with no GUS gene expression after 24 h bombardment. Bar= 0.3cm.
87
+
Biolistic transformation using pRQ6 (uid A+, hph ) for hygromycin
3.3.8
resistant
3.3.8.1 Minimal lethal dose of hygromycin
Minimal lethal dose was determined for the concentration used to distinguish the
transformed and untransformed plant in later experiment. In order to obtain the minimal
lethal dose, the unbombarded explants in various ages were tested in different
concentrations of hygromycin. The minimal lethal dose refers to the explants in 75
mg/L hygromycin with 100% mortality of explants after five weeks in culture with
significant different (P<0.05) between concentrations used (25 mg/L hygromycin and 50
mg/L hygromycin). The explants in 25 mg/L and 50 mg/L hygromycin survived even
when the explants were cultured continuously for another five weeks. However, shoot
regeneration of the surviving explants was inhibited when continuously cultured with
hygromycin treatment.
Control experiment was conducted with 0 mg/L hygromycin showed 100%
growth of shoots in all ages. Minimum concentration of hygromycin to inhibit growth
and cause mortality of explants was 75 mg/L (Table 3.3 and Figure 3.9). The significant
difference at 0.05 levels (P < 0.05) between hygromycin treatment and without treatment
showed hygromycin has an effect on mortality of explants. Each plate contained ten
explants with number of cotyledons explants used for each treatment was 60 explants.
88
Table 3.3 The effect of different concentrations of hygromycin in various ages of
explants on the mortality of explants using the unbombarded explants for minimal lethal
dose after five weeks in culture.
Hygromycin
Explant age
Percentage of mortality
(mg/L)
(days)
explants (%)
25
1
0
7
0
21
0
35
0
1
25
7
10
21
0
35
0
1
100
7
100
21
100
35
100
1
100
7
100
21
100
35
100
1
0
7
0
21
0
35
0
50
75
100
0
Note: Control with 0 mg/L hygromycin
89
A
B
C
Figure 3.9 The effect of different concentrations of hygromycin on explants after five weeks in culture.
A. 50 mg/L. Bar= 4cm.
B. 75 mg/L. Bar=4cm.
C. Control without hygromycin treatment. Bar=4cm.
90
3.3.8.2 The effect of delay selection on regeneration of transformed plant in
hygromycin media
This experiment was carried out to determine the time before the exposure of
explants to 75 mg/L hygromycin. The cotyledon explants were bombarded with pRQ6
(uidA+, hph+) plasmid. Transient gene expression was observed 24 h post-bombardment
showed GUS spots on explants to confirm the delivery of DNA plasmid. Timing of
selection on bombarded explant has effect on number of regenerating transformed plants.
This was supported by data analysis showed that there was a significant difference (P<
0.05) between selection using explants (1 day and 7 days) and delay selection using
shooted explants (21 days and 35 days) on percentage of regenerating transformed plants.
The bombarded cotyledon explants were transferred in various ages of 1 day, 7
days, 21 days and 35 days post-bombardment on MS media supplemented with 75 mg/L
hygromycin. Direct exposure of bombarded cotyledons explants 1 day and 7 days after
bombardment to hygromycin resulted on 100% mortality of explants. In contrast, delay
selection using 21 days and 35 days of shooted explants were resistant to hygromycin
with 28% and 54% percentage of plantlets, respectively after five weeks in culture.
This observation was achieved because of the hph gene action to inactivate the
hygromycin via a 4-O- phosphotransferase. Hph gene catalyzes the phosphorylation of
the 4-hydroxyl group on the cyclitol ring (hyosamine), thereby producing 7’-O-
91
phosphoryl-hygromycin B which totally lacks biological activity both in vivo and in
vitro (Pardo et al., 1985).
Therefore, delay selection was the optimal condition for plants production in 75
mg/L hygromycin. Table 3.4 shows the summary of the regenerating plants in which the
highest percentage of regeneration (54%) was obtained after five weeks in 75 mg/L
hygromycin. Figure 3.10(a) shows untransformed plant that could not survive in 75
mg/L hygromycin whereas Figure 3.10(b) shows plant resistant to hygromycin treated
after five weeks.
92
Table 3.4 The effect of selection on number of regenerating plantlets when cultured on
75 mg/L hygromycin and 100 mg/L hygromycin after five weeks in culture.
Hygromycin
Number
Explant
Number
Percentage
(mg/L)
of
age
of
of
explants
(days)
regenerating
regenerating
plantlets
plantlets
bombarded
(%)
100
75
80
1
0
0
80
7
0
0
80
21
5
7
80
35
18
23
80
1
0
0
80
7
0
0
80
21
16
20
80
35
43
54
80
1
80
100
80
7
80
100
80
21
80
100
80
35
80
100
0
Note: Control with 0 mg/L hygromycin.
93
A
C
B
D
Figure 3.10 The effect of hygromycin on plantlet regeneration and GUS assay of I. balsamina.
A. Untransformed plants in MS media supplemented with 75 mg/L hygromycin. Bar= 4cm.
B. Transformed plants in MS media supplemented with 75 mg/L hygromycin. Bar= 4cm.
C. Chimeric on young leaf. Bar=1cm.
D. Chimeric on roots. Bar=0.5cm.
94
3.3.8.3 Chimera expression of I. balsamina
The transformation via meristematic region led the chimera pattern that could
affect on resistancy of plants in selection system. In this study, the chimera pattern of I.
balsamina was observed using the GUS assay. However, over than 70% of blue stains
on leaves were observed and led the plants to survive when cultured in 75 mg/l
hygromycin after five weeks in culture. Figure 3.10 (c) and Figure 3.10 (d) show
chimera pattern on leaves and roots of I. balsamina, respectively.
3.3.8.4 The effect of hygromycin in selection system on regeneration of I. balsamina
The effect of optimal concentration of hygromycin (75 mg/L) on regeneration of
I. balsamina was studied. The age of shooted cotyledons explants of 21 days and 35
days exhibited percentage of regenerating plantlets with 20% and 54%, respectively in
the presence of 75 mg/L hygromycin. However, when the hygromycin was increased to
100 mg/L, the percentage of survival plants started to decrease up to 50% resulted only
7% (21 days of shooted explants) and 23% (35 days of shooted explants) percentage of
regenerated plantlets (Table 3.4 ). Therefore, in any experiment 75 mg/L hygromycin
will be used to obtain the transformed plant in delay selection method.
95
3.3.8.5 Transformation frequency
Transformation frequency was determined from number of GUS and polymerase
chain reaction (PCR) positive plants per number of explant bombarded as described by
Lee et al., (2003) (Section 3.2.15). Table 3.5 shows transformation frequency with
18.3% analyzed from GUS positive plant. Therefore, the success in biolistic
transformation of I. balsamina when using hph gene could be used as a basic system and
procedure for further study with other genes.
Transformation frequency = (14/40) x 84 x 100
160
= 18.3 %
Table 3.5 The effect of delay selection using 35 days shooting explants in 75 mg/L
hygromycin on regenerating of the transformed plantlets after five weeks in culture.
Treatment
Number of
Number of
Number
explants
regenerated
of GUS
bombarded
plants
+
plants/
Transformation
frequency
(%)
Total
plants
analyzed
75mg/L
160
84
hygromycin
96
14/40
18.3
3.3.8.6 Polymerase chain reaction (PCR) analysis of transformed plants
This experiment was carried out to see whether the hygromycin resistant plants
+
carry pRQ6 (hph ) transformed plasmid DNA. From a total of 40 GUS positive plants
analyzed, only 14 plants found to be PCR positive. The specific primers were designed
as mentioned earlier in section 3.2.16.3. Figure 3.11 also shows a representative from
14 plants sample of with approximately 0.8 kb DNA amplified as expected.
97
1
2
3
4
5
6
7
8
2 kb
1 kb
0.8 kb
0.6 kb
Figure 3.11 Agarose gel analysis of polymerase chain reaction (PCR) product of
transformed I. balsamina. A 0.8 kb DNA was amplified using specific primers for hph
gene.
Lane 1 and Lane 8: 1 kb DNA marker (Promega, Malaysia).
Lane 2: non-transformed plant cell (Negative control).
Lane 3-6: transformed plants showing 0.8 kb hph gene.
+
Lane 7: PCR of pRQ6 (hph ) plasmid (Positive control).
98
3.3.9
Biolistic transformation of bar gene for Basta (phosphinothricin) resistant
3.3.9.1 The effect of herbicide Basta (phosphinothricin) on I. balsamina
Initial experiment was carried out to determine whether Basta can react with
adult I. balsamina. From the experiment, I. balsamina (7 month old) showed 100%
mortality of plants after 48 h of exposure by spraying using Basta aqueous solution
(0.1% (v/v). The leaves started to change color to brown (necrosis) followed by the
stem (Figure 3.12).
A
B
Figure 3.12 The effect of Basta on I. balsamina after 48 h of exposure by spraying.
A. Control (without treatment). Bar= 13 cm
B. 0.1% of Basta. Bar= 15 cm
99
3.3.9.2 The effect of herbicide phosphinothricin (PPT) on plant tissue culture of I.
balsamina
This experiment was carried out to determine minimal lethal dose of herbicide
phosphinothricin (PPT) on tissue culture of I. balsamina. The untransformed explants in
various ages were tested at different concentrations of phosphinothricin (PPT) (0.5 mg/L,
1.0 mg/L, 5 mg/L and 10 mg/L) and the minimal lethal dose was determined. The
phosphinothricin (PPT) has effect on mortality of explants and data analysis showed that
there was significance difference (P < 0.05) on explants treated with or without (control)
of phosphinothricin (PPT).
When the explants were cultured on 1 mg/L
phosphinothricin (PPT), 100% mortality of explants was observed showed significant
difference (whereas, explants applied with 0.5 mg/L phosphinothricin (PPT) did survive)
(Table 3.6 and Figure 3.13). The explants turn to the black spots on the surface of
cotyledons due to inhibition of glutamine synthethase, a key enzyme in nitrogen
metabolism and leads to ammonium accumulation in explants (Eugene et al., 1991).
100
Table 3.6 The effect of different concentrations of phosphinothricin (PPT) on
unbombarded explants ages for minimal lethal dose after five weeks in culture.
Phosphinothricin
Explant age (days)
(mg/L)
0.5
1.0
5
10
0
Percentage of mortality
explants (%)
1
0
7
0
21
0
35
0
1
100
7
100
21
100
35
100
1
100
7
100
21
100
35
100
1
100
7
100
21
100
35
100
1
0
7
0
21
0
35
0
Note: Control with 0 mg/L phosphinothricin (PPT).
101
A
B
Figure 3.13 The effect of phosphinothricin (PPT) in different concentrations on shoots
for minimal lethal dose.
A. 0.5 mg/L phosphinothricin (PPT). Bar=2cm.
B. 1 mg/L phosphinothricin (PPT). Bar=2cm.
3.3.9.3 The effect of selection on phosphinothricin (PPT) media and GUS gene
expression.
The explants were co-transformed with pRQ6 (hph+) and pAHG11 (bar+). This
experiment was carried out to determine level of plant resistant and assess gene
expression in all ages of explant (1 day - 35 days old).
The bombarded explants with co-transformation of pRQ6 (hph+) and pAHG11
(bar+) resulted in 100% mortality for early (1 day and 7 days old explant) and delay
selection (21 days and 35 days old shooted explant) in media supplemented with 1 mg/L
102
phosphinothricin (PPT). However, GUS expression was observed in all explants (38%94%) depending on the age of explants and showed the presence of GUS gene from
pRQ6 (hph+) (Table 3.7). Therefore, the transformation was successfully achieved.
However bar gene was not expressed. It was maybe due to the bar gene driven by
ubiquitin promoter showed the ubiquitin promoter was not effective in dicots plants
compared to Cauliflower Mosaic Virus (CaMV) promoter. In control experiment, all
explants without the presence of phosphinothricin (PPT) in media were successfully
regenerated into plantlets.
103
Table 3.7 The effect of 1 mg/L phosphinothricin (PPT) on bombarded explants with cotransformation of pRQ6 (hph+, uid+) and pAHG11 (bar+) in regenerating transformed
plants.
Phosphinothricin Explant Percentage
(PPT)
age
(mg/L)
(days)
of GUS
Number of
Number of
Percentage
explants
regenerating
of
bombarded
explants
regenerating
+
explants
plants
(%)
(%)
1
0
1
94
80
0
0
7
87
80
0
0
21
49
80
0
0
35
38
80
0
0
1
0
40
40
100
7
0
40
40
100
21
0
40
40
100
35
0
40
40
100
Note: Control with 0 mg/L phosphinothricin (PPT).
104
3.4
Discussion
Transient GUS expression in the present study showed that cotyledon was the
most regenerative tissue (with axillary meristem at proximal part) and potentially useful
as the target tissues for biolistic transformation. The present investigation on biolistic
transformation of I. balsamina was carried out using pRQ6 (hph+) and co-transformation
of pRQ6 (hph+, uidA+) with pAHG11 (bar+) encoding for hygromycin and Basta
resistance, respectively.
The DNA is commonly bound to the tungsten or gold particles by calcium
chloride and spermidine co-precipitation. The sensitivity of the particles (toxic or nontoxic) against organism is varied. The E. coli plasmid was reported to be sensitive to the
tungsten compared to other bacteria plasmids (Russell et al., 1992). In the present study,
the plasmid DNA was coated using gold particles (1.0 µm) before bombardment due to
the toxicity of tungsten and the sensitivity of the plasmid to the tungsten. The toxicity of
the particles also contributed to the cell damage and reduced the ability of the transient
expression study in the optimization of biolistic parameters. In other reports, gold
particles were preferred as microcarriers for biolistic because of the uniformity,
spherical shape and inert nature (Jain et al., 1996; Russell et al., 1992).
The successful of using biolistics technique depends on the physical and
biological parameters, therefore the optimization of both parameters (physical and
biological parameters) are maximally effective for transformation study (Kikkert et al.,
2003). This study suggested bombardment using the biological and physical factors on
105
GUS gene expression showed high average number of GUS spots per explants with
149.3 spots and 128.1 spots when using plasmid pRQ6 (hph+) and co-transformation
with pAHG11 (bar+), respectively. The necessity to optimize the velocity using the
target distance and helium pressure is for optimal transformation rates with different
tissues types, depending on the cell wall thickness and the need to penetrate several
layers (Birch and Bower, 1994). In the present study, the GUS gene expression was
assessed at different target distances and helium pressure. The highest GUS gene
expression was observed when using 9 cm target distance and 1100 psi helium pressure.
These results were achieved as the organized tissues with thicker cells walls required
higher particle velocities for penetration than thin walled cells from suspension cultures
(Birch and Bower, 1994). This concurs with the single bud of banana (Sreeramanan et
al., 2005), sorghum (Tadesse et al., 2003), meristematic explants of cowpea (Ikea et al.,
2003) and in embryogenic suspension cells of rice (Jain et al., 1996).
Multiple bombardments can be achieved from empirical investigations. The
balance between increased gene transfer and cell injury determined from it was based on
specific target tissue and bombardment conditions (Sreeramanan et al., 2005; Wong,
1994; Birch and Bower, 1994). However, the two times bombardments in this study
caused the explants injury although the GUS gene expression was higher than using one
time bombardment (King and Kasha, 1994). In contrast, the two times bombardments
were sufficient to give high transient GUS gene expression in single buds of banana with
100 of average number GUS expressing spots (Sreeramanan et al., 2005). This finding
supported by Ikea et al., (2003) who reported that bombarding the explants twice
increased the number of GUS gene expression and this could be due to the fact that
106
multiple bombardments allowed better coverage of the target areas and compensated for
misfire from faulty and poorly set rupture disc.
The precipitation of different concentrations DNA onto gold particles (1.0 µm)
was tested to obtain the potential amount for DNA delivery.
The use of high
concentrations of DNA (1.5 µg) gave the highest GUS gene expression. However,
increasing the concentrations of DNA precipitated increased transient expression
frequencies. The particle aggregation will occurs due to high amount of DNA and
resulted on poor dispersal and increased cell damage (Birch and Bower, 1994). There
was also no significant effect in increasing the plasmid concentration to 1.5 µg DNA.
Therefore, the use of 1.0 µg of DNA also resulted in the maximum GUS gene
expression.
Osmoticum has also shown to have major effect on transformation efficiency in
bacteria and for both the chloroplast and the nucleus in plants (Birch, 1997). This was
proved with the previous reports on the use of osmotic media in pre-culture treatment
before bombardment.
In order to test the effects of osmotic agent on GUS gene
expression, cotyledons explants were cultured in various types (mannitol, sorbitol,
mixture of mannitol and sorbitol) and concentrations (0.2 - 0.6 M) with the highest GUS
gene expression (110.2 spots) was obtained using 0.4 M mannitol and sorbitol. Osmotic
treatment using higher concentration at 0.6 M mannitol and sorbitol decreased number
of GUS spots per explant (71 spots) in this study due to osmotic adjustment, which
allowed the cells to return to hypertonic state (Santos et al., 2002). This is in agreement
with Ye et al., (1990) who found that biolistic transformation of higher chloroplast
107
plants using mixture of sorbitol and mannitol at 0.1 M increased dramatically (20-fold)
the number of GUS spots per plate (100 spots) compared to the used of higher
concentrations at 1.5 M significantly reduced to lower spots (5 spots). Lee et al., (2003)
reported that mixture of 0.2 M sorbitol and 0.2 M mannitol resulted in significant 3.2
fold increase in transient expression frequency of GUS gene of rice callus culture. In
contrast, the used of different between osmotic treatments (mannitol, sorbitol and
mixture of mannitol and sorbitol) at 0.25 - 1.0 M on calli of cocoa resulted on the
highest GUS gene expression (40 spots) when using 0.25 M of mannitol alone with no
significance different between treatments (Santos et al., 2002). The use of mannitol
alone as the osmotic treatments in bombardment was also reported in bluegrass (Gao et
al., 2005), orchid (Men et al., 2003) and rice (Jain et al., 1996). Osmotic treatments
involved the reduction of the volume of the vacuoles increased the possibility to reach
the nucleus, resulting in larger number of cells successfully expressing the introduced
gene (Birch and Bower, 1997). The pre-culture treatment using osmotic treatment for 4
h and 32 h prior to bombardment in this study lower average number of GUS spots per
explant with 5.8 spots and 31.4 spots, respectively.
The observation on physical
appearance of cotyledon in this study showed that cotyledon could not regenerate shoots
as effect on damage at meristem site of cotyledon due to the insufficient time for the
cells to uptake the osmotic treatment to prevent the damage to surrounding cells by
shock wave during bombardment (Birch and Bower, 1997). This finding was supported
by Santos et al., (2002) who found when pre-culture time was reduced to 4 h, lower
GUS spots per explant (10 spots) on calli of cocoa was observed compared to 16 h preculture time (40 spots per explant). The pre-culture osmotic treatment (0.4 M sorbitol
and mannitol) for 16 h prior bombardment in this study gave higher GUS gene
108
expression (110.7 spots). Similar finding was reported before on calli of rice with high
GUS spots per cells ( 893 spots) when pre-culture using osmotic treatment (0.4 M
sorbitol and mannitol) for 16 h prior bombardment (Lee et al., 2003)
In the present study, 24 h incubation post-bombardment showed higher average
number of GUS spots (110.7 spots) as compared to 4 h (19 spots) incubation time. This
is could be due to the cells recovery from injuries caused by bombardment. Other
results showed prolonged post-bombardment incubation time from two to six days for
had improved transformation frequency of Vitis vinifera L. (56%) (Vidal et al., 2003).
Sreeramanan et al., (2005) reported that it took at least six days for banana cells to
recover from the injuries of bombardment. Using the optimal bombardment conditions
as described in Section 3.3.1 to Section 3.3.7, bombarded explants showed high GUS
gene expression on explants including at the axillary meristem, the region that started
the regeneration of multiple shoots.
In order to find out the minimal lethal dose of hygromycin and phosphinothricin
(PPT), the effect of different concentrations of hygromycin and phosphinothricin (PPT)
on explants were conducted. The results showed all explants became necrosis at 75
mg/L of hygromycin and 1 mg/L phosphinotricin (PPT). In contrast, 50 mg/L
hygromycin was the optimal concentrations that inhibited the growth of calli in rice (Lee
et al., 2003; Li et al., 1993) and cotyledons of pepper (Li et al., 2003).
109
Delay the exposure time of selective agents (hygromycin; phophinothricin) at
later regeneration phase (Jones et al., 2005) was reported before on pepper explants
using delay selection within 2 to 7 days post-bombardment (Li et al., 2003).
In the
present study, delay selection using 35 days of shooted explants produced after
bombardment resulted on 84 regenerated plantlets at 75 mg/L hygromycin. However,
early selection (1 day and 7 days of explant post-bombardment) at 75 mg/L contributing
100% of explants mortality. This finding concurs with Li et al., (2003) who reported
that delay selection led to an increased in differentiation efficiency for all genotype with
transformation frequency in range 70% to 87% of pepper (Capsicum annuum L.).
Ghosh et al., (2002) reported on delay selection (6 weeks post-bombardment) when
culturing shooted explants of jute (Corchorus capsularis L.) on selection medium
containing 2 mg/L bialaphos resulted on 34 bialaphos-resistant shoots PCR positive of
bar gene out of 248 bombarded explants. In contrast, Men (2003) reported that delay
selection (20 days) resulted in relatively lower transformation efficiency (0.5%) of
orchids as compared to early selection using 10 days of post-bombardment explants with
(4%).
In order to obtain the transformed plants in the present study, the bombarded
shooted explants of 35 days were cultured on MS medium containing 75 mg/L
hygromycin produced 54% of hygromycin resistant plantlets. Only 40 GUS positive
plants were used for polymerase chain reaction (PCR) analysis. Results showed only 14
plants were PCR positive suggested the presence of hph gene. Initially, the GUS spots
were detected in cotyledons explants however, in development of leaves, the spots
disappeared and the expression was nearly on the whole leaf. This finding concurs with
110
Gobert et al.,(2006) in Arabidopsis reported that initially, expression occurs in small
clusters of the cells that tend to concentrate near the leaf vasculature and cotyledon
periphery, creating the spotty pattern. However, during the development of leaf, the
expression becomes more ubiquitous around vascular bundles.
In this study, GUS positive explants exhibited chimera pattern. However, the
blue staining of GUS was over 70% of the entire leaf surface and roots surface led to the
survival of plants in the hygromycin selection. According to Birch and Bower (1994),
the advantage of meristematic target tissue is that it can be excised and regenerated to
plants with minimal time in tissue culture as observed in this study when plantlets were
produced within eight weeks in culture. However, transformed plants are likely to be
chimeric as observed in biolistic transformation of soybean and cotton.
The genomic DNA in this study was isolated using cetyl metyl ammonium
bromide (CTAB) modified method (Zidani et al., 2005).
The extraction process
involves digesting away the cell walls in order to release the cellular constituents
followed by disruption of the cell membranes to release DNA into the extraction buffer
using cetyl metyl ammonium bromide (CTAB). The released DNA was protected from
endogenous nuclease by the use of EDTA. The DNA quality was estimated at ratio of
A260nm / A280nm which varied between 1.8 to 2 and low samples results on lower DNA
contents with lower ratio below 1.8. The initial DNA extracts often contain a large
amount of RNA, proteins, polysaccharides and tannins which may interfere with the
extracted DNA and may be difficult to separate.
However, denaturation and
precipitation from the extract could remove the proteins. The ultra centrifugation step is
111
very efficient in removing all impurities such as proteins, oligonucleotide and
polysaccharides. On the other hand, the heat treatment of RNAase was used to remove
the RNA (Zidani et al., 2005). Other DNA extraction technique in the present study was
not carried out.
Khan et al., (2001) documented the first report regarding the successful use of
monocot (maize) derived ubiquitin promoter for the insectidal cry gene of Bacillus
thuringiensis (Bt gene) expression in dicot system. Therefore, there is a possibility of
the used of ubiquitin promoter for bar gene expression in I. balsamina. Using the
biolistic particles delivery, the transformation using bar gene driven by different
promoters (ubiquitin promoter in monocots and Cauliflower Mosaic Virus (CaMV)
promoter in dicot) has been achieved in monocots and dicotyledonous such as Kentucky
bluegrass (Gao et al., 2005), cowpea (Ikea et al., 2003), pearl millet (Goldman et al.,
2003), jute (Ghosh et al., 2002), rice (Jain et al., 1996) and barley (Stiff et al., 1995).
The co-transformation was carried out using the pRQ6 (hph+, uidA+) and pAHG11
(bar+). The GUS positive explants indicated the successful co-transformation of pRQ6
(hph+, uidA+) with pAHG11 (bar+) into the explants.
However, none of the
regenerating plantlets survive in media selection containing 1 mg/L phosphinotricin
(PPT). It was maybe due to no bar gene expression because of the used of ubiquitin
promoter.
Birch and Bower (1994) reported the rate of conversion from transient
expression to stable incorporation of DNA delivery in plant cells range from 1% up to
9%.
The high frequency of GUS spots was very important to achieve the stable
112
expression as only a few percent of transiently expressing cells integrate and stably
express the foreign gene. Currently, the transformation frequency of 18.3% was
achieved from PCR positive plants (14) out of 40 analyzed plants (GUS positive) from
regenerated plantlets (84) in 75 mg/L hygromycin. The transformation frequency was
calculated based on number of resistant plant (GUS and PCR positive) out of number of
explants bombarded (n=160 explants). This results showed that the possibility of I.
balsamina to achieve the stable transformation via biolistics.
113
3.5
Conclusion
Maximum GUS expression with 149.3 average numbers of GUS spots per
explants and 95% percentage of GUS positive explants was obtained when the explants
were cultured in media with osmotic treatment containing MS medium supplemented
with 1 mg/L BAP and 0.4 M mannitol and sorbitol for 16 h of pre-culture treatment.
Then, the explants were bombarded using 1.0 µg plasmid DNA, 9 cm target distance,
1100 psi with one time bombardment. The post-bombardment explants incubation time
for 24 h enhanced the transient expression assay. Regenerating of the transformed
plants was achieved with delay selection. The 35 days shooted explants after
bombardment were cultured in MS medium supplemented with 75 mg/L hygromycin in
five weeks. The transformation frequency of I. balsamina with 18.3% proved the
possibility of the cotyledonous explants in regenerating transformed plants towards
stable transformation.
114
CHAPTER 4
General Conclusion
In conclusion, the regeneration system of I. balsamina with high percentage of
regenerating plants was achieved using cotyledons explants of I. balsamina on MS
medium supplemented with 1 mg/L BAP for shoots induction and half strength MS
media supplemented with 0.1 mg/L indole acetic acid (IAA) for roots induction. The
optimization of biolistics conditions (physical and biological parameters) was used on
biolistic transformation using plasmid pRQ6 (hph+, uidA+) and co-transformation of
pRQ6 (hph+, uidA+) and pAHG11 (bar+), which resistance to antibiotic (hygromycin)
and herbicide (Basta), respectively. The GUS positive explants obtained in 24 h postbombardments showed the success for transformation and co-transformation. However,
there was no shoots regenerated when explants were cultured on phosphinothricin (PPT)
media.
In contrast, timing on selection showed that delay selection gave the high
number of regenerating plants (GUS positive) on hygromycin selection media. The
115
transformed plants resistant to hygromycin exhibited the GUS gene expression in
chimeric pattern. The GUS positive explants and shoots showed the presence of uidA
gene while PCR positive plants showed the presence of hph gene for hygromycin
resistant. Transformation of I. balsamina was successful (18.3% transformation
frequency) with the possibility of obtaining stable transformation.
116
FUTURE WORK
The bombardments conditions and the selection system in the present study
could be used for future work. The chimeral study of I. balsamina may also be carried
out for further study to enhance the transformation efficiency.
The production of
plantlets from adventitious shoots of I. balsamina should be used in biolistic
transformation. The use of adventitious shoots in transformation possibly could reduce
the chimera in I. balsamina and stable transformation could be achieved. There was no
report regarding stable transformation on I. balsamina and this work could be used for
further study to produce transgenic I. balsamina.
The establishment of biolistic technique in this study could be used for producing
herbicide resistant of I. balsamina. This technique also could be used for other herbicide
resistant gene expression in plant including dehalogenase gene that resistant to herbicide
Dalapon. The lack of success to produce Basta resistant plants in the present study
could be due to the use of the ubiquitin promoter. Transgenic I. balsamina may be
produced using the bar gene driven by cauliflower mosaic virus (CaMV) promoters as
reported earlier in other dicotyledons plants.
117
I. balsamina has been used as a model in inflorescence study of research to
enhance the yield in floral industry. The biolistic method in this study was used instead
of A. tumefaciens due to the explant type (multiple shoot) that produced high number of
shoots. The multiple shoots arise from axillary meristem without wound side that
available for the infection of A. tumefaciens. There is possibility of transformation using
A. tumefaciens by creating the wound side of meristem however it may cause injury at
the axillary meristem and could affect on the shoot regeneration at latter stage which
decreased number of shoots (Saini and Jawal, 2005). The data in this study will also be
useful for developing bombardments transformation methods for other plants using other
genes. This study had also provided information towards understanding various aspect
of biolistic-mediated transformation.
118
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APPENDICES
Appendix A
Murashige and Skoog (1962) media containing the macro element, micro element ,
iron source and vitamins.
Macro element
CaCl2
KH2PO4KNO3
MgSO4
NH4NO3
Concentrations (mg/L)
332.02
170.00
1900.00
180.54
1650.00
Micro element
CoCl2. 6H2O
CuSO4. 5H2O
HBO3
KI
MnSO4. H2O
Na2 MoO4.2H2O
ZnSO4.7H2O
0.025
0.025
6.20
0.83
16.9
0.25
8.6
Vitamins
Glycine
Myo-Inositol
Nicotinic acid
Pyridoxine HCl
Thiamine HCl
2.00
100.00
0.50
0.50
0.10
Iron Source
FeSO4.7H2O
Na2 EDTA .2H2O
278.00
373.00
136
Appendix B
Plant growth regulators and the solvents used for stock preparation.
Plant Growth Regulator
Solvents
Benzylaminopurine (BAP)
Ethanol/ 0.1M Natrium hydroxide (NaOH)
Thidiazuron (TDZ)
0.1M Natrium hydroxide(NaOH)
Indole acetic acid (IAA)
0.1M Natrium hydroxide (NaOH)
Indole butyric acid (IBA)
Ethanol/ 0.1M Natrium hydroxide (NaOH)
137
Appendix C
Plant growth regulators and the chemical names.
Plant growth
Abbreviation
Chemical name
regulators
Auxin
Cytokinin
IAA
Indole acetic acid
IBA
Indole butyric acid
NAA
α-naphthalene acetic acid
2,4-D
2,4-dichlorophenoxyacetic acid
BAP
Benzylaminopurine
Kinetin
Furfurylaminopurine
TDZ
1-phenyl-3-(1,2,3,-thiadiazol-5-yl)urea
Zeatin
(Thidiazuron)
4-hydroxy-3-methyl-trans-2butenlaminopurine
Appendix D
Luria -bertani Media
(g/L)
Bacto tryptone
10
Bacto yeast
5
Natrium chloride (NaCl)
10
pH was adjusted to 5.7 and for agar plates,
15g/L Bacto agar was added. Media were
autoclaved at 121°C
138
Appendix E
The bacterial growth with different plasmid in LB media with ampicilin.
Bacterial
Plasmid
Antibiotic
Escherichia coli (E.coli)
pRQ6 (hph )
50 mg/ml ampicilin
Escherichia coli (E.coli)
pAHG11 (bar )
+
50mg/ml ampicilin
+
139
Appendix F
Plasmid pRQ6 (8.25kb) with uidA ß-glucuronidase (GUS) gene and hph gene encoding
for hygromycin resistant.
140
Appendix G
Plasmid pAHG11 (7.3 kb) with bar gene confers to Basta (PPT) resistant.
`
141