Allah Bakhsh

Home , Bakhsh

EXPRESSION OF TWO INSECTICIDAL GENES IN COTTON

ALLAH BAKHSH

NATIONAL CENTRE OF EXCELLENCE IN MOLECULAR BIOLOGY UNIVERSITY OF THE PUNJAB LAHORE, PAKISTAN 2010

Expression of two insecticidal genes in Cotton

A DISSERTATION

Submitted to

UNIVERSITY OF THE PUNJAB

In fulfillment of the requirement for the Degree

DOCTOR OF PHILOSOPHY

MOLECULAR BIOLOGY

Allah Bakhsh

Supervisor:

Dr. Tayyab Husnain

Professor & Head of Plant Biotechnology Lab National Centre of Excellence in Molecular Biology University of the Punjab Lahore, Pakistan

2010

In The Name Of Allah the Most Merciful the Most Compassionate and the Most Beneficent

iii

CERTIFICATE

This is to certify that the research work described in this thesis is the original work of the author and has been carried out under my direct supervision. I have personally gone through all the data/results/materials reported in the manuscript and certify their correctness/authenticity. I further certify that the material included in this thesis have not been used in part or full in a manuscript already submitted or in the process of submission in partial/complete fulfillment of the award of any other degree from any other institution. I also certify that the thesis has been prepared under my supervision according to the prescribed format and I endorse its evaluation for the award of

Ph.D degree through the official procedures of the university.

In accordance with the rules of the centre, data book nos. 626,787 and 835 are declared as unexpendable documents that will be kept in the registry of the centre for a minimum period of three years from the date of thesis defense examination.

(DR. TAYYAB HUSNAIN) Professor and Supervisor

DEDICATED TO MY

FATHER & MOTHER

ACKNOWLEDGEMENTS

All praises are for The Almighty Allah and The Holy Prophet Muhammad (peace be upon him).

It seems impossible to pay thanks to my Kind and worthy supervisor “Dr. Tayyab

Husnain” Professor, National Centre of Excellence in Molecular Biology, University of the

Punjab, whose kind behaviour, keen interest and guidance throughout the course of this study

helped me to do the task in time The work presented in this manuscript was accomplished under

his enthusiastic guidance, sympathetic attitude and intellectual supervision.

It is my honor and pleasure due for Dr. S. Riazuddin (Ex- Director) & Dr. Amin Athar

(Director CEMB), University of the Punjab for providing me facilities to carry out my research

work. Their kind behavior coupled with friendly attitude was a source of inspiration for me.

I would like to express my sincere thanks to Dr. Ahmad Ali Shahid (Incharge MPhil/PhD

programme) for their inspiring guidance and support that I gained from him in theory and practical

research work.

Special thanks are due to Dr. Idress Nasir, Dr. Munir ud Khan and Dr. Ghazanfar Ali Khan

for their technical guidance during field studies of transgenic crop. Dr. Kausar Malik, Dr. Zia ur

Rehman, Dr. Abdul Qayyum Rao, Dr. Bushra Rashid and Mr. Zafar Saleem whose sympathetic

and aspiring attitude gave me perseverance to continue my struggle without being deterred and

disappointed by initial failures.

I am obliged to acknowledge the Higher Education Commission Pakistan for granting me a

fully-funded merit scholarship to meet my finances during PhD studies.

I would love to acknowledge the contribution of my school teachers who trimmed my potential for further studies. Special thanks go to Sir Ajmal Khan, Sir Saghir Ahmad, Sir Waheed

Ahmad, Sir Shahid and Sir Ameer (Late).

I am enormously grateful to my hostel fellows (Zeeshan Shamim & Wakil Ahmad, Shahid

Yar, Imran, Ali, Tanveer, Atiq, Amjid, Shahu, Zubair, Safdar, Shareef, Usman, Qasim, Nazim,

Sajjad, Noman, Rashid and Sarshik) for their company and continuous encouragement during the

study.

I have been very lucky for having cooperative lab fellows whose accommodative and

friendly behaviour made my work less laborious. Thanks to Ayesha Latif, Saima Babar, Dr.

Muhammad Irfan, Imran Ali, Mehmood ur Rehman, Tahir Rehman Samiullah, Dr. Asma Maqbool,

Dr. Muzna Zahur, Younas Khan, Saira Nadeem, Sana Khalid, Fatima Batool, Sarfraz Kiani,

Kamran Shahzad, Shahid Rehman, Azmat ullah Khan, Aleem Ashraf, Dr. Anisuzamaan, Abdul

Hafiz Dahab, Buhsra Tabassum, Zahida, Abdul Munim Farooq, Usman Aslam, Puspito, Siddique and Tanveer. The services rendered by Mr. Ilyas Tabassum, Nisar Ahmad, Muhammad Azam,

Khaliq, Nazir, Raza Ali, Karim and Munir are also worthy to be acknowledged.

How can I forget to acknowledge my chums whose encouragement and moral support has

been endless for me! Thanks to Ashiq Waqar, Imran Mani, Mazhar Bhai, Arshad Jamil, Zaman

Khan, Mudasir khan. Akmal, Sajjad, Dilawar, Boota, Siddiq, Asif Raza, AD Foji and Javed Iqbal.

I want to express my gratitute for my parents for seeding in my character the strong

motivation, the dedication, and the strong will for giving me the oppurtunity to travel along to

follow my dreams, for my believing in me, for their patience, for their suffering throughout the

years, for their love and sacrifices and continued encouragement for counting the days and years to

have me in their arms. I am also highly thankful to my uncles Faraz Hussain, Allah Ditta and

Muhammad Zafar, my cousins, my brothers Tariq, Ashiq, Waseem, Nadeem, my sisters and Kinza

Tariq whose prayers and support enabled me to complete my studies.

ALLAH BAKHSH

vii

SUMMARY

Agriculture serves as a jugular vein in the economy of Pakistan and a good chunk of the

population in the country depends directly and indirectly on agriculture. It is the source of

livelihood of almost 44.7 percent of the total employed labour force in the country. With the

present contribution to GDP at 21.8 percent, agriculture sector is the mainstay of the rural economy

around which socio-economic privileges and deprivation revolve.

The importance of cotton can hardly be over emphasized in the economy of Pakistan.

Pakistan is one of the ancient home of cultivated cotton, 5th largest producer of cotton, the 3rd

largest exporter of raw cotton, 4th largest consumer of cotton, and a leading exporter of yarn in the

world. Transgenic cotton expressing Bt (Bacillus thuringiensis) toxins is currently cultivated on a

large commercial scale in many countries, but data has shown that it behaves variably in toxin

efficacy against target insects under field and green house conditions.

The present study was conducted to determine stable integration and spatio -temporal

expression of two insecticidal genes (Cry1Ac and Cry2A) in advance lines of transgenic cotton

variety CIM-482. The quantitative levels of these insecticidal genes were determined. Seasonal

decline in expression differed significantly among different cotton lines tested in green house and

field conditions., the aim of study was to quantify the expression of these insecticidal genes with

the age of plants as well in different plant parts because the sustainable expression of Bt genes is crucial for its effectiveness to control the lepidopteron insects pest especially American bollworms.

The leaves of the Bt cotton plants were found to have the highest levels of toxin expression followed by squares, anthers, petals and bolls. Expression of Bt genes decreased consistently with the age of plants.

viii

Compared with the temporal or spatial-specific expression of the toxin, constitutive

expression of foreign proteins in transgenic plants may cause adverse effects, such as the metabolic

burden imposed on plants for constant synthesis of foreign gene products, and these may increase

the potential risk of resistance of the target insects to Bt. There is also concern about the food

safety of genetically modified plants (Kuiper et al. 2001; Shelton et al. 2002; Conner et al. 2003).

Therefore, in certain circumstances, it is desirable to use expression-specific promoters which only

express the foreign gene in specific plant tissues or organs (Cai et al. 2007).

Generally Genetically Modified (GM) crops contain the viral 35S CaMV promoter to drive

insecticidal and herbicidal resistance genes. In cotton the reductions in the efficacy of Bacillus

thuringiensis toxin (Bt) expressing plants has been attributed to reductions in promoter activity.

Sustained expression of these endotoxins through out the life of cotton plant is possible by using a

promoter with robust activity.

For this purpose, a promoter from RuBisCo (Ribulose-1,5-bisphosphate carboxylase

oxygenase) was isolated from Gossypium arboreum. RuBisCo is the bifunctional enzyme found in the chloroplasts of plants that catalyzes the initial carbon dioxide fixation step in the Calvin cycle and functions as an oxygenase in photorespiration. RuBisCo is the most abundant protein found in plant leaves, representing up to 50% of the soluble protein. These promoters and their transit peptides are attractive candidates for expression of genes at high levels. A construct was developed by isolating RbcS (RubisCo small subunit) promoter from Gossypium arboretum. This RbcS promoter was fused with Cry1Ac gene; followed by NOS terminator (construct was named as Rb-

Ac in which Cry1Ac had been cloned under RbcS promoter). After the formation and confirmation of construct, a local cotton variety NIAB-846 was transformed with this construct using

Agrobacterium tumefaciens strain LB4404. Same cotton variety was transformed with another construct pk2Ac harboring Cry1Ac under 35S promoter. Transformation efficiency remained

0.20% in case of Rb-Ac construct while it was 0.15% in case of pk2Ac plants. The putative

transgenic plants were screened using GUS assay. Furthermore, these transgenic plants were

subjected to PCR and southern blot to confirm gene integration. The comparative study for insecticidal gene expression in Rb-Ac and pk2Ac plants showed that RbcS is efficient promoter to drive the expression of Cry1Ac gene consistent throught out the life of cotton plant as compared to

35S promoter. As promoter activity would be restricted to green tissues, there would no expression

of Bt gene in soil and seed; hence no threat to soil organisms, environment and to users of cotton products and by products.

TABLE OF CONTENTS

Certificate ii

Acknowledgements iv

Summary vi

List of Figures xiii

List of Tables xv

Abbreviations xvi 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 7 2.1 Insect Resistance Management (Two Genes Strategy) 9 2.2 Temporal and Spatial Expression in Insecticidal Protein 14 2.3 Factors contributing to endotoxin variability 18 2.4 RuBisCo gene as Promoter 24 2.4.1 Agrobacterium mediated Transformation 30 2.4.2 Genus Agrobacterium 32 Agrobacterium mediated transformation in Cotton 3 MATERIALS AND METHODS 39 3.1 Experimental Material 39 3.2 Confirmation of Gene Integration and Expression 39 3.2.1 Genomic DNA isolation 40 3.2.2 Polymerase Chain Reaction (PCR) 41 3.2.3 Southern Blot Analysis 42 3.2.3.1 Probe Labeling 43 3.2.4 Western Blot Analysis 43 3.2.4.1 Protein Extraction 43 3.2.4.2 SDS Polyacrylamide Gel Electrophoresis 44 3.2.4.3 Western Blotting 44 3.2.4.4 Temporal and Spatial Expression of Cry1Ac and Cry2A entotoxins 44 3.3 Leaf Biotoxicity Assay 46

3.4 Isolation of RbcS promoter from Gossypium arboretum 46 3.4.1 Total RNA isolation 46 3.4.2 Agarose Gel Electrophoresis 47 3.4.3 Quantification of Total RNA 47 3.2.4 cDNA synthesis 47 3.4.5 Polymerase Chain Reaction (PCR) 48 3.4.6 Purification of DNA Fragment from Agarose Gel 49 3.5 TA Cloning and Transformation of Eluted DNA 49 3.5.1 Ligation of insert in TA Vector 49 3.5.2 Transformation in E. Coli 49 3.5.3 Screening of Positive Clones 50 3.5.4 Plasmid DNA isolation of Positive Clones 51 3.5.5 Restriction Analysis of Cloned DNA 51 3.5.6 Sequencing of Positive Clones 52 3.5.7 Preparing a product for Sequencing 52 3.5.8 Removal of vector Sequences from Sequence Results 53 3.5.9 Promoter Analysis 53 3.6 Cloning of RbcS and Cry1Ac in pCAMBIA 1301 54 3.6.1 Amplification of Cry1Ac+Nos from PIA-2 vector 55 3.6.2 Restriction digestion of PCR product and pCAMBIA 1301 56 3.6.3 Ligation of insert (Cry1Ac+NOS) into pCAMBIA 1301 57 3.6.4 Transformation of Plasmid in E.Coli 57 3.6.5 Plasmid DNA isolation 58 3.6.6 Restriction Analysis of Cloned Fragment (Cry1Ac+NOS) 58 3.6.7 PCR confirmation of (Cry1Ac+NOS) integration in pCAMBIA 1301 59 3.6.8 Restriction Digestion of RbcS product and pCAMBIA 1301 59 3.6.9 Ligation of insert (RbcS) in pCAMBIA 1301 60 3.6.10 Transformation of plasmid in E.Coli 60 3.6.11 Plasmid DNA isolation 60 3.6.12 PCR Confirmation of RbcS integration in pCAMBIA 1301 61 3.6.13 Restriction Analysis of cloned RbcS+Cry1Ac+NOS fragment 61 xii

3.6.14 Orientation confirmation of Cassette in pCAMBIA 1301 62 3.7 Electroporation of plasmid in Agrobacterium 63 3.8 Transformation of Gossypium hirsutum Var. 846 63 3.8.1 Delinting of Cotton Seed 63 3.8.2 Seed Sterilization 63 3.8.3 Isolation of Embryo 64 3.8.4 Medium Preparation 64

3.8.5 Bacterium Inoculum Preparation 64 3.8.6 Agrobacterium Culture Treatment 65 3.8.7 Co Cultivation 65 3.8.8 Selection of Transformants 65 3.8.9 Transient Expression of GUS gene 66 3.8.10 Selection on Antibiotic medium 66 3.8.11 Transformation Efficiency 66 3.8.12 Root Formation 67 3.8.13 Transformation of Transgenic plants to soil 67

3.9 Molecular Analysis of Transgenic Plants (T0 Progeny) 68 3.9.1 Genomic DNA isolation 68 3.9.2 Polymerase Chain Reaction (PCR) 68 3.9.3 Southern Blot Analysis 68 3.9.4 Temporal and Spatial Expression of Cry1Ac gene 68

3.10 Molecular Analysis of T1 Progeny 69

3.10.1 Polymerase Chain Reaction (PCR) 69 3.10.2 SDS Polyacrylamide Gel Electrophoresis 69 4 RESULTS 70 4.1 Confirmation of gene integration and Expression 70 4.1.1 PCR Analysis of Insecticidal genes 70 4.1.2 Southern Blot Analysis 72 4.1.3 Western Blot Analysis 73 4.2 Temporal and Spatial Expression of Cry1Ac and Cry2A 74

4.2.1 Temporal Expression of Cry1Ac gene 74 xiii

4.2.2 Spatial Expression of Cry1Ac gene 76 4.2.3 Temporal Expression of Cry2A gene 77

4.2.4 Spatial Expression of Cry2A gene 79

4.3 Leaf Biotoxicity Assay 80

4.4 Isolation of RbcS promoter from Gossypium arboreum 82

4.4.1 RNA extraction and cDNA synthesis 82 4.4.2 Amplification and Gel Elution of RbcS promoter 83 4.4.3 Sequencing of RbcS promoter Region 84

4.5 Cloning of insecticidal gene under RbcS in pCAMBIA 1301 85 4.5.1 Amplification and Gel Elution of Cry1Ac+NOS Fragment 85 4.5.2 Ligation and Transformation 86 4.5.3 Confirmation of Cry1Ac+NOS Fragment in pCAMBIA 1301 86 4.5.4 Ligation and Transformation 88 4.5.5 Confirmation of RbcS+Cry1Ac+NOS Fragment in pCAMBIA 1301 88 4.5.6 Orientation of RbcS+Cry1Ac in pCAMBIA 1301 91 4.6 Electroporation and Agrobacterium mediated Transformation 92 4.6.1 Transient Expression of GUS gene 92 4.6.2 Transformation Efficiency 93

4.7 Molecular Analysis of Transgenic Plants (T0 progeny) 95 4.7.1 PCR Analysis of Transgenic Plants 95 4.7.2 Southern Blot Analysis of Transgenic Plants 97 4.7.3 Temporal and Spatial Expression of Cry1Ac gene 98

4.8 Molecular Analysis of Transgenic Plants (T1 progeny) 100 4.8.1 Polymerase Chain Reaction 100

4.8.2 SDS Polyacrylamide Gel Electrophoresis 100 5 DISCUSSION 103 6 LITERATURE CITED 115 7 PUBLICATIONS 144 8 APPENDICES 145

xiv

LIST OF FIGURES

Figure-3.1 Map of vector pCAMBIA 1301 adapted from (http://www.cambia.org) 54 Figure-3.2 Construct Map PIA-2 55 Figure-3.3 Cassette showing Cry1Ac under RbcS promoter in pCAMBIA 1301 62 Figure-3.4 Cassette showing Cry1Ac under 35S promoter (pk2Ac ) 62 Figure-4.1 PCR amplification of Cry1Ac gene from cotton transgenic lines 70 Figure-4.2 PCR amplification of Cry2A gene from cotton transgenic lines 71 Figure-4.3 Southern Blot of Cry1Ac in Transgenic Plants 72 Figure-4.4 Western blot showing 68Kda band in Transgenic Plants 73 Figure-4.5 Temporal Expression of Cry1Ac in transgenic cotton lines 74 Figure-4.6 Spatial Expression of Cry1Ac in transgenic cotton lines 76 Figure-4.7 Temporal Expression of Cry2A in transgenic cotton lines 77 Figure-4.8 Spatial Expression of Cry2A in transgenic cotton lines 79 Figure-4.9 Leaf Biotoxicity Assay with 2nd instar Heliothis Larvae 80 Figure-4.10 Mortality rates of Heliothis larvae at different stages of crop 81 Figure-4.11 Total RNA extracted from Gossypium arboreum var. NIAB-846 82 Figure-4.12 PCR amplification of RbcS promoter from Gossypium arboreum 83 Figure-4.13 Promoter Sequence isolated from Gossypium arboreum 84 Figure-4.14 PCR amplification of Cry1Ac+NOS from PIA-2 85 Figure-4.15 PCR and Digestion confirming integration of Cry1Ac+ NOS insert 87 Figure-4.16 PCR Amplification of RbcS and Cry1Ac from pCAMBIA1301 89 Figure-4.17 Digestion confirming integration of RbcS+Cry1Ac+ NOS insert 90 Figure-4.18 Cassette showing Cry1Ac under RbcS promoter in pCAMBIA 1301 90 Figure-4.19 Orientation confirmation of RbcS+Cry1Ac in pCAMBIA 1301 91 Figure-4.20 GUS histochemical Assay of Transgenic Embryos and leaves 92 Figure-4.21 Schematic representation of cotton transformation steps 94 Figure-4.22 PCR amplification of RbcS and Cry1Ac in Rb-Ac transgenic plants 95 Figure-4.23 PCR amplification of 35S and Cry1Ac in pk2Ac transgenic plants 96 Figure-4.24 Southern Blot of Cry1Ac in putative transgenic plants 97 Figure-4.25 Temporal Expression of Cry1Ac in Rb-Ac and pk2Ac plants 98

Figure-4.26 Spatial Expression of Cry1Ac in Rb-Ac and pk2Ac plants 99

Figure-4.27 Amplification of Cry1Ac in Rb-Ac plants (T1 Progeny) 101

Figure-4.28 Amplification of Cry1Ac in pk2Ac plants (T1 Progeny) 101

Figure-4.29 SDS PAGE showing 68Kda band of Cry1Ac protein 102

xvi

LIST OF TABLES Table-3.1 Primer List for the amplification of Cry1Ac and Cry2A genes 41 Table-3.2 Primer list for the amplification of RbcS Primer 48 Table-3.3 Reaction Mix of Ligation 50 Table-3.4 Reaction mix of Digestion 51 Table-3.5 Reaction Mix for Sequencing 52 Table-3.6 Primer List for the amplification of Cry1Ac +NOS fragment 55 Table-3.7 Reaction mix of Digestion with Kpn1 and BamH1 56 Table-3.8 Reaction Mix of Ligation 57 Table-3.9 Reaction mix of Digestion 58 Table-3.10 Reaction mix of Digestion with Kpn1 59 Table-3.11 Reaction Mix of Ligation 60 Table-3.12 Reaction mix of Digestion with EcoR1 and BamH1 61 Table-3.13 Primer list for the confirmation of insert orientation 62 Table-4.1 ANOVA of Cry1Ac protein among transgenic lines and sampling dates 75 Table-4.2 ANOVA of Cry2A protein among transgenic lines and sampling dates 78 Table-4.3 Transformation Efficiency of NIAB-846 with Rb-Ac and pk2Ac 93 Table-4.4 Chi Square test to evaluate goodness of fit test of Cry1Ac in Rb-Ac 102 Table-4.5 Chi Square test to evaluate goodness of fit test of Cry1Ac in pk2Ac 102

xvii

ABBREVIATIONS Rb-Ac Cry1Ac gene under RbcS promoter

Pk2Ac Cry1Ac gene under Cry1Ac promoter

μL micro litre

μg micro gram

°C degree centigrade

% Percent

ATP Adenosine Triphosphate

BSA Bovine Serum Albumin

Bt Bacillus thuringiensis

BCIP/NBT 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium cm Centimeter

Cry Crystal

DNA Deoxyribonucleic Acid dNTPs Dinucleotide Triphosphate

EDTA Ethylene diamine tetra acetic acid

ELISA Enzyme Linked Immunosorbent Assay g Gram

GUS Glucuronidase

HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid

HgCl2 Mercuric Chloride

Kb Kilobase

KCl Potassium Chloride

KDa kilo Dalton

L Litre

SSU Rubisco small sub unit

xviii

SSL Rubisco Large sub unit

Na2CO3 Sodium Carbonate

NaCl Sodium Chloride

NaOH Sodium Hydroxide

M Molar mg Milligram mgL-1 Milligram per litre mL milli litre mM milli molar

MS Murashige and Skoog

N2 Nitrogen

Na Sodium

NADP Nicotinamide adenine dinucleotide phosphate ng nano gram nm nano meter (wavelength)

NPT Neomycin phosphotransferase

OD Optical Density

PBS Phosphate Saline Buffer pH power of Hydrogen pmol pico moles

RNA Ribonucleic Acid rpm rounds per minute

SDS Sodium Dodecyl Sulphate

TE Tris Ethylene diamine tetra acetic acid

Ti Tumor inducing

U Units

YEP Yeast Extract Peptone

xix

1. INTRODUCTION

“Cotton is the most important cash crop and backbone of textile industry of the world.

Pakistan is the fifth largest producer of cotton in the world, the third largest exporter of raw cotton, the fourth largest consumer of cotton, and the largest exporter of cotton yarn. 1.3 million Farmers

(out of a total of 5 million) cultivate cotton over 3 million hectares, covering 15 per cent of the cultivable area in the country. Cotton and cotton products contribute about 10 per cent to GDP and

55 per cent to the foreign exchange earnings of the country. Taken as a whole, between 30 and 40 percent of the cotton ends up as domestic consumption of final products (Economic Survey of

Pakistan 2008-09). “

“Cotton is susceptible to attack by more than 15 economically important insects, the major lepidopteron being, american Boll worm (Heliothis armigera), pink Boll worm (Pectinophora gossypiella), spotted Boll worm (Earius insulana/vitella), army Bollworm (spodoptera lithura). At

present, crop protection in agricultural system relies almost exclusively on the use of broad

spectrum highly toxic agrochemical that has led to serious environmental problems and human

health concerns, leading to make efforts towards developing its biological control measures

(Bakhsh et al. 2009). To combat losses from insect pests, insecticides are used excessively every

year in developing countries like Pakistan. In 2006-07, 16,495.00 tons of pesticides were consumed

which cost 66 Millions US Dollars (Economic Survey of Pakistan 2008-09). “

“Cotton breeders have continuously sought to improve cotton through conventional plant breeding which has introduced numerous improvements in crop yield during past centuries.

However, resistance to insect pests and diseases does not exist in available germplasm. This has led to a limit in availability of new genetic information into plants and to create plant varieties with novel characters through plant breeding techniques (Hussain et al. 2000). Current approaches to 1

cotton improvements include use of genetic engineering that has gained momentum in developed as well as developing countries. “

“One of the goal of plant breeder is to pyramid the genes expressing agriculturally desirable characteristics. This strategy has also been adopted by the biotechnologists. In order to increase the protective efficacy, the spectrum of activity and durability of resistance, it is envisaged that packages of different genes will be introduced into the crops. For this purpose cotton has been genetically engineered by insecticidal genes taken from Bacillus thuringienesis. “

“Bacillus thuringiensis (Bt) is a gram positive, spore-forming, soil-dwelling bacterium that produces crystalline protein inclusions known as δ-endotoxins. The number of sequenced crystal proteins in Bt is more than 100, encoding Cry and Cyt proteins (Schnepf et al. 1998). These crystal proteins are toxic to larvae of different orders of insects e.g. lepidoptera, coleoptera and diptera.

These are being widely used to develop insect resistance in various crops (Gasser and Fraley,

1992). The traditional breeding program could successfully accomplish pyramiding the foreign Bt genes with native insect resistant trait, in a single genetic background (Altman et al. 1996 and

Sachs et al. 1998). “

“The most significant breakthrough in plant biotechnology is the development of the techniques to transform genes from unrelated sources into commercially important crop plants to develop resistance against insect pests (Lycett and Grierson, 1990; Dhaliwal et al. 1998). Bacillus thuringiensis (Bt) is perhaps, the most important source of insect resistant genes. Genes from B. thuringiensis encode for crystal proteins, which are toxic against larvae of different insects, e.g.

Lepidopterans (Höfte and Whiteley, 1989; Cohen et al. 2000), Coleopterans (Krieg et al. 1983;

Herrnstadt et al. 1986) and Dipteran insects (Andrews et al. 1987). These genes are generally safe for human consumption (BANR, 2000). “ 2

“Genetically modified (GM) crops were grown on 800 million ha globally (James 2008).

Among them, transgenic cotton expressing insecticidal proteins from Bacillus thuringiensis (Bt) has been one of the most rapidly adopted GM crops in the world (James 2002, Barwale et al. 2004,

Dong et al. 2005) containing Cry gene(s) such as Cry1Ac, Cry1Ac + Cry2Ab or Cry1Ac + Cry1F,

Bt cotton is considerably effective in controlling lepidopteran pests, and is highly beneficial to the

grower and the environment by reducing chemical insecticide sprays and preserving population of

beneficial arthropods (Gianessi and Carpenter, 1999; Tabashnik et al. 2002). “

“Mechanism of endotoxins to kill the targeted insect is actually the action of the Bt Cry

proteins involving solubilization of the crystal in the insect midgut, when ingested by larvae, toxin

proteins bind to specific receptors in the midgut region, and toxin binding in susceptible insects

disrupts midgut epithelium, thereby causing overall toxic effects and ultimately resulting in death

of the larvae (Kranthi et al. 2005). This strategy proved to be beneficial in many cotton growing countries of the world in order to get rid of heavy pest pressure which was really costly and laborious in terms of spraying schedule and was not environmental friendly as well. “

“Transgenic cotton expressing Bt (Bacillus thuringiensis) toxins is currently cultivated on a large commercial scale in many countries, but data has shown that it behaves variably in toxin efficacy against target insects under field and green house conditions conditions (Kranthi et al.

2005). Understanding of the temporal and spatial variation in efficacy and the resulting mechanisms is essential for cotton protection and production. “

“For the Bt cotton to be sustainable, it is important that the toxin protein be expressed in adequate quantities in appropriate plant parts at the requisite time of the season to afford protection against major target insect pests. However, a number of studies conducted in Australia, USA,

China and India have indicated that the levels of Bt protein in cotton tissues fluctuate during the

whole growing season, and may logically cause variation in efficacy of Bt cotton against lepidopteron pests (Benedict et al. 1996; Chen et al. 2000; Greenplate et al. 2001; Mahon et al.

2002, Kranthi et al. 2005). “

“Constitutive promoters are widely used in insect-resistant transgenic rice to express Bt

genes, such as the 35S CaMV promoter (Cheng et al. 1998; Alam et al. 1999), the ubiquitin promoter (Chen et al. 2005; Tang et al. 2006) and the actin promoter (Wu et al. 1997; Tu et al.

2004). Nearly all transgenic crops around the world utilize the CaMV 35S promoter (Odell et al.

1985) (or similar promoters from closely-related viruses) to drive transgenes. It is only now

becoming clear that this promoter is not as robust as laboratory and glasshouse studies have

suggested and its function is influenced by as yet undefined physiological and perhaps

environmental factors (Sunilkumar et al. 2002). “

“The second most common group of promoters after viral promoters for plant

biotechnology have come from highly-expressed plant genes, such as those for seed storage

proteins, photosynthetic proteins or housekeeping genes, all of whose mRNAs were easily cloned

and characterized (Potenza et al. 2003). Actin, ubiquitin and tubulin gene promoters have all been

used in various plant species for expressing transgenes or selectable markers. As sophistication in

biotechnology improves, the need for more developmentally or environmentally regulated promoters has become evident and considerable effort is going into the discovery of specific tissue or biotic, hormonal or abiotic stress responsive genes and promoters (Potenza et al. 2003). “

“RuBisCo is the bifunctional enzyme found in the chloroplasts of plants that catalyzes the initial carbon dioxide fixation step in the Calvin cycle and functions as an oxygenase in photorespiration. In higher plants it consists of eight each of two subunits, a large subunit (LSU) encoded by the chloroplast genome and the small subunit (SSU) polypeptide encoded in the

nuclear genome (Ellis, 1981). The SSU polypeptides are formed as precursors containing an

amino-terminal extension termed a transit peptide that is involved in the transport of the SSU

polypeptide into the chloroplast during which the transit peptide is removed. Rubisco is the most

abundant protein found in plant leaves, representing up to 50% of the soluble protein. Thus, the

SSU promoters and their transit peptides are attractive candidates for expression of genes at high

levels in green tissue and for targeting of different proteins into the chloroplast (Ellis, 1981). “

“The small subunits promoters and their transit peptides have been used for expression of

genes at high levels and have shown promising expression results in driving the expression of

introduced genes (Wong et al. 1992; Fujimoto et al. 1993; Datta et al. 1998: Song et al. 2000; Jang et al. 2002; Liu et al. 2005; Panguluri et al. 2005; Amarasinghe et al. 2006; Anisimov et al. 2007;

Suzuki et al. 2009; Kim et al. 2009). “

“A study was conducted by Amarasinghe et al. (2006) in which he used RbcS (Rubisco small subunit) promoter to drive the expression of Bt gene and he found that the Bt expression was consistent in plants in green house and field conditions. Anisimov et al. 2007 studied activity of the highest expressing rubisco small subunit (RbcS) promoters (pRbcS) from the cotyledons of germinating seedlings of Brassica rapa var. oleifera to drive high-level and preferably stage specific transgenic protein expression in Brassicaceae plants. The mRNA level of RbcS and of gusA quantified in transformed plants. The results demonstrated that promoter most active in seedlings under native conditions was also most active in transgenic constructs at the same stage of plant development. “

“A locally approved cotton cultivar CIM-482 was transformed with two insecticidal genes

Cry1Ac and Cry2A using Agrobacterium mediated transformation. A construct pk2Ac was

developed by isolating Cry1Ac and Cry2A genes from locally soil dwelling bacterium and further 5

transformed in cotton (Rashid et al. 2008). Both genes were driven by constitutively expressing

CaMV 35S Promoter that enables the expression of both endotoxins in almost all plant parts. “

The present study was conducted on transgenic cotton lines derived from locally transformed CIM-482 cotton variety with two insecticidal genes Cry1Ac and Cry2A. The study was carried out at campus of National Centre of Excellence in Molecular Biology (CEMB), University of the Punjab, and Lahore, Pakistan with the purpose to determine stable integration and spatio temporal expression of insecticidal genes in advance cotton lines. Variation in endotoxins expression levels was found at different growth stages of the crop and also in different plant parts.

A strategy was devised out to make the endotoxins levels consistent in cotton plant. For this purpose, a promoter from photosynthetic genes (RuBisCo small subunit) was isolated from

Gossypium arboreum and a construct was developed in which Cry1Ac gene was driven by RbcS promoter, followed by NOS terminator. The transformation of this construct was carried out in local cotton variety (NIAB-846) using Agrobacterium mediated transformation. The same cotton varity was transformed with another construct pk2Ac having Cry1Ac gene under 35S promoter.

Molecular analysis of the putative transgenic plants showed the integration and expression of

Cry1Ac gene. The further results showed that endotoxin was being produced in higher quantity in

Rb-Ac plants; also the expression was consistent as compared to plants transformed with pk2Ac construct.

2. LITERATURE REVIEW

“Bacillus thuringiensis is a gram positive, spore forming, soil dwelling bacterium that

produces crystalline protein inclusions known as δ-endotoxins (Martin and Travers, 1989). These

crystalline proteins and spores have been used as microbial insecticide for over forty years (Flexner

et al. 1986) and are highly insecticidal at even low concentrations (Schnepf et al. 1998). As these

proteins are non-toxic to mammals and other organisms, Bt strains and their insecticidal crystal

proteins (ICPs) have acquired acceptability as eco-friendly biopesticides all over the world. Bt

protein is under extensive use in agriculture, horticulture, forestry, animal health and mosquito control from the time of its discovery (Schnepf et al. 1995). These ICPs are classified on the basis

of amino acid sequence homology. The ICPs fall under 40 different classes with some toxins

exhibiting specificity to multiple insect orders (Crickmore et al. 1998). “

“It has been concluded that the mechanism of action of the Bt Cry proteins involves

solubilization of the crystal in the insect midgut, when ingested by larvae, toxin proteins bind to

specific receptors in the midgut region, and toxin binding in susceptible insects disrupts midgut

epithelium, thereby causing overall toxic effects and ultimately resulting in death of the larvae

(Kranthi et al. 2005). “

“The presence or absence of the appropriate receptors is believed to play a role in the

specificity of the mode of action of the Bt proteins. There have been two main receptor types, the

cadherins or cadherin-like proteins (Dorsch et al. 2002; Xie et al. 2005) and aminopeptidase N

(Knight et al. 1994; Luo et al. 1997). Hossain et al. (2004) hypothesized an interaction between the

two types of above mentioned receptors for Bt protein, in the mid gut of susceptible insects. “

“With the advent of molecular biology and genetic engineering, it has become possible to

use Bt more effectively and rationally by introducing the ICPs of Bt in crop plants (Kumar et al.

1996). The first transgenic plants using Bt genes were developed in 1987. The tobacco plants

engineered with truncated genes encoding cry1Aa and cry1Ab toxins were found to be resistant to

the larvae of tobacco hornworm (Jouanin et al. 1998). However, the levels of Cry protein

expression in the plant tissues were not very high. “

“A significant breakthrough was made in 1990 by researchers at Monsanto Company

(USA) who modified the Bt genes (Cry1Ab and Cry1Ac) for better expression in plant cells (Perlak et al. 1991). Expression of such modified genes in crop plants, Cry1Ac in cotton and cry3Aa in potato, conferred considerable protection against lepidopteran and coleopteran pests respectively.

Subsequently, many crop plants which include rice, maize, peanut, soybean, canola, tomato and cabbage were transformed with various modified Bt genes (De Maagd et al. 1999). Selvapandiyan et al. (1998) provided an interesting example of native gene (cry1IA5) expression resulting in significant resistance to Helicoverpa armigera in transgenic tobacco. “

“Genetically modified (GM) crops were grown on 800 millionth hectares globally in 2008

(James 2008). Among them, transgenic cotton expressing insecticidal proteins from Bacillus thuringiensis (Bt) has been one of the most rapidly adopted GM crops in the world (James 2002;

Barwale et al. 2004; Dong et al. 2005). Containing Cry gene(s) such as Cry1Ac, Cry1Ac + Cry2Ab

or Cry1Ac + Cry1F, Bt cotton is considerably effective in controlling lepidopteran pests, and is

highly beneficial to the grower and the environment by reducing chemical insecticide sprays and

preserving population of beneficial arthropods (Gianessi and Carpenter, 1999; Tabashnik et al.

2002). “

2.1 Insect Resistance Management (Two Genes Strategy)

“It was believed in the past that insects will not develop resistance to B. thuringiensis

toxins. However, a number of insect populations of several different species with different levels of resistance to B. thuringiensis crystal proteins were obtained by laboratory selection experiments in mid 1980s using either laboratory adapted insects or insects collected from wild populations (Ferré et al. 1995; Tabashnik 1994). Examples of laboratory-selected insects resistant to individual Cry toxins include the Indianmeal moth (Plodia interpunctella) (McGaughey, 1985), the almond moth

(Cadra cautella) (McGaughey and Beeman 1988), the tobacco budworm (H. virescens) (Gould et al. 1992), the Colorado potato beetle (Leptinotarsa decemlineata) (Whalon et al. 1993), the cabbage looper (T. ni) (Estada and Ferré, 1994), the beet armyworm (S. exigua) (Moar et al.1995),

European corn borer (O. nubilalis) (Bolin et al.1995), the cotton leafworm (Spodoptera littoralis)

(Cohn et al.1996). It is important, however, to keep in mind that selection in the laboratory may be very different from selection that occurs in the field. Insect populations maintained in the laboratory presumably have a considerably lower level of genetic diversity than field populations.

The potential for fast evolution of insect resistance to Bt crops threatens the benefits achieved by planting the transgenic Bt crops (Tabashnik, 1994; Gould, 1994). Hence, evolution of resistance to the single toxin plants will not only have immediate negative consequences but also far reaching implications for integrated pest management.”

“Resistance management strategy generally relies heavily on the theoretical assumptions and on computer models simulating insect population growth under different conditions (Mallet and Porter, 1992; Roush, 1994; Gould, 1994; Tabashnik, 1994; Alstad and Andow, 1995; Ives et al. 1996). The proposed strategy includes the use of multiple toxins i.e. pyramiding or stacking

(Salm, et al. 1994). Cry toxins that recognize different receptors in same target species can be

deployed in this strategy: since they are very less prone to cross-resistance. “

“Cohen et al. (2000) reported that there is always a risk that insects could become resistant

to Bt toxin after prolonged and repeated field exposures. “The most practical approach to prolong

the effectiveness of Bt crops has been refugia strategy and pyramiding of two or more genes in the

same cultivar (Cohen et al. 2000).”

“Insect populations, which are relatively resistant against one crystal protein, show wild type sensitivity towards crystal proteins of other classes (Salm et al. 1994). Therefore, simultaneous application of different crystal proteins is a necessary component of sound resistance management. So strategies to avoid the evolution of resistance include expressing multiple Bt toxins at high doses or fusing Bt toxins together, with resistance in both approaches requiring the unlikely acquisition of multiple simultaneous mutations. Theoretical models also suggest that plants containing two dissimilar Bt toxin genes (pyramided plants) have the potential to delay resistance more effectively than single-toxin plants used sequentially or in mosaics (Zhao et al.

2003).”

“Bt cry toxins could be combined with other insecticidal proteins. The multiple-attack strategy assumes that within a population, if insects homozygous for one resistance gene are rare, then insects homozygous for multiple resistance genes are extremely rare. Crops or sprays deploying multiple toxins would still control even insects homozygous for one or two resistance genes yet heterozygous for another gene. “Simultaneous introduction of three insecticidal genes cry1Ac, cry2A and GNA into indica rice basmati 370 have been reported (Maqbool et al. 2001) while cry1Ab and cry 1Ac (Cheng et al. 1998), “tobacco was transformed with cry1Ac and GNA

(Zhao et al. 2001), tomato with cry1Ab and cry1Ac (Salm et al. 1994). “Cotton larvae which were

fed fresh plant tissue indicated that dual toxin B. thuringiensis cultivars expressing cry1Ac and

cry2A endotoxin were more toxic to bollworm (Helicoverpa zea) army worm (Spodoptra

frugiperda) and beet worm (Spodoptra exigua) than single toxin (Stewart et al. 2001). Crop

rotation, high or ultrahigh dosages, and spatial or temporal refugia have also been recommended

(McGaughey and Whalon, 1992; Tabashnik, 1994).”

“A unique model system consisting of Bt transgenic broccoli plants and the diamondback

moth, (Plutella xylostella) a greenhouse study was conducted using an artificial population of

diamondback moths carrying genes for resistance to the Bt toxins cry1Ac and cry1C. After 24

generations, resistance to pyramided two-gene plants was significantly delayed as compared with

resistance to single-gene plants deployed in mosaics, and to cry1Ac toxin when it was first used in

a sequence. These results have important implications for the development and regulation of

transgenic plants.”

“A plant expression vector containing Cry1Ac protein and the proteinase inhibition gene

API-B was introduced into an elite cotton cultivar by Agrobactertium tumefaciens (Wu et al. 2003).

Based on the results of kanamycin resistant test and insect bioassay using Heliothis armigera, PCR

detection and Southern-blot, they found that the inheritance and segregation of Bt gene were

complicated, some transformants were in accordance with mendelian patterns of inheritance, yet

others were non-Mendelian patterns. “

“Greenplate et al. (2003) performed laboratory studies to characterize the lepidopteran

toxicity of cotton plants expressing two different toxin proteins from Bt, Cry1A only, Cry2Ab

only, or both toxins. In all cases, the Cry2Ab component was the larger contributor to total toxicity

in the two-toxin isoline. Toxin-specific, ELISA tests confirmed that the levels of each toxin in tissues of the two-toxin isoline were not statistically different from the levels found in the corresponding tissues of the respective single-toxin isoline. Considering the additive interaction of toxins, a relatively simple insect resistance monitoring procedure was proposed for the monitoring of commercial cotton varieties expressing both toxins.”

” “Efficacy of the transgenic cotton, Gossypium hirsutum (L.), genotype, Bollgard II

(Monsanto 15985), which expressed two Bt proteins (Cry1Ac+Cry2Ab) active against lepidopterous pests was compared with Bollgard (DP50B), which expressed only one Bt protein

(Cry1Ac), and, in all tests, the conventional variety, DP50, was used as a non-Bt control. Dual- toxin Bollgard II genotype was found highly effective against lepidopterous pests that were not adequately controlled by the current single-toxin Bollgard varieties (Chitkowski et al. 2003). “

“The double construct Bollgard® II Bt cotton, with the two genes cry1Ac and cry2Ab was approved in Australia in 2002. It was the first approval for the product globally, with clearance in the US. Bollgard II is more effective than Bollgard I and will further reduce insecticide requirements and most importantly provide more durable resistance (Bosch et al. 2002). The two genes together in the Bollgard II package deliver an effective punch against lepidopteran pests. The second toxin expresses itself in high levels in the leaves, squares, bolls and flowers. There are added benefits of the two genes working in concert together.”

Second generation dual- Bt gene cottons Bollgard II® (Cry 1Ac + Cry 2Ab) and

WideStrike™ (Cry 1Ac + Cry 1F) express two Bt endotoxins and were introduced in order to raise the level of control for H. zea, which was not satisfactorily controlled by the Cry 1Ac toxin alone

(Jackson et al. 2003; Ferry et al. 2004; Bates et al. 2005; Gahan et al. 2005). The Cry 1Ac and Cry

2Ab toxins have different binding sites in the larval midgut and are considered to be a good

combination to deploy in delaying resistance evolution. This is due to the fact that a species cannot

easily evolve resistance to both toxins because that would require two simultaneous, independent

mutations in genes encoding the receptors (Jackson et al. 2003). This argument was reinforced by

Bird and Akhurst (2005) when they found that insects developed resistance to one crystal protein

group by changing particular receptor sites but were still fully susceptible to other crystal proteins.

For the sustainable resistance in cotton plants, Rashid et al. (2008) transformed a locally

approved cotton variety CIM-482 with two insecticidal genes Cry1Ac and Cry2A using sonication assisted Agrobacterium mediated transformation. Cry1Ac and Cry2A genes were isolated from locally soil dwelling bacterium and further transformed in cotton. This strategy delayed the targeted insect pest resistance in cotton.

“The Single-gene Bt plants are most prevalent but from a resistance management standpoint they are inferior to dual-gene plants. Fiber quality, the agronomic characteristics of the plant and seed composition remain unchanged. Industry trends suggest that pyramided plants may be favored in the future. Along with effective insect control, pyramided plants have an added advantage of requiring a smaller refuge, a part of the field where non-Bt plants are grown. Refuges create opportunities for Bt-resistant insects to mate with other insects that do not have resistance. The offspring of such a mating will be susceptible to the toxins. New cotton technology is being developed to provide improved insect control and a wider spectrum of activity (Perlak et al.

2001).”

Selection pressure in transgenic plants can be reduced by restricting the expression of the crystal protein genes to certain tissues of the crop, the remainder providing a form of spatial refuge

(Schnepf et al. 1998; Shelton et al. 2000). This strategy is based on differential toxin expression 13

across the crop’s phenology. Material from the extremes of the plant (the tips, bolls and leaves

adjacent to the bolls) results in high mortality to insects whilst lower mortality was observed from feeding on leaf tissue in the 2nd to 4th node and in the flowers (Harris et al. 1998).

“Transgenic cotton expressing Bt (Bacillus thuringiensis) toxins is currently cultivated on a large commercial scale in many countries, but observations have shown that it behaves variably in

toxin efficacy against target insects under field conditions. Understanding of the temporal and

spatial variation in efficacy and the resulting mechanisms is essential for cotton protection and

production (Bakhsh et al. 2009). “

2.2 Temporal and Spatial Variations in Insecticidal Protein

“It is important that the toxin protein be expressed in adequate quantities in appropriate

plant parts at the requisite time of the season to afford protection against major target insect pests

to contain sustainability. A number of studies conducted in Australia, USA, China and India have

indicated that the levels of Bt protein in cotton tissues fluctuate during the whole growing season,

and may logically cause variation in efficacy of Bt cotton against lepidopteron pests (Benedict et

al. 1996; Chen et al. 2000; Greenplate et al. 2001; Mahon et al. 2002; Kranthi et al. 2005). “

“Fitt et al. 1998 reported that cotton plants carrying Cry1Ac have shown significant

declines in efficacy against Helicoverpa spp. During the growing season, particularly from

flowering onwards (Greenplate et al. 1998). It was reported that toxins to Helicoverpa armigera in

leaves of a commercial Chinese cotton variety GK-12 that contains Cry1Ac were significantly

decreased as the crop approached maturation (Wu et al. 2003), while insecticidal protein levels in

GK-19, a transgenic Bt cotton cultivar carrying a Cry1Ac/Cry1Ab fused gene, were higher during

the early stages of cotton growth and significantly declined hereafter, behaving more variably than

in GK-12 during the whole period of cotton growth and development (Wan et al. 2005). “

“The season-long differences in expression of Cry1Ac among cultivars can vary as much as

twofold throughout the growing season (Adamczyk et al. 2001; Adamczyk and Sumerford, 2001;

Greenplate et al. 2001). “Resistant power to the targeted boll worm in Bt transgenic hybrid cotton

remained only for 110 days, after which the crop can be exposed to bollworm attacks. The Cry1Ac

level declined as the plant grew and was found to drop below its lethal level of 1.9µg /g within 110

days after sowing (Kranthi et al. 2005). “

“Many researchers reported that it seems to be a common phenomenon that the efficacy is

relatively high in early growing season, but significantly declines during late season for most

commercialized Bt cotton varieties (Greenplate, 1999; Greenplate et al. 2000; Xia et al. 2005). “

“Chen et al. (2000) reported that Bt protein concentrations also differ in different parts of

the plant. Toxin protein content in the fully expanded leaves was significantly higher than those in

roots, stems and petioles during the seedling stage, while ovaries at anthesis expressed

considerably more toxin protein than pistils and stamens at the flowering stage. It was further

indicated that in a seven to nine leaf-stage plant, the fully expanded leaves on main stem were

considerably higher than older basal leaves in toxin protein concentration, while the young leaves

near the stem terminal expressed the lowest levels of toxin proteins. “

“Olsen and Daly et al.( 2000) concluded that not only is there less Bt protein in older

plants, it appears that the protein is either less available or less toxic to neonates. The concentration

of Cry1Ac protein, as a proportion of total protein, also declines during the season (Holt et al.

1998). “

“Guo et al. (2001) introgressed transgenic lines containing Bt genes among each other as

well as with non transgenic conventional lines. Efficacy of transgenic lines against bollworms was

evaluated at different growing stages and it was found that there was a declining level of efficacy

with the plant age as the mortality (%) of Helicoverpa and Bt toxin protein expressional level

decreased gradually. “

“Mahon et al. (2002) reported that endotoxin protein concentrations in Bt cotton plants

decline markedly after squaring, and molecular analyses pinpointed the change to the production of

mRNA. Finnegan et al. (1998) found that Cry1Ac levels decreased consistently throughout the

growing season, and concluded that part of the decline in Cry1Ac expression was related to

reduction in the levels of mRNA production. “

“Kranthi et al. (2005) reported that in Bt transgenic hybrid cotton containing Cry1Ac, the leaves were found to have the highest levels of Cry1Ac expression followed by squares, bolls and flowers. The toxin expression was the lowest in the ovary of flowers and rinds of green bolls which

are the most favoured sites of bollworm attack. “

“Olsen et al. (2005) reported that the developmental decline in bioefficacy in field-grown plants was associated with reduced Cry1Ac transcript levels and Bt toxin levels in post-squaring cotton. Changes in the efficacy of Bt toxin were attributed to changes in plant chemistry associated with the maturation of the cotton plant. “

“Xia et al. (2005) investigated the changes of Bt gene and its expression at different developmental stages at DNA, mRNA and protein levels in the R4 generation of GK139-20 insect-

resistant transgenic materials. It was found that the expression of Bt toxin gene was in a temporal

or spatial manner, and the content of Bt crystal protein in the same tissue decreased along with the

growth of the transgenic cotton plants because of the decrease in full-length Bt toxin gene

transcripts. The over expression of Bt toxin gene at earlier stages led to gene regulation at the post-

transcription level and contributed to the consequent gene silencing that was developmentally

regulated. Lower expression level of Bt insecticidal gene at late development stages correlated with

changes in the methylation state of the 35S promoter region. “

“Bakhsh et al. (2009) studied the spatio -temporal expression of two insecticidal genes

(Cry1Ac and Cry2A) in transgenic cotton. The quantitative levels of both Cry1Ac and Cry2A genes

were found variable among the cotton lines and also varied between different plant parts. The expression of both genes declined gradually with the age of plant as well as in different plant parts through out the season. The maximum endotoxins expression was found in leaves of Bt cotton followed by squares, bolls, anthers and petals. Toxin level in fruiting part was less as compared to other parts showing the inconsistency in toxin level inspite of using 35S CaMV promoter which is constitutively expressing promoter. “

Manjunatha et al. (2009) studied four Bt cotton hybrids (MRC-7201, MRC-6918, RASI

XL-708 and SP-11) for Cry1Ac protein profiling in fully opened terminal leaf, first terminal pre- candle square and first terminal bolls at 80-90, 110-115, 135-140 and 150-165 days after sowing.

The results indicated that the expression of Cry1Ac was declined over season independent of the genotype. Among the hybrids the expression level was different.

“Adamczyk et al. (2009) developed a method to determine if differences in the overall level

of Cry1Ac among Bollgard lines could be correlated to the mRNA transcripts using quantitative

real-time polymerase chain reaction (qPCR). The commercial cultivars of Bollgard cotton,

Gossypium hirsutum L., used in this study differed in the amount of expressed Cry1Ac protein. He

found that Cry1Ac mRNA transcript differs among Bollgard lines and are correlated with

corresponding Cry1Ac protein levels. The plant-mechanism for which this occurs is still unknown.

2.3 Factors Contributing to Endotoxin Variability

“Several factors might be responsible in gene expression variation ie. Change nucleotide

sequence of the gene, promoter, and the insertion point of the gene in the DNA of the transgenic

variety, transgene copy number, the internal cell environment, as well as several external factors in

the environment (Hobbs et al. 1993; Guo et al. 2001; Rao, 2005). Therefore, investigation at

molecular, genetic, as well as physiological levels should help in understanding the differential

expression of transgene and the quantitative changes in insecticidal proteins in Bt cotton plants. “

“The full expression of transgene(s) in a transgenic crop variety is crucial to agricultural

production, but the expression levels of a gene in the transgenic crop may decrease as the age of

the crop advances, vary between young and older parts such as the leaves or between comparable

parts in vegetative and reproductive phases. Factors such as soil characteristics, rainfall, the

severity of pests and diseases, and adequate, appropriate and timely farming management have

direct or indirect influences on the performance of the crop and may affect the expression of the

transgenes. All these factors, inherent in the varieties and the environment, vary from crop season

to season, make the difference between optimal or below optimal performance of a crop or its

failure (Rao, 2005). “

“Several environmental factors in genetically modified crops have been reported to affect

the expression of transgenes such as water stress in Bt Maize (Traore et al. 2000) or by nitrogen

deficiency (Bruns and Abel, 2003), transgenic petunia that contains the gene encoding a

dihydroflavonol reductase by high light intensity and temperature (Meyer et al. 1992), transgenic

tomato that carries a gene encoding polygalacturonase and pectin methylesterase by high

temperature (Lurie et al. 1996), and transgenic peas containing a seed-specific r-amylase inhibitor

by water deficit (Sousa-Majer et al. 2004). “

“Although the molecular mechanism in the differential expression of Cry genes in Bt cotton

is not fully documented, a number of studies have indicated a close relationship between levels of

Bt toxin and the nitrogen as well as carbon metabolism. “

“Nitrogen is an essential nutrient for cotton production, but the traditional rate of nitrogen

fertilization for cotton may not have been optimized for the Bt transgenic cotton. It is assumed that

the pattern of allocation to defensive compounds depends on the relative availability of carbon and

nutrients as well as their relationship with the plant growth rate (Bryant et al. 1983). In Bt

transgenic cotton, the production of toxin protein was affected by an interaction between CO2 and nitrogen, and elevated CO2 deceased N allocation to Bt toxin (Coviella et al. 2002). A significant

correlation of Bt toxin concentration with whole-plant N concentration in Bt transgenic maize was

found by Bruns and Abel et al. (2003). “

“The increasing levels of available N may increase protein levels in plant tissues, especially

in vegetative cells in most plant species including cotton (Tisdale and Nelson et al. 1975). The

increased protein is mostly in the form of enzymes and can be used for further growth and

development. Therefore, as availability of N to the plant is increased, greater quantities of the

endotoxin-synthesizing enzymes and/or mRNA are likely produced, thus greater quantities of the

Bt toxin protein will be synthesized (Bruns and Abel et al. 2003). “

“The leaf tissue with low chlorophyll does not fully express Cry1A (Abel and Adamczyk et

al. 2004). It was further suggested that photosynthesis-regulating factors related to mRNA

transcription and translation should have effects on Cry1A production and insect control. Pettigrew

and Adamczyk et al. 2006 found that through applying varied rates and sources of nitrogen

fertilization to three commercial cotton varieties including two transgenic varieties, that higher

rates of nitrogen fertilization did not increase lint yield but produced higher leaf chlorophyll

concentrations and increased the expression of the Bt endotoxin protein. “

“By practicing the foliar applications of chaperone, a plant growth regulator, greatly

improved late-season endotoxin levels through the enhancement of protein status in Bt cotton,

particularly in the squares which resulting in increased mortality of neonate bollworms feeding on

the treated plants (Oosterhuis and Brown et al. 2003& 2004). It was further suggested that

chaperone appears to increase protein concentration and the efficiency of endotoxin expression

even at high-temperature stress (Brown and Oosterhuis et al. 2003). “

“Decline in endotoxin proteins in cotton tissues may also result from degradation of the

proteins or remobilization (translocation) of total N for further growth and development. It was

suggested that high temperature might result in degradation of total and endotoxin protein in the

leaf, and thus reduced the amount of the toxin protein and Bt cotton efficacy (Chen et al. 2005a). “

“The reduced levels of the toxin protein in early-planted cotton leaves were presumably

caused by remobilization of the leaf N to support the larger developing boll load compared with

late-planted cotton (Pettigrew and Adamczyk et al. 2006). Rui et al. 2005 detected a large amount

of endotoxin protein in xylem sap of Bt cotton plants and leaves of non-Bt cotton plants that were

grafted to the Bt plant, respectively, providing strong evidence that the endotoxin protein is

transportable. “

“A significant decline in glutamic-pyruvic transaminase (GPT) activity and soluble protein

contents was found (Chen et al. 2005) suggesting that high temperature may result in the

degradation of soluble protein in the leaf, with a resulting decline in the level of the toxin Cry1A. “

“Thirteen commercial varieties of transgenic Cry1Ac Bacillus thuringiensis Berliner (Bt)

cotton were examined across two sites know about the potential factors that impact endotoxin

expression. It was found that two varieties (NuCOTN 33B and DP458B/RR) having same parental

background (DP 5412) expressed Cry1Ac at significantly higher levels compared to the 11 other

Cry1Ac varieties. The data strongly suggest that parental background has stronger impact on the

expression of Cry1Ac than the environment (Adamczyk et al. 2001). However studies also show that variability in endotoxins expression is independent of genotypes who tested eight commercial hybrids and found the variation in endotoxins expression (Kranthi et al. 2005). “

“Constitutive promoters are widely used in insect-resistant transgenic rice to express Bt genes, such as the 35S CaMV promoter (Cheng et al. 1998; Alam et al. 1999), the ubiquitin promoter (Chen et al. 2005; Tang et al. 2006) and the Actin promoter (Wu et al. 1997; Tu et al.

2004). “

“Regulation can occur at many different stages of gene expression, and can be particularly important during transcription. The promoters that drive transgene expression ensure this control.

Promoters are regions of the DNA upstream of a gene’s coding region that contain specific sequences recognized by proteins involved in the initiation of transcription (Buchanan et al., 2000).

““ “Nearly all transgenic crops around the world utilize the CaMV 35S promoter (Odell et al.

1985) (or similar promoters from closely-related viruses) to drive transgenes. It is only now becoming clear that this promoter is not as robust as laboratory and glasshouse studies have suggested and its function is influenced by as yet undefined physiological and perhaps environmental factors (Sunilkumar et al. 2002). “

“Nilsson et al. (1992) transformed hybrid aspen (Populus tremula x P. tremuloides) with

fused bacterial luxF2 gene under cauliflower mosaic virus 35S promoter via Agrobacterium

tumefaciens. The luciferase expression in all transformants was determined by destructive 21

enzymatic assay as well as by non-destructive image analysis in leaves left attached to intact plants.

Variation in luciferase expression was found by both measurement techniques. It was found that

enzymatically assayed luciferase activity in leaves was notably lower in transgenic hybrid aspen plants than in tobacco plants transformed with the same vector. This was not due to a difference in luciferase enzyme activity between the two species, and therefore it indicated that the 35S promoter is not as active in hybrid aspen as in tobacco. “

“A study was conducted by Pauk et al. (1995) in which gene fusions between the β- glucuronidase (GUS) reporter gene and the promoters of the cauliflower mosaic virus 35S RNA transcript (CaMV 35S) and the mannopine synthase (mas) genes were introduced into rapeseed varieties via Agrobacterium-mediated transformation. Fluorometric assay of β-glucuronidase activity indicated that both promoters were functional in the majority of the studied organs of transgenic rapeseed plants, but the promoter activity varied considerably between the organs at different developmental stages. “

“Wessel et al. (2001) reported the variation in expression patterns of three promoters

(Cauliflower Mosaic Virus (CaMV) 35S, modified CaMV 35S and the promoter of an Arabidopsis thaliana Lipid Transfer Protein gene) using firefly luciferase reporter system. The expression of luciferase gene varied not only among independent transformatants but also between leaves on the same plant and within a leaf. Imaging of luciferase activity in the same leaves over 50 days period showed that individual transformatants show different types of temporal regulation and also this spatial and temporal pattern is inherited by the next generation. “

“Though 35S CaMV is presumed to be a constitutive promoter, some reports suggest that it is not expressed in all cell types. The expression profile of this promoter was investigated using the green fluorescent protein (GFP) gene as a reporter system in cotton during embryo development,

and in all the vegetative and floral cell and tissue types. After germination, varying levels of

promoter activity were observed in all cell and tissue types in the hypocotyl, cotyledon, stem, leaf,

petiole, and root. The promoter was also expressed in all floral parts. Although cotton pollen

exhibited a low level of greenish autofluorescence, Thus it is concluded that the expression of the

35S promoter was developmentally regulated being expressed in most cell and tissue types in

cotton albeit at different levels (Sunilkumar et al. 2002). “

“The binary transformation vector pVW432 carrying the GUS gene was fused to the CaMV

35S promoter to test transformation efficiencies and promoter activity in B. napus. Having studied the transformation efficiencies, different developmental stages of plants were analysed by protein assay to analyze promoter activity. β-glucuronidase activity altered from maturity to senescence

indicating that the CaMV 35S promoter has different activity at different developmental stages in

B.napus (Haddad et al. 2002). “

“Yang et al. (2005) transformed soybean (Glycine max L.) with chimeric -glucuronidase

(GUS) gene under the control of the 355 CaMV promoter using direct DNA transfer via electric

discharge particle acceleration. Expression of GUS gene in plants was localized using thin tissue

sections. Different levels of GUS were obtained in various tissues, being maximum in leaf, parenchyma cells in xylem, and phloem tissues of stem while very low or no GUS activity was detected in cortex cells of root, vascular cambium cells in stem, and the majority of cortex cells in leaf midrib. They concluded that 35S CaMV promoter is cell type specific and is developmentally regulated in soybean. “

“We must rely on conventional breeding and selection to solve these problems of variable efficacy of transgenic cotton, but it will be useful, in the longer term, to identify other gene promoters that can drive strong expression of transgenes throughout the season. It is also important

to have such promoters available for the next generation of transgenic cotton so that different traits

can be stacked without relying on the same promoter so as to avoid transcriptional gene silencing

induced by multiple copies of a single promoter such as the CaMV 35S promoter (Fagard &

Vaucheret, 2000). “

“The second most common group of promoters after viral promoters for plant

biotechnology have come from highly-expressed plant genes, such as those for seed storage

proteins, photosynthetic proteins or housekeeping genes, all of whose mRNAs were easily cloned

and characterized (Potenza et al. 2003). Actin, ubiquitin and tubulin gene promoters have all been

used in various plant species for expressing transgenes or selectable markers. As sophistication in

2.3 Ribulose-1, 5-Bisphosphate Carboxylase/Oxygenase Gene as Promoter

“RuBisCo is the bifunctional enzyme found in the chloroplasts of plants that catalyzes the

initial carbon dioxide fixation step in the Calvin cycle and functions as an oxygenase in

photorespiration. In higher plants it consists of eight each of two subunits, a large subunit (LSU)

encoded by the chloroplast genome and the small subunit (SSU) polypeptide encoded in the

nuclear genome (Ellis, 1981). The SSU polypeptides are formed as precursors containing an

amino-terminal extension termed a transit peptide that is involved in the transport of the SSU

polypeptide into the chloroplast during which the transit peptide is removed. RuBisCo is the most

abundant protein found in plant leaves, representing up to 50% of the soluble protein. Thus, the

SSU promoters and their transit peptides are attractive candidates for expression of genes at high

levels in green tissue and for targeting of different proteins into the chloroplast (Ellis, 1981). 24

“Tissue-specific promoters have been shown to work efficiently in dicots, and a few reports are available describing their expression in monocots (Matsuoka et al. 1994; Yin and Beachy, 1995). “

“The small subunit of ribulose-1, 5-bisphosphate carboxylase (rbcS) is encoded by gene families in most plants. Promoters from one group of these genes contain two cis-acting elements, the I-box and the G-box, that are important for tissue-specific expression (Donald and Cashmore,

1990; Manzara et al., 1991). Analysis of transgenic tomato plants expressing a rbcS - promoter/GUS fusion gene confirmed that promoter fragments ranging from 0.6 to 3.0 kb of rbcS1, rbcS2, and rbcS3A genes were sufficient to confer the organ-specific expression pattern (Manzara et al. 1993; Meier et al. 1995). In these genes, the I-box and G-box are located within -600 to -100 bp upstream of the transcription initiation site. The 560-bp promoter fragment from the cotton rbcS gene used to assemble the GUS reporter gene construct includes putative I-box (-287 to -274 base pair) and G-box (-260 to -252 base pair) sequences confers expression comparable 35S CaMV promoter (Song et al. 2000). “

“Truncated cry1Ab gene has been introduced into several cultivars of rice (indica and japonica) by microprojectile bombardment and protoplast system. The expression was driven by two constitutive promoter (35S from CaMV and Actin-1 from rice) and two tissue specific promoters (pith tissue and PEPC for green tissue from maize). The results demonstrated that

PEPCP in general and 35SP in some lines act as strong promoters in CryIA(b) expression. The level of Bt protein was generally high in the leaves for the PEPC promoter which was comparable with the levels of a few high Bt protein plants with the 35SP or Actin-1 promoter. (Datta et al.

1998). “

“The expression of the modified gene for a truncated form of the cryIA(c) gene, encoding the insecticidal portion of the lepidopteran-active CryIA(c) protein from Bacillus thuringiensis var.

kurstaki (B. t.k.) HD73, under control of the Arabidopsis thaliana ribulose-l, 5-bisphosphate

carboxylase (RuBisCo) small subunit atsiA promoter with and without its associated transit peptide was analyzed in transgenic tobacco plants. Examination of leaf tissue revealed that the atslA promoter with its transit peptide sequence fused to the truncated CryIA(c) protein provided a 10- fold to 20-fold increase in cryIA(c) mRNA and protein levels compared to gene constructs in which the cauliflower mosaic virus 35S promoter with a duplication of the enhancer region (CaMV-

En35S) was used to express the same cryIA(c) gene (Wong et al. 1992). “

“Fujimoto et al. (1993) transformed rice plants with tissue specific promotor. The transformed plants had nearly 0.05% toxin of the total soluble leaf protein, and showed resistance to the rice leaf folder (Cnaphalocrosis medinalis) and yellow stem borer (Chilosuppressalis sp). “

“A 5'-upstream regulation region of rice RuBisCo small subunit gene (rbcS) was cloned

from a Chinese cultivar Wuyunjing 8, and its sequences were confirmed by comparison with the

known genome sequences of both japonica and indica rice. The cloned rbcS promoter was fused to

the 5'-upstream of GUS (Beta glucuronidase) coding region in a binary vector, and was introduced

into rice by Agrobacterium-mediated transformation. The results of both histochemical staining

and quantitative analysis of GUS activity showed that the expression level of GUS fusion gene was

significantly stronger in leaf blade and sheath than in other organs of transgenic rice plants, and the

GUS activity was restricted to the mesophyll cells of leaf tissue, which showed that the rice rbcS

promoter could control not only the tissue- but also the cell-specific expression of foreign genes in

transgenic rice. The rice rbcS promoter might be very useful for the expression of target genes in

transgenic rice, with particularly high efficiency in leaf tissues (Liu et al. 2005). “

“Panguluri et al. 2005 isolated green tissue-specific promoter of RbcS gene family from

pigeon pea by PCR and studied the expression of uidA gene encoding β-glucuronidase in the

transgenic tobacco plants under the control of pigeon pea RbcS promoter. The results showed that

this promoter was as strong as pea RbcS3A promoter characterized earlier. “

“Zheng et al. 2005 applied agrobacterium-mediated genetic transformation to produce beet

armyworm (Spodoptera exigua Hubner) resistant tropical shallots (Allium cepa L. group

Aggregatum). A cry1Ca or a H04 hybrid gene from Bacillus thuringiensis, driven by the

chrysanthemum ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (Rubisco SSU)

promoter, along with the hygromycin phosphotransferase gene (hpt) driven by the CaMV 35S

promoter, was used for genetic transformation. Molecular Analysis confirmed the integration and

expression of foreign genes. The amount of Cry1Ca expressed in transgenic plants was higher than

the expression levels of H04 (0.39 vs. 0.16% of the total soluble leaf proteins, respectively). There

was a good correlation between protein expression and beet armyworm resistance. “

Amarasinghe et al. (2006) isolated RbcS promoter from Gossypium hirsutum L. cv. Coker

315 and transformed Arabidopsis and cotton with GUS reported gene liked with isolated RbcS

promoter to determine its expression level. They reported that RbcS had the highest expression

throughout the vegetative and reproductive stages. Histochemical results in T2 lines revealed that

cotyledons and leaves had very high expression of the GUS gene. The roots had almost no

detectable GUS expression. Young stem and the young floral buds also had low levels of GUS

expression. In mature open flowers staining was observed in the green bract surrounding the

flower, in the green ovary wall, and in the stigma, and pollen, but not in ovules. “

“Lee et al. (2006) reported the integration and expression of Cry1Ab insecticidal genes

under RbcS promoter in transformed oil palm. A Biolistic method was used to transform immature

embryos (IEs) of oil palm. More than 700 putative transformed IEs from independent transformation events were generated. Transient transformation efficiency of 81-100 % was achieved. The presence of Cry1Ab mRNA transcripts showed that it is fully functional in oil palm, at the transcriptional level. “

“Anisimov et al. 2007 studied activity of the highest expressing RuBisCo small subunit

(RbcS) promoters (pRbcS) from the cotyledons of germinating seedlings of Brassica rapa var. oleifera and Nicotiana tabacum plants that were transformed using an Agrobacterium-mediated transformation strategy to drive high-level and preferably stage specific transgenic protein expression in plants. The mRNA level of RbcS and of gusA quantified in transformed plants. The results demonstrated that promoter most active in seedlings under native conditions was also most active in transgenic constructs at the same stage of plant development. “

“Two truncated versions of the pRbcS-2 (360 bp and 624 bp), a full version of pRbcS-2

(1.6kb) and 35S CaMV promoter were fused to the gusA gene to determine minimal length of the most active promoter in transgenic tobacco plants after Agrobacterium transformation. GUS protein expression was determined quantitatively in recovered shoots by an enzymatic assay in vitro. GUS expression levels under both truncated pRbcS versions did not differ significantly from each other; however, it appeared to be more than fourfold lower than that of gusA under the 1.6-kb pRbcS-2 promoter, while being higher than 35S-gusA expression (Anisimov et al. 2007). “

“RuBisCo contents were substantially increased in Rice (Oryza sativa L.) plants after

Agrobacterium-mediated transformation with the rice rbcS sense gene under the control of the rice rbcS promoter. The primary transformants were screened for the ratio of Rubisco to leaf-N content, and the transformants with >120% wild-type levels of RuBisCo were selected. In the progeny of the selected lines of the transformants, the mRNA levels of one member of the rbcS gene family were increased from 3.9- to 6.2-fold, whereas those of other members of the rbcS gene family were 28

unchanged. The total levels of rbcS mRNA were increased from 2.1- to 2.8-fold. The levels of rbcL

mRNA were increased from 1.2- to 1.9-fold. RuBisCo protein content was significantly increased

by 30% on a leaf area basis (Suzuki et al. 2007). “

“Cia et al. (2007) isolated a tissue specific promoter (PD54O) from rice and used it to drive

the expression of Cry1Ac encoding Bacillus thuringiensis endotoxin against rice leaf-folders. No

Cry1Ac protein found in endosperm or embryo. A reporter gene regulated by a series of truncated

PD54O showed various tissue-specific expression patterns. “

“Suzuki et al. 2009 reported that four out of five members of the RbcS multigene family

(OsRbcS2-Os RbcS5) were highly expressed in leaf blades of rice (Oryza sativa L.) irrespective of

plant growth stage, whereas accumulation of all RbcS mRNAs in leaf sheats, roots and developing

spikelets were quite low. A highly positive correlation was observed between total RbcS and RbcL

mRNA levels and RuBisCo content at their maxima, irrespective of tissues and growth stage. The

results indicated that the total RbcS mRNA levels may be a primary determinant for maximal

RuBisCo protein content and that RuBisCo gene expression is well coordinated through the whole

life of rice. “

“The insect resistant gene Cry1C under rice rbcS promoter was transformed in Zhonghua

11 (Oryza sativa L. ssp. japonica) via Agrobacterium-mediated transformation to confer resistance against yellow stem borer (Tryporyza incertulas Walker), striped stem borer (Chilo suppressalis

Walker) and leaf folder (Cnaphalocrocis medinalis Guenec). An elite transgenic line RJ5 was selected which possessed high resistance to leaf folders and stem borers and had very good agronomic performance. The levels of Cry1C were at undetectable in endosperm. It was noted as only 2.6 ngg-1 in the endosperm (Ye et al. 2009). “

“A synthetic truncated Cry1Ac gene was linked to the rice rbcS promoter and its transit

peptide sequence (tp) and was transformed in rice using Agrobacterium-mediated transformation

method. Use of the rbcS-tp sequence increased the Cry1Ac transcript and protein levels by 25- and

100-fold, respectively, with the accumulated protein in chloroplasts comprising up to 2% of the

total soluble proteins. The high level of Cry1Ac expression resulted in high levels of plant resistance to three common rice pests, rice leaf folder, rice green caterpillar, and rice skipper, as

evidenced by insect feeding assays. Transgenic plants were also evaluated for resistance to natural

infestations by rice leaf folder under field conditions. Throughout the entire period of plant growth,

the transgenic plants showed no symptoms of damage, whereas non transgenic control plants were

severely damaged by rice leaf folders (Kim et al. 2009). “ “

2.4 Agrobacterium -Mediated Transformation

2.4.1 Genus Agrobacterium

“The genus Agrobacterium has been divided into a number of species on the basis of disease symptomology and host range. Thus, Agrobacterium radiobacter is an “avirulent” species,

Agrobacterium tumefaciens causes crown gall disease, Agrobacterium rhizogenes causes hairy root

disease and Agrobacterium rubi causes cane gall disease. A new species, Agrobacterium vitis has

been proposed that causes galls on grape and a few other plant species. (Otten et al. 1984). “

“Agrobacterium has the ability to transfer DNA in a wide range of group of organisms including many monocot and dicot gymnosperms (Loopstra et al., 1990; Levee et al., 1999;

McAfee et al., 1993; Yibrah et al., 1996; Wenck et al., 1999) and angiosperm species (DeCleene

and DeLey 1976; Anderson and Moore 1979; Porter 1991). Agrobacterium has the capability to transform fungi including yeasts (Bundock et al. 1995; Bundock and Hooykaas, 1996 ; Piers et al.

1996), basidiomycetes (De Groot, 1998) and ascomycetes (Abuodeh et al. 2000; De Groot, 1998)

and it has been reported that Agrobacterium can transfer DNA to human cells (Kunik et al. 2001).

“ “Plant transformation employs the use of Agrobacterium tumefaciens in most of the cases.

This bacterium is a naturally occurring soil borne pathogenic bacterium that causes crown gall

disease. Agrobacterium tumefaciens naturally infects the wound sites in dicotyledonous plant

causing the formation of the crown gall tumors. The first evidences indicating this bacterium as the

causative agent of the crown gall was reported in 1907 (Smith and Townsend, 1907). The crown

gall disease is due to the transfer of a specific fragment “T-DNA” (transfer DNA) from a large

tumor-inducing (Ti) plasmid within the bacterium to the plant cell (Zaenen et al. 1974). “

“T-DNA gets integrated into the plant genome and ultimately leading to the development of

the crown gall phenotype (Chilton et al. 1977). Two bacterial genetic elements are required for T-

DNA transfer to plants. The first element is the T-DNA border sequences that consist of 25bp

direct repeats flanking and defining the T-DNA. The borders are the only 12 sequences required in

cis for T-DNA transfer (Zambryski et al. 1983). The second element consists of the virulence (vir)

genes encoded by the Ti plasmid in a region outside of the T-DNA. The vir genes encode a set of

proteins responsible for the excision, transfer and integration of the T-DNA into the plant genome

(Godelieve et al. 1998). “

“The three important facts make the use of T-DNA in plant transformation. Firstly, the tumor

formation in the plant cells is the result of T-DNA transfer and integration of and the subsequent

expression of T-DNA genes. Secondly, the T-DNA genes are transcribed only in plant cells and do

not play any role during the transfer process. Thirdly, any foreign DNA placed between the T-

DNA borders can be transferred to plant cells, no matter where it comes from (Maqbool, 2009). “

“Several Agrobacterium tumefaciens and plant tissue specific factors influence the transfer of T-DNA and its integration into the plant genome. These include plant genotype, explant, 31

vectors-plasmid, bacteria strain, addition of vir-gene inducing synthetic phenolics compounds, culture media composition, tissue damage, suppression and elimination of A. tumefaciens infection after co-cultivation (Alt-morbe et al. 1989; Bidney et al. 1992; Hiei et al. 1994; Komari et al.

1996; Nauerby et al. 1997; Klee, 2000). “

“These well-established facts, allowed the construction of the first vector and bacterial strain systems for plant transformation (Hooykaas and Schilperoort, 1992; Deblaere et al. 1985;

Hamilton, 1997; Torisky et al. 1997). “

2.4.2 Agrobacterium Mediated Transformation in Cotton

“Agrobacterium-mediated transformation is the most widely used method to transfer genes into plants. Transformation is typically done on a small excised portion of a plant known as an explant. The small piece of transformed plant tissue is then regenerated into a mature plant through tissue culture techniques. The first reported plant transformation by Agrobacterium was in 1983

(Fraley et al. 1983). Since then, major advances have been made to increase the number of plant species that can be transformed and regenerated using Agrobacterium. “

“The first genetically engineered cotton plant was reported in 1987 (Firoozabady et al.

1987; Umbeck et al. 1987). In the report by Umbeck et al. (1987), hypocotyl explants of

G.hirsutum cv. Coker 312 were transformed by Agrobacterium tumefaciens strain LBA4404 with neomycin phosphotransferase II (NPT II) and chloramphenicol acetyltransferease (CAT) genes driven by nopaline synthase promoter (NOS). Molecular analysis confirmed that the genes were in the primary plants, but progeny evaluation was not reported. “

“An agronomically valuable gene Cry1A was transformed in cotton cultivar Coker 312 by using A208 strain of Agrobacterium strain for the first time. This gene was isolated from Bacillus

thuringiensis and was driven by CaMV 35S promoter. The expression of the Bt protein in the

primary transgenic plant was confirmed through immunological (Western) analysis and insect feeding bioassays. The Bt gene was inherited as a single dominant Mendelian trait in the progeny

and plants were phenotypically normal. Field tests of these transgenic plants in 1992 showed good

protection against cotton bollworm (Pectinophora zea), the pink bollworm (Perlak et al. 1990). “

“Transgenic cotton resistant to the herbicide 2, 4-D was reported by Bayley et al. (1992).

Transgenic primary plants and progeny were tested by spraying with 2, 4-D and damage was recorded at 3 weeks. Molecular analysis was done using PCR analysis. Progeny were also assayed for 2, 4-D monooxygenase activity and a 3:1 segregation pattern of inheritance was confirmed. “

“A method for rapid genotype-independent transformation as well as regeneration of shoots from germinating seedlings of cotton (Gossypium spp.) was reported by Gould et al. (1998).

Isolated shoots were inoculated with a super-virulent strain of Agrobacterium tumefaciens. This strain was used for inoculation in isolated shoots, subjected to a mild selection of antibiotic then regenerated directly as shoots in vitro. “

“Zapata et al. (1999) solved the problem of genotype dependent regeneration in cotton by using shoot apices as explants. “The seedling shoot apex was transformed using Agrobacterium tumefaciens LBA4404 to transfer the nptII and GUS genes driven by a CaMV 35S promoter.

Transformation was confirmed by the Kanamycin resistant phenotype in progeny and by Southern hybridization analysis of the progeny. The transformation efficiency was low (only 0.8%).“

“Zhao et al. (2001) fused a synthetic Bt Cry1Ac gene with a secretary signal coding sequences at 5' end and a modified GNA gene were used to construct a plant expression vector pBSGS1M+ and this vector was transferred into tobacco (Nicotiana tabacum L.) by

Agrobacterium-mediated transformation method. Results of PCR, Southern blot and Slot blot analysis indicated that both the chimeric Bt Cry1Ac and GNA genes were integrated into the

genomes of transformed plants. Western blot analysis indicated that at least the Cry1Ac protein was

produced in transgenic plants. “

“A protocol for efficient transformation and regeneration of cotton was reported by

Leelavathi et al. (2004). Embryogenic calli co-cultivated with Agrobacterium carrying cry1Ia5

gene were cultured under dehydration stress and antibiotic selection for 3-6 weeks to generate

several transgenic embryos. About 83% of these plants have been confirmed to be transgenic by

Southern blot analysis. An efficiency of ten kanamycin-resistant plants per Petri plate of co-

cultivated embryogenic callus was obtained. The method is extremely simple, reliable, efficient,

and much less laborious than any other existing method for cotton transformation. “

Ikram ul Haq (2004) reported a different method of recovery of putative transformants in

cotton (Gossypium hirsutum L.) cv. Cocker-312using Agrobacterium-mediated gene transfer

system. The hypocotyl-derived embryogenic calli (two month- old) were infected through

agroinfiltration for 10 min at 27 psi in a suspension of Agrobacterium tumefaciens strain GV3101

carrying tDNA with the GUS gene, encoding β-glucuronidase (GUS), and the neomycin

phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after,

the histochemical GUS assay revealed 46.6% and 20% GUS activity respectively. The putative

transgenic plants were developed via somatic embryogenesis. In 4 independent experiments, up to

28.23% transformation efficiency was achieved. To confirm the integration and expression of

transgenes, PCR amplification and Southern blot analysis were performed. “

“To get a high transformation frequency, Wu et al. (2005) developed a simple protocol for

transformation of cotton (Gossypium hirsutum L.) through Agrobacterium mediation coupled with

the use of embryogenic calli as explants. Two Chinese cotton cultivars, cv. Ekang 9 and Jihe 321

were inoculated with Agrobacterium tumefaciens strain LBA4404 having binary vector pBin438

carrying a synthetic Bacillus thuringiensis-active Cry1Ac and API-B. The forty five regenerated

plants were successfully transferred in soil, among these 12 proved to have the active Cry1Ac and

API-B gene. The transgenic plants were highly resistant to cotton bollworm (Heliothis armigera) larvae. The mortality rate was ranging from 95.8 to 100%.“

“Zhao et al. (2006) transformed the most economically significant Chinese cotton cultivar

(Gossypium hirsutum L. cv. Zhongmian 35) via Agrobacterium tumefaciens-mediated DNA transfer. The aroA-M1 gene that confers resistance to the glyphosate was fused with a chloroplast- transit peptide of Arabidopsis thaliana 5-enolpyruvyl-3-phosphoshikimate synthase (ASP) and expressed in cotton plants under the control of a CaMV35S promoter. Transgenic plants were directly selected on medium containing glyphosate. Thirty-four independent transgenic lines were obtained after selection, giving a maximal 1.9% transformation frequency. DNA hybridization,

Western blot and PCR techniques were used to confirm the integration and expression of the aroA-

M1 gene in T0 plants and T1 progenies. There was an increased resistance of T0 and T1 transgenic

plants towards glyphosate. “

“An elite Indian genotype (Bikaneri Nerma) of cotton (Gossypium hirsutum L.) was

transformed with a synthetic gene encoding Cry1Ac δ-endotoxin of Bacillus thuringiensis via

Agrobacterium-mediated genetic transformation. The shoor apical meristems were isolated from seedlings as explants. Regeneration of shoots was carried out in selection medium containing kanamycin after co-cultivation of the explants with Agrobacteriumtumefaciens (strain EHA 105).

Progeny obtained by selfing T0 plants was grown in the greenhouse and screened for the

integration of neomycin phosphotransferase (nptII) and Cry1Ac genes by polymerase chain

reaction (PCR) and Southern hybridization. The quantity of Cry1Ac was measured by Quan-T

ELISA kits. Insect bioassays and field tests of the promising lines (T2 and T3 generations) ensured

potential of the transgenic cotton for resistance against cotton bollworm (Katageri et al. 2007). “

“Sonication assisted Agrobacterium mediated transformation (SAAT) procedure has shown

promising for the transformation of plants. This method has the potential for uniform

transformation of meristematic tissues. Furthermore, the SAAT procedure produced a 100-1400

fold increase in gene expression in a variety of tissues (Trick & Finer, 1997). “

“Hussain et al. (2007) reported a new procedure for cotton transformation based on

cavitations caused by sonication which results in thousands of micro wounds on and below the

surface of plant tissue and allow Agrobacterium to travel deeper and completely throughout the

tissue. This wounding fashion increases the probability of infecting plant cells lying deeper in

tissue. Many parameters were optimized for the enhancement of GUS transient expression in

cotton using mature embryos as explant. GUS was first detected 24h following incubation of the

explants and by 48h, GUS expression was very intense which served as a useful indicator of

successful transformation of the cotton explant following sonication assisted Agrobacterium

mediated transformation (SAAT) procedure. The study also showed the competitive advantage of

this procedure over other transformation procedures being routinely used.

Rashid et al. (2008) transformed a locally approved cotton variety CIM-482 with two Bt

genes (Cry1Ac and Cry2A) using sonication assisted Agrobacterium mediated transformation.

Putative transgenic plants were confirmed via PCR, Southern hybridization, and western-blotting.

Those showing mortality of 75 to 100% for the second instar ofHeliothis armigera (compared with

0% for the control) were considered Bt-positive. This strategy has delayed the targeted insect pest

resistance in cotton. A local cotton variety CIM-482 was transformed with Arabidopsis thaliana

PhyB gene under the control of CaMV 35S promoter, cloned in pBinPhyB vector through 36

Agrobacterium tumefaciens strain LBA 4404 to enhance photosynthetic rate in Gossypium hirsutum L. After Successful transformation of Phy B gene, transgenic plants showed increased photosynthetic rates (Qayyum et al. 2009).

“The four upland cotton cultivars, Ekangmian10, Emian22, Coker201 and YZ1 were

transformed with novel Bacillus thuringiensis genes (Cry1C, Cry2A, Cry9C) and bar gene via

Agrobacterium-mediated transformation. With the bar gene as a selectable marker, about 84.8 % of resistant calli have been confirmed positive by polymerase chain reaction (PCR) tests, and totally 50 transgenic plants were regenerated. Southern blot was performed to confirm the integration of inserts. Bioassay showed 80 % of the transgenic plantlets generated resistance to both herbicide and insect. “

“In order to produce transgenic cotton resistance to insects, hypocotyl explants were transformed with Agrobacterium tumefaciens strain LBA4404 harboring the recombinant binary vector pBI121 containing the cry1Ab gene under the control of CaMV 35S promoter. The selectable marker used was neomycin phosphotransferase. Inoculated tissue sections were placed onto co-cultivation medium. Transformed calli were selected on MS medium and cefotaxime.

Plantlets were subsequently regenerated from putative transgenic calli. Polymerase chain reaction

(PCR) and southern blot analysis were used to confirm the integration of cry1Ab and nptII transgenes into the plant genome. Western lot analysis of transgenic plants revealed the presence of a 67kDa band in transgenic cotton lines using the anti-Cry1Ab polyclonal anti-serum. A homozygous line in T2 progeny plants (Line 61) showed promising performance against Heliothis

armigera larvae (Tohidfar et al. 2008). “

Li et al. (2009) reported the transformation of two cotton genotypes, Simian 3 (SM 3) and

WC. These genotypes were co-transformed using a mixture of four Agrobacterium tumefaciens

cultures of strain LBA4404, each carrying a plasmid harboring the following genes, Bt + sck (for

Bacillus thuringenesis protein and modified Cowpea trypsin inhibitor), bar (for glufosinate), keratin, and fibroin. The frequency of callus induction, embryogenesis, and plant regeneration were notably different between the two genotypes. However, there were no differences between the two genotypes for number of plantlets carrying multiple gene copies of different gene combinations as well as transformation frequency for different gene combinations. The results PCR analysis indicated that more than 80% of plantlets carried the nptII gene for kanamycin resistance. Overall, the co-transformation frequency of two or more genes was about 35%. The integration of the target genes into the cotton genome was confirmed by southern blot analysis. “

3. MATERIALS AND METHODS

3.1 Experimental Material

Seed cotton of transgenic lines of T4 progeny was taken from CEMB cotton Store. It was

delinted with commercial H2SO4 at a rate of 100ml/kg of seed. After removing the fuzz, seed was

washed with tape water four to five times to remove the remaining acid from seeds. The advance

homozygous lines 3001-1, 3009-8, 3010-14, 3013-3, 3016-1, 3010-2, 3033-4, 3012-3, 3043-7,

3010-14, 3038-3, 3043-4 were selected on the basis of agronomic and morphological performance

under field conditions and insect resistance being good in expression of endotoxins ( data

obtained in T3 generation), lesser boll damage %age in field after being artificially infested. These

lines were sown along with their parent variety (untransformed CIM-482) according to randomized

complete block design (RCBD) with three replications. Only insecticides not active on lepidoptera

were applied to all plots throughout the season.

Experimental fields were surrounded with rows of untransformed CIM-482 and MNH-93

(another locally approved cotton cultivar) to serve as refugia to prolong insect resistance. Sorghum

bicolor was grown around the field to isolate field from surroundings following recommendations

of National Biosafety Committee guidelines (NBC, 1999).

3.2 Confirmation of Gene Integration and Expression

Molecular Analysis i.e PCR and Southern blot were performed to confirm the stable

integration of insecticidal genes Cry1Ac and Cry2A in advance cotton lines.

3.2.1 Genomic DNA Isolation

“Transgenic plants were subjected to DNA isolation along with non transgenic control

plants. Saha et al. (1997) method of DNA isolation was used with some modifications. The leaf of

cotton plant was grinded to fine powder in liquid nitrogen in pestle and mortar. “One gram fresh

young leaves were grinded to very fine powder in the presence of liquid nitrogen. Then 1mL of

extraction buffer (Appendix-I) was added and centrifuged at 3000xg for 20min. The supernatant

was discarded and pellet was resuspended in 700µL of lysis buffer (Appendix-I). The tubes were

incubated at 65°C for 30 min-1hour.After that equal volume of Chloroform: isoamyl alcohol was

added, mixed and centrifuged at 14K for 20min at 4°C. This step was repeated again. The aqueous

phase was shifted to new tube and 0.6volume of isopropanol was added and incubated at room

temperature for 15-30 min. The tubes were centrifuged at 14K for 15 min. The supernatant was

discarded and the pellet was washed with 70% ethanol and air dried after it. The tubes were

incubated at 4°C for 30min-1 hour. The tubes were centrifuged at 14K for 15 min. The pellet was

resuspended in 100µL of TE buffer and incubated at 65°C for 30 min. The tubes were centrifuged

at 14K for 15 min. The supernatant was shifted in another 1.5 mL tube. RNase was added and

incubated at 37°C for 15 min. Two volume of 100% ethanol+ 0.1 volume of 3M sodium acetate

was added and placed at -20°C for 1 hour. The DNA was pellet down at 12K for 15-20 min. “

“After washing with 70% ethanol, pellet was air dried and finally dissolved in water. DNA

was resolved by running on 0.8% agarose gel on the basis of molecular weight. Ethidium bromide

of concentration 0.5-1µg/mL was added. Gel was run at 70-80V for 1-2 hours. “

3.2.2 Polymerase Chain Reaction (PCR)

Genomic DNA was isolated from fresh cotton leaves using the method described by Saha et

al. (1997). PCR was run for the detection of integrated Cry1Ac and Cry2A genes by amplifying

internal fragments of 565bp and 600bp respectively by a modification of the method by Saiki et al.

(1988). DNA extracted from untransformed plants was used as negative control and that of

plasmid pk2Ac as positive control. PCR was carried out in reaction volume of 20 µL containing the

genomic DNA template 100 ng, forward and reverse primers 50 pM each (Cry1Ac and Cry2A

specific primers , dNTPs 200 µM, 1X PCR Buffer (50 mM KCl, 1.5mM MgCl2 and 10mM Tris-

HCl) and Taq Polymerase 1 unit. “

The PCR was performed at 94ºC for 4 minutes 94ºC for 1 minutes 54ºC for 1and 72ºC

for 1 minutes followed by 35 times. The amplified PCR fragments were resolved on 1% agarose

gel and observed under UV light. For amplification of genes, gene specific primers sequences were used (Table-3.1)

Table-3.1: Primer list for PCR amplification of Cry1Ac and Cry2A genes

. Primer Name Primer Sequence Annealing Temp. Product Size

Cry1Ac Forward 5’-ACAGAAGACCCTTCAATATC-3’ 52°C 565bp

Cry1Ac Reverse 5’GTTACCGAGTGAAGATGTAA-3’ ------

Cry2A Forward 5’-AGATTACCCCAGTTCCAGAT-3’ 52°C 600bp

Cry2A Reverse 5’-GTTCCCGAAGGACTTTCTAT-3’ ------

3.2.3 Southern Blot Analysis

Southern analysis was performed on genomic DNA from leaves of transgenic plants and

from non transgenic control plants as described by Southern (1975). DNA was extracted as

described in the section 3.2.1. Genomic DNA (20μg) was digested with Hind111 for Cry1Ac gene

to excise from the cassette. The digested DNA samples were run in 1% agarose gel in 1X TAE

buffer at 10 V and 20oC for 18-20h. Gel was stained with ethidium bromide and photographed.

Gel was denatured with 1.5M NaCl + 0.5M NaOH for 30 minutes and depurinated with

1.5M HCl for 10 minutes. Gel was neutralized with 1.5M NaCl and 0.5M Tris-HCl, pH 7.5.

Digested DNA samples were transferred from gel to Hybond-N+ nylon membrane (Amersham)

with 20X SSC (Appendix-I) by capillary action. The DNA on membrane was fixed by exposing to

UV light for 3-5 minutes.

The membrane was put in the pre-hybridization solution (Appendix-I) for one hour at 65oC in hybridization tube. Hybridization with probe was carried out for 16-18 hours. After hybridization, detection procedure was followed according to the Fermentas Biotin Chromogenic

Detection Kit (Cat# K0661). The membrane was washed in 30mL of 1X blocking/washing buffer for 5 min at room temperature with moderate shaking. Then the membrane was submerged in blocking solution for 30 min with moderate shaking. 20µL of diluted streptavidin AP conjugate was prepared and added to the membrane for 30 min after removing blocking solution. Afterwards, the membrane was again washed with 1X blocking/washing buffer. The washing buffer was removed and 30mL of detection buffer was added for 30 min. Finally enzymatic reaction was performed by incubating the membrane in 20mL of freshly prepared 1X NBT/BCIP dissolved in detection buffer.

3.2.3.1 Probe Labeling

Probe for the detection of Cry1Ac was labeled by Fermentas Biotin DecaLabel™ DNA

Labeling Kit (Cat #K0651). According to the instruction given in the manual, added 10μL of template (100ng), 10μL of decanucleotide in 5x reaction buffer and nuclease free water (40μL) in a

1.5mL tube. The tube was vertexed and spun down. The tube was incubated in boiling water bath

for 5-10 min and cooled it on ice quickly. After it, 5μL of biotin labeled mix was added in the tube

along with 1μL of Klenew fragment (5U). The contents of the tube were spun down. The tube was incubated at 37oC for one two hours and reaction was stopped by adding 1μL of 0.5M EDTA (pH

8.0). The labeled DNA (probe) was estimated by spotting it on nitrocellulose membrane along with control labeled DNA (provided in the kit) in serial dilutions and was finally detected by using

Fermentas Biotin Chromogenic Detection Kit (Cat# K0661).

3.2.4 Western Blot Analysis

3.2.4.1 Protein Extraction

Fresh leaves of transgenic and non-transgenic plants were taken and approximately 200-

500mg weighed leaves were taken and grinded in liquid nitrogen in pre-chilled pestle and mortar.

Ground material was transferred to autoclaved 1.5mL tube and 400 μL of protein extraction buffer

(Appendix-II) was added. Samples were homogenized, incubated at 4oC (30 minutes to 1 hour) and

then centrifuged at 4°C for 10 minutes at 14000 rpm. Supernatant was taken in another 1.5mL tube

and protein was quantified with the help of BioRad reagent (Bradford, 1976).

For further study of the expression of introduced genes in transgenic plants, western blot

analysis was performed.

3.2.4.2 SDS Polyacrylamide Gel Electrophoresis

SDS-PAGE was performed in a BioRad Mini gel apparatus following basic procedure

described by Laemmli (1970). 50-100 μg of total plant protein was separated on 12% SDS-

polyacrylamide gel (Appendix-II).

3.2.4.3 Western Blotting

Proteins separated by SDS-PAGE were blotted on to Nitrocellulose membrane (Hybond-

C Amersham) using a Trans-Blot Semi-Dry Transfer Cell (Bio-Rad). Western blot analysis was

done as described by Towbin et al. (1979). Nitrocellulose membrane with gel was sandwiched

between 5 pieces of whatman blotting papers, three at the lower side and two on the upper side.

The nitrocellulose sheet was towards the cathode. After transfer membrane was blocked for an

hour at room temperature or overnight at 4ºC in blocking solution (5% skim milk in 1X PBST,

Appendix-II) followed by one-hour incubation at room temperature in the respective primary

antibodies diluted at 1:10000 in the blocking solution. After three washings, 10 minutes each wash,

with PBST, the membrane was incubated at room temperature with alkaline phosphatase

conjugated anti-rabbit antibody (secondary antibody) diluted at 1:5000 in blocking solution for one

hour. The membrane was again washed three times (10 minutes each wash) with PBST. Color was

developed with Sigma Fast NBT/BCIP tablet dissolved in 10mL autoclaved water as alkaline

phosphatase substrate. The membrane was air dried after final washing with distilled water.

3.2.4.4 Temporal and Spatial Expression of Cry1Ac and Cry2A Endotoxins

The amount of insecticidal protein (Cry1Ac& Cry2A) present among twelve different

transgenic lines was determined throughout the season. Because differential expression of Cry1Ac

occurs among different plant structures (Greenplate, 1999; Adamczyk et al., 2001), a single

structure was selected for quantification. For each sample date and for all transgenic lines, a single

main-stem terminal leaf was collected from five plants/transgenic line. The samples were transported to the laboratory and were processed same day.

Expression of both Cry1Ac and Cry2A insecticidal genes in cotton transgenic lines was quantified by Enzyme Linked Immunosorbent Assay using Envirologix Kit (Cat # AP 007). The whole procedure was followed as provided in the kit. Negative and positive controls were added to

the wells along with test samples. The titer of both the toxins was measured along with the age of

crop as well as in different plants of plant. For this purpose, after every fifteen days intervals after

the crop has 5-6 leaves, plant samples were brought to the laboratory, ground in liquid nitrogen.

The one third of grinded powder (100mg) from leaves and from other plant parts was taken in

1.5mL tube. Added 300 μL protein extraction buffer (Appendix-II). After 1hr of ice incubation,

centrifuged at 13000 rpm for 25 min, Supernatant was used for further analysis.

ELISA was performed according to procedure given in the kit and quantification of both

Cry1Ac and Cry2A endotoxins was done by plotting absorbance values of Cry1Ac and Cry2A test

samples on the standard curve generated with purified Cry1Ac standards on each of ELISA plates

and expresses as ng of Cry1Ac and Cry2A per gram of fresh tissue weight. The different fruiting

parts i.e anthers, petals, ovary and bolls were undertaken for this study.

Protein expression data of both genes (Cry1Ac and Cry2A) was further subjected to

statistical analysis using SPSS software (version 11.0, SPSS Inc) to evaluate the differences among

transgenenic lines, sampling dates and interaction of transgenic lines and sampling dates at 5%

level of significance.

3.3 Leaf Biotoxicity Assay

To check the efficacy of endotoxins against targeted insect pests, laboratory biotoxicity

assays of transgenic and control cotton leaves with Heliothis larvae (2nd instar) were conducted.

The five leaves from the upper, middle and the lower portion of each line were detached in Petri plates after 30, 60 and 90 days of crop age, placed on moist filter papers and brought back to the laboratory. The 2nd instar Heliothis larvae were fed to them. After 2-3 days, mortality rates of

Heliothis larvae were noted alongwith the control CIM-482. The mortality rates were calculated as

follows:

%Mortality = No.of dead larvae *100 Total no. of larvae 3.4 Isolation of RbcS Promoter from Gossypium arboreum L

3.4.1 Total RNA Extraction

For total RNA extraction the method of Jaakola et al. (2001) with some modifications was

used .Fully expanded young leaves were collected and pulverized tissue to a fine powder in a pre

cooled mortar. To each 1g of grinded sample, 15mL of pre-warmed (70ºC) extraction buffer

(Appendix-III) was added and vortexed for two minute. The tubes were incubated at 70ºC for 20

minutes, during incubation sample were vortexed after every 5 min and centrifuged at 12,000g for

10 minutes at 4ºC. The supernatant was shifted to 1.5mL tubes and centrifuged at 13,000rpm for 20

min. Supernatant was transferred to new 1.5mL tubes and extracted twice with an equal volume of

Chloroform: isoamyl alcohol. Phases were separated at 13000rpm at room temperature. To the

aqueous phase supernatant 1/4 volume of 10 M Lithium Chloride was added and mixed gently.

RNA was precipitated by incubating samples overnight at 4ºC. The tubes were centrifuged at

13000rpm for 20 min at 4ºC. The pellet was washed with 500μL of 70% ice cold ethanol. After air

drying the pellet was dissolved in 100ul of pre-warmed (65 ºC) SSTE buffer (Appendix-III). The

contents of tubes were extracted once with an equal volume of Phenol: Chloroform: Isoamyl

alcohol. To the supernatant, two volume of ice cold absolute ethanol was added to precipitate RNA

at -20ºC overnight. The tubes were centrifuged at 13000rpm for 20 min at 4ºC. The pellet was washed with 500μL of 70% ice cold ethanol. The pellet was air dried and resuspended in DEPC treated deionized water.

3.4.2 Agarose Gel Electrophoresis

Agarose gel electrophoresis was used to check integrity of RNA. Agarose gel of 1% was prepared in 1X TAE buffer (Appendix-I). Ethidium bromide of concentration 0.5-1µg/mL was added. Gel was run at 80 -100V for 30-45 min.

3.4.3 Quantification of Total RNA

RNA concentration was measured with spectrophotometer. Samples were prepared by adding 5μL of total RNA into quartz cuvette and 495μL of water, spectrophotometer was run and results were taken at A260 and A280. These results were calculated by following equation

Concentration in μg/μL =A260*40*100*dilution factor

Dividing A260/A280 checked purity of RNA; ratio should be equal to 2.00.

3.4.4 cDNA Synthesis

cDNA was synthesized by using Fermentas cDNA synthesis kit. For cDNA synthesis the following reagents were used.

DNase treated RNA 1µg

Anchored oligo dt primers (20 μM) 1µL

DEPC-treated water to 12µL

Reaction mixture was incubated at 70ºC for five minutes and quickly chilled by placing on ice. Then 4µL of 5X reaction buffer, 1µL of ribonuclease inhibitor (20µ/µL) and 2µL of 10mM

dNTP mix was added and incubated at 37ºC for 5 min. Finally 1µL of H minus M-MuLV reverse

transcriptase (200µ/µL) was added and mixture was incubated at 42 ºC for 60 min. The reaction

was stopped by heating at 70ºC for 10 min.

3.4.5 Polymerase Chain Reaction (PCR)

cDNA was used to amplify 445bp fragment of RbcS promoter (RuBisCo Small Subunit).

Primers for the amplification of RbcS promoter (445 bp) were designed from sequence of

Gossypium hirsutum RbcS gene (NCBI Accession No. X54091). PCR was carried out in reaction

volume of 20 µL containing the genomic DNA template 100 ng, forward and reverse primers 50

pM each (RbcS specific primers), dNTPs 200 µM, 1X PCR Buffer (50 mM KCl, 1.5mM MgCl2 and 10mM Tris-HCl) and Taq Polymerase 1 unit. The PCR was performed at 94ºC for 4 minutes,

94ºC for 1 minutes, 56ºC for 1 min and 72ºC for 1 minutes followed by 35 times. The amplified PCR fragments were resolved on 1% agarose gel and observed under UV light. For amplification of genes, following primer sequences were used (Table-3.2).

Table-3.2: Primer list for PCR amplification of RbcS Primers

Primer Name Primer Sequence Annealing Product Temp. Size

RbcS Forward 5’-GCGGTACCCAAAAGAGTCTCACTGATCC-3’ 54°C 445bp

RbcS Reverse 5’-GCGGTACC CTCTTTGTGGTCATAATGGT-3’ ------

3.4.6 Purification of DNA fragment from Agarose Gel

PCR product was purified from agarose gel with Fermentas DNA purification Kit (Cat# k

0513) in such a way that the required band was cut from the gel, weighed and incubated with 3

volumes of DNA binding solution in a 1.5mL tube at 550C temperature till gel slice get dissolved.

3µL of beads were added in 1.5mL tube after thorough mixing and put at room temperature for 5

minutes. Supernatant was removed after centrifugation and pellet was washed three times with

washing buffer provided in the kit. Again supernatant was removed, the pellet was air dried and

resuspended in TE buffer (pH.8.0) and put at room temperature for 10 minutes.

3.5 TA Cloning and Transformation of the Eluted DNA

Eluted PCR products were cloned into Invitrogen TA vector (pCR®2.1) according to the

protocol given in TA cloning kit (Invitrogen, Cat # K 4500-01).

3.5.1 Ligation of Insert in TA Vector

The eluted DNA insert was added into TA vector solution. The reagents that were used for ligation

are given in (Table-3.3). Samples incubated at 140C overnight.

Table-3.3: Reaction Mix of Ligation

Reagents Required

Template 20-50ng

Vector 50ng

10X Ligation buffer 1µL

Ligase 1µL (4.0 Weiss units)

Total volume 10µL

3.5.2 Transformation in E. coli

The E. coli strain DH5α was used for various plasmid and ligation mixture transformations.

The 2µL ligated product was transformed in to DH5α chemically competent (50µL) and mixed by tapping. These cells were incubated on ice for one hour and were subjected to heat shock at 42°C for 40-60 seconds. SOC medium (Appendix-V) was added to them, and incubated in 37°C shaking incubator for 1 hour. 40µL IPTG (100mg/mL) and X-gal (40mg/mL) solutions were spread on pre- warmed LB-kana plates (50µg/mL). Cells were then plated on these plates and incubated at 37ºC for overnight.

3.5.3 Screening of Positive Colonies

The blue and white colonies appeared on plates after overnight incubation. White colonies were assumed to contain the insert and blue colonies contained self-ligated vector. So white colonies

were picked by a sterilized tooth- pick and used for inoculate 3mL of LB broth (Appendix-IV). The

cultures were incubated for sixteen hours at 37°C with shaking at 250 rpm.

3.5.4 Plasmid DNA Isolation from Positive Colones

“The cells were harvested at 5000rpm for 1 minute. 150µL of solution-1 (Appendix-VI) was added to resuspend the pallet and samples were incubated in ice for 10 min; 200µL of solution-2 (Appendix-VI) was added and incubated at room temperature for 10 min. until it became transparent. Then 200µL of solution-3 (Appendix-VI) was added and kept in ice for 10 min.

Centrifuged the tubes for 10 min. at 13000rpm. Supernatant was recovered in new tubes and extracted with equal volume of phenol: chloroform: isoamyl alcohol. Supernatant was taken carefully and shifted in new tubes. DNA was ethanol precipitated, pellet was washed with 70% ethanol, air dried and dissolved in sterilized water. The samples were treated with RNAase by using 1 µL of RNase A (sigma). “

3.5.5 Restriction Analysis of Cloned DNA

Insert was confirmed with restriction digestion of DNA with EcoR1. Reaction mix used is given in (Table-3.4)

Table-3.4: Reaction mix of Digestion

Reagents Required

DNA to be digested 1µg

10X EcoR1 buffer 2µL

EcoR1 (10µ/µL) 1uL

H2O made volume up to 20 uL

Samples were incubated at 37°C for 2 hour. The results were analyzed by running the digested

samples on 1 % agarose gel.

3.5.6 Sequencing of Positive Clones

The reagents used for sequencing of the positive clones are given in (Table-3.5). The sequencing PCR was performed at 96ºC for 20seconds, 96ºC for 20seconds, 52ºC for 15 seconds, 60ºC for 4 min followed by 35 cycles.

Table-3.5: Reaction mix for Sequencing

Reagents Required

DNA 100-200ng

M13 Primers (3.3µM) 1µL

Big Dye Sequencing Mix 1µL

5X dilution buffer 1µL

H2O made volume up to 10ul

3.5.7 Preparing a Sequenced Product for Loading on ABI Prism 3100 Sequencer

After sequencing reactions PCR products were precipitated by adding 20 µL of 75%

isopropanol. The reactions were mixed by vortex and left for precipitation at room temperature for

30 minutes. The samples were centrifuged at 14,000 rpm for 20 minutes at room temperature. The

supernatant was fully discarded. The pellets were washed with 50 µL of 75% isopropanol. The

pellets were air dried. Furthermore, the pellet was suspended in 15 µL of formamide. The samples

were shifted in 96-wells plate and denatured at 95°C for 5 minutes and quick chilled by placing in

ice for ten minutes before loading on the ABI PRISM 3100 sequencer genetic analyzer according to the manufacturer’s instructions given in technical manuals. Sequence was analyzed manually by

using Chromas software version (v 1.45).

3.5.8 Removal of Vector Sequences From Sequence Results

Analyzing the Invitrogen TA vector (pCR®2.1) map showed that the sites of M13 primers

are away from the insertion site of PCR- Product, so the sequence reads with M13 primers contain

additional sequences of vector along with the sequence of PCR-Product. Vector was screened both

manually and NCBI’s online software VecScreen. The vector sequences were removed and pure

sequences were obtained.

3.5.9 Promoter Analysis

The pure promoter sequences obtained after the sequencing were subjected to bioinformatics

tools ie. “ChloroP 1.1” (devised by Technical University of Denmark, available online) for the

predication of transit peptide in RbcS promoter. The ChloroP server predicts the presence of

chloroplast transit peptides (cTP) in protein sequences and the location of potential cTP cleavage

sites. The transit peptide is required for their transport across the relevant membranes from their

site of synthesis in the cytoplasm.

3.6 Cloning of RbcS and Cry1Ac Gene into Plant Expression Vector pCAMBIA-1301

The binary vector pCAMBIA-1301 (11837bp) was used for cloning of Cry1Ac gene under

RbcS promoter in plant expression vector; one of the main objective of study. The map of vector is

shown in (Figure-3.1). The vector contains kanamycin gene for bacterial selection and hygromycin

for plant selection resistance genes. “This vector contains a fully functional gusA reporter

construct, the construct uses E.coli gusA (N358Q — to avoid N-linked glycosylation) with an

intron (from the castor bean catalase gene) inside the coding sequence to ensure that expression of

glucuronidase activity is derived from eukaryotic cells, not from expression by residual

Agrobacterium tumefaciens cells”

Figure-3.1: Map of vector pCAMBIA-1301. Adaped from http://www.cambia.org/)

3.6.1 Amplification of Cry1Ac+NOS Terminator from PIA-2 vector

For cloning of genes in plant expression vector pCAMBIA-1301, insecticidal gene Cry1Ac

(1.82 kb) was amplified from PIA-2 construct along with NOS terminator (0.270 kb) under kpn I

and BamH I sites. PIA-2 is pGEM-4Z based vector harboring Cry1Ac gene under ubiquitin

promoter (Figure-3.2). To amplify Cry1Ac+NOs fragment, folwoing primers were used (Table-6).

HindIII BamHI EcoRI BamHI

Ubiquitin Cry1Ac gene NOS

2.02Kb 1.845Kb 0.270Kb

Figure-3.2: pGEM-4Z based PIA-2 vector having Cry1Ac under Ubiquitin

Table-3.6: Sequence of primers used for amplification of Cry1Ac+NOS terminator from PIA-2 vector

Primer Sequence Annealing Product Temp. Size

Cry-NOS F 5’-GCGGTACCAGGAGGCCATTGCTATTACT-3’ 54oC 2.11 Kb

Cry-NOS R 5’-CGGGATCCAATTCCTATAATCGACATCAAAA-3’ ------

These primers introduced Kpn1 and BamH I restriction sites upstream and downstream

respectively. PCR was carried out in reaction volume of 20 µL containing the genomic DNA

template 100 ng, forward and reverse primers 50 pM each (Cry1Ac + NOS terminator-specific

primers (Table-3.6), dNTPs 200 µM, 1X PCR Buffer (50 mM KCl, 1.5mM MgCl2 and 10mM Tris-

HCl) and Taq Polymerase 1 unit. The PCR was performed at 94ºC for 4 minutes, 94ºC for 1

minutes, 54ºC for 1 min and 72ºC for 1 minutes followed by 35 times.

The amplification products were run on 1% agarose gels, visualized with UVP gel documentation system (Molecular Probes, Inc., USA), excised gel band was purified by using

Fermentas DNA purification kit (Cat# k0513).

3.6.2 Restriction Digestion of PCR Products and pCAMBIA-1301

The purified PCR products and pCAMBIA-1301 vector were double digested with Kpn I and BamH I restriction enzymes to produce overhangs in desired fragments of Cry1Ac+NOS and

pCAMBIA-1301 vector. Reaction mix used is given in (Table-3.7).

Table-3.7: Reaction Mix of Digestion with Kpn1 and BamH I

Ingredients Required

DNA to be digested 1- 5µg

10X Buffer Tango 4.0µL

Kpn I (10µ/µL) 1.0µL

BamH I(10µ/µ) 1.0µL

H2O made volume up to 20µL.

The samples were incubated for 2 hour at 37°C. The digested vector was run on 0.8%

agarose gel and PCR products on 1% agarose gel at 80V for 90 min and analyzed under UV-light.

The gel slices containing digested product were cut with sterilized blade and were eluted according

to the protocol of Fermentas Gel purification kit (Cat # K0513) to elute the DNA.

3.6.3 Ligation of Insert (Cry1Ac+NOS) into pCAMBIA 1301 Vector

Both insert and vector overhangs were ligated by using T4 DNA Ligase (Fermentas)

according to manufacture protocol. Vector to insert ratio was 1:1 and ligation temperature was

22°C for 2 hours. Reaction mix used for ligation is given in (Table-3.8).

Table 3.8: Reaction Mix of Ligation

Ingredients Required

Insert (Cry1Ac+NOS) 100ng

10X Ligation buffer 1.0µL

Vector 500ng

T4 Ligase 1.0µL

H2O made volume up to 10uL

3.6.4 Transformation of plasmid in E. coli

Ligated product was transformed in E. coli competent cells strain DH5α by heat shock

method as mentioned in section 3.5.2.

3.6.5 Plasmid DNA Isolation

Plasmid DNA was prepared by inoculating single bacterial colony in 3ml LB broth

containing the kanamycin 50μg/mL from the overnight grown plate. The samples were incubated at

37ºC at 250 rpm for 12-16 hours. The whole process of plasmid DNA isolation (miniprep) was repeated as mentioned in 3.5.4 Section. The isolated recombinant plasmids DNA of construct were confirmed by double restriction digestion with enzymes Kpn I and BamH I and by PCR analysis.

3.6.6 Restriction Analysis of Cloned Fragment of Cry1Ac+NOS terminator

Insert was confirmed with restriction digestion of plasmid DNA with Kpn I and BamH I. Reaction mix used is given in (Table-3.9).

Table 3.9: Reaction mix of Digestion

Reagents Required

DNA to be digested 5µg

10X buffer Tango 2µL

Kpn I (10µ/µL) 1µL

BamH I (10µ/µL) 2µL

H2O made volume up to 20µL

Samples were incubated at 37°C for 2 hour.

3.6.7 PCR Confirmation of Cry1Ac+NOS Integration in pCAMBIA 1301

PCR reaction was conducted to amplify 2.01 kb fragment of Cry1Ac+NOS from plasmid

DNA. PCR eluted fragment of Cry1Ac+NOS fragment was used as positive control. The PCR was

performed at 94ºC for 4 minutes 94ºC for 1 minutes 54ºC for 1and 72ºC for 1 minutes followed

by 35 times. The amplified PCR fragments were resolved on 1% agarose gel and observed under

UV light.

3.6.8 Restriction Digestion of RbcS PCR Products and pCAMBIA-1301

The purified PCR products of RbcS (promoter region) and pCAMBIA-1301 (carrying

Cry1Ac+NOS terminator cloned under Kpn1 and BamH1 sites) vector were digested with Kpn I to

produce overhangs in desired fragments of RbcS and pCAMBIA-1301 vector. Reaction mix used

is given in (Table-3.10)

Table 3.10: Reaction Mix of Digestion with Kpn1

Ingredients Required

DNA to be digested 1- 5µg

10X Buffer 2.0µL

Kpn I (10µ/µL) 1.0µL

H2O made the volume up to 20µL

The samples were incubated for 4 hour at 37°C. The digested vector was run on 0.8%

agarose gel and PCR products on 2% agarose gel at 80V for 60 min and analyzed under UV-light.

The gel slices containing digested products were cut with sterilized blade and were eluted according to the protocol of Fermentas Gel purification kit (Cat # K0513) to elute the DNA.

3.6.9 Ligation of Insert (RbcS) into pCAMBIA-1301 Vector

Both insert and vector overhangs were ligated by using T4 DNA Ligase (Fermentas)

according to manufacture protocol. Vector to insert ratio was 1:1 and ligation temperature was

22°C for 2 hours. Reaction mix used for ligation is given in (Table-3.11).

Table 3.11: Reaction Mix of Ligation

Ingredients Required

Insert (RbcS) 100ng

10X Ligation buffer 1.0µL

Vector 50ng

T4 Ligase 1.0µL

H2O made volume up to 10µL

3.6.10 Transformation of plasmid in E. coli

Ligated product was transformed in E. coli competent cells strain DH5α by heat shock

method as mentioned in section 3.5.2.

3.6.11 Plasmid DNA Isolation

Plasmid DNA was prepared by inoculating single bacterial colony in 3mL LB broth containing the kanamycin 50μg/ml from the overnight grown plate. The samples were incubated at

37ºC at 250 rpm for 12-16 hours. The whole process of plasmid DNA isolation (miniprep) was

repeated as mentioned in section 3.5.4. The isolated recombinant plasmids DNA of construct were

confirmed by double restriction digestion with enzymes Kpn I and BamH I and by PCR analysis.

3.6.12 PCR Confirmation of RbcS Integration in pCAMBIA 1301

PCR reaction was conducted to amplify 445 bp fragments of RbcS from plasmid DNA.

PCR eluted fragment of RbcS fragment was used as positive control. The PCR was performed at

94ºC for 4 minutes 94ºC for 1 minutes 54ºC for 1and 72ºC for 1 minutes followed by 35 times.

The amplified PCR fragments were resolved on 1% agarose gel and observed under UV light.

3.6.13 Restriction Analysis of Cloned Fragment of RbcS+ Cry1Ac+NOS terminator

To confirm the integration of cloned fragments, a digestion reaction was performed to

excise 2.56 Kb fragment of RbcS+ Cry1Ac+NOS terminator from pCAMBIA-1301 vector. As

RbcS promoter was cloned under Kpn I sites and Cry1Ac+NOS fragment was cloned under Kpn I

and BamH I sites, so the excision of 2.56 fragment of RbcS+Cry1Ac+ NOS was not possible

(Figure-3.3). That’s why the unique restriction site of EcoR I present in the vector before Kpn I

was used to excise cloned fragment of RbcS+ Cry1Ac+NOS terminator (Table-3.12)

Table 3.12: Reaction Mix of Digestion with EcoR I and BamH I

Ingredients Required

DNA to be digested 1- 5µg

10X Buffer 2.0µL

EcoR I(10µ/µL) 1.0µL

BamHI (10µ/µL) 1.0µL

H2O made volume up to 20µL

3.6.14 Orientation Confirmation of Cassette in pCAMBIA 1301

Primers were designed from the promoter region (RbcS) and Cry1Ac gene to confirm the

orientation of cassette (RbcS+Cry1Ac+NOS). The primer sequences designed to confirm the

orientation of integrated genes are given in (Table-3.13).

Table-3.13: Primer list for PCR amplification of Orientation Primers

Primer Name Primer Sequence Annealing Product Temp. Size

Rb-CryF 5’-ACCAAAGATACCCCAGATG -3’ 56°C 350bp

Rb-CryR 5’-CCAAAGATACCCCAGATGAT -3’ ------

Agrobacterium tumefaciens strain LBA 4404 containing Cry1Ac insecticidal gene driven by RbcS promoter was used for transformation. A construct pk2Ac carrying Cry1Ac and Cry2A

under 35S CaMV promoter was also transformed in cotton (Figure-3.4). The purpose was to

compare expression of insecticidal gene Cry1Ac driven by RbcS and 35S CaMV promoter.

KpnI KpnI BamHI

RbcS Cry1Ac NOS

Figure-3.3: Cry1Ac under under RbcS promoter in pCAMBIA1301 (Rb-Ac) HindIII HindIII PvuII PvuII

35S Cry1Ac NOS 35S Cry2A NOS

Figure-3.4: pk2Ac cassette showing Cry1Ac and Cry2A under 35S CaMV

3.7 Electroporation of Plasmid in Agrobacterium

Electroporation of plasmid Rb-Ac in Agrobacterium competent cells (LBA4404) was done

using Bio-Rad electroporation device (# 165-2105). Competent cells (100μL) were mixed with

2μL of Rb-Ac plasmid (100ng) and pk2Ac plasmid and placed on ice. The electroporation device

was set at a capacitance of 25μF, voltage 2.2kV with a 200 ohms resistance. After electroporation,

1mL of SOC (Appendix-V) medium was added and incubated at 28ºC for 2-3 hours at 200 rpm.

Culture was spread on plates containing YEP (Appendix-VII) medium and 50 µg/mL of kanamycin for selection of transformed cells. The plates were incubated at 28ºC for 24-48 hours. Transformed

Agrobacterium cells were selected in the form of distinct colonies. Cultures were prepared from these colonies in YEP broth containing kanamycin 50µg/L at 28ºC for 24 hours and glycerol stocks were prepared and stored at -70ºC.

3.8 Transformation of Gossypium hirsutum Var. NIAB-846

3.8.1 Delinting of Cotton Seeds

To remove lint from cotton seeds (Gossypium hirsutum L. Var. NIAB-846), concentrated

H2SO4 (95-98%) was used at the rate of 100 mL/kg of seeds. The seeds were continuously stirred

with the help of spatula after adding H2SO4 for 10-15 minutes until all lint was removed from seeds

and shiny surface of seeds appeared. Seeds were washed 5-6 times with tap water to remove the

acid completely. The seeds, which floated at the surface of water, were removed.

3.8.2 Seed Sterilization

After washing, seeds were sterilized by adding few drops of Twene 20 in water washed by

vigorous shaking. After that seeds were washed three times with autoclaved water. For surface

sterilization, seeds were dipped in 0.1% HgCl2 and 0.1% SDS for 15 minutes with constant

shaking. After that seeds were washed five times with autoclaved distilled water. The whole

washing was done in the laminar air flow cabinet to maintain the sterilize conditions. Then the

seeds were soaked in autoclaved distilled water for one hour. After one hour excess water was

removed and the seeds were kept in the dark for germination at 30°C overnight.

After the vector transformation and confirmation, mature embryos of local cotton variety

NIAB-846 were transformed with Rb-Ac and pk2Ac constructs. The method used for this purpose

was shoot cut apex method (CEMB modified shoot apex cut method as done by Gould et al.,

1998).

3.8.3 Isolation of Embryo

Next mornings, with a forceps, testa of the seeds was removed carefully and cotyledonary

leaves were excised with surgical blades. Mature cotton embryos were isolated from the

germinating seeds. The isolated embryos were kept on moist filter paper so that they may not

become dry.

3.8.4 Medium Preparation

To culture the transformed embryos, MS (Murashige and Skoog, 1962) broth (Appendix-

VIII) was used. The medium was sterilized at 121ºC and 15 lbs psi for 20 minutes in an autoclave

chamber. After autoclaving, medium was allowed to cool down to 50°C and antibiotics

hygromycin and cefotaxime were added as 75µg/mL and 250µg/mL respectively to the medium for

selection of transformants. Then medium was poured in glass culture jars and petri plates and

allowed to solidify at room temperature.

3.8.5 Bacterial Inoculum Preparation

Agrobacterium strain LBA4404 containing plasmid Rb-Ac and pk2Ac individually was

streaked on solidified agar medium containing kanamycin 50µg/mL and incubated for 24-48 hours

at 28oC. Single colony was picked and inoculated 10mL of YEP (Appendix-VII) broth containing

50µg/mL of kanamycin in 50 ml culture tube. The samples were incubated on rotary shaker at

temperature 28ºC for 24 hours with 200rpm. After completion of incubation period bacterial

culture was centrifuged at 3000xg for 15 minutes. Supernatant was discarded and resuspended the

pellet in 10mL of MS broth.

3.8.6 Agrobacterium Culture Treatment

Shoot tips of the isolated embryos were injured by using sharp treat blade held on petri

plate, shoots were highlighted by using light microscope. After cutting, embryos were immediately

shifted to the Agrobacterium inoculum suspension for treatment with bacterial culture and

incubated for 1 hour on a rotary shaker at very slow speed. Total 4, 000 embryos were used in the

transformation experiments.

3.8.7 Cocultivation

After bacterial innoculum treatment, the culture was removed and embryos were shifted to

MS+kinetin (1mg/mL) medium and cocultivated for 72 hours. At this stage antibiotics were not

added to the media. The cultures were kept in growth room at a temperature of 25°C± 2°C and a

photoperiod at 16 hours light and 8 hours dark.

3.8.8 Selection of Transformants

The survival percentage of the embryos at different growth stages e.g. after cocultivation,

antibiotic selection, shoot and root formation was observed on MS media containing (Appendix-

VIII).

3.8.9 Transient Expression of GUS Gene

Transient expression of GUS gene was studied through histochemical GUS assay. GUS solution was prepared containing 25mg/L X-gluc, 10mM EDTA, 100mM NaH2PO4, 0.1% Triton

X-100 and 50%methanol, (pH was adjusted to 8.0). The GUS solution was protected from light.

After 72 hours of cocultivated embryos were dipped in GUS solution in an eppendorf and kept at

370C overnight and viewed under microscope for blue spots. The tissues were incubated with X- glu solution at 37 0C for 16 hours. The blue spots were observed. Transformed plant tissues were

checked with GUS assay using non-transformed plants as negative control.

3.8.10 Selection on Antibiotic Medium

After three days (72hrs) of cocultivation, plantlets were shifted to selection medium i.e. MS

plus hygromycin (75µg/mL) and ceftriaxone (250μg/mL). Medium was also supplemented with

different growth hormones i.e. Kinetin (1mg/mL), Zeatin (1mg/mL) and BAP (1mg/mL). Control

plants were maintained on simple MS media. Plants were subcultured to fresh selection medium

after every 10 days till 6-8 weeks. The transformation efficiency was calculated after eight weeks

of transformants growing on selection medium. For this purpose number of plants surviving after

eight weeks on selection medium was counted and percentage was calculated in comparison with total number of embryos cocultivated in all the experiments. This was evaluated on the basis of plant formation on 75μg/mL hygromycin and ceftriaxone 250μg/mL. The hygromycin level was

selected high to reduce the number of false positive plants or escapes.

3.8.11 Transformation Efficiency

Total 4,000 embryos were used in the transformation experiments. The transformation

efficiency after eight weeks of transformants growing on selection medium was calculated. This

was evaluated on 75mg/mL Kanamycin. 66

3.8.12 Root Formation

After 6-8 weeks, the selected plants with well developed shoots were subcultured to

selection free medium i.e. antibiotics were not added to the medium. At this stage MS medium as

supplemented with different growth hormones in combinations or alone to observe the rooting

(kinetin 1mg/mL+1mg/mL), (kinetin 1 mg/mL+IBA1mg/mL), (IAA1mg/mL). Plants were

continued to subculture on the selection free medium supplemented with growth hormones for 4-6

weeks.

3.8.13 Transformation of Transgenic Plants to Soil

A soil mixture was prepared to shift the transformed plants from glass culture tubes to soil.

The mixture was composed of clay + sand + peat moss in ratio of 1:1:1, mixed well, moistened

properly with distilled water, filled in a bag and autoclaved. After autoclaving the composite soil

was allowed to cool and filled in plastic pots having at the bottom drain hole. Then rooted shoots

were taken out of the glass tubes, the root was washed with autoclaved water, dipped into IBA

(1mg/mL) and then planted into the pot filled with soil mixture. None transformed were shifted to

soil without IBA treatment and pots were covered with polythene bags. Plants were kept in a

growth room at 30ºC+2ºC for 16 hours photoperiod in light intensity (250-300µmol m-2 S-1). After

3-4 days the covers were removed occasionally two times daily. After 1-2 weeks the cover were removed completely. The plants were ready to shift to glass house.

3.9 Molecular Analysis of Transgenic Plants (T0 Progeny)

3.9.1 Genomic DNA Isolation

Genomic DNA was isolated from putative transgenic plants after two weeks of shifting in

green house and then to the field conditions. The leaves of putative transgenic cotton plants was

grinded to fine powder in liquid nitrogen in pestle and mortar. Saha et al (1997) method of DNA

isolation was used with some modification mentioned in section 3.2.1.

3.9.2 Polymerase Chain Reaction (PCR)

Genomic DNA was isolated from putative transgenic cotton plants using the method

described by Saha et al. (1997). PCR was run for the detection of integrated Cry1Ac genes by

amplifying internal fragments of 565bp by a modification of the method by Saiki et al. (1988)

DNA extracted from untransformed plants was used as negative control and that of plasmid pk2Ac

as positive control. PCR conditions were kept the same as mentioned in the section 3.2.2.

3.9.3 Southern Blot Analysis

PCR positive transgenic plants of Rb-Ac and pk2Ac were further subjected to southern

blot analysis as described by Southern (1975). DNA was extracted as described in the section

3.2.1. Genomic DNA (20μg) was digested with EcoRI and BamHI restriction enzymes to excise

Cry1Ac gene from Rb-Ac plants. The restriction enzyme HindIII was used to excise Cry1Ac gene

from pk2Ac plants. The protocol for southern blot was followed as mention in the section 3.2.3.

3.9.4 Temporal and Spatial Expression of Cry1Ac gene

The Expression of Cry1Ac in transgenic plants of Rb-Ac and pk2Ac was estimated at

different intervals using the procedure as mentioned in the section 3.2.4.4. Enzyme Linked

Immunosorbent Assay was performed using Envirologix Kit (Cat # AP 007) and concentration of

Cry1Ac gene in Rb-Ac and pk2Ac plants was quantified and expressed as ng per gram of fresh tissue. The whole procedure was followed as mentioned in section 3.2.4.4.

3.10 Molecular Analysis of Transgenic Plants (T1 Progeny)

The transgenic cotton plants in T0 progeny were carefully picked from the field and were

raised in green house conditions for further multiplication of seed and confirmation of Cry1Ac

integration in next progeny. 15 plants of Rb-Ac and plants of Rb-Ac were raised in pots in green

house.

3.10.1 Polymerase Chain Reaction (PCR)

Genomic DNA was isolated from transgenic plants at 5-6 leaf stage in green house. DNA

was isolated using the procedure as described by Saha et al. (1997) with some modification as

mentioned mentioned in section 3.2.1. PCR was run for the detection of integrated Cry1Ac genes

by amplifying internal fragments of 565bp by a modification of the method by Saiki et al. (1988)

DNA extracted from untransformed plants was used as negative control and that of plasmid pk2Ac

as positive control. PCR conditions were kept the same as mentioned in the section 3.2.2. The PCR

results were subjected to Chi Sqaure test to check the segregation of Cry1Ac segregation in T1 progeny.

3.10.2 SDS Polyacrylamide Gel Electrophoresis

SDS-PAGE of transgenic Rb-Ac and pk2Ac plants was carried out by the method of

Laemmli (1970). About 50-100 µg of isolated protein was mixed with loading dye denatured by heat shock in boiling water bath for 5 minutes and quick chilled on ice. Prepared 12% resolving, and 4% stacking gel (Appendix-II) in gel assembly. Stained the gel with Commassie stain for 30 minutes and destained it with destaining solution.

4. RESULTS

4.1 Confirmation of Gene Integration and Expression

The different molecular analysis were performed to confirm the integration of insecticidal genes Cry1Ac and Cry2A in advance lines of transgenic cotton being grown in the field condition.

4.1.1 PCR Analysis of Insecticidal Genes

Polymerase chain reaction (PCR) of both genes Cry1Ac and Cry2A was performed for all

transgenic cotton plants including positive and negative controls. Few plants were taken as

representative from each transgenic lines. PCR analysis confirmed the stable integration of Cry1Ac

and Cry2A genes in these cotton lines. A 565 bp and 600 bp internal fragments for Cry1Ac and

Cry2A respectively were amplified (Figure-4.1& 4.2) in all transgenic plants along with that of

plasmid (pk2Ac) DNA as positive control . No amplification was detected in negative control.

Figure-4.1: PCR amplification of Cry1Ac gene from cotton transgenic lines

Lane 1 : Lamba HindIII Marker

Lane 2-13 : Transgenic plants of cotton lines

Lane 14 : +ve control (pk2Ac plasmid)

Lane 15 : -ve Control (Non transgenic CIM-482)

1 2 3 4 5 6 7 8 9

600 bp

Figure-4.2: (A&B) PCR amplification of Cry2A gene from cotton transgenic lines

A B

Lane1 : 1Kb plus ladder (Ferments) Lane1 : 100 bp plus ladder (Ferments)

Lane 2- 7 : Transgenic plants of cotton lines Lane 2- 7 : Transgenic plants of cotton lines Lane 8 : -ve control (Non transgenic CIM-482) Lane8 : +ve control (Pk2Ac plasmid)

Lane9 : +ve control (Pk2Ac plasmid) Lane 9 : -ve control (Non transgenic CIM-482)

4.1.2 Southern Blot Analysis

Few of the PCR positive plants were further preceded and the presence and stable

integration of Cry1Ac gene in cotton genome was confirmed by Southern blot. These transgenic

plants were belonging to 3001, 3010, 3016 and 3033 lines. Gene integration was detected by a

Biotin DecaLabel™ gene specific probe. Plant genomic DNA was digested with HindIII

restriction enzyme along with plasmid DNA (pk2Ac) to release 3 Kb fragment. The digested DNA

was transferred to Nitrocellulose membrane (Hybond-N) Amersham and hybridized with Cry1Ac

specific probe. It showed the integration of Cry1Ac gene in cotton genome of few transgenic lines.

Non transgenic CIM-482 plant DNA was used as negative control that showed no any signal while

that of plasmid DNA pk2Ac was used as positive control (Figure-4.3).

3 kb

Figure-4.3: Southern blot of Cry1Ac gene in transgenic plants

Lane 1 : Negative Control (Non transformed CIM-482 plant)

Lane 2 : Positive Control (Plasmid DNA Pk2Ac)

Lane 3-5 : Transgenic Plants of cotton lines

4.1.3 Western Blot Analysis

The Western blot is the authentic way to check the expression of exogenous genes

introduced in plant genome. The plants showing the integration of Cry1Ac gene in southern blot

were subjected to western blot. Polyclonal anti-rabbit antibodies were used to detect 68Kda band.

A Bacillus thuringiensis strain HD-73 was used as a positive control while the protein isolated

from the transgenic plant (CIM-482) was used as negative control. Transgenic lines showed 68Kda

band while no band was observed in negative sample (Figure-4.4).

68Kd

Figure-4.4: Western blot showing 68Kda band of Cry1Ac protein in transgenic plants

Lane 1 : Positive control (Bacillus thuringiensis strain HD-73

Lane 2 : Negative Control (Non transformed plants CIM-482)

Lane 3-7 : Transgenic Plants of cotton lines

4.2 Temporal and Spatial Expression of Cry1Ac and Cry2A

4.2.1 Temporal Expression of Cry1Ac gene

Enzyme Linked Immunosorbent Assay (ELISA) is sound technique to detect and quantify

any target protein present in transgenic plants. The one of the main objective of the study was to

quantify the insecticidal protein levels along with the age of transgenic cotton plants. For this

purpose, Expression of Cry1Ac gene was quantified in these lines after 15 days interval using

Evirologix ELISA kit (Cat # AP 007). Result showed that the toxin level declined along with the

age of plant (Figure- 4.5) being maximum at vegetative stage, 30 days crop. As the crop progressed

towards the maturity, there was a gradual decline in expression. Similar expression pattern was

found in all these transgenic lines. Statistical analysis revealed that these Cry1Ac differences

among transgenic lines, sampling dates and their interaction were significant at 5% level of

significance (Table-4.1).

Figure-4.5: Temporal expression of Cry1Ac gene in transgenic cotton lines

Table-4.1: ANOVA for Cry1Ac Expression among Transgenic lines and Sampling dates

Source df Sum of Squares Mean Square F Sig. Transgenic lines 11 71110.507 6464.592 39.047 0 Sampling date 5 160351.93 32070.386 193.708 0 Lines * Dates 55 27186.734 494.304 2.986 0 Error 216 35761.075 165.561 Total 288 1240383.778

To establish the level of significance among various transgenic lines, sampling dates ane Lines *

Dates interaction, Least Significance Difference (5% level) was applied.

4.2.2 Spatial Expression of Cry1Ac gene

Enzyme Linked Immunosorbent Assay (ELISA) was used to quantify the spatial expression

of Cry1Ac gene in transgenic lines. For this purpose, different plant parts i.e. Leaves, square buds,

bolls, anthers, petals and ovary were sampled from the field and subjected to assay for the

quantification of protein being produced in these plant parts. Result revealed that the expression of

Cry1Ac was variable in different plant parts, being maximum in the leaves followed by square

buds, bolls and anthers respectively (Figure-4.6). Petals were showing less toxin expression in

every plant as compared to other plant parts being only 8-10 ng per gram of fresh tissues weight

while Cry1Ac expression in ovary remained undetectable.

Spatial Expression of cry1Ac in transgenic cotton lines

50 45 40 35 30 25 20 15 10 5 wt.) fresh (ng/g Conc. Protein 0 3001-1 3009-8 3013-3 3016-1 3033-4 3038-3 Transgenic Lines

Leaf Square Petal Anther Boll

Figure-4.6: Spatial expression of Cry1Ac gene in transgenic cotton lines

4.2.3 Temporal Expression of Cry2A gene

The expression of Cry2A insecticidal gene was also quantified in these transgenic lines using Enzyme Linked Immunosorbent Assay. As these transformed lines were transformed with

two genes Cry1Ac and Cry2A, so the same protein samples were used to quantify the expression of

Cry2A protein. Result showed that the Cry2A toxin level also declined along with the age of plant

(Figure-4.7) being maximum at the vegetative stage of crop plants. Afterwards, the protein levels of Cry2A declined slowly as the crop age increased. Statistical analysis revealed that these differences among transgenic lines, sampling dates and their interaction were significant at 5% level of significance (Table-4.2).

Figure-4.7: Temporal expression of Cry2A gene in cotton lines

Table-4.2: ANOVA for Cry2A Expression among Transgenic lines and Sampling dates

Source df Sum of Squares Mean Square F Sig. variety 145051.11 11 13186.464 374.6520 date 201738.87 5 40347.774 1.15E+030 variety * date 12954.876 55 235.543 6.692 0 Error 7602.462 216 35.197 Total 1503763.6 288

To establish the level of significance among various transgenic lines, sampling dates ane Lines *

Dates interaction, Least Significance Difference (5% level) was used.

4.2.4 Spatial Expression of Cry2A gene

Cry2A protein levels were also quantified spatially by Enzyme Linked Immunosorbent

Assay. The different plant parts i.e. Leaves, square buds, bolls, anthers, petals and ovary were

undertaken for this study to quantify the protein being produced in these plant parts. Result showed that the expression of Cry12A was variable in different plant parts, being maximum in the leaves followed by square buds, bolls and anthers respectively (Figure-4.8). Petals were showing less

toxin expression in every plant as compared to other plant parts being only 8-10 ng per gram of

fresh tissues weight while Cry2A expression in ovary remained undetectable.

3001-1 3009-8 3013-3 3016-1 3033-4 3038-3

Transgenic Lines

Figure-4.8: Spatial expression of Cry2A gene in cotton lines

4.3 Biotoxicity Leaf Assay

Laboratory Biotoxicity assays with 2nd Instar Heliothis larvae were conducted to confirm

the variation in expression of these insecticidal genes along with the age of plant. In this assay,

mortality %age of insect larvae was checked by detaching ten leaves of transgenic plants in petri

plants along with control plants at 30,60 and 90 days of crop age . The 2nd Instar Heliothis larvae were fed to these leaves. Larvae dead or alive were counted in each petri plate (Figure-4.9).

Transgenic lines showed a varying mortality rate of Heliothis larvae that ranged between 60-90 %.

The varying mortality rates indicated a gradual decline in insecticidal proteins levels. Temporal change in the efficacy of these transgenic lines against target insect pest has been shown in (Figure-

4.10). A transgenic lines 3016 that showed 100 mortality rate at 30 days crop age, had 80% mortality rate at 90 days crop age. Similar was the case with other transgenic lines while in case of non transformed control CIM-482, no any larval mortality was noted.

Figure-4.9: Leaf biotoxicity assay with 2nd instar Heliothis larvae

Figure-4.10: Mortality of Heliothis Larvae in different lines at 30, 60 and 90 days of crop

4.4 Isolation of RbcS Promoter from Gossypium arboreum

4.4.1 RNA Extraction and cDNA Synthesis

Total RNA was extracted from leaves of Gossypium arboreum by method of Saha et al

(1997). RNA pellet was resuspended in DEPC treated water. The quality of RNA was checked by

running on 1.2% agarose gel in TAE buffer and samples were mixed with RNA loading dye prior

to loading. The products were visualized as a smear with ethidium bromide staining (Figure-4.11)

along with two distinct ribosomal RNA bands, 28S and 18S respectively. RNA quantification was

done by nanodrop spectrophotometer. A total of 1µg of mRNA isolated was reverse transcribed in

a 20 µL reaction volume using fermentas cDNA synthesis kit (Cat #K1612) according to the

instruction given by the manufacturer. The integrity of cDNAs was evaluated by running in 1.2% agarose at 50 volts for 3 hours.

1 2 3 4 5 6

28S

18S

Figure-4.11: Total RNA extracted from Gossypium arboreum var. FDH-786

Lane1-5 : RNA of leaf samples of Gossypium arboreum

Lane 6 : 100 bp Ladder (Fermentas)

4.4.2 Amplification and Gel Elution of RbcS Promoter

RuBisCo small subunit (RbcS) promoter region was amplified by using Gossypium arboreum cDNA as template. Using gene specific primers, a band of 445 bp was obtained as required (Figure-4.12). This required band was amplified in all samples of Gossypium arboreum.

While designing the primers, KpnI restriction sites were introduced in both forward and reverse

primers along with additional sequences for proper sitting and functioning of the enzyme.

Amplified product was eluted from the gel by (DNA extraction kit Fermentas). Eluted DNA pellet

was dissolved in TE buffer. Size and quality was estimated by running 4uL of eluted fragment on

1.5% agarose gel at 90V for 20 min.. This fragment was further cloned in Invitrogen TA vector

(pCR®2.1).

Figure-4.12: PCR Amplification of RbcS promoter from desi cotton (Gossypium arboreum)

var.FDH-786.

4.4.3 Sequencing of RbcS Promoter Region

Cycle sequencing of the purified products was performed with an ABI prims 3100

automatic sequencer using Big Dye TM terminator cycle sequencing ready reaction kit (Perkin

Elmer, Branchburg, NJ, USA). The data obtained in CD from the sequencing lab. was analyzed.

The pure promoter sequence (445bp) obtained after removing Invitrogen TA vector (pCR®2.1)

sequences. The promoter sequence obtained was subjected to transit peptide prediction server

(ChloroP 1.1) to confirm the presence of transit peptide in the sequence (Figure-4.13).

Figure-4.13: Promoter sequence of RbcS isolated from desi cotton (Gossypium arboreum) var. FDH-786

4.5 Cloning of Insecticidal Gene under RbcS promoter in Plant Expression

Vector pCAMBIA 1301

4.5.1 Amplification and Gel Elution of Cry1Ac+NOS Fragment

One of the main objective of this work was to clone the Cry1Ac gene under RbcS promoter in plant expression vector. For this purpose, Cry1Ac+NOS terminator fragment was amplified from

PIA-2 construct; pGEM-4Z based construct already available in CEMB, Plant Molecular Biology

Lab. (Figure-4.14). While designing the primers, KpnI and BamHI restriction sites were introduced in both forward and reverse primers respectively to facilitate its removal and insertion in plant expression vector along with additional sequences for proper sitting and functioning of the enzyme.

A fragment of 2.11 kb (Cry1Ac 1.845kb and NOS terminator 0.279 Kb) was amplified (Figure-

4.14). The `PCR product was gel eluted using Fermentas DNA purification kit.

1 2 3 4 5 6 7 8 9

HindIII BamHI EcoRI BamHI

Ubiquitin Cry1Ac gene NOS

2.02Kb 1.845Kb 0.270Kb

PIA-2

Lane 1-8 : Plasmid DNA PIA-2

Lane 9 : Lamba HindIII Marker

Figure-4.14: PCR Amplification of Cry1Ac+NOS terminator from PIA-2 construct

4.5.2 Ligation & Transformation

The insert (Cry1Ac+NOS) and pCAMBIA 1301 were digestion individually with KpnI and

BamHI enzymes to nicks for proper ligation of insert into plant expression vector. The digested pCAMBIA 1301vector and digested Cry1Ac + NOS fragment were subjected to ligation after knowing the concentration of vector and insert. The concentration of both fragments was determined by spectrophotometer. As the size of pCAMBIA vector was large (~11kb) so the vector to insert ratio was taken as 1:1. The ligated product was transformed into DH5 alpha competent cellsusing heat shock method of transformation. A good quantity (~1µg/µL) of plasmid DNA

(having cloned Cry1Ac+NOS insert) was recovered which was further used for the confirmation of cloned fragments.

4.5.3 Confirmation of Insert (Cry1Ac+NOS Fragment) in pCAMBIA 1301

To confirm the integration of Cry1Ac+ NOS fragment in the pCAMBIA 1301 under KpnI and BamH I restriction sites, PCR reaction was performed using gene specific primers for Cry1Ac+

NOS insert. Also, plasmid DNA was digested using KpnI and BamH I restriction enxzymes to excise 2.11 Kb fragment of Cry1Ac+ NOS. PCR assay amplified a 2.11 kb fragment of Cry1Ac+

NOS terminator from samples. Similarly, a fragment of same length was excised as a result of digestion reaction which confirmed the integration of insert (Cry1Ac+ NOS) in pCAMBIA 1301

(Figure-4.15).

1 2 3 4 1 2 3 4

2.11 Kb

Figure-4.15: PCR and Restriction digestion confirming the integration of Cry1Ac+ NOS insert

Lane 1 : Lamba HindIII Marker Lane 1-3 : Excised fragment Cry1Ac+NOS

Lane 2-3: Plasmid DNA from pCAMBIA 1301

Lane 4 : +ve control (Gel Elueted Cry1Ac+NOS ) Lane 4 : Lamba HindIII Marker

4.5.4 Ligation & Transformation

The Cry1Ac+ NOS cloned pCAMBIA 1301vector was further used in cloning to insert the

RbcS promoter ahead of Cry1Ac insecticidal genes under KpnI restriction site. For this purpose,

Cry1Ac+ NOS cloned pCAMBIA-1301vector was digested with KpnI and likewise, RbcS fragment

(freshly gel eluted) was also digested with KpnI. The digested fragments were gel sliced and ligation was preceded. Before ligation, the concentration of both fragments was determined by spectrophotometer. The vector to insert ratio was taken as 1:1. The ligated product was transformed into DH5 alpha competent cells. Miniprep yielded in good concentration of plasmid DNA.

4.5.5 Confirmation of Insert (RbcS+ Cry1Ac+NOS Fragment) in pCAMBIA 1301

PCR and Restriction digestion reaction were conducted to confirm the integration of RbcS in pCAMBIA 1301 already cloned with Cry1Ac+ NOS terminator. PCR reaction was conducted using gene specific primers for RbcS promoter and Cry1Ac gene to yield 445bp and 565bp fragments respectively. A digestion reaction was also conducted to excise a fragment of 2.56kb

(RbcS + Cry1Ac+ NOS Fragment) using EcoRI and BamHI restriction sites. The PCR product was

run on 1.5% agarose gel while digestion gel was run on 1% agarose gel. The amplification of

445bp fragment of RbcS and 565bp fragment of Cry1Ac confirmed the integration of these inserts

in pCAMBIA 1301 (Figure-4.16). The digestion results further confirmed the results as 2.56kb

fragment was released from pCAMBIA 1301 (Figure-4.17). The insecticidal genes Cry1Ac was

cloned under RbcS promoter (Figure-4.18).

445bp

565bp

Figure-4.16: PCR Amplification of RbcS promoter and Cry1Ac gene from vector pCAMBIA-1301

Lane 1 : Lamba HindIII Marker Lane 1 : Lamba HindIII Marker

Lane 2-7 : Plasmid DNA Lane 2-6 : Plasmid DNA

Lane 8 : +ve control (Gel Elueted RbcS ) Lane 7 : +ve control (PIA-2 plasmid DNA)

1 2 3 4 5 6 7

2.5 Kb

Figure-4.17: Restriction digestion excised a fragment of 2.5kb confirming the integration of RbcS+ Cry1Ac+ NOS insert in pCAMBIA 1301

Lane 1 : 1Kb plus ladder (Fermentas)

Lane 2-7 : Digested samples of plasmid DNA

KpnI KpnI BamHI

RbcS Cry1Ac NOS

Figure -4.18: Cassette in pCAMBIA 1301 showing Cry1Ac under RbcS promoter (Rb--Ac)

4.5.6 Orientation of Rbcs and Cry1Ac in pCAMBIA 1301

The proper orientation of RbcS promoter and Cry1Ac gene in pCAMBIA 1301 vector was

further confirmed by designing primers from RbcS as well as Cry1Ac gene sequences. Primer 3 software was used to design primers that would amplify a band of 350 bp from RbcS to Cry1Ac

region. PCR assay confirm the orientation of inserted fragments by amplifying a band of 350 bp

size in all samples of plasmid (Figure-4.19).

1 2 3 4 5 6 7 8 9

350 bp

Figure-4.19: Orientation confirmation of RbcS and Cry1Ac integration in pCAMBIA 1301

Lane 1 : 1kb Plus ladder (Fermentas)

Lane 2-9 : Plasmid DNA samples

4.6 Electroporation and Agrobacterium Mediated Transformation

After the confirmation of construct, a commercially approved local cotton variety NIAB- 846 was transformed with this construct having Cry1Ac under RbcS promoter. The same variety was transformed with another construct harboring Cry1Ac under 35S promoter. After electroporation of competent cells of Agrobacterium tumefaciens strain LB4404, few colonies were confirmed through PCR with respective gene primer and further preceded for transformation in cotton.

4.6.1 Transient expression of GUS gene

After Agrobacterium mediated transformation, transient expression of GUS gene was studied through histochemical GUS assay. The (X-gluc) 5- bromo, 4- choloro, 3- indolyl glucoronide, gives a blue precipitate, a very insoluble and highly indigos formed by oxidative dimerization of colourless indoxyl derivative that result from hydrolysis of X-gluc by β- glucoronidase enzyme. After 72 hours of cocultivation, embryos and leaves were incubated with x- gluc solution and kept 37ºC overnight. The blue colour indicated the transformation of GUS gene into transformed embryos and leaves as compared with non transformed embryos and leaves where no blue colour were observed (Figure-4.20). 1 2 3 4 5 6

Figure-4.20: GUS Immunohistochemical assay of transgenic embryos and leaves

Lane 1-2: Embryo transformed with Rb-Ac and pk2Ac construct respectively

Lane 3 : Non transformed embryo as –ve control

Lane 4-5: Transgenic leaves transformed with Rb-Ac and pk2Ac construct resp.

Lane 6 : Non transgenic leaf as –ve control 92

4.6.2 Transformation Efficiency

A total of 4,000 mature embryos were used in all the transformation experiments. After

eight weeks of selection on 75mg/mL hygromycin, 50 plants of Rb-Ac and 43 plants for Pk2Ac

plants were obtained. Among these 50 plants of Rb-Ac plants, only 4 plants were shifted in the

field while only 3 out of 43 pk2Ac plants were shifted in the field. Therefore, the overall

transformation efficiency in both types of transgenic plants remained 0.175% (Table-4.3).

Table 4.3: Transformation efficiency of cotton variety NIAB-846 with Rb-Ac and Pk2Ac constructs

Construct No. of Survival % Survival % Plants in Transformation

Embryos (3-4 weeks) (7-8 weeks) soil Efficiency (T.E %)

Rb-Ac 2000 225 50 4 0.20%

Pk2Ac 2000 735 43 3 0.15%

4.6.3 Establishment of Transgenic Plants in Soil

Out of 50 Rb-Ac putative transgenic plants obtained after MS hygro selection, only 4

Finally two plants of Rb-Ac and two plant of pk2Ac established in the soil properly and were

subjected to molecular analysis to confirm the introduced gene integration and expression in cotton

genome.

A B

C D

A: Isolated mature embryos of cotton

B : Selection of transformed shoots on MS medium

C: Transformed plants growing in vitro

D: Transfer of rooted plants in the soil

Figure-4.21: Schematic representation of cotton transformation steps

4.7 Molecular Analysis of Transgenic Cotton Plants (T0 Progeny)

4.7.1 PCR analysis of Transgenic plants

A total of 2 Rb-Ac and 2 pk2Ac putative transformants were shifted to field conditions and further analyzed for the integration and expression of Cry1Ac gene at the molecular level. Analysis revealed that both plants of Rb-Ac and plants of pk2Ac were positive. The integration of RbcS promoter and Cry1Ac gene in Rb-Ac putative transgenic plants was checked by PCR of genomic

DNA of transformants. The amplified fragment of 445 bp of RbcS promoter and 565 bp fragment

of Cry1Ac suggested the successful transformation in these cotton plants (Figure-4.22). Hence two

types of the transgenic plants were obtained; plants with Cry1Ac under RbcS promoter and other

plants with Cry1Ac under 35S promoter. All the transgenic plants were phenotypically normal and

grew well.

1 2 3 4 1 2 3 4 5

445bp

565bp

Figure-4.22: Amplification of RbcS promoter and Cry1Ac in putative Rb-Ac transgenic plants

Lane 1-2: Amplification of RbcS promoter Lane 1 : 1Kb plus ladder (Fermentas) in transgenic cotton plants Lane2-3: Amplification of Cry1Ac in transgenic Lane 3 : Positive control (RbcS) cotton plants

Lane 4 : 1Kb plus ladder (Fermentas) Lane 4 : Negative control (NIAB-846)

Lane5 : Positive control (plasmid DNA) 95

1 2 3 4 5 1 2 3 4 5

565bp

190bp

Figure-4.23: Amplification of 35S promoter and Cry1Ac in pk2Ac putative transgenic cotton plants

Lane 1 : 50bp plus ladder (Fermentas) Lane 1 : 1Kb plus ladder (Fermentas)

Lane 2-3: Amplification of 35S promoter in Lane 2 : Negative control (NIAB-846) transgenic cotton plants Lane3-4: Amplification of Cry1Ac in transgenic Lane 3 : -ve Control plants

Lane 4 : Positive control (plasmid DNA) Lane5 : Positive control (plasmid DNA) :

4.7.2 Southern Blot Analysis of Transgenic plants

PCR positive plants of Rb-Ac and pk2Ac were further subjected to southern blot to

confirm the integration of Cry1Ac gene in transgenic plants. Rb-Ac plants were digested with

EcoR1 and BamHI enzymes while pk2Ac plants were digested with HindIII enzyme. A 3kb

fragment (35S+Cry1Ac+NOS terminator) in case of pk2Ac plants was detected while a fragment of

2.5Kb (RbcS+Cry1Ac+NOS terminator) was detectected in Rb-Ac plants showing the integration

of Cry1Ac in cotton genome (Figure-4.24)

1 2 3 4 5 6

Figure-4.24: Southern Blot of Cry1Ac in putative transgenic plants

Lane 1 : Negative control (untransformed NIAB-846)

Lane 2 : Positive control (plasmid DNA pk2Ac)

Lane 3-4: Transgenic plants of pk2Ac in T0 progeny

Lane 5-6: Transgenic plants of Rb-Ac in T0 progeny

4.7.3 Temporal and Spatial Expression of Cry1Ac Gene

The transgenic plants of Rb-Ac and pk2Ac were subjected to Enzyme linked

immunosorbent assay for the quantification of insecticidal protein Cry1Ac levels along with the age of transgenic cotton plants. Expression of Cry1Ac in both types of transgenic plants after every 15 days intervals showed that Rb-Ac plants were producing maximum expression of Cry1Ac protein and no decline in expression was observed in these plants. While in case of pk2Ac, Cry1Ac levels declined as the crop progressed towards the maturity (Figure-4.25). Expression in Rb-Ac plants was upto 250ng per gram of fresh tissue weight even when the plant was 90 days old.

Figure-4.25: Temporal Expression of Cry1Ac in Rb-Ac and pk2Ac transgenic cotton plants

Cry1Ac protein was also quantified in different plant tissues of Rb-Ac and pk2Ac plants.

Plant tissue ie. leaf, boll, square, anthers, petals and ovary were taken for this purpose. A result

showed that leaves of both Rb-Ac and pk2Ac plants were having maximum Cry1Ac concentration

followed by bolls and squares. No expression was detected in petals and ovary. Rb-Ac plants

showed more expression of Cry1Ac in bolls and squares as compared to pk2Ac plants while no

expression in anthers (Figure-4.26).

Figure-4.26: Spatial Expression of Cry1Ac in Rb-Ac and pk2Ac transgenic cotton plants

4.8 Molecular Analysis of Transgenic Cotton Plants (T1 Progeny)

4.8.1 Polymerase Chain Reaction

A total of 15 Rb-Ac and 15 pk2Ac transgenic plants were shifted to the green house conditions and further analyzed for the integration of Cry1Ac gene in plant genome. PCR assay showed that all Rb-Ac plants were positive while 10 plants of pk2Ac out of 15 plants were showing a Cry1Ac band of 565 bp. 11 positive plants of Rb-Ac and 9 positive plants of pk2Ac have been shown in gel pictures respectively (Figure-4.27 & Figure-4.28). Chi Square test was applied on PCR results to check the segregation of Cry1Ac in Rb-Ac and pk2Ac plants. Chi square calculated value was higher than tabulated value at 5% level of significance indicating that Cry1Ac gene was following the distorted medelian ratio in T1 Rb-Ac plants (Table-4.4) while Chi square

calculated value was lower that tabulated value at 5% level of significance indicating that Cry1Ac

gene was following the medelian inheritance pattern in T1 pk2Ac plants (Table-4.5).

4.8.2 SDS Polyacrylamide Gel Electrophoresis

Expression of Cry1Ac protein in transgenic plants of Rb-Ac and pk2Ac in T1 progeny was confirmed by SDS Polyacrylamide Gel Electrophoresis. A 68 kDa band of Cry1Ac protein was observed in all the transgenic plant samples including positive control while no such band was present in negative control (Figure-4.29) thereby confirming the expression of introduced gene

(Cry1Ac) in transgenic plants.

100

565bp

Figure-4.27: Amplification of Cry1Ac in Rb-Ac transgenic plants (T1 Progeny)

Lane 1 : Lamba HindIII Marker

Lane 2-12 : Transgenic plants of Rb-Ac (T1 Progeny) Lane 13 : Negative Control (Non Transformed NIAB-846)

Lane 14 : Positive control (plasmid DNA)

565bp

Figure-4.28: Amplification of Cry1Ac in pk2Ac transgenic plants (T1 Progeny)

Lane 1 : Lamba HindIII Marker

Lane 2-8, 10-11: Transgenic plants of pk2Ac (T1 Progeny) Lane 9, 12 & 13: Undetected plants of pk2Ac

Lane 14 : Negative Control (Non Transformed NIAB-846) Lane 15 : Positive control (plasmid DNA)

101

Table-4.4: Chi Square test to evaluate goodness of fit test of Cry1Ac gene in Rb-Ac plants

Rb-Ac Observed ( O) Expected (E) (O-E)2/E χ2 =∑ (O-E)2/E Tabulate value (df=1) at 5% probability level Cry1Ac 15 11.25 1.25 5.00 3.84 Detected

Not Detected 0 3.75 3.75

Table-4.5: Chi Square test to evaluate goodness of fit test of Cry1Ac gene in pk2Ac plants

pk2Ac Observed ( O) Expected (E) (O-E)2/E χ2 =∑ (O-E)2/E Tabulate value (df=1) at 5% probability level

Cry1Ac 10 11.25 0.138 0.554 3.84 Detected

Not Detected 5 3.75 0.416

102

1 2 3 4 5 6 7 8 9 10 11

68kd

Figure-4.29: SDS page showing 68Kda band of Cry1Ac protein in transgenic plants of Rb-Ac and pk2Ac

Lane 1 High Molecular wt. Protein Marker

Lane 2-5 Transgenic plants of Rb-Ac (T1 progeny)

Lane 6-9 Transgenic plants of pk2Ac (T1 progeny) Lane 10 Negative Control (Non Transform CIM-846) Lane 11 Positive Control (Bacillus thuringiensis var. HD-73)

102

5. DISCUSSION

Transgenic cotton lines encoding insecticidal crystalline proteins from Bacillus

thuringiensis can reduce the use of conventional broad- spectrum pesticides dramatically against

targeted pests. There is always a risk that insects could become resistant to Bt toxin after prolonged

and repeated field exposure. The most practical approach to prolong the effectiveness of Bt crops

has been refugia strategy and pyramiding of two or more genes in the same cultivar (Cohen et al.

2000).

This study was undertaken to determine the integration and spatio temporal expression of

insecticidal genes Cry1Ac and Cry2A in transgenic cotton lines. Molecular approaches revealed

that genes remained stable in subsequent generation as it was confirmed by PCR (Figure-4.1& 4.2)

and Southern blot analysis of the transgenic plants (Figure-4.3) while expression level of these

genes varied temporally and spatially (Figure-4.5- 4.8).

To confirm the expression results further in field, biotoxicity assays were conducted to

determine the mortality %age of Heliothis larvae (2nd instar) at different time intervals of crop age.

Cotton leaves were collected for biotoxicity assay after 30, 60 and 90 days of crop age which

showed that there was gradual decline in efficacy of insecticidal genes expression against targeted insects after 30 days time intervals as some of larvae survived after 90 days. These results are in

accord with Karanthi et al. (2005) who found that toxin titer in different fruiting parts in 110 days

crop cannot confer full protection against target insect pest.

Study of toxin titer in cotton plant is very crucial as it must be in sufficient quantity at any

time to protect the crop against lepidopterans especially boll worms. In the present study, a gradual decline in endotoxin expression was found along with the passage of plant growth and most

103

importantly, the expression level was lower in reproductive parts of plants which are the most susceptible parts for attack of boll worms that is an alarming sign for the cotton scientist to combat against these insect pests.

Expression level of Cry1Ac and Cry2A genes declined progressively over the crop growth with toxin level falling to 15-20 ng/g of fresh tissue weight ((Figure-4.5& 4.7). These results are in agreement with the previous studies conducted by Finnegan et al. (1998), Fitt et al. (1998),

Greenplate et al. (1998), Chen et al. (2000), Mahon et al. (2002), Karanthi et al. (2005), Xia et al.

(2005), Olsen et al. (2005), Bakhsh et al. (2009), Manjunatha et al. (2009) and Adamczyk et al.

(2009) who have reported inconsistency in insecticidal gene expression over the crop growth period. Expression levels of insectidal genes were also found variable in different plant parts. The leaves of Bt cotton were having maximum expression as compared to certain fruiting parts

(Greenplate, 1999; Greenplate et al. 2000; Adamczyk et al. 2001; Gore et al. 2001). In this study, the lowest expression was found to be in the petal of plants while the expressions in ovary remained undetectable.

Adamczyk et al. (2001) examined 13 commercial varieties of transgenic Cry1Ac Bacillus thuringiensis Berliner (Bt) cotton across two sites to know about the potential factors that impact endotoxin expression. He found that two varieties (NuCOTN 33B and DP458B/RR) expressed

Cry1Ac at significantly higher levels compared to the 11 other Cry1Ac varieties while no significant differences were found among these 11 varieties derived from a single transformation event 531. But in this study variation in expression pattern of insecticidal genes was observed in all transgenic lines.

It was found by Olsen et al. (2005) that initiation of squaring is associated with decrease in the levels of Cry1Ac transcripts detected in cotton plants in a controlled environment while in the

104

field major decline in efficacy occurred when the plants were flowering. In our study there was a

gradual decline in efficacy, being less in reproductive parts as compared to leaves.

Olsen et al. (2005) also mentioned that reduction in Cry1Ac transcripts was most likely due

to failure of 35S promoter at post squaring cotton rather than developmentally induced post

transcriptional gene silencing, as two other transgene under the same promoter also showed a

decline in transcript level at post squaring.

A recent study was conducted by Adamczyk et al. (2009) in which author has correlated the overall differences of Cry1Ac levels to mRNA transcripts in Bollgard cotton lines using

quantitative real-time polymerase chain reaction (qPCR). He found that mRNA transcript levels

varied among Bollgard lines ultimately leading to decline in Cry1Ac protein levels. Such results

were also previously described by Mahon et al. (2002), Olsen et al. (2005) and Xia et al. (2005).

However it was also mentioned that plant mechanism for which this occurs in still unknown.

The commercial cultivars of Bollgard cotton differ in the amount of expressed Cry1Ac

protein. Many researchers have correlated this decline in efficacy to the increased survival rates of

H. armigera (Holt, 1998; Adamczyk and Gore, 2004; Kranthi et al. 2005; Olsen et al. 2005; Wan et

al. 2005).

Gene expression varies with the nucleotide sequence of the gene, promoter, and the

insertion point of the gene in the DNA of the transgenic variety, transgene copy number, the

internal cell environment, as well as several external factors in the environment (Hobbs et al. 1993;

Guo et al. 2001; Rao, 2005). Therefore, investigation at molecular, genetic, as well as

physiological levels should help in understanding the differential expression of transgenes and the quantitative changes in insecticidal proteins in Bt cotton plants.

105

Adamczyk et al. (2001) and (2004) reported that the expression of Cry1Ac δ-endotoxins in commercial cultivars remained variable and the genetic background of these varieties is the main contributing factor towards this differential expression of Cry1Ac. However, the variation in

Cry1Ac expression has also been observed in varieties having dissimilar genetic background suggesting that genetic background may not be the contriburting factors towards this variation

(Khan et al. 2008; Bakhsh et al. 2009; Manjunatha et al. 2009).

The expression level of a gene may be influenced by its copy number (Hobbs et al. 1993;

Agaisse et al. 1995; Guo et al. 2001; Rao, 2005). All the transgenic lines under investigation showed similar pattern of gradual decline in Cry1Ac expression in spite of having different copy numbers ((Noreen et al. 2008). Shirsat et al. (2003) reported that the number of gene copies of the introduced legA genes could not be correlated with expression levels in the same plants, suggesting that the variation in expression resulted from other factors.

Nearly all transgenic crops around the world utilize the CaMV 35S promoter (Odell et al.

Wessel et al. (2001) reported the variation in expression patterns of three promoters

(Cauliflower Mosaic Virus (CaMV) 35S, modified CaMV 35S and the promoter of an Arabidopsis thaliana (Lipid Transfer Protein gene) using firefly luciferase reporter system. The expression of luciferase gene varied not only among independent transformatants but also between leaves on the same plant and within a leaf. Imaging of luciferase activity in the same leaves over 50 days period

106

showed that individual transformatants show different types of temporal regulation and also this spatial and temporal pattern is inherited by the next generation.

“ In the present study, expression of insecticidal genes under 35S CaMV promoter has been variable in different tissues and at different crop growth stages indicating that 35S promoter is developmental promoter. These results are in accord with Yang et al. (2005) who transformed soybean (Glycine max L.) with chimeric -glucuronidase (GUS) gene under the control of the 355

CaMV promoter using direct DNA transfer via electric discharge particle acceleration and found that expression of GUS gene in R1 plants were localized using thin tissue sections. Different levels of GUS expression were obtained in various tissues, being maximum in leaf, parenchyma cells in xylem, and phloem tissues of stem while very low or no GUS activity was detected in cortex cells of root, vascular cambium cells in stem, and the majority of cortex cells in leaf midrib. He further concluded that 35S CaMV promoter is cell-type-specific and is developmentally regulated (Nilsson et al. 1992; Pauk et al. 1995; Wessel et al. 2001; Sunilkumar et al. 2002; Haddad et al. 2002; Yang et al. 2005; Amarasinghe et al. 2006; Bakhsh et al. 2009).

Transgene promoter activity can be characterized by the distribution of different expression levels with in a plant, each level occurring with its own frequency. The temporal and spatial expression feature of a transgene in different independent transgenic lines must be intrinsic to the promoter activity. Since every independent transformant shows minor or major differences in spatial and temporal regulation of a transgene, apparently in every transformant, there is a different influence from flanking plant DNA sequences. Thus, the promoter driving the expression of transgene specifies the cell types in which transgene is expressed and defines the temporal regulation of transgene promoter activity with in these cells (Wessel et al. 2001).

107

Keeping in view the decline in efficacy of insecticidal genes, the present study actually

triggered research to find possible new promoter that can induce more consistent expression of insecticidal genes throughout the growth period of the cotton plant.

We must rely on conventional breeding and selection to solve these problems of variable efficacy of transgenic cotton, but it will be useful, in the longer term, to identify other gene promoters that can drive strong expression of transgenes throughout the season. It is also important to have such promoters available for the next generation of transgenic cotton so that different traits can be stacked without relying on the same promoter so as to avoid transcriptional gene silencing

induced by multiple copies of a single promoter such as the CaMV 35S promoter (Fagard &

Vaucheret, 2000).

The second most common group of promoters after viral promoters for plant biotechnology

have come from highly-expressed plant genes, such as those for seed storage proteins,

photosynthetic proteins or housekeeping genes, all of whose mRNAs were easily cloned and

characterized (Potenza et al. 2003). Actin, ubiquitin and tubulin gene promoters have all been used

in various plant species for expressing transgenes or selectable markers. As sophistication in

biotechnology improves, the need for more developmentally or environmentally regulated

promoters has become evident and considerable effort is going into the discovery of specific tissue

or biotic, hormonal or abiotic stress responsive genes and promoters (Potenza et al. 2003).

RuBisCo is the bifunctional enzyme found in the chloroplasts of plants that catalyzes the

initial carbon dioxide fixation step in the Calvin cycle and functions as an oxygenase in

photorespiration. In higher plants it consists of eight each of two subunits, a large subunit encoded

by the chloroplast genome and the small subunit polypeptide encoded in the nuclear genome (Ellis,

1981). The SSU polypeptides are formed as precursors containing an amino-terminal extension 108

termed a transit peptide that is involved in the transport of the SSU polypeptide into the chloroplast during which the transit peptide is removed. RuBisCo is the most abundant protein found in plant leaves, representing up to 50% of the soluble protein. Thus, the SSU promoters and their transit peptides are attractive candidates for expression of genes at high levels in green tissue and for targeting of different proteins into the chloroplast (Ellis, 1981).

The small subunits promoters and their transit peptides are attractive candidates for expression of genes at high levels and have shown promising expression results in driving the expression of introduced genes (Wong et al. 1992; Fujimoto et al. 1993; Datta et al. 1998; Song et al. 2000; Jang et al. 2002; Liu et al. 2005; Panguluri et al. 2005; Amarasinghe et al. 2006;

Anisimov et al. 2007; Suzuki et al. 2009; Kim et al. 2009).

The expression of the modified gene for a truncated form of the cryIA(c) gene, encoding the insecticidal portion of the lepidopteran-active CryIA(c) protein from Bacillus thuringiensis var. kurstaki (B.t.k.) HD73, under control of the Arabidopsis thaliana ribulose-l, 5-bisphosphate carboxylase (Rubisco) small subunit atsiA promoter with and without its associated transit peptide was analyzed in transgenic tobacco plants. Examination of leaf tissue revealed that the atslA promoter with its transit peptide sequence fused to the truncated CryIA(c) protein provided a 10- fold to 20-fold increase in cryIA(c) mRNA and protein levels compared to gene constructs in which the cauliflower mosaic virus 35S promoter with a duplication of the enhancer region (CaMV-

En35S) was used to express the same cryIA(c) gene (Wong et al. 1992).

Anisimov et al. (2007) studied activity of the highest expressing Rubisco small subunit

(RbcS) promoters (pRbcS) from the cotyledons of germinating seedlings of Brassica rapa var. oleifera to drive high-level and preferably stage specific transgenic protein expression in

Brassicaceae plants. The mRNA level of RbcS and of gusA quantified in transformed plants. The 109

results demonstrated that promoter most active in seedlings under native conditions was also most active in transgenic constructs at the same stage of plant development. In the present study, the results of protein expression found in case of Rb-Ac plants (Cry1Ac under RbcS promoter) were much higher than pk2Ac plants (Cry1Ac under 35S CaMV). These results are in accordance with the results found by Wong et al. (1992), Amarasinghe et al. (2006) and Anisimov et al. (2007).

RbcS (RuBisCo small subunit) promoter region was isolated from Gossypium arboreum by reverse transcribing RNA isolated from leaves of desi cotton (Figure-4.12) and was cloned in a plant expression vector (Figure-4.16& 4.17). After successful cloning, the further objective was to transform a cotton cultivar with insecticidal gene (Cry1Ac) driven by RbcS promoter. Also the same cotton variety was transformed with insecticidal gene (Cry1Ac) driven by 35S CaMV promoter. In the present study pCAMBIA 1301 vector was used to transform as it has been used by many researcher to transform the genes like cbnA gene in rice (Shimizu et al. 2002), acsA and acsB genes, USP-1 and HSP-26 genes in cotton (Li et al. 2005; Maqbool 2009), HMGR gene (3- hydroxy-3-methylglutaryl-CoA reductase) in Taraxacum plants (Bae et al. 2005).

Plant transformation is powerful techniques that can be used to knock out genes or to introduce in plants. Plant transformation provides an opportunity to introduce traits of own choice from different species, adding characteristics which would be difficult or even impossible to acquire using traditional breeding techniques, such as insect resistance, herbicide resistance, cup quality and tolerance to abiotic stress like drought or frost. Difficulties are encountered in transferring genes between species because of natural barriers, which result in reduction in fertility after genetic transfer (Ribas et al. 2006).

Most of the successful strategies of the cotton transformation involve development of embryogenic callus and subsequent plant regeneration. Cotton is a recalcitrant crop and the success 110

is limited to only those varieties which are regenerable like Coker lines and Siokra 1-3 cultivars

(Trolinder and Xhixan, 1989; Stewart, 1991). Although to-date, no Pakistani variety has been identified with such regeneration property yet attempts were made to regenerate any variety and to learn more about the regeneration process of Coker as standard using different explants and regeneration procedures.

Agrobacterium strains play an important role in the transformation process, as they are responsible not only for infectivity but also for the efficiency of gene transfer. The suitability of

LB4404 strain harboring various plasmids for the transformation of cotton was observed in this experiment. Agrobacterium tumefaciens strain LB4404 was found to be infective with respect to transformation.

An efficient transformation system is an important tool for gene manipulation. In the present study, a modified shoot cut apex method as described by Qayyum (2009) was used. Four thousands embryos of cotton variety NIAB-846 (commercially approved local cotton variety) were isolated. Under 75 mg/mL hygromycin selection pressure, a total of 965 transgenic (after 3-4 weeks) and 93 transgenic plants (after 6-8 weeks) were recovered. Finally 4 transgenic plants (2 plants of Rb-Ac and 2 plants of pk2Ac construct) were shifted to the field. The overall transformation rate in the present study was 0.175%.

The root formation in transgenic plants is inhibited by antibiotic so an intermediate level of hygromycin (75mg/mL) is important to minimize the false positives (Zapata et al. 1999; Zhang et al, 2001; and Wu et al. 2006). Moreover, after transplant root regeneration is minimized due to antibiotic so growth and low survival rate make the final plant formation much slower (Hazra et al.

2000). Maximum rooting was observed when black portion of growing root on selection medium was cut, treated with IBA 1mg/mL and then cultured on simple MS medium. This practice reduced

111

transformation period from one year to 3-4 months (Firoozabady et al. 1987; Umbeck et al. 1987).

The plants obtained in the present study were phenotypically normal. The plants were shifted to the

field after they had developed 5-6 leaves. After that, the transgenic plants were subjected to

different molecular analysis.

The different molecular analysis ie GUS assay; PCR, Southern blot and ELISA were

performed to confirm the integration and expression of introduced genes in putative transgenic plants. The functional GUS gene produces blue coloration when placed in GUS substrate (X Gluc) which indicates the expression of introduced genes (Basu et al. 2004). GUS expression in mature embryos and leaves of cotton variety NIAB-846 was studied through histochemical GUS assay.

The blue spots on the embryos and leaves confirm the transformation system and hence indicate the integration and expression of GUS gene in recipient host. Non transformed embryo and leaves had no blue color (Figure-4.20).

The newly transformed plants were subjected to PCR to confirm the integration of RbcS promoter and Cry1Ac gene in Rb-Ac plants. Similarly integration of 35S promoter and Cry1Ac

gene was confirmed in pk2Ac plants. Amplification of 445bp and 565 bp confirmed the integration

of RbcS promoter and Cry1Ac gene respectively in Rb-Ac plants (Figure-4.22) while amplification

of 190bp and 565bp confirmed the successful transformation of 35S promoter and Cry1Ac gene respectively in pk2Ac plants (Figure-4.23).

PCR positive plants were further subjected to southern blot. A detection of ~2.5 kb fragment on membrance confirmed the integration of Cry1Ac gene in Rb-Ac plants and ~3 kb in case of pk2Ac plants (Figure-4.24). The presence of the expected hybridization signals in the transformed plants showed that the promoter and Cry1Ac remained intact when integrated into the

cotton genome in both Rb-Ac and pk2Ac plants. 112

The titer of Cry1Ac toxin in Rb-Ac and pk2Ac quantified at different developmental stages

indicated clearly that Rb-Ac plants were expressing Cry1Ac toxin at high concentration as

compared to pk2Ac plants. This further suggests that RbcS is efficient promoter driving the

expression of Cry1Ac consistent in cotton plant as a decline in Cry1Ac was observed in pk2Ac

plants (Figure-4.25). Spatial expression of Cry1Ac gene in different plant parts further confirmed

that promoter activity is limited to green parts of plants ie, leaf, stems, square buds and anthers.

The high expression of Cry1Ac protein was quantified in Rb-Ac plants being maximum in leaf as

compared to pk2Ac plants that were showing less concentration of Cry1Ac titer. Expression of

insecticidal protein is most critical in leaves as majority of the newly hatched lepidopterans larvae

initially feed by scraping chlorophyll in the tender leaves and, as they grow, moves over to the

squares and bolls for further feeding and development (Jayaraj, 1982; Gore at al. 2002).

There are many reports regarding the inheritance and expression stability of foreign genes

in transgenic crops. The mendelian inheritance of the foreign genes in transgenic plants has been

observed in alfalfa (Micallef et al. 1995) in rice (Duan et al. (1996) in maize (Fearing et al. 1997)

in cotton (Canming et al. 2000; Zhang et al. 2000; Xia et al. 2007; Zhang et al. 2007; Daud et al.

2009) and in cow pea (Ivo et al. 2008). However in many cases, the foreign gene displayed

complicated segregation profiles instead of Mendelian segregation (Spencer et al. 1992; Wan and

Lemaux 1994; Somers et al. 1994; Altman et al. 1996; Wu et al. 2002; Rashid et al. 2008). In the

present study, the same cotton variety was transformed with two construct having Cry1Ac under

different promoters. T1 progeny analysis of Rb-Ac plants showed the distorted medelian

inheritance of Cry1Ac gene (Table-4.2) while it followed mendelian inheritance in case of pk2Ac plants (Table-4.3). So in case of Rb-Ac plants, we can postulate about the integration of Cry1Ac to a particular position on chromosome that remained linked during meiotic division and stably inherited in next progeny. 113

Efforts could be focused on developing transgenic cotton varieties with tissue-specific promoters to enhance the expression of toxin genes in fruiting parts that are more susceptible to attack. In this study, variability in expression of insecticidal genes was made consistent by the use of promoter (RbcS) that would drive the expression of insecticidal genes throughout life of a cotton plant. The use of this promoter is also useful for adressing the biosafety issues as this promoter activity is limited to green parts of plants, hence no gene expression in roots and in cotton seed, ultimately no threat to soil organisms and safety of cotton products and by products would also be assured.

114

6. LITERATURE CITED

Abel, C. A., and Adamczyk J. J. (2004) Relative concentration of Cry1A in maize leaves and cotton bolls with diverse chlorophyll content and corresponding larval development of fall armyworm (Lepidoptera: Noctuidae) and southwestern corn borer (Lepidoptera Crambidae) on maize whorl profiles. J. Econ. Entomol. 97: 1737-1744.

Abuodeh, R. O., Orbach, M. J., Mandel, M. A., Das, A. and Galgiani, J. N. (2000) Genetic transformation of Coccidioides immitis facilitated by Agrobacterium tumefaciens. J Infect Dis 181:2106-2110.

Adamczyk, J. J., Hardee, D. D., Adams, L. C, Sumerford, D. V. (2001). Correlating differences in larval survival and development of bollworms (Lepidoptera: Noctuidae) and fall armyworms (Lepidoptera: Noctuidae) to differential expression of Cry1Ac(c) δ-endotoxin in various plant parts among commercial cultivars of transgenic Bacillus thuringiensis cotton. Journal of Economic Entomology 94: 284-290.

Adamczyk, J. J., and Sumerford, D.V. (2001) Potential factors impacting season-long expression of Cry1Ac in 13 commercial varieties of Bollgard cotton. J. Insect Sci. 1: 1-6.

Adamczyk, J. J ., Gore, J. (2004) Development of bollworms, Helicoverpa zea (Boddie), on two commercial Bollgard cultivars that differ in overall Cry1Ac level. J Insect Sci 4:32. Available online at http://insectsceince.org/4.32.

Adamczyk, J. J, Meredith, W. R. (2004). Genetic Basis for Variability of Cry1Ac Expression Among Commercial Transgenic Bacillus thuringiensis (Bt) Cotton Cultivars in the United States. The Journal of Cotton Science 8:17–23.

Adamczyk, J. J, Perera, J. O, Meredith, W. R. (2009) Production of mRNA from the Cry1Ac transgene differs among Bollgard lines which correlate to the level of subsequent protein. Transgenic Res (2009) 18:143-149.

115

Agaisse, H., Lereclus, D. (1995). How does bacillus thuringiensis produce so much insecticidal crystal protein? journal of bacteriology. 177(21): 6027–6032.

Alam, M. F., Datta K., Abrigo, E., Oliva, N., Tu, J., Virmani, S. S., et al (1999). Transgenic insect resistant maintainer line (IR68899B) for improvement of hybrid rice. Plant Cell Rep 18:572–575.

Alstad, D.N. and Andow, D.A. (1995). Managing the evolution of insect resistance to transgenic plants. Science, 268, 1894-1896.

Altman, D.W., Benedict, J.H. and Sach, E.S. (1996) Transgenic plants for the development of durable insect resistance, a case study for cotton and Bacillus thuringiensis. Ann. N.Y. Acad. Sci. 792:106-114.

Alt-Morbe J., Kithmann, H., Schroder, J., (1989). Differences in induction of Ti-plasmid virulence genes virG and virD and continued control of vir D expression by four external factors. Mol. Plant-Microbe Interact 2: 301-308.

Amarasinghe, B. H. R., Nitschke, E. F., Wu,Y., Udall, J.A ., Dennis, E.S., Constable, G and Llewellyn, D. J. (2006) Genomic approaches to the discovery of promoters for sustained expression in cotton (Gossypium hirsutum L.) under field conditions: expression analysis in transgenic cotton and Arabidopsis of a Rubisco small subunit promoter identified using EST sequence analysis and cDNA microarrays. Plant Biotechnology 23:437–450.

Anderson, A., and Moore, L. (1979) Host specificity in the genus Agrobacterium. Phytopathol 69:320–323.

Andrews, R.W., Fausr, R., Wabiko, M. H., Roymond, K.C and Bulla, L.A. (1987). Biotechnology of Bt: A critical review. Bio/Technology, 6: 163-232.

116

Anisimov, A., Kimmo, K., Anne, K., Seppo, K., Kari, J and Viktor, K. (2007) Cloning of new rubisco promoters from Brassica rapa band determination of their activity in stably transformed Brassica napus and Nicotiana tabacum plants. Mol Breeding 19:241–253.

Bae, T.W., Park, H.R., Kwak, Y.S., Lee, H.Y and Ryu, S.B. (2005) Agrobacterium tumefaciens- mediated transformation of a medicinal plant Taraxacum platycarpum Plant Cell, Tissue and Organ Culture 80: 51–57.

Bakhsh, A., Rao, A. Q., Shahid, A. A, Husnain, T and Riazuddin S. (2009). Insect Resistance and Risk Assessment Studies in Advance Lines of Bt Cotton Harboring Cry1Ac and Cry2A Genes. American-Eurasian J. Agric. & Environ. Sci., 6 (1): 01-11.

Bakhsh, A., Rao, A. Q., Shahid, A. A., Husnain, T and Riazuddin, S. (2009). CaMV35S is a Developmental Promoter Being Temporal and Spatial in Expression Pattern of Insecticidal Genes (Cry1Ac & Cry2A) in Cotton. Research Journal of Cell and Molecular Biology; 3(1): 56-62.

Barwale, R. B., Gadwa, V.R., Zehr, U and Zehr, B (2004) Prospects for Bt cotton technology in India. AgbioForum 7, 23-26.

Basu, C., Albert, A., Kauschb, P and Chandlee, J. M. (2004) Use of β-glucuronidase reporter gene for gene expression analysis in turfgrasses. Biochem Bioph Res Co 320 (1): 7-10.

Bates, S.L., Zhao, J., Roush, R.T., Shelton, A.M. (2005). Insect resistance management in GM crops: past, present and future.Nat. iotechnol. 23: 57-62.

Bayley, C., Trolinder, N., Ray, C., Morgan, M., Quisenberry, J.E and Ow, D.W (1992) Engineering 2,4-D resistance into cotton. Theor Appl Genet 83: 645-649.

Benedict, J. H., Sachs, E. S., Altman, D. W., Deaton, W. R., Kohel, R. J and Berberich, S. A. (1996) Field performance of cottons expressing transgenic Cry1A insecticidal proteins for

117

resistance to Heliothis virescens and Helicoverpa zea (Lepidoptera: Noctuidae). J. Econ. Entomol. 89, 230-238.

Bidney, D., Scelonge, C., Martich, J., Burus, M., Sims, L and Huffman, G. (1992). Microprojectile bombardment of plant tissues increased transformation frequency of Agrobacterium tumefaciens. Plant Mol. Biol. 18: 301-313.

Board on Agriculture and Natural Resources (BANR ). Genetically modified pestprotected plants: science and regulation. Washington D.C., USA, National Academy Press, 2000. 292 p. ISBN 0-30- 906930-0. Available from Internet: http://fermat.nap.edu/books/0309069300/html.

Bolin, P. C., Hutchison, W.D. and Andow, D. A. (1995). Selection for resistance to Bacillus thuringiensis endotoxin in a Minnesota population of European corn borer. In proc. 26th Ann. Meeting Soc. Invert. Pathol. Cornell Univ. Ithaca, NY.

Bosch, J., Fuchs, S., Pustejonsky, D., Molt, E., Albers, D. (2002). Yield and economic comparison of Bollgard varieties in the Tebax Gulf Coast Region. In Proc. Beltwise Cotton Conf. Part-2, Jan. 8-12, 4 pages.

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem.72: 248-254.

Brown, R.S., and Oosterhuis D. M. (2003). Effect of foliar ChaperoneTM applications under elevated temperatures on the protein concentrations and physiological responses of cotton. Ark. Agri. Exp. Sta. Res. Ser. 521:108-111.

Bruns, H.A., and Abel, C.A. (2003) Nitrogen fertility effects on Bt ∂-endotoxin and nitrogen concentrations of maize during early growth. Agron. J. 95, 207-211.

118

Bryant, J.P, Chapin, F. S and Klein, D.R. (1983) Carbon nutrient balance of boreal plants in relation to vertebrate herbivory. Qikos 40, 357-368.

Buchanan, B. B., Gruissem, W., Jones, R. L., Eds. Biochemistry and molecular biology of plants. Rockville, MD: American Society of Plant Physiologists; 2000:340–342.

Bundock, P., and Hooykaas. P. J. J. (1996) Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae genome by illegitimate recombination. Proc Natl Acad Sci USA 93:15272–12175.

Bundock, P., Dulk-Ras, A. D., Beijersbergen, A and Hooykaas, P. J. J. (1995) Trans-kingdom T- DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J 14:3206–3214.

Canming, T., Jing, S., Xiefi, Z., Wangzhen, G., Tianzhen, Z., Jinlian, S., Congfen, G., Weijun, Z., Zhiian, C. and Sandui, G. (2000). Inheritance of resistance to Helicoverpa armigera of 3 kinds of transgenic Bt strains available in upland cotton in china. Chinese Science Bulletin. 45(90): 363-367.

Chen, D., Ye, G., Yang, C., Chen, Y and Wu, Y. (2005a) The effect of high temperature on the insecticidal properties of Bt cotton. Environ. Exp. Bot. 53:333-340.

Chen, H., Tang, W., Xu, C.G., Li, X.H., Lin, Y.J and Zhang, Q. F. (2005) Transgenic indica rice gene of Bacillus thuringiensis exhibit enhanced כplants harboring a synthetic Cry2A resistance against rice lepidopteran pests. Theor Appl Genet 111:1330–1337.

Chen, S., Wu, J., Zhou, B., Huang, J and Zhang R. (2000) On the temporal and spatial expression of Bt toxin protein in Bt transgenic cotton. Acta Gossypii Sin. 12, 189-193.

119

Cheng, X., Sardana, R., Kaplan, H and Altosaar, I. (1998). Agrobacterium tumefaciens- transformed rice plants expressing synthetic cryIAb and cryIAc genes are highly toxic to striped stem borer and yellow stemborer. Proc Natl Acad Sci USA 95:2767–2772.

Chilton, M.D., Drummond, M.H., Merlo, D.J., Sciaky, D., Montoya, A.L., Goprdon, M.P and Nester, E.W. (1997) Stable incorporation of plasmid DNA into higher plant cell: the molecular basis of crown gall tumorigenesis. Cell 11:263-271.

Chitkowski, R. L., Turnipseed, S. G., Sullivan, M. J. and Bridges, W. C. (2003). Field and laboratory evaluation of transgenic cottons expressing one or two Bacillus thuringiensis var. Kurstaki Berliner proteins for management of noctuid (Lepidoptera) pests. J. Econ. Entomol. 96, 755-762.

Cohen, B.M., Gould, F and Bentur, J.C. (2000) Bt rice: practical steps to sustainable use. International Rice Research Notes, 2: 4-10.

Cohn, M. J., Chaufaux, J., Buisson, C., Gilois, N., Sanchis, V. and Lereclus, D. (1996). Spodoptera littoralis (Lepidoptera Noctuidae) resistance to cry1C and cross resistance to other Bacillus thuringiensis crystal toxins. J. Econ. Entomol. 89, 791-797.

Coviella, C. E., Stipanovic, R. D and Trumble, J. T. (2002) Plant allocation to defensive compounds: interactions between elevated CO2 and nitrogen in transgenic cotton plants. J. Exp. Bot. 53:323-331.

Crickmore, N., Zeigler, D.R., Feitelson, J., Schnepf, E., VanRie, J., Lereclus, D., Baum, J., Dean, D.H. (1998). Revision of the Nomenclature for the Bacillus thuringiensis Pesticidal Crystal Proteins. Microbiol Mol Biol Rev. 62(3):807–813.

Datta, K., Vasquez, Z., Tu, J., Torrizo, L., Alam, MF., Oliva, N., Abrigo, E., Khush, GS., Datta, S.K. (1998) Constitutive and tissue-specific differential expression of the cryI(A)b gene in

120

transgenic rice plants conferring resistance to rice insect pests. Theor Appl Gene. 97(2):20- 30. Daud, M. K., Variath, M. T., Ali, S., Jamil, M., Khan, M. T., Shafi, M. and Shuijin, Z. (2009). Genetic transformation of bar gene and its inheritance and segregation behavior in the resultant transgenic cotton germplasm (br001). Pak. J. Bot., 41(5): 2167-2178.

De Groot, M.J.A., Bundock, P., Hooykaas, P.J.J and Beijersbergen, A.G.M. (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol 16:839–842.

De Maagd, R. A., Bosch, D and Stiekema, W. (1999). Bacillus Thuringiensis Toxin-Mediated Insect Resistance in Plants. Trends Plant Sci., 4: 9–13.

De Rocher, E.J., Vargo-Gogola, T.C., Diehn, S.H., Green, P.J (1998). Direct evidence for rapid degradation of Bacillus thuringiensis toxin mRNA as a cause of poor expression in plants. Plant Physiol. 117: 1445-1461

Deblaere, R., Bytebier, B., De Greve, H., Deboeck, F., Schell, J., Van Montagu, M. and Leemans, J. (1985) Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants. Nucleic Acid Research 13: 4777-4788.

DeCleene, M and DeLey, J. (1976) The host range of crown gall. Bot Rev. 42:389–466.

Dhaliwal, H.S., Kawai, M and Hirofumi, U. (1998) Genetic Enginnering for abitoic stress tolerance in plants. Plant Biotechology, 15: 1-10.

Diehn, S.H., Chiu, W.L., De Roche, E.J and Green, P.J. (1998) Premature adenylation at multiple sites with in Bacillus thuringiensis toxin gene encoding region. Plant physiol. 117: 1433- 1443.

121

Donald, R.G.K and Cashmore, A.R. (1990) Mutation of either G box or I box sequences profoundly affects expression from the Arabidopsis rbcS-1A promoter. EMBO J. 9: 1717- 1726.

Dong, H.Z, Li, W.J, Tang, W and Zhang, D.M (2005) Increased yield and revenue with a seedling transplanting system for hybrid seed production in Bt cotton. J. Agron. Crop Sci. 191, 116- 124.

Dong, H.Z., Li, W.J. (2007) Variability of endotoxin expression in Bt transgenic cotton. J Agron Crop Sci 193:21–29.

Dorsch, J.A, Candas, M., Griko, N.B., Maaty, W.S.A., Midboe, E.G., Vadlamudi, R.K., Bulla, L.A. (2002) Cry 1A toxins of Bacillus thuringiensis bind specifically to a region adjacent to the membrane-proximal extracellular domain of Bt-R1 in Manduca sexta: involvement of a cadherin in the entomopathogenicity of Bacillus thuringiensis. Insect Biochem. Mol. Biol. 32: 1025-1036.

Duan, X., Li, X., Xue, Q., Abo-El-Saad, M., Xu, D and Wu, R. (1996) Transgenic rice plants harbouring an introduced potato proteinase inhibitor II gene are insect resistant. Nature Biotechnol 14: 494-498.

Economic survey of Pakistan 2008-09. Agriculture Chapter #2. Ministry of Finance, Govt. of Pakistan.

Ellis, R.J. (1981) Chloroplast proteins: Synthesis, transport, and assembly. Annu Rev Plant Pbysiol 32:111-137 Estada, U. and Ferre, J. (1994). Binding of insecticidal crystal proteins of Bacillus thuringiensis to the midgut brush border of the cabbage looper, Trichoplusia ni (Hubner) ( Lepidoptera Noctuidae) and selection for the resistance to one of the crystal proteins. Appl. Environ. Microbiol. 60, 3840-3846.

122

Fagard, M and Vaucheret, H. (2000) (Trans) gene silencing in plants: how many mechanisms? Ann Rev Plant Physiol Plant Mol Biol 51: 167–194.

Fearing PL, Brown D, Vlachos D, Vlachos D, Meghji M, Privalle L (1997) Quantitative analysis of CryIA(b) protein expression in Bt maize plants, tissues, and silage and stability of expression over successive generations. Mol Breed 3:169-176.

Ferré, J., Escriche, B., Bel, Y. and Van, R. J. (1995). Biochemistry and genetics of insect resistance to Bacillus thuringiensis insecticidal crystal proteins. FEMS Microbiology Letters 132, 1-7. Ferry, N., Edwards, M.G., Mulligan, E.A., Emami, K., Petrova, A.S,, Frantescu, M., Davison, G.M., Gatehouse, A.M.R. (2004). Engineering resistance to insect pests. In: Christou P, Klee H (eds). Handbook of Plant Biotechnology. Vol. 1. John Wiley and Sons, Chichester, pp. 373-394.

Finnegan, E. J., Lllewellyn, D. J and Fitt, G. P. (1998) what’s happing to the expression of the insect protection in field-grown Ingard cotton. In: ed. Proceedings 9th Australian Cotton Conference, 12– 14 August 1998, Broadbeach, Queensland. Australian Cotton Growers_ Research Association, Wee Waa, Australia.

Firozabady, E., Deboer, D.L and Merlo, D.J. (1987). Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants. Plant Mol Biol 10: 105-116.

Fitt, G. P., Daly, J. C., Mares, C. L and Olsen, K. (1998) Changing Efficacy of Transgenic Cotton Plant Patterns and Consequences. Sixth Australian Entomological Research Conference, Brisbane, Australia.

Flexner, J.L., Lighthart, B and Croft, B.A (1986) The effects of microbial pesticides on non-target, beneficial arthropods. Agri. Ecosys. Environ. 16: 203-254.

123

Fraley, R.T., Rogers, S.G., Horsch, R.B., Sanders, P.R., Flick, J.S., Adams, S.P., Bittner, M.L., Brand, L.A., Fink, C.L., Fry, J.S., Galluppi, G.R., Goldberg, S.B., Hoffmann, N.L and Woo, S.C. (1983) Expression of bacterial genes in plants. Proc Natl Acad Sci (USA) 80:4803-4807. Gahan, L.J., Ma, Y.T., Cobble, M.L.M., Gould, F., Moar, W.J., Heckel, D.G. (2005). Genetic basis of resistance to Cry 1Ac and Cry 2Aa in Heliothis virescens (Lepidoptera: Noctuidae). J. Econ. Entomol. 98: 1357-1368.

Gasser, C. S and Fraley, R.T. (1992) Transgenic crops. Sci. Am. 226: 67-70.

Gianessi, L.P and Carpenter, J.E. (1999) Agricultural Biotechnology: Insect Control Benefits. National Center for Food and Agricultural Policy, Washington DC, USA.

Godelieve, G., Angenon, G and Van, M.M. (1998) Agrobacterium-mediated plant transformation a scientifically intriguing story with significant applications, Transgenic plant research, Keith Lindsey. Harwood Academic Publishes: 1-34.

Gore, J., Leonard, B.R., Adamczyk, J.J. (2001) Bollworm (Lepidoptera: Noctuidae) survival on Bollgard® and Bollgard II® cotton flower buds (squares) and flowers. Journal of Economic Entomology. 94(6): 1446-1451.

Gore, J., Leonard, B.R., Church, C.E and Cook, D.R. (2002). Behavior of bollworm (Lepidoptera: Noctuidae) larvae on genetically engineered cotton. J. Econ. Entomol. 95:763–769.

Gould, F. (2003). Potential and problems with high dose strategies for pesticidal engineered crops. Biocontrol Sci. Technol. 4, 451-461.

Gould, F., Martinez-Ramirez, A., Anderson, A., Ferre, J., Silva, F.J. and Moar, W.J. (1992). Boroad spectrum resistance to Bacillus thuringiensis toxins in Heliothis virescens. In Proc. Nat. Acad. Sci. USA.89, 7986-7990.

124

Gould, J and Magallanes-Cedeno, M. (1998) Adaptation of Cotton Shoot Apex Culture to Agrobacterium-Mediated Transformation. Plant Mol Biol Repor 16: 1–10.

Greenplate, J. T. (1999) Quantification of Bacillus thuringiensis insect control protein Cry1Ac over time in Bollgard cotton fruit and terminals. J. Econ. Entomol. 92, 1377-1383.

Greenplate, J. T., Head, G. P., Penn, S. R and Kabuye V.T. (1998) Factors potentially influencing the survival of Helicoverpa zea on Bollgard cotton. In: P. Dugger, and D. A. Richter, eds. Proceedings, 1998 Beltwide Cotton Conference, pp. 1030-1033. National Cotton Council of America, Memphis, TN.

Greenplate, J. T., Penn, S. R., Mullins, J. W and Oppenhuizen M. (2000) Seasonal Cry1Ac levels in DP50B: the Bollgard basis for Bollgard II. In: P. Dugger, and R. Richter, eds. Proceedings, Beltwide Cotton Conference, San Antonio, TX, 4–8 Jan. 2000, pp. 1039— 1041. National Cotton Council of America, Memphis, TN.

Greenplate, J., Mullins, W., Penn, S. R and Embry, K. (2001) Cry1Ac Bollgard® varieties as influenced by environment, variety and plant age: 1999 gene equivalency field studies. In P. Dugger, and R. Richter, eds. Proceedings of the Beltwide Cotton Conference, Volume 2, pp. 790-793. National Cotton Council of America, Memphis, TN.

Greenplate, J., Mullins, W., Penn, S. R., Dahm, A., Reich, B. J., Osborn, J. A., Rahn, P. R., Ruschke, L. and Shappley, Z. W. (2003). Partial Characterization of cotton plants expressing two toxins proteins from Bacillus thuringiensis, relative toxins contribution, toxins interaction and resistance management. J. Appl. Entomol. 127 (6). 340-347.

Guo, W. Z., Sun, J., Guo, Y. F and. Zhang, T. Z. (2001) Investigation of different dosage of inserted Bt genes and their insect-resistance in transgenic Bt cotton. Acta Genet. Sin. 28: 668-676.

125

Guo, X., Huang, C., Jin, S., Liang, S., Nie, Y., Zhang, X. (2007). Agrobacterium-mediated transformation of Cry1C, Cry2A and Cry9C genes into Gossypium hirsutum and plant regeneration. Biologia plantarum. 51 (2) :242-248

Haddad, R., Morris, K. and Buchanan-Wollaston, V. (2002). Regeneration and Transformation of Oilseed (Brassica napus) using CaMV 35S Promoter- β-glucuronidase Gene. J. Agric. Sci. Technol, 4: 151-160.

Hamilton, C.M. (1997) A binary-BAC system for plant transformation with high-molecular- weight DNA. Gene 200:107-116.

Harris, J.G., Hershey, C.N., Watkins, M.J. (1998). Theusage of Karate (α-cyhalothrin) oversprays in combination with refugia, as a viable andsustainable resistance management strategy for Bt otton. Proceedings of the Beltwide Cotton Conference, Nat. Cotton Council, Memphis, TN. Vol. 2: 1217-1220.

Hazra, S., Kulkarni, A. V., Nalawade, S. M., Banerjee, A. K,. Afrawal, D. C and Krishnamurthy, K. V. (2000) Influence of explants, genotype and culture vessels on sprouting and proliferation of pre-existing meristems of cotton (Gossypium hirsutum L. and Gossypium arboreum L). In Vitro Cell Dev Biol Plant. 36:505-10.

Herrnstadt, G. Soares, R.W., Edward, L., Edwards D. (1986) A new strain of Bacillus thuringiensis with activity against coleopteran insects. Bio/Technology, 4 (4) : 305-308.

Hiei, Y., Ohta, S., Komari, T., Kumashiro, T. (1994). Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T- DNA Plant Journ. 6: 271-282.

Hobbs, S. L. A., Warkentin, T. D and Delong C. M. O. (1993) Transgene copy number can be positively or negatively associated with transgene expression. Plant Mol. Biol. 21, 17-26.

126

Hofte, H and Whitely, H.R. (1989) Insecticidal crystal protein of Bacillus thuringenesis . Microbiological reviews, June, 53: 242-255.

Holt, H. E. (1998) Season-long monitoring of transgenic cotton plants-development of an assay for the quantification of Bacillus thuringiensis insecticidal protein. In: Proceedings 9th Australian Cotton Grower Research Association, pp. 331-335. Queensland, Wee Waa, Australia.

Hooykaas, P. J. J and Shilperoort, R.A. (1992). Agrobacterium and plant genetic engineering. Plant Mol Biol 19:15-38.

Horner, T. A. and Dively, G. P. (2003). Effect of MON 810 Bt field corn on Helicoverpa zea (Lepidoptera Noctuidae) cannibalism and its implications to resistance development. J. Econ. Entomol. 96, 931-934.

Hossain, D. M., Shitomi, Y., Moriyama, K., Higuchi, M., Hayakawa, T., Mitsui, T., Sato, R., Hori, H. (2004) Characterization of a novel plasma membrane protein, expressed in the midgut epithelia of Bombyx mori, that binds to Cry1A toxins. Appl. EnViron. Microbiol. 70 (8): 4604-4612.

Hussain, S. (2002) Genetic Transformation of Cotton with Galanthus Nivalis Agglutinin (GNA) gene. PhD Thesis, The University of Punjab, Lahore, Pakistan.

Hussain, S.S., Husnain, T., Riazuddin, S. (2007). Sonication assisted Agrobacterium mediated transformation (SAAT): an alternative method for cotton transformation. Pak. J. Bot., 39(1): 223-230.

Ikram ul Haq (2004). Agrobacterium-Mediated Transformation of Cotton (Gossypium hirsutum L.) via Vacuum Infiltration. Plant Molecular Biology Reporter 22: 279-288.

127

Ives, A. R., Alstad, D.N. and Andow, D.A. (1996). Evolution of insect resistance to Bacillus thuringiensis transformed plants. Sci. 273, 1412-1413.

Ivo, N.L., Nascimento, C.P., Vieira, L.S., Campos, F.A.P and Aragão, F.J.L (2008) Biolistic- mediated enetic transformation of cowpea ( Vigna unguiculata ) and stable Mendelian inheritance of transgenes. Plant Cell Reports. 27(9). 1475-1483.

Jaakola, L., Pirttila, A. M., Halonen, M and Hohtola, A. (2001) Isolation of high quality RNA from Bilberry (Vaccinium myrtillus L.) fruit. Mol Biol 19: 201-203.

James, C (2002) Global Review of Commercialized Transgenic Crops: 2001 Feature: Bt Cotton. ISAAA Briefs No. 26. ISAAA, Ithaca, NY.

James, C (2008): Executive Summary of Global Status of Commercialized Biotech/GM Crops: 2008. ISAAA Briefs No. 34. ISAAA, Ithaca, NY.

Jang, I.C., Lee, K.H., Nahm, B.H., Kim, J.K. (2002) Chloroplast targeting signal of a rice rbcS gene enhances transgene expression. Mol Breed 9:81-91.

Jayaraj, S (1982) Proc. Workshop on Heliothis Management, 15-20 November.

Jackson, R. E., Bradley, J.R., Van Duyn, J.W. (2003). Field performance of transgenic cottons expressing one or two Bacillus thuringiensis endotoxins against bollworm, Helicoverpa zea (Boddie). J. Cotton Science 7: 57-64.

Jouanin, L., Bonade-Bottino, M., Girard, C., Morrot, G and Giband, M. (1998) Transgenic plants for insect resistance. Plant Sci., 131: 1–11.

Katageri, I.S., Vamadevaiah, H.M., Udikeri1, S.S., Khadi, B.M and Kumar, P.A. (2007) Genetic transformation of an elite Indian genotype of cotton (Gossypium hirsutum L.) for insect resistance. Current Science 93(12): 1843-1847.

128

Kim, E.H., Suh, S.C., Park, B.S., Shin, K.S., Kweon, S.J., Han, E.J., Park, S.H., Kim, Y.S and Kim, J.K. (2009). Chloroplast-targeted expression of synthetic Cry1Ac in transgenic rice as an alternative strategy for increased pest protection. Planta; 230:397–405.

Klee, H (2000) A guide to Agrobacterium binary Ti vectors. Trends in Plant Science 5: 446-451.

Knight, P.J., Crickmore, N., Ellar, D.J. (1994) The receptor for Bacillus thuringiensis Cry1A(c) ä- endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N. Mol. Microbiol. 11: 429-436.

Komari, T., Hiei, Y., Saito, Y., Murai, N., Kumashiro, T. (1996). Vector caring two separate T- DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selective markers. Plant J. 10: 165-174.

Kranthi, K. R., Naidu, S., Dhawad, C. S., Tatwawadi, A., Mate, K ., Patil, E., Bharose, A. A., Behere, G. T., Wadaskar, R. M and Kranthi, S. (2005) Temporal and intraplant variability of Cry1Ac expression in Bt-cotton and its influence on the survival of the cotton bollworm, Helicoverpa armigera. Curr. Sci. 89: 291-298.

Krieg, A., Huger, A.M., Langenbruch, G.A and Schnetter, W. (1983) Bacillus thuringiensis var tenebrionis: a new pathotype effective against larvae of coleopteran. Journal of Applied Entomology, 96: 500-508.

Kumar, P.A., Sharma, R.P., Malik, V. (1996). Insecticidal proteins of Bacillus thuringiensis. Adv Appl Microbiol 42: 1–43.

Kung, S.D (1976) Tobacco fraction I protein: A unique genetic marker. Science 191:429.434.

Kunik, T., Tzfira, T., Kapulnik, Y., Gafni, Y., Dingwall, C and Citovsky. V. (2001) Genetic transformation of HeLa cells by Agrobacterium. Proc Natl Acad Sci USA 98:1871–1876.

129

Laemmli, UK. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685.

Lee, M., Yeun, L and Abdullah, R. (2006). Expression of Bacillus thuringiensis insecticidal protein gene in transgenic oil palm. Electronic Journal of Biotechnology .9 (2): 117-126.

Leelavathi, S., Sunnichan, S.G., Kumria, R., Vijaykanth, G.P., Bhatnagar, R.K., Reddy, V.S. (2004) A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.): Embryogenic calli as a source to generate large numbers of transgenic plants. Plant Cell Rep 22: 465-470.

Levee, V., Garin, E., Klimaszewska, K and Seguin, A. (1999) Stable genetic transformation of white pine (Pinus strobus L.) after cocultivation of embryogenic tisues with Agrobacterium tumefaciens. Mol Breeding 5:429–440.

Li, F., Wu, S., Chen, T., Zhang, J., Wang, H., Guo, W and Zhang, T. (2009). Agrobacterium- mediated co-transformation of multiple genes in upland cotton. Plant Cell, Tissue and Organ Culture. 97(3): 225-235

Li, X., Wang, X.D, Zhao. X and Dutt, Y. (2004) Improvement of cotton fiber quality by transforming the acsA and acsB genes into Gossypium hirsutum L. by means of vacuum infiltration. Plant cell reports 22(9): 691-697.

Liu, Q.Q., Yu, H.X., Zhang, W.J., Wang, H., Gu, M.H. (2005) Journal of plant physiology and molecular biology (Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao); 31 (3): 247-53.

Loopstra, C. A., Stomp, A. M and Sederoff, R. R. (1990) Agrobacteriummediated DNA transfer in sugar pine. Plant Mol Biol 15:1–9.

130

Luo, K. E., Sangadala, S., Masson, L., Mazz, A., Brousseau, R., Adang, M. J. (1997) The Heliothis Virescens 170 kDa aminopeptidase function as “receptor A” by mediating specific Bacillus thuringiensis Cry1A ä-endotoxin binding and pore formation. Insect Biochem. 27: 735-743.

Lurie, S., Handros, A., Fallik, E., and Shapira, R. (1996) Reversible inhibition of tomato fruit gene expression at high temperature. Plant Physiol. 110, 1207-1214.

Lycett, G.W and Grierson, D. (1990) Genetic engineering of crop plants. Butterworth Heinemann, London, UK, 303 p. ISBN 0-40-804779-8.

Mahon, R., Finnergan, J., Olsen, K and Lawrence, L. (2002) Environmental stress and the efficacy of Bt cotton. Aust. Cotton grower 22: 18-21.

Mahon, R., Finnergan, J., Olsen, K and Lawrence, L. (2002) Environmental stress and the efficacy of Bt cotton. Aust. Cotton grower 22:18-21.

Mallet, J and Porter, P. (1992). Preventing insect adaptation to insect resistance crops, are seed mixtures or refugia the best strategy? In Proc. R. Soc. Lond. Ser. B. 250, 165-169.

Manjunatha, R., Pradeep, S., Sridhar, S., Manjunatha, M., Naik, M. I., Shivanna, B.K., Hosamani, V. (2009). Quantification of Cry1Ac protein over time in different tissues of Bt cotton hybrids. Karnataka J. Agric. Sci., 22(3-Spl. Issue): 609-610.

Manzara, T., Carrasco, P and Gruissem W. (1993) Developmental and organ-specific changes in DNAprotein interactions in the tomato rbcS1, rbcS2, and rbcS3 promoter regions. Plant Mol. Biol. 21:69-88.

Manzara, T., Carrasco, P and Gruissem, W. (1991) Developmental and organ-specific changes in promoter DNA-protein interactions in tomato rbcS gene family. Plant Cell 3:1305-1316.

131

Maqbool, A (2009). Search for drought tolerant genes through differential display. PhD Thesis, The University of Punjab, Lahore, Pakistan.

Maqbool, S. B., Riazuddin, S., Loc, T.N., Gatehouse, J. A. and Chritou, P. (2001). Expression of multiple insecticidal genes confers broad resistance against a range of different insect pests. Mol. Breed.7, 85-93.

Martin, P.A and Travers, R.S. (1989) Worldwide abundance and distribution of Bacillus thuringiensis isolates. App. Environ. Microbiol. 55:2437-2442.

Matsuoka, M., Kyozuka, J., Shimamoto, K., Kano-Murkami, Y. (1994) The promoter of two carboxylases in a C4 plant (maize) direct cell-speciÞc, light regulated expression in a C 3 plant (rice). Plant J 6: 331-319.

McAfee, B. J., White, E. E., Pelcher, L. E and Lapp, M. S. (1993) Root induction in pine (Pinus) and larch (Larix) spp. using Agrobacterium rhizogenes. Plant Cell Tissue Organ Cult 34:53–62.

McGaughey, W. H. and Whalon, M. E. (1992). Managing insect resistance to Bacillus thuringiensis toxins. Sci. 258, 1451-1455.

McGaughey, W.H. and Whalon, M.E. (1992). Managing insect resistance to Bacillus thuringiensis toxins. Sci.258, 1451-1455.

Meier, I., Kristie, L., Fleming, J.A and Gruissem, W. (1995). Organ-specific differential regulation of a promoter subfamily for the ribulose-1, 5-bisphosphate carboxylase/oxygenase small subunit genes in tomato. Plant Physiol. 107:1105-1118.

Meyer, P., Linn F., Heidmann I., Meyer, Z. A. H., Neiedenhof, I and Sedler, H (1992) Endogenous and environmental factors influence 35 s promotor methylation of maize A1 gene construct in transgenic plant petunia and its color phenotype. Mol. Gen. Genet. 231, 345-352.

132

Micallef, M.C., Austin, S and Bingham, E.T. (1995) Improvement of transgenic alfalfa by backcrossing. In Vitro Cell Dev Biol Plant 31:187-192.

Molar, W.J., Pusztai-Carey, M., Van-Faassen, H., Bosch, D., Frutos, R., Rang, C., Luo, K. and Adang, M.J. (1995). Development of Bacillus thuringiensis cry1c resistance by Spodoptera exigua (Hubner) ( Lepidoptera Noctuidae). Appl. Environ. Microbiol. 61, 2086-2092.

National Biosafety Committee (NBC) Biosafety guidelines in genetic engineering and biotechnology. Ministry of Environment Local Government, Rural Development, Government of Pakistan, 1999.

Nauerby, B., Billing, K., Wyndaele, R. (1997). Influence of the antibiotic timentin on plant regeneration compared to carbernicillin and cefotaxime in concentration suitable for elimination of Agrobacterium tumefaciens. Plant Science 123: 169-177.

Nilsson, O., Aldén, T., Sitbon F., Anthony, C.H., Chalupa, V., Sandbergand, G and Olsson, O. (1992). Spatial pattern of cauliflower mosaic virus 35S promoter-luciferase expression in transgenic hybrid aspen trees monitored by enzymatic assay and non-destructive imaging. Transgenic Research .1(5): 209-220.

Noreen, S. (2008). Detection of Bt genes in Transgenic Cotton. MPhil Thesis, The University of Punjab, Lahore, Pakistan.

Odell, J.T., Nagy, F., Chua , N.H. (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313: 810–812.

Olsen, K. M and Daly, J. C. (2000) Plant-toxin interactions in transgenic Bt cotton and their effects on mortality of Helicoverpa armigera. Entomol. Soc. Am. 93, 1293-1299.

133

Olsen, K. M., Daly, J. C., Holt, H. E and Finnegan, E. J. (2005) Season-long variation in expression of Cry1Ac gene and efficacy of Bacillus thuringiensis toxin in transgenic cotton against Helicoverpa armigera (Lepidoptera: Noctuidae). J. Econ. Entomol. 98, 1007-1017.

Oosterhuis, D. M and Brown, R. S. (2003) Increased plant protein, insect mortality and yield with Chaperone TM. Ark. Agri. Exp. Sta. Res. Ser. 521, 101-107.

Oosterhuis, D. M., and Brown R. S. (2004) Effect of foliar Chaperone TM applications on endotoxin and protein concentration, insect mortality and yield response of cotton. Ark. Agri. Exp. Sta. Res. Ser. 533, 51-56.

Otten, L., DeGreve, H., Leemans, J., Hain, R., Hooykaas, P. and Schell, J. (1984) Restoration of virulence of vir region mutants of Agrobacterium tumefaciens strain B6S3 by coinfection with normal and mutant Agrobacterium strains. Mol Gen Genet 195:159–163.

Panguluri, S.K., Sridhar, J., Jagadish, B., Sharma, P.C and Kumar, P.A. (2005) Isolation and characterization of a green tissue-specific promoter from pigeonpea (Cajanus cajan L. Millsp.) Indian Journal of Experimental Biology. 43: 369-372.

Pauk, J., Stefanov, I., Fekete, S., Bögre, L., Karsai, I., Fehér, A. and Dudits, D. (1995). A study of different (CaMV 35S and mas ) promoter activities and risk assessment of field use in transgenic rapeseed plants. Euphytica. 85(3): 411-416.

Perlak, F.J., Deaton, R.W., Armstrong, T.A., Fuchs, R.L., Sims, S.R., Greenplate, J.T and Fischhoff, D.A. (1990). Insect resistant cotton plants. Biotechnol, 8: 939-943.

Perlak, F.J,, Fuchs, R.L., Dean, D.A., McPherson, S.L., Fischhoff, D.A. (1991). Modification of the coding sequence enhances plant expression of insect control protein genes. Proc Natl Acad Sci U S A. 88(8):3324–3328.

134

Perlak, F. J., Oppenhuizen, M., Gustafson, K., Voth, R., Sivasupramaniam, S., Heering, D., Carey, B., Ihrig, R. A. and Roberts, J. K. (2001). Development and commercial use of BollgardR cotton in USA-Early promises versus todays reality. The Plant J. 27, 489-501.

Pettigrew, W. T and Adamczyk J. J. (2006) Nitrogen fertility and planting date effects on lint yield and Cry1 ac (bt) endotoxin production. Agron. J. 98, 691-697.

Piers, K. L., Heath, J. D., Liang, X., Stephens, K. M and Nester, E. W (1996) Agrobacterium tumefaciens-mediated transformation of yeast. Proc Natl Acad Sci USA 93:1613–1618.

Porter, J. R. (1991) Host range and implications of plant infection by Agrobacterium rhizogenes. Crit Rev Plant Sci 10:387–421.

Potenza, C., Aleman, L., Sengupta-Gopalan, C. (2003) Targeting transgene expression in research, agricultural, and environmental applications: promoters used in plant transformation. In Vitro Cell Devel Biol—Plant 40: 1–22. Publ. Co., New York.

Qayyum, A. (2009) Expression of PhytoB gene in Cotton . PhD Thesis, The University of Punjab, Lahore, Pakistan.

Rao, A.Q., Bakhsh, A., Kiani, S., Shahzad, K., Shahid, A.A., Husnain, T and Riazuddin, S. (2009) The Myth of Plant Transformation. Biotechnology advances; 27(6):56-62.

Rao, C.K. (2005) Transgenic Bt Technology: 3. Expression of Transgenes. http://www.monsanto. co.uk/news/ukshowlib.phtml?uid¼9304.

Rashid, B., Zafar, S., Husnain T and Riazuddin, S. (2008). Transformation and inheritance of Bt genes in Gossypium hirsutum. Journal of Plant Biology, 51(4):248-254..

Ribas, A. F., Pereira, P. F. L and Vieira, L. G. E. (2006) Genetic transformation of coffee. Braz J Plant Physiol 18(1):83-94.

135

Roush, R. T., (1994). Managing pests and their resistance to Bacillus thuringiensis, can manage crops better than sprays? Biocontrol Sci. Technol. 4, 501-516.

Rui, Y., Zhu, B and Luo Y. (2005) Long distance transportation of Bt-toxin through xylem sap in Bt cotton (Gossyposium). Chinese Bull. Bot. 22, 320-324.

Sachs, S.E., Benedict, H.J., Taylor, F.J., Stelly, M.D., Altman, W.D., Berberich, A.S., Davis , K.S. (1998) Expression and segregation of genes encoding Cry1A insecticidal proteins in cotton. Crop Science 38:1-11.

Saha, S., Callahan, F. E., Douglas, A. D and Creech, J. B., (1997) Localization of cotton tissue on quality of extractable DNA, RNA and protein. J.Cotton Sci. 1:10–40.

Saiki, R., Chang, C.A., Levenson, C.H., Warren,T.C., Boehm, C.D., Kazazian, H.H.J and Erlich H.A. (1988) Diagnosis of sickle cell anemia and beta-thalassemia with enzymatically amplified DNA and nonradioactive allele-specific oligonucleotide probes.New Engl. J. Med. 319: 537−541.

Salm, T., Bosch, D., Hone, G., Feng, L., Munstreman, E., Bakker, P., Stiekems, W.J. and Visser, B. (1994). Insect resistance of transgenic plants that express modified Bacillus thuringiensis cry1Ab and cry1C genes. A resistance management strategy. Plant Mol. Biol.26, 51-59.

Schnepf, E (1995) Bacillus thuringiensis toxins: regulation, activities and structural diversity. Curr. Opin. Biotechnol, 6:305-312.

Schnepf, E., Crickmore, N., Lereclus, D., Baum, J., Feitelson, D., Zeigler, R and Dean, D.H. (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and Molecular Biology Reviews. 62(3): 775-806.

136

Selvapandiyan, A., Reddy, V.S., Kumar, P.A., Tewari, K.K., Bhatnagar, R.K. (1998) Transformation of Nicotiana tabacum with a native cry1Ia5 gene confers complete protection against Heliothis armigera. Mol Breed 4:473-478.

Shelton, A.M., Tang, J.D., Roush, R.T., Metz, T.D., Earle, E.D. (2000). Field tests on managing resistance to Bt-engineered plants. Nat. Biotechnol. 18:339-342.

Shimizu, M., Kimura, T., Koyama, T. Suzuki, T., Ogawa, N., Miyashita, K., Sakka, K and Ohmiya, K. (2002) pCambia vector Molecular Breeding of Transgenic Rice Plants Expressing a Bacterial Chlorocatechol Dioxygenase Gene. Appl Environ Microbiol. 68(8): 4061–4066.

Shirsat, A.H., Wilford, N., Croy, R.R.D. (2003). Gene copy number and levels of expression in transgenic plants of a seed specific gene. Plant Science. 61(1): 75-80.

Smith, E.F and Towsend, C.O. (1907). A plant tumor of bacterial origin. Science 25:671-673.

Somers, D.A., Torber, K.A., Pawlowski, W.P and Rines, H.W. (1994) Genetic engineering of oat. In: enry RJ, Ronald JA (ed) improvement of cereal quality by genetic engineering. Plenum Press, New York, pp 37-46.

Song, P., Heinen, J.L., Burns, T.H and Allen, R.D. (2000). Expression of Two Tissue-Specific Promoters in Transgenic Cotton Plants. The Journal of Cotton Science 4:217-223.

Sousa-Majer, M. J., Turner N.C., Hardie, D. C., Morton R. L., Lamont, B and Higgins T. J. V. (2004) Response to water deficit and high temperature of transgenic peas (Pisum sativum L.) containing a seedspecific r-amylase inhibitor and the subsequent effects on pea weevil (Bruchus pisorum L.). survival. J. Exp. Bot. 55, 497-505.

Southern, E.M. (1975) Detection of Specific sequencee among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517.

137

Spencer, T.M., O’Brien, J.V., Start ,W,G and Adams, T.R. (1992) Segregation of transgene in maize. Plant Mol Biol 18:201–210.

Stewart, J.M (1991). Biotechnology of cotton. ICAC Review article on cotton production C.A.B international 8-25.

Sunilkumar, G., Mohr, L., Lopata-Finch, E., Emani, C and Rathore, K.S. (2002) Developmental and tissue-specific expression of CaMV 35S promoter in cotton as revealed by GFP. Plant Mol Biol 50: 463474.

Suzuki, Y., Ohkubo, M., Hatakeyama, H., Ohashi, K., Yoshizawa, R ., Kojima, S., Hayakawa, T., Yamaya, T., Mae, T., Makino, A. (2007) Increased Rubisco content in transgenic rice transformed with the ‘sense’ rbcS gene. Plant Cell Physiol. 48: 626 -637.

Suzuki, Y., Kaori. N., Ryuichi, Y., Tadahiko, M and Amane, M. (2009) Differences in Expression of the RBCS Multigene Family and Rubisco Protein Content in Various Rice Plant Tissues at Different Growth Stages.Plant and Cell Physiology 2009 50(10):1851- 1855;doi:10.1093/pcp/pcp120.

Tabashnik, B.E. (1994). Evolution of resistance to Bacillus thuringiensis. Annual Review of Entomology. 39:47–79.

Tabashnik, B.E., Dennehy, T.J., Sims, M.A., Larkin, K., Head, G.P., Moar, W.J and Carriere, Y. (2002) Control of resistant pink bollworm that produces Bacillus thuringiensis toxin Cry2Ab. Appl. Environ. Microbiol. 68:3790-3794.

Tang, W., Chen, H., Xu, C.G., Li, X.H., Lin, Y.J and Zhang, Q.F. (2006) Development of insect- .gene. Mol Breed 18:1–10 כresistant transgenic indica rice with a synthetic Cry1C

Tisdale, S. L and Nelson ,W. L. (1975) Soil Fertility and Fertilizers, 3rd edn. Macmillan.

138

Tohidfar, M., Ghareyazie, B., Mosavi, M., Yazdani, S., Golabchian, R. (2008). Agrobacterium- mediated transformation of cotton (Gossypium hirsutum) using a synthetic cry1Ab gene for enhanced resistance against Heliothis armigera. Iranian Journal of Biotechnology 6(3):164- 173.

Torisky, R.S., Kovacs, L., Avdiushko, S., Newman, J.D., Hunt, A.G and Collins, G.B. (1997) Development of a binary vector system for plant transformation based on supervirulent Agrobacterium tumefaciens strain Chry5. Plant Cell Reports 17:102-108. Towbin, H., Staehelin, T and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gel to nitrocellulose sheets: Procedures and some applications. Proc. Nalt. Acad. Sci. USA. 76: 4350-4354.

Traore, S. B., Carlson, R. E., Pilcher, C. D and Rice, M. E. (2000) Bt and non-Bt maize growth and development as affected by temperature and drought stress. Agron. J. 92:1027-1035. Trick, H.N and Finer, J.J (1997) Sonication-assisted Agrobacterium mediated transformation. Transgenic Research, 6: 329-336.

Trolinder, N.L and Xhixian, C. (1989) Genotype specificity of the somatic embryogenesis response in cotton. Plant Cell Reports. 8: 133-136.

Tu, J., Zhang, G., Datta, K., Xu, C., He, Y., Zhang, Q. (2004) Field performance of transgenic elite commercial hybrid rice expressing Bacillus thuringiensis δ-endotoxin. Nat Biotechnol 18:1101–1104.

Umbeck, P., Johnson, G., Barton, K and swain, W (1987) Genetically transformed cotton (Gossypium hirsutum L) plants. Bio technol; 5:263-266.

Wan, P., Zhang, Y ., Wu, K and Huang, M. (2005) Seasonal expression profiles of insecticidal protein and control efficacy against Helicoverpa armigera for Bt cotton in the Yangtze River valley of China. J. Econ. Entomol. 98, 195-201.

139

Wan, Y., Lemaux, P.G. (1994) Generation of large numbers of independently transformed fertile barley plants. Plant Physiol 104: 37–48.

Wenck, A. R., Quinn, M., Whetten, R. W., Pullman, G and Sederoff, R. (1999) High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda). Plant Mol. Biol 39:407–416.

Wessel, V.l., Tom, R., Antoinette, B., Linus, V.P and Alexander, V.K. (2001) Characterization of position-induced spatial and temporal regulation of transgene promoter activity in plants. Journal of Experimental Botany, 52: 949-959.

Whalon, M,E,, Miller, D,L., Hollingworth, R.M., Grafius, E.J. and Miller, J.R. (1993). Selection of a Colorado potato beetle (Coleoptera: Chrysomelidae) strain resistant to Bacillus thuringiensis. Journal of Economic Entomology 86: 226-233.

Wong, E. Y., Hironaka, C.M and Fischhoff, D.A. (1992) Arabidopsis thaliana small subunit leader and transit peptide enhance the expression of Bacillus thuringiensis proteins in transgenic plants Plant Molecular Biology 20(1): 81-93.

Wu G ; Cui H ; Ye G ; Xia Y ; Sardana R ; Cheng X ; Li y ; Altosaar I ; Shu Q (2002). Inheritance and expression of the cry1Ab gene in Bt (Bacillus thuringiensis) transgenic rice. Theoretical and Applied Genetics, 104(4): 727-734.

Wu, C., Fan, Y., Zhang, C., Oliva, N and Datta, S.K. (1997) Transgenic fertile japonica rice plants expressing a modified cry1A(b) gene resistant to yellow stemborer. Plant Cell Rep 17:129– 132.

Wu, J. H., Zhang, X. L., Luo, X. L., and Tian, Y. C. (2003). Inheritance and segregation of transformants in cotton with two types of insect resistance genes. Article in Chinese, Yi Chaun Xue Bao, 30, 631-636.

140

Wu, J., Zhang, X., Nie, Y and Luo, X. (2005) Agrobacterium tumefaciens and regeneration of insect-resistant plants. Plant Breeding 124(2):142–146.

Wu, K., Guo, Y., Greenplate, J. T and Deaton, R. (2003) Efficacy of transgenic cotton containing Cry1Ac gene from Bacillus thuringiensis against Helicoverpa armigera in northern China. J. Econ. Entomol. 96: 1322-1328.

Wu, Y., Chen, Y., Liang, X and Wang, X. (2006) An experiment assessment of the factors influencing agrobacterium mediated transformation in tomato. Russ J Plant Physol 53 (2):252-6.

Xia, L., Xu, Q and Guo, S. (2005) Bt insecticidal gene and its temporal expression in transgenic cotton plants. Acta Agron. Sin. 31: 197-202.

Xia, L.H., He, Y.R., Chun, L.U., Yang, Z.Y and Hong, Z.J. (2007) Inheritance and resistance to insects in CryIA(c) transgenic cabbage. Chinese Journal of Agricultural Biotechnology 4: 57-61.

Xie, R., Zhuang, M., Ross, L. S., Gomez, I., Oltean, D. I., Bravo, A., Soberon, M., Gill, S. S. (2005). Single amino acid mutations in the cadherin receptor from Heliothis Virescens affect its toxin binding ability to Cry1A toxins. J. Biol. Chem. 280 (9): 8416- 8425.

Yang, N.S and Christou, P. (2005) Cell type specific expression of a CaMV 35S-GUS gene in transgenic soybean plants. Developmental Genetics 11: 289-293.

Yang, Q., Yang, L., Liu, P., Li, S and Song, J. (2007) Agrobacterium tumefaciens-mediated transformation of Trichoderma harzianum. KMITL Sci. Tech.7 (2):185-191.

Ye, R., Huang, H., Yang, Z., Chen, T., Liu, L., Li, X., Chen, H., Lin, Y. (2009) Development of insect-resistant transgenic rice with Cry1C-free endosperm. Pest Manag Sci. 65(9):1015-20.

141

Yibrah, H. S., Gronroos, R., Lindroth, A., Franzen, H., Clapham, D and von Arnold, S. (1996) Agrobacterium rhizogenes-mediated induction of adventitious rooting from Pinus contorta hypocotyls and the effect of 5-azacytidine on transgene activity. Transgenic Res. 5:75–85.

Yin, Y and Beachy, R. (1995) The regulatory regions of the rice tungro bacilli form virus promoter and interacting nuclear factor in rice (Oryza sativa ¸.). Plant J 7 : 969-980.

Zaenen, I., Larebeke, V. N., Teuchy, H., Van, M. M and Schell, J. (1974) Supercoiled circular DNA in crown gall inducing Agrobacterium strains. J Mol Biol 86: 109-127.

Zambryski, P., Joos, H., Genetello, C., Leemans, J., Van, M. M and Schell, J. (1983) Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO J. 2: 2143-2150.

Zapata, C., Park, S.H., El-Zik, K.M and Smith, R.M (1999) Transformation of a Texas cotton cultivar by using Agrobacterium and the shoot apex. Theor Appl Genet 98:252–256.

Zhang B. H., Guo, T.L and Wang Q. L. (2000) Inheritance and segregation of exogenous genes in transgenic cotton. J. Genet. 79, 71-75.

Zhang, L. G., Cheng, L. M., Xu, N., Zhao, N. M., Li, C. G., Jing, Y and Jia, S. R. (2001) Efficient transformation of tobacco by ultasonication. Bio/Tech. 9:273-277.

Zhang, Lei., Wu, D., Zhang, Li and Yang, C. (2007) Agrobacterium-mediated transformation of Japanese lawngrass (Zoysia japonica Steud.) containing a synthetic cryIA(b) gene from Bacillus thuringiensis. Plant Breeding 126(4): 428-432.

Zhao, C.Y., Yuan, Z.Q., Qin, H.M and Tian, Y.C. (2001) Studies on transgenic tobacco plants expressing two kinds of insect resistant genes. Sheng Wu Gong Cheng Xue Bao. 17(3):273- 7.

142

Zhao, F.Y., Li, Y.F and Xu, P. (2006) Agrobacterium-mediated transformation of cotton (Gossypium hirsutum L. cv. Zhongmian 35) using glyphosate as a selectable marker. Biotechnol Lett. 28(15):1199-207. Zhao, J. Z., Cao, J., Li, Y., Collins, H. L., Roush, R. T., Earle, E.D. and Shelton, A.M. (2003). Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution. Nat. Biotechnol. 21, 1493-1497.

Zhao, T., Ni, Z., Dai, Y., Yao, Y., Nie, X and Sun, O. (2006) Characterization and expression of 42 MADS-box genes in wheat (Triticum aestivum L.) Mol Genet Genomics 276(4):334- 350.

Zheng, S.J., Henken, B., Maagd, R.A., Purwito, A., Krens, F.A and Kik, C. (2005). Two different Bacillus thuringiensis toxin genes confer resistance to beet armyworm (Spodoptera exigua H¨ ubner) in transgenic Bt-shallots (Allium cepa L.). Transgen Res 14:261–272.

143

7. PUBLICATIONS

Following are the publications out of this research work.

1. Allah Bakhsh, Abdul Qayyum Rao, Ahmad Ali Shahid, Tayyab Husnain and S. Riazuddin (2009) “35S CaMV as developmental promoter in being temporal and spatial expression of Cry1Ac and Cry2A genes in Cotton”. Research Journal of Cell and Molecular Biology:3(1): 52-62. 2. Allah Bakhsh, Abdul Qayyum Rao, Ahmad Ali Shahid, Tayyab Husnain and S. Riazuddin (2009) “Insect resistance and Biosafety Studies in advance lines of Bt Cotton”. American Eurasian Journal of Agriculture and Environmental Sciences: 6(1): 01-11. 3. Abdul Qayyum, Allah Bakhsh, Kamran Shahzad, Sarfraz Kiani, Ahmad Ali Shahid Tayyan Husnain and S. Riazuddin (2009). The Myth of Plant Transformation. Biotechnology Advances; 27 (6): 753-763.

144

8.APPENDICES

APPENDIX-I

DNA Extraction Buffer

0.5M glucose.

0.2M Tris-Hcl

5mM Ethylenediaminetetraacetic acid (EDTA)

2% Polyvinylpyrrolidone (PVP)

0.2% Mercaptoethanol

DNA Lysis Buffer

0.2% Mercaptoethanol

1.4 M NaCl

25mM Ethylenediaminetetraacetic acid (EDTA)

2% Hexadecyltrimethylammonium bromide (CTAB)

2% Polyvinylpyrrolidone (PVP)

5x TBE Buffer

Trizma base 54g

Boric acid 27.5g

0.5M EDTA 20mL

Adjust volume to 1 liter with distilled water.

6x DNA Loading Dye

Ficoll 20 % EDTA 0.1M 145

SDS 1 % Bromophenol blue 0.25 % Xylene Cyanol 0.25 %

20x SSC

NaCl 350.6g

Na-Citrate 176.4g

Adjust pH to 7.0 with NaOH and make up volume to 2 liter with distilled water

Pre-Hybridization Buffer

SSC 5x

Blocking reagent 1%

Sarkosyl 0.1%

SDS 0.02%

Dissolve blocking reagent at 65-80ºC with constant shaking and store at –20ºC.

Hybridization Buffer

Add 5-20ng of probe per mL of pre-hybridization buffer. Store at –20ºC.

APPENDIX-II

Protein Extraction Buffer (5mL)

Glycerol 0.5mL

0.5M EDTA 0.4mL (pH 7.5)

5M NaCl 0.15mL

1M TrisCl 0.05mL (pH 7.5)

NH4Cl 26.7mg

DTT 15mg

PMSF 2mM

146

Carbonate Buffer

Na2CO3 0.04M

NaHCO3 0.06M

Adjust pH of Na2CO3 to 9.6 with NaHCO3 and store at 4ºC.

Phosphate Buffered Saline (PBS)

NaCl 8 g

KCl 0.2 g

KH2PO4 0.24 g

Na2HPO4 1.44 g

Dissolve in 1 liter of distilled water; adjust pH to 7.4 and autoclaved.

Phosphate Buffered Saline-Tween 20 (PBST)

Tween-20 500 μL (0.05 %)

Dissolved in 1 liter of 1x PBS.

12% separating gel

Water 2.8mL

Acrylamide (30%) 3.2mL

4xTris-SDS 2.5mL (pH8.8)

APS (10 %) 26.7μL

TEMED 5.3μL

4% stacking gel

Water 2.5mL

Acrylamide (30%) 0.533mL

147

4xTris-SDS 1mL (pH6.8)

APS (10 %) 31.5μL

TEMED 6.3μL

10% APS (ammonium per sulphate)

APS 0.1g

Water 1mL

4x TRIS-SDS Buffer pH 8.8.

Trizma base 18.2g

SDS 0.4g

Adjust pH to 8.8 with concentrated HCl, make up volume to 100mL, filter sterilize and store at 4ºC.

4x TRIS-SDS Buffer pH 6.8

Trizma base 6.05g

SDS 0.4g

Adjust pH to 6.8 with concentrated HCl, make up volume to 100mL, filter sterilize and store at 4ºC.

10x Running Buffer

Trizma base 30.05g

Glycine 142.5g

SDS 10g

Dissolve in 1 liter of distilled water

148

Transfer Buffer

Trizma base 3.032g

Glycine 14.416g

Methanol 200mL

Adjust volume upto 1 liter.

Coomassie Stain

Coomassie blue 2.5g

Methanol 455mL

Glacial acetic acid 91mL

Adjust volume to 1 liter with distilled water.

Coomassie Destain

Methanol 250mL

Glacial acetic acid 70mL

Adjust volume to 1 liter with distilled water.

2x Sample Buffer

4x TRIS-SDS Buffer 2.5mL (pH 6.8)

Glycerol 2mL

SDS 0.4g

β-mercaptoethanol 200uL

Bromophenol blue 200uL

Adjust volume upto 10 mL with distilled water.

149

Blocking Solution

Skim milk 1.25g

1x PBS 25mL

APPENDIX-III

RNA extraction buffer

2% Hexadecyltrimethylammonium bromide (CTAB) 2% Polyvinylpyrrolidone (PVP) 100 mM Tris HCl pH:8 25mM EDTA Spermidine .5g/L 2% Mercaptoethanol 2M NaCl SSTE buffer

1M NaCl

0.5% SDS

10mM Tris HCl pH:8

1mM EDTA pH:8

APPENDIX-IV

LB Broth (1L)

Yeast extracts 5g

Bactotrypton 10g

NaCl 10g

Dissolved in 1 liter of distilled water, adjusted pH 7.5 and autoclaved.

LB AGAR

LB containing 15 g / liter of Bacto Agar

150

APPENDIX-V

S.O.B. MEDIUM Tryptone 20 g Yeast Extract 5 g NaCl 0.584 g KCl 0.186 g Dissolved in 1 liter of distilled water, adjusted pH: 7.0 and autoclaved.

S.O.C. MEDIUM 2M Mg++ STOCK

MgCl2. 6H2O 20.33 g

MgSO4.7H2O 24.65 g Dissolved in 100mL of distilled autoclaved water.

APPENDIX-VI

Solution-1

25mM Tris HCl 10Mm EDTA 50mM Glucose Solution-2:

0.2M NaOH 1% SDS Solution 3 3M Potassium Acetate (pH 4.8)

APPENDIX-VII

YEP Broth

Yeast extract 10g/L

Bactopeptone 5g/L

NaCl 10g/L

151

pH 7.5

YEP Agar

YEP containing 15 g / liter of Bacto Agar

APPENDIX-VIII

MS Medium (Murashige and Skoog, 1962) Composition

MS Salts 4.33g/L

MS Vitamins 1mL/L

Sucrose 30g/L

Gelrite 3g/L

pH 5.6-5.8

MS Vitamins 1000X

Nicotinic acid 50mg/100mL

Thiamine hydrochloride 50mg/100mL

Pyridoxine hydrochloride 50mg/100mL

Myo-inositole 100mg/L (of medium)

152