DYNAMICS OF OsRGLP1 GENE DURING FUNGAL EXPOSURE

IN TRANSGENIC POTATO

NADIA MAJEED

00-arid-946

Department of Biochemistry

Faculty of Sciences

Pir Mehr Ali Shah

Arid Agriculture University Rawalpindi

Pakistan

2016

DYNAMICS OF OsRGLP1 GENE DURING FUNGAL EXPOSURE

IN TRANSGENIC POTATO

by

NADIA MAJEED

(00-ARID-946)

A thesis submitted in partial fulfillment of

the requirements for the degree of

Doctor of Philosophy

in

Biochemistry

Department of Biochemistry

Faculty of Sciences

Pir Mehr Ali Shah

Arid Agriculture University Rawalpindi

Pakistan

2016

ii

CERTIFICATION

I hereby undertake that this research is an original one and no part of this thesis

falls under plagiarism. If found otherwise, at any stage, I will be responsible for the

consequences.

Name: NADIA MAJEED Signature: ____________________

Registration No. 00-arid-946 Date: 2016

Certified that the contents and form of thesis entitled “ Dynamics of

OsRGLP1 Gene During Fungal Exposure in Transgenic Potato ” submitted by Ms.

Nadia Majeed have been found satisfactory for the requirement of the degree.

Supervisor: _____________________________ (Prof. Dr. S. M. Saqlan Naqvi)

Co-supervisor: _____________________________

(Dr. Iqbal Hussain)

Member: _____________________________

(Dr. M. Javaid Asad)

Member: _____________________________

(Prof. Dr. Abdul Rauf)

Chairperson: _______________________________________

Dean, Sciences: _____________________________________

Director, Advanced Studies: ___________________________

iii

Dedicated to my Family

iv

CONTENTS

Page

LIST OF FIGURES ix

LIST OF PLATES x

LIST OF TABLES xi

LIST OF ABBREVIATIONS xii

ACKNOWLEDGEMENTS xiii

ABSTRACT xv

1. INTRODUCTION 1

2. REVIEW OF LITERATURE 7

2.1 GERMIN AND GERMIN LIKE PROTEINS (GLPs) 7

2.1.1 Germins 7

2.1.2 Germin like Proteins (GLPs) 8

2.2 BIOCHEMICAL/ENZYMATIC PROPERTIES 8

2.2.1 Oxalate Oxidase Activity 8

2.2.2 ADP-glucose Phosphodiesterase/Pyrophosphatase Activity 9

2.2.3 Superoxide Dismutase Activity 9

2.3 GLPs IN RESPONSE TO ABIOTIC STRESSES 10

2.4 GERMINS & GLPs IN DEFENSE AGAINST

PATHOGENESIS

10

2.5 GERMIN & GLPs IN DEFENSE AGAINST FUNGAL

PATHOGENS 11

2.6 POTATO AN IMPORTANT AGRONOMICAL CROP 11

2.7 POTATO PRODUCTION IN PAKISTAN 12

2.8 POTATO VARIETIES GROWN IN PAKISTAN 13

2.8.1 Desiree 13

v

2.9 MAJOR FUNGAL DISEASES OF POTATO 13

2.9.1 Potato Late Blight 14

2.9.2 Black Dot 14

2.9.3 Potato Pink Rots 15

2.9.4 Black Scurf 15

2.9.5 Fusarium Dry Rot and Wilt 15

2.10 AGROBACTERIUM-MEDIATED DNA TRANSFER 16

2.11 GENETIC ENGINEERING, AS TOOL FOR CROP

IMPROVEMENT 17

2.12 MOLECULAR RESISTANCE IN POTATO 19

3 MATERIALS AND METHODS 22

3.1 PLANT MATERIAL 22

3.2 PLANT TRANSFORMATION 22

3.2.1 Electrocompetent Cell Preparation 22

3.2.2 Transformation of Agrobacterium tumefaciens Strain GV3101 22

3.2.3 Confirmation of Selected Clones of Agrobacterium tumefaciens

GV3101 by PCR 25

3.2.4 Pre-Culturing or Pre-Conditioning of Explants 25

3.2.5 Infection and Co-culturing of Explants with Transformed

GV3101

26

3.2.6 Culturing of Explants on Pre Selection Medium 26

3.2.7 Culturing on Selection Medium Containing Suitable Antibiotic 26

3.2.8 Shifting of Explants to Rooting Medium 26

3.3 CONFIRMATION OF GENE TRANSFER 27

3.3.1 Genomic DNA Isolation from Selected Plants 27

3.3.2 Confirmation of Transfer of OsRGLP1 by PCR 28

vi

3.4 EXPRESSION ANALYSIS 28

3.4.1 RNA Isolation 28

3.4.2 DNA Contamination Removal 29

3.4.3 One Step RT-PCR with Platinum Taq 29

3.5 RELATIVE QUANTIFICATION OF TRANSGENE IN REAL

TIME

31

3.5.1 RNA Isolation 31

3.5.2 cDNA synthesis 32

3.5.3 Routine PCR with cDNA 32

3.5.4 Real Time PCR 32

3.6 PLANT ACCLIMATION 33

3.7 MORPHOLOGY AND GROWTH ANALYSIS OF

TRANSGENIC LINES IN COMAPRISON WITH WILD TYPE

CONTROL

33

3.7.1 Plant Height 34

3.7.2 Number of Shoots per Plant 34

3.7.3 Number of Leaves per Plant 34

3.7.4 Number of Tubers Harvested per Plant 34

3.8 FUNCTIONAL EVALUATION OF TRANSGENE 34

3.8.1 Protein Estimation of Plant Leaf Samples before SOD Assay 34

3.8.2 In Solution Superoxide Dismutase Activity 35

3.8.3 Detection of Localized H2O2 Level by DAB uptake method 36

3.8.4 Oxalate Oxidase (OXO) Activity Assay 36

3.9 ANTI-FUNGAL ASSAYS WITH SELECTED HIGHLY

EXPRESSED LINES

36

3.9.1 Disease Incident Assays of Transgenic Potato with F. oxysporum

f.sp .tuberosi 36

vii

3.9.1.1 Multiplication of Fungal Culture on PDA 37

3.9.1.2 Inoculum Preparation 37

3.9.1.3 Inoculation and Disease Incidence Scoring 37

3.10 DATA HANDLING AND STATISTICAL ANALYSIS 38

4 RESULTS AND DISCUSSION 39

4.1 TRANSFORMATION OF AGROBACTERIUM STRAIN GV3101

WITH RECOMBINANT VECTORS pC:OsRGLP1 AND

pG:OsRGLP1

39

4.2 AGROBACTERIUM MEDIATED TRANSFORMATION OF

POTATO

39

4.2.1 Transformation of Potato with GV3101 harboring expression

vectors 41

4.2.2 Selection and Regeneration of Plants 41

4.3 MOLECULAR ANALYSIS 42

4.3.1 Confirmation of Presence of Transgene by PCR

44

4.3.2 Transcription/Expression Analysis

44

4.3.2.1 One Step Reverse Transcriptase PCR using Platinum Taq

44

4.4 COMPARISON OF MARKER ASSISTED

TRANSFORMATION EFFICIENCES FOR OsRGLP1

49

4.5 RELATIVE QUANTIFICATION OF OsRGLP1

49

4.6 SHIFTING OF PLANTS TO GREEN HOUSE CONDITION

50

4.7 COMPARATIVE ANALYSIS OF MORPHOLOGY AND

GROWTH 50

4.7.1 Plant Height

54

4.7.2 Number of Shoots per Plant

54

4.7.3 Leaves number per Plant

56

4.7.4 Number of Tubers Harvested per Plant

56

4.8 FUNCTIONAL EVALUATION OF TRANSGENE

56

viii

4.8.1 Oxalate Oxidase (OXO) Activity Assay

56

4.8.2 In Situ Detection of H2O2 in Potato Leaves

60

4.8.3 Total Protein Estimation of Plant Leaf Samples before SOD

Assay

62

4.9 SUPEROXIDE DISMUTASE ACTIVITY DETERMINATION

62

4.9.1 High Temperature Effect on SOD Activity

65

4.9.2 Effect of KCN and H2O2 on SOD Activity

67

4.10 ANTIFUNGAL ASSAYS WITH SELECTED HIGHLY

EXPRESSED LINES WITH F. OXYSPORUM F. SP. TUBEROSI

70

4.10.1 Inoculation and Disease Incidence Scoring

70

SUMMARY 80

LITERATURE CITED 82

ANNEXURES 100

ix

LIST OF FIGURES

Fig. No. Page

1 A) Map of the pCAMBIA1301, the expression vector B) Modified

T-DNA region 23

2 Map of the pH7WG2, the expression vector B) Modified T-DNA

region 24

3 Transcript analysis of OsRGLP1 in untransformed control and

transgenic lines using EF-1α as internal control 52

4 Comparison of plant height 55

5 Comparison of number of shoots per plant 55

6 Comparison of number of leaves 57

7 Comparison of tubers harvested per plant 57

8 Calibration Curve with 1mg/ml BSA 63

9 Measurement of SOD activity in leaf extracts of untransformed

control and transgenic potato plants.

66

10 High temperature effect on SOD activity in control and transgenic

samples

68

11 KCN treatment effect on SOD activity in untransformed control

and transgenic samples

69

12 H2O2 treatment effect on SOD activity in untransformed control

and transgenic samples

71

13 Percentage survival rate of potato plants infected with Fusarium

oxysporum f.sp. tuberosi

76

14 Percentage survival rate of potato plants infected with Fusarium

oxysporum f.sp. tuberosi

77

15 Survival rate of untransformed control plants (D-WT) 78

x

LIST OF PLATES

Plate. No. Page

1 Confirmation of presence of OsRGLP1 through Colony PCR 40

2 Confirmation of OsRGLP1 through Colony PCR 40

3 Confirmation of presence of OsRGLP1 in transgenic lines from

pC:OsRGLP1 transformation

45

4 Confirmation of presence of OsRGLP1 in transgenic lines from

pG:OsRGLP1 transformation

45

5 One step RT PCR of EF-1α with independent transgenic lines

from pC:OsRGLP1

47

6 One step RT PCR of OsRGLP1 with independent transgenic lines

from pC:OsRGLP1

47

7 One step RT PCR of EF-1α with independent transgenic lines

from pG:OsRGLP1

48

8 One step RT PCR of OsRGLP1 with independent transgenic lines

from pG:OsRGLP1

48

9 Reverse transcriptase PCR with Ef-1α 51

10 Reverse transcriptase PCR with Ef-1α 51

11 Different stages of potato transformation from tissue culturing to

greenhouse condition

53

12 Oxalate oxidase activity determination in transgenic and

untransformed control plants

59

13 Detection of H2O2 by DAB staining in leaf disks 61

14 Different Stages of Antifungal Assay in Desiree potato plants 72

15 Visual comparison between infected transgenic potato plants

expressing OsRGLP1 and untransformed control plants

75

xi

LIST OF TABLES

Table. No. Page

1 Comparison of two recombinant vectors for OsRGLP1 transfer

efficiency in Potato cv Desiree

43

2 Calculated Protein Concentration by Lowry Method 64

3 Disease scores on potato plants infected with Fusarium

oxysporum f. sp. tuberosi

73

xii

LIST OF ABBREVIATIONS

µL Micro liter

Bp Base Pair

cDNA Complementary DNA

CTAB CetylTrimethyl Ethyl Ammonium Bromide

DNA Deoxyribonucleic Acid

E. coli Escherichia coli

EDTA Ethyl dimethyl tetra acetic acid

G Gram

GA3 Gibberellic Acid

GLP Germin like protein

IAA Indole Acetic Acid

L Liter

LB Lauria-Bartani

M Molar

Mg Mili gram

mL Mili Litre

mM Mili molar

MS Murashige and Skoog

NaCl Sodium chloride

NBT Nitro Blue Tetrazolium

OD Optical density

OxO Oxalate oxidase

PCR Polymerase Chain Reaction

PDA Potato Dextrose Agar

RNA Ribonucleic Acid

RNAse Ribonuclease

ROS Reactive Oxygen Species

SAGE Serial analysis of Gene Expression

SOD Super Oxide Dismutase

TAE Tris acetate EDTA

T-DNA Transfer DNA

ZR Zeatin Riboside

xiii

ACKNOWLEDGEMENTS

All the praises for ALMIGHTY ALLAH, WHO is the creator of the Universe,

most beneficent, gracious and merciful. He created man to pursue knowledge, find

truth and unhide the secretes of the Universe, Who gave me power of vision to witness

and mind to think and judge. My wholehearted gratefulness to our beloved Prophet

Hazrat Muhammad (PBUH) who guided us to learn and explore.

I find myself enormously privileged to work under the supervision of Prof. Dr.

S. M. Saqlan Naqvi, Dean Faculty of Sciences, Department of Biochemistry, Pir

Mehr Ali Shah Arid Agriculture University Rawalpindi. His professional skills,

valuable suggestions, constructive criticism, sincere encouragement and fervent

interest have made this project possible. His guidance helped me in all time of research

and writing of this thesis.

I would like to thank my overseas supervisor Dr. David S. Douches, Professor

and Director, Potato Breeding and Genetics Program, Michigan State University, USA

for providing me research facilities and guidance. I would also like to say thanks to Dr.

Daniel Zarka, Donna Kells, Dr. Radin Sadre and Saltanat Mambatova at Michigan

State University, USA.

I greatly appreciate genuine assistance, valued suggestion and co-operation of

my co-supervisor Dr. Iqbal Hussain who helped me a lot to complete this

undertaking. I feel honor to express my earnest thanks to the members of my

supervisory committee, Dr. M. Javaid Asad, Associate Professor, Department of

Biochemistry, and Prof. Dr. Abdul Rauf, Chairman Department of Plant Pathology

for their encouraging behavior, valuable suggestions and professional guidance.

xiv

I would like to thank Dr. Muhammad Gulfraz, Chairman, Department of

Biochemistry, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi (PMAS

AAUR) for his encouragement and insightful comments.

I thank my fellow lab mates and colleagues especially Bushra Javaid, Dur-e-

Shahwar, Tasawar Sultana, Shahzad Hussain Shah, Amna Muhammad, Rabia

Khalid, Iffat Kiani, Rakhshanda Shamim and Afshan Akram for their invaluable

help and cooperation.

My sincere thanks also goes to my colleagues and friends from Animal

Biotechnology lab especially, Rizwana Abdul Ghani and Shagufta Jabeen. The

assistance provided by technical staff of the lab is highly appreciative.

This research work could have not been completed without the prayers,

invariable support, and cooperation of my family In particular, my mother, who is

always a source of encouragement for me. I am deeply obliged to all of them.

May ALLAH honour them all and JazakALLAH khair for their help (ameen).

Nadia Majeed

xv

ABSTRACT

Germin like proteins (GLPs) are large group of related and ubiquitous plant

proteins. These proteins are considered to be involved in most of the processes

important for the development of plant and defense mechanism. Although multiple

copies of this gene family have been reported in a number of species (wheat, barley,

rice, soybean, mosses and liverwort), even then the up-regulated expression of GLP

transgenes has demonstrated additional defense capabilities. Due to reported role of

GLPs in conferring fungal resistance, there is a need to explore their antifungal

activity. For this purpose potato was selected as experimental material. It is a vegetable

crop and produces, on average, additional food energy and protein than cereals. Potato

with immense nutritive value, large yield, and significant cash return to farmers, has

become a major crop for both farmers and consumers in Pakistan. Potato is susceptible

to many kinds of diseases, especially to fungal pathogens, therefore genetic

engineering of potato for disease resistance is an important strategy study and apply

disease resistance. GM technology can be an effective tool for crop improvement. The

transgenic approach was pursued to introduce rice (Oryza sativa) germin like protein

gene OsRGLP1 using two recombinant vectors pC:OsRGLP1 and pG:OsGLP1 via

Agrobacterium mediated transformation. Confirmation of presence of gene was carried

out by polymerase chain reaction (PCR). One-step reverse transcriptase PCR was used

for transcriptional analysis and expression was quantified in real time in all PCR

positive putative transgenic lines. Functional status of transgene was studied in

selected high expression lines from two vectors. It was observed that OsRGLP1

possesses super oxide dismutase activity and it is heat resistant and is sensitive to

H2O2. These characteristics make it a Fe like SOD. A high H2O2 level was detected in

transgenic lines. Fungal assay with Fusarium oxysporum f. sp. tuberosi showed

xvi

enhanced foliar resistance in transgenic lines in comparison to untransformed control

plants. OsRGLP1 may be used as source for nonspecific fungal resistance in plants and

the antioxidant activity of heat resistant SOD may be explored for abiotic stress

tolerance.

1

Chapter 1

INTRODUCTION

Germins and germin-like proteins (GLPs) are a large and extremely diverse

family of plant proteins that are present ubiquitously. Wheat germin was first detected

in germinating wheat grains. Proteins from GLPs family present in all organs and at all

developmental stages and many of them are also involved in several stress related

responses. It is thus obvious that these proteins may be involved in many of the

processes important for plant development and defense mechanism (Bernier and

Berna, 2001). Germins make a group of similar proteins reported only in rice, oat,

wheat, maize, rye and barley (Lane, 2002). GLPs have a maximum of 40-70%

sequence similarity to wheat germin; however on average the level of similarity is

about 50% ( Dunwell et al., 2001).

Germins and GLPs are apoplastic proteins and are said to be the part of the

cupin superfamily that are identified in numerous eukaryotes as well as prokaryotes.

The mutual feature of all these proteins is a preserved 3D structure that forms a six

stranded beta barrel (Dunwell et al., 2004).

Germins and germin-like proteins (GLPs) have been associated to the

solidification of plant cell wall thereby deliver resistance to environmental stresses in

plants. There are numerous illustrations available linking GLP expression to plant

defense and recommend GLPs as markers in the defense mechanism of plant.

GLPs/SOD catalyzes the conversion of superoxide to H2O2 and O2, is of major

importance in protecting living cells from toxicity produced by superoxide anion under

oxidative stress conditions. Several SODs might be involved in controlling oxidative

2

stress in many cell compartments such as chloroplast, peroxisomes and mitochondria

(Alscher et al., 2002).

Park et al. (2004) associated GLPs in response to viral pathogens by isolating

cloned cDNA from hot pepper plant that display high sequence homology to a GLP

and suggested that isolated CaGLP1 gene may be involved in defense to viral

pathogens and classified it as novel PR protein family.

León-Galván et al. (2011) proposed that the CchGLP gene codes for a GLP

with Mn-superoxide dismutase activity and suggested a probable role for CchGLP in

pathogen resistance, and likely with salicylic acid and ethylene signal pathways

involved in these events. When transgenically expressed in Nicotiana tabacum cv

xanthi plants showed increased resistance to giminiviruses (Guevara-Olvera et al.,

2012). Wang et al., 2013 for the first time investigated the expression of AhGLPs in

peanut and studied their roles under various stresses, both abiotic (salt, H2O2, wound)

and biotic (leaf spot, mosaic and rust), and during development.

The down-regulation of OsGLP1 in an indica rice cultivar by siRNA-mediated

gene silencing showed significant reduction in its expression and exhibited decreased

height and were extremely affected by fungal pathogens, while its increased expression

in transgenic tobacco plant has recognized its relationship with cell wall and enzymatic

activity as SOD also suggests its involvement in plant height regulation and disease

resistance (Banerjee et al., 2010). Over expression of root-expressed germin like

protein OsRGLP1 in tobacco have shown that this GLP possess heat resistant SOD

activity and when studied for its sensitivity to inhibitors it behaved like FeSOD

(Yasmin, 2009).

3

The introduction and expression of defense related proteins into plant genomes

have shown that the progress of phytopathogenic fungi can be reduced significantly.

Disease control can never be accomplished absolutely, the level of disease lessening

depends on the approach employed and features of the fungal pathogen. Application of

signals that induce salicylic acid, cytokinin and ethylene levels have shown improved

disease tolerance or susceptibility in transgenic plants. The communication between

the expressed gene product, plant species, and phytopathogen is complex mechanism

which designate that the response of transgenic plants to this stress cannot be easily

expected. Introduction of more than one defense gene have shown extensively more

potential in providing disease resistance than single transgene introgression (Punja,

2001).

Potato is one of the world’s fourth agronomically significant crop in terms of

demand and production. Therefore strategies that allow betterment of commercial

potato varieties are of distinctive relevance for developed and developing countries

(Chakravarty et al., 2007).

Potato belongs to family Solanaceae which comprises of about 90 genera and

2,800 species. This family is found all over the world but mostly reside in tropical

regions of Latin America (Correll, 1962). Potato belongs to genus Solanum which

comprises of about 2,000 species out of which 150 are tuber bearing (Ward, 1991).

This genus is subdivided into several subsections of which potatoe containing only all

tuber bearing potatoes (Hawkes, 1990). The potato has complicated and variable

genetic makeup. These variations not only include wild potato species but also semi-

cultivated plants and local land races (Ross, 1986).

4

Potato (Solanum tuberosum) is ranked third among food crops after rice and

wheat and fifth for its total production in Pakistan. It is an important vegetable crop

because of its natural prospects for high production, cost-effective income and

nutritious values (Bhutta, 2008). In Pakistan, people mostly consume at least one tuber

a day while in Europe and South America it is considered as a essential food (Haq et

al., 2008). Potato crop faces loss in production due to pests that include fungal, viral

and bacterial pathogen (Malik, 1995 and Bhutta et al., 2004), because of increase in

current potato areas, unavailability of disease free hybrid seed and lack of information

about incorporated disease management (Bhutta and Hussain, 2002; Bhutta et al.,

2005).

In potato tissue culture the initial plants are obtained from pathogen free

portion by specific pathogen free shoots established in vitro by using meristem tip

culture with or without thermotherapy. Micropropagation of potato has been described

by many scientists (Goodwin and Adisarwanto, 1980). Since potato is susceptible to

many kinds of diseases, especially potato late blight caused by fungus Phytophthora

infestans, transformation of potato for disease resistance is considered an important

objective now a day.

Development of disease resistance is one of the several classical approaches to

disease control. There are two types of resistance, genetic and induced. Genetic

transformation of plants is frequently used as a means of plant improvement, but has

not been readily recognized as a mean for examining the plant gene function. Most of

the plants have been found to be very challenging to transform, and so various genetic

transformation techniques were established. These include: Agrobacterium mediated

transformation, PEG, electroporation and various biolistic methods. Agrobacterium

5

mediated transformation is nevertheless main technology used for the production of

genetically modified plants (Beaujean et al., 1998).

Plant regeneration/transformation in potato has been stated using different

explants types like leaf, stem, tuber and microtuber discs (Mitten et al., 1990; Visser,

1991; Dale and Hampson, 1995; Chang et al., 2002). Research has also been

accomplished to identify genes involved in the growth cycle of potato, to increase its

productivity and to increase the production of potato to various environments.

The genetically modified (GM) varieties of main agroeconomic crops,

especially rape (canola) cotton, soybean and maize were among the first grown

commercially in 1996. General impression of GM crops in the developed and

developing countries has been positive. Due to increased pest and weed control yield

per unit area has increased. Environmentally low pesticide use in turn benefit non-

target and helpful organisms have, water pollution on surface and underground

becomes reduced and lowers the chances of accidents and health issues to farm

workers (Mannion and Morse, 2013).

A major socioeconomic influence of GM crops is the better income for large

and small scale farmers. Health betterment have also been attained, particularly

through reduced use of pesticide. Additional potential benefits of GM crops for health

improvement are achieved by supplementing particular nutrients. Golden rice is the

example of bio-fortification which intensify the accessibility of vitamin A (Dubock,

2013). The insufficiency of this vitamin in diet is the major reason of night blindness in

millions of Asians. It is suggested that the advantages and complications linked with

GM are not exclusive and certainly are comparable to those that have extensively been

blamed for older technologies. Generally, GM crops have evidenced to be an

6

optimistic addition to most of the technologies which consist of up-to-date agriculture

(Mannion and Morse, 2013).

It is proposed that GLPs may induce fungal resistance in transgenic plants; so

the present study was aimed to

1. Generate transgenic potato lines using a rice root-expressed GLP (OsRGLP1) and to

assess the potential of GLPs as inhibitor of fungal pathogenesis and to evaluate its

effectiveness.

7

Chapter 2

REVIEW OF LITERATURE

Germin and GLPs are huge and diverse family of plant proteins. Over the

year’s evidence have accumulated that germin and germin like proteins have a

defensive role against phytopathogen. Although multiple copies of this gene family

have been reported in a number of species (wheat, barley, rice soybean mosses and

liverwort) even then the upregulated expression of GLP transgenes has established

supplementary defense capabilities. Potato is an economically important crop plant and

is susceptible to many diseases especially fungal diseases. There has always been a

need to further improve defense capabilities of potato cultivars which are important for

good yield but not evolved in the environment of target areas.

2.1 GERMIN AND GERMIN LIKE PROTEINS (GLPs)

2.1.1 Germins

Germin is a protein marker that belongs to a group of superfamily. The name

‘germin’ was given because the first discovered in the germinating wheat seed (Lane

1991). All of them have germin motif which forms predictable β-barrel core that binds

metal ions. (Requena and Bornemann 1999). Years later the protein was categorized as

a glycosylated protein which is homopentameric (Jaikaran et al., 1990) that possess

OXO activity (Lane et al., 1993). This oxalate oxidase enzyme (OXO) catalyses the

conversion of oxalate and dioxygen to CO2 and hydrogen peroxide (H2O2) (Lane

1994). The suggested role of enzyme in fighting against fungal pathogens led to the

generation of transgenic plants with enhanced OXO activity and hence increased

phytopathogen resistance (Dunwell, 1998).

8

2.1.2 Germin Like Proteins (GLPs)

These proteins are germin related proteins and are part of cupin super family

(Lane et al., 1994; Woo et al., 2000) that form homohexameric complexes

(Christensen et al., 2004). It is well-known that member of this family may be present

in all organs and developmental stages of a plant and some are involved in response to

different stresses (Bernier and Berna, 2001). The largest number of germin like protein

have been found in rice among all cereal plants. (Dunwell 1998). Cannon et al., (2004)

reported that the genome of higher plants contain more than 30 GLP genes as multiple

copies.

2.2 BIOCHEMICAL/ENZYMATIC PROPERTIES

Different enzymatic activities are proposed to be related to these proteins.

Oxalate oxidase activity which is linked to germins (Berna and Bernier, 1997; Lane et

al., 1993); SOD activity which is found in some GLPs as well as germins

(Zimmermann et al., 2006) and the ADP-glucose pyrophosphatase activity, barley

GLP possess this property (Rodriguez-Lopez et al., 2001).

2.2.1 Oxalate Oxidase Activity

The oxidoreductase enzyme which converts OA to CO2 and H2O2 and hence

possess oxalate oxidase activity. The OXO activity was first identified by Zaleski and

Reinhard (1912) in wheat grain powder. After almost 80 years when barley sequence

became available it was exposed that germin was a protein that possess OXO activity.

Zhang et al., 1995 reported that germin like OXO is an oligomeric protein and studied

its increased activity and H2O2 level upon attack of fungal pathogen Erysiphe graminis

f.sp. hordei in Barley. Requena and Bornemann (1999) found that barley OXO is a

manganese containing enzyme. Overexpression of wheat gemin like protein in poplar

9

leaves showed increased resistance to phytopathogenic fungi Septoria musiva (Liang et

al., 2001). A Wheat gf-2.8 OXO when transformed in sunflower showed increased

resistance to oxalic acid releasing fungus Sclerotinia sclerotiorum (Hu et al., 2003).

2.2.2 ADP-Glucose Phosphodiesterase /Pyrophosphatase Activity

ADP-glucose phosphodiesterase or pyrophosphatase (AGPPase) activity has

been known in a barley GLP. Isolation and characterization of two isoforms of

AGPPase using barley leaves has revealed that, AGGPPase1 (SAGPPase1) is able to

be solubilized in ionic buffer of low strength while SAGPPase2 is extracted in high

concentration of salt solution. Internal sequence and N-terminal analysis studies

exposed that both SAGPPase1 and SAGPPase2 are different oligomers of the HvGLP1

(Rodriguez Lopez et al., 2001).

2.2.3 Superoxide Dismutase Activity

SOD catalyzes the conversion of superoxide radical O2•- to molecular oxygen

and hydrogen peroxide and is therefore involved in major defense mechanism against

the O2•- radical (Halliwell, 1978). Yamahara et al., (1999) isolated a GLP with

manganese SOD activity from Barbula unguiculata commonly known as moss. It was

the first GLP that was proven to be a metalloprotein with Mn-SOD activity but no

OXO activity was found to be associated with it. The first known nodule associated

GLP with SOD activity has been shown to shares sequence homology with a presumed

bacterial attachment protein receptor rhicadhesin (Gucciardo et al., 2007). The GLPs

with super oxide dismutase activity in wheat and barley are important element of

enhanced resistance against Blumeria graminis (Christensen et al., 2004). Yasmin,

(2009) reported that rice germin like protein gene 1 (OsRGLP1) possess H2O2

sensitive FeSOD activity which is heat stable and studied the involvement of

OsRGLP1 upstream regulatory elements in biotic and abiotic stresses.

10

2.3 GLPs IN RESPONSE TO ABIOTIC STRESSES

There are a number of reports available on the association of GLPs with

response to abiotic stresses. Hurkman group (1991; 1994; 1996) for the first time

studied the involvement of germin and GLPs expression in establishing mechanism of

protection of barley during salt stress. Gucciardo et al., (2007) when transiently

expressed coding sequence of PsGER1 from pisum sativum in tobacco leaves, found

this GLP/SOD resistant to abiotic stresses including high temperature, high level of

H2O2 and denaturation by detergent.

2.4 GERMINS & GLPs IN DEFENSE AGAINST PATHOGENESIS

Over the years sufficient evidence has accumulated that shows germin and

germin like proteins to have a role in defense against pathogens. In wheat and barley

these proteins were demonstrated to possess oxalate oxidase activity. The germin gene

that was only studied in detail was expressed in germinating and adult plant exposed to

infection (Berna and Bernier, 1999).

Plants attacked by the pathogens exhibit a difficult defense response that stops

the pathogen at early stage or limits pathogen to minute necrotic area, resulting in

prevention of spore formation and distribution of contagious agent. Many defense

reactions have been detected including production of reactive oxygen species locally,

hypersensitive cell death, formation of papillae at the area of attempted pathogen

attack, accumulation of phenolic compounds and induction of defense related genes

(Thordal-Christensen et al., 1997).

The involvement of germin like proteins in peanut AhGLPs in abiotic and

biotic stresses was chracterised by Wang et al., (2013). In a mini review by Breen and

Bellgard (2010) the dynamic role of GLPs in plant defense mechanism has been

11

discussed. Park et al., (2004) categorized pepper germin-like CaGLP1 as a novel

pathogenesis-related protein that may be involved in defense response of plant to

viruses.

2.5 GERMIN & GLPs IN DEFENSE AGAINST FUNGAL PATHOGENS

Germin and GLPs are said to be involved in defense against broad range of

pathogenic fungi (Zimmermann et al., 2006). Wei et al. (1998) isolated from barley, an

OXO like protein, which expresses resistance to this disease. Christensen et al. (2004)

described HvGLP4 as a functional SOD, and studied the transient expression of

TaGLP4 and HvGLP4, and observed their contribution in resistance to fungal diseases

in wheat and barley. A BvGLP1 gene from sugar beet when transformed into

Arabidopsis thaliana showed enhanced resistance to soil born phytopathogen

Rhizoctonia solani (Knecht 2010). Banerjee et al., 2010 documented OsGLP1 as

defense related protein that possess SOD activity and accumulates H2O2 when

transgenically expressed in Tobacco.

The study of QTLs on chromosome 8 of rice (Manosalva et al., 2009) have

exhibit the difficult nature of GLPs and their participation in high disease resistance

against pathogenic fungus, the Magnaporthe oryzae and Rhizoctania solani. Rietz et

al., (2012) showed the GLP family in rape (Brassica napus) for the first time, and

strongly suggested that members of BnGLP with SOD activity in B. napus may add to

decrease the exposure of fungal pathogen Sclerotinia sclerotiorum.

2.6 POTATO AN IMPORTANT AGRONOMICAL CROP

Potato is known to be a crop of agronomical importance. It is tuberous crop full

of starch, from the Solanaceae family. Potatoes are rich in nutrients such as

carbohydrates, dietary fibers, vitamins and minerals (e.g. potassium, magnesium, iron).

12

About 8,000 years ago, the Potato was originated in the Andes of South America. Inca

Indians cultivated them for first time around 6,000 years ago (The Potato Council,

2008-2010).

Currently potatoes are cultivated on average of 19.5 million hectares around the

World. The biggest potato producer is China now. World’s major potato producing

region are Asia and Europe. The potato is rich in starch and provide nourishing food

for the deprived and starving therefore plays a strong role in developing countries. It is

best at the places where land is inadequate and labor is plentiful, situations that present

in many developing world.

2.7 POTATO PRODUCTION IN PAKISTAN

Potato being the fourth most significant crop with large yield, immense

nutritive value and huge cash return to farmer has become principle crop for both

farmers and consumers in the country. In 1947 the area under potato production was

3,000 hectare with a yield of 9 metric tonne/hectare (Profile of Potato in Pakistan

2015). Pakistan is now producing 3.5 million tonnes of potato from 161.9 thousand

hectares however a 7.8 percent decline have been observed in potato production from

year 2013 to 2014 due to the decrease in area sown comparing to corresponding period

2012 to 2013 (Pakistan Economic Survey 2013-14).

Despite 7.8% decline in production when compared with other crops it is still

fast emerging cash crop for farmers of potato producing district in central Punjab

because of the better hybrid seed and rapid high yield. Due to non-availability of

internationally accepted seed locally, farmers import it from Holland and India with

investment of over Rs 120,000 and Rs 90,000 per acre respectively. The cost of the

seed therefore makes it a highly expensive business.

13

2.8 POTATO VARIETIES GROWN IN PAKISTAN

Both red and white skin potato varieties are grown in the country. The red skin

varieties include Desiree, Ultimas, Cardinal, Kuroda, Oscar and Symphonia. The white

skin varieties Santé, Diamant, Multa, Hermes, Lady Rosetta, Ajax and Patrones are

being cultivated commercially (Awan 2012).

2.8.1 Desiree

It is a red skinned and was first bred in the Netherlands in 1962. It is the most

popular red potato all over the world and is liked by potato consumers in Pakistan. It

has yellowish flesh with a characteristic flavor and is suitable for all cooking purposes.

2.9 MAJOR FUNGAL DISEASES OF POTATO

Fungi form a group of eukaryotic saprophytic heterotrophs most of which are

pathogenic because they are able to cause disease (Knogge, 1996). Fungi alone cause

more than 70% of all major crop diseases (Agrios, 2005). Almost all of the

flowering plants species are confronted by fungal attack.

Fungi can also be adaptable parasites, which invade plants through wounds or

natural openings for intrusion and proliferation. Fungi from this group with

comparatively less virulence have wide host range and minor disease symptoms.

Narrow host range fungi includes true pathogens that are dependent on their host plants

but under certain conditions may also survive outside of the hosts. plant pathogens are

found in this group but they have restricted number of host species. The last group is

obligate pathogens, who need living host plant to complete their life cycle. (Knogge,

1996)

Fungal penetration into plant is likely to be controlled by many factors of

which one factor is the release of enzymes; cutinases, cellulases, pectinases, and

14

proteases. All of them may not necessarily take part in penetration (Knogge, 1996).

Once get into the plant, fungi secretes toxins, also known as phytotoxins. Fungal

toxins goal the H+-ATPase enzyme localized in plant plasma membrane (Wevelsiep, et

al., 1993) that plays a central role in “uphill” solute transport process and in the

intracellular pH regulation (Briskin, and Hanson. 1992).

2.9.1 Potato Late Blight

The air borne oomycete Phytophthora infestans causes a foliar and tuber

disease in potatoes called late blight. Black/brown lesions of the leaves or stem expand

very soon and become necrotic. Sporangia could be seen as white growth on the lower

side of the leaves. The tubers become infected in the field as well as in storage, tuber

tissue discolor first and then become reddish brown/purplish. Temperature and

humidity are the most significant factors generating the late blight epidemic (Harrison,

1992). Marketable potato farmers mostly trust on fungicide use for the phytophthora

infestans, control and the study demonstrated that weekly sprays were more cost-

effective and reasonable for the management of late blight of potato (Ghazanfar et al.,

2010). Younis et. al., (2009) evaluated fifty-seven lines/varieties of potatoes for late

blight disease resistant out of which twenty-seven varieties / lines were rated as

resistant to this disease.

2.9.2 Black Dot

It is a very serious problem of potatoes in some countries, which is triggered by

the soil borne fungus Colletotrichum coccodes (Lees and Hilton 2003). Infection by the

pathogen develops on all underground parts i.e. tuber, root, stolon (Andrivon et al.,

1998) and some basal regions, leaves and stem. (Johnson, 1994). Infected tubers

15

develop silvery lesions which develop black microsclerotia (Dillard, 1992).

2.9.3 Potato Pink Rots

Soil-borne fungus Phytophthora erythroseptica causes pink rot of potato. It

kills plants, thereby reduce produce and storing possibility. A characteristic color

change is observed within five to twenty minutes after cutting the tuber. Disease is

mainly related with the potatoes grown in poor soils, but has also been found in well

managed soils (International Potato Center, 1996).

2.9.4 Black scurf

Rhizoctonia solani is a soil born fungus, common in potato crops, it causes

black scurf disease that results in poor quality tubers and reduced yield (International

Potato Center 1996). R. solani is a diverse species and is categorized in 13 genetically

different anastomosis groups (Woodhall et al., 2013). Tariq et al., (2010) studied the

potential of plant rhizosphere related bacteria insulated from healthy and diseased

potato plants grown in the soil of Faisalabad and Naran, Pakistan and suggested that

two bacterial isolates have exceptional possibility to be used as operational biocontrol

of potato black scurf disease caused by Rhizoctonia solani Kuhn AG-3.

2.9.5 Fusarium Dry Rot and Wilt

Different Fusarium species cause serious problems to potato. Dry rot is serious

storage problem of potato. In the start tubers develop dark lesions which later expand

and contain mycelia. Infection originates at the surface wound during harvest. Plant

either fails to emerge from infected tubers or may wilt and die subsequently. In grown

plants it cause yellowing of lower and upper leaves than subsequent wilting occur and

tuber become discolored. Fusarium strains become systemic and can be transmitted

through potato seeds (International Potato Center, 1996).

16

Fusarium wilt is potential major threat to potato in Pakistan. Shafique and

Sobiya (2012) studied pathogenic potential of F. solani, by injecting different strains

of F. solani in potato and conducted antifungal bioassays to assure the mycotoxic

potential of different plant parts of Parthenium hysterophorus.

2.10 AGROBACTERIUM-MEDIATED DNA TRANSFER

In plant biotechnology, the most widely utilized technique to produce

transgenic plants is Agrobacterium-mediated transformation, which is heavily

patented. Substantial research has been done to comprehend and refine the molecular

machinery of Agrobacterium which is capable of the generation and incorporation of

the T-DNA into the host cell. This led to the creation of many recombinant

Agrobacterium strains, vectors and intriguing technology is used presently as a

major tool for the successful transformation of numerous crop plants, flowers and trees

(Tzfira and Citovsky 2006).

Agrobacterium tumefaciens is a soil-dewelling bacterium which has the

exceptional capability to transfer a well-defined DNA segment of its Ti plasmid into

the nucleus of cells that are infected infected where it is firmly incorporated into the

host genome and transcribed (Binns and Thomashaw, 1988). T-DNA of the bacterium

contains a set of oncogenes, that codes for an enzyme responsible for the formation of

auxins and cytokinins hence cause tumor or crown gall cells formation and the genes

that synthesize opines that are used as nitrogen and carbon source by the bacterium.

Genes positioned exterior to the T-DNA are responsible for opine catalysis and T-

DNA transfer/integration procedures (Zupan and Zambrysky, 1995).

Applied use of T-DNA transfer system to plant cells exhibit three main

evidences; First is the transmission and incorporation of T-DNA and its consequent

17

expression that cause tumor formation which is considered to be the transformation

process of plant cells. Secondly, the genes of T-DNA are only able to transcribe in

plant cells. Thirdly, any alien DNA placed within the T-DNA borders can be

transmitted to plant cell. Considering these congenital facts, for plant transformation,

researchers were able to engineer the first recombinant vector/plasmid and bacterial

strain systems (Deblaere et al., 1985).

There is a significant increase in the number of reports on successful

Agrobacterium mediated transformation of various plants species and cultivars

(Herrera-Estrella et al., 2005).

2.11 GENETIC ENGINEERING, AS TOOL FOR CROP IMPROVEMENT

Using genetic engineering one can integrate more than one or more trait into a

plant. According to James (2012), about 170 million hectares was planted with

transgenic crops and these include crops with high market cost, such as herbicide

tolerant maize, canola and soybean cotton; insect resistant maize, cotton, rice and

potato; and virus resistant papaya and squash and the area has been increasing

constantly. Commercially accessible herbicide resistant and insect tolerant maize and

cotton are the example of transgenic crops with collective characters.

The first FDA (US Food and Drug Administration) approved GM crop FLAVR

SAVR tomato was created by Calgene (Martineau, 2001), these tomatoes in contrast to

their non-GM counterpart were modified so that they could stay longer on the plant

and increase their flavor before harvesting.

There is a list of prominent biotech crop producers of which USA is at the top

followed by Argentina and Brazil; and Pakistan is at number 6 with an area of 2.8

18

million hectares under Bt-cotton (James 2012). A crop with more than one engineered

attributes has been introduced with the name of SmartStax maize by Monsanto and

Dow Agrosciences which is broad range herbicide tolerant and resistant to number of

insects (Mannion and Morse 2013).

GM technology is principally aimed to increase crop productivity. Crops have

not been genetically modified to improve productivity, it mainly increases due to

decreased losses as the crop predators/competitors (i.e. insects, weeds, fungi, viruses)

are significantly decreased (Mannion and Morse 2013). The increase in productivity

has been seen in both developing and developed countries (James, 2010).

Another major advantage of GM technology is the reduced pesticide usage by

producing insect resistant crops. Barfoot and Brooks (2008) have revealed how insect

resistant crops have benefited specially in terms of amount of pesticide used,

environmental welfares and price savings.

Numerous authors such as Qaim (2009), Carpenter (2010) and Finger et al.

(2011) have reviewed the overall financial benefits of GM crops. Farm revenue have

been increased by $33.8 billion for the period 1996-2006, with $6.9 billion being

added in 2006 by GM crops (Mannion and Morse 2013).

GM technology also proposes health benefits by engineering the increased

production of important vitamins and nutrients in principal crops which could hold

huge population (Sakakibara and Saito, 2006; Sauter et al., 2006). A bio-fortified

golden rice is an example which comprehend high vitamin A level (Dubock,

2013).

19

Societal outlook of GM crops range beyond health uncertainties. Qaim (2010)

illustrated advantages of GM technology specifically in terms of small scale income,

nutrition and health of poor, by exemplifying Bt cotton and beta-carotene rich Golden

Rice. These examples specify the involvement of this technology in poverty decline

and food safety in developing countries.

The benefits of GM crops dominate the risks therefore should be treasured and

could be used to increase worldwide food production (Mannion and Morse 2013).

A private company J. R. Simplot has developed genetically engineered

potato which yield reduced amounts acrylamide during frying and chipping which

otherwise is suspected to cause cancer in people has been approved by USDA for

commercial planting (The New York Times Nov 7, 2014).

2.12 MOLECULAR RESISTANCE IN POTATO

Recombinant DNA technology in advance breeding promises to combat major

agronomical problems. Late blight is deadly epidemic of potatoes caused by oomycete

phytophthora infestans, so developing resistant potato varieties is of prime importance

in breeding potatoes. More than one resistant gene introduction into potato varieties is

required to meet the need.

Jo et al., (2014) used cisgenic (Jacobsen and Schouten 2000) approach to

introduce cloned potato R genes for late blight resistance, Rpi-sto1 and Rpi-vnt1.1

from the wild potato Solanum stoloniferum and Solanum venturii, respectively, into

three different potato varieties; Atlantic, Bintje and Potae9 and observed durable late

blight resistance. They also studied marker free transformation and concluded that it is

20

less genotype dependent and affected by vector backbone incorporation as compared to

marker-assisted transformation.

Liu et al., (2012) developed Verticillium wilt resistant potatoes by genetically

modifying it with StoVe1 gene and concluded the role of this gene in providing

resistance to fungal pathogen Verticillium dahlia.

Hoshikawa et al., (2012) isolated, cloned and transformed five novel thionin

genes from different Brassicaceae species, into potato cv Waseshiro and observed

enhanced resistance to gray mold and demonstrate that thionin genes could be

used to produce resistant breeds against various pathogenic fungi.

Kuhl et al., (2007) generated transgenic potato lines by incorporating RB gene

and observed increased resistance to late blight both in field condition and in detached

leaf bioassay.

Zarka et al., (2010) introduced and characterized the cry1Ia1 gene in the potato

cultivar Spunta to create resistance to insect potato tuber moth.

Hemavathi et al., (2010) developed marker assisted transgenic potatoes by

introducing GLOase gene which when constitutively expresses results in higher AsA

(ᶫ-ascorbic acid) content, which is directly associated with increased resistance to

abiotic (salt, oxidative, and drought) stresses.

Potatoes engineered with GalUR gene, results in development of salt tolerant

crop because the overexpression of the gene triggers ascorbate pathway enzymes

(Upadhyaya et al., 2011).

21

Potato expressing CuZnSOD, APX, NDPK2 genes (Kim et al., 2010) under the

control of stress inducible SWPA2 promoter from sweetpotato showed improved

tolerance to oxidative and temperature stress in parallel to transgenic potatoes

expressing both CuZnSOD APX genes, only NDPK2 gene or non-transgenic plants.

More than 18 potato diseases have been described in Pakistan out of which

seven are caused by fungal pathogens Phytophthora infestans, Alternaria solani,

Erysiphe cichoracearum, Erwinia carotovora spp. carotovora, Fussarium spp

Streptomyces scabies, and Verticillium dahlia (Awan, 2012). Potato variety Desiree is

the most popular variety in the country and is susceptible to many of these

phytopathogen. As reviewed earlier researchers have used this variety to study

molecular resistance of many genes for biotic and abiotic stresses. GLPs have been

proposed to exhibit a crucial role in numerous characteristics of plant development or

stress resistant and expected extensive responsiveness for their potential involvement

to plant basal host resistance. Due to their Oxalate oxidase and/or superoxide

dismutase activity they generate higher levels H2O2 and can serve as a cofactor for

strengthening of the cell wall by papilla formation due to their cross-linking with plant

cell wall proteins at the site of pathogen infection (Wei et al., 1998), and can initiate

defense responces by acting as a signaling molecule in either way (Lane 1994; Zhou et

al. 1998).

To further establish the role of GLPs in fungal resistance this study was

designed to transfer a rice root-expressed GLP into Desiree, a commonly grown potato

variety in Pakistan.

22

Chapter 3

MATERIALS AND METHODS

3.1 PLANT MATERIAL

Potato plantlets of cv Desiree were obtained and transformed at Potato

Breeding and Genetics Program laboratory, Michigan State University, USA and were

used for molecular and functional analysis.

3.2 PLANT TRANSFORMATION

3.2.1 Electrocompetent Cell Preparation

Three ml of LB liquid medium (Annexure I) was inoculated with single colony

of GV3101 and was incubated two days at 28◦C with continuous shaking at 250 rpm.

Then 100 ml LB medium was inoculated with 1 ml saturated culture and was

incubated at 28◦C with shaking at 250 rpm until the OD600 of the culture reached 0.4-

0.5. Bacterial cells were immediately palleted with 3,000 rpm at 4◦C for 30 minutes.

All the steps were performed on ice. Pallet was washed twice with ice cold 10%

glycerol at 2,500 rpm spin at 4◦C for 10 minute and then 5 minutes. After washing, the

cells were suspended in 1 to 2 ml ice cold glycerol depending upon the density of the

cells. Cells were dispensed into 50 µl aliquot in pre-chilled sterile eppendorf tubes and

were immediately stored at -80◦C.

3.2.2 Transformation of Agrobacterium tumefaciens Strain GV3101

Electrocompetent cells of Agrobacterium strain GV3101 were transformed

with the plasmid pC:OsRGLP1 and pG:OsRGLP1. Two µL of each plasmid

preparation was mixed with 50 µl of competent cells in eppendorf separately. Total

reaction mixture of 52 µl was placed in electroporator cuvette. All the steps were

23

B

A

Figure 1: A) Map of the pCAMBIA1301, the expression vector B) Modified

T-DNA region

A) The map of pCAMBIA1301 expression vector showing the location of

important genes, MCS (multiple cloning sites) and regulatory parts. GUS

gene is under the control of CaMV35S promoter, hygromycin is plants

selection marker and kanamycin is bacterial selection marker. B) T-DNA

region modified by replacing GUS gene with root-expressed rice germin like

protein gene (OsRGLP1).

24

Figure 2: A) Map of the pH7WG2, the expression vector B) Modified T-DNA

region

A) The map of pH7WG2 expression vector showing the location of important

genes, MCS (multiple cloning sites) and regulatory parts. ccdB killer gene is under

the control of CaMV35S promoter, hygromycin is plants selection marker and

streptomycin is bacterial selection marker. B) T-DNA region modified by

replacing ccdB gene with root-expressed rice germin like protein gene

(OsRGLP1).

B

A

25

performed on ice. Electroporation was carried out each time at 1,900 volts for 15

milliseconds in Electroporator (BTX, USA model ECM 399). Electroporated cells

were suspended in 1000 ml LB liquid medium immediately. The transformed mixture

was incubated at 28°C for 45 minutes on constant shaking of 250 rpm. About 250 µl of

each transformed mixture was then spread on LB agar (Annexure II) plates having 50

mg/L kanamycin and were incubated for two days at 28°C.

3.2.3 Confirmation of Selected Clones of Agrobacterium tumefaciens GV3101

by PCR

Colony PCR of antibiotic selected clones was done using gene specific primers

to confirm the presence of gene. Following primer pairs were used for plasmids

pC:OsRGLP1 and pG:OsRGLP1 respectively

Forward Primer 5ʹ-ATCTAGATCT CATCTCAAACACACCACC-3ʹ

Reverse Primer 5ʹ-CTCGAGGTGACC GTCACAAAGAACACTG-3ʹ

Forward Primer 5ʹ- CACCATGGCTTCGTCTTCCTTC-3ʹ

Reverse Primer 5ʹ-TCAGTAATGGTTGTTCTCCCAG-3ʹ

The thermal profile adopted for PCR of OsRGLP1 for both plasmids was 94oC

initial denaturation for 3 minutes followed by thirty five cycles each of 94oC for thirty

seconds, 55oC for thirty seconds and 72oC for 1 minute, and extension at 72oC finally

for 10 minutes. The amplified product was run on 1% agarose gel prepared in TAE

(Tris acetate ethyldimethyl tetra acetic acid) buffer (Annexure III). Bromophenol blue

was used for tracking of amplicon and after staining with ethidium bromide visualized

by UV illuminator. 100bp ladder was used as a marker to identify the size of the band.

3.2.4 Pre-Culturing or Pre-Conditioning of Explants

Stem internodes were cut on moist sterilized filter paper in a petri dish and

26

were placed in ZIG liquid medium (Modified MS medium (Murashige and Skoog,

1962) ; Annexure IV) and incubated at room temperature for two days.

3.2.5 Infection and co-culturing of Explants with Transformed GV3101

Single confirmed colony of GV3101 transformed with plasmid pC:OsRGLP1

and pG:OsRGLP1 was inoculated in 3ml of LB liquid medium supplemented with 3µl

of kanamycin (50mg/L) and 6µl of streptomycin (25mg/L) respectively. Cultures were

allowed to grow at 28°C with 250 rpm shaking for 48 hrs. Grown cultures were

centrifuged and the pellets were resuspended in sterile water. Two hundred µL of

resuspended cell culture was added to a single pre-culture plate and mixed to make

sure that all the explants came in contact with Agrobacterium. Infected explants were

incubated at 25°C for two to three days in restricted light.

3.2.6 Culturing of Explants on Pre Selection Medium

Infected and co-cultured explants were blot dried with sterilized filter paper and

shifted to petri dishes containing ZIG pre-selection medium (Annexure V)

supplemented with cefotaxime (500mg/L). Plates were placed at 25±3°C in incubator

providing light intensity of approximately 2000 lux with a photoperiod of 16 hrs.

3.2.7 Culturing on Selection Medium Containing Suitable Antibiotic

One week after pre-selection explants were shifted to ZIG selection medium

(Annexure VI). Medium composition was same as for pre selection except hygromycin

15mg/L was added because the plasmids pC:OsRGLP1 and pG:OsRGLP1

contain hygromycin in their T-DNA region and the antibiotic was used for plant

selection.

3.2.8 Shifting of Explants to Rooting Medium

After getting shoots of about 1.5 to 2 cm were carefully excised from callus

27

nodules and were shifted to rooting medium (Annexure VII) in test tubes and were

placed at 25±3°C in incubator in a light intensity of approximately 2000 lux with a

photoperiod of 16 hrs. Roots were developed after 2 to 3 weeks.

3.3 CONFIRMATION OF GENE TRANSFER

Plants after selection on hygromycin and before shifting to soil were assessed

for presence of transgene OsRGLP1 in their genomic DNA.

3.3.1 Genomic DNA Isolation from Selected Plants

Genomic DNA was extracted from fresh leaves of selected transformed as well

as untransformed control plants using DNeasy plant mini Kit (QIAGEN Valencia,

CA). Approximately 100mg of plant tissue was ground under liquid nitrogen to a fine

powder with the help of pestle and mortar. Tissue powder was shifted to 2 mL

microfuge tube containing 400 µL of Buffer AP1 and 4 µL of RNase A stock solution

and vortexed vigorously. Mixture was incubated at 65°C for 10 min and was mixed 2

to 3 times by inverting tube during incubation. 130 µl of Buffer AP2 was added to the

lysate, mixed, and incubated for 5 min on ice and centrifuged for 5 minutes at 14,000

rpm (20,000xg) at room temperature. After centrifugation, the lysate was transferred to

the QIAshredder spin column (lilac) sitting in a 2 ml collection tube and was

centrifuged at 14,000 rpm (20,000xg) for 2 minutes at room temperature. Flow-

through was then shifted to a new 2 mL microfuge tube to which 675 µL Buffer AP3/E

was added and mixed. 650 µL of mixed solution was added to new QIAshredder spin

column (lilac) sitting in a 2 mL collection tube and centrifuged at 8,000 rpm for 1

minute at room temperature. Filtrate was discarded and the step was repeated with

remaining sample. 500 uL of Buffer AW was added in the DNeasy spin column and

centrifuged at 14,000 rpm (20,000xg) for 2 minutes at room temperature to dry the

membrane. DNeasy Mini spin column used in the previous steps was put in a new 1.5

28

mL microfuge tube to which 100 μL of Buffer AE was added and incubated at room

temperature for 5 minutes. And centrifuged at 8,000 rpm for 1 minute at room

temperature. DNeasy Mini spin column was removed from the tube. The eluted

genomic DNA solution in the tube was preserved at -80°C until used.

3.3.2 Confirmation of Transfer of OsRGLP1 by PCR

Presence of OsRGLP1 in transgenic plants was confirmed through PCR with

specific pair of primers, forward primer for 35S (CaMV) promoter and reverse primer

specific for gene.

Forward Primer 5ʹ-CTATCCTTCGCAAGACCCTTC-3ʹ

Reverse Primer 5ʹ-CTCGAGGTGACC GTCACAAAGAACACTG-3ʹ

Forward Primer 5ʹ-CTATCCTTCGCAAGACCCTTC-3ʹ

Reverse Primer 5ʹ-TCAGTAATGGTTGTTCTCCCAG-3ʹ

The thermal profile for PCR amplification was same used in section 3.2.3.

3.4 EXPRESSION ANALYSIS

Over expression of mRNA for OsRGLP1 gene was analyzed RT-PCR and the

procedure was executed for all putative PCR positive lines from both plasmids using

primers that were specific for OsRGLP1 and housekeeping gene EF-1α.

3.4.1 RNA Isolation

RNA from transformed as well as untransformed plants was isolated using

RNeasy plant mini Kit (QIAGEN Valencia, CA). Weighed amount of fresh leaves

(100 mg) was immediately placed in liquid nitrogen, and ground thoroughly with a

mortar and pestle. Tissue powder and liquid nitrogen was decanted into an RNase-free,

liquid-nitrogen–cooled, 2 ml microcentrifuge tube and liquid nitrogen was allowed to

evaporate, 450 µl Buffer RLT (containing 10µL β Marceptoethanol per 1 mL of

buffer) was added and vortexed vigorously. A short 1 to 3 minute incubation at 56°C

29

was given to disrupt the tissue. The lysate was transferred to a QIAshredder spin

column (lilac) placed in a 2 ml collection tube, and was centrifuged for 2 minutes at

14,000 rpm (20,000xg) at room temperature. The supernatant of the flow-through was

carefully transferred to a new microcentrifuge tube without disturbing the cell-debris

pellet in the collection tube. 0.5 volume of ethanol (96–100%) was added to the

cleared lysate, and mixed immediately. The sample was transferred to an RNeasy spin

column (pink) placed in a 2 ml collection tube and centrifuged for 15 s at 10,000 rpm

(8000xg). Flow-through was discarded after centrifugation. 700 µl of Buffer RW1 was

added to the RNeasy spin column centrifuged for 15 s at 10,000 rpm to wash the spin

column membrane. RNeasy spin column was placed in a new 2 ml collection tube and

centrifuged at 14,000 rpm (20,000xg) for 1 min. The RNeasy spin column was placed

in a new 1.5 ml collection tube to which 30 to 50 µl RNase-free water was added

directly to the spin column membrane and centrifuge for 1 min at 10,000 rpm (8000xg)

to elute the RNA. The RNA preparation was subjected to DNA contamination removal

afterwards.

3.4.2 DNA Contamination Removal

Possibility of DNA contamination in RNA preparation was eliminated by

using TURBO DNA-free™ Kit (Catalog Number AM1907). 10x TURBO DNase

Buffer (0.1 volume) and 1 µL TURBO DNase was added to the RNA, and was mixed

gently and incubated at 37°C for 30 minutes. After that re-suspended DNase

Inactivation Reagent (typically 0.1 volume) was added and mixed well and incubated

for 5 minutes at room temperature and was mixed occasionally. After incubation

sample was then centrifuged at 10,000×g for 1.5 minutes and the RNA was transferred

to a fresh tube.

3.4.3 One step RT-PCR with Platinum Taq

For detection of expression of OsRGLP1 one step RT-PCR was done using

30

platinum taq system (Invitrogen by Life Technologies). Thermal cycler was

programmed so that cDNA synthesis was followed immediately with PCR

amplification automatically. Components for both cDNA synthesis and PCR were

combined in a single tube, using gene specific primers and total RNA. Reaction was

carried out in a volume of 20 µL containing 10 µL 2X Reaction Mix, 2 µL template

RNA, 1 µL forward primer (10 µM), 1 µL reverse primer (10 µM), 1 µL RT/Platinum

Taq Mix and 5 µL autoclaved distilled water. The thermal profile included three steps,

cDNA synthesis and pre-denaturation, PCR amplification and final extension. First

step included 1 cycle of 45oC for 30 minutes followed by pre denaturation for 2

minutes on 94oC. Second step included 35 cycles of: denaturation at 94oC for 15

second, annealing at 55oC for 30 seconds and extension at 72oC for 40 seconds. Third

step included 1 cycle of 72oC for 10 minutes for final extension. Reaction was cooled

down and stored at 4oC until used.

Following primer pairs were used for whole gene OsGLP1, for cds (coding)

region of OsGLP1 and housekeeping gene elongation factor 1 subunit α (EF-1α)

respectively.

Forward Primer 5ʹ-ATCTAGATCT CATCTCAAACACACCACC-3ʹ

Reverse Primer 5ʹ-CTCGAGGTGACC GTCACAAAGAACACTG-3ʹ

Forward Primer 5ʹ- CACCATGGCTTCGTCTTCCTTC-3ʹ

Reverse Primer 5ʹ-TCAGTAATGGTTGTTCTCCCAG-3ʹ

EF-1α Forward 5 ʹ - GGTGGTTTTGAAGCTGGTATCTC-3 ʹ

EF-1α Reverse 5 ʹ -CCAGTAGGGCCAAAGGTCACA-3 ʹ

Amplified products were analyzed by electrophoresis using 1% agarose gel

prepared in Tris acetate EDTA (TAE) buffer (40 mM Tris acetate, 1 mM EDTA pH

8.1) according to Sambrook and Russell (2001). Bromo-phenol blue was used as

31

loading/tracking dye. After staining with ethedium bromide gel was visualized by UV

trans-illuminator. DNA bands were compared with 1kb/100bp gene ruler for size

estimation.

3.5 RELATIVE QUANTITATION OF TRANSGENE IN REAL TIME

3.5.1 RNA Isolation

RNA from control as well as PCR positive transformed lines was isolated using

Thermo Scientific GeneJET Plant RNA Purification Mini Kit. 100 mg of plant tissue

was placed into liquid nitrogen and grinded thoroughly with help of a mortar and

pestle and was transferred immediately into 1.5 microcentrifuge containing 500 µL of

Plant RNA Lysis Solution, supplemented with 10 µL of 2M DTT. Contents were

thoroughly mixed by vortexing for 10 to 20 seconds, incubated for 3 minutes at 56°C

and centrifuged at 14,000 rpm for five minutes at room temperature. The supernatant

(450 to 550 µL) was collected and transferred to the clean microcentrifuge tube to

which 250 µL of 96% ethanol was added and mixed by pipetting. The prepared

mixture was transferred to a purification column inserted in a collection tube. The

column was centrifuged for 1 min at 11,000 rpm. The column and collection tube were

reassembled after discarding the flow-through solution. 700 µL of Wash Buffer WB 1

was added to the purification column and centrifuged for 1 min at 11,000 rpm. After

discarding the flow-through and collection tube the purification column was placed

into a clean 2 mL collection tube. 500 µL of Wash Buffer 2 was added to the

purification column and centrifuged for 1 min at 11,000 rpm. Column and collection

tube were reassembled after discarding the flow-through. The step was repeated and

the column was re-spined for 1 min at maximum speed i.e. 14,000 rpm. Purification

column was transferred to RNase-free 1.5 mL microcentrifuge tube after discarding the

collection tube containing the flow-through solution. To elute the RNA, 50 µL of

nuclease-free water was added to the center of the purification column membrane and

32

centrifuged for 1 min at 11,000 rpm. Purification column was discarded and RNA was

stored at -80°C till use.

3.5.2 cDNA Synthesis

The extracted total RNA was used directly for cDNA synthesis which was

synthesized by using RevetAid First Strand cDNA Synthesis Kit (Thermo Scientific

cat #K1621). Template RNA 2 µL was placed in a nuclease free micro-centrifuge tube

to which; 1 µL Oligo(dT)18 primer, , 4 µL 5X Reaction Buffer , 1 µL RiboLock RNase

Inhibitor (20u/µL), 2 µL 10 mM dNTP Mix, 1 µL RevertAid M-MuLV Reverse

Transcriptase (200u/µL) was added. Total volume of 20 µL was obtained by using 9

µL of Nuclease-Free water. The reaction was setup on ice. The reaction mixture was

mixed gently and centrifuged briefly. The reaction contents were incubated at 42oC for

60 minutes followed by heating at 70°C for 5 min to stop reaction. The cDNA was

stored at -80°C for longer storage.

3.5.3 Routine PCR with cDNA

The cDNA was used as template and routine PCR was done to check the

presence of cDNA using EF-1α primers designed for real time PCR. The thermal

profile used for PCR was initial denaturation at 94oC for 3 minutes followed by thirty

five cycles each of 94oC for 30 seconds, 56oC for 30 seconds and 72oC for 40 seconds,

and final extension at 72oC for 10 minutes. The amplified PCR product was

fractionated through 1% agarose gel. The band size was identified using 100bp ladder

as marker. The following primer pair was used for EF-1α amplification.

Forward primer 5ʹ-AGAAGGTCGGTTACAACCCTGA-3ʹ

Reverse primer 5ʹ-TACC ACCAGTAGGGCCAAAG-3ʹ

3.5.4 Real Time PCR

Relative quantification of expression of OsRGLP1 in independent transgenic

33

lines generated from two plasmid was done by Real-time PCR. Maxima SYBR Green

qPCR Master Mix (2X) kit (Fermentas Life Sciences Cat# K0221) was used for the

purpose. Reaction volume was 12 µL containing 1 µL of cDNA (1:10 dilution), 6 µL

SYBR Green qPCR Master Mix, 1 µL 25 pM primer mix (Forward & Reverse), and 4

µL nuclease free water. Thermal profile for real time PCR included one cycle of pre-

amplification denaturation at 95oC for ten minutes, followed by forty cycles of

denaturation at 95oC for 15 seconds, annealing at 56oC for 30 seconds and extension at

72oC for 30 seconds. The signal was detected after extension of each cycle. Line-Gene

K Fluorescence Quantitative PCR Detection System (BIOER) was used for relative

quantification. Following primers were used for this purpose:

RTGLP Forward 5ʹ-CACTCCTCGGAAGACGAAC-3ʹ

RTGLP Reverse 5ʹ-CCCACAGGGAATACGAACAC-3ʹ

EF-1α Forward 5ʹ-AGAAGGTCGGTTACAACCCTGA-3ʹ

EF-1α Reverse 5ʹ-TACCACCAGTAGGGCCAAAG-3ʹ

3.6 PLANT ACCLIMATION

PCR confirmed lines from both plasmids were then shifted to greenhouse

condition. Potato plantlets grown on MS medium after one week were shifted to pots

containing peat moss vermiculite and sand (1:1:1, v:v:v) mixture. The pots were roofed

by polythene bags to preserve the moisture for 3-4 days after transfer. Moisture was

controlled by removing bags from plants. Plants were watered every other day. Six

plants for each independent line as well as untransformed plants were grown this way.

3.7 MORPHOLOGY AND GROWTH ANALYSIS OF TRANSGENIC

LINES IN COMPARISON WITH WILD TY CONTROL

Morphology of 3 months old untransformed control plants and independent

34

transgenic lines was observed. Forty two plants were grown in greenhouse, 6 for each

type and data was collected for plant height, number of shoots, number of leaves and

tubers harvested per plant.

3.7.1 Plant Height

Plant height was measured in centimeter using scale and recorded for

untransformed control as well as transgenic lines.

3.7.2 Number of Shoots per Plant

Number of shoots were counted per plant and data was recorded for

statistical analysis.

3.7.3 Number of Leaves per Plant

Number of leaves per plant was recorded for each replicate and the averages

were taken for statistical analysis.

3.7.4 Number of Tubers Harvested per Plant

Numbers of tubers harvested per plant were counted from both control and

transgenic lines for each replicate.

3.8 FUNCTIONAL EVALUATION OF TRANSGENE

Accumulation of H2O2, superoxide dismutase and oxalate oxidase activity in

transgenic plants was studied to establish any correlation with over expression of

OsRGLP1.

3.8.1 Protein Estimation of Plant Leaf Samples before SOD Assay

Protein concentration of untransformed control and transgenic plants was

determined by Lowry’s method (Lowry et al., 1951) before doing SOD assay.

Different dilutions of BSA were prepared from BSA stock (1mg/ml). To these

35

dilutions, sequentially add the extraction buffer and then 2 mL of copper sulphate

alkaline reagent (Annexure VIII). After thorough mixing according to the method the

contents were incubated for 10 minutes at room temperature in the dark. Folin

Ciocalteau solution 0.1 mL was then added to each tube, vortexed and again incubated

at room temperature for 30 minutes again in the dark. Absorbance was then recorded at

600 nm and standard curve was drawn using absorbance vs concentration of protein.

Protein concentration of plant samples was determined by plotting their OD600 on

standard curve.

3.8.2 In Solution Superoxide Dismutase Activity

Leaves from control plants as well as transgenic lines were homogenized using

pestle and mortar in the presence of liquid nitrogen. The soluble proteins were

extracted in 0.2 M Tris-HCl extraction buffer (pH 8) followed by centrifugation at

10,000 rpm for 10 minutes and the supernatant was transferred to a new microfuge

tube.

The assay was based on Beauchamp and Fridovich method (1971) with some

modifications. To make 1500µl activity solution, 696.5µl reaction mixture (Annexure

IX) and 596.5 distilled water was added to 1.5 mL cuvette. Then 5.5µl (75µM/L)

Nitroblue Tetrazolium (NBT) and 200µl enzyme extract was added to the reaction

mixture. Finally 1.5µl (2µM) riboflavin was added. The reaction was then initiated by

inserting the cuvettes in an illumination chamber. The reaction was stopped after 15

minutes by taking out the cuvettes from illumination chamber. The assay was carried

out in triplicates. Similarly, the replicate was placed in dark to determine the %

inhibition. The blanks of both light and dark reactions, lacking protein gave the

maximum reduction of NBT by highest absorbance at OD595. The SOD activity was

determined by taking the difference between the absorbance of the samples in light and

dark. The % inhibition was calculated by taking the difference between OD of blank

36

and OD of sample and divided that by OD of blank. ‘The 1 unit of SOD was defined as

the amount of SOD required to inhibit the photochemical reduction of NBT by 50%.

3.8.3 Detection of Localized H2O2 Level by ‘DAB-uptake method’

Hydrogen peroxide level was detected through 3, 3`-diaminobenzidine (DAB)

staining in untransformed and transgenic leaf samples (Thordal-Christensen et al.

1997). Leaf disks ten of each type were placed in DAB solution (1mg/ml) and

incubated overnight. Chlorophyll was removed by heating the leaf discs with 96%

ethanol for 10 minutes for chlorophyll removal. Leaf tissues were observed for H2O2

level.

3.8.4 Oxalate Oxidase (OXO) Activity Assay

OXO assay was executed on leaf, stem and root of untransformed control and

transgenic plants. Tissue localization of oxalate oxidase activity was determined by

histochemical assay according to the method established by Liang et al. (2001). Leaf

discs, sliced roots and stem from control and transgenic plants were incubated with

oxalic acid (2.5 mM) in a succinate buffer (25 mM succinic acid, 3.5 m M EDTA, pH

4.0) containing 4-chloro-1-naphthol (0.6 mg/ml) as staining reagent. The incubation

was carried out at room temperature in dark for 24 hours. Germinating wheat seeds

were used as positive control. Plant tissues were photographed with a digital camera.

The experiment was repeated three times with at least 6 explants in each trial.

3.9 ANTI-FUNGAL ASSAYS WITH SELECTED HIGHLY EXPRESSED

LINES

Pathogen resistance of transformed and non-transformed potato plants was

determined against Fusarium oxysporum f. sp. tuberosi.

3.9.1 Disease Incidence Assays of Transgenic potato with F. Oxysporum f spp.

37

Fusarium oxysporum f. sp. tuberosi isolates were purchased from First Fungal

Culture Bank of Pakistan (FCBP), Institute of Agricultural Sciences (IAGS), Punjab

University Lahore. Transformed and untransformed control plants were grown in

greenhouse for fungal assay.

3.9.1.1 Multiplication of Fungal culture on PDA

Cultures were multiplied on petri dishes containing PDA (Annexure X)

medium supplemented with 250mg/ml streptomycin in order to stop bacterial growth.

Plates were incubated at 25oC for 3 to 4 days. Microscopic characteristics were

observed under light microscope.

3.9.1.2 Inoculum Preparation

Fungal inoculum was prepared using sorghum seeds according to the method

of (Akhtar et al., 2005). Sorghum seeds were washed with tap water and soaked

overnight. Excessive water was drained and were placed in plastic bags plugged with

cotton and sterilized by autoclaving for 30 minutes at 1 kg/cm² pressure. Sterilized

sorghum seeds were inoculated with 1cm diameter PDA discs punched from the

periphery of actively growing 5 days old culture of F. oxysporum f spp. and then

placed in an incubator at (27±1) °C and the fungi were allowed to grow so that the

surface of all sorghum seeds was colonized.

3.9.1.3 Inoculation and Disease Incidence Scoring

The potting mixture (clay and sand 1:1) was sterilized by autoclaving for 30

minute at 1 kg/cm² pressure. The sterilized clay sand mixture was inoculated with

weighed inoculum of F. oxysporum f spp. mixed and incubated for one week at 25°C.

The pots were filled with infected clay sand mixture. Six weeks old potato plants from

greenhouse control as well as transgenic line from both vectors were shifted to the pots

and watered as required. Plants in pots without inoculum served as negative control.

Pots were kept in greenhouse at 25°C under natural light. Symptoms were scored at

38

12, 15, 18 and 21 days after planting based on modified scale of Silva and Bettiol

(2005).

3.10 DATA HANDLING AND STATISTICAL ANALYSIS

Data was scored for statistical analysis. ANOVA/ DMRT was used as per

needed using software MSTATC.

39

Chapter 4

RESULTS AND DISCUSSION

4.1 TRANSFORMATION OF AGROBACTERIUM STRAIN GV3101 WITH

RECOMBINANT VECTORS pC:OsRGLP1 AND pG:OsRGLP1

The confirmed recombinant plasmids pC:OsRGLP1 and pG:OsRGLP1

containing OsRGLP1 cDNA were used to transform Agrobacterium strain (GV3101)

via electroporation. Transformed and kanamycin resistant GV3101 cells were

subjected to colony PCR to confirm presence of desired fragment of OsRGLP1 (Plate

1 & 2). PCR positive colonies were afterwards used to introduce OsRGLP1 gene into

potato plants.

4.2 AGROBACTERIUM MEDIATED TRANSFORMATION OF POTATO

Potato variety Desiree was transformed with two expression constructs for

functional evaluation of root-expressed rice germin like protein OsRGLP1 especially

in terms of resistance against pathogenic fungi in potato. The Agrobacterium mediated

transformation was done through Agrobacterium strain GV3101 harboring OsRGLP1

gene using internode/stem pieces as explants. Agrobacterium-mediated genetic

transformation has long been used and a preferred method to generate transgenic

plants. This phytopathogenic bacterium has the unique capability to deport a specific

DNA fragment (T-DNA) into the nucleus of infested cells, where it is stably

incorporated into the genome of the host and transcribe (Binns and Thomashaw, 1988).

Agrobacterium tumefaciens is the only cellular organism which can infect broad range

of plant species mostly dicots (De Cleene and De Ley, 1976) and some monocots (De

Cleene, 1985). Potato being a dicot plant is a natural host to this bacterium.

40

Plate1: Confirmation of presence of OsRGLP1 through Colony PCR

Lane 1: DNA ladder 100bp, Lane 2: NTC (no template control) Lane3: Plasmid

pC:OsRGLP1 as positive control Lane 4 to 6: 958 bp amplified OsRGLP1gene

from three colonies

Plate 2: Confirmation of OsRGLP1 through Colony PCR

Lane 1: 1kb ladder, Lane 2: NTC (no template control) Lane 2 &3: 676bp

amplified OsRGLP1 from two colonies Lane 4: Plasmid pG:OsRGLP1 as

positive control.

41

4.2.1 Transformation of Potato with GV3101 harboring expression vectors

About 200 stem pieces were co-cultured with GV3101 harboring pC:OsRGLP1

and pG:OsRGLP1 for each on ZIG medium, co-cultured for two days under same

growth conditions as mentioned in section 3.2.5. After 48 hours of co-culturing the

bacterial growth appeared on the bottom surface of the petri dish and around the

periphery of stem pieces/internodes.

Successful Agrobacterium mediated transformation involves two major steps;

one is the selection and regeneration of transformed tissues and second is the

elimination of Agrobacterium (Teixeira da Silva and Fukai, 2001). Cefotaxime,

carbenicillin, vancomycin and timentin are generally used antibiotics that can

efficiently exclude Agrobacterium growth (Nauerby et al., 1997). Cefotaxime is a β-

lactam antibiotic that attaches to the penicillin binding proteins thereby inhibiting cell

wall synthesis of bacteria. A number of researchers used 500 mg/L cefotaxime in

Agrobacterium mediated transformation (Lowe et al., 1993; Renou et al., 1993,

Dolgov et al., 1997). Cefotaxime is cephalosporin antibiotic which is very less toxic to

plants, even at very high concentration (500 mg/L). After co-culture, infected

internodes were transferred to sterile filter paper, blot dried and were shifted to ZIG

medium supplemented with 500 mg/L cefotaxime to stop Agrobacterium growth.

Plates were wrapped with parafilm and were incubated at 25±3°C in incubator and

covered with four layer of cheese cloth to maintain extremely low light condition for

one week.

4.2.2 Selection and Regeneration of Plants

Blochlinger and Diggelmann (1984) constructed a vector by placing cds

region of hygromycin B phosphotransferase gene under the control of the regulatory

42

sequences of Moloney sarcoma virus, hence offered a dominant marker for selection of

eukaryotic cells that are not naturally resistant to this antibiotic.

Both pCAMBIA1301 and pH7GW2 vectors contain hygromycin B

phosphotransferase gene in T-DNA region under the control of CaMV 35S promoter,

which is a good plant selection marker. It provides resistance to hygromycin when

expressed in transformed cells. A discussed earlier eukaryotic cells lack the ability to

resist to this antibiotic; hence it is a good selection marker and enlarged opportunity

for regeneration of only transgenic cells on selection medium. Plants after one week on

selection medium were then shifted to ZIG medium supplemented with 500mg/L

cefotaxime and 15mg/L hygromycin and were shifted to fresh ZIG medium after every

7-10 days. Regeneration was seen as small green calli around both cut sides of stem

pieces after three to four weeks on selection medium (3.2.7). The hygromycin resistant

calli ultimately started organogenesis and small shoots appeared. When about 2 cm

long shoots were carefully cut and shifted transferred to rooting media mentioned in

section (3.2.8) for root formation and propagation. All the media used from selection

till regeneration and rooting were supplemented with cefotaxime to avoid the bacterial

growth which appeared in its absence.

4.3 MOLECULAR ANALYSIS

A total of 200 internodes for each transformation were co-cultured with

GV3101. From pC:OsRGLP1 transformation about 114 internodes were regenerated

on selection medium containing 15mg/L hygromycin while 150 explants regenerated

from pG:OsRGLP1 transformation (Table1). Plants selected on antibiotic hygromycin

were confirmed by PCR. Among regenerated rooted shoots, 35 plantlets from

pC:OsRGLP1 and 15 plantlets from pG:OsRGLP1 transformation were selected for

43

Table1: Comparison of two recombinant vectors for OsRGLP1 transfer efficiency in Potato cv Desiree

Vector

Parent/recombinant

Explant

No.

Regeneration

time (days)

Hygromycin

selected

explant #

Regeneration

%

Shoot *

%

Shoot

No.

Rooting

%

PCR+

%

Transformation

frequency

%

Transgenic

lines

selected

No.

RT-PCR +

lines

%

pCAMBIA1301/

pC:OsRGLP1

200 60-120 114 100 71 35 100 75 53 9 77

pH7WG2,0/

pG:OsRGLP1

200 60-120 150 100 75 15 100 80 60 5 100

*Shoot %; percentage of number of shoots over number of explants, rooting %; percentage of number of rooted shoots over number of

shoots, PCR+ %; percentage of PCR positive shoots over the number of shoots, transformation frequency %; calculated as shoot % × rooting

% × PCR+ %. Among all regenerated shoots, 35 plants from pC:OsRGLP1 and 15 from pG:OsRGLP1 were tested by PCR for gene

presence. RT-PCR+ lines %; percentage of transcript positive lines over selected PCR+ lines tested through one step reverse transcriptase

PCR.

44

DNA isolation and detected for presence of gene of interest by PCR.

4.3.1 Confirmation of Presence of Transgene by PCR

DNA of both selected transgenic and untransformed control plants was isolated

through DNeasy plant mini Kit (QIAGEN Valencia, CA). Extracted DNA was used as

a template in PCR to confirm the presence of gene of interest. Amplification was done

using specific primer set mentioned in material method section 3.3.2, using thermal

profile mentioned in section 3.2.3.

When separated on 1 % agarose gel, about 1 kb and nine 900bp band appeared

in genomic DNA of transgenic plants from pC:OsRGLP1 (Plate 3) and pG:OsRGLP1

(Plate 4) transformation respectively while DNA from control plant did not show any

amplification. Water was used as no template or negative control and confirmed

plasmid for both vectors was used as positive control.

4.3.2 Transcription /Expression Analysis

Nine PCR confirmed independent transgenic lines of pC:OsRGLP1 and five of

pG:OsRGLP1 were subjected to reverse transcriptase PCR for expression analysis.

RNA from putative transgenic lines from both plasmids was isolated using RNeasy

plant mini Kit (QIAGEN Valencia, CA). TURBO DNA-free™ Kit was used to remove

DNA contamination from RNA preparation.

4.3.2.1 One step Reverse Transcriptase-PCR using Platinum Taq

For detection of expression of OsRGLP1 in potato one step reverse

transcriptase PCR was performed using platinum taq system (Invitrogen by life

technologies) as a substitute approach to estimate the copy number. Sets of primer

pairs used and thermal profile is as mention in section 3.4.3.

45

Plate3: Confirmation of presence of OsRGLP1 in transgenic lines from pC:OsRGLP1

transformation

Lane 1: 100bp DNA marker, Lane 2: NTC (no template control), Lane 3: Positive

control (plasmid), Lane 4: internal control (untransformed), Lane 5 to 13: amplified

product using p35S forward and gene specific

Plate4: Confirmation of presence of OsRGLP1 in transgenic lines from pG:OsRGLP1

transformation

Lane 1: 100bp DNA marker, Lane 2: NTC (no template control), Lane 3: positive

control (plasmid), Lane 4 to 7 and 9: amplified product using p35S forward and gene

specific reverse primer confirms the presence of OsRGLP1 in 5 independent transgenic

lines, Lane 8: internal control (untransformed)

46

PCR amplification of all selected independent transgenic lines from two

expression vectors (pC:OsRGLP1 and pG:OsRGLP1) showed bands of 400bp with

primers used for housekeeping gene (Plate 5 & 7). EF-1α (AB061263) which is a

ubiquitous protein that facilitates the attachment of aminoacyle-tRNA to ribosome

during translation process (Ursin et al., 1991). To confirm the presence of RNA and to

normalize the expression in different samples EF-1α primers were used in PCR. It has

previously been used as internal control by different researchers (Nicot et al., 2005,

Kuhl et al., 2007, Chen et al., 2010).

The amplification with gene specific primers showed interesting results. Out of

nine PCR positive independent lines 7 lines were OsRGLP1 transcript positive while

two showed no expression (Plate 6). The absence of OsRGLP1 transcript in two PCR

positive lines could be described by a fractional deletion/rearrangement of the

OsRGLP1 transgene, leaving intact the 958bp DNA region, but inactivating or

truncating transcription (Kuhl et al., 2007). Felcher et al., (2003) detected post-

transcriptional silencing in other transgenic lines this also explains the transcript

absence.

One of the most important step in the study of gene function in plants is the

cloning of that gene into a plasmid vectors. Gateway is a new reliable technology for

cloning of sequences in larger plasmids. All five PCR positive transgenic lines selected

from pG:OsRGLP1 transformation were OsRGLP1 transcript positive showing a 676bp

band which represents the coding sequence of OsRGLP1 gene (Plate 8). Based on these

observation it may be safely concluded that Gateway recombinant vectors are good

system to integrate T-DNA into the plant genome and for their efficient transcription.

47

Plate5: One step RT PCR of EF-1α with independent transgenic lines from

pC:OsRGLP1

Lane 1: 100bp DNA marker Lane 2: NTC (no template control) Lane 3: Desiree

untransformed as internal control lane 4 to 12: 400bp amplicon confirms the

presence of housekeeping gene and cDNA from 9 independent transgenic lines

Plate 6: One step RT PCR of OsRGLP1 with independent transgenic lines

from pC:OsRGLP1

Lane 1: 100bp DNA marker, Lane 2: Negative control (Desiree untransformed),

Lane 3 to11: 958bp amplicon confirms the presence of OsRGLP1 transcript in

independent transgenic lines

48

Plate8: One step RT PCR of OsRGLP1 with independent transgenic lines

from pG:OsRGLP1

Lane 1: 1kb plus DNA marker, Lane 2: internal control (untransformed desiree

plant) Lane 3 to 7: 650bp amplicon confirms the presence of OsRGLP1

transcript of housekeeping gene in cDNA from 5 independent transgenic lines

Plate7: One step RT PCR of EF-1α with independent transgenic lines from

pG:OsRGLP1

Lane 1: 1kb ladder Lane 2: internal control (desiree untransformed) Lane 3 to 7:

400bp amplicon confirms the presence of EF-1α transcript in cDNA from 5

independent transgenic lines

49

4.4 COMPARISON OF MARKER-ASSISTED TRANSFORMATION

EFFICIENCIES FOR OsRGLP1

Marker-assisted transformation efficiency calculated as 75% for pC:OsRGLP1

and 82% for pG:OsRGLP1 when depicted as the percentage of PCR positive rooting

shoots (Table 1).

In order to do a more appropriate comparison of two recombinant vectors,

transformation efficiency was calculated according to Jo et al., (2014) which also

counts shoot regeneration efficiency. Vector pC:OsRGLP1 had a transformation

frequency 53%, while transformation frequency calculated for pG:OsRGLP1 was 56%.

Nine independent transgenic line were generated from pC:OsRGLP1 and five from

pG:OsRGLP1 while percentage of RT-PCR positive even was 77% and 100%

respectively (Tables 1). No significant difference was observed in transformation

frequency calculated for plant transformation for both recombinant vectors but

difference was observed when percentage for reverse transcriptase PCR+ lines was

calculated .

4.5 RELATIVE QUANTITATION OF OsRGLP1

Real Time-PCR is consider as a very sensitive method for the detection and

relative quantification of low abundance mRNAs (Bustin, 2000), and can be used for

the analysis expression of gene at specific tissue (Bustin et al., 2000), and for plant

studies (Gachon et al., 2004). To analyze gene expression precise, sensitive and

reproducible measurements are essential for specific mRNA sequences. Therefore

choice of proper internal control is very essential to normalize the expression level.

According to Nicot et al., (2005) the expression EF-1α did not seem to be influenced

during salt, cold and late blight stresses, in the present study it was selected to use as

reference gene to normalize the expression levels of OsRGLP1.

50

Before quantifying the expression, the cDNA from all PCR positive lines from

two vectors and untransformed control were subjected to routine PCR using primers

for EF-1α and the result was seen on 1% agarose gel in the form of 281bp bands

(Plates 9 & 10).

Five out of 7 were observed as high expression lines from pC:OsGRLP1 and

three out of 5 transgenic lines were found to be high expression lines from

pG:OsGRLP1. Two lines from pC:OsGRLP1 transformation showed negligible

expression with line DC-8 exhibiting lower expression (Figure 3). These results

coincide with the results from one step RT-PCR (Plate 6 & 8).

4.6 SHIFTING OF PLANTS TO GREENHOUSE CONDITION

PCR confirmed transgenic lines from both plasmids were maintained in tissue

culture laboratory. These lines were then shifted to greenhouse for morphological

studies. Potato plantlets were first re-propagated on MS medium and then after one

week were shifted to pots containing peat moss vermiculite and sand (1:1:1) mixture.

Six plants for each independent line as well as control plants were grown this way

(plate11). Almost all the plants from tissue culture were established and survived in the

green house condition, and the data was collect to study the effect of gene (OsRGLP1)

on morphology and growth.

4.7 COMPARITIVE ANALYSIS OF MORPHOLOGY AND GROWTH

GLPs may play regulatory role in different development stages of plants such

as leaf, root, flower, seed and fruit development (Dunwell et al., 2008). Constitutive

expression of OsRGLP1 in potato could affect its morphology. Experiments were

designed to study different morphological characteristic of high expression lines from

both vectors. The parameters included plant height, number of leaves shoots and tubers

51

Plate 9: Reverse transcriptase PCR with Ef-1α

Lane1: 100 bp marker lane 2: NTC Lane3: internal control (untransformed) lane 4 to

12 independent transgenic lines from pC:OsRGLP1 transformation

Plate 10: Reverse transcriptase PCR with Ef-1α

Lane1: 1kb ladder, Lane2: NTC Lane 3: internal control (untransformed), Lane 4 to

8: five independent transgenic lines from pG:OsRGLP1 transformation

52

Figure 3: Transcript analysis of OsRGLP1 in untransformed control and transgenic

lines using EF-1α as internal control

Untransformed potato variety Desiree plants served as control. Transcript level for

OsRGLP1 gene in control and transgenic lines was normalized with the EF-1α

messenger RNA level. Transcript levels were significantly higher in transgenic lines

than in the control. Significance determined with the DMRT (p < 0.05) was indicated

by letters.

53

Plate11: Different stages of potato transformation from tissue culturing to greenhouse

condition.

A: two week old explants on ZIG selection medium B: four week old explant on ZIG

selection medium C: Desiree transformed with pCAMBIA1301-OsRGLP1 D: Desiree

transformed with pH7WG2:OsRGLP1 E and F: Soil acclimatized transgenic plants

transformed with plasmid pCAMBIA1301-OsRGLP1 and pH7WG2:OsRGLP1

respectively G: Soil acclimatized untransformed control plant H: untransformed

control plant on MS medium I and J: Minitubers from transgenic lines from both

plasmids K: Minitubers from control plants

54

harvested per plant. Plants were initially sub-cultured on MS medium for a week and

were shifted to greenhouse. Observations were made on 3 months old untransformed

control plants and high expression lines 6 plants for each type.

4.7.1 Plant Height

Plant height was measured in centimeter using scale and recorded for

untransformed control as well as transgenic lines. All high expression lines from both

pG:OsRGLP1 and pC:OsRGLP1 transformation showed significant difference in plant

height as compared to untransformed control plants. Significance was observed with

DMRT (P< 0.05) and is shown by small letters (Figure 4). Vector backbone integration

did not play a this increase in plant height in transgenic line from both vectors could be

due to OsRGLP1 expression, as GLPs are identified as protein which are involved in

developmental regulation of plants. It has already been proposed that production of

OH- from H2O2 may take part of OsRGLP1 that resulted in the generation of high

H2O2 which may be one of the reason of expansion in cell wall. Banerjee and Maiti

(2010) exposed the efficient role of rice OsGLP1 in plant height regulation by

establishing transgenic rice plants through gene silencing.

4.7.2 Number of Shoots per Plant

Number of shoots were counted per plant and data when analyzed through

DMRT (P < 0.05), significant difference was observed in two out of three transgenic

lines from both vectors when compared with untransformed controls (Figure 5).A

number of properties/functions have been associated with GLPs, one of which is their

participation in regulation of different stages of plant development. (Dunwell et al.,

2008). The increase in shoots per transgenic plants can be attributed to the

involvement and over expression of OsRGLP1 gene.

55

Figure 4: Comparison of plant height

Comparison of plant height of 3 month old untransformed control (D-wt) and 6

independent transgenic lines from two recombination vectors (DG-1, DG-2, DG-3 of

pG-OsRGLP1 and DC-1, DC-2, DC-7 of pC-OsRGLP1). Data is an average of 6

plants for each line as well as control. Significance was tested with DMRT (P < 0.05).

Figure 5: Comparison of number of shoots per plant

Comparison of number of shoots of 3 month old untransformed control (D-wt) and 6

independent transgenic lines from two recombination vectors (DG-1, DG-2, DG-3 of

pG-OsRGLP1 and DC-1, DC-2, DC-7 of pC-OsRGLP1). Data is average 6 plants for

each line as well as control. Significance was tested with DMRT (P < 0.05).

56

4.7.3 Leaves number per Plant

Expression of germin/GLPs is not limited to cereal, and is not specific to

germination. SAGE technologies (Gibbings et al., 2003), study of ESTs and

microarrays have shown a variety of expression pattern of GLPs in different species.

An increase in leaves number per each plant was observed in comparison to

untransformed control. Significance was indicated by small letters when the data was

analyzed by DMRT (P < 0.05) (Figure 6). Increase in leaf number can be correlated to

the role of GLPs in plant height as one report have shown that OsGLP1 regulate plant

height development (Banerjee and Maiti, 2010).

4.7.4 Number of Tubers Harvested per Plant

Germin like proteins are most ubiquitous plant proteins and their expression is

linked to number of trait, they are differentially expressed during particular periods of

plant growth and development. Over expression of germin like protein gene in

transgenic plant may play a role in the better development of morphological

characteristics. Numbers of tubers harvested for each plant were counted from both

untransformed control and transgenic lines for each replicate. Data was analyzed by

DMRT (P < 0.05), significant increase in tuber number was observed for transgenic

lines compared to untransformed control (Figure 7).

4.8 FUNCTIONAL EVALUATION OF TRANSGENE

Oxalate oxidase activity (OXO), H2O2 increase determination, superoxide

dismutase activity and fungal assays were done to evaluate OsRGLP1 function in

highly expressed transgenic potato lines.

4.8.1 Oxalate Oxidase (OXO) Activity Assay

OXO is an oxidoreductase enzyme which in the presence of O2 converts oxalic

57

Figure 6: Comparison of number of leaves

Comparison of number of leaves of 3 month old untransformed control (D-wt) and 6

independent transgenic line from two recombination vectors (DG-1, DG-2, DG-3 of

pG-OsRGLP1 and DC-1, DC-2, DC-7 of pC-OsRGLP1). Data is average 6 plants for

each line as well as control plants. Significance was tested with DMRT (P < 0.05).

Figure 7: Comparison of tubers harvested per plant

Comparison of tubers harvested per plant of 3 month old untransformed control (D-wt)

and 6 independent transgenic line from two recombination vectors (DG-1, DG-2, DG-3

of pG-OsRGLP1 and DC-1, DC-2, DC-7 of pC-OsRGLP1). Data is average 6 plants

for each line as well as control plants. Significance was tested with DMRT (P < 0.05).

58

acid into CO2 and H2O2 (Lane 1994). Barley germin and wheat germin are OXO,

(Requena and Bornemann 1999, Jaikaran et al., 1990, Lane 1994, Lane 2000).

In order to characterize OsRGLP1 protein for the presence of any OXO activity

associated, transgenic potato plants were examined through the procedure developed

by Liang et al. (2001). Leaf discs, stem and root sections from untransformed control

and high expression plants were used for this assay. Germinating wheat seeds served

as positive control, while plant tissues incubated in solution without oxalic acid served

as negative control. The location of OXO was achieved by incubating plant tissues in a

buffer comprising oxalic acid and dye 4-chloro-1-naphthol as substrate.

The degradation of oxalic acid by OXO produces H2O2, which is used by

endogenous peroxidases, causing the development of a dark blue precipitate. No

detectable OXO activity was observed in plant tissues after incubation with activity

creator solution in the presence or absence of oxalic acid, while the control wheat

seeds exhibited a dark blue color in embryonic region (Plate 12). Similar results were

observed when OsRGLP1 was overexpressed in a model plant Nicotiana tabacum

(Yasmin, 2009). It has been previously described by many scientists that germinating

wheat seeds over express germin like OXO (Liang et al., 2001; Patnaik and Khurana,

2001).

Germins are generally described OXO while germin like proteins are mostly

SODs (Zimmermann et al., 2006; Godfrey et al., 2007). Reactive oxygen species such

as superoxide (•O2−), hydroxyl radical (OH−), hydrogen peroxide (H2O2), singlet

oxygen, and lipid hydro peroxides are produce when partial reduction of oxygen takes

place. These very unstable reactive oxygenspecies (ROS) that are able to detect only

by end product measurement. These end products are the result of their reaction with a

59

Plate 12: Oxalate oxidase activity determination in transgenic and untransformed

control plants.

Four days old germinating wheat seed (A) when kept in activity creator solution with

oxalic acid. Dark blue color produced on seed surface displaying the presence of OXO

activity. Four days old germinating wheat seed (B) when kept without oxalic acid in

activity creator solution. Untransformed control Leaf disks, Stem cross sections and

root cutting were kept solution with (C, I & O respectively) and without oxalic acid

(D, J, and P respectively) in activity creator. Transgenic 1 (single highly expressed line

from pC:OsRGLP1 transformation) leaf disks, stem cross sections and root cutting

were kept with (E, K and Q respectively) and without oxalic acid (F, L and R

respectively) in activity developer solution. Transgenic 2 (single highly expressed line

from pG:OsRGLP1 transformation) leaf disks, stem sections and root cutting were

kept solution with (G, M and S respectively) and without oxalic acid (H, N and T

respectively) in activity creator. Samples were placed in staining solution and

incubated at 25˚C for 24 hours. Three independent experiments were conducted.

Blue precipitates

showing OXO

activity

60

particular substance can be measured by changes in their color fluorescence, or

luminescence.

4.8.2 In Situ Detection of H2O2 in Potato leaves

Reactive oxygen species such as hydrogen peroxide and superoxide have been

detected usually by staining methods (Jambunathan 2010). H2O2 can be

histochemically detected in plants tissues by staining through 3, 3′- diaminobenzidine

(DAB). When react with DAB in the presence of endogenous peroxidases, H2O2

produce dark brown polymer which can be visualized easily. (Thordal-Christensen et

al., 1997; Daudi et al., 2012).

After DAB staining leaf cuttings from control and transformed plants

developed brown color on leaf surface that represents the polymerization product of

DAB which ultimately displays the localization of H2O2. High H2O2 accumulation was

detected in transgenic leaf samples as compared to control (Plate13), these results are

in accordance with the previous study of OsRGLP1 in tobacco by Yasmin, (2009).

The high production of H2O2 and superoxide radical (•O2−) is a common

characteristic that plant displays during defense responses generated by the plant

whenever challenged to elicitors and microbial pathogens (Lamb and Dixon, 1997). It

has been suggested that a fast increase in both extra been suggested that a fast increase

in both extra and intra cellular H2O2 is involved in the initiation and/or implementation

of the hyper sensitive response (Levine et al., 1994; Bestwick et al., 1998). There is

significant evidence that H2O2 has extensive role in resistance responses, because it is

essential for crosslinking of cell wall constituents as part of structural defence

reactions and may also control gene expression connected with phytoalexin

61

Plate 13: Detection of H2O2 by DAB staining in leaf disks.

Transgenic A(High expression line from pC:OsRGLP1 transformation).Transgenic B

(High expression line from pG:OsRGLP1 transformation). Leaf disks were transferred

in Leaf discs were soak in 0.1 % DAB staining solution for 14-18 hours, chlorophyll

pigmentation was removed by boiling in 96% ethanol for 5-10 minutes. Six leaf discs

were made for each type of plant for three consecutive experiments.

62

biosynthesis, antioxidant defences and improvement of systemic acquired resistance

(Lamb and Dixon, 1997).

The increased levels of H2O2 generation as identified through DAB staining in

transgenic potato plants clearly specifies the contribution of enzymatic activities to

some extent. The most shared GLPs activities comprise OXO and SOD, both enzymes

produce H2O2 from different chemical reactions. These explanations suggested that

OsRGLP1 might have both or one of these activities.

4.8.3 Total Protein Estimation of Plant Leaf Samples before SOD Assay

Total protein quantification is prevalent to many applications in molecular

research. It is very important to normalize the biological activity in protein content

before its measurement. The most employed protocols to assess total protein are based

on the reduction of copper in the presence of a color generating reagent (Lowry et al.,

1951; Smith et al.,1985). To measure the specific activity of super oxide dismutase

enzyme in different plant sample it was important to estimate total protein before

performing activity assay. The concentration of protein was measured by Lowry’s

assay (Lowry et al., 1951). The standard curve (Figure 8) was obtained using 1mg/ml

BSA as a standard and concentration of protein from 1gm sample of selected

transgenic line from two vectors as well as untransformed control plants were

calculated are mentioned in Table 2.

4.9 SUPEROXIDE DISMUTASE ACTIVITY DETERMINATION

During normal cellular metabolism ROS are generated that contain H2O2,

superoxide anion (O2•-), singlet oxygen (1O2), free hydroxyl radical (OH•) and

hydroxyl anion (OH-) cause impairment to living tissues by oxidizing cellular

components such as nucleic acids, protein, carbohydrate and lipids. Their leve

63

Figure 8: Calibration Curve with 1mg/ml BSA

64

Table 2: Calculated Protein Concentration by Lowry Method

S. No Plant Type Amount of Sample in

Grams

Protein Concentration µg/µl

1 D-WT 1gm 0.92 µg

2 DG-1 1gm 0.78µg

3 DG-2 1gm 1.1µg

4 DG-3 1gm 1.0 µg

5 DC-1 1gm 1.0 µg

6 DC-2 1gm 0.97 µg

7 DC-7 1gm 0.96 µg

Protein concentration in gm sample of original genotype potato (Desiree) and 6

independent transgenic line from two recombination vectors (DG-1, DG-2, DG-3 of

pG:OsRGLP1 and DC-1, DC-2, DC-7 of pC:OsRGLP1).

65

increases as a result of extreme environmental stresses, for example intense light

(Fryer et al., 2002), drought (Price et al., 1989), extreme temperatures (McKersie et

al., 1993), and exposure to herbicides, bacteria, viruses and fungi (Apel and Hirt,

2004; Tertivanidis et al., 2004; Mullineaux et al., 2006).

How plant protects itself when it experiences oxidative burst is a complex

mechanism and involves together enzymatic and non-enzymatic antioxidant

components. One of the main enzyme is superoxide dismutase that plays an important

role in plant defence mechanism, through detoxifying superoxide radicals by

converting them into H2O2 (Bowler et al., 1992). The SOD activity was measured as

the inhibition of photoreduction of nitroblue tetrazolium (NBT) by recording OD of

solution at 595 nm. In the present study the transgenic potato lines were subjected to

SOD assay to assess the contribution of OsRGLP1. SOD activity was performed based

on Beauchamp and Fridovich method (1971) with some modifications in fresh leaf

samples of control and transgenic plants.

One SOD unit was calculated as the amount of enzyme required for 50%

inhibition in photoreduction of the NBT in contrast with tubes missing the plant

extract, and expressed as units of enzyme activity per milligram of protein. Increased

SOD level was observed in transgenic potato lines contrast to control plants (Figure 9),

earlier reported by (Yasmin, 2009) in transgenic tobacco.

4.9.1 High Temperature Effect on SOD Activity

Germin like proteins exhibiting SOD activity are known to be heat stable

(Carter and Thornburg, 2000; Yasmin et al., 2008; Yasmin, 2009). Added SOD

activity in transgenic potato lines was evaluated for its heat tolerance, by preheating

66

Figure 9: Measurement of SOD activity in leaf extracts of untransformed control

and transgenic potato plants.

A) SOD activity in total protein extract from leaf samples of untransformed control

and transgenic potato plants. B) SOD activity in leaf extract of transgenic plants

after deduction of endogenous SOD activity of control samples. Bars represent

standard error (SE) based on three independent experiment. The letters designate

significance when compared with control according to the DMRT test (P < 0.05)

A

B

67

leaf extracts before measuring SOD activity at 90°C for 15 minutes.

It was observed that the SOD activity of transgenic samples remained

significantly higher than the control after heat treatment (Figure 10A). SOD activity

native to the plant decreased 60% in untransformed control plants. In case of

transgenic potato lines SOD activity after subtraction of the native activity, was same

in treated and untreated samples (Figure 10B). The results suggested that the added

SOD activity in transgenic potato plants may be attributed to some heat stable SOD

and thus may be because of OsRGLP1.

4.9.2 Effect of KCN and H2O2 on SOD Activity

Super Oxide Dismutase (SOD) are ubiquitous metalloenzymes and plants

generally possess three types of SODs. Based on their prosthetic metal ion they are

named as: manganese SOD, iron SOD, and copper/zinc SOD. These SOD isoforms are

located in different subcellular compartments; FeSODs are generally present in

chloroplasts, MnSODs in peroxisomes and mitochondria, and Cu/ZnSODs are found in

the cytosol, peroxisomes, chloroplasts and apoplasts. It is reported that FeSOD is

sensitive to H2O2 and is resistant to KCN treatment. (Alscher et al., 2002).

To foresee the metal ion cofactor attached to OsGLP1/SOD, its sensitivity to

KCN and H2O2 was studied. The transgenic potato lines and untransformed control

samples were subjected to 3mM KCN and 10mM H2O2 treatment before SOD assay.

SOD activity of treated and untreated samples from transgenic plants showed no

significant difference (Figure 11A), while it was reduced to 50% in control samples

treated with KCN demonstrating the existence of endogenous Cu/ZnSOD which is

known to be KCN sensitive. Added SOD activity detected in samples from transgenic

plants was insensitive to KCN, proposing that it was not Cu/ZnSOD (Figure 11B).

68

Figure 10: High temperature effect on SOD activity in control and transgenic samples.

SOD activity of untransformed control and transgenic samples (A) after high

temperature treatment. SOD activity of transgenic samples (B) after deduction of

endogenous SOD activity of control samples. Bars represent standard error (SE) based

on three independent experiment. The letters designate significance according to the

DMRT test (P < 0.05).

A

B

A

69

Figure 11: KCN treatment effect on SOD activity in untransformed control and

transgenic samples.

SOD activity of leaf samples (A) from control and transgenic plants. SOD activity

of transgenic samples (B) after deduction of endogenous SOD activity of control

samples. Bars represent standard error (SE) based on three independent experiment.

The letters designate significance in each group according to the DMRT test (P <

0.05)

B

A

70

Transgenic potato and untransformed control samples were treated with 10mM H2O2

before performing SOD assay to see its effect on SOD. The results demonstrated that

SOD activity in H2O2 treated transgenic samples was significantly decreased in

comparison to untreated samples, while the activity remained unaffected in control

samples (Figure 12 A&B). These results suggested that added SOD activity conferred

by transgene OsRGLP1 in transgenic plants sample is sensitive to H2O2 and tabacum

exhibited similar results (Yasmin, 2009).

4.10 ANTI-FUNGAL ASSAYS WITH SELECTED HIGHLY EXPRESSED

LINES WITH F. OXYSPORUM F SP. TUBEROSI

To assess the role of OsRGLP1 in pathogenic fungi interaction the high

expression selected transformed lines and non-transformed potato plants were infected

with Fusarium oxysporum f. sp. tuberosi. Transformed and untransformed plants were

grown in greenhouse for fungal assay. Fungal culture were multiplied on PDA and

were incubated at 25oC for 3 to 4 days and the fungal inoculum was prepared using

sorghum seeds according to the method of (Akhtar et al., 2005) (Plate 14).

4.10.1 Inoculation and Disease Incidence Scoring

The sterilized potting mixture was inoculated with Fusarium oxysporum f. sp.

tuberosi mixed and incubated at 25°C for a week. Six weeks old untransformed control

as well as transgenic potato plants from both vectors were shifted to the pots and

watered as required. Plants in pots without inoculum served as negative control. The

infected plants were observed daily and were scored after every four day and

categorized into the six classes from 1 to 6, established on the expansion of the disease

symptoms (Table 3). Strong differences were detected in the progression of the disease

71

Figure 12: H2O2 treatment effect on SOD activity in untransformed control and

transgenic samples

SOD activity in untransformed control and transgenic leaf sample: (A) after treatment

with 10mM H2O2. SOD activity of transgenic samples. (B) after deduction of

endogenous SOD activity of control samples. Bars represent standard error (SE) based

on three independent experiment. The letters designate significance in each group

according to the DMRT test (P < 0.05)

A

B

72

Plate 14: Different Stages of Antifungal Assay in Desiree potato plants.

A & B Fusarium oxysporum f. sp. tuberosi C Sterilized sorghum seeds, D Fungal

inoculum prepared using sorghum seeds E Sterilized sand and clay mixture (1:1) with

weighed amount of fungal inoculum F & G Plants subjected to infection, H & I Plant

grown in greenhouse for fungal assay

73

Table3 : Disease scores on potato plants infected with Fusarium oxysporum f. sp.

tuberosi

Disease scores at days post inoculation

Line No. of Plantlets 12 15 18 21

D-WT uninfected 18 1 1 1 1

D-WT Infected 18 3 4 4 to 5 5 to 6

DG-1 uninfected 18 1 1 1 1

DG-1 Infected 18 1 1 1 to 2 2

DG-2 uninfected 18 1 1 1 1

DG-2 Infected 18 1 2 2 2 to 3

DG-3 uninfected 18 1 1 1 1

DG-3 Infected 18 1 1 to 2 2 2

DC-1 uninfected 18 1 1 1 1

DC-1 Infected 18 1 1 1 to 2 2

DC-2 uninfected 18 1 1 1 1

DC-2 Infected 18 1 1 1 to 2 2

DC-7 uninfected 18 1 1 1 1

DC-7 Infected 18 1 1 to 2 2 2

Plants of three high OsRGLP1 expression lines from each vector and untransformed

control D-WT were infected with Fusarium oxysporum f. sp. tuberosi. Disease

symptoms scoring induced by F. oxysporum were based on modified scale of Silva and

Bettiol (2005): 1, no symptoms; 2, plant showing yellowing of leaves and wilting (1 to

20 %) ; 3, plant showing yellowing of leaves and wilting (21 to 40 %); 4, plant

showing yellowing of leaves and wilting (41 to 60 %); 5, plant showing yellowing of

leaves and wilting (61 to 80 %); 6, plant showing yellowing of leaves and wilting (80

to 100 %) or die. Three independent experiments were carried out. Plants without

infection considered as control.

74

symptoms between transgenic and untransformed plants (Table 3; Plate 15). Twelve

days after infection, disease symptoms were seen on untransformed control plants, in

the form of yellowing of leaves, mainly on older leaves. Transgenic leaves showed no

symptoms after 12 days, slight yellowing of leaves was seen 21 days after infection

(Table 3; Plate 15). After 21 days untransformed plants suffered seriously from

fungal infection, and more than 70% of the leaves displayed severe infection

symptoms (score 5 to 6) while for transgenic plants very minor infection symptoms

were noticeable on the oldest leaves represented by scoring 1 to 2 (Table 3). Survival

rate percentage was calculated 12, 15, 18 and 21 days post inoculation. Significant

difference was observed between transgenic lines and untransformed control plants

when data was analyzed using DMRT test (Figure 13 & 14). Survival rate percentage

when analyzed for control plants at each four day interval, significance in difference

was observed (Figure 15). These findings advocate that the over expression of

OsRGLP1 in potato lessens the susceptibility of the potato plants to Fusarium

oxysporum f. sp. tuberosi. Zimmermann et al., 2006 improved the tolerance in barley

by transient overexpression of HvGER5 gene in epidermal cells and detected that

added resistance is reliant on its SOD activity. A promising description for the

antifungal function of OsRGLP1 in potato plant could be the production of H2O2 due

to its enzymatic activity (SOD or OxO). H2O2 helps in papillae formation by

crosslinking cell wall proteins at the infection sites. This cell wall strengthening helps

the cell to protect itself against fungal penetration (Wei et al., 1998; and Christensen et

al., 2004).H2O2 produced by OsRGLP1 may also function as a signaling molecule by

triggering other defense responses in plants so OsRGLP1 may

75

Plate 15. Visual comparison between infected transgenic potato plants expressing

OsRGLP1 and untransformed control plants.

D-WT(control), transgenic1 (high expression line from pC:OsRGLP1

transformation) and transgenic 2 (high expression line from pG:OsRGLP1

transformation) plants infected with Fusarium oxysporum f. sp. tuberosi in soil 21

days after post-inoculation. Uninfected plants served as control.

A B C D E

Transgenic 2

Uninfected

Infected

D-WT Transgenic 1

76

Figure 13: Percentage survival rate of potato plants infected with Fusarium

oxysporum f. sp. tuberosi

Plants of six independent OsRGLP1 transgenic potato lines (bars with different

patterns) and untransformed control D-WT (blue) were infected with F.

oxysporum f. sp. tuberosi. Six week old potato plantlets were shifted to infection

mixture (sand + clay + inoculum) and the survival rate in % was calculated 12

and 15 days post-inoculation. Bars denote standard errors, based on three

independent experiments. The data represents significant differences in plant

survival rates for transgenic lines and the untransformed control plants 12 and 15

days after inoculation based on DMRT test (p < 0.05), as indicated by letters.

F G H I

77

Figure 14: Percentage survival rate of potato plants infected with fusarium

oxysporum f. sp. tuberosi

Plants of six independent OsRGLP1 transgenic potato lines (bars with different

patterns) and untransformed control D-WT (blue) were infected with F. oxysporum

f. sp. tuberosi. Six week old potato plantlets were transferred for infection to soil

(sand and clay) and the rate of plant survival was scored 18 and 21 days post-

inoculation. Standard errors are represented by bars based on three independent

experiments with 6 plants each. Plant survival rates show significant differences

between transgenic lines and control 18 and 21 days post inoculation based on

DMRT test (p < 0.05), as indicated by letters.

78

Figure 15: Survival rate of untransformed control plants (D-WT)

Survival rate of control plants on 12, 15, 18 and 21 days post-inoculation. Bars

represent standard errors based on three independent experiments with 6 plants

each. The data show significant differences of survival rate of the control plants on

each four day interval after inoculation based on DMRT test (p < 0.05), as indicated

by letters.

79

be employed for general defense against fungal infections.

The results from the present study showed that the protein product of the gene

OsRGLP1 is a heat stable iron like SOD that may play a role in abiotic stresses

including drought, salinity, high and low temperature and high light intensity that

generate ROS (reactive oxygen species). The high expression transgenic potato plants

were not completely protected against infection by F oxysporum f spp. however, the

delay in disease progression was observed, over-expression of this gene may provide a

general defense system against fungi at least in potato. It may be concluded that the

OsRGLP1 could be a favorable candidate gene for crop improvement through genetic

engineering strategy.

80

SUMMARY

Germins and germin like proteins (GLPs) together create a huge and extremely

diverse family of plant proteins. All germins and GLPs are glycoproteins someway

linked with the extracellular matrix. Mostly three functions are said to be predicted for

such proteins: some possess an enzymatic activity (OXO, SOD, AGPPase), others

appear to be structural proteins whereas some others act like receptors. Potato is one of

the world’s fourth agronomically significant crop in terms of demand and production.

The introduction and expression of defense related proteins into plant genomes have

shown that the progress of phytopathogenic fungi can be reduced significantly.

Development of disease resistance is one of the several classical approaches to disease

control. Generally, genetically modified crops revealed to be an optimistic addition to

other technologies which used in up-to-date agriculture. (Mannion and Morse, 2013).

Agrobacterium tumefaciens strain GV3101 was transformed with two expression

vectors pC:OsRGLP1 and pG:OsRGP1 and transformed clones were confirmed with

PCR which were used to introduce gene of interest (OsRGLP1) in potato cv Desiree.

Transgenic potato lines were generated via marker assisted Agrobacterium mediated

transformation as both vectors contain hygromycin gene as marker. Transformed

plants were selected on hygromycin (15mg/L) and confirmed by PCR amplification of

OsRGLP1. Transgene positive lines from two independent transformation experiments

were subjected to transcript analysis. RNA was isolated from PCR positive transgenic

lines and one step RT-PCR amplification resulted in all PCR positive lines as

transcript positive from pG:OsRGP1 vectors while out 9 PCR positive independent

lines obtained from pC:OsRGLP1, 7 were transcript positive while 2 were transcript

negative. Expression of all independent PCR positive lines from two transformation

was quantified by normalizing the expression to a well reputed housing keeping gene

for potato EF-1α. The results from quantification experiment coincide with the results

81

obtained from one step RT-PCR. Three high expression lines were selected from two

transformations to check the effect of transgene OsRGLP1 on morphology and growth

and were grown in green house condition. Plant height, shoot number, number of

leaves and tubers harvested were documented and data was analyzed by

ANOVA/DMRT. Significant difference was observed in each parameter in comparison

with untransformed control. Transgenic potato lines were assessed for superoxide

dismutase and oxalate oxidase activity and H2O2 level was determined. Detection of

H2O2 by 3, 3′ -diaminobenzidine (DAB) staining showed increased accumulation in

transgenic potato plants when compared with untransformed control. High expression

potato lines were also valuated for oxalate oxidase activity, no visual OXO activity

was found in transgenic as well as control plants germinating wheat seeds were used as

positive control and where activity was seen as blue precipitates. SOD activity in leaf

samples of transformed potato lines and untransformed control plants was measured as

inhibition of reduction of NBT by variation in absorbance taken at 595 nm. Transgenic

lines demonstrated significant increase in SOD activity as compared to control. Heat

treatment of this SOD activity indicated that it is a heat stable SOD and might be due

to OsRGLP1, as GLPs are previously reported as heat resistant. Effect of inhibitor

treatment on SOD activity was assessed it was observed and the added SOD activity in

transformed plants was inactive to KCN and sensitive to H2O2 proposing it to be Fe

like SOD. Fungal assay with Fusarium oxysporum f. sp. tuberosi revealed that the

survival rate % of transgenic lines was significantly high as compared to control

plants, the significance was measured by DMRT.

82

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Akhtar, H., S. Anita and S. P. Kumar. 2005. Studies on the management of root-knot

nematode, Meloidogyne incognita-wilt fungus, Fusarium oxysporum disease

complex of green gram, Vigna radiata cv. ML-1108. J. Zhejian. Univ. Sci., 6(8):

736-742.

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ANNEXURES

Annexure I

LB Liquid Medium

Tryptone 10g/L

Yeast Extract 10 g/L

NaCl 5 g/L

pH was adjusted to 7.0

Annexure II

LB Agar Medium

Tryptone 10g/L

Yeast Extract 10 g/L

NaCl 5 g/L

Agar 7g/L

pH was adjusted to 7.0

Annexure III

10X TAE (Tris acetate ethyldimethyl tetra acetic acid) Buffer

Tris base 48.5g/L

Glacial acetic acid 11.4ml

EDTA, disodium salt 3.7 g

Tris base, glacial acetic acid and EDTA were dissolved in 800 ml of deionized water

before diluting buffer up to 1 L.

The pH of diluted solution was ~8.

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Annexure IV

ZIG Liquid Medium

MS salts 4.44g/L

Sucrose 20g/L

Gibberellic acid (GA3) 0.2mg/L

Indole 3-acetic acid (IAA) 0.1mg/L

Zeatin Riboside (ZR) 3.33mg/L

pH was adjusted to 5.7-5.9 before autoclaving.

Annexure V

ZIG pre-selection medium

MS salts 4.44g/L

Sucrose 20g/L

Agar 7g/L

pH was adjusted to 5.7 to 5.9 and medium was autoclaved

Gibberellic acid (GA3) 0.2mg/L

Indole 3-acetic acid (IAA) 0.1mg/L

Zeatin Riboside (ZR) 3.33mg/L

Cefotaxime 250-500mg/L

Annexure VI

ZIG selection medium

MS salts 4.44g/L

Sucrose 20g/L

Agar 7g/L

pH was adjusted to 5.7 to 5.9 and medium was autoclaved

Gibberellic acid (GA3) 0.2mg/L

Indole 3-acetic acid (IAA) 0.1mg/L

102

Zeatin Riboside (ZR) 3.33mg/L

Cefotaxime 250-500mg/L

Hygromycin 15mg/L

Annexure VII

Rooting Medium

MS salts 4.44g/L

Sucrose 20g/L

Agar 7g/L

pH was adjusted to 5.7-5.9 before autoclaving.

Annexure VIII

Copper sulphate alkaline reagent (Lowry Solution)

Solution A

NaOH 2.8598 g

Na2CO3 14.3084 g

Solution B

CuSO4.5(H2O) 1.4232 g

Solution C

C4H4KNaO.4H2O 2.85299 g

Annexure IX

Reaction mixture for SOD assay

Enzyme extract 100µl

methionine 13 mM

nitro-blue tetrazolium (NBT) 75 µM

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EDTA 0.1 mM

potassium phosphate buffer (pH 7.8) 50 mM

Riboflavin 2 µM

Annexure X

PDA medium

Potato Dextrose Agar 39.9g

Distilled Water up to 1000 mL

Dissolved before autoclaving