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    Agrobacterium Mediated Genetic Transformation of Potato

    By

    WASEEM AHMAD

    Department of Biochemistry Faculty of Biological Sciences Quaid-i-Azam University Islamabad, Pakistan

    2010

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    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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    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