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i
Agrobacterium Mediated Genetic Transformation of Potato
By
WASEEM AHMAD
Department of Biochemistry Faculty of Biological Sciences Quaid-i-Azam University Islamabad, Pakistan
2010
ii
Agrobacterium Mediated Genetic Transformation of Potato
Submitted by
WASEEM AHMAD
Thesis Submitted to
Department of Biochemistry
Quaid-i-Azam University, Islamabad
In the partial fulfillment of the requirements for the degree of
Doctor of Philosophy
in
Biochemistry / Molecular Biology
Department of Biochemistry Faculty of Biological Sciences
Quaid-i-Azam University Islamabad, Pakistan
2010
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To my beloved brother Nadeem Ahmad
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Contents
Chapter 1
1.1
1.2
1.3
1.4
1.5
1.5.1
1.6
1.6.1
1.6.1.1
1.6.2
1.6.2.1
1.6.3
1.7
1.7.1
1.7.2
1.7.3
1.7.4
1.7.5
1.7.6
1.8
1.8.1
1.8.2
1.8.3
1.8.4
1.9
Acknowledgements
List of Tables
List of Figures
List of Abbreviations
Abstract
Introduction and Review of Literature
Uses of potato and its nutritional value
Production of potato
Diseases of potato
Measures for diseases control
In vitro regeneration
In vitro regeneration of potato
Genetic transformation
Biolistic transformation
Biolistic transformation of potato
Agrobacterium-mediated transformation
Agrobacterium mediated transformation of potato
Genetic modifications of potato for disease resistance
Defense mechanisms in plants
Hypersensitive response
Cell wall fortification
Pathogen related proteins
Salicylic acid and Benzoic acid
Phytoalexins
Phytoanticipins
Plant defense mechanisms and role of secondary
metabolites
Role of antimicrobials in plant defense
Role of antioxidants in plant defense
Role of phenolics in plant defense
Role of flavonoids in plant defense
rol genes of Agrobacterium rhizogenes
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3
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1.9.1
1.9.2
1.10
1.11
Chapter 2
2.1
2.2
2.3
2.3.1
2.3.2
2.3.3
2.3.4
2.4
2.4.1
2.4.2
2.4.3
2.4.4
2.4.5
2.4.6
2.5
2.5.1
2.5.2
2.6
2.7
2.7.1
2.7.2
2.8
2.8.1
2.9
Contents
Functions of rolA gene in transformed plants
Functions of rolC gene in transformed plants
rol genes and secondary metabolites production
Objectives
Materials and Methods
Glassware and chemicals
Plant material
In vitro regeneration
Callus induction
Shoot induction
Root induction
Plant acclimatization
Biolistic gene transfer
Agrobacterium maintenance and culture
Plasmid isolation
Preparation and coating of gold particles
Optimization of biolistic transformation
Effect of osmotic treatment
Histochemical gus assay
Agrobacterium mediated transformation
Optimization of Agrobacterium mediated transformation
Effect of antibiotics on explant survival
Agrobacterium mediated stable transformation with gus
gene
Stable transformation with rol genes
Gene sequencing
Agrobacterium mediated stable transformation of potato
with rolA and rolC gene
PCR analysis of the transformants
Agarose gel electrophoresis
Southern blot analysis
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2.9.1
2.9.2
2.9.3
2.9.4
2.9.5
2.10
2.11
2.12
2.13
2.14
2.15
Chapter 3
3.1
3.1.1
3.1.1.1
3.1.1.2
3.1.1.3
3.1.1.4
3.1.1.5
3.1.2
3.1.2.1
3.1.2.2
3.1.2.3
3.1.2.4
3.1.2.5
3.1.3
Contents
DNA restriction
Agarose gel electrophoresis
Transfer of restriction fragments to membrane
Labeling of DNA using [α-32 P]
Hybridization process
Extraction of transgenic plants
Antifungal activity
Antibacterial Activity
Determination of antioxidant activity
Determination of total phenolics
Determination of total flavonoids
Results
Optimization of in vitro culture system
Callus induction
Effect of medium on callogenesis
Effect of cultivar on callogenesis
Effect of explant on callogenesis
Effect of interaction among callus induction medium,
cultivar and explant type on percentage callus induction
Effect of Interaction among callus induction medium,
cultivar and explant type on number of days to form callus
Shoot induction
Effect of medium on shoot induction
Effect of cultivar on shoot induction
Effect of explant on shoot induction
Effect of interaction among shoot induction medium,
cultivar and explant type on percentage shoot induction
Effect of interaction among shoot induction medium,
cultivar and explant type on number of days to shoot
induction
Root induction
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3.1.3.1
3.1.3.2
3.1.3.3
3.1.4
3.2
3.2.1
3.2.2
3.2.3
3.2.4
3.2.5
3.2.6
3.2.7
3.3
3.3.1
3.3.2
3.3.3
3.3.4
3.3.5
3.3.6
3.3.6.1
3.3.6.2
3.4
3.4.1
3.4.2
Contents
Effect of media on root induction
Effect of cultivar on root induction
Effect of interaction between medium and cultivar on
initiation time, length and number of roots
Plant acclimatization
Optimization of transformation through biolistic gun
Effect of helium pressure on transient gus expression
Effect of target distance on transient gus expression
Effect of particle size on transient gus expression
Effect of explant type on transient gus expression
Effect of interaction among helium pressure, target
distance, particle size and explant type on transient gus
expression
Effect of osmotic treatment on percentage transient gus
expression
Effect of osmotic treatment on percentage callus formation
Optimization of Agrobacterium-mediated transformation
Effect of bacterial density on transient gus expression
Effect of inoculation time on transient gus expression
Effect of co-cultivation period on transient gus expression
Effect of explant type on transient gus expression
Effect of interaction among bacterial density, inoculation
time, co-cultivation period and explant type on transient
gus expression
Effect of antibiotics on explant survival
Effect of cefotaxime on explant survival
Effect of kanamycin on explant survival
Agrobacterium-mediated stable transformation of potato
with gus reporter gene
Histochemical gus assay of the putative transformants
Polymerase chain reaction (PCR) analysis of the putative
gus transformants
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3.5
3.5.1
3.5.1.1
3.5.1.2
3.5.2
3.5.2.1
3.5.2.2
3.6
3.7
3.8
3.9
3.10
Chapter 4
4.1
4.2
4.3
4.4
4.5
4.6
Contents
Agrobacterium-mediated stable transformation of potato
with rolA and rolC gene
Agrobacterium-mediated stable transformation of potat
