IN THE NAME OF ALLAH, THE COMPASSIONATE, THE MERCIFUL

INHERITANCE OF TRANSGENE(S) IN COTTON (GOSSYPIUM HIRSUTUM L.)

A THESIS SUBMITTED TO THE UNIVERSITY OF THE PUNJAB IN COMPLETE FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN MOLECULAR BIOLOGY

BY

GHAZANFAR ALI KHAN

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

(2007)

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 our direct supervision. We have personally gone through all the data/results/materials reported in the manuscript and certify their correctness/authenticity. We 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. We also certify that the thesis has been prepared under our supervision according to the prescribed format and we endorse its evaluation for the award of Ph.D. degree through the official procedures of the university.

(DR. S. RIAZUDDIN) Co-Supervisor

(DR. TAYYAB HUSNAIN) Supervisor

ii

I DEDICATE THIS HUMBLE EFFORT, THE FRUIT OF MY THOUGHTS AND STUDY TO MY AFFECTIONATE MOTHER AND FATHER, WHO INSPIRED ME TO HIGHER IDEALS OF LIFE AND HEREAFTER.

iii

ACKNOWLEDGEMENTS All praises are for The Almighty Allah and The Holy Prophet Muhammad (peace be upon him). I acknowledge with a deep sense of gratitude the help that I have received from Dr. S. Riazuddin (H.I., S.I., T.I.), National Professor and Director, National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore. To him, I am greatly indebted for technical skilful supervision, much valuable advice and for a great many suggestions. It was the highest honour for me to work with the great man whose wisdom and services have been recognized nationally and internationally. All necessary facilities of scientific provisions in the laboratories and field, provided to me by Dr. S. Riazuddin, were unprecedented. The huge amounts of money required by me for travelling, insect collection and all necessary purchases were always available. It was due to his personal interest in my work and unshakable trust on me that I always thought myself to be the luckiest person in the world. I am really unable to encircle all aspects of kindnesses of Dr. S. Riazuddin, and feel myself helpless in expressing my sincere thanks to him. It is indeed my honour and pleasure to acknowledge the contributions of my reverend supervisor Dr. Tayyab Husnain, Professor, Centre for Molecular Biology, Lahore. Indeed it was a blessing of God on me having such a nice teacher one can only imagine. His behaviour was so kind, his interest was so keen, his confidence in me was so immense, his guidance was so perfect, his attitude was so friendly and his supervision was so intellectual that can not be explained in mere words. I would really be proud of declaring myself to be his obedient servant. I would also express my sincere thanks to Dr. Syed Sadaqat Mehdi (Professor of Plant Breeding and Genetics, University of Agriculture, Faisalabad), presently serving Virtual University of Pakistan as Registrar (Academics). In spite of his busy schedules, he always welcomed me whenever I demanded his kind help. It was impossible to complete and comprehend statistical analyses without his guidance. He has been a source of illumination and knowledge to me since my graduation days. My M.Sc (Hons) research work was also completed under his inspiring and intelligent guidance. I am obliged to acknowledge the Higher Education Commission of Pakistan for granting me a fully-funded merit scholarship for PhD studies. The personal and sympathetic interest of Mr. Junaid Iqbal, former Secretary to the Government of the Punjab, Department of Agriculture, Lahore and Dr. Noor-ul-Islam

iv

khan, Director, Cotton Research Institute, Faisalabad in granting me study leave on full pay for three years for PhD studies, is also acknowledged. I have been the lucky one having very nice lab fellows whose accommodative and friendly behaviour made my work less laborious. The worth-mentioning include Dr. Asifa Majeed, Dr. Sarfaraz Hussain, Dr. Idrees Ahmed Nasir, Dr. Ahmed Ali Shahid, Mrs. Bushra Rashid, Dr. Kausar Malik, Mr. Zafar Saleem, Farah Naz, Muhammad Irfan and Allah Bakhsh. The services offered by Mr. Ilyas Tabassum, Mr. Raza Ali Zaidi, Kashif, Karim, Munir, Khaliq and Nazir are also worthy to be acknowledged. Last but not least, I wish to submit my sincere and earnest thanks to my wife Dr. Munazza Ghazanfar without whose support and sincere efforts, it would have been impossible for me to complete this work. My loving daughters Arva Sarosh and Zoha Ghazanfar and sons Asadullah Khan and Saadullah Khan have suffered a lot due to my extremely busy time-table; they are especially thanked for their innocent and undoubtedly sincere prays for my success. I am also highly thankful to my brothers and sisters whose good wishes and support enabled me to complete my studies.

(GHAZANFAR ALI KHAN)

v

SUMMARY
A local cotton variety MNH-93 was transformed with the Bacillus thuringiensis (Bt) gene Cry1Ab through Agrobacterium-mediated transformation method using mature cotton embryos as explant and kanamycin as a selectable marker at a concentration of 50mgL-1. The transformation efficiency remained 0.26%. The plants were analyzed for transgene integration through PCR and Southern Blotting. The gene copy number was also found through Southern Blotting. The plants were analyzed for gene expression through ELISA, Western Dot Blot and Bioassays. The Bt protein being produced in the transgenic plants was quantified using ImageQuant software, which ranged from 0.00 to 1.35% of the total protein. The progenies of the positive plants were raised under field conditions. Single Plant Selections were made upto five generations and consequently, four homozygous lines were developed. The transgene presence and expression was reconfirmed at each stage through molecular analyses and bioassays. Homozygous lines thus obtained were evaluated for field performance and insect resistance, and also used in producing hybrids. The inheritance of Bt gene was studied in five successive selfed generations. It was concluded that the transgene was faithfully inherited in the progenies. To study the inheritance pattern in filial generations, the four transgenic lines were crossed to two nonBt varieties to produce six hybrids. It was concluded that the Bt gene was inherited as a dominant trait and there was no difference among reciprocal crosses at F1 level. It was further found that the segregation of the gene was not always in Mendelian fashion at F2 level. The heterosis and heterobeltiosis was computed in all crosses for various characteristics. The heterosis and heterobeltiosis ranged from -15.19 to 107.07% and vi

18.58 to 98.79%, respectively for yield per plant; from -20.34 to 81.36% and -20.34 to 81.36%, respectively for number of bolls per plant; from -6.96 to 21.38% and -9.30 to 9.99%, respectively for boll weight; from 13.02 to 26.44% and -0.52 to 26.17%, respectively for ginning outturn; and from -8.11 to 36.23% and -5.56 to 23.68%, respectively for mortality %age of Heliothis larvae in laboratory bioassays. The Broad Sense Heritability and Genetic Advance for insect resistance in Bt versus non-Bt crosses were calculated. Both of these were high in four out of six hybrids. The lower values were found to be in those combinations where non-Bt parent belonged to a different genetic background. The correlation of Bt trait with other traits was also calculated. The Bt trait had a strong and significant negative correlation with natural infestation of Spotted Bollworm, highly significant and strong negative correlation with plant height, and significant and strong positive correlation with Ginning Outturn Percentage. The correlation of Bt with yield, number of monopodial branches per plant, number of sympodial branches per plant, number of bolls per plant, boll weight, staple length and fibre fineness was statistically non-significant. The observations on some important qualitative characters of cotton were also taken during the present studies. The plant shape, boll shape, boll opening and reaction to virus of the variety remained unchanged after transformation. The plant reaction to insects was found to be susceptible in case of non-Bt cotton and tolerant in case of Bt cotton. The only qualitative character that showed deterioration was leaf hairiness which, after transformation, became smooth to sparsely hairy from profusely hairy. In field bioassays, thirty 2nd instar Heliothis larvae were artificially infested, in three installments, to each plant. The transgenic lines showed upto 67% lesser Heliothis population as compared to control. Furthermore, the transgenic lines showed upto 30% vii

lesser counts of naturally occurring Spotted Bollworm than in control lines. In laboratory bioassays, the transgenic lines gave upto 88% higher mortality of Heliothis larvae than the control. In the field trials where no chemical insecticide was applied, the transgenic lines gave upto 23% more Seed Cotton Yield, 11% increase in Ginning Outturn %age, 42% increase in Number of Sympodial Branches per Plant, and 28% reduction in Plant Height. All other characters of the variety viz. Number of Monopodial Branches, Number of Bolls per Plant, Boll Weight, Staple Length and Fibre Fineness remained intact after transformation. It was further found that Bt cotton required 41% lesser chemical insecticides to control Lepidopteran insects. At this statistically economical usage of insecticides, the Bt line CEMB-3 gave 28% more Seed Cotton Yield than the non-Bt line of the same parentage.

viii

TABLE OF CONTENTS

CERTIFICATE ACKNOWLEDGEMENTS SUMMARY LIST OF FIGURES LIST OF TABLES LIST OF ANNEXURES ABBREVIATIONS

ii iv vi xiv xv xvi xvii

1
1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1.10 1.11

INTRODUCTION
BIOTECHNOLOGY AND THE CONVENTIONAL TECHNOLOGY AGRICULTURE IN PAKISTAN COTTON SITUATION IN PAKISTAN PLANT PROTECTION ADVANCES IN COTTON RESEARCH INSECT RESISTANCE THROUGH Bt GENES Bt SAFETY STUDIES WORLDWIDE ADOPTION OF Bt CROPS BENEFITS OF Bt CROPS NEED OF THE DAY OBJECTIVES

1
2 3 4 5 5 7 8 11 13 15 16

2
2.1 2.2 2.3 2.4

REVIEW OF LITERATURE
BACILLUS THURINGIENSIS PLANT TRANSFORMATION FIELD STUDIES INHERITANCE

17
18 22 28 33

3
3.1 3.1.1 3.1.2

MATERIALS AND METHODS
AGROBACTERIUM TRANSFORMATION Agrobacterium tumefaciens Competent Cells Preparation Agrobacterium Transformation with pKMAB By Heat Shock Method

42
43 43 43

ix

3.1.3 3.1.4 3.1.4.1 3.1.4.2 3.2 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 3.3 3.3.1 3.3.2 3.3.3 3.3.3.1 3.3.3.1.1 3.3.3.1.2 3.3.3.1.3 3.3.3.1.4 3.3.3.2 3.3.3.3 3.3.3.4 3.3.3.5 3.3.3.6 3.3.4 3.3.4.1 3.3.4.2 3.3.4.3 3.4 3.5 3.5.1 3.5.1.1

Long and Short Term Storage of Bacterial Strain Confirmation of Agrobacterium Transformation Plasmid Isolation Confirmation of Transformation Through PCR COTTON TRANSFORMATION Selection of a Suitable Variety Seed Delinting Seed Sterilization Bombardment with Tungsten Particles Agrobacterium Mediated Transformation MOLECULAR ANALYSES OF TRANSGENIC PLANTS Genomic DNA Isolation Polymerase Chain Reaction Southern Hybridization Probe Making/DNA Labelling Plasmid Digestion Gel Elution DNA Labeling with Biotin-11-dUTP Probe Estimation Genomic DNA Digestion Gel Running for Southern Hybridization Gel Transfer Assembly Blot Processing Copy Number Estimation Immunological Assay of Transgenic Plants Isolation of Protein from Cotton Leaves Enzyme Linked Immunosorbent Assay Western Dot Blot INSECT BIOASSAYS FIELD STUDIES Development of Transgenic Pure Lines 1st Generation x

44 44 44 45 45 45 46 46 46 47 48 48 49 50 50 50 50 51 51 52 52 52 53 53 53 54 54 54 55 55 56 56

3.5.1.2 3.5.1.2.1 3.5.1.3 3.5.1.4 3.5.2 3.5.2.1 3.5.2.2 3.5.2.2.1 3.5.2.2.2 3.6 3.6.1 3.6.2 3.6.2.1 3.6.2.1.1 3.6.2.1.2 3.6.2.1.3 3.6.2.2 3.6.2.3 3.7 3.8 3.9 3.10 3.11 3.11.1 3.11.2 3.11.3 3.11.4 3.11.5 3.11.6 3.11.7 3.11.8

2nd Generation Selection Criteria 3rd Generation 4th & 5th Generations Field Trials Bt trials 2004-2005 Comparative Study of Insecticide Applications on Bt and non-Bt Cotton Lines 2004-2005. Insecticide Application Trial, 2004 Insecticide Application Trial, 2005 Bt INHERITANCE STUDIES Bt Inheritance in Transgenic Selfed Generations Bt Inheritance in Filial Generations Crossing among Bt and non-Bt Lines Emasculation Pollination Combinations of Crosses Inheritance Studies in F1 Generation Inheritance Studies in F2 Generation HETEROSIS AND HETEROBELTIOSIS STUDIES HERITABILITY AND GENETIC ADVANCE STUDIES CORRELATION STUDIES COMPARISON OF SOME QUALITATIVE CHARACTERS OF Bt AND NON-Bt COTTON STATISTICAL ANALYSES Analysis of Variance t-test Assuming Unequal Variances Chi Square Test Estimation of Heterosis and Heterobeltiosis t-Test For Heterosis t-test for Heterobeltiosis Heritability Estimates Genetic Advance Estimates

57 58 58 59 59 59 63 63 64 65 65 65 66 66 66 67 67 67 68 69 70 71 72 72 72 73 73 74 74 74 75

xi

3.11.9 3.11.10

Estimation of Correlation t-test For Correlation

75 76

4
4.1 4.1.1 4.1.2 4.1.3 4.1.4 4.1.5 4.1.6 4.1.7 4.1.8 4.2 4.2.1 4.2.2 4.2.3 4.2.4 4.3 4.3.1 4.3.1.1 4.3.1.2 4.3.1.3 4.3.1.4 4.3.1.5 4.3.1.6 4.3.1.7 4.3.1.8 4.3.1.9 4.3.1.10 4.3.1.11 4.3.1.12 4.3.1.13

RESULTS AND DISCUSSION
COTTON TRANSFORMATION Selection of a Suitable Variety Agrobacterium Transformation with pKMAB Agrobacterium Mediated Transformation of Cotton with pKMAB Cotton Genomic DNA Isolation Polymerase Chain Reaction Southern Blot Analysis Enzyme Linked Immunosorbent Assay Western Blot Analysis DEVELOPMENT OF TRANSGENIC PURE LINES 1st Generation 2nd Generation 3rd Generation 4th & 5th Generations FIELD STUDIES ON Bt COTTON Bt Trials 2004-05 Bt Protein %age Natural Infestation of Spotted Bollworm Field Bioassay with Heliothis Lab Bioassay Seed Cotton Yield per Plant Plant Height Number of Monopodial Branches per Plant Number of Sympodial Branches per Plant Number of Bolls per Plant Boll Weight Ginning Outturn Percentage Staple Length Fibre Fineness xii

77
78 78 78 81 81 81 84 84 84 87 87 92 98 101 101 101 103 103 109 113 113 114 115 115 115 118 118 118 121

4.3.2 4.3.2.1 4.3.2.2 4.4 4.4.1 4.4.2 4.4.2.1 4.4.2.2 4.5 4.5.1 4.5.2 4.5.3 4.5.4 4.5.5 4.6 4.6.1 4.6.2 4.7 4.8 4.9 4.9.1 4.9.2 4.9.3 4.9.4 4.9.5 4.9.6 4.9.7

Comparative Study of Insecticide Applications on Bt and non-Bt Cotton Lines 2004-2005. Studies During the Year, 2004 Studies During the Year, 2005 Bt INHERITANCE STUDIES IN TRANSGENIC GENERATIONS Bt Inheritance Studies in Selfed Generations Bt Inheritance Studies in Filial Generations Inheritance of Transgene in F1 Generation Mendelian Inheritance Studies in F2 Generation STUDIES ON HETEROSIS AND HETEROBELTIOSIS IN F1 GENERATION Seed Cotton Yield per Plant Number of Bolls per Plant Boll Weight Ginning Outturn Percentage Lab Bioassay Results (Mortality %age of Heliothis Larvae) HERITABILITY AND GENETIC ADVANCE STUDIES IN Bt COTTON Heritability for Bt Resistance Genetic Advance for Bt Resistance CORRELATION OF Bt TRAIT WITH OTHER ECONOMIC TRAITS COMPARISON OF SOME QUALITATIVE CHARACTERS OF Bt AND NON-Bt COTTON DISCUSSION Transformation Development of Transgenic Pure Lines Field Studies Bt Inheritance Heterosis and Heterobeltiosis Heritability and Genetic Advance Correlation

121 121 123 130 130 130 130 132 135 135 138 138 139 140 141 141 144 146 148 150 150 152 154 158 160 161 162 164

5

LITERATURE CITED

xiii

LIST OF FIGURES
Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Figure 19 Figure 20 Figure 21 Figure 22 Figure 23 Figure 24 Figure 25 Figure 26 Figure 27 Figure 28 Schematic Diagram of the construct pKMAB PCR Confirmation of Transformation of Agrobacterium tumefaciens C58C1 Cotton Genomic DNA Isolation PCR of Transformed Plants Southern Blot Analysis of Transformed Plants ELISA of Transformed Plants Western Dot Blot of Transformed Plants Comparative View of Damaged and Healthy Cotton Bolls Laboratory Bioassay with Heliothis Larvae Layout Plan of Progeny Rows Grown during Kharif, 2003 Data on Different Characters of All Plants of 2nd Generation, Kharif, 2003 3rd Generation Progeny Plants in Green House Cotton Field 2004 & 2005 Bt Protein Content Spotted Bollworm Insect Release Method for Field Bioassay Field Bioassay Laboratory Bioassays Yield per Plant Plant Height Number of Monopodial Branches per Plant Number of Sympodial Branches per Plant Number of Bolls per Plant Boll Weight Ginning Outturn Percentage Staple Length Fibre Fineness Yield Comparisons of Bt and non-Bt Genotypes 79 80 82 83 85 86 88 89 91 93 95 99 102 104 104 111 111 112 112 116 116 117 117 119 119 120 120 129

xiv

LIST OF TABLES
Table 1 Table 2 Table-3 Table 4 Table-5 Table-6 Table 7 Table 8 Table 9 Table 10 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16 Table 17 Table 18 Table 19 Table 20 Table 21 Insect Resistance and Number of Bolls of 1st Generation Plants Characteristics of Selected Five Plants from 2nd Generation Bt Protein %age in 3rd Generation Plants 2003-2004 Analysis of Variance: Mean Squares for Different Characters of the Bt Trials 2004-2005 Analysis of Variance: Mean Squares for Different Characters of the Bt Trials 2004-2005 Mean Comparisons for Different Characters of the Bt Trials 20042005 Mean Comparisons for Different Characters of the Bt trials 20042005 Comparative Study of Insecticide Applications, 2004 Seed Cotton Yield Comparisons of Bt and non-Bt Genotypes under Different Insecticide Application Treatments Analysis of Variance for Seed Cotton Yield Comparative Study of Insecticide Applications, 2005 Comparison of Insecticide Use and Seed Cotton Yields on Bt and non-Bt Cotton Lines. History Sheet of Transgenic Pure Lines Developed at CEMB Molecular Analysis of F1 Plants Segregation of Bt Gene in F2 Populations of Six Crosses Analysis of Variance: Mean Squares of F1 Hybrids for Different Characters Mean Performance of F1 Hybrids and Their Parents for Different Characters Estimates of Heterosis and Heterobeltiosis for Different Characters of F1 Hybrids Heritability and Genetic Advance of Bt Resistance in the Crosses between Bt and non-Bt Cotton Lines Correlation of Bt Insect Resistance Trait with the Economic Traits of Cotton Comparison of Some Important Qualitative Characters of Bt and non-Bt Cotton var. MNH-93 90 97 100 105 106 107 108 122 125 126 127 128 131 133 134 137 142 143 145 147 149

xv

LIST OF ANNEXURES

ANNEXURE-I ANNEXURE-II ANNEXURE-III

RECIPES OF VARIOUS MEDIUMS RECIPES OF VARIOUS SOLUTIONS RECIPES OF VARIOUS BUFFERS

I III V

xvi

ABBREVIATIONS

µl µg °C % A° ATP BSA Bt BTK

micro litre micro gram degree centigrade Percent Angstrom Adenosine Triphosphate Bovine Serum Albumin Bacillus thuringiensis Bacillus thuringiensis var. kurstaki

BCIP/NBT 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium cm Cry DNA dNTPs EC EDTA ELISA ems et al. ETL exo F1 F2 g Centimeter Crystal Deoxy Ribonucleic Acid Dinucleotide Triphosphate Electric Conductivity Ethylene diamine tetra acetic acid Enzyme Linked Immunosorbent Assay error mean square (et alii) and others Economic Threshold Level Exonuclease First Filial Generation Second Filial Generation Gram

xvii

GCA GOT GUS HEPES HgCl2 Kb KCl KDa kg L lbs/in2 Na2CO3 NaCl NaOH No. M mg mgL-1 ml mM MS N N2 Na NADP ng nm

General Combining Ability Ginning Outturn Glucuronidase N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid Mercuric Chloride Kilobase Potassium Chloride kilo Dalton Kilogram Litre pounds per square inch Sodium Carbonate Sodium Chloride Sodium Hydroxide Number Molar Milligram Milligram per litre milli litre milli molar Murashige and Skoog Normal Nitrogen Sodium Nicotinamide adenine dinucleotide phosphate nano gram nano meter (wavelength) xviii

NPT OD PCR PBS pH p.mol q/ha RNA rpm SCA SDS SSC TE Ti U UV viz. YEP

Neomycin phosphotransferase Optical Density Polymerase Chain Reaction Phosphate Saline Buffer power of Hydrogen pico moles quintals per hectare Ribonucleic Acid rounds per minute Specific Combining Ability Sodium Dodecyl Sulphate Standard Sodium Citrate Tris Ethylene diamine tetra acetic acid Tumor inducing Units ultra violet Namely Yeast Extract Peptone

xix

CHAPTER 1

INTRODUCTION

1

1.1

BIOTECHNOLOGY AND THE CONVENTIONAL TECHNOLOGY
The application of biotechnology tools to agriculture has allowed scientists to

transform plants without the need for sexual compatibility between species, thus establishing the possibility of rapidly producing new crop varieties with traits beneficial to human health and the environment. Plants have been transformed successfully to improve their pest and disease resistance, herbicide tolerance, nutritional qualities, and stress tolerance. The rapid transformation of plants with enhanced traits holds great promise for increased efficiency of land use, a development that can help feed the expanding world population using sustainable growing practices (Mackey and Santerre, 2000; Royal Society, 1998). The doubling or possible tripling of global food demand by the mid twenty-first century (Mackey and Santerre, 2000) necessitates deployment of appropriate technologies that are culturally acceptable and environmentally sustainable (James and Krattiger, 1996; Royal Society, 2000). The deficiency in efficient and adequate food production is greatest in developing countries because they have the largest population growth rates but tend to be in climates with comparatively poor soil and water resources and the greatest pest pressures. The magnitude of the problem is starkly illustrated by the demographics: Approximately 4.6 billion people live in developing countries, with a growth rate of 1.9 %, compared with 1.2 billion people who live in the industrial countries, with a growth rate of 0.1 % (James, 1997). Food production increases resulting from the Green Revolution of the 1960s and 1970s have helped to close the gap between food supply and demand. But conventional plant breeding techniques may not be adequate to keep pace with demand for both production increases and improvements in land use and

2

environmental quality. As Nobel Laureate Norman Borlaug has said, “If we grow our food and fibre on the land best suited to farming with the technology we have and what’s coming, including proper use of genetic engineering and biotechnology, we will leave untouched vast tracts of land with all of their plant and animal diversity.” The international scientific community concurs that conventional technology alone will not support sufficient growth in the nutritional quality (availability of nutrients and micronutrients) and nutritional quantity (caloric input) of food production by 2050, when total world population is estimated to be approximately 11 billion (James and Krattiger, 1996). Thus, to ensure both nutritional adequacy and environmental health of the world’s poorest people in the twenty-first century, plant biotechnology must be investigated and deployed in both developed and developing countries (Conway and Toenniessen, 1999).

1.2

AGRICULTURE IN PAKISTAN
Agriculture accounts for nearly 23 percent of Pakistan’s national income (GDP)

and employs 42 percent of its workforce. Agriculture also supplies raw material to Pakistan’s industries, notably textile industry, the largest industrial sub-sector of the economy. Most importantly, 67.5 percent of country’s population living in rural areas is directly or indirectly dependent on agriculture for their livelihood. Given its importance to national economy, the Government attaches high priority to raising agricultural productivity with a view to promoting faster agricultural growth and hence, raising farmers income. Pakistan witnessed unprecedented drought during the first two yeas of the decade of 2000 (2000-01 and 2001-02) which resulted in contraction of agricultural value added. In other words, agriculture registered negative growth in these two years. The next two years (2002-03 and 2003-04) witnessed a modest recovery in agricultural growth at the back of improvement in the availability of water for irrigation purpose. A

3

stronger – than expected – performance of agriculture has been one of the hallmarks of the fiscal year 2004-05 on account of unprecedented increase in cotton production (14.6 million bales) and a near bumper wheat crop of the size 21.1 million tons. Major crops, accounting for 37.1 percent of agricultural value added registered stellar growth of 17.3 percent as against 1.8 percent last year. Stellar performance of these two crops helped agriculture staging an impressive recovery in 2004-05. The agriculture sector grew by 7.5 percent in 2004-05, which is higher than actual growth of 2.2 percent last year and a target of 4.0 percent (Economic Survey of Pakistan, 2004-05).

1.3

COTTON SITUATION IN PAKISTAN
Cotton is an important non-food cash crop and a significant source of foreign

exchange earning. It accounts for 10.5 percent of the value added in agriculture and about 2.4 percent to GDP. In addition to providing raw material to the local textile industry, the surplus lint cotton is exported. The area and production target for cotton crop during the current fiscal year were 3140 thousand hectares and 10720 thousand bales, respectively. The crop was however, sown on an area of 3221 thousand hectares – 2.6 percent more than the target and 7.8 percent more than last year (2989 thousand hectares). The production of cotton is estimated at 14.618 million bales for 2004-05, the highest ever recorded in the country’s history, and up by 45.5 percent over the last year’s production of 10.0 million bales. Factors responsible for the unprecedented rise in cotton production include: a 7.8 percent rise in area under the crop; higher boll bearing; use of improved quality of pesticide resulting in low pest pressures; and favourable weather condition for growth and development of the crop. (Economic Survey of Pakistan, 2004-05).

4

1.4

PLANT PROTECTION
Plant protection is an important factor amongst the agricultural inputs. Though it

can not induce higher yields on its own but without effective protection against the attack of pests and diseases, the beneficial outcome of other inputs may not be realized either. In this regard, the Department of Plant Protection provides facilities, such as, Locust Survey and Control, Aerial Pest Control, Pesticide Registration and Testing etc. while the private sector carries plant protection measures including ground sprays. No aerial activity was undertaken during the current year. (Economic Survey of Pakistan, 2004-05). Cotton is susceptible to attack by more than 15 economically important insects, mainly belonging to the insect order Lepidoptera, Coleoptera and Homoptera. On overall basis, 13% of the cotton crop is lost to insects (Gatehouse and Hilder, 1994). These insects are being controlled by the chemical insecticides, which otherwise have serious environmental and human health threats. During July-March (2004-05), 34.4 thousand tons of agricultural pesticides were imported while 23.0 thousand tons locally formulated. The approximate value of the pesticides used during this period is about Rs.10000 million. (Economic Survey of Pakistan, 2004-05). Moreover, insects have also been evolving resistance against these chemicals. Over 500 species of insects have become resistant to one or multiple synthetic chemical insecticides (Schnepf, E. et al., 1998).

1.5

ADVANCES IN COTTON RESEARCH
Cotton is the main world fibre crop, and has an immense importance in Pakistan’s

economy. Therefore the cotton plant has always been subjected to extensive research aimed at improving its genetic architecture to obtain greater benefits. As a result of concerted efforts of the cotton breeders in the country, numerous high yielding varieties

5

have been evolved through selection and breeding. Although cultivation of newly developed cotton varieties has increased the overall production of seed cotton in Pakistan, the increasing demand for raw material in the expanding textile industry and more edible oil to feed the growing population necessitated the research workers to further exploit the available genetic resources by using conventional and non-conventional techniques. Besides other techniques, one way of increasing cotton production is by increasing average yield of seed cotton by changing genetic architecture of the varieties. Before developing such an important breeding program, availability of information on the genetic controlling mechanism of various plant traits related to insect resistance, seed cotton yield and fibre quality is necessary. There are numerous approaches to handle the breeding population for the purpose of making selection of desirable genotypes. Inheritance and correlation studies provide dependable information about different plant characters. Variation in any character in a segregating or mixed population is due to both genetic and environmental factors. The genetic factor is of most importance in plant breeding since it can be used to improve the population. The greater the proportion of total variability that is due to environmental factors, the more difficult it will be to select for inherited differences. If environmental variability is small in relation to genetic differences, selection will be more efficient. Therefore, heritability (the inherited portion of the variability) is a statistic that may be used to evaluate the effectiveness of selection during segregation generations following hybridization. Heritability is a measure of the value of selection of a particular character and an index of transmissibility of the genes controlling the character (Khan, M.A. et al., 2001). Thus an understanding about genetic behaviour of variation is of prime importance for the use of appropriate selection protocols.

6

Estimates of genetic parameters obtained from well designed and executed genetic experiments provide the breeder with the information necessary to determine the best breeding procedures, for a particular species. Moreover, when heritability estimates are available, progress from selection can be predicted for any breeding system, since expected gain is a function of heritability. Therefore such guided selection produces Genetic Advance. This change is of great interest to the plant breeders, since it changes the population mean. The magnitude of Genetic Advance from selection for a character in a cross is determined by the total variation in the population, the heritability of the character and the selection pressure, i.e. the proportion of the population that is selected (Khan, M.A. et al., 2001).

1.6

INSECT GENES

RESISTANCE

THROUGH

Bt

The conventional breeding has been aimed at developing insect resistant varieties, but no variety has so far been released exhibiting complete insect resistance. The modern techniques of biotechnology offer potential to overcome this problem by the introduction of genes encoding insecticidal proteins from Bacillus thuringiensis into plants to develop insect resistance. Bacillus thuringiensis (Bt) is a gram positive, spore-forming, soildwelling bacterium that produces crystalline protein inclusions known as δ-endotoxins. Bacillus thuringiensis (Bt) was first isolated in 1901 from a diseased silkworm moth in Japan. In 1911, Berliner, E. isolated a similar microbe from a diseased flour moth in Germany and gave Bt its current scientific name (Van Frankenhuyzen, 1993). The association of Bt with insect pathogenicity suggested its application as an insecticide to control the European corn borer (Ostrinia nubialis) in Europe during the late 1920s. Inquiries of the factors responsible for Bt’s pathogenicity did not begin until the 1950s 7

and culminated in the late 1980s with an understanding of the molecular basis of its toxic mechanism (Gill et al., 1992). However, Bt microbial preparations were used to control pests prior to understanding how they worked. When nutrients are plentiful and pH and temperature are favourable (as in an insect body), Bt grows rapidly and reproduces asexually by simple cell division (vegetative growth). As nutrients in their immediate environment become limiting, Bt cells produce a spore that only germinates when conditions become favourable again. At the time of sporulation, Bt also produces a crystalline proteinaceous inclusion called the parasporal body. When certain insect species incidentally ingest the sporulated Bt cells with their parasporal body, the alkaline midgut (i.e. insect digestive tract) solubilizes the crystalline parasporal body releasing protein toxins known collectively as delta-endotoxins (Gill et al., 1992). The endotoxins are actually protoxins that must be cleaved by insect midgut proteases into the molecular form that eventually kills the insect. The toxic protein fragment binds to specific molecular receptors on susceptible insects’ midgut cells, causing the membranes to lose their integrity and the gut tissue to swell up (Gill et al., 1992). The insect stops feeding and eventually starves to death. A dying insect is probably the most favorable environment for Bt growth and reproduction. As the insect body completely decays due to bacterial septicemia, the spores and proteins disperse into the environment where they can be ingested by other unsuspecting insects.

1.7

Bt SAFETY STUDIES
There have been a number of concerns raised from different people regarding the

use of Bt crops. For example, the resistance to pest insects in biotechnology-derived crops may pose new or different human safety concerns in comparison to conventionally bred crops with similar traits. Bt plants could be harmful to non-target organisms. This could

8

reduce the number of beneficial organisms that would naturally help control the pest species. Bt crops may be problematic for soil health. An additional environmental hazard of insect resistant crops is that targeted pests could develop resistance to the effects of Bt. This is because constant exposure to the Bt toxin produced by these plants encourages the survival of individual pests which have a genetic immunity to Bt. Similarly, the Bt gene could have broad ecological impacts following its spread throughout the population (introgression). Cry proteins generally have little or no effect on natural insect predators and parasites, as indicated by laboratory and field studies conducted with lady beetles, green lacewing, damsel bugs, big-eyed bugs, parasitic wasps, and other arthropods (for example, Dogan et al., 1996; Amer et al., 1999). This allows beneficial organisms to survive in Bt-protected crops where the beneficial insects can help control secondary pests. Secondary pests can often become a problem when predator and parasite populations are reduced by conventional broad-spectrum insecticides. For instance, beneficial arthropods alone kept aphids below damaging levels in commercial New Leaf Plus potato fields which had not been treated to control aphids; Beneficial insects and spiders were more abundant in these fields; This appears to provide an additional benefit of preventing economic outbreaks of spider mites (Feldman et al., 1992; Reed et al., 1993). Similarly, use of Bt cotton in China, with a concomitant reduction in insecticide use, resulted in an average increase of 24% in the number of insect predators over what was found in conventional cotton fields (Xia et al., 1999). Thus, to the extent that Bt crops require fewer applications of externally applied insecticides, populations of beneficial organisms are more likely to be preserved, which result in less crop damage, requirement for fewer chemical insecticides, and the potential for higher yields.

9

The U.S. EPA has determined that the numerous toxicology studies conducted with Bt microbial products show no adverse effects and has concluded that these products are not toxic or pathogenic to humans (McClintock et al., 1995; EPA, 1998a). EPA, in its 1998 re-registration eligibility decision, concluded that microbial Bt products pose no unreasonable adverse effects to humans or the environment and that all uses of those products are eligible for re-registration (EPA, 1998a). The World Health Organization’s (WHO) International Program on Chemical Safety Report on Environmental Health Criteria for Bt concluded that: “Bt has not been documented to cause any adverse effects on human health when present in drinking water or food” (IPCS, 2000). There have only been two reports of potential adverse effects in humans from the use of microbial Bt products, neither of which was attributable to exposure to Cry proteins (EPA, 1988; McClintock et al., 1995). Cry proteins are rapidly degraded under conditions which simulate the gastrointestinal conditions of the mammalian system. Therefore, these Cry proteins will be rapidly degraded and inactivated upon consumption. Finally, receptor-mediated binding to the brush-border membrane in midgut epithelium cells leads to membrane-bound forms of the Cry protein. This is believed to take place in three steps: binding to midgut receptor proteins, partitioning into the brush border membrane, and finally, forming channels and pores. Binding to these receptors is required for a Cry protein to exert any activity (English and Stalin, 1992). If receptor binding does not occur, the Cry protein will have no effect on that organism. Noteborn et al., (1993) detected no specific binding of Cry1Ab protein to mouse and rat gastrointestinal tract tissue in vivo. The Cry and NPTII-selectable marker proteins have been shown to pose no significant allergic concerns. Commonly allergenic proteins are typically prevalent in food, stable to the acidic and proteolytic conditions of the digestive system and stable to

10

food processing and are glycosylated (Taylor and Lehrer, 1996). None of the three classes of Cry proteins (Cry 1, Cry 2, or Cry 3 classes) nor the NPTII-selectable marker protein share any of these characteristics. Overall, Cry proteins are characterized as being practically non-toxic to non-target organisms (EPA Fact Sheet, 1996a).

1.8

WORLDWIDE ADOPTION OF Bt CROPS
Growers sustain billions of dollars in crop loss or reduced yield due to pests,

which have the potential to be controlled by Cry proteins (Gianessi and Carpenter, 1999). In cases such as European corn borer, stalk damage caused by second generation borers which have entered the inside of the corn stalks is difficult to control with externally applied pesticides. In addition, important chemical insecticides, such as synthetic pyrethroids used on cotton to control budworm, are losing their effectiveness due to the onset of pest resistance (Smith, 1999). During the five years since their commercial introduction, growers have rapidly adopted Bt-protected crops as an effective tool to enhance high yield sustainable agriculture. Total planted acreage in the United States for Bt-protected cotton, corn, and potato exceeded 16 million acres in 1998 (Gianessi and Carpenter, 1999), comprising 17 and 18% of the total corn and cotton acreage, respectively. According to reports by James (1997, 1998, 1999), the global acres of Bt-protected plants have increased from approximately 10 million acres in 1997 to 20 million acres in 1998 and 29 million acres in 1999. The latest position of Bt crops worldwide is shown in the graph below:-

11

12

1.9

BENEFITS OF Bt CROPS
The benefits of decreased pest management costs, increased yields, and greater

crop production flexibility are responsible for the rapid adoption of these crops (Marra et al., 1998; Culpepper and York, 1998). The Economic Research Service of the U.S. Department of Agriculture reports (Klotz-Ingram et al., 1999) that the use of certain Bt crops is associated with “significantly higher yields” and “fewer insecticide treatments for target pests.” A study conducted by the U.S. National Center for Food and Agricultural Policy (Gianessi and Carpenter, 1999) examined the impact of planting Bt-protected crops. The authors concluded that: “rapid adoption of this technology is directly tied to benefits of greater effectiveness in pest control technology and very competitive cuts in farmer’s costs.” Gianessi and Carpenter (1999) reported that Bt cotton created an estimated $92 million in additional value in the United States in 1998. In summary, the benefits of using Bt protected crops include the following: (a) reduced chemical insecticide treatments for target pests; (b) highly effective pest control; (c) increased crop yields; (d) supplemental pest control by preserving or enhancing populations of beneficial organisms; and (e) reduced levels of fungal toxin. The adoption of Bt-protected plants has led to significant reductions in chemical insecticide use. Plantings of Bt-protected cotton in 1996 helped Alabama growers use the least amount of insecticides on cotton since the1940s (Smith, 1997). In 1998, an

13

estimated 2 million pounds less chemical insecticide was used for bollworm/budworm control in six key cotton-producing USA states compared to 1995 usage. Following the introduction of Bt-protected cotton in 1996, a total average of 2.4 insecticide applications were made to control budworm/bollworm across all cottonproducing states (Williams, 1997). Pre-1996 insecticide use was significantly higher (2.9 to 6.7 applications) in the six states where the Bt cotton has been most widely adopted (Williams, 1999). During the three years in which Bt-protected cotton has been planted, the number of insecticide treatments for budworm/bollworm in these states fell to an overall average of 1.9 applications. The reduced number of insecticide treatments corresponds to a 12% decline in the total pounds of chemical insecticides applied. Of course, some insecticide applications may be necessary to control those insects, which are not controlled by the specific Bt protein expressed in the plant. Comparable surveys of cotton growers in Australia during 1998–1999 also showed substantial reductions in insecticide use following the introduction of Btprotected cotton. Depending on the growing region, reductions in chemical insecticide use varied from 27–61%, with an average of 43% reduction. This corresponded to 7.7 fewer insecticide sprays on the Bt-protected cotton than on conventional cotton fields. In China, insecticide reductions associated with Bt protected cotton have been even greater. In four years of testing, the use of insecticides has decreased by 60–80% compared with chemical insecticide use in conventional cotton (Xia et al., 1999). Most European and southwestern corn borer larvae that attempt to feed on Btprotected corn are only able to make a slight scar on the corn leaf and die within 72 hours. Bt corn hybrids express Cry protein in all plant parts throughout the season and provide

14

essentially 100% protection from European and southwestern corn borer. A survey by Weinzierl et al., (1997) found only two corn borer survivors on about 325 acres. Bt crop protection translates to significant yield increases. Annual corn loss due to European corn borer fluctuates widely, 33 to 300 million bushels per year (USDA, 1975). In 1997, Bt-protected corn was planted on 4 million acres (USDA, 1998) and European corn borer infestation was typical to heavy. That year, Bt corn provided a yield premium of almost 12 bushels per acre (Gianessi and Carpenter, 1999). One year later, European corn borer infestation was extremely light and Bt-protected corn was planted on 14 million acres.

1.10

NEED OF THE DAY
The number of sequenced crystal proteins in Bt is more than 100, encoding Cry

and Cyt proteins (Schnepf, E. 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, Halcomb et. al., 1996 and Sachs et. al., 1998). Transgenic plants do have some weaknesses like other technologies- insects can develop resistance to them thereby eliminating their effectiveness (Cohen et. al., 2000). However a number of solutions have been developed to overcome this problem such as Bt gene at high doses, cultivation of 5% crop having no Bt gene (non-Bt refugia) and transformation of multiple genes in plants. In view of the above discussion, it may be concluded that there is an urgent need to evaluate local cotton varieties as regards to transformation, inheritance of transgene(s) and correlation studies on transgene(s) with other economic traits. The development of

15

insect resistant varieties of cotton would mean saving of millions of rupees spent on the import of chemical insecticides, annually. If new transgenic lines of cotton are successfully developed, these may become base for a number of insect resistant varieties in future through conventional breeding by selection and crossing transgenic lines with non-transgenic lines i.e. the development of stable varieties by using the proven breeding tools.

1.11

OBJECTIVES
The present study was conducted with the following objectives:-

1. Transformation of cotton with insecticidal gene; 2. Molecular analysis for the transgene integration and expression in plants; 3. Studies on transfer of gene(s) of transgenic line into non-transgenic line through crossing; 4. Studies of inheritance pattern of transgene(s) in cotton; 5. Studies on correlation of transferred gene(s) with other economic characters.

16

CHAPTER 2

REVIEW OF LITERATURE

17

2.1

BACILLUS THURINGIENSIS
Berliner isolated Bacillus thuringiensis in 1911, from the flour moth collected in

the German province of Thuringia. This organism had already been discovered by Ishwata in 1901 in Japan where it carried wilt disease in silkworm caterpillar. Bacillus thuringiensis is a ubiquitous gram positive, spore forming, soil-dwelling bacterium which produces crystalline protein inclusions known as δ-endotoxins (Martin and Travers, 1989; Hofte and Whitely, 1989).). These endotoxins have insecticidal activity which can be described as ingestion, solubilization, proteolytic activation, penetration of peritrophic membrane, receptor-binding (reversible and irreversible), membrane-insertion, ion-channel formation and cell-lysis (Schwartz et. al., 1991, Lee et. al., 1992). The inclusion bodies consist of proteins (referred to as Cry proteins) which are selectively active against a narrow range of insects and, as a class of proteins, are effective against a wide variety of insect pests. Cry proteins are produced as protoxins that are proteolytically activated upon ingestion (Hofte and Whitely, 1989). Cry proteins bind to specific sites (i.e. receptors) in the midgut cells of susceptible insects and form ion-selective channels in the cell membrane (English and Slatin, 1992). The cells swell due to an influx of water which leads to cell lysis and ultimately the death of the insect (Knowles and Ellar, 1987). These crystalline inclusions are effective against a variety of Lepidopteran, Dipteran and Coleopteran insects (Beegle et al., 1992). The composition of the crystalline proteins in different Bt isolates varies considerably and each presents a unique combination of several different proteins that exhibit different insect specificities (Crickmore et al., 1998). After solubilization and proteolytic activation of the crystal protein inclusions, by sensitive midgut proteases, an active protease resistant core of 55-70kDa is generated.

18

This activation can be achieved in vitro by trypsin digestion. The activated toxin binds to specific receptor molecules located in the microvillar brush border membranes (Hoffman et al., 1988; Thomas and Ellar, 1983) where it alters the electrochemical potential gradient across the midgut by generating pores or selective/non-selective channels (Knowles and Dow, 1993; Wolfersberger, 1992) destroying the osmotic balance of cell membrane and causing the cell lysis and swell. Many Bt strains, which contain mixtures of up to six or eight different Cry proteins, have been widely used as microbial pesticides since 1961. These products account for about 1 to 2% of the global insecticide market (Baum et al., 1999). Bt microbial products have, and continue to be, the preferred insect control choice for organic growers. Cry protein-encoding genes were an obvious choice for plant expression as a means to protect crops against insect pests. In 1981, the first Cry gene was cloned and expressed in Escherichia coli (Schnepf and Whiteley, 1981). With more than 100 Cry genes described (Crickmore et al., 1998) and dozens of plants transformed to produce Cry proteins, there is significant potential for expanding the role of Bt-mediated plant protection. The next generation of Bt-protected plants will contain multiple Cry genes, thereby providing growers with a product that offers a broader spectrum of pest control and reduced susceptibility for insects to develop resistance (Fred et al., 2000). Until recently, the technical means to produce Bt protected plants were not available. Now, however, the combination of plant cell tissue culture and modern molecular methods allows for a greater diversity of traits, including Bt genes, to be efficiently introduced and deployed in plants for insect control. Because they are proteins and the difficulty of expressing this class of protein in plants has been overcome, Bt proteins are now relatively straightforward to produce in plants (Perlak et al., 1990).

19

Several characteristics, inherent to Bt-protected plants, provide these products with a degree of safety that is unmatched by any other pest control product. First, proteins as a class are generally not toxic to humans and animals, nor are they likely to bioaccumulate in fatty tissue or to persist in the environment like some halogenated chemical pesticides. Proteins which are toxic to humans and animals have been well studied and are readily identified in short-term laboratory studies with surrogate species (Sjoblad et al., 1992). Second, Cry proteins exhibit a high degree of specificity for the target and closely related insect species and must be ingested to be effective. The Cry proteins have no contact activity. Each Cry protein affects relatively few insect species and then, only when ingested by early larval instars; later instars are generally less sensitive. Third, the potential for human and non-target exposure to Cry proteins is extremely low. Unlike pesticides applied to leaves, Cry proteins are contained within the plant tissue in microgram quantities and are produced at low levels in the pollen. In addition to these inherent safety factors, product safety has been established by an extensive safety database on and experience with microbial Bt products (McClintock et al., 1995; EPA, 1988, 1998a, b). Cry proteins generally have little or no effect on natural insect predators and parasites, as indicated by laboratory and field studies conducted with lady beetles, green lacewing, damsel bugs, big-eyed bugs, parasitic wasps, and other arthropods for example (Dogan et al., 1996; Amer et al., 1999). This allows beneficial organisms to survive in Btprotected crops where the beneficial insects can help control secondary pests. Secondary pests can often become a problem when predator and parasite populations are reduced by conventional broad-spectrum insecticides. Feldman et al. (1992) and Reed et al. (1993) observed in research plots that beneficial arthropods alone kept aphids below damaging

20

levels in commercial New Leaf Plus potato fields which had not been treated to control aphids. Beneficial insects and spiders were more abundant in these fields. This appears to provide an additional benefit of preventing economic outbreaks of spider mites. Similarly, use of Bt cotton in China, with a concomitant reduction in insecticide use, resulted in an average increase of 24% in the number of insect predators over what was found in conventional cotton fields (Xia et al., 1999). Thus, to the extent that Bt crops require fewer applications of externally applied insecticides, populations of beneficial organisms are more likely to be preserved, which result in less crop damage, requirement for fewer chemical insecticides, and the potential for higher yields. Bt microbial products are the most widely used biopesticide in the world, comprising 1 to 2% of the global insecticide market in the 1990s (Baum et al., 1999). Cry proteins are highly specific to their target insect pest. Cry proteins have little or no effect on other organisms. In almost 40 years of widespread use, microbial Bt products have caused no adverse human health or environmental effects (EPA, 1998a; Mc-Clintock et al., 1995). The reasons for the rapid adoption of Bt crops are primary benefits of increasing yields due to elimination of losses by European corn borer (Carpenter and Gianessi, 2001). Other benefits of modified plants were emphasized by several authors like reduced environmental impact of insecticides, potential of higher yields and better food supply in the developing countries, better food safety due to reduced fungal infections and remediation of polluted soils (Borlaug, 2000; Mackey and Santerre, 2000; Munkvold and Hellmich, 2000; Mendelsohn et al., 2003; Kasha, 2000). However, the new modified crops could not be the panacea for solving all the pest problems due to specific mode of actions of toxins against the target pests (Sharma et al., 2000). 21

2.2

PLANT TRANSFORMATION
Transformation is a technique of integration and expression of foreign genes into

the nuclear genome of plants via different methods. Introduction of specific foreign genes into plants provide a best way to resolve difficulties about plant physiology that cannot be solved by any other biochemical approach. The essential requirements in a gene transfer system for production of transgenic plants are (a) availability of target tissues including cells competent for plant regeneration (b) a method to introduce DNA into cells (c) a procedure to select and regenerate transformed plants at a high frequency. There are several methods to introduce foreign genes into plant genome i.e. Agrobacterium-mediated transformation (Fraley et al., 1983), microprojectile bombardment (Songstad et al., 1995) microinjection (Potrykus, I., 1991), DNA delivery into intact cells by electroporation (Dekeyser et al., 1990). Agrobacterium-mediated transformation is a most common method to transform dicotyledonous plants. The other method being used to transform cotton is microprojectile bombardment (Finer and McMullen, 1990). Agrobacterium tumefaciens causes crown gall (neoplastic diseases) tumors on many dicotyledonous and some monocotyledonous plants naturally. It transfers its segments of DNA called T-DNA from its tumor inducing plasmid (Ti) to the plant genome. The most important region of Ti plasmid is virulence involved in the processing and transfer of T-DNA (Zupan and Zambryski, 1995). The first evidences indicating this bacterium as the causative agent of the crown gall goes back to more than ninety years (Smith and Townsend, 1907). T-DNA contains two types of genes: the oncogenic genes, encoding for enzymes involved in the synthesis of auxins and cytokinins and responsible for tumor formation: and the genes encoding for the synthesis of opines. These compounds, produced by condensation between amino acids

22

and sugars, are synthesized and excreted by the crown gall cells and consumed by Agrobacterium tumefaciens as carbon and nitrogen sources (Riva et al., 1998). The 30kb virulence (vir) region consists of six operons that are essential for T-DNA transfer (virA, virB, virC, virD, virE and virG) or for the increasing of transfer efficiency (virC and virE). The virD and virE are most important proteins in T-DNA integration (Hooykaas and Schilperoort, 1992; Jeon et al., 1998). Different chromosomal-determined genetic elements have shown their functional role in the attachment of Agrobacterium tumefaciens to the plant cell, chvA and chvB, involved in the synthesis and excretion of the β-1, 2 glucan, chvE required for the sugar enhancement of vir genes induction and bacterial chemotaxis (Ankenbauer et al., 1990). In 1983, the era of plant transformation was initiated when Agrobacteriummediated gene delivery was used for producing transgenic plants. Fraley et al. (1983) reported the Agrobacterium-mediated transformation of petunia and tobacco. Chimeric antibiotic resistant gene was inserted into Agrobacterium tumefaciens and then to the plant cells by in vitro transformation techniques. Protoplast cells were inoculated with Agrobacterium tumefaciens. The chimeric genes contain nopaline synthase region joint to gene for neomycin phosphotransferase I and II. They checked the expression of chimeric genes by the ability of transformed cells to proliferate on medium containing 50mgL-1 kanamycin. They discussed that expression of NPTase I and II enzymes in plants depends on transcription from the nopaline synthase promoter. They observed that useful range of chimeric antibiotic resistance gene was quite broad and most plants within the host range of Agrobacterium tumefaciens could be transformed and identified. They also concluded that there is possibility that Ti plasmid (non-oncogenic) can be used to obtain kanamycinresistant transgenic plants.

23

People

used

different

tissue

sources

to

enhance

Agrobacterium-based

transformation efficiency. Leaf discs transformation with Agrobacterium tumefaciens provides a source of genetically uniform cells that have capacity to regenerate whole plant (Horsch et al., 1985). The leaf discs of petunia, tobacco and tomato were inoculated with a strain containing a modified tumor-inducing plasmid, cultured for 2 days and transferred to selective medium containing kanamycin. They reported that shoot development occurred within 2-4 weeks and transformants were confirmed by their ability to form roots on kanamycin medium. They tested the mesophyll protoplast for kanamycin resistance and found all cells were resistant. This showed that mesophyll cells were transformed. Cotton is a recalcitrant crop and not easy to regenerate. There is only one variety regenerated and transformed through callus i.e. Coker. Firoozabady et al. (1987) and Umbeck et al. (1987) first time reported transformation of G.hirsutum L var. Coker 201 using Agrobacterium method. Cotyledon pieces were co-cultivated with Agrobacterium tumefaciens strain containing Ti plasmid with a chimeric gene encoding kanamycin resistance. Kanamycin containing callus induction medium was used for selection. They obtained high frequency callus and 80% of which induced somatic embryos and normal plants germinated. They reported that 25-35mgL-1 kanamycin concentration is sufficient for the discrimination of transformed and non-transformed plants. Their results showed that high titer of bacteria resulted in overgrowth of bacteria and low titer ~108 cells ml-1 should be used. This method could be used with cis and binary disarmed vector system. Transformation was confirmed by opines products kanamycin resistance, DNA hybridization and immunoassay. Meristematic and callus transformation in sunflower and siokara through Agrobacterium has also been reported by Schrammeijer et al. (1990); Cousins et al.

24

(1991). The tissues were co-cultivated with disarmed Agrobacterium tumefaciens strain harboring a binary vector carrying genes encoding GUS (β-glucuronidase) and NPT-II (neomycin phosphotransferase-II) activity. They analyzed the influence of media conditions, time of co-cultivation and stage of seeds on shoot development and meristem transformation. Transformants were selected by their ability to grow on selection medium i.e. containing kanamycin. Transformation was also confirmed by assay for GUS and NPT-II. GUS positive shoots were rooted on rock wool and transferred to soil. Integration of foreign DNA was confirmed by PCR. They reported that shoot meristem transformation has the advantage that shoots develop directly from primary and secondary meristem without an intervening callus phase and Agrobacterium based method is most suitable to insert foreign gene with high efficiency. Kolganova et al. (1991); Shrivastava et al. (1991) studied the morphogenetic potential of transformed cotton callus tissues. They used hypocotyls of 4-6 days old seedlings and inoculated with Agrobacterium tumefaciens, carrying the kanamycin resistance gene (neomycin phosphotransferase-II). They observed the formation of morphogenetic structures in calluses growing on a selective medium with kanamycin. Transfer of the marker gene was confirmed by the test for neomycin phosphotransferase activity. They concluded that it was possible to activate the morphogenetic potential of the transformed callus tissues and high concentration of kanamycin is toxic to plants. Transgenic cotton (Gossypium hirsutum L.) plants of a Texas cultivar were obtained using Agrobacterium-mediated transformation coupled with the use of shoot apex explants. After inoculation with Agrobacterium tumefaciens strain LBA4404 containing the plasmid PBI121, regeneration of primary plants was carried out in a medium containing kanamycin 100mgL-1. Progeny was checked for expression of the TDNA marker gene encoding neomycin phosphotransferase-II by painting kanamycin

25

(2%) on the leaves. Plants that survived the leaf painting were analyzed by DNA blots. Evidence for integration of the transgenes was observed in two successive generations from the regenerants (T0). The transformed plants appeared to have more than one copy of the T-DNA (Zapata et al., 1999; Gould and Magallanes-Cedeno, 1998). Transformation of plants via particle bombardment (tungsten/gold) is widely used method after Agrobacterium-mediated transformation. Genetically engineered DNA coated particles are accelerated in plant cells. This method has advantage of its ability to deliver DNA in regenerable plant cells and transient gene expression. This was first reported by Klein et al. (1987) and refined by Christou et al. (1998). Stable transformation of cotton (Gossypium hirsutum L.) at a high frequency has been obtained by particle bombardment of embryogenic cell suspension cultures (Finer and McMullen, 1990). Transient and stable expression of the β-glucuronidase (GUS) gene was monitored in cell suspension cultures. Transient expression, measured 48h after bombardment, was abundant and stable expression was observed in over 4% of the transiently expressing cells. The high efficiency of stable expression is due to the multiple bombardment of rapidly dividing cell suspension cultures and the selection for transformed cells by gradually increasing the concentrations of the antibiotic Geneticin (G418) and Hygromycin. Southern analysis indicated a minimum transgene copy number of one to four in randomly selected plants. Fertile plants were obtained from transformed cell cultures less than three months old. However, transgenic and control plants from cell cultures older than 6 months produced plants with abnormal morphology and a high degree of sterility. McCabe and Martinell (1993); Chlan et al. (1995) used gold beads coated with DNA to deliver foreign genes in the meristem tissue of the embryogenic axis. They used plasmid harboring NPT-II (neomycin phosphotransferase) gene. 26 They selected the

plantlets on kanamycin containing medium and obtained plants. They used phytohormone Indole Acetic Acid for rapid root formation. Plants got from that process carried the foreign genes in one or more of their tissue layers. Mendelian segregation was observed by molecular and genetic characterization. Bidney et al. (1992) have shown that efficiency of Agrobacterium-mediated gene transfer could be increased by wounding the explants by bombardment with naked particles. The virus resistant cotton var. CIM-443 was transformed with Cry1Ab gene by using Agrobacterium and biolistic method in combination (Majeed et al., 2000). The transformation efficiency obtained was more than 9% after two months selection on 100mgL-1 kanamycin medium. The integration of Bt and NPT-II gene was confirmed by Dot Blot and Western Blot Analysis. These plants also showed remarkable resistance against Helicoverpa armigera and >80% mortality was observed in T0 plants. The expression of Bt Cry1Ac and Cry1Ab genes has been reported in cotton by Perlak et al. (1990) and Barton, (1989). Total protection from insect damage of leaf tissue from these plants was observed in laboratory assays when tested with Lepidopteran insects. Cry1Ab had 5-fold higher unit activity for Pink Bollworm than for Cotton Bollworm and Tobacco Budworm, and Cry1Ac had 5-fold higher activity for both Cotton Bollworm and Tobacco Budworm than for Pink Bollworm. Reports revealed that truncated Cry1Ac, Cry1Ab expressed highly than wild type gene. Modification of key regions of the structural gene without changing amino acids sequences resulted in the dramatic increase in the levels of protein.

27

2.3

FIELD STUDIES
Field trials of transgenic cotton (BTK) lines have been studied (Benedict et al.,

1996; Altman et al., 1996) against Lepidopterans. These plants were carrying a gene that codes Cry1Ac and Cry1Ab delta-endotoxin from Bacillus thuringiensis var. kurstaki. It was found that these insect resistance lines showed a reduction of the insecticide application for Tobacco Budworm, Bollworm, Cabbage Looper and increased farm profit. Pyramiding the transgene with host plant resistance trait could substantially enhance Bt trait. Transgene have the potential for environmental control of insect pest without reliance on insecticides. Bt transgene involves their inheritance in subsequent generation. Analysis showed that cotton plants of both genetic backgrounds that possessed the Cry1Ab insecticidal protein or high terpenoid glanding or both were more resistant to Tobacco Budworm larvae than plants with other traits. Cry1Ab insecticidal protein with high terpenoid provided the highest level of resistance than Cry1Ab only and improved the durability of Cry1Ab in commercial cotton (Sachs et al., 1998; 1996). The epistatic and environmental factors affect the foreign gene expression in cotton (Gossypium hirsutum L.). These effects could influence the stability, breeding, durability and efficacy of foreign genes. The Cry1A gene expression was variable and influenced by genetic and environmental factors. Site of gene insertion and cotton background were significant sources of variation for Cry1A gene expression. The somaclonal/epistatic effects increased plant to plant variation and caused Cry1A gene expression to behave as a quantitative trait. These effects were heritable and caused similar effects in several different genetic backgrounds of F2 families. Breeding pest resistant plants using plant genetic engineering technique is an effective strategy in integrated pest management (IPM). Increasing the expression level of

28

foreign insecticidal protein by using a strong promoter is a useful method. The expression of Bt toxin in individual plant can be upto 0.255% of total soluble proteins (Chunlin et al., 1999; Zeng et al., 2002). Bioassay showed that synthetic Cry1Ac gene with a stronger promoter like ubiquitin or OM could be effective strategy to enhance expression in plants. This report suggests that chimeric OM and ubiquitin are stronger promoters than the CaMV35S promoter that was widely used in plant genetic engineering. Pest insect resistance bioassay indicated that some of the homozygous Cry1Ac transgenic rice plants of T2 progeny showed high level resistance against striped stem borer (Chilo suppressalis) at field trial. There are a few reports illustrating that in some cases Bt genes were less toxic to first instar Helicoverpa armigera after the plant is producing fruit. The plant’s physiological state and age explained changes in toxicity. Differences in LC50 varied from 2.4 to 726 fold, depending on the source of toxin and conventional plant material. These results suggest that plant toxin interactions in fruiting cotton are reducing the toxicity of the Cry1Ac protein. As transgenic Bt insect resistant cotton, temporal difference of resistance existed in plants converging on different dosages of inserted Bt genes i.e. there was a declining level of efficacy with plant age as the mortality (%) of Helicoverpa and Bt toxin protein expression level decreased gradually, when analyzed with leaves from the main stem (Olsen and Daly, 2000; Guo et al., 2001). Insect pests are major cause of damage to the world’s commercially important agricultural crops. The insecticidal activity of Bt is mainly due to the production of crystal insecticidal proteins. Feeding behavior of bollworm (Helicoverpa zea) and tobacco budworm (Heliothis virescens) was evaluated in pure and mixed stands of non-transgenic and transgenic (BTK) cotton (Gossypium hirsutum L.) expressing an insecticidal protein Cry1Ac. Five plant stands composed of BTK and non-BTK plants were evaluated; two

29

pure stands and three mixed stands. Percentage ratios of BTK to non-BTK plants in the stands were 100:0, 75:25, 50:50, 25:75 and 0:100, respectively. The attack of Bollworm and Tobacco Budworm larvae was found less on BTK plants than non-BTK plants 24h after infestation with 3rd instars. At 48h, bollworm larvae were completely diminished on BTK plants. Mortality %age was greater in case of 1st instar to 4th instar larvae of Bollworm when fed on transgenic cotton plants as compared to 5th instar while no significant difference observed in case of Tobacco Budworm fed both on BTK cotton and non-BTK cotton plants (75.3%-48%, 73.3%-41.3%). Cry1Ac insecticidal proteins are more effective against 1st to 4th instar larvae of Tobacco Budworm and Bollworm when expressing in cotton (Gossypium hirsutum L) plants as compared to 5th instar larvae. The data also suggested that larvae of both species frequently moved among plants, feeding indiscriminately on BTK and non-BTK plants (Estruch et al., 1997; Halcomb et al.. 1996; Halcomb et al., 2000; Van Rie.J., 2000). Bt transgenic variety of upland cotton (Gossypium hirsutum L.) expressing the insecticidal protein Cry1Ac from Bacillus thuringiensis Berliner sp. Kurstaki was evaluated for resistance to Helicoverpa armigera and Pink Bollworm (Pectinophora gossypielli). The results indicated that there was no significant difference in egg densities between transgenic and non-transgenic varieties during the season, although survival of the larvae on Cry1Ac expressing plants was significantly reduced. The larval population of Helicoverpa armigera was significantly higher on non-transformed plants as compared to transgenic. The annual ginned cotton yield was also significantly higher than those in non-Bt cotton. However resistance in H.armigera against Cry1Ac had also been observed and damage percentage was reported higher in few plants (Wu et al., 2003). Similar results were obtained in Pectinophora gossypielli, both resistant and susceptible strains, observed on Cry1Ac expressing cotton. The survival of resistant

30

larvae on transgenic cotton producing Cry1Ac (Bt cotton) was 46% relative to their survival on non-Bt cotton. Compared with susceptible, the resistant strains showed increased ability to survive and develop on Bt cotton and on Cry1Ac-treated diet. In contrast, Bt cotton killed all susceptible larvae tested. F1 hybrid progeny of resistant and susceptible adults did not survive on Bt cotton, which indicates recessive inheritance of resistance. Compared with resistant or susceptible larvae reared on non-Bt cotton, resistant larvae reared on Bt cotton had lower survival and slower development, and achieved lower pupal weight and fecundity (Liu, et al., 2001). Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. To encounter potential problems with resistance, second generation transgenic cotton that produces B.thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed (Stewart et al., 2001; Tabashnik et al., 2002). The assay performed on several Lepidopteran pests on fresh plant tissue indicated that dual toxin B.thuringiensis cultivars, expressing both Cry1Ac and Cry2Ab endotoxins of B.thuringiensis were more toxic to bollworms, Helicoverpa armigera (Boddie), Fall Armyworms, Spodoptera frugiperda (J.E.Smith), and Beet Armyworms, Spodoptera exigua (Hubner), than single-toxin cultivars expressing Cry1Ac. Bollworm and Tobacco Budworm (Heliothis virescens) growth was reduced by Bt cotton, particularly in the dualtoxin cultivars. In contrast, Cry1Ac-resistant Pink Bollworm had little or no survival on second generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong crossresistance to Cry2Aa. Strains with >90% larval survival on diet with 10µg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10µg of Cry2Aa per ml. However, the average

31

survival of larvae fed on a diet with 1µg of Cry2Aa per ml was higher for Cry1Acresistant strains (2-10%) than for susceptible strains (0%) (Chitkowski et al., 2003). Shelton et al., (2000) conducted field tests on managing resistance to Btengineered plants. Present resistance management strategies rely on a “refuge” composed of non-Bt plants to conserve susceptible alleles. They have used Bt-transgenic broccoli plants and the diamondback moth as a model system to examine resistance management strategies. The higher number of larvae on refuge plants in field tests indicated that a “separate refuge” was more effective at conserving susceptible larvae than a “mixed refuge” and reduced the number of homozygous resistant (RR) offspring. Adamczyk and Gore (2004) conducted research to quantify the development of the corn earworm (bollworm), Helicoverpa zea (Boddie), on two different transgenic cotton cultivars (DP 50B and NuCOTN 33B) that contained different levels of the Cry1Ac endotoxin from the soil bacterium, Bacillus thuringiensis Berliner. Using a field cage, an inverse relationship between the amount of Cry1Ac among cultivars versus the weight of bollworm larvae was observed. Larvae that were recovered from the DP 50B cultivar expressing lower Cry1Ac weighed significantly more than larvae collected from the higher expressing NuCOTN 33B cultivar. Cotton plants from NuCOTN 33B were measured as expressing 300% more Cry1Ac than DP 50B plants. The distribution of larval weights indicated that more late-instars (> 200mg) were collected from the lower expressing DP50B cultivar than the higher expressing NuCOTN 33B cultivar. Within a single population, bollworm larvae were highly variable in their development when feeding on Bollgard cotton.

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2.4

INHERITANCE
Zhang et al. (2000) studied inheritance and segregation of foreign Bt (Bacillus

thuringiensis) toxin and tfdA genes in cotton. The transformed cotton varieties CCRI30 and NewCott 33B expressing the Bt Cry1A gene, and cotton line TFD expressing tfdA gene were crossed with CCRI19, CCRI12 and Lumian 6. The results confirmed inheritance and segregation of (i) the exogenous Bt gene in transgenic CCRI 30 and NewCott 33B, governing resistance to bollworm, and (ii) the exogenous tfdA gene in transgene TFD, governing resistance to the herbicide 2,4-D. Both resistance characters were governed by a single dominant nuclear gene, and were not affected by cytoplasm. They concluded that foreign traits encoded by single genes are inherited and expressed in Mendelian fashion in cotton. They also indicated that a practical backcross breeding programme could be used to develop cotton cultivars combining one or more resistance traits from foreign and native gene sources. Altman et al. (1996) analyzed F2 progenies to ascertain the inheritance pattern of Bt genes Cry1Ab and Cry1Ac. Their data indicated that the mode of inheritance was not always Mendelian in different genetic backgrounds. They stated that this situation should not be considered unusual for cotton if transgenes were regarded as another type of exotic gene. This conclusion about exotic genes is generally recognized by cotton breeders and geneticists who normally work with such material. Maluf et al. (2002) studied inheritance of resistance to the root-knot nematode in lettuce. The loose leaf lettuce ‘Grand Rapids’ is resistant to both M.incognita and M.javanica. Resistance to M.incognita has a high heritability, under the control of single gene locus, in which the ‘Grand Rapids’ allele, responsible for resistance (Me), has predominantly additive gene action, and has incomplete penetrance and variable

33

expressivity. The authors studied the inheritance of the resistance of ‘Grand Rapids’(P2) to M.javanica in a cross with a standard nematode-susceptible cultivar Regina-71 (P1). F1 (Regina-71 x Grand Rapids) and F2 seeds were obtained, and the F2 inoculated, alongwith the parental cultivars with a known isolate of M.javanica to evaluate nematode resistance. A high broad sense heritability estimate (0.798) was obtained for gall indices. Class distributions of gall indices for generations P1, P2, and F2 were in agreement with theoretical distributions based on monogenic inheritance model. Bonos (2006) studied the dollar spot disease incited by Sclerotinia homoeocarpa an important disease of creeping bentgrass (Agrostis stolonifera). The objectives of this study were to (i) determine narrow-sense heritability and predicted gain from selection for dollar spot resistance in creeping bentgrass and (ii) evaluate inheritance characteristics of dollar spot disease resistance. Differences in progeny means between crosses were observed over both years. Progeny from resistant × resistant crosses had significantly less disease severity than resistant × susceptible and susceptible ×susceptible crosses. High narrow-sense heritability estimates (0.79 [2002], 0.79 [2003]) and large mean squares for general combining ability supported the idea that additive gene action plays a significant role in disease resistance and support previous research that dollar spot resistance is most likely quantitatively inherited. Panhwar (2002) conducted heterosis studies in six intra specific hybrids of G.hirsustum L. for number of sympodial branches, number of bolls, boll weight and seed cotton yield per plant on an average performance. All hybrids gave better results than their parents. Highest increase of hybrids 69.23% for boll weight over their parents was observed followed by 64.24% for seed cotton yield, 22.97% for number of bolls and 19.62% for number of sympodia per plant.

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Ahmad et al. (2005) studied heterosis and inbreeding depression in 7 x 7 diallel crosses in sunflower. Significant genetic differences were observed among the parents, their F1 hybrids and F2 populations for all characters under study. Yield and leaf area showed highly significant heterosis in F1 hybrids ranging from 102 to 309% and 46.3 to 163.9%, respectively, while inbreeding depression in the F2 population ranged from 17 to 71% and -9.7 to 43%, respectively. Leaves per plant showed level of heterosis in F1 hybrids (-0.9 to 39.7%), whereas the effect of inbreeding depression in F2 population was comparatively high (1.1 to 22.2%) for this character. The parent RHA-822 proved itself to be a good general combiner by making higher contribution towards heterosis both in F1 hybrids and in F2 populations. Iqbal (2003) conducted Generation Mean analysis for seed cotton yield and number of sympodial branches per plant in cotton (Gossypium hirsutum L.). The results showed that 5 crosses over mid and 4 crosses over better parent showed significant heterosis for number of sympodial branches per pant whereas only 4 crosses exhibited inbreeding depression for this character. The generation mean analysis indicated the presence of additive gene action in 3 crosses. Significant marked inbreeding depression from F1 to F2 generation was observed in all the crosses except one for yield of seed cotton per plant. The scaling test revealed involvement of epistasis in all crosses except one for yield of seed cotton. Meredith and Brown (1998) conducted research to determine if parental region of origin was related to mid parent and useful heterosis. They also explored the use of molecular markers (restriction fragment length polymorphisms, RFLPs) and coefficients of parentage in identifying heterotic effects. Significant heterosis over all crosses for total and first harvest yield, lint percentage, boll weight, and 50% span length were detected. For total yield, the specific combining ability and specific combining ability by location

35

interaction components accounted for 79% of the total genetic variance components. General combining ability effects accounted for the remaining 21%. Frick and Bauman (1978) worked on heterosis in maize as measured potassium uptake properties of seedling roots. It appeared to be of adaptive value to the plant and was physiologically related to dent maize hybrid vigour, although a casual relationship was not implied. It was successfully used to predict single-cross hybrid yield to within 5.75 q/ha, over a range of 38.125 q/ha, in more than 75% of the cases. Bourland (1978) used a multiple regression variable selection analysis in cotton. The results demonstrated that low seed weight and short roots on day 9 were important in explaining variability in stand establishment. Biradar and Borikar (1984) working on path analysis for seedling vigour in sorghum, observed that plumule length, radicle length and 100-grain weight were the most highly correlated with seedling dry weight. Stamp (1987) predicted that maize seedling growth was better under fluctuating than under constant temperatures. Heterosis was least at low fluctuating temperatures. Chlorophyll content, ribulose bisphosphate carboxylase activity and phosphoenolpyruvate carboxylase activity varied between temperature regime but not genotypes. At constant 14°C and at three days 18°C, three days 10°C, NADP malate dehydrogenase activity was low and heterosis for the trait high; genotype-specific reactions to temperature regime were marked for this enzyme activity. Magoja and Palacios (1987) studied the hybrids between Zea diploperennis and sweet corn variety Evergreen, and compared with their parents for growth characteristics at 10 to 30 days intervals after planting. Total dry weight increment, a character taking into account the differences in initial seed dry weight between the hybrid and the parents,

36

was always higher in the hybrids than in the either parent. After 30 days, the dry weight of the hybrids was almost twice that of maize and more than twice that of Z. diploperennis. Hoskinson et al. (1964) observed marked varietal differences in cotton, some experimental lines being vigorous and tolerant of the early season disease-insect complex. Alam et al. (1992) performed an experiment involving ten genetically diverse Gossypium hirsutum L. and their 45 F1 hybrids. The data recorded for plant height, seed cotton yield, number of sympodia per plant, number of bolls per plant and ginning outturn percentage were analyzed for combining ability and heterosis over better parent. For all the characters, variance due to GCA and SCA was highly significant. The higher magnitude of the GCA:SCA ratio indicated that additive gene effects controlled plant height, seed cotton yield, number of sympodia per plant and number of bolls per plant. Non-additive type of gene action was observed for ginning outturn percentage. Haq and Khan (1993) conducted a 4x4 diallel cross experiment and genetic analysis of the data showed that plant height was affected with partial dominance whereas overdominance gene action was noted for the number of bolls per plant, boll weight, seed cotton yield and ginning outturn percentage. Carvalho et al. (1994) used diallel cross technique involving six Gossypium hirsutum varieties to produce 30 hybrids in order to study combining ability and heterosis. GCA was observed for all the traits except three fibre quality traits. The values of GCA indicated that additive gene effects played a part in conditioning variability. The seed cotton yield was controlled by non-additive type of gene action. Carvalho et al. (1995) conducted a 6x6 diallel cross experiment to study inheritance of number of bolls per plant, plant height and fibre maturity. The results

37

showed that both dominance and additive effects were more pronounced. In case of yield and boll weight, dominance effects were more dominant. Hussein et al. (1998) conducted an eight-parent diallel analysis and observed the failure of regression analysis, which suggested the involvement of dominance and epistasis for this trait. Additive with partial dominance was reflected from the graphic analysis for seed and lint indices. Additive dominance model was inadequate for lint index in F2 generation. Zhang et al. (2001) studied inheritance of stripe rust resistance by crossing three resistant cultivars of wheat LB, SP, XN4 with one susceptible MX169 and evaluating the resistance of parental, F1, F2 and F3 plants in the field. Transgressive segregation for resistance was observed in the resistance by resistance crosses of LB x XN4 and XN4 x SP but not in cross LB x SP. Broad sense heritability was high in all crosses except LB x SP. Garcia et al. (2002) studied the relationship between the genetic distance measured using RAPD markers, among parental lines, and the heterosis, observed as yield of their F1 hybrids. Estimations of GCA, SCA and heterosis were performed using seven elite lines and their F1 hybrids of “Serrano” pepper. The genotypes tested were statistically different for fruit yield. Among all the hybrids and parental lines, the F1 (P05 x P01) produced the highest yield. Also, GCA and SCA were statistically significant, with P07 showing the highest GCA effect, and the F1 (P05 x P01) the highest SCA. The F1 (P06 x P0) showed the highest heterosis (178.5%). Genetic distances calculated on RAPD markers produced a dendogram with seven nodes for the parental lines. However, the correlation between the matrix of genetic distances among parental lines and the matrix of heterosis was low (r = 0.3281) and not significant.

38

Ahmad et al. (2003) studied a wide range of average performance and genetic variability estimated for F crosses of nine commercial varieties of cotton viz., CIM-443, MNH-147, FH-682, N-1 Karishma, SLS-1, CIM-446, CIM-448, FVH-53 and MNH-552 for bolls and seed cotton yield per plant, boll weight, staple length, ginning out turn (%) and virus infestation (%). The highest genotypic variability was recorded for virus infestation (94.61%) followed by bolls per plant (29.84%). The highest estimates of heritability associated with highest genetic advance for bolls per plant (97.8 and 60.78), virus infestation % (95.0 and 189.9) and boll weight (97.39 and 10.99) suggested selection for improvement of these traits due to presence of sufficient genotypic variability. However, low estimates of these parameters for staple length showed slow progress through selection. Canming et al. (2000) studied 3 kinds of transgenic Bt strains, Shanxi 94-24, Zhongxin-94 and R-19 in upland cotton in China. Genetic studies indicated that the resistance of the three transgenic Bt cotton strains to Helicoverpa armigera is controlled by one pair of non-allelic dominant genes. Linkage relationship between the resistant genes of R-19 and Shanxi 94-24 transgenic Bt strains show that they may be inserted in the same chromosome. F1 hybrids crossed among the 3 strains show that high levels of protection from feeding damage are the same as that of their parents. Therefore, there is no co-suppression phenomenon in many transgenic plants. The results presented afford a fundamental reliance in developing transgenic Bt insect-resistant cultivars and exploiting the heterosis of hybrids in upland cotton. Wu et al. (2002) reported the inheritance and expression patterns of the Cry1Ab gene in the progenies derived from different Bt transgenic japonica rice lines under field conditions. Both Mendelian and distorted segregation ratios were observed in some selfed and crossed F2 populations. From the seedling to the maturing stage, the content of the

39

Cry1Ab produced in the uppermost leaves gradually increased and peaked at the booting stage, then decreased to the lowest at the heading stage. This changing tendency of Cry1Ab content was similar from R4 to R6 generation. The content of the Cry1Ab protein in leaves of transgenic rice reached 0.9% to 0.14% of the total soluble protein in 1998 and 1999, respectively. Husnain et al. (2002) studied expression of an insecticidal gene Cry1Ab under three different promoters in leaves, stem and panicles to determine organ-specificity in Basmati rice. Enhanced Resistance against two Lepidopteran insects, stem borer and leaf folder was observed. The result of Western Hybridization and insect bioassays demonstrated that all these promoters express the Cry1Ab gene at similar levels in leaves and panicles. The Cry1Ab gene was expressed in stems at 0.05% of the total protein under the control of the PEPC promoter alone or in combination with the pollen-specific promoter. On the other hand, it was expressed at 0.15% under the control of ubiquitin promoter. Southern Blot indicated integration of the complete plant transcriptional unit at multiple insertion sites. The results demonstrated that a specific promoter could be used to limit the expression of Cry1Ab in the desired parts of Basmati rice. Bashir et al. (2004) conducted field trials of indica basmati rice (B-370). Sixty neonate larvae of yellow stem borer were artificially infested to each plant in three installments. Data were recorded in terms of dead hearts and white heads at vegetative and flowering stage, respectively. Transgenic lines exhibited inherent ability to protect from target insects. The presence of Cry genes was observed with Dot Blot, PCR and Southern analysis, while ELISA and Western Blot analysis confirmed the presence of Cry proteins. The expression level of Cry1Ac varied from 0.21% to 1.03% and 0.95% to 1.13% of the total protein during 1st and 2nd year, respectively. The transgenic lines had no effect on non-target insects.

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Milicia et al. (1966) found no correlation between germination behavior and earliness of maturity in the hybrids of maize. Burris (1973) reported that seed size and mechanical integrity were correlated with dry matter yield components in most crops. Rajanna et al. (1975) observed significant correlations between the growth analysis parameters and all other seed seedling vigour indices in maize. Kronstad (1977) found that ten barley seedling vigour characteristics were significantly correlated with rate of emergence in the field. From a step-wise multiple regression analysis it appeared that seed weight, three old seedlings ATP content, total adenosine phosphate content in the hydrated embryo, and seven day old seedling dry weight should be good seedling vigour indices for predicting field emergence rate.

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CHAPTER 3

MATERIALS AND METHODS

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3.1
3.1.1

AGROBACTERIUM TRANSFORMATION
Agrobacterium Preparation
The Agrobacterium tumefaciens strain C58C1 was grown in 15ml YEP broth at

tumefaciens

Competent

Cells

300rpm at 30°C for 24 hours. A flask containing 1.0 liter YEP broth was inoculated with the grown culture and placed on an orbital shaker at 350rpm at 30°C for 16 hours. The OD value was recorded at 595nm to confirm the optimal bacterial growth (0.6 to 0.8). The culture was cooled down at 4°C and transferred to two pre-washed 500ml plastic bottles. The cells were harvested through 15 minutes centrifugation at 4000rpm at 4°C. The pellets were twice washed with 500ml HEPES solution. Finally each pellet was dissolved in 1.0ml 10% glycerol solution under ice-cold conditions. The aliquots of 50µl were prepared and stored at -70°C.

3.1.2

Agrobacterium Transformation with pKMAB by Heat Shock Method
The plasmid pKMAB (containing CaMV35S promoter, Cry1Ab gene and T-DNA

terminator) was transformed into Agrobacterium tumefaciens strain C58C1 by heat shock method. A quantity of 2.0µl of plasmid DNA was added to an aliquot of the competent cells of Agrobacterium C58C1. After incubation on ice for 1 hour, heat shock was given at 42°C for 30 minutes. The material were incubated on ice for 2 minutes and diluted with 1.0ml SOC solution. The culture was grown at 28±2°C for 3 hours at 200rpm. The SOC solution was spreaded on YEP medium supplemented with 50mgL-1 kanamycin.

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3.1.3

Long and Short Term Storage of Bacterial Strain
Agrobacterium tumefaciens strain C58C1 containing pKMAB was grown in 10ml

YEP broth containing 50mgL-1 kanamycin for 16-24 hours at 200rpm at 30°C. For short term storage, the culture was streaked on YEP medium supplemented with 50mgL-1 kanamycin. The plates were incubated at 30°C for 24-48 hours and then stored at 4°C for two months. For long term storage, glycerol stocks were prepared. The bacterial culture was mixed with sterile 50% glycerol + 50% YEP broth, in equal volume. The aliquots were made and stored at -70°C for two years.

3.1.4
3.1.4.1

Confirmation of Agrobacterium Transformation
Plasmid Isolation

A few single colonies were picked from the transformed Agrobacterium-streaked plate and put separately in the tubes containing 5ml YEP broth supplemented with 50mgL-1 kanamycin. The tubes were kept on an orbital shaker at 200rpm at 30°C for 48 hours. The culture was centrifuged at 14000rpm at 4°C for 15 minutes and the pellet resuspended in 200µl solution of 50mM Glucose, 10mM EDTA, 25mM Tris-HCl pH 8.0 and 50ng/µl Lysozyme. The mixture was incubated at room temperature for 10 minutes. Another 400µl solution containing 1% SDS and 0.2N NaOH was added. The tubes were inverted several times and incubated at room temperature for 10 minutes. A 60µl solution containing 2 volumes of 0.2N NaOH and 1 volume of Phenol was further added followed by the addition of 300µl 5M Na-Acetate pH 7.9. The mixture was incubated at -20°C for 20 minutes and then centrifuged at 5000rpm for 5 minutes at 4°C. The supernatant was taken and an equal volume of Phenol:Chlorofom:Isoamyl Alcohol (25:24:1) was added

44

followed by centrifugation at 5000rpm for 5 minutes at 4°C. The upper phase was taken and two volumes of ice-cold 95% Ethanol were added to it. The tubes were inverted several times and centrifuged at 5000rpm for 10 minutes at 4°C. The pellet was washed with ice-cold 70% Ethanol, air dried and finally resuspended in T.E. Buffer.

3.1.4.2

Confirmation of Transformation through PCR

The PCR was performed to confirm the Agrobacterium tumefaciens strain C58C1 transformation. The primers specific to Cry1Ab (19 mer Cry1AbF: GTT ACC CTG ATT GAT AGG C and 20 mer Cry1AbR: ACA GAA GAC CTT TCA ATA TC) were used to amplify a specific 550 bp region. The PCR parameters were initial denaturation at 94°C for 2 minutes followed by35 cycles of 94°C for 45 seconds, 52°C for 45 seconds and 72°C for 45 seconds and a final extension of 10 minutes at 72°C. The reaction mixture was prepared using 100ng plasmid DNA, 1.0p.mol/µl forward primer, 1.0p.mol/µl reverse primer, 0.1mM dNTPs, 1X PCR Buffer and 1.0 unit Taq polymerase.

3.2
3.2.1

COTTON TRANSFORMATION
Selection of a Suitable Variety
A cotton (Gossypium hirsutum L.) variety MNH-93 was locally developed at

Cotton Research Station, Multan and released for commercial cultivation in cotton zone of Punjab province. This variety has a good regeneration potential through tissue culture. It has high yield potential and desired fibre characteristics. Moreover, it has shown better genetic stability at field level. Hence the variety was selected for transformation. The seed

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of the variety MNH-93 was obtained from the Directorate of Cotton Research Institute, Faisalabad.

3.2.2

Seed Delinting
The cotton seeds were delinted by applying commercial Sulphuric Acid in a

minimum with sufficient quantity to wet all seed grains. The seeds were vigorously stirred with the help of a spatula until total fuzz was removed and the seed surface became shiny black. The delinted seeds were then rinsed thoroughly in running tap water in order to completely remove the acid which otherwise could affect seed germination. The complete removal of the acid was confirmed with the help of litmus paper.

3.2.3

Seed Sterilization
The seeds were surface sterilized with tap water + Tween-20 for 3 minutes. Seeds

were rinsed with distilled water 4-8 times. Then the seeds were put in a 2.0L glass flask containing 0.1% HgCl2 and 0.1% SDS solution mixture. The seeds were kept on a 200rpm orbital shaker at room temperature for 15 minutes. The seeds were rinsed 4-8 times with autoclaved distilled water. The sterilized seeds were soaked in a minute quantity of distilled water and placed in dark at 30°C overnight. The mature embryos were isolated from the seeds.

3.2.4

Bombardment with Tungsten Particles
The bombardment with Tungsten particles was done to create small wounds on

the surface of the embryos which would facilitate DNA transfer from Agrobacterium. Tungsten particles M10 (60mg) were taken in an eppendorf and washed twice with absolute alcohol and distilled water. The tungsten particles were dissolved in 100ml 50% glycerol, vortexed and stored at -20°C. The frozen stocks of Tungsten were thawed at 4°C

46

and vortexed at high speed for 1 minute. An aliquot of 60µl was taken in an eppendorf and centrifuged for 0.5-1.0 minute at 3000rpm. The pellet was resuspended in 20µl T.E. Buffer. The prepared tungsten particles were then coated on a filter assembly and allowed to dry for 1-2 minutes in a laminar hood. The filter assembly was fixed in leur-lock of Particle Bombardment Gun. The mature embryos were placed at a pre-optimized distance of 22cm and bombardment done under vacuum using helium gas at a pressure of 60lbs/in2.

3.2.5

Agrobacterium Mediated Transformation
The embryos were co-cultivated with Agrobacterium tumefaciens strain C58C1

harboring pKMAB plasmid. The overnight culture of Agrobacterium was grown in YEP broth containing 50mgL-1 kanamycin at 200rpm for 16-24 hours. An aliquot of 1.0ml of culture was taken to estimate the OD of the culture at 595A°. The required OD was 0.5-1. The culture was centrifuged at 3000rpm for 30 minutes at 4°C to get pellet. The supernatant was decanted and the pellet dissolved in 5ml MS broth. The bombarded embryos were co-cultivated with this prepared culture for 30 minutes at 70rpm. The embryos were blot-dried and cultured on MS medium. Twenty five non-transformed embryos were cultured on MS medium as control. The plates were kept at 28±2°C for 3 days. After 3 days, plantlets were sub-cultured on selection medium i.e. MS medium containing 50mgL-1 kanamycin. Cefotaxime (250mgL-1) was also added to inhibit bacterial overgrowth. Sub-culturing was done after every 10 days. After 2 months selection, these plants were shifted to kanamycin-free MS medium and kept in it till fully developed seedlings were obtained. Another method was also applied for co-cultivation. The wounded embryos were directly co-cultivated with 15ml transformed-Agrobacterium culture in a petri plate placed on an orbital shaker at 70rpm for 15 minutes at 30°C. The

47

embryos were blot-dried and implanted on MS medium plus kanamycin (50mgL-1) as a selectable marker. The growing embryos were sub-cultured after every four days on MSkanamycin medium. On attaining a sufficient length of the growing embryos, these were sub-cultured in magenta boxes containing the same selection medium. The plantlets remained on selection medium for a total of two months after which they were shifted to selection free medium and then to soil.

3.3

MOLECULAR

ANALYSES

OF

TRANSGENIC PLANTS
3.3.1 Genomic DNA Isolation
A combination of two methods (Saha et al., 1997 and Zhang et al., 2000) with modifications was optimized and followed for DNA isolation from cotton leaves. About 0.5gm fresh terminal leaves were taken and ground in liquid N2. The ground powder was put in eppendorf and incubated on ice for 1-2 minutes. 1.0ml DNA extraction buffer was added to it. The eppendorfs were centrifuged at 5000-6000rpm in a benchtop centrifuge. The supernatant was removed and 0.5ml DNA Lysis Buffer was added. The tubes were incubated at 65°C for 30 minutes. An equal volume of Chloroform:Isoamyl Alcohol (24:1) was added and centrifuged at 14000rpm for 20 minutes at 4°C. The aqueous phase was transferred to new tubes. The above step was repeated. An equal volume of Isopropanol was added and incubated at room temperature for 1 hour. The DNA was spooled out in new eppendorfs and air-dried. 1.0ml solution containing 80% Ethanol and 0.2M Na-Acetate pH 5.2 was added to each eppendorf. The tubes were incubated at 4°C for 30-60 minutes and centrifuged at 14000rpm for 15 minutes. The pellet was washed with 70% Ethanol. The pellet was air-dried and resuspended in 200µl TE Buffer. The

48

tubes were incubated at 65°C for 1 hour. The tubes were centrifuged at 12000rpm for 15 minutes. The supernatant was taken and 2.0µl RNAase (10mg ml-1 stock) was added. The tubes were incubated at 37°C for 15 minutes. 400µl 100% Ethanol (2 volumes) and 20µl 3M Na-Acetate pH5.2 (0.1 volume) were added to each tube. The tubes were incubated at -20°C for 1 hour. The DNA was spooled out and air-dried. The DNA was finally resuspended in 100µl low EC water or TE Buffer. The DNA concentration was estimated by running samples on 0.7% Agarose gel and comparing with the λ-uncut marker or λ/HindIII bands of known concentrations.

3.3.2

Polymerase Chain Reaction
The PCR was performed to screen positive plants with the following set of

primers:Cry1Ab forward primer 5´-GTT ACC CTG ATT GAT AGG C-3´ (nucleotide 1106-1125) Cry1Ab reverse primer 5´ACA GAA GAC CTT TCA ATA TC-3´ (nucleotide 1636-1656) The total amplified portion was 550 base pairs. The PCR parameters were initial denaturation at 94°C for 2 minutes followed by 35 cycles of 94°C for 45 seconds, 52°C for 45 seconds and 72°C for 45 seconds and a final extension of 10 minutes at 72°C. The reaction mixture was prepared as described above in section 3.1.4.2.

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3.3.3
3.3.3.1
3.3.3.1.1

Southern Hybridization
Probe Making/DNA Labelling
Plasmid Digestion

The 20µg plasmid DNA (pKMAB) was digested with 120 units each of the enzymes EcoRI and BamHI to release 1.845kb Cry1Ab gene fragment. The reaction was carried out in the presence of 1X Buffer of BamHI at 37°C for 4 hours. The DNA was precipitated by adding 3 volumes 100% Ethanol, 0.8 volume 7.5M Ammonium Acetate and 10µl glycogen followed by incubation at -20°C for one hour. The DNA was finally resuspended in 40µl water. 3.3.3.1.2 Gel Elution

The desired DNA fragment (1.845kb) was eluted using DNA Extraction Kit of Fermentas. The digested DNA was run on 0.8% TAE-Agarose gel till the gene fragment was completely separated. The gel slice containing the gene fragment was excised and weighed on an electronic balance. The approximate volume of the gel slice was determined by taking 1.0g equal to 1.0ml. The gel slice was put in three volumes of Binding Solution (6M Sodium Iodide) and incubated at 55°C for 15 minutes to dissolve the gel. The Silica Powder suspension was added to the tube at the rate of 2µl per µg DNA and again incubated at 55°C for 5 minutes. The tube was vortexed every 1-2 minutes. The tube was centrifuged at 13000rpm for 5 seconds. The supernatant was removed. The pellet was washed thrice with 500µl ice-cold wash buffer (containing Tris, NaCl and EDTA). The pellet was air-dried and resuspended in water or TE Buffer using a volume equal to the initial volume of the silica powder suspension added. The tube was incubated at 55°C for 5 minutes. The tube was centrifuged at 13000rpm for 10 seconds. The supernatant was transferred to a new tube. The new tube was centrifuged again at 50

13000rpm for 30 seconds to remove small amounts of silica powder. The supernatant was transferred to a new tube. The amount of DNA was quantified either with the help of a spectrophotometer or running an aliquot on 1% Agarose gel. The final concentration of DNA was maintained at 100ng per µl with the help of DNA concentrator. 3.3.3.1.3 DNA labeling with Biotin-11-dUTP

The labeling was done using Biotin DecaLabel™ DNA Labeling Kit of Fermentas. 10µl DNA template (100ng per µl), 10µl Decanucleotide in 5X buffer and 44µl water were mixed in an eppendorf by vortexing. The eppendorf was centrifuged at 10000rpm for 3-5 seconds followed by incubation in boiling water for 5-10 minutes. The eppendorf was cooled down on ice and spun down quickly in microcentrifuge. The 5µl Biotin Labeling Mix (1mM dGTP, 1mM dATP, 1mM dCTP, 1mM dTTP and 0.35mM Biotin-11-dUTP aqueous solution) and 1µl (5U) Klenow fragment (exo¯) were added to the eppendorf. The eppendorf was vortexed briefly and centrifuged at 10000rpm for 3-5 seconds. The eppendorf was incubated at 37°C for 20 hours. The reaction was stopped by adding 1.0µl 0.5M EDTA pH 8.0. 3.3.3.1.4 Probe Estimation

Four serial dilutions (containing 1.0µl, 0.1µl, 0.01µl and 0.001µl DNA) each of labeled DNA and control labeled DNA were spotted on Hybond-N membrane. The membrane was air-dried. The DNA cross-linking was performed by exposing the membrane to UV light for 3 minutes. The detection was done as given in section 3.3.3.5 below.

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3.3.3.2

Genomic DNA digestion
The DNA (27µg) of transformed plants was digested with EcoRI (160U) and

BamHI (160U) enzymes. The reaction was incubated at 37°C overnight. The samples were checked for complete digestion on 0.8% Agarose gel.

3.3.3.3

Gel Running for Southern Hybridization

The completely digested samples were loaded on 1cm thick 1% Agarose gel and run at 30 volts overnight. The gel was immersed in Depurination solution and kept on shaking for 10 minutes until Bromophenol blue turned yellow. The Depurination solution was replaced with the Denaturation solution and kept on shaking for 45 minutes until yellow color turned blue. The Denaturation solution was discarded and Neutralization solution was added. The gel was kept on agitation for 35 minutes.

3.3.3.4

Gel Transfer Assembly

A 3mm filter paper wick pre-soaked with 2X SSC (Standard Sodium Citrate) solution was placed on a ceramic tile mounted on a steel tray filled with10X SSC solution. The sides of the paper wick were immersed in the solution. The three layers of Whatman Sheets were placed on the wick onto which the gel was placed upside down. The Hybond-N membrane was placed on the gel above which a pile of blotting papers and newsprint were placed. The size of the membrane, blotting papers and the newsprint was exactly same as that of the gel. A 1.5 kg weight was put on top of the assembly. The assembly was left overnight to facilitate DNA transfer from the gel to the membrane through capillary action.

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3.3.3.5

Blot Processing

The transfer assembly was disassembled and the membrane air-dried. The membrane was wrapped in saran wrap and UV cross-linked for 3 minutes. The membrane was incubated with Pre-hybridization solution at 65°C for 2 hours. The Pre-hybridization solution was discarded and the membrane was incubated with Biotin-labelled Cry1Ab Probe solution at 65°C for 12 hours. The Probe solution was denatured in boiling water for 10 minutes before use. The detection was done using Biotin Chromogenic Detection Kit (Fermentas). The membrane was kept on shaking in 1X Blocking/Washing Buffer for 5 minutes at room temperature. The Washing Buffer was replaced with 1% Blocking Solution and kept on shaking for 30 minutes. The Blocking Solution was replaced with Streptavidin-AP conjugate. The membrane was kept on shaking at room temperature for 30 minutes. The membrane was twice washed with Blocking/Washing Buffer by placing it on shaking for 15 minutes each time. The membrane was incubated in 1X Detection Buffer for 10 minutes. The membrane was finally incubated in 1X Substrate solution (BCIP/NBT) at room temperature in the dark for 30 minutes.

3.3.3.6

Copy Number Estimation
To estimate copy number of the transgene in the transformed plants, 27µg

genomic DNA was digested with 320 units (about 12U/µg) of the enzyme SstI (SacI) which has a unique site in the plasmid pKMAB. The Southern Hybridization was done as described above in the sections 3.3.3.3 to 3.3.3.5.

3.3.4

Immunological Assay of Transgenic Plants
The transgenic cotton plants were analyzed through ELISA and Western Dot Blot

for protein expression studies.

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3.3.4.1

Isolation of Protein from Cotton Leaves

About 0.5gm fresh terminal leaves were ground in liquid nitrogen. 1.0ml Protein Extraction Buffer was added and centrifuged at 14000rpm at 4°C. The supernatant was taken. The protein concentration was estimated by taking OD value at 595A° and calculated comparing with the already plotted curve of known concentrations of BSA (Bovine Serum Albumin).

3.3.4.2

Enzyme Linked Immunosorbent Assay

50µg total protein was loaded on a 96-well ELISA plate. The Carbonate Buffer (100µl) was added to each well. The plate was incubated at 4°C overnight. The wells were thrice washed with 1X PBS. 5% skimmed milk (200µl) was put in each well. The plate was incubated at 37°C for 30 minutes. The wells were thrice washed with 1X PBS. The primary antibodies (200µl) already diluted in 5% skimmed milk (2µl/ml) were added to each well. The plate was incubated at 37°C for 2 hours. The wells were thrice washed with 1X PBS. 200µl of skimmed milk (5%) was added in each well and the plate was incubated at 37°C for 30 minutes. The wells were thrice washed with 1X PBS. The secondary antibodies (200µl) already diluted in 5% skimmed milk (0.5µl/ml) were added to each well. The plate was incubated at 37°C for 1 hour. The wells were thrice washed with 1X PBS. The color substrate NBT/BCIP (1 tablet dissolved in 10ml water) was added to each well. The plate was incubated at room temperature for 45 minutes. The reaction was stopped with 2M Na2CO3.

3.3.4.3

Western Dot Blot

The Hybond-C membrane was used for Western Dot Blot. 10ng protein was loaded on the membrane. The membrane was completely air-dried and incubated in Blocking Buffer/Reagent for 30 minutes at room temperature or overnight at 4°C. The 54

membrane was thrice washed with 1X PBS. The membrane was probed with the diluted primary antibodies (1:2500) for 1 hour. The membrane was washed thrice with 1X PBS. The membrane was probed with the diluted secondary antibodies (1:2000) for 1 hour. The membrane was washed thrice with 1X PBS. The color was developed in AP Buffer (NBT/BCIP). The Bt contents were quantified after scanning the blots by using software Image Quant TL supplied by Amersham Biosciences Corporation (Pvt) Limited.

3.4

INSECT BIOASSAYS
The transgenic cotton plants were subjected to lab bioassays with American

Bollworm (Heliothis armigera). Five fresh leaves from each plant were taken and placed on wet filter paper in petri plates accommodating one leaf per plate. One 1st/2nd instar larva, pre-fasted for 4-6 hours, was released in the each plate and allowed to feed on the leaf. The data on insect mortality were taken on daily basis upto seventh day. The plants were categorized as under:Resistant plants Susceptible plants 40% or more insect mortality Less than 40% % insect mortality

3.5

FIELD STUDIES
For the purpose of field studies, the transgenic cotton plants were planted in the

experimental area of National Center of Excellence in Molecular Biology, University of the Punjab, Lahore during the years 2002, 2003, 2004 and 2005. All

standard/recommended practices for raising plants were adopted. Besides raising cotton crop during the kharif season, an additional crop per year was obtained in the green house during November-March. The temperature and humidity 55

conditions in the green house were maintained at 25-40°C and 30-70%, respectively. The field studies were multifarious in nature and are described below, in seriatim.

3.5.1

Development of Transgenic Pure Lines
Each transformation event results in a different change in the genetic architecture

of the cells. The site of gene insertion and the copy number are always varying. There may be up-regulation or vice-versa of some native genes due to the insertion of a foreign gene. Similarly, the expression of one or more gene(s) may be masked or enhanced otherwise. Some native genes may become silenced or the silenced genes may become active due to the pleiotropic effects of foreign gene. In brief, each transgenic plant has its unique genetic architecture and level of expression. It was therefore imperative that each transformed plant may be studied separately.

3.5.1.1

1st Generation

The plants of cotton variety MNH-93, transformed with the gene Cry1Ab, were grown first time under field conditions during kharif, 2002. The data on the following characters were recorded:i) Number of Bolls per Plant

The total number of bolls was counted at the end of season. Since cotton is an indeterminate crop and new bolls keep on forming throughout the year, both mature and immature bolls were included in counting. ii) Percent Boll Damage Under Natural Conditions

The transgenes were meant to control Lepidopteran insects, therefore no insecticidal spray against the Lepidopteran insects was applied. The plants were monitored for degree of damage due to Lepidopteran insects during the entire season. The

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Boll Damage was calculated in percentage by dividing the number of damaged bolls with the total number of bolls on the plant, multiplied by 100. iii) Laboratory Bioassay

The lab bioassays of the plants were done as described in the section 3.4.

3.5.1.2

2nd Generation

The progenies of the 1st generation plants were grown during Kharif, 2003. The field layout was planned according to the international guidelines for Bt-crop growing. Each plant progeny consisted of two rows (each row of 10m length) accommodating 66 plants. The plant to plant and row to row distances were kept at 30 and 75cm, respectively. The transgenic plants were surrounded by 4.5m wide belt of non-Bt cotton as refugia. The whole cotton field was further surrounded by 4.5m wide sorghum belt, as an isolation boundary. The data were recorded on individual plant basis for the following characteristics 1. 2. 3. 4. 5. 6. 7. 8. Boll Damage Percentage due to natural infestation of bollworms Insect Mortality Percentage in lab bioassay with Heliothis larvae Seed Cotton Yield (g) Plant Height (cm) Number of Monopodial Branches per Plant Number of Sympodial Branches per Plant Confirmation of transgene presence through PCR, and Confirmation of transgene expression through Western Dot Blot.

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3.5.1.2.1

Selection Criteria

Single plant selection was made on the basis of the following criteria:1. The plant must be positive in molecular screening tests viz. PCR and Western Dot Blot; 2. 3. The plant must be higher yielding in terms of seed cotton; The plant must bear less than 5% boll damage due to natural infestation of bollworms; 4. The plant must have shown at least 40% insect mortality in the lab bioassays; 5. The plant should bear low number of monopodial branches and high number of sympodial branches; 6. The plant should give Ginning Outturn Percentage equal or more than the control plants; and 7. The plant should be equal or short in stature than control.

3.5.1.3

3rd Generation

The progenies of the selected plants from 2nd generation were raised in the green house during winter 2003-04. The plants were grown in tumbler-shaped earthen pots of 60cm height and 45cm upper diameter. Each pot accommodated a single plant. Each progeny consisted of ten plants. The light, temperature and humidity conditions of the green house were maintained by using high-powered lights, heaters and humidifiers. During the entire plant growing period, the temperature and humidity ranged between 2540ºC and 30-70%, respectively.

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The plants were again subjected to individual studies and molecular analysis. Plant selections were made on the basis of the criteria stated above. The plants in this generation showed 50% to 100% homozygosity, therefore the seed of selected plants from each progeny row was bulked to have four pure lines.

3.5.1.4

4th & 5th Generation

The four pure lines mentioned above were further analyzed at molecular level, for two successive generations to eliminate false positive plants and to check for errors in selection. A sample of 30 plants, at random, was taken from each pure line during every year and analyzed at molecular level through PCR and Western Dot Blot.

3.5.2
3.5.2.1

Field Trials
Bt TRIALS 2004-2005

The field trials comprising of four pure lines/Bt genotypes viz. CEMB-3, CEMB-11, CEMB-16 and CEMB-17 and one non-Bt genotype/variety MNH-93, as control, were conducted for two consecutive years during 2004-2005. The trials were planted using Randomized Complete Block Design with three replications. The plant to plant and row to row distance was kept 30 and 75cm, respectively. The plot size was 3m x 6m. No insecticidal spray against Lepidopteran insects was applied. The crop was however, protected from the attack of sucking pests by applying suitable insecticides. All agronomic practices were adopted as per recommendation of the Punjab Agriculture Department.

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The data were recorded on the following characteristics:1. Bt Protein Percentage The Bt contents in all genotypes were quantified to have a true picture of variation in insect resistance of the plants due to Bt concentration. The total protein was isolated from fresh leaves of the plants. The concentration of total protein was estimated with the help of spectrophotometer at 595 nm. The reading was compared with the BSA standard curve. An equal amount of protein of each sample (10ng) was loaded directly onto a Hybond-C membrane and the procedure of Western Blotting was followed. After processing, the blot was scanned and the Bt contents quantified using ImageQuant TL software of the Amersham BioSciences (Pvt) Limited. 2. Natural Infestation of Spotted Bollworm The pest scouting was done on weekly basis in the cotton trials. The number of spotted bollworms was counted. Occasionally, a few larvae were found dead on the transgenic lines due to Bt effect. The dead larvae were not included in the data. The results were averaged at the end of the season to have a comparison of the occurrence of the insects. 3. Field Bioassays Different methods were tried to conduct field bioassays with 1st/2nd instar American Bollworm (Heliothis armigera) e.g. placing the larvae on leaves of the plants with the help of camel hair brush; releasing pupae to pupate in the field, grow into adult, lay eggs and damage the plants at larval stage; and releasing the lab-reared larvae through glass vials in the field. The method of glass vials was proved to be the better one. For the purpose of field bioassays, a large number of Heliothis larvae were reared in the CEMB insectary. Ten larvae of 1st/2nd instar were placed in a small glass vial and 60

tied to the middle of the stem of each plant in the experiment. The glass vial was opened afterwards to let the insects travel to all parts of the plant. The number of live insects per plant were recorded daily in the field upto seventh day of artificial infestation. 4. Insect Resistance Shown In Lab Bioassays The laboratory bioassays were conducted as described in section 3.4. 5. Seed Cotton Yield (g) The seed cotton picked from ten randomly selected plants from each plot during all pickings was mixed and weighed on an electronic balance. Average yield of seed cotton per plant for each genotype in each replication was calculated by dividing the total yield with the number of plants and expressed in grams. 6. Plant Height (cm) The plant height was recorded in centimeters from base to the apex by using a meter rod. 7. Number of Monopodial Branches per Plant At maturity, the number of monopodial branches (indirect fruiting branches) of ten randomly selected plants from each plot were counted and averaged for the purpose of statistical analysis. 8. Number of Sympodial Branches At maturity, the number of sympodial branches (direct fruiting branches) of ten randomly selected plants from each plot were counted and averaged for the purpose of statistical analysis.

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9. Number of Bolls per Plant Ten plants in each plot were randomly selected for data recording. The actual count of effective bolls on the plant to be sampled was recorded and summed up for all pickings. The mean was calculated by dividing total number of bolls with the number of plants. 10. Average Boll Weight (g) The average boll weight was obtained by dividing the total seed cotton yield of ten randomly selected plants in each plot by the respective total number of effective bolls. 11. Ginning Outturn (%age) The produce of each plant was cleaned and dried. A sample of 100 grams from each plant was taken and ginned separately with a Single Roller Electric Gin. The lint obtained was weighed and the following formula was used to calculate Ginning Outturn (GOT):GOT %age = (Weight of lint / Weight of seed cotton in the sample) x 100 12. Staple Length (mm) The Staple Length was measured by Fibrograph Model 530 (electronic). The “Tuft Method” was also used to measure Staple Length. A representative sample of lint of each plant was taken and turned into a sliver and run through a draw box till a uniform band of parallel fibers was obtained. These fibers were then mounted on a set of metallic comb, fixed parallel to each other on a stand. One end of the processed sample was aligned using tweezers, and two tufts were drawn from each sample. The tufts were placed on a velvet covered tuft board and two lines were drawn, one on the even end of the tuft just beneath the grip mark of the tweezers and second on the opposite end of

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the tuft where the rate of change of visual density of fibers was found to be maximum. The distance between the two lines was measured with a fine millimeter scale. The average staple length was calculated by taking the mean of the two tufts of a sample. 13. Fibre Fineness(µg/in) The Fibre Fineness was measured with the help of “Sheffield Micronairecomplete with Air Compressor” and expressed in microgram per inch. The data collected on the above-mentioned characters were subjected to analysis of variance and mean comparisons as described in the section 3.11 below.

3.5.2.2

Comparative Study of Insecticide Applications on Bt and Non-Bt Cotton Lines, 2004-2005.

The experiments were conducted to assess the possible reduction in number of spray applications against Lepidopteran insects on Bt lines in comparison with the number of spray applications on non-Bt cotton. The experiments were conducted during kharif seasons of 2004 and 2005. Regular pest scouting was done and insecticide was applied against Lepidopteran insects as and when needed according to the experimental treatments. 3.5.2.2.1 Insecticide Application Trial, 2004

A simple experiment was conducted during the year 2004. The Bt genotype CEMB-3 (transformed MNH-93) and its non-Bt counterpart CEMB-C (non-Bt MNH-93) were sown in adjacent plots of the same size (3m x 6m). The plots were separated completely by planting 3m wide sorghum belt between the two plots to eliminate the possibilities of target insect travelling from one plot to another, and to avoid the effect of insecticidal sprays of one plot on the other. The experiment was thrice replicated. The

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non-Bt lines were regularly sprayed with suitable insecticides to control the Lepidopteran insects, whereas no insecticide was applied to the Bt lines throughout the season to control Lepidopteran insects. The seed cotton yield per plot was recorded and the means were statistically compared by using “t-test assuming unequal variances” as explained in the statistical analysis section 3.11. 3.5.2.2.2 Insecticide Application Trial, 2005

.In the year 2005, the experiment was conducted using Split Plot Randomized Complete Block Design with four replications having genotypes in the main plots and treatments (insecticide applications) in the sub-plots. The trial comprised of one Bt genotype CEMB-3 and one non-Bt genotype MNH93. There were four insecticide treatments as follows:L0 L1 No insecticide application during the entire season Insecticide Application at an ETL (economic threshold level) of 3 larvae per 25 plants – general recommendation by the Punjab Agriculture Department L2 L3 Insecticide Application at an incidence of 5 larvae per 25 plants Insecticide Application at an incidence of 8 larvae per 25 plants

The following four distinct classes on the basis of number of applied insecticidal sprays (against Lepidopteran insects) in each genotype were framed;Class 1 Class 2 Class 3 Class 4 0 sprays 1.75 spray 2.5 sprays 3.0 sprays 64

All classes of one genotype were compared, one by one, with all four classes of the other genotype. In this way, a total of 16 combinations were made. The corresponding yields of the classes were also compared, likewise. The combinations exhibiting significant differences in number of spray applications as well as yields were identified. The yield data were subjected to analysis of variance and the comparisons were made using t-test, as described in the statistical analysis section 3.11.

3.6

Bt INHERITANCE STUDIES
The inheritance of Bt gene was studied in two ways:1. 2. Inheritance of Bt gene in selfed transgenic generations, and Inheritance of Bt gene in filial generations.

3.6.1

Bt Inheritance in Transgenic Selfed Generations
The Bt gene inheritance studies were done in five successive selfed generations of

Bt plants. The plants were analyzed through different molecular techniques. The stable integration and faithful transmission of the gene in successive generations was studied with the help of PCR. The expression studies were done with the help of Western Dot Blot technique. Proper history-sheets of each plant and its progenies were maintained.

3.6.2

Bt Inheritance in Filial Generations
To study the Mendelian inheritance of the gene, filial generations were developed

through crossing between transgenic and non-transgenic lines.

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3.6.2.1

Crossing among Bt and Non-Bt Lines

The transgenic pure lines CEMB-3 and CEMB-11 alongwith two non-transgenic varieties MNH-93 (the original parent of the transformed lines having the same genetic architecture except the Bt gene) and CIM-482 (the non-Bt variety having genetic background altogether different from Bt lines) were sown in 60cm x 45cm earthen pots in the green house on 1st January, 2004. The temperature and humidity of the green house ranged between 25-40°C and 30-70%, respectively. The electric heaters and highpowered lights were used as and when required to maintain the temperature. Similarly, humidifiers were used to maintain the humidity in the green house. When the parent plants initiated flowering, crossing work was started. Maximum number of flower emasculations were attempted to obtain sufficient quantity of F1 hybrid seed. 3.6.2.1.1 Emasculation

Emasculation was done daily in the afternoon. The petals of the squares (unopened flower buds) were removed carefully with the help of a scalpel and forceps. The anthers were removed with the forceps. The stigma was covered by using paper straw tube closed at the upper end to avoid any natural pollination. The emasculated square was tagged with a colored tag to make it distinguished from the other flowers. 3.6.2.1.2 Pollination

The pollination was done in the morning during 9.00 to 10.00 a.m. A freshly opened flower from the male parent was detached from the plant. Its pollen were either collected in a butter paper bag and dusted on the stigma of the emasculated flower with the help of a camel hair brush, or the pollen were dusted directly on the pre-selected stigma by rubbing flower over the stigma. After pollination, the stigma was again covered

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with a paper straw tube closed at the upper end. The colored tag was replaced with the white tag to distinguish it from the un-pollinated flowers. 3.6.2.1.3 Combinations of crosses

The following six combinations of crosses were made:Cross.No. 1. 2. 3. 4. 5. 6. FEMALE PARENT MNH-93 CEMB-3CIM-482 CEMB-3 MNH-93 CEMB-11 MALE PARENT CEMB-3 MNH-93 CEMB-3 CIM-482 CEMB-11 MNH-93

3.6.2.2

Inheritance Studies in F1 Generation

The F1 plants were grown in green house during winter, 2004-05. The plants were analyzed in the laboratory through PCR and Western Dot Blot. The transfer of Bt gene from a Bt plant to a non-Bt plant was checked. The percentage of positive plants was calculated to infer the behaviour of the transgene whether dominant or recessive.

3.6.2.3

Inheritance Studies in F2 Generation

The F2 generation was grown in the CEMB experimental area during kharif, 2005. The progeny of each F1 hybrid was grown using maximum seed available. The purpose of sowing F2 was to study segregation pattern of Bt gene in the second filial generation i.e. whether the transgene was being inherited in Mendelian fashion or otherwise. The plants were analyzed at molecular level by the technique of Western Dot Blot. The plants were clearly classified as Bt-positive or Bt-negative. The results were analyzed statistically by chi-square goodness of fit test as described in the section 3.11.

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3.7

HETEROSIS AND HETEROBELTIOSIS STUDIES
Heterosis is increased vigour of different characteristics in hybrids; the possibility

to obtain a “better” individual by combining the virtues of its parents. This is commonly known as “hybrid vigour” or “outbreeding enhancement”. It is often the opposite process of inbreeding depression, which increases homozygosity. Heterosis is an example of heterozygous advantage. The estimation of hybrid vigour has been of prime importance to the plant breeders. It is defined as the percent increase or decrease of a hybrid over its mid-parent whereas percent increase of a hybrid over its better parent is called “Heterobeltiosis”. The seed of four parental lines and six crosses was sown on 7th October, 2004 in the green house. The sowing was done in 30cm x 45cm earthen pots using a Randomized Complete Block Design with three replications. One replication comprised of ten plants of each genotype. Five plants were randomly selected from each genotype for recording data on the following characters 1. 2. 3. 4. 5. Seed Cotton Yield (g) Number of bolls per Plant Boll Weight (g) Ginning Outturn Percentage (GOT %age) Lab Bioassay Results (Mortality %age of Heliothis larvae)

The data were subjected to analysis of variance technique followed by means comparison through New Duncan’s Multiple Range test. The estimates of heterosis and

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heterobeltiosis and their respective tests of significance were calculated as described in the statistical analysis section 3.11.

3.8.

HERITABILITY AND GENETIC ADVANCE STUDIES
The variation in any character in a segregating or mixed population is due to both

genetic and environmental factors. The genetic factor is foremost important in plant breeding since it can be used to improve the population. The greater the proportion of total variability that is due to environmental factors, the more difficult it will be to select for inherited differences. If environmental variability is small in relation to genetic differences, selection will be more efficient. The inherited portion of the variability is called “heritability”. Therefore, heritability is a statistic that may be used to evaluate the effectiveness of selection during segregation generations. It is a measure of the value of selection of a particular character and an index of transmissibility of the genes controlling the character. Genetic variance is the only component of the phenotypic variance that is heritable and useful in crop improvement. Therefore, the ratio of genetic variance to the phenotypic variance is called “Broad Sense Heritability”. When heritability estimates are available, progress from selection can be predicted for any breeding system, since expected gain is a function of heritability. When a genetically variable population is subjected to selection, a proportion of the population with extreme phenotype is selected or saved, the rest of the population being discarded. The mean performance of the progeny from the selected plants is expected to be higher than that of the original or base population. This increase in performance per generation is called “Genetic Advance” or “Genetic Gain”.

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The ‘Broad Sense Heritability’ (BSH), ‘Genetic Advance’ and ‘Relative Expected Gain’ (in order to make comparison in gain in selection) were calculated as described in the statistical analysis section 3.11.

3.9

CORRELATION STUDIES
The strength and direction of a linear relationship between two random variables

is called “Correlation”. In general statistical usage, correlation or co-relation refers to the departure of two variables from independence and measured with correlation coefficient (r) with range (-1) ≤ (r) ≤ (+1). Therefore, Correlation is a bivariate measure of association (strength) of the relationship between two variables. It varies from 0 (random relationship) to 1 (perfect linear relationship) or -1 (perfect negative linear relationship). In the present studies, correlation of Bt insect resistance character (Bt content in the genotypes expressed as percent of the total protein) with the following economic characters of cotton was studied:1. 2. 3. 4. 5. 6. 7. 8. 9. Seed Cotton Yield (g) Plant Height (cm) Number of Monopodial Branches per Plant Number of Sympodial Branches per Plant Number of Bolls per Plant Boll Weight (g) Ginning Outturn Percentage Staple Length (mm) Fibre Fineness (µg/in)

70

10.

Natural infestation of Spotted Bollworm (An important parameter, other

than the morphological plant characteristics, was also included in the study i.e. intensity of natural infestation of Spotted Bollworm in the field. The data were recorded on Bt and non-Bt cotton genotypes sown in the field. The number of live insects per plant were counted during the season and averaged at the end. The data thus generated were associated with the Bt content in the genotypes to compute correlation). The correlation was calculated as described in the statistical analysis section 3.11.

3.10

COMPARISON OF SOME QUALITATIVE CHARACTERS COTTON
The observations on the following qualitative characters of Bt and non-Bt cotton

OF

Bt

AND

NON-Bt

were also taken during the present studies:1. 2. 3. 4. 5. 6. Plant Shape Leaf Hairiness Boll Shape Boll Opening Reaction to Virus Reaction to Bollworms

The changes occurred in the Bt cotton after transformation were compared with the original characters.

71

3.11

STATISTICAL ANALYSES
The data recorded in various experiments were statistically analyzed as described

below:-

3.11.1

Analysis of Variance

The analysis of variance was done with the help of the techniques mentioned by Steel and Torrie (1980). To establish the level of significance among various genotypes, New Duncan’s Multiple Range Test (5% level) was applied to compare the means for all parameters.

3.11.2

t-test Assuming Unequal Variances

The t-test assuming unequal variances was computed by the following formula given by Snedecor and Cochran (1989):-

where Y1 = Σ Y1 /n1 Y2 = Σ Y2 /n2 s12 = Σ (Y1- Y1)2/ (n1-1) s22 = Σ (Y2- Y2)2/ (n2-1)

72

The degrees of freedom were calculated by the following formula:-

F-test was applied to see whether the variances were equal or not by the following formula:F = s12 / s22

3.11.3

Chi Square Test

The chi square test (χ2 )was applied to the data recorded from F2 generation using the following formula:χ2 = ∑ (O-E)²/ E where O = observed values E = expected values

3.11.4

Estimation of Heterosis and Heterobeltiosis

Heterosis was calculated as percent increase (+) or decrease (-) exhibited by the hybrids over mid parent. Heterobeltiosis was calculated as percent increase (+) or decrease (-) exhibited by the F1 hybrid over better parent. Both were calculated by using the following formulae:Heterosis (%) = ( F1 - Mid Parent ) X 100 Mid Parent Heterobeltiosis (%) = ( F1 - Better Parent ) X 100 Better Parent

73

The “t” test was employed to determine whether F1 hybrid means were statistically significant from mid parent and better parent values or otherwise. The “t” values were calculated by the following formulae narrated by Wynne et al. (1970).

3.11.5

t-test for Heterosis
“t” = (F1ij – MPij ) / √⅜ems

where F1ij = the mean of ijth F1 cross, MPij = the mid parental value of ijth F1 cross, and ems = the error mean square.

3.11.6

t-test for Heterobeltiosis

“t” = (F1ij – MPij ) / √½ems where F1ij = the mean of ijth F1 cross, MPij = the mid parental value of ijth F1 cross, and ems = the error mean square.

3.11.7

Heritability Estimates

The Broad Sense Heritability was estimated by the following formula:Broad Sense Heritability (%) = [(VF2-VF1)/VF2 X 100] where VF2 = The variance of F2 population of a particular cross, and

74

VF1 = The variance of F1 population of a particular cross

3.11.8

Genetic Advance Estimates

The Genetic Advance was calculated by the following formula:GA = (k) x √Vp x (H) where k = the selection differential in standard deviation units (the value of ‘k’ at 5% selection intensity (2.06) was used in the calculations), Vp = the phenotypic variance of the base population for the trait in question, and H = the heritability of the trait expressed in fraction. The Relative Expected Gain (REG) was calculated by the following formula:REG = Genetic Advance x 100 Population Mean

3.11.9
The

Estimation of Correlation
correlation was calculated by the following formula:-

where X = the value(s) of 1st variable Y = the value(s) of 2nd variable, and N = the no. of observations.

75

3.11.10

t-test for correlation

The “t” test was employed to determine whether the correlation was statistically significant or not. The “t” values were calculated by the following formula:t = (r√n-2 )/(√1-r2 ) where r = the correlation value n = the no. of observations

76

CHAPTER 4

RESULTS AND DISCUSSION

77

4.1
4.1.1

COTTON TRANSFORMATION
Selection of a Suitable Variety
A local cotton variety MNH-93, evolved by the Cotton Research Station, Multan

was selected for transformation. This variety is high yielding with the desirable fibre characteristics. It has been recommended for general cultivation in the cotton zone of Punjab province.

4.1.2

Agrobacterium Transformation with pKMAB
The construct pKMAB contains two genes, neomycin phosphotransferase

(kanamycin resistance) under Nos promoter and OCS terminator, and cry1Ab under CaMV35S promoter and T-DNA gene terminator. The construct was transferred into Agrobacterium tumefaciens strain C58C1 to make co-integrate vector PGV2260:pKMAB (Fig 1). The plasmid isolation was done through Phenol:Chloroform:Isoamyl Alcohol method and the Agrobacterium transformation was confirmed by PCR (Fig 2).

78

Figure 1

SCHEMATIC DIAGRAM OF THE CONSTRUCT pKMAB
NcoI TatI SacI EcoRI XbaI

Border Sequence

Pnos Promoter

Neo Gene

3َ OCS

35S Promoter

CAB 22L Leader Sequence

Bt Gene Cry1Ab

T DNA Gene 7

Border Sequence

ScaI Bam HI

Pnos Promoter Neo Gene OCS 35S Promoter CAB 22L

Nopaline Synthase Promoter Neomycin Phosphotransferase (nptII) gene Octapine Synthase Terminator Gene Cauliflower Mosaic Virus 35S Promoter Enhancer

79

Figure 2

PCR Confirmation of Transformation of Agrobacterium tumefaciens C58C1
1 2 3 4 5 6 7

550bp

The recombinant construct pKMAB (containing CaMV35S promoter, Cry1Ab gene, T-DNA terminator and a Kanamycin resistance gene as a selectable marker) was transformed to Agrobacterium tumefaciens strain C58C1 by heat shock method. The plasmid isolation was done through Phenol:Chloroform:Isoamyl Alcohol method and the Agrobacterium transformation was confirmed by PCR by amplifying 550 base pair region of the gene. The Lane 1 shows λ/HindIII Marker, Lane 2-5 show Agrobacterium plasmids, Lane 6 shows Negative Control and Lane 7 shows Positive Control.

80

4.1.3

Agrobacterium-Mediated Transformation of Cotton with pKMAB
The mature embryos of cotton were subjected to tungsten particle bombardment

for micro-wound production and co-cultivation with the transformed Agrobacterium. A total of 10000 embryos were used in transformation. After eight weeks of selection on 50 mgL-1 kanamycin, 26 plants were obtained. The transformation efficiency was 0.26%. These plants were transferred to antibiotic free medium to enhance plant growth. These plants were then shifted to soil.

4.1.4

Cotton Genomic DNA Isolation
The isolation of DNA from cotton cells is much tedious than many other crops.

The cotton genomic DNA was isolated following Saha et al., 1997 and Zhang & Stewart, 2000. However, the problems of protein contamination and DNA degradation were observed. Therefore both of the above-mentioned protocols were used in combination with slight modifications at extraction buffer and precipitation steps. In this way, high quality and high yielding DNA was obtained (Fig 3).

4.1.5

Polymerase Chain Reaction
PCR analysis of newly transformed plants including control was done for the

detection of Cry1Ab. The amplification of the 550bp band was achieved in all plants showing that these plants had been transformed successfully (Fig 4). The plasmid

pKMAB was used as positive control whereas the DNA isolated from control plant was used as negative control. The PCR analysis was repeated for confirmation purpose.

81

Figure 3

Cotton Genomic DNA Isolation

1

2

3

4

5

6

7

8

9 10 11 12 13 14 15

16

The cotton genomic DNA was isolated following Saha et al., 1997 and Zhang et al., 2000 in combination with slight modifications at extraction buffer and precipitation steps to obtain high quality and high yielding DNA. The concentration of the plant DNA shown in Lane 2 to 16 was quantified by comparing with λ/HindIII Marker shown in Lane 1.

82

Figure 4

PCR OF TRANSFORMED PLANTS

1 2

3

4 5 6

7

8

9 10 11 12 13

550 bp

PCR analysis of the putative transformed plants including controls was done for the detection of Cry1Ab. The amplification of the 550bp band was achieved in all plants shown in Lane 2-11 besides positive control shown in Lane 13. The plasmid pKMAB was used as positive control. The DNA isolated from control plant was used as negative control shown in Lane 12.

83

4.1.6

Southern Blot Analysis
Southern hybridization was performed to confirm the integration of Cry1Ab gene

in the plants. The plant genomic DNA and plasmid pKMAB were digested with EcoRI and BamHI enzymes to release 1.845 kb cassette. The plants transformed with Cry1Ab gene were shown positive with Cry1Ab probe. These plants were also positive in PCR. No signal was detected in negative plants. The digested plasmid pKMAB was used as positive control. To assess copy number of the transgene in the transformed plants, genomic DNA of four selected plants was digested with a unicutter enzyme SstI (SacI) and probed with Cry1Ab labeled DNA. It was revealed that the plant CEMB-3 had two copies of the transgene in its genome whereas the plants CEMB-11, CEMB-16 and CEMB-17 had three copies of the transgene (Figure 5).

4.1.7

Enzyme Linked Immunosorbent Assay
The objective of the plant transformation experiments was to produce

transgenic plants expressing Cry1Ab gene. Enzyme Linked Immunosorbent Assay was used to screen the plants for expression of Cry1Ab. Total protein was isolated from all plants. The protein samples were bound to 96-wells microtitre plate and after treatment with specific antibodies, presence of Cry protein was detected by colour-reaction. All plants gave yellow colour showing expressed gene (Figure 6).

4.1.8

Western Blot Analysis
The transformed plants were further analyzed through Western Dot Blot

technique. The total protein from fresh leaves of transformed cotton plants was extracted

84

Figure 5

SOUTHERN BLOT ANALYSIS OF TRANSFORMED PLANTS
CEMB-11 CEMB-16 CEMB-17 CEMB-11 CEMB-16 B CEMB-17 CEMB-3 CEMB-3 85

A

(A) Genomic DNA of four selected plants was digested with a unicutter enzyme SstI and probed with Cry1Ab labeled DNA to assess copy number of the transgene in the transformed plants. (B) The number of bands showed that the plant CEMB-3 had two copies of the transgene in its genome whereas the plants CEMB-11, CEMB-16 and CEMB-17 had three copies of the transgene.

Figure 6

ELISA OF TRANSFORMED PLANTS

Enzyme Linked Immunosorbent Assay was used to screen the plants for expression of Cry1Ab. The total protein isolated from plants was bound to microtitre plate and after treatment with specific antibodies, presence of Cry protein was detected by colour-reaction. The protein samples A1 to H1 and A2 to C2 giving yellow colour showed expressed gene. The positive control protein samples were loaded in the wells E2 to G2 whereas the protein from un-transformed plant was used as negative control in D2.

86

and loaded on Hybond-C membrane. The purified Cry1Ab protein was used as positive control. The blocking was done with 5% skimmed milk + 0.05% Tween-20 dissolved in 1X PBS. The blot was first probed with specific primary antibodies and then with secondary antibodies. The colour was developed using NBT/BCIP tablets. The presence of Cry protein was detected in all samples except negative control (Fig 7).

4.2

DEVELOPMENT OF TRANSGENIC PURE LINES

4.2.1

1st Generation
The field studies were to be done on the plants transformed with Bt gene Cry1Ab.

It was therefore, imperative to develop pure lines which could be used as breeding material. For this purpose, all 26 plants transformed with Cry1Ab were grown under field conditions at CEMB, Lahore during Kharif, 2002. Since the gene Cry1Ab was supposed to produce toxic proteins in the plant tissue against Lepidopteran insects, the plants were not sprayed with any insecticide against Lepidopteran insects, during the whole season. Only three insecticidal sprays were done to control the sucking insects like whitefly, jassids and aphids. The data were recorded on various plant characteristics; the most important at this stage were boll damage (%age) due to natural infestation of bollworms (Fig 8). A boll which was completely or partially damaged (at least 10%) by any bollworm was counted as the damaged one. The bolls which were totally undamaged were counted as healthy bolls. The boll damage ranged from 8% to 100% in the plants (Table 1). The plants were also subjected to insect bioassays with 2nd instar Heliothis larvae under laboratory conditions (Fig 9). The insect mortality in lab bioassay ranged from 20% to 100% (Table 1).

87

Figure 7

WESTERN DOT BLOT OF TRANSFORMED PLANTS

A 1 2 3

B

C

4 5

6

7

The transformed plants were screened on the basis of Bt gene expression assessed through Western Dot Blot. Among the plant samples (A1 to A7; B1 to B7 and C1 to C5) which developed colour using NBT/BCIP tablets, the presence of Cry protein was detected. The Bt contents were quantified using Image Quant TL software of the Amersham BioSciences (Pvt). The purified Cry1Ab protein was used as positive control (C7) whereas the protein from un-transformed plant was loaded as Negative Control (C6).

88

Figure 8

COMPARATIVE VIEW OF DAMAGED AND HEALTHY COTTON BOLLS

A

B

C

D

Among various plant characteristics; the most important one was Boll Damage (%age) due to natural infestation of bollworms. A boll which was damaged at least 10% by any bollworm was counted as the damaged one (A&C). The totally undamaged bolls were counted as healthy bolls (B&D). The boll damage in the 1st generation plants ranged from 8% to 100% in the plants

89

Table 1

Insect Resistance and No. of Bolls of 1st Generation Plants
Insect mortality % shown in lab bioassay with Heliothis 90 50 60 80 80 20 50 90 50 70 60 80 90 70 60 70 100 100 80 100 80 100 70 80 70 100 20 No. of Total Bolls (Mature + Immature) 72 63 71 79 51 19 11 2 10 22 60 52 65 22 13 7 46 40 39 29 24 37 41 57 30 36 8 Average Boll Damage %age During the Peak Infestation Period (August-October) 24 14 24 8 37 26 64 100 40 86 18 12 34 50 92 29 17 15 46 24 21 19 27 18 37 25 63

S.No. NAME

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

CEMB-1 CEMB-2 CEMB-3 CEMB-4 CEMB-5 CEMB-6 CEMB-7 CEMB-8 CEMB-9 CEMB-10 CEMB-11 CEMB-12 CEMB-13 CEMB-14 CEMB-15 CEMB-16 CEMB-17 CEMB-18 CEMB-19 CEMB-20 CEMB-21 CEMB-22 CEMB-23 CEMB-24 CEMB-25 CEMB-26 Control

The boll damage due to naturally occurring bollworms in the field ranged from 8% to 100% in the plants. The insect mortality in lab bioassay ranged from 20% to 100%.

90

Figure 9

LABORATORY BIOASSAY WITH HELIOTHIS LARVAE

A

B

C

D

The transgenic cotton plants were subjected to lab bioassays with American Bollworm (Heliothis armigera). Five fresh leaves from each plant were taken and placed on wet filter paper in petri plates accommodating one leaf per plate. One 1st/2nd instar larva, prefasted for 4-6 hours, was released in the each plate and allowed to feed on the leaf. After 4872 hours feeding, the transgenic plants A, B and C showed insect mortality whereas the insect survived on control plant D.

91

The progeny rows of all 26 plants were desired to be grown but the seed setting in most of the plants did not take place under field conditions. The unfavourable weather conditions during the season besides late sowing hindered the bolls to mature. Moreover, most of the bolls which matured late season produced lighter seeds, which were discarded during seed selection for next sowing. At the end, sufficient seed of nine plants only could be obtained for further studies in progeny rows.

4.2.2

2nd Generation
The progeny of the selected nine plants was grown in Kharif, 2003. The field

layout was planned according to the international guidelines for Bt crop growing. Each plant progeny consisted of two rows accommodating 66 plants. The plant to plant distance was kept at 30cm and row to row at 75cm. The transgenic plants were surrounded by 4.5m wide belt of non-Bt cotton as refugia. The whole cotton field was further surrounded by 4.5m wide sorghum belt as an isolation boundary. A layout plan has been shown in Fig 10. The progeny plants grown during kharif, 2003 showed a considerable heterozygosity in plant morphology and insect resistance. The data have been given graphically in Fig 11. The data were recorded on individual plant basis on various characteristics viz. boll damage %age due to natural infestation of bollworms, percent insect mortality in lab bioassay with Heliothis larvae, yield, plant height, number of monopodial branches per plant and number of sympodial branches per plant. The plants were also analyzed at molecular level through PCR and Western Dot Blot.

92

Figure 10

LAYOUT PLAN OF PROGENY ROWS GROWN DURING KHARIF, 2003
.
Sorghum Non-Bt Refugia Path

CEMB-11

CEMB-24

CEMB-22

CEMB-17

CEMB-10

The field layout was according to the international guidelines for Bt crop growing. The transgenic plants were surrounded by 4.5m wide belt of non-Bt cotton as refugia. The whole cotton field was further surrounded by 4.5m wide sorghum belt as an isolation boundary.

93

CEMB-16

CEMB-3

CEMB-9

CEMB-4

Control

The Boll Damage (%age) ranged from 0.0% to 100%. The Heliothis larval mortality %age in the lab bioassay ranged from 10% to 100%. The yield per plant ranged from 0.0 g to 165.1g. The plant height ranged from 47 to 230cm. The number of monopodial branches per plant ranged from 1 to 6 per plant. Similarly, the number of sympodial branches ranged from 4 to 40 per plant (Fig 11). All of these plant characteristics were considered simultaneously for plant selections. The criteria were set as follows: 1. the plant should be positive in molecular screening tests viz. PCR and Western Dot Blot; 2. the plant should be higher yielding; 3. the plant should bear less than 5% damage due to natural infestation of bollworms; 4. the plant should be showing at least 40% insect mortality in the lab bioassays; 5. the plant should bear low number of monopodial branches and high number of sympodial branches; 6. the plant should give Ginning Outturn %age equal or more than the control plants; and 7. the plant should be equal or short in stature than control.

94

Figure 11

DATA ON DIFFERENT CHARACTERS OF ALL PLANTS OF 2nd GENERATION, KHARIF, 2003

The data on different characteristics of 2nd generation plants were recorded on individual plant basis. 95

On the basis of the criteria stated above, selection of the best five plants was done. The data pertaining to the best five plants is given in the Table 2. The 1st plant CEMB 3-2 was positive in PCR and Western Dot Blot; it gave 87.8g seed cotton yield and 43.96% GOT; it had 4 monopodial branches, 17 sympodial branches, 195cm height; it showed 3.33% boll damage under natural conditions and killed 60% Heliothis larvae in the laboratory bioassay. The 2nd plant CEMB 11-2 was positive in PCR and Western Dot Blot; it gave 102.3g seed cotton yield and 39.6% GOT; it had 3 monopodial branches, 33 sympodial branches, 105cm height; it showed 2.86% boll damage under natural conditions and killed 40% Heliothis larvae in the laboratory bioassay. The 3rd plant CEMB 16-10 was positive in PCR and Western Dot Blot; it gave 109.3g seed cotton yield and 41.0% GOT; it had 1 monopodial branch, 12 sympodial branches, 172cm height; it showed 2.63% boll damage under natural conditions and killed 50% Heliothis larvae in the laboratory bioassay. The 4th plant CEMB 16-15 was positive in PCR and Western Dot Blot; it gave 88.6g seed cotton yield and 42.0% GOT; it had 4 monopodial branches, 22 sympodial branches, 117cm height; it showed 0.0% boll damage under natural conditions and killed 60% Heliothis larvae in the laboratory bioassay. Similarly, the 5th plant CEMB 17-25 was positive in PCR and Western Dot Blot; it gave 95.3g seed cotton yield and 38.5% GOT; it had 5 monopodial branches, 19 sympodial branches, 170cm height; it showed 3.45% boll damage under natural conditions and killed 50% Heliothis larvae in the laboratory bioassay.

96

Table 2

CHARACTERISTICS OF SELECTED FIVE PLANTS FROM 2nd GENERATION
Insect S. No. PCR Result Western Dot Blot Result Mortality shown in the Lab Bioassay (%) Boll Damage due to Natural Infestation (%age) No. of Monopodial Branches No. of Sympodial Branches Plant Height (cm) Yield (gm) Ginning Outturn (%age)

Plant No.

1 2 3 4 5 6

CEMB 3-2 CEMB 11-2 CEMB 16-10 CEMB 16-15 CEMB 17-25 Control

+ + + + + -

+ + + + + -

60 40 50 60 50 20

3.33 2.86 2.63 0.00 3.45 20

4 3 1 4 5 2

17 33 12 22 19 18

195 105 172 117 170 190

87.8 102.3 109.3 88.6 95.3 46.8

43.96 39.6 41.0 42.0 38.5 38.0

All plants of 2nd generation were subjected to screening on the basis of the data recorded regarding the above-mentioned traits. The data pertaining to finally selected five plants are shown here. 97

4.2.3

3rd Generation
The progenies of the selected five plants were raised in the green house during

winter 2003-04 (Figure 12). The plants were grown in tumbler-shaped earthen pots of 60cm height and 45cm upper diameter. Each pot accommodated a single plant. Each progeny consisted of ten plants. The temperature and humidity conditions of the green house were maintained artificially. During the entire plant growing period, the temperature and humidity ranged between 25-40ºC and 30-70%, respectively. The plants were again subjected to individual studies and molecular analysis. The progeny plants were analyzed through PCR and Western Blotting. The plants of two progenies viz. CEMB 3-2 and CEMB 16-10 showed 100% positive results in PCR and Western Dot Blot. The plants of three progenies viz. CEMB 16-15, CEMB 11-2 and CEMB 17-25 showed 90% positive results in PCR and Western Dot Blot. By this time, the facility of Bt Quantification through scanning had been arranged. The Bt contents were quantified using Image Quant TL software of the Amersham BioSciences (Pvt). The Bt content ranged from 0.09% to 0.88% in the CEMB 3-2 plants, from 0.00% to 1.18% in the CEMB 11-2 plants, from 0.26% to 1.35%. in the CEMB 1610 plants, from 0.00% to 0.83% in the CEMB 16-15 plants and from 0.00% to 0.76% in the CEMB 17-25 plants (Table-3).

98

Figure 12

3RD GENERATION PROGENY PLANTS IN GREEN HOUSE

A
A A night-time view of the green house

B
B Plants inside the green house

Besides raising cotton crop during the kharif season, an additional crop per year was obtained in the green house during November-March. The temperature and humidity conditions in the green house were maintained at 25-40°C and 3070%, respectively.

99

Table-3

Bt Protein %age in 3rd Generation Plants 2003-2004
CEMB 3-2 Plant No.1 Plant No.2 Plant No.3 Plant No.4 Plant No.5 Plant No.6 Plant No.7 Plant No.8 Plant No.9 Plant No.10 Standard Deviation 0.56 0.88 0.55 0.40 0.70 0.09 0.11 0.20 0.60 0.46 0.26 CEMB 11-2 1.07 1.02 0.16 1.18 0.94 0.85 0.61 0.20 1.06 0.00 0.44 CEMB 16-10 0.53 0.32 1.35 0.26 0.27 0.32 0.49 0.39 0.33 0.30 0.33 CEMB 16-15 0.40 0.44 0.40 0.39 0.62 0.62 0.26 0.00 0.83 0.68 0.24 CEMB 17-25 0.76 0.54 0.56 0.58 0.00 0.39 0.63 0.40 0.52 0.55 0.20

The plants were subjected to individual studies and molecular analysis. The Bt contents were quantified using Image Quant TL software of the Amersham BioSciences (Pvt).The seeds of the positive plants were picked in each descent and bulked at this stage. Since the progenies of the plant nos. 16-10 and 16-15 were identical and also share a common ancestor, the seed of these two lines was bulked jointly.

100

The seeds of the positive plants were picked in each descent and bulked at this stage. Since the progenies of the plant nos. 16-10 and 16-15 were identical and also share a common ancestor, the seed of these two lines was bulked jointly. The plants in the 3rd generation were phenotypically similar and had been screened at molecular level; there was no need for further selfing. The bulked seed was thus declared to be pure lines and named as CEMB-3, CEMB-11, CEMB-16 and CEMB-17.

4.2.4

4th & 5th Generation
Besides included in the field trials, the pure lines mentioned above were further

analyzed at molecular level, for two successive generations to eliminate false positive plants and to check for errors in selection. A sample of 30 plants, at random, was taken from each line during every year and analyzed at molecular level through PCR and Western Dot Blot. All plants in both generations were found to be positive in molecular screening. The results have been given in Table-13.

4.3
4.3.1

FIELD STUDIES ON Bt COTTON
Bt Trials 2004-05
The field trials comprising of four Bt genotypes/pure lines viz. CEMB-3, CEMB-

11, CEMB-16 and CEMB-17 and one non-Bt genotype/variety MNH-93 as control, were conducted for two consecutive years during 2004-2005 (Figure-13). The primary objective was to evaluate various transgenic lines regarding their insect resistance level in comparison with the non-transgenic control. The data were recorded on various characters and the results are described below, in seriatim.

101

Figure 13

COTTON FIELD 2004

C B A

COTTON FIELD 2005

A B C
The field trials comprising of four Bt genotypes/pure lines viz. CEMB-3, CEMB11, CEMB-16 and CEMB-17 (A) and one non-Bt genotype/variety MNH-93 as control (B) were conducted for two consecutive years. The fields were further surrounded by an isolation boundary (C).

102

4.3.1.1

Bt Protein %age

The Bt contents in all genotypes were quantified to have a true picture of the variation due to Bt concentration in insect resistance of the plants. The total protein was isolated from fresh leaves of the plants. The concentration of total protein was estimated with the help of spectrophotometer at 595nm. The reading was compared with the BSA standard curve. An equal amount of protein of each sample (10ng) was loaded directly onto a Hybond-C membrane and the procedure of Western Blotting was followed. After processing, the blot was scanned and the Bt contents quantified using Image Quant TL software of the Amersham BioSciences (Pvt). The Bt protein estimates have been shown graphically in Figure-14. The data were subjected to Analysis of Variance and it was revealed that the genotypes had highly significant differences among themselves (Table-5). The mean comparisons given in the Table-7 showed that all transgenic lines were statistically different from the control. Among the transgenic lines, CEMB-17 had the highest level of Bt content (0.298% of the total protein). The genotypes CEMB-11 and CEMB-16 had 0.287% and 0.261% of the total protein respectively, and were statistically not different from CEMB-17. Similarly, CEMB-3 had the lowest level of Bt content (0.216%) among all transgenic lines but it was statistically not different from CEMB-11 and CEMB-16.

4.3.1.2

Natural Infestation of Spotted Bollworm

The number of naturally occurring Spotted Bollworms in the Bt trials have been presented graphically in Figure-15. The mean squares are given in Table-4 from which it is clear that the lines differ highly significantly in the natural infestation of Spotted Bollworm. The means were separated using DMR test (Table-6).

103

Figure 14

Bt PROTEIN CONTENT
0.45 0.40 0.35

PERCENTAGE

0.30 0.25 0.20 0.15 0.10 0.05 0.00 CEMB-3 CEMB-11 CEMB-16 CEMB-17 CEMB-C

GENOTYPES An equal amount of protein of each sample (10ng) was loaded directly onto a Hybond-C membrane and the procedure of Western Blotting was followed. Bt contents were quantified using Image Quant TL software of the Amersham BioSciences (Pvt). Figure 15

SPOTTED BOLLWORM
1.20

NO.OF INSECTS PER PLANT

1.00 0.80 0.60 0.40 0.20 0.00

CEMB-3

CEMB-11

CEMB-16

CEMB-17

CEMB-C

GENOTYPES The number of live insects per plant were counted on weekly basis during the season and averaged at the end. 104

Table 4

ANALYSIS OF VARIANCE: MEAN SQUARES FOR DIFFERENT CHARACTERS OF THE Bt TRIALS 2004-2005

SOURCE OF VARIATION

YIELD PER PLANT

PLANT HEIGHT

NO. OF MONOPODIAL BRANCHES

NO. OF SYMPODIAL BRANCHES

NATURAL INFESTATION OF SPOTTED BOLLWORM

FIELD BIOASSAY (Heliothis)

GOT %age

Replication

224.20 *

413.22 *

12.89 *

9.33 *

0.19 **

0.01 ns

0.10 ns

Genotypes

24.59 ns

653.30 **

6.09 ns

13.11 **

0.09 **

0.01 ns

10.04 **

Error

58.96

614.24

2.23

2.59

0.02

0.01

2.11

** indicates significant differences at P< 0.01 probability level. * indicates significant differences at P< 0.05 probability level. ns = Non-significant The analysis of variance was done with the help of the techniques mentioned by Steel and Torrie (1980).

105

Table 5

ANALYSIS OF VARIANCE: MEAN SQUARES FOR DIFFERENT CHARACTERS OF THE Bt TRIALS 2004-2005

SOURCE OF VARIATION

NO. OF BOLLS PER PLANT

BOLL WEIGHT

STAPLE LENGTH

FIBRE FINENESS

Bt PROTEIN CONTENT

LAB BIOASSAY (Heliothis)

Replication

34.77 ns

0.69 ns

0.65 ns

0.05 ns

0.004070 ns

240.00 *

Genotypes

12.3 ns

1.15 ns

0.84 ns

0.03 ns

0.045343 **

181.73 *

Error

13.07

0.42

0.54

0.04

0.001435

43.33

** indicates significant differences at P< 0.01 probability level. * indicates significant differences at P< 0.05 probability level. ns = Non-significant The analysis of variance was done with the help of the techniques mentioned by Steel and Torrie (1980).

106

Table 6

MEAN COMPARISONS FOR DIFFERENT CHARACTERS OF THE Bt TRIALS 2004-2005*
YIELD PER PLANT (g) NO. OF MONOPODIAL BRANCHES PER PLANT NO. OF SYMPODIAL BRANCHES PER PLANT NATURAL INFESTATION OF SPOTTED BOLLWORM (no. of insects per plant) 0.69 b 0.76 b 0.76 b 0.72 b 0.99 a FIELD BIOASSAY (Heliothis) (no. of insects per plant) 0.10 a 0.05 a 0.07 a 0.12 a 0.15 a

GENOTYPES

PLANT HEIGHT (cm)

GOT %age

CEMB-3 CEMB-11 CEMB-16 CEMB-17 CEMBCONTROL • •

24.44 a 21.70 a 19.31 a 22.04 a 19.88 a

110.00 b 95.17 b 102.50 b 96.83 b 131.47 a

4.08 a 3.98 a 5.82 a 4.42 a 6.12 a

12.32 b 12.47 b 11.82 b 13.50 b 9.52 a

34.23 a 34.28 a 34.15 a 34.04 a 31.29 b

To establish the level of significance among various genotypes, New Duncan’s Multiple Range Test (5% level) was applied to compare the means for all parameters. * Means followed by the same letter are statistically non-significant

107

Table 7

MEAN COMPARISONS FOR DIFFERENT CHARACTERS OF THE Bt TRIALS 2004-2005*

NO. OF BOLLS GENOTYPES PER PLANT

BOLL WEIGHT (g) 2.93 a 2.91 a 4.02 a 2.70 a 2.38 a

STAPLE LENGTH (mm) 24.96 a 25.91 a 26.13 a 25.87 a 26.35 a

FIBRE FINENESS (µg/in) 4.63 a 4.59 a 4.80 a 4.64 a 4.82 a

Bt PROTEIN (%age of total protein) 0.215987 b 0.287177 ab 0.260833 ab 0.298383 a 0.000000 c

LAB BIOASSAY (Heliothis mortality %age) 41.333 a 33.333 ab 40.667 a 34.667 ab 22.000 b

CEMB-3 CEMB-11 CEMB-16 CEMB-17 CONTROL • •

15.00 a 12.50 a 11.03 a 13.37 a 16.17 a

To establish the level of significance among various genotypes, New Duncan’s Multiple Range Test (5% level) was applied to compare the means for all parameters. * Means followed by the same letter are statistically non-significant

108

The transgenic lines had significantly lower infestation of Spotted Bollworm than the control line. However, the transgenic lines had non-significant differences among themselves. As regards the aim of developing transgenic lines after transformation, this parameter (data on natural infestation of Spotted Bollworms) bears the paramount importance. The transgene was hypothesized to produce enough toxin to control the target insects. The data provided ample evidence of the transgene producing Bt toxin at a level controlling the target insects like Spotted Bollworm.

4.3.1.3

Field

Bioassay

with

American

Bollworm

(Heliothis

armigera)
The field trials were conducted in the experimental area of CEMB, Lahore. The Lahore District is situated outside of the cotton zone. There is a marked difference between weather conditions of Lahore and the cotton belt i.e. the districts of Rahim Yar Khan, Multan, Khanewal, Vehari, etc. The Lahore weather does not permit all cotton insects to flourish. Similarly, the growing of cotton at Lahore also requires special skills and attention towards its critical stages. Among the Lepidopteran insects, only Spotted Bollworm infests naturally in the Lahore area. The occurrence of American Bollworm is very rare at Lahore, whereas the occurrence of Armyworm is occasional everywhere. Under these circumstances, it was planned to artificially rear American Bollworm in the laboratory and release in the transgenic field to study the insect resistance level of the Bt cotton lines in comparison with the control lines. For this purpose, the moths were, time and again, collected from the Districts of Multan, Khanewal, Sahiwal and Okara during both years of field studies. The 2nd instar larvae were obtained in the insectary under controlled conditions and

109

released in the field. A total of 72,000 insects were released in the field in six installments (three per year) using glass vials. One glass vial, accommodating 10 insect, was tied to the centre of main stem of each plant and opened to allow the insects come out and travel over the plant to feed (Figure-16). Astonishingly, the first release of insects in both the years completely vanished within two days after release. After thorough deliberations, it was concluded that the presence of large number of predators in the field and high temperature at the insect releasing time might be the responsible factors. The number of surviving Heliothis larvae after one week of 2nd and 3rd field infestations respectively during both the years was recorded (Figure 17). The number of surviving larvae in control line was much higher than Bt lines. The Bt line CEMB-11 showed 67% less surviving Heliothis population than control. The lines CEMB-3, CEMB-16 and CEMB-17 had 33%, 53% and 20% less insect population of surviving Heliothis larvae than the control line. The mean squares data have been presented in Table-4 which however, indicated that there were statistically non-significant differences among the genotypes. Moreover, in spite of the artificial release of Heliothis larvae at a high rate of 10 insects per plant, the number of surviving Heliothis larvae per plant was much less than Spotted Bollworm.

110

Figure 16

INSECT RELEASE METHOD FOR FIELD BIOASSAY

A glass vial containing ten 2nd instar Heliothis larvae was tied to the main stem of the plant and opened to allow the insects to feed on the plant. Figure 17

FIELD BIOASSAY
0.20
NO. OF SURVIVING HELIOTHIS LARVAE

0.18 0.16 0.14
0.12

0.10 0.08 0.06 0.04 0.02 0.00 CEMB-3 CEMB-11 CEMB-16 CEMB-17 CEMB-C

GENOTYPES The number of surviving Heliothis larvae after one week of 2nd and 3rd field infestations respectively during both the years was recorded. 111

Figure 18

LABORATORY BIOASSAYS
60

MORTALITY PERCENTAGE OF HELIOTHIS LARVAE

50 40 30 20 10 0

CEMB-3

CEMB-11

CEMB-16

CEMB-17

CEMB-C

GENOTYPES The data on insect mortality were taken on daily basis upto seventh day. Figure 19

YIELD PER PLANT
30 25 20

YIELD (g)

15 10 5 0

CEMB-3

CEMB-11

CEMB-16

CEMB-17

CEMB-C

GENOTYPES Average yield of seed cotton per plant picked from ten randomly selected plants was calculated by dividing the total yield with the number of plants and expressed in grams. 112

This may be attributed to a number of factors such as 1. there was a lesser chance of survival in the field for artificially reared insects; 2. there were a large number of predators in the field at the time of release of insects because the trials were kept immune to chemical insecticides; 3. the insects were forced to live outside their natural habitat which caused insect mortality at a higher rate.

4.3.1.4

Laboratory Bioassay

The results obtained on insect mortality percentage after conducting laboratory bioassays using 2nd instar Heliothis larvae have been presented graphically in Figure-18. The control line showed a lower mortality percentage of insects than the transgenic lines. The mean squares are given in Table-5 according to which the genotypes had significant differences among themselves at 0.05 probability level. When the means were compared using Duncan’s New Multiple Range test, it was revealed that the control line showed significantly less mortality %age of 2nd instar Heliothis larvae than the transgenic lines CEMB-3 and CEMB-16. The mortality percentage was however not significantly different from two lines CEMB-17 and CEMB-11 (Table-7).

4.3.1.5

Seed Cotton Yield per Plant

The seed cotton yield data have been given graphically in Figure-19. The transgenic line CEMB-3 gave 22.93 % more yield than the control line. Similarly, CEMB-11 and CEMB-17 gave 9.15% and 10.86% more yield than control, respectively. The line CEMB-11, however, showed 3% less yield than the control. Statistically, the genotypes had non-significant differences among themselves in yield per plant (Table-4).

113

It means that the Bt gene had exerted no significant positive or negative effect on seed cotton yield. It was as per expectations because the Bt gene is not a yield contributing gene. However, theoretically the Bt genotypes might have higher yield than the non-Bt check due to the enhanced insect resistance capability of the Bt plants-an indirect effect. The results indicated that although there was an increase in seed cotton yield (upto 23%) but it was non-significant statistically. This phenomenon occurred probably due to the fact that the trial was kept immune to any supplemental insect control-chemical insecticide. Moreover, the natural pest pressure remained low during both years of study. These results are also supported by the results obtained from the experiments ‘Comparative Study of Insecticide Applications on Bt and non-Bt Cotton Lines 20042005’ and ‘Correlation of Bt Trait with Other Economic Traits-Yield’ which have been reported in the following pages.

4.3.1.6

Plant Height

The plant height of different genotypes has been represented graphically in Figure-20. The data were subjected to analysis of variance which revealed that differences among the genotypes were highly significant statistically (Table-4). The means were separated using DMR test and it was shown that the control line had significantly more plant height than all transgenic lines. The reduction in cotton plant height is always desirable to the plant breeders. In this context, the reduction in height after transformation is a positive change in the Bt cotton.

114

4.3.1.7

Number of Monopodial Branches per Plant

A perusal of the Table-4 indicates that the lines did not differ in respect of number of monopodial branches per plant. The number of monopodial branches per plant, however, ranged from 3.98 in CEMB-11 to 6.12 in control (Figure-21). The results clearly indicated that the transformation had not affected this character of the plants. The unalteration of the characters other than for which transformation was done is highly desirable.

4.3.1.8

Number of Sympodial Branches per Plant

The number of sympodial branches per plant ranged from 9.52 in control to 13.50 in CEMB-17 (Figure-22). The analysis of variance showed that the genotypes differed highly significantly in number of sympodial branches per plant (Table-4). The Duncan’s New Multiple Range Test was applied to separate the means. The mean comparison presented in Table-6 showed that the means of all transgenic lines differ from the mean of the control. The results indicated that the number of sympodial branches per plant had increased after transformation, and this is also a positive sign towards selection of plants.

4.3.1.9

Number of Bolls per Plant

The average number of bolls per plant of all genotypes have been presented graphically in Figure-23. It ranged from 11.03 in CEMB-16 to 16.17 in CEMB-control. The analysis of variance data have been presented in Table-5. The number of bolls per plant differed non-significantly among the genotypes. The data showed that the transformation of cotton with a foreign gene had not altered its character of number of bolls per plant.

115

Figure 20

PLANT HEIGHT
160 140 120

CENTIMETRES

100 80 60 40 20 0 CEMB-3 CEMB-11 CEMB-16 CEMB-17 CEMB-C

GENOTYPES The plant height was recorded in centimeters from base to the apex by using a meter rod. Figure 21

NUMBER OF MONOPODIAL BRANCHES PER PLANT
8 7 6

NUMBER

5 4 3 2 1 0 CEMB-3 CEMB-11 CEMB-16 CEMB-17 CEMB-C

GENOTYPES At maturity, the number of monopodial branches of ten randomly selected plants from each plot were counted and averaged for the purpose of statistical analysis. 116

Figure 22

NUMBER OF SYMPODIAL BRANCHES PER PLANT
16 14 12

NUMBER

10 8 6 4 2 0 CEMB-3 CEMB-11 CEMB-16 CEMB-17 CEMB-C

GENOTYPES At maturity, the number of sympodial branches of ten randomly selected plants from each plot were counted and averaged for the purpose of statistical analysis. Figure 23

NUMBER OF BOLLS PER PLANT
20 18 16 14

NUMBER

12 10 8 6 4 2 0 CEMB-3 CEMB-11 CEMB-16 CEMB-17 CEMB-C

GENOTYPES The actual count of effective bolls on ten randomly selected plants was recorded and summed up for all pickings. The mean was calculated by dividing total number of bolls with the number of plants. 117

4.3.1.10

Boll Weight

The boll weight ranged from 2.38g to 4.02g. A comparison of the average boll weight of the genotypes has been presented in Figure-24. The lowest boll weight (2.38g) was recorded in control line whereas the highest (4.02g) was recorded in the CEMB-16 (Table-7). However, the genotypes had non-significant differences among themselves regarding Boll Weight (Table-5).

4.3.1.11

Ginning Outturn Percentage

The Ginning Outturn Percentage ranged from 31.29 (control) to 34.28 (CEMB-3) (Figure-25). The mean squares given in the Table-4 shows that the genotypes differed highly significantly among themselves. When the means were compared following DMR test, it was revealed that CEMB-3 had highest GOT %age (34.28), followed by CEMB-11 (34.23), CEMB-16 (34.15) and CEMB-17 (34.04). The transgenic lines were statistically non-significantly different from each other. The lowest GOT %age (31.29) was recorded in the control line which was significantly different from all transgenic lines. The results clearly showed that the transformation had caused a highly positive and desirable change of increasing GOT %age in the transgenic lines.

4.3.1.12

Staple Length

The staple length ranged from 24.96mm (CEMB-3) to 26.35mm (control) as shown in Figure-26. The mean squares presented in the Table-5 showed that the genotypes differed non-significantly from one another in respect of staple length. The data showed that the transformation event had not caused any significant changes in the staple length controlling genes.

118

Figure 24

BOLL WEIGHT
5.0 4.5 4.0 3.5

GRAMS

3.0 2.5 2.0 1.5 1.0 0.5 0.0 CEMB-3 CEMB-11 CEMB-16 CEMB-17 CEMB-C

GENOTYPES The average boll weight was obtained by dividing the total seed cotton yield of ten randomly selected plants in each plot by the respective total number of effective bolls. Figure 25

GINNING OUTTURN PERCENTAGE
36.00 35.00 34.00

GOT %age

33.00 32.00 31.00 30.00 29.00

CEMB-3

CEMB-11

CEMB-16

CEMB-17

CEMB-C

GENOTYPES A sample of 100 grams from each plant was taken and ginned separately with a Single Roller Electric Gin. The lint obtained was expressed as GOT %age. 119

Figure 26

STAPLE LENGTH
27.50 27.00 26.50

MILLIMETERS

26.00 25.50 25.00 24.50 24.00

CEMB-3

CEMB-11

CEMB-16

CEMB-17

CEMB-C

GENOTYPES The Staple Length was measured by Fibrograph Model 530 (electronic). Figure 27

FIBRE FINENESS
5.00 4.90

MICROGRAM PER

4.80 4.70 4.60 4.50 4.40

CEMB-3

CEMB-11

CEMB-16

CEMB-17

CEMB-C

GENOTYPES The Fibre Fineness was measured with the help of “Sheffield Micronaire” and expressed in microgram per inch.

120

4.3.1.13

Fibre Fineness

The fibre fineness ranged from 4.59 µg/in (microgram per inch) for CEMB-11 to 4.82 µg/in for CEMB-Control (Figure-27). The mean squares for fibre fineness presented in Table-5 showed that the genotypes did not differ significantly from each other. The results after study of this character are encouraging as there had been no change observed in fibre fineness in cotton after transformation. The original character of fibre fineness has thus remained intact.

4.3.2

Comparative Study of Insecticide Applications on Bt and Non-Bt Cotton Lines 2004-2005.

4.3.2.1

Studies during the Year 2004

A simple experiment was conducted during the year 2004. The Bt genotype CEMB-3 (transformed MNH-93) and its non-Bt counterpart CEMB-C (non-Bt MNH-93) were sown in adjacent plots of the same size. The plots were separated completely by growing 3m wide sorghum belt between the plots to eliminate the possibilities of insect travelling from one plot to another, and to avoid the effect of insecticidal sprays of one plot to another. The experiment was thrice replicated. The non-Bt lines were regularly sprayed with suitable insecticides to control the Lepidopteran insects, whereas no insecticide to control Lepidopteran insects was applied to the Bt lines throughout the season. The seed cotton yield data were recorded and means were statistically compared by using “t-test assuming unequal variances”.

121

Table 8

COMPARATIVE STUDY OF INSECTICIDE APPLICATIONS, 2004 †
SEED COTTON YIELD (gm) GENOTYPE CEMB-3 CEMB-C R1 494.10 1296.10 R2 337.70 1051.50 R3 639.90 1517.00 TOTAL 1471.70 3864.60 MEAN 490.56 1288.20

t-TEST: TWO-SAMPLE ASSUMING UNEQUAL VARIANCES Variable 1 Mean Variance Observations Hypothesized Mean Difference Df t Stat P(T<=t) one-tail t Critical one-tail P(T<=t) two-tail t Critical two-tail 490.57 22840.57 3.00 0.00 3.00 -4.98 ** 0.01 2.35 0.02 3.18 Variable 2 1288.20 54219.37 3.00

The t-test for two samples assuming unequal variances was applied to compare the treatments.

** indicates significant differences at P< 0.01 probability level.

122

The null hypothesis was that “the means for seed cotton yield of unsprayed Bt and sprayed non-Bt lines are equal”. It means Bt-line could give statistically equal yield without any insecticide application as compared to the non-Bt line having all necessary spray applications. The results however, revealed that the control-line gave significantly more seed cotton yield than Bt-line (Table-8). It was concluded that the total abstinence from insecticide application to Bt cotton was not a feasible strategy and the possible reduction in number of insecticide applications may be sought, instead. Therefore the experiment was modified during the next year.

4.3.2.2

Studies during the Year 2005

.In the year 2005, the experiment was conducted using Split Plot Randomized Complete Block Design with four replications having genotypes in the main plots and treatments (insecticide applications) in the sub-plots. The results have been given in Table-9. A perusal of Table-9 revealed that CEMB-3 gave 42.0% more seed cotton yield in L1 i.e. highest economic threshold level treatment (3 insects per 25 plants) as compared to its yield in L0 i.e. no spray treatment. Similarly, it gave 10.90% and 19.93% more yield in the treatments L2 (5insects per 25 plants) and L3 (8 insects per 25 plants), respectively as compared to its yield in L0. The control line CEMB-C gave 31.81% more yield in L1 i.e. highest economic threshold level treatment as compared to its yield in L0 i.e. no spray treatment. Similarly, the control line gave 27.79% and 17.13% more yield in the treatments L2 and L3, respectively in comparison with its yield in L0. These results have also been shown graphically in Figure-28. Both of the genotypes gave lowest yield at L0 level i.e. no spray and the highest yield at L1 i.e. spray application at the highest and recommended economic threshold level (ETL) of 3 insects per 25 plants. The analysis of

123

variance of the experiment has been given in Table-10. A perusal of the ANOVA table reveals that the genotypes differed non-significantly among themselves. During the season, the spray applications were decided on the basis of the pest scouting data. Although the experiment was conducted according to the Split Plot Design having levels in the sub-plots but the objective was not to ascertain the best ETL level. The primary objective was to assess how many number of spray applications could be reduced in case of Bt cotton giving a yield comparable with the non-Bt cotton having significantly more number of spray applications. Therefore, at the end of the experiment, four distinct classes on the basis of number of applied insecticidal sprays in each genotype were framed (Table 11). Every class of one genotype was compared, one by one, with four classes of the other genotype. In this way, a total of 16 combinations were made. These classes were compared using t-test. Only one combination on the basis of spray applications could be found where the number of spray applications significantly differed in Bt and non-Bt lines (excluding class-1 i.e. L0), in which no spray was done). When the corresponding yields were compared, it was found that the yields differed nonsignificantly, although the Bt line gave 27.65% more seed cotton yield than control(Table-12). It was thus concluded that the Bt line CEMB-3 at 1.75 insecticidal spray applications, gave the same yield as the CEMB-C gave at 3.0 applications of insecticide against bollworms. In this way, a statistically significant and practical saving of 1.25 sprays or 41% of insecticides (meant for Lepidopteran, only) can be done by the use of this line of Bt cotton in comparison with its non-Bt counterpart.

124

Table 9

SEED COTTON YIELD COMPARISONS OF Bt AND NON-Bt GENOTYPES UNDER DIFFERENT INSECTICIDE APPLICATION TREATMENTS
L0 GENOTYPES/ YIELD L1 %AGE INCREASE/ DECREASE OVER L0 42.00 31.81 L2 %AGE INCREASE/ DECREASE OVER L0 10.90 27.79 L3 %AGE INCREASE/ DECREASE OVER L0 19.93 17.13

YIELD (g)

YIELD (g)

YIELD (g)

YIELD (g)

CEMB-3 CEMB-C

41.19 29.36

58.49 38.70

45.68 37.52

49.40 34.39

The crop was sprayed when the insect population reached Economic Threshold Level on the basis of the pest scouting data. Economic Threshold Levels (ETLs) / Treatments L0 No Spray (Control) L1 Spray at an incidence of 3 insects per 25 plants L2 Spray at an incidence of 5 insects per 25 plants L3 Spray at an incidence of 8 insects per 25 plants

125

Table 10

ANALYSIS OF VARIANCE FOR SEED COTTON YIELD
(COMPARATIVE STUDY OF INSECTICIDE APPLICATIONS, 2005)

SOURCE OF VARIATION TREATMENTS GENOTYPES LEVELS LXV ERROR TOTAL ns non-significant

df

SS

MS

F.RATIO

7.00 1.00 3.00 3.00 24.00 31.00

2357.55 1501.41 710.18 145.96 13122.38 15479.94

336.79 1501.41 236.73 48.65 546.77

0.62 ns 2.75 ns 0.43 ns 0.09 ns

The analysis of variance was done with the help of the techniques mentioned by Steel and Torrie (1980).

126

Table 11 COMPARATIVE STUDY OF INSECTICIDE APPLICATIONS, 2005 NO. OF SPRAY APPLICATIONS Class-1 (L0) CEMB-3 CEMB-C Class-2 (L1) CEMB-3 CEMB-C Class-3 (L2) CEMB-3 CEMB-C Class-4 (L3) CEMB-3 CEMB-C

R1 R2 R3 R4

0

0

4

4

3

4

2

2

0

0

4

3

3

3

2

3

0

0

2

3

2

2

2

1

0

0

2

2

2

1

1

1

AVERAGE

0

0

3

3

2.5

2.5

1.75

1.75

Four distinct classes on the basis of number of applied insecticidal sprays in each genotype were framed.

127

Table 12

COMPARISON OF INSECTICIDE USE AND YIELDS ON Bt AND NON-Bt COTTON LINES.

Bt (CEMB-3)

NON-Bt (CEMB-C)

No. of sprays against bollworms

1.75 *

3.0

Yield (gm/plant)

49.40 ns

37.52

* ns

Mean values differ significantly from those of non-Bt counterparts. Mean values differ non-significantly from those of non-Bt counterparts

A statistically significant and practical saving of 1.25 sprays or 41% of insecticides (meant for Lepidopteran, only) was by the use of CEMB-3 line of Bt cotton in comparison with its non-Bt counterpart.

128

Figure 28

YIELD COMPARISONS OF Bt AND NON-Bt GENOTYPES
70.00

CEMB 3
60.00

CEMB C

GRAMS PER PLANT

50.00

*
40.00

*
30.00

20.00

10.00

0.00

L0 (No spray)

L1 (3 sprays)

L2 (2.5 sprays)

L3 (1.75 sprays)

TREATMENTS Economic Threshold Levels (ETLs) / Treatments L0 L1 L2 L3 No Spray (Control) 3 insects per 25 plants 5 insects per 25 plants 8 insects per 25 plants

* Significantly different in number of spray applications but non-significant in yield
The experiment was conducted according to the Split Plot Design having levels in the sub-plots but the objective was not to ascertain the best ETL level. The primary objective was to assess how many number of spray applications could be reduced in case of Bt cotton giving a yield comparable with the non-Bt cotton having significantly more number of spray applications.

129

4.4

Bt

INHERITANCE

STUDIES

IN

TRANSGENIC GENERATIONS
4.4.1 Bt Inheritance Studies in Selfed Generations
The inheritance of Bt transgene was studied in five successive selfed generations through molecular analyses during development of transgenic pure lines, as described above in section 4.2. The techniques of PCR and Western Dot Blot were used. In each generation, the seeds of only those plants which were positive in molecular analyses were picked. It was concluded that the transgene is faithfully inherited in progenies. The pattern of inheritance however, depended upon its hemizygosity, homozygosity or heterozygosity. Once the pure lines have been developed, there was no problem revealed in faithful transmission of the transgene in next selfed generations. The history sheet of the four pure lines developed during these studies is given in Table-13.

4.4.2 4.4.2.1

Bt Inheritance Studies in Filial Generations Inheritance of Transgene in F1 Generation

Two transgenic lines viz. CEMB-3 and CEMB-11 were crossed to two nontransgenic lines viz. MNH-93 and CIM-482 to produce six hybrids namely: MNH-93 X CEMB-3 and its reciprocal CEMB-3 X MNH-93, CIM-482 X CEMB-3 and its reciprocal CEMB-3 X CIM-482, MNH-93 X CEMB-11 and its reciprocal MNH-93 X CEMB-11.

130

Table 13

HISTORY SHEET OF TRANSGENIC PURE LINES DEVELOPED AT CEMB
S. NO. LINE NO. 1ST GENERATION
Total Plants = 1 PCR positive = 1 ELISA positive = 1 Southern Blot Positive = 1 Western Blot Positive =1 Total Plants = 1 PCR positive = 1 ELISA positive = 1 Southern Blot Positive = 1 Western Blot Positive =1 Total Plants = 1 PCR positive = 1 ELISA positive = 1 Southern Blot Positive = 1 Western Blot Positive =1 Total Plants = 1 PCR positive = 1 ELISA positive = 1 Southern Blot Positive = 1 Western Blot Positive =1

2ND GENERATION
Total Plants Analyzed = 17 PCR positive = 17 Western Dot Blot Positive =17 Total Plants Analyzed = 18 PCR positive = 12 Western Dot Blot Positive =12 Total Plants Analyzed = 17 PCR positive = 4 Western Dot Blot Positive =5 Total Plants Analyzed = 30 PCR positive = 26 Western Dot Blot Positive =26

3RD GENERATION
Total Plants Analyzed = 10 PCR positive = 10 Western Dot Blot Positive =10 Total Plants Analyzed = 10 PCR positive = 9 Western Dot Blot Positive =9 Total Plants Analyzed = 10 PCR positive = 9 Western Dot Blot Positive =9 Total Plants Analyzed = 10 PCR positive = 9 Western Dot Blot Positive =9

4TH GENERATION
Total Plants Analyzed = 30 PCR positive = 30 Western Dot Blot Positive =30 Total Plants Analyzed = 30 PCR positive = 30 Western Dot Blot Positive =30 Total Plants Analyzed = 30 PCR positive = 30 Western Dot Blot Positive =30 Total Plants Analyzed = 30 PCR positive = 30 Western Dot Blot Positive =30

5TH GENERATION
Total Plants Analyzed = 30 PCR positive = 30 Western Dot Blot Positive =30 Total Plants Analyzed = 30 PCR positive = 30 Western Dot Blot Positive =30 Total Plants Analyzed = 30 PCR positive = 30 Western Dot Blot Positive =30 Total Plants Analyzed = 30 PCR positive = 30 Western Dot Blot Positive =30

1.

CEMB-3

2.

CEMB-11

3.

CEMB-16

4.

CEMB-17

The inheritance of Bt transgene was studied in five successive selfed generations through molecular analyses during development of transgenic pure lines The history sheets of all Bt plants were maintained for screening purpose. The history of four pure lines developed during these studies is given above. 131

The F1 generations along with the parental lines were grown in green house. A total of 108 F1 plants were analyzed through PCR and Western Dot Blot to study the transmission and expression of the transgene in F1 generation. It was found that all 108 plants were positive in PCR as well as Western Dot Blot (Table-14). It was thus concluded that • • the gene was stably integrated in the genome of the transgenic plants; the transgene could be successfully transferred from Bt lines to non-Bt lines; and • the gene was dominant

4.4.2.2

Mendelian Inheritance Studies in F2 Generation

The F2 generations of all six crosses were raised during Kharif, 2005. A fairly large sample was taken from each F2 family and analyzed through Western Dot Blot. Since the genes segregate in F2 generation, it was imperative to clearly differentiate between negative and positive plants regarding Bt expression. The test of Western Dot Blot was found to be the most suitable in order to rapidly analyze large number of plants in lesser time and to differentiate between the two groups. The plants were distinctly categorized into two types viz. positive and negative. The data were subjected to chisquare goodness of fit test against the Mendelian ratio 3:1. The data have been given in Table-15.

132

Table 14

MOLECULAR ANALYSIS OF F1 PLANTS

S.NO

NAME OF CROSS

TOTAL PLANTS ANALYZED

NUMBER OF PLANTS POSITIVE IN PCR

NUMBER OF PLANTS POSITIVE IN WESTERN DOT BLOT

1

MNH-93 X CEMB-3

20

20

20

2

CEMB-3 X MNH-93

19

19

19

3

CIM-482 X CEMB-3

11

11

11

4

CEMB-3 X CIM-482

19

19

19

5

MNH-93 X CEMB-11

20

20

20

6

CEMB-11 X MNH-93

19

19

19

All plants of F1 generation were analyzed through PCR and Western Dot Blot to study the transmission and expression of the transgene in F1 generation. It was concluded that the gene was stably integrated in the genome of the transgenic plants; the transgene could be successfully transferred from Bt lines to non-Bt lines; and the gene was dominant

133

Table 15

SEGREGATION OF Bt GENE IN F2 POPULATIONS OF SIX CROSSES
Total Plants No. of Negative Plants No. of Positive Plants Chisquare value for 3:1 ratio 32.061 0.004 0.356 45.449 47.382 2.194

S.No.

Combinations

Ratio

1. 2. 3. 4. 5. 6.

MNH-93 X CEMB-3 CEMB-3 X MNH-93 CIM-482 X CEMB-3 CEMB-3 X CIM-482 MNH-93 X CEMB-11 CEMB-11 X MNH-93 Total

88 93 60 92 103 67 503

45 23 13 51 56 22 210

43 70 47 41 47 45 293

Distorted (Non-Mendelian) 3:1 (Mendelian) 3:1 (Mendelian) Distorted (Non-Mendelian) Distorted (Non-Mendelian) 3:1 (Mendelian)

The data were subjected to chi-square goodness of fit test against the Mendelian ratio 3:1. 134

A perusal of Table-15 revealed that Mendelian segregation ratios existed in 3 out of 6 cross combinations. The first cross MNH-93 X CEMB-3 showed non-Mendelian segregation while its reciprocal combination gave Mendelian ratio 3:1. Similarly, the 3rd cross CIM-482 X CEMB-3 showed Mendelian segregation while its reciprocal gave nonMendelian segregation ratio. Likewise, the 5th cross combination MNH-93 X CEMB-11 depicted non-Mendelian segregation ratio whereas its reciprocal showed Mendelian ratio 3:1.

4.5

STUDIES

ON

HETEROSIS

AND

HETEROBELTIOSIS IN F1 GENERATION
The superiority of an F1 hybrid over both of its parents in terms of yield or some other character is called heterosis. By definition, heterosis is over-dominance and is synonymous with the term hybrid vigour. Average heterosis refers to the superiority of an F1 over the mid-parent value; Heterobeltiosis describes the superiority of an F1 to its better parent; whereas economic heterosis is the superiority of an F1 hybrid over the best commercial variety of the crop in question. The plant breeder is generally interested in higher grain yield or increased vegetative growth, but heterosis may result in greater cell size, plant height, leaf area, root development, ear or grain size, grain number, and so on. Keeping in view the great importance of the subject especially in context of the genetically engineered Bt cotton, heterosis was studied in a few important characters in F1 hybrids of Bt and non-Bt cotton.

4.5.1

Seed Cotton Yield per Plant (g)
The data were collected on seed cotton yield per plant (g) regarding F1 progenies

and their parents and were subjected to the analysis of variance technique. The mean

135

squares presented in Table-16 indicated highly significant differences among genotypes. The genotypes were further partitioned into parents, crosses and parents versus crosses. The parents showed non-significant variation, the crosses showed highly significant variation whereas the parents versus crosses showed significant variation. The mean values of parental lines and those of crosses are presented in Table-17. Duncan’s Multiple Range test applied to the means indicated that among crosses, the cross CEMB-3 X CIM482 gave the maximum yield (48.09 gm/plant) and differed significantly from all other crosses. The cross CEMB-11 X MNH-93 showed the lowest value (15.37 gm/plant) and varied significantly from others except MNH-93 X CEMB-3, CIM-482 X CEMB-3 and CEMB-3 X MNH-93. Four crosses namely MNH-93 X CEMB-3, CIM-482 X CEMB-3, CEMB-3 X MNH-93 and MNH-93 X CEMB-11 had a range of 19.70 to 31.68 gm for this character and indicated non-significant differences among themselves. Since the parents showed non-significant differences among themselves in the analysis of variance, the DMR test was not applied to the parents. The estimates of heterosis and heterobeltiosis are presented in Table-18. The heterosis and heterobeltiosis ranged from -15.19% to 107.07% and -18.58% to 98.79%, respectively. One F1 hybrid CEMB-3 X MNH-93 showed significant and positive heterosis and heterobeltiosis, two hybrids CEMB-3 X CIM-482 and MNH-93 X CEMB11 showed highly significant heterosis and heterobeltiosis while the remaining hybrids showed non-significant heterosis and heterobeltiosis.

136

Table 16

ANALYSIS OF VARIANCE: MEAN SQUARES OF F1 HYBRIDS FOR DIFFERENT CHARACTERS
Mean Squares
Source of Variation Replications Genotypes Parents Crosses Parents vs Crosses Error Reps x Parents Reps x Crosses Reps X P vs C df 2 9 3 5 1 18 6 10 2 Yield per Plant 13.30 ns 290.38 ** 51.93 ns 410.47 ** 405.30 * 42.79 27.02 58.23 12.88 No. of Bolls per Plant 0.64 ns 10.63 ** 1.19 ns 15.19 ** 16.18 * 1.44 0.33 2.22 0.83 Boll Weight 0.04 ns 0.38 * 0.47 ns 0.31 ns 0.43 ns 0.12 0.13 0.11 0.14 GOT %age 0.26 ns 63.99 ** 84.42 ** 12.42 ** 260.59 ** 0.95 0.91 0.76 1.95 Lab Bioassay (Mortality %age) 0.45 ns 5.03 ** 1.78 ** 5.17 ** 14.08 ns 0.82 0.06 0.05 6.93

** indicates significant differences at P< 0.01 probability level. * indicates significant differences at P< 0.05 probability level. ns = Non-significant The analysis of variance was done with the help of the techniques mentioned by Steel and Torrie (1980).

137

4.5.2

Number of Bolls per Plant
The analysis of variance presented in Table-16 indicated highly significant

differences among genotypes for number of bolls per plant. When genotypes were further partitioned into parents, crosses and parents versus crosses, the crosses showed highly significant variation whereas the parents versus crosses showed significant variation. The mean values of parental lines and those of crosses are presented in Table-17. Duncan’s Multiple Range test applied to the means indicated that among crosses, the cross CEMB-3 X CIM-482 gave the maximum number of bolls per plant (10.17) and differed significantly from all other crosses except MNH-93 X CEMB-11 (8.92). The cross CEMB-11 X MNH-93 showed the lowest value (3.92) and varied significantly from others except CIM-482 X CEMB-3. Three crosses namely MNH-93 X CEMB-3, CIM482 X CEMB-3 and CEMB-3 X MNH-93 had a range of 5.83 to 6.75 for this character and indicated non-significant differences among themselves. Since the parents showed non-significant differences among themselves in the analysis of variance, the DMR test was not applied to the parents. The estimates of heterosis and heterobeltiosis are presented in Table-18. The heterosis and heterobeltiosis ranged from -20.34% to 81.36% and -20.34% to 81.36%, respectively. One F1 hybrid CEMB-3 X MNH-93 showed significant and positive heterosis but non-significant heterobeltiosis, two hybrids CEMB-3 X CIM-482 and MNH-93 X CEMB-11 showed highly significant heterosis and heterobeltiosis while the remaining hybrids showed non-significant heterosis and heterobeltiosis.

4.5.3

Boll Weight(g)
The analysis of variance presented in Table-16 indicated significant differences

among genotypes. When genotypes were further partitioned into parents, crosses and 138

parents versus crosses, none of them showed significant variation. The mean values of parental lines and those of crosses are presented in Table-17. Duncan’s Multiple Range test was applied to the means which indicated that among crosses, the cross CEMB-3 X CIM-482 gave the maximum boll weight (4.35 gm) and differed significantly from all other crosses except CEMB-11 X MNH-93 (3.91 gm) and CEMB-3 X MNH-93 (4.13 gm). The cross CIM-482 X CEMB-3 showed the lowest value (3.59 gm) and varied nonsignificantly from MNH-93 X CEMB-11, CEMB-3 X MNH-93, CEMB-11 X MNH-93 and MNH-93 X CEMB-3. The DMR test was also applied to the parents. Among parents, MNH-93 had the lowest value for this character (3.05) and differed significantly from others while the other parental lines differed non-significantly among themselves with a range of 3.72 to 3.96 gm. The estimates of heterosis and heterobeltiosis are presented in Table-18. The heterosis and heterobeltiosis ranged from -6.96% to 21.38% and -9.30% to 9.99%, respectively. One F1 hybrid CEMB-3 X MNH-93 showed highly significant positive heterosis and significant heterobeltiosis, two hybrids CEMB-11 X MNH-93 and CEMB-3 X CIM-482 showed significant heterosis and heterobeltiosis while the remaining hybrids showed non-significant heterosis and heterobeltiosis.

4.5.4

Ginning Outturn Percentage
The analysis of variance presented in Table-16 indicated highly significant

differences among genotypes. When genotypes were further partitioned into parents, crosses and parents versus crosses, all of them showed highly significant variation. The mean values of parental lines and those of crosses are presented in Table-17. Duncan’s Multiple Range test was applied to all means. Among crosses, the cross CIM-482 X CEMB-3 gave the maximum GOT (46.47%) and differed significantly from all other

139

crosses except CEMB-3 X CIM-482 (45.13). The cross CEMB-3 X CIM-482 (45.13%) also differed non-significantly from MNH-93 X CEMB-3. The cross CEMB-11 X MNH93 showed the lowest value (41.08%) and varied non-significantly from CEMB-3 X MNH-93. The cross MNH-93 X CEMB-11 having a GOT value 42.84% differed significantly from others except CEMB-3 X MNH-93 and MNH-93 X CEMB-3. The DMR test was also applied to the parents. Among parents, CIM-482 had the highest value for this character (45.37%) and differed significantly from all others while the other parental lines differed non-significantly among themselves with a range of 34.35% to 35.62%. The estimates of heterosis and heterobeltiosis are presented in Table-18. All hybrids exhibited highly significant heterosis and heterobeltiosis. The heterosis and heterobeltiosis ranged from 13.02% to 26.44% and -0.52% to 26.17%, respectively. Only one F1 hybrid CEMB-3 X CIM-482 showed negative heterobeltiosis while all other hybrids showed positive heterosis and heterobeltiosis.

4.5.5

Lab Bioassay Results (Mortality %age of Heliothis larvae)
The analysis of variance presented in Table-16 indicated highly significant

differences among genotypes. When genotypes were further partitioned into parents, crosses and parents versus crosses; parents versus crosses showed non-significant variation whereas the parents and crosses showed highly significant variation among themselves. The mean values of parental lines and those of crosses are presented in Table-17. Duncan’s Multiple Range test was applied to all means. Among crosses, the cross MNH-93 X CEMB-3 gave the maximum mortality %age (94%) and differed significantly from all other crosses except CEMB-3 X MNH-93 (90%) and CIM-482 X

140

CEMB-3 (86%). The cross CIM-482 X CEMB-3 (86%) also varied non-significantly from MNH-93 X CEMB-11 and CEMB-11 X MNH-93. The cross CEMB-3 X CIM-482 gave the lowest mortality %age (68%) and differed significantly from others except CEMB-11 X MNH-93 and MNH-93 X CEMB-11. The DMR test was also applied to the parents. Among parents, CEMB-3 had the highest value for this character (76%) and differed significantly from MNH-93 and non-significantly from CEMB-11 and CIM-482. The lines CEMB-11, MNH-93 and CIM-482 differed non-significantly among themselves. The estimates of heterosis and heterobeltiosis are presented in Table-18. The heterosis and heterobeltiosis ranged from -8.11% to 36.23% and -5.56% to 23.68%, respectively. Two F1 hybrids MNH-93 X CEMB-3 and CEMB-3 X MNH-93 showed highly significant positive heterosis and heterobeltiosis. One F1 hybrid CIM-482 X CEMB-3 showed significantly positive heterosis and heterobeltiosis. The remaining three hybrids exhibited non-significant heterosis and heterobeltiosis.

4.6

HERITABILITY AND GENETIC ADVANCE STUDIES IN Bt COTTON

4.6.1

Heritability for Bt Resistance
Since the efficiency of selection would depend upon the magnitude of variability

that is heritable and caused by genetic factors, the higher values, therefore, of Heritability accompanied by higher Genetic Advance for the character studied should be quite valuable.

141

Table 17

MEAN PERFORMANCE OF F1 HYBRIDS AND THEIR PARENTS FOR DIFFERENT CHARACTERS
Seed Cotton Yield per Plant (g) Crosses MNH-93 X CEMB-3 CEMB-3 X MNH-93 CIM-482 X CEMB-3 CEMB-3 X CIM-482 MNH-93 X CEMB-11 CEMB-11 X MNH-93 Parents CEMB 3 CEMB 11 MNH 93 CIM 482 22.26 a 18.23 a 14.88 a 24.19 a 5.92 a 4.92 a 4.92 a 6.08 a 3.76 a 3.72 a 3.05 b 3.96 a 34.50 b 35.62 b 34.35 b 45.37 a 76 a 72 ab 62 b 72 ab 21.63 bc 27.89 bc 19.70 bc 48.09 a 31.68 b 15.37 c 6.17 b 6.75 b 5.83 bc 10.17 a 8.92 a 3.92 c 3.60 b 4.13 ab 3.59 b 4.35 a 3.60 b 3.91 ab 43.52 bc 41.80 cd 46.47 a 45.13 ab 42.84 c 41.08 d 94 a 90 a 86 ab 68 c 76 bc 74 bc No. of Bolls per Plant Boll Weight (g) GOT %age Lab Bioassay (Mortality %age)

Duncan’s Multiple Range test was applied to all means after Analysis of Variance. The means followed by the same letter (s) in a column are not statistically significant at 5% level of probability

142

Table 18

ESTIMATES OF HETEROSIS AND HETEROBELTIOSIS FOR DIFFERENT CHARACTERS OF F1 HYBRIDS
GENOTYPES Seed Cotton Yield per plant (g) Ht(%) MNH-93 X CEMB-3 CEMB-3 X MNH-93 CIM-482 X CEMB-3 CEMB-3 X CIM-482 MNH-93 X CEMB-11 16.49ns 50.19* -15.19ns 107.07** 91.35** Hbt(%) -2.81ns 25.31* No. of Bolls per plant Ht(%) 13.81ns 24.58* Hbt(%) 4.17ns 14.02ns -4.11ns 67.12** 81.36** Boll Weight (g) Ht(%) 5.89ns 21.38** -6.96ns 12.83* 6.42ns Hbt(%) -4.08ns 9.96* -9.30ns 9.99* -3.19ns 5.09* GOT %age Ht(%) 26.44** 21.43** 16.38** 13.02** 22.45** 17.43** Hbt(%) 26.17** 21.17** 2.43** -0.52** 20.27** 15.33** Lab Bioassay (Mortality %age) Ht(%) 36.23** 30.43** 16.22* -8.11ns 13.43ns 10.45ns Hbt(%) 23.68** 18.42** 19.44* -5.56ns 5.56ns 2.78ns

-18.58ns -2.80ns 98.79** 73.78** 69.40** 81.36**

CEMB-11 X -7.19ns -15.71ns -20.34ns -20.34ns 15.52* MNH-93 ** indicates significant differences at P< 0.01 probability level. * indicates significant differences at P< 0.05 probability level. ns = Non-significant

The “t” test was employed to determine whether F1 hybrid means were statistically significant from mid parent and better parent values or otherwise. The “t” values were calculated by the formulae narrated by Wynne et al. (1970). 143

The data pertaining to Broad Sense Heritability (BSH) of Bt insect resistance character has been presented in Table-19. A perusal of the table reveals that the Broad Sense Heritability was high in three cross combinations CEMB-11 X MNH-93 (69.30), MNH-93 X CEMB-11 (66.96) and CEMB-3 X MNH-93 (65.38). The BSH was moderate in the cross combination MNH-93 X CEMB-3 (47.03), low in the cross combination CIM-482 X CEMB-3 (38.012) and very low in the cross combination CEMB-3 X CIM482 (22.10).

4.6.2

Genetic Advance for Bt Resistance
The response to selection can be predicted with the help of Genetic Advance

values. A perusal of the Table-19 revealed that the for the character of Bt insect resistance, the cross combination CEMB-11 X MNH-93 showed the highest Genetic Advance value of 42.08 followed by its reciprocal MNH-93 X CEMB-11 with a value of 39.18. The cross combination CEMB-3 X MNH-93 and its reciprocal MNH-93 X CEMB3 also showed high values of Genetic Advance i.e. 36.12 and 28.62, respectively. The cross combination CIM-482 X CEMB-3 and its reciprocal CEMB-3 X CIM-482 showed comparatively lower values of GA i.e. 21.07 and 12.08, respectively In order to make comparison in gain in selection, Relative Expected Gain (REG), expressed as percentage of mean was also estimated. It is more useful for identification of better traits. A perusal of the Table-19 revealed that the cross combination CEMB-11 X MNH-93 showed the highest REG value of 90.89 followed by its reciprocal MNH-93 X CEMB-11 with a value of 80.91. The cross combination CEMB-3 X MNH-93 and its reciprocal MNH-93 X CEMB-3 showed high values of REG (77.05 and 50.50, respectively). The cross combination CIM-482 X CEMB-3 and its reciprocal CEMB-3 X CIM-482 showed low values of REG.

144

Table 19

HERITABILITY AND GENETIC ADVANCE FOR Bt RESISTANCE IN THE CROSSES BETWEEN Bt AND NON-Bt COTTON LINES
S.No. Cross combination
(lab bioassay results)

F1 Variance

(lab bioassay results)

F2 Variance

Broad Sense Heritability (%) 47.03

Genetic Advance 28.62

Relative Expected Gain 50.50

1

MNH-93 X CEMB-3

462.2222

872.6003

2

CEMB-3 X MNH-93

248.8889

718.9791

65.38

36.12

77.05

3

CIM-482 X CEMB-3

448.8889

724.1495

38.012

21.07

41.21

4

CEMB-3 X CIM-482

604.4444

775.9131

22.10

12.68

25.74

5

MNH-93 X CEMB-11

266.6667

807.0058

66.96

39.18

80.91

6

CEMB-11 X MNH-93

266.6667

868.6599

69.30

42.08

90.89

On the basis of heritability estimates and increase in mean performance per generation, progress from selection can be predicted.

145

The crosses bearing high values of heritability and REG may give better progenies. The high values of Heritability and Genetic Advance also indicate an additive type of gene action.

4.7

CORRELATION

OF

Bt

TRAIT

WITH

OTHER ECONOMIC TRAITS.
The correlation of Bt insect resistance character with some other economic characters of cotton was computed. The results have been given in Table-20. A perusal of the table revealed that the correlation existed between Bt and all characters but in most of the cases, it was statistically non-significant. The correlation of Bt with yield, number of monopodial branches per plant, number of sympodial branches per plant, number of bolls per plant, boll weight, staple length and fibre fineness was statistically non-significant. The Bt trait had highly significant but negative correlation with plant height. The correlation of Bt with plant height (0.99) was found to be strong and linear (Table-20). A perusal of Table-20 further revealed that Bt had significantly positive and strong correlation with Ginning Outturn Percentage (0.90). An important parameter, other than the morphological plant characteristics, was included in the study i.e. intensity of natural infestation of Spotted Bollworm in the field. The data were recorded on Bt and non-Bt cotton genotypes sown in the field. The number of live insects per plant were counted during the season and averaged at the end. The data thus generated were compared with the Bt content in the genotypes to compute correlation. The Bt trait had a strong negative correlation (-0.83) with natural infestation of Spotted Bollworm (Table-20).

146

Table 20

CORRELATION OF Bt INSECT RESISTANCE TRAIT WITH THE ECONOMINC TRAITS OF COTTON
Natural Infestation of Spotted Bollworm (number of insects per plant) -0.83 * No. of Monopodial Branches per Plant No. of Sympodial Branches per Plant

Bt Protein Content (%)

0.40 ns

-0.99 **

-0.43 ns

0.77 ns

-0.77 ns

0.49 ns

0.90 *

0.34 ns

-0.68 ns

*

significant

Correlation is a bivariate measure of association (strength) of the relationship between two variables. Zero value shows a random relationship whereas 1or -1 shows perfect linear relationship. 147

Fibre Fineness (µg/in)

Ginning Outturn (%)

No. of Bolls per Plant

Staple Length (mm)

Plant Height (cm)

Boll Weight (g)

Yield (g)

4.8

COMPARISON OF SOME QUALITATIVE CHARACTERS COTTON
The observations on some important qualitative characters of cotton were also

OF

Bt

AND

NON-Bt

taken during the present studies. The observations were recorded in all generations and trials conducted during the period under report. The comparisons, in general, of Bt and non-Bt cotton have been given in Table-21. The monopodial plant shape of the variety remained unchanged after transformation. Similarly, boll shape remained Roundish even after transformation. The boll opening was good fluffy for both Bt and non-Bt cotton lines. The reaction to virus was also found to be the same in both types. The plant reaction to insects was found to be susceptible in case of non-Bt cotton. It was however, tolerant in case of Bt cotton. The only qualitative character that showed deterioration was leaf hairiness. The leaf hairiness was found to become smooth to sparsely hairy after transformation, whereas it was profusely hairy in non-Bt parent. All lines of cotton showed the same hairiness pattern suggesting strong pleiotropic effects of Bt gene on the hairiness gene(s). The decline in hairiness in Bt cotton may require a slight increase in dose or number of insecticide applications to control sucking pests. Further scientific studies on this aspect may however, prove or disprove this supposition.

148

Table 21

COMPARISON OF SOME IMPORTANT QUALITATIVE CHARACTERS OF Bt AND NON-Bt COTTON VAR. MNH-93

CHARACTER

Bt MNH-93

Non-Bt MNH-93

Plant Shape

Monopodial

Monopodial

Smooth to Sparsely Leaf Hairiness Hairy Profusely Hairy

Boll Shape

Roundish

Roundish

Boll Opening

Good Fluffy

Good Fluffy

Reaction to Virus

Susceptible

Susceptible

Reaction to Bollworms

Tolerant

Susceptible

The qualitative characters were also taken into account while screening plants in different generations

149

4.9
4.9.1

DISCUSSION
Transformation
A number of local varieties have been screened at CEMB, on the basis of their

regeneration capability through tissue culture for subsequent transformation with foreign genes (Hussain, 2002). However, the lengthy procedures of transformation and development of pure lines had caused a considerable loss in reaping full benefits afterwards. The main reasons to this loss have been the replacement of old varieties with the new high yielding varieties, or the lower genetic stability of the varieties at field level. It was therefore imperative to select for transformation, such a cotton variety that had shown better adaptability and more genetic stability at field level besides having a good yield and regeneration potential. In the light of the data available regarding area under different cotton varieties during 1980-2000 (Economic Surveys of Pakistan, 1980-2000) and screening data of Hussain (2002), the variety MNH-93 was thus the most suitable material for transformation purpose. In the present transformation, CaMV35S promoter was used with Cry1Ab. Chunlin et al. (1999) and Zeng et al. (2002) conducted bioassays and showed that synthetic Cry1Ac gene with a strong promoter like ubiquitin or OM could be the effective strategy to enhance expression in plants. This report suggested that chimeric OM and ubiquitin were stronger promoters than the CaMV35S promoter that was widely used in plant genetic engineering. In the present studies, transformation method used was primarily Agrobacteriummediated. However, it was supplemented with bombardment of tungsten particles through biolistic gun. The tungsten particles were bombarded just to create micro-wounds on the embryos to facilitate DNA transfer by Agrobacterium. Our strategy was in line with 150

earlier research workers such as Finer and McMullen (1990), Fraley et al. (1983) and Bidney et al. (1992). According to Finer and McMullen (1990), Agrobacterium-mediated transformation is a most common method to transform dicotyledonous plants. The other method being used to transform cotton is microprojectile bombardment. Agrobacterium tumefaciens causes gall (neoplastic diseases) tumors on many dicotyledonous and some monocotyledonous plants naturally. It transfers its segments of DNA called T-DNA from its tumor inducing plasmid (Ti) to the plant genome. Earlier Fraley et al. (1983) had reported the Agrobacterium-mediated transformation of petunia and tobacco. Bidney et al. (1992) have shown that efficiency of Agrobacterium-mediated gene transfer could be increased by wounding the explants by bombardment with naked particles. The transformation efficiency in the present studies has been 0.26% which was however very low as compared to Majeed et al. (2000) who transformed cotton variety CIM-443 with Cry1Ab gene by using Agrobacterium and biolistic method in combination and obtained a high transformation efficiency of 9% after two months selection on 100mgL-1 kanamycin medium. This may be attributed to the regeneration capability through tissue culture of the variety MNH-93 which was good but comparatively lower than the variety CIM-443 used by Majeed et al. (2000). In the present studies, regeneration of plants was obtained using mature embryos of cotton at a concentration of 50mgL-1 kanamycin in selection medium. This was in line with many earlier research workers such as Firoozabady et al. (1987) and Umbeck et al. (1987) who first time reported transformation of cotton (G.hirsutum L) using Agrobacterium method. They co-cultivated cotyledon pieces with Agrobacterium containing Ti plasmid with a chimeric gene encoding kanamycin resistance. They however regenerated plants through callus using 25-35mgL-1 kanamycin concentration for discrimination of transformed and non-transformed plants. Schrammeijer et al. (1990)

151

and Cousins et al. (1991) also used Agrobacterium-mediated transformation in sunflower and siokara. They also selected transformants by their ability to grow on selection medium i.e. containing kanamycin. The transformed plants were analyzed for DNA integration through PCR and Southern Blotting. The copy number was also found through Southern Blotting. Our studies are in line with several research workers including Schrammeijer et al. (1990) and Cousins et al. (1991) who confirmed the integration of foreign DNA through PCR. Zapata et al. (1999) and Gould and Magallanes-Cedeno (1998) have also reported the transformed plants analyzed through DNA blots for evidence of integration of transgenes and their copy number. The transgene expression was confirmed through ELISA and Western Dot Blot. Our findings were in line with Bashir et al. (2004) who confirmed the expression of Cry proteins through ELISA and Western Blotting while evaluating transgenic lines of indica Basmati rice. According to Perlak et al. (1990), the technical means to produce Bt protected plants were not available, until recently. Now however, the combination of plant cell tissue culture and modern molecular methods allow for a greater diversity of traits, including Bt genes, to be efficiently introduced and deployed in plants for insect control. He has further stated that because they are proteins and the difficulty of expressing this class of proteins in plants has been overcome, Bt proteins are now relatively straightforward to produce in plants.

4.9.2

Development of Pure Transgenic Lines
The primary objective of developing transgenic pure lines was to develop an

insect resistance source. The breeding for high yield or for other morphological and quality characters was not the basic aim. It was however, preferable during the plant

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selections in each generation, to choose those plants in which high resistance was also accompanied by such other characteristics as are desirable by the plant breeders. The methods adopted during selections were according to the standard procedures laid down in the books by Poehlman, J.M. (1978); Khan, M.A. (2001) and Singh, B.D. (2005). Our studies also supported Benedict et al. (1996) and Altman et al. (1996) who while studying field trials on transgenic cotton (BTK) lines found that the transgenes involved their inheritance in subsequent generations. Shelton et al. (2000) conducted field tests on managing resistance to Btengineered plants. They reported that the present resistance management strategies rely on a “refuge” composed of non-Bt plants to conserve susceptible alleles. They have used Bt-transgenic broccoli plants and the diamondback moth as a model system to examine resistance management strategies. The higher number of larvae on refuge plants in field tests indicated that a “separate refuge” was more effective at conserving susceptible larvae than a “mixed refuge” and reduced the number of homozygous resistant (RR) offspring. Our strategy of Resistance Management during the generations planting and conducting field trials also included a separate refuge. In the present studies, Bt contents were quantified and expressed as percent of total protein. The Bt contents showed variation in successive generations. In the 3rd generation, the Bt contents ranged from 0.09% to 1.35% of the total protein whereas in the successive two generations, the Bt contents ranged from 0.21% to 0.29% of the total protein. The possible explanation to this behaviour may be that the 3rd generation plants were grown in green house under controlled conditions whereas the next two generations were grown in field under more fluctuating temperatures. Our findings were in confirmation of the findings of Sachs et al. (1998) who found that Cry1A gene expression was variable and strongly influenced by environmental factors. The expression level of

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Cry1Ab gene, under CaMV35S promoter, in our Bt cotton lines was found to be generally higher in comparison with Husnain et al. (2002) who reported that the Cry1Ab gene was expressed in Basmati rice stems at 0.15% under the control of ubiquitin promoter. However, the expression of transgene in our lines varied in different generations. This changing tendency have been reported by various research workers such as Wu et al. (2002) reported changing tendency of Cry1Ab content at different developmental stages from R4 to R6 generation. They reported that the content of the Cry1Ab protein in leaves of transgenic rice reached 0.9% to 0.14% of the total soluble protein in 1998 and 1999, respectively. Similarly, Chunlin et al. (1999) and Zeng et al. (2002) found that the expression of Bt toxin in individual plant can be upto 0.255% of total soluble proteins. Also according to Bashir et al. (2004), the expression level of Cry1Ac varied from 0.21% to 1.03% and 0.95% to 1.13% of the total protein during 1st and 2nd year of rice field trials, respectively. There was a varying behaviour of transgenic progeny plants in respect of resistance shown in lab bioassays against Heliothis larvae. This has been in confirmation of many research workers. Chunlin et al. (1999) and Zeng et al. (2002) reported that some of the homozygous Cry1Ac transgenic rice plants of T2 progeny showed high level resistance against striped stem borer (Chilo suppressalis) at field trial.

4.9.3

Field Studies
The Bt field trials were conducted for two consecutive years at CEMB, Lahore to

study the performance of Bt lines in comparison with non-Bt control. The results exhibited that the lines containing Bt gene produced enough toxin to kill and/or repel the Lepidopteran insects. The data recorded on natural infestation of Spotted Bollworm gave a clear proof of the achievement of the goal of transformation. In the present studies,

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while evaluating the lines for insect resistance, the data on natural infestation of Spotted Bollworm was the most reliable criteria because only the spotted bollworm occurs naturally in Lahore environment. These results were in accordance with the results obtained after Bt protein quantification. The results obtained after conducting lab bioassays also supported significant differences among control and Bt lines. The DMR test clearly indicated the Bt lines to be better in insect resistance than the non-Bt counterpart. The data obtained after much hectic and laborious exercise of conducting Field Bioassays/Artificial Infestation of Heliothis in the field were not so encouraging. This may be attributed to the reasons already described i.e. the insect moths were collected from entirely different climatic zones than that of Lahore area, reared artificially in the labs under controlled conditions of temperature and humidity, and released in the field directly. Consequently, the mortality rate of the insects had been higher due to abrupt change in their environment. The method of insect release therefore, needed to be improved further. Our findings that the control line showed higher survival percentage of Heliothis than the Bt lines were similar to the results already reported by various research workers, a few of which are described below:The expression of Bt Cry1Ac and Cry1Ab genes have been reported in cotton by Perlak et al. (1990) and Barton (1989). They reported total protection from insect damage of leaf tissue from these plants in laboratory assays when tested with Lepidopteran insects and that Cry1Ab had 5-fold higher unit activity for pink bollworm than for cotton bollworm and tobacco budworm. Sachs et al. (1996; 1998) reported that cotton plants of both genetic backgrounds that possessed the Cry1Ab insecticidal protein or high terpenoid glanding or both were more resistant to Tobacco budworm larvae than plants with other traits. Estruch et al. (1997); Halcomb et al. (1996); Halcomb et al. (2000) and Van Rie. J.

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(2000) evaluated plant stands of BTK and non-BTK. They found that the attack of bollworms and tobacco budworm was less on BTK plants as compared to non-BTK plants and that mortality %age was greater in case of bollworm while no significant difference was observed in case of Tobacco budworm fed both on BTK and non-BTK plants. Our findings were also in confirmation of Wu et al. (2003) who reported that the survival of larvae was significantly reduced in Cry1Ac expressing plants in spite of the fact that egg densities between transgenic and non-transgenic plants varied nonsignificantly. Similarly, Liu et al. (2001) reported that Bt cotton killed all susceptible larvae tested. The survival of resistant larvae was 46% relative to their survival on non-Bt cotton. The Bt lines were also studied for a number of other characters besides the primary insect resistance traits. The lines were found to be statistically different at a significant level from the control in Number of Sympodial Branches per Plant, Plant Height and Ginning Outturn Percentage (GOT). A careful study of the data showed that after transformation, the Bt plants had got other positive effects as well in addition to insect resistance, i.e. the GOT percentage and Number of Sympodial Branches per Plant had increased significantly from the non-Bt parent. The increase in these two characters is much desirable from the breeders’ point of view. Similarly, the Bt plants had shown a significant reduction in height which is also a desirable change. The Bt lines were not differing significantly from their non-Bt counterpart in the other characteristics studied viz. Number of Monopodial Branches per Plant, Number of Bolls per Plant, Boll Weight, Staple Length and Fibre Fineness. It is thus evident that these characteristics of the plants were not affected by the transformation event and remained almost at their parental level. This is also much desirable from the breeder’s point of view. A plant breeder always wishes to get a variety transformed for insect

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resistance but keeping the other characters intact/unaltered. Since the seeds used for transformation were of an approved variety, it was therefore a highly positive aspect of this transformation that most of the varietal characters remained un-altered. The most important character in a crop-improvement programme is yield. The data presented in Table-5 indicated that the genotypes had non-significant differences among themselves regarding yield. The crop was kept immune to insecticidal sprays against Lepidopteran insects. Theoretically, the Bt lines should give higher yields than control. In fact, the Bt lines generally gave higher yields than the control line, in both years of study; however, the increased yields were non-significant statistically. This behaviour of the transgenic lines may be attributed to the lower natural infestation of the Lepidopteran insects during the years of study. In the comparative studies of insecticide applications on Bt and non-Bt cotton, it was concluded that a saving of 41% of insecticides (meant for Lepidopteran, only) can be done by the use of Bt cotton line CEMB-3 in comparison with its non-Bt counterpart MNH-93. Our findings are in confirmation of many earlier research workers. Field trials of transgenic cotton (BTK) lines have been studied against Lepidopterans by Benedict et al (1996) and Altman et al (1996). Their plants were carrying genes that code Cry1Ab and Cry1Ac delta-endotoxins from Bacillus thuringiensis var. kurstaki. They found that these insect resistance lines showed a reduction of the insecticide application for tobacco budworm, Bollworm, Cabbage looper and increased farm profit. Similarly, Stewart et al. (2001) and Tabashnik et al. (2002) reported that crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. Our findings are also in confirmation of Xia et al. (1999) who reported use of Bt cotton in china with a concomitant reduction in insecticide use. They concluded that Bt cotton required fewer

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chemical insecticides and a potential for higher yields. Carpenter and Gianessi (2001) reported the primary benefits of increasing yields due to elimination of losses by European corn borer. Other benefits of modified plants were emphasized by several authors like reduced environmental impact of insecticides, potential of higher yields and better food supply in the developing countries, better food safety due to reduced fungal infections and remediation of polluted soils (Borlaug, 2000; Mackey and Santerre, 2000; Munkvold and Hellmich, 2000; Mendelsohn et al., 2003; Kasha, 2000). However, the new modified crops could not be the panacea for solving all the pest problems due to specific mode of actions of toxins against the target pests (Sharma et al., 2000).

4.9.4

Bt Inheritance
In the selfed-generations studies, the Cry1Ab gene was found to be behaving

differently in different progenies in respect of integration and expression. This may be attributed to the epistatic and environmental factors besides segregation of the gene. According to the earlier reports of Sachs et al. (1996; 1998) epistatic and environmental factors affect the foreign gene expression in cotton (Gossypium hirsutum L); these effects could influence the stability, breeding, durability and efficacy of foreign genes. They reported site of gene insertion and cotton background to be the significant sources of variation for Cry1A gene expression. These effects were heritable and caused similar effects in several different genetic backgrounds of F2 families. In the present studies, varietal differences were observed in various characters. This has been in confirmation of Hoskinson et al. (1964) who observed marked varietal differences in cotton, some experimental lines being vigorous and tolerant of the early season disease-insect complex.

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In the F1 generation studies, the Bt gene was found to be successfully transferred from Bt to non-Bt lines and that it was dominant. Our results are in confirmation of Canming et al. (2000) who found that the resistance of the three transgenic Bt cotton strains to Helicoverpa armigera is controlled by one pair of non-allelic dominant genes. Carvalho et al. (1995) conducted a 6x6 diallel cross experiment and to study inheritance of number of bolls per plant, plant height and fibre maturity. Their results showed that both dominance and additive effects were more pronounced. In case of yield and boll weight, dominance effects were more dominant. In the present studies, the segregation of Bt gene was found to be in Mendelian fashion in three out of six crosses. This has been in confirmation of various earlier research reports e.g. Wu et al. (2002) reported that both Mendelian and distorted segregation ratios were observed in some selfed and crossed F2 populations. Canming et al. (2000) also reported segregation of resistant and susceptible plants in Mendelian 3:1 ration in six F2 populations whereas in one F2 population the segregation was nonMendelian. On the other hand, Zhang et al. (2000) studied inheritance and segregation of foreign Bt (Bacillus thuringiensis toxin) and tfdA genes in cotton. Their results confirmed that inheritance and segregation of both resistance characters was governed by a single dominant nuclear gene, and was not affected by cytoplasm. Their data supported the conclusion that foreign traits encoded by single genes are inherited and expressed in Mendelian fashion in cotton. However, the situation has been otherwise in the studies of Altman et al. (1996) who analyzed F2 progenies to ascertain the inheritance pattern of Bt genes Cry1Ab and Cry1Ac. Their data indicated that the mode of inheritance was not always Mendelian in different genetic backgrounds. They stated that this situation should not be considered unusual for cotton if transgenes were regarded as another type of exotic

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gene. According to them, this conclusion about exotic genes is generally recognized by cotton breeders and geneticists who normally work with such material.

4.9.5

Heterosis and Heterobeltiosis
The phenomenon of heterosis and heterobeltiosis was studied in varying degrees

of magnitude in various characters. The heterosis ranged from -20.34% in CEMB-11 X MNH-93 for number of bolls per plant to 107.07% in MNH-93 X CEMB-11 for yield of seed cotton per plant. Similarly, the heterobeltiosis ranged from -20.34% in CEMB-11 X MNH-93 for number of bolls per plant to 98.79% in MNH-93 X CEMB-11 for yield of seed cotton per plant. Different crosses showed significant and non-significant hybrid vigour in different characters. Our results were in confirmation of many earlier reports on heterosis. Panhwar et al. (2002) conducted heterosis studies in six intra specific hybrids of G. hirsutum L. for number of sympodial branches, number of bolls, boll weight and seed cotton yield per plant on an average performance. They concluded that highest increase of hybrids 69.23% over their parents was observed for boll weight followed by 64.24% for seed cotton yield, 22.97% for number of bolls and 19.62% for number of sympodia per plant. Similarly, Ahmad et al. (2005) found highly significant heterosis in yield and leaf area of F1 hybrids ranging from 102 to 309% and 46.3 to 163.9%, respectively. Iqbal and Nadeem (2003) conducted Generation Mean analysis for seed cotton yield and number of sympodial branches per plant in cotton (Gossypium hirsutum L.). Their results showed that 5 crosses over mid and 4 crosses over better parent showed significant heterosis for number of sympodial branches per pant. They reported involvement of epistasis in all crosses except one for yield of seed cotton.

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4.9.6

Heritability and Genetic Advance
The Broad Sense Heritability of Bt insect resistance character was found to be

high in three out of six crosses and moderate in one. It was also observed that the crosses of Bt lines with their original non-Bt parent (MNH-93) had higher values of Genetic Advance as compared to the crosses made with a different variety (CIM-482). Our findings are in agreement with Zhang et al. (2001) who reported high Broad Sense Heritability in all crosses except one. Similarly Maluf et al. (2002) studied inheritance of resistance to the root-knot nematode in lettuce. They reported that resistance to M.incognita has a high heritability (0.798), under the control of single gene locus. Similarly, Bonos (2006) determined Narrow Sense Heritability and predicted gain from selection for dollar spot resistance in creeping bentgrass. He reported high Narrow Sense Heritability estimates (0.79 [2002], 0.79 [2003]) and large mean squares for General Combining Ability and supported the idea that additive gene action plays a significant role in disease resistance. Ahmad et al. (2003) reported very high estimates of Heritability associated with high Genetic Advance for Bolls per Plant (97.8 and 60.78), Virus Infestation %age (95.0 and 189.9) and Boll Weight (97.39 and 10.99). Their data suggested selection for improvement of these traits due to presence of sufficient genotypic variability. Since, the efficiency of selection would depend upon the magnitude of variability that is heritable and caused by genetic factors, the higher values, therefore, of heritability accompanied by high genetic advance for the characters studied should be quite valuable. In the present studies, high Heritability accompanied with high Genetic Advance was observed in three crosses whereas moderately high Heritability and high Genetic Advance was noted in one cross. The findings of the present study suggest that the crosses

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exhibiting high heritability estimates have sufficient genetic variability worth of exploitation and effective selections can be done in early generations for Bt insect resistance character. The low heritability does not mean that there would be no progress, but improvement, in this case, would be slow and gradual for this trait.

4.9.7

CORRELATION
It was found that the correlation of Bt was statistically non-significant with a

number of characters including yield, number of monopodial branches per plant, number of sympodial branches per plant, number of bolls per plant, boll weight, staple length and fibre fineness. These results are similar to Milicia et al. (1966) who found no correlation between germination behaviour and earliness of maturity in the hybrids of maize. In our studies, it was observed that the Bt trait had a strong negative correlation with natural infestation of spotted bollworm and plant height. These results were encouraging and depicted that an increase in the Bt contents in the plants strongly enhanced their inbuilt insect resistance capability, thereby reducing the counts of naturally occurring Lepidopteran insect-spotted bollworm. Similarly, an increment in Bt protein would reduce the plant height proportionately. In fact, the reduction in plant height in cotton is highly desirable by the cotton breeders due to its many advantages. Therefore, the introduction of Bt gene (Cry1Ab) in cotton variety MNH-93 gave an advantage of reduction in height in addition to enhanced insect resistance. These results of negative correlation in our studies are similar to Adamczyk and Gore (2004) who reported an inverse relationship between the amounts of Cry1Ac among cultivars versus the weight of bollworm larvae. An increase in GOT %age is one of the primary objectives of cotton breeding. In the present studies, it was found that the Bt insect resistance trait had a significantly

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positive correlation with Ginning Outturn %age. In this way, introduction of Bt gene in cotton yielded a comparative additional benefit of higher GOT %age. These results of significant correlation are similar to Kronstad (1977) who found various barley characteristics to be significantly correlated. Our results are also in line with Biradar and Borikar (1984) who observed high correlations among different characteristics in sorghum. The correlation data presented here give a good picture of the relationship of Bt insect resistance with different characters which would be much helpful to the plant breeders while making selections and planning further experiments of crop improvement.

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CHAPTER 5

LITERATURE CITED

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LITERATURE CITED
Adamczyk, Jr. J. J. and Gore, J. (2004). Development of bollworms, Helicoverpa zea, on two commercial Bollgard cultivars that differ in overall Cry1Ac levels. Journal of Insect Science, 4:32. Ahmad, S., Khan, M. S., Swati, M. S., Shah, G. S. and Khaliq, I. H. (2005). A study on heterosis and inbreeding depression in Sunflower (Helianthus annuus L.). Songklanakarin J. Sci. Technol. 27(1): 1-8. Ahmad, S., Karim, A., Jabbar, A., Hassan, M., Muhammad, T. and Iqbal, M. (2003). Genetic analysis for some characteristics in cotton (Gossypium hirsutum L.). Online Journal of Biological Sciences. 3(2): 228-232. Alam, A. K. M. R., Roy, N. C. and Islam, H. (1992). Heterosis and combining ability in upland cotton (Gossypium hirsutum L.). Ann. Bangladesh Agric. 2(1): 31-39. 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 Amer, C., Berry, A. R. E. and Kogan, M. (1999). Effects of phtyophagous Heteropteran predators of feeding on transgenic Bt potato plants. Submitted for publication. Ankenbauer, R. G. and Nester, E. W. (1990). Sugar-mediated induction of Agrobacterium tumefaciens virulence genes: structural specificity and activities of monosaccharides. J. Bacter. 172: 6442-6446. Barton, K. (1989). Development of insect resistant cotton. AgBiotech 89-Proceedings of a conference held in Arlington- Virginia USA. 28-30: 168-171. Bashir, K., Husnain, T., Fatima, T., Latif, Z., Mehdi, S. A. (2004). Field evaluation and risk assessment of transgenic indica basmati rice. Molecular Breeding. 13: 301312. Baum, J. A., Johnson, T. B., and Carlton, B. C. (1999). Bacillus thuringiensis- natural and recombinant bioinsecticide products. In Methods in Biotechnology. Vol 5. Biopesticides: Use and Delivery (F. R. Hall and J. J. Mean, Eds.), pp. 189–209. Humana Press, Inc., Totowa, NJ.

165

Beegle, C. C. and Yamanoto, T. (1992). History of Bacillus thuringiensis Berliner Research and Development. Can. Entomol. 124: 587-616. Berliner, E. (1911). Uber die schlaffsucht der Mehlmottenraupe Z. Ang. Entomol. 2: 2956. Bidney, D., Schelonge, C., Mrtich, J., Burrus, M., Sims, L. and Huffman, G. (1992). Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens. Plant Mol. Biol. 18: 301-313. Biradar, S. G. and Borikar, S. T. (1984). Path analysis for seedling vigour in sorghum. J. Maharashtra Agric. Univ. 9(2): 228-229. Bonos, S. A. (2006). Heritability of dollar spot resistance in creeping bentgrass. Phytopathology. 96:808-812. Borlaug, N. E. (2000). Ending world hunger: the promise of biotechnology and the threat of antiscience zealotry. Plant Physiol. 124: 487-490. Bourland, F. M. (1978). Inheritance and inter-relationship of several seed and seedling characteristics of cotton (Gossypium hirsutum L.). Diss. Abst. Inter. B. 39(3): 1124-1125B. Burris, J. S. (1973). The evaluation of various indices of seedling vigour and field performance. Agron. Madison, Wisconsin, U.S.A. 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. Carpenter, J. E. & Gianessi, L. (2001). Agricultural biotechnology: updated benefit estimates. National Center for Food and Agricultural Policy (NCFAP). 1-48. Carvalho, L. P. D. E., Moraes, C. F. D. E., Cruz, C. D., Carvalho, L. P. and Moraes, C. F. (1994). Combining ability and Heterosis in upland cotton. Revista-ceres. 41(237): 514-527. Carvalho, L. P. D. E. (1995). Genetic control of fibre percentage and boll weight in cotton. Revista-Ceres. 419(244): 626-636.

166

Chlan, C. A., Lin, J., Cray, J. W. and Cleveland, T. E. (1995). A procedure of biolistic transformation and regeneration of transformed cotton for meristematic tissues. Plant Mol. Biol. Rep. 13: 31-37. Chitkowski, R. L., Turnipseed, S. G., Sullivan, M. J. and Bridges, W. C. F. (2003). Field and laboratory evaluations of transgenic cottons expressing one or two Bacillus thuringiensis var. kurstaki Berliner proteins for management of Noctuid (Lepidoptera) pests. J. Econ. Entomol. 96(3): 755-762. Christou, P., Swain, W. F., Yang, N. S. and McCabe, D. E. (1998). Inheritance and expression of Soybean callus by DNA-coated gold particles. Plant Physiol. 87: 671-674. Chunlin, S., Zhen, Z., Guifang, X., Altosaar, I. and Pingzhang, F. (1999). Construction of plant vectors with high level expression of Bt toxin gene and studies on their expression behaviour in transgenic tobaccos. Chin. J. Biotechnol. 15(4): 203-210. Cohen, B. M., Gould, F and Benture, J. S. (2000). Bt rice: Practical steps to sustainable use. Intern. Rice Res. Notes. 25(2): 4-10. Conway, G. and Toenniessen, G. (1999). Feeding the world in the twenty-first century. Nature. 402 Suppl: C55–C58. Cousins, Y. L., Lyon, B. R. and Liewelly, D. J. (1991). Transformation of Australian cotton cultivars: prospects for cotton improvement. Aust. Jour. Plant Physiol. 18: 481-491. Crickmore, N., Ziegler, D. R., Feitelson, J., Schnepf, E., Van Rie, J., Lereclue, R., Baum, J. and Dean, D. H. (1998). Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62: 807–813. Culpepper, A. S. and York, A. C. (1998). Weed management in glyphosate-tolerant cotton. J. Cotton Sci. 4: 174–185. Dekeyser, R. A., Claes, B., De Rycke, R. M. U., Habets, M. E., Van Montagu, M. M. and Caplan, A. (1990). Transient gene expression in intact and organized rice tissues. The Plant Cell. 2: 591-602.

167

Dogan, E. B., Berry, R. E., Reed, G. L. and Rossignol, P. A. (1996). Biological parameters of convergent lady beetle (Coleoptera; Coccinellidae) feeding on aphids (Homoptera; Aphididae) on transgenic potato. J. Econ. Entomol. 89: 1105– 1108. Economic Survey of Pakistan. (2004-05). Ministry of Finance. Government of Pakistan. Islamabad. Chapter 02: 9-15. English, L., and Slatin, S. L. (1992). Mode of action of delta-endotoxin from Bacillus thuringiensis: A comparison with other bacterial toxins. Insect Biochem. Mol. Biol. 22: 1–7. EPA (1988). EPA guidance for the re-registration of pesticide products containing Bacillus thuringiensis as the active ingredient, Reregistration Standard 540; RS89-023. EPA (1996a). EPA Fact Sheet for Bacillus thuringiensis subspecies kurstaki Strain EG 7841, September 1996 (Ecogen). EPA (1998a). EPA Registration Eligibility Decision (RED) Bacillus thuringiensis, EPA 738-R-98-004, March 1998. EPA (1998b). (RED Facts) Bacillus thuringiensis. EPA-738-F-98-001. Estruch, J. J., Carozzi, N. B., Desai, N., Duck, N. B., Warren, G. W. and Koziel, M. G. (1997). Transgenic plants: an emerging approach to pest control. Nat. Biotechnol. 15(2): 137-141. Feldman, J., Reed, G. L., Wyman, J. A., Stewart, J. and Stone, T. B. (1992). Genetically Modified Colorado Potato Beetle Resistant Potato plants, Foliar-Applied microbial Bt and conventional insecticides: Comparative impacts on non-target arthropods. Appendix 1. New Leaf Public Interest Document, EPA. Finer, J. J. and McMullen, M. D., (1990). Transformation of cotton (Gossypium hirsutum L.) via particle bombardment. Plant Cell Rep. 19: 586-589. Fraley, R. T., Rogers, S. G., Horsch, R. B., Sanders, I. R., Flick, J. S., Adams, S. P., Bittner, M. L., Brand, L. A., Fink, C. L., Fry, J. S., Gallupi, G. R., Goldberg, S. B., Hoffman, N. L., and Woo, S. C. (1983). Expression of bacterial genes in plant cells. Proc. Natl. Acad. Sci. USA. 50: 4803-4807.

168

Fred, S. B., Bruce, G. H. and Roy, L. F. (2000). Safety and advantages of Bacillus thuringiensis-protected plants to control insect pests. Regulatory Toxicology and Pharmacology. 32: 156–173. Firoozabady, E., DeBoer, D. L., Merio, J. D., Halk, E. I., Amerson, L. N., Rashka, K. E. and Murry, E. (1987). Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants. Plant Mol. Biol. 10: 105-116. Frick, H. and Bauman, L. F. (1978). Heterosis in maize as measured by K uptake properties of seedling roots. Crop Sci. 18(1): 99-103. Garcia, B. F. G. E., Salinas G., O. Pozo C., H. Reyes V., M. Ramírez M., J.A. López S., M. Aguirre B., and O. Salazar S. (2002). Estimation of genetic distances among green pepper (Capsicum annuum. L.) lines using RAPD markers and its relationship with heterosis Proceedings of the 16th International Pepper Conference Tampico, Tamaulipas, Mexico. November 10–12, 2002 Gasser, C. S. and Fraley, R. T. (1992). Transgenic crops. Sci. Am. 226: 67-70. Gatehouse, A. M. R. and Hilder, V. (1994). Genetic manipulation of crops for insect resistance. In: Marshall G, Walter D (eds) Molecular Biology in Crop Protection. pp. 176-201. Chapman and Hall, London. Gianessi, L. P. and Carpenter, J. E. (1999). Agricultural Biotechnology: Insect Control Benefits. National Center for Food and Agricultural Policy. Gill, S. S., E. A. Cowles, and P. V. Pietrantonio. (1992). The mode of action of Bacillus thuringiensis endotoxins. Ann. Rev. Entomol. 37:615–636. Gould, J.H. and Magallanes-Cedeno, M. (1998). Adaptation of cotton shoot apex culture to Agrobacterium-mediated transformation. Plant Mol. Bio. Rep. 16(3): 283-285. Guo, W. Z., Sun, J., Guo, Y. F. and Zhang, T. Z. (2001). Investigations of different dosages of inserted Bt genes and their insect resistance in transgenic Bt cotton. Yi Chuan Xue Bao. 28(70): 668-676. Halcomb, L. J., Benedict, H. J., Cook, B. and Ring, R. D. (1996). Survival and growth of bollworm and tobacco budworm on non-transgenic and transgenic cotton expressing a Cry1A insecticidal protein (Lepidoptera: Noctuidae). Environ. Entomol. 25(2): 253-255. 169

Halcomb, L. J., Benedict, H. J., Cook, B., Ring, R. D. and Correa, J. C. (2000). Feeding behaviour of bollworm and budworm (Lepidoptera: Noctuidae) larvae in mixed stands of non-transgenic and transgenic cotton expressing an insecticidal protein. J. Econ. Entomol. 93(4): 1300-1307. Haq, M. I. and Khan, M. A. (1993). Genetic analysis of some agronomic characters in upland cotton. Pak. J. Agri. Res. 14(4): 283-289. Hoffman, C., Vanderbruggen, H., Hofte, H., Van Rie, J., Jansens, S. and Van-Mellaert, H. (1988). Specificity of Bt delta-endotoxin is correlated with the presence of the brush border membrane of target insects midguts. Proc. Natl. Acad. Sci. USA. 85: 7844-7848. Hofte, H. and Whitely, H. R. (1989). Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol. Rev. 53: 242–255. Hooykaas, P. J. J. and Schilperoort, R. A. (1992). Agrobacterium and plant genetic engineering. Plant Mol. Biol. 19:15-38. Horsch, R. B., Fry, J. E., Hoffman, N. L., Eichholtz, D., Rogers, S. G. and Fraley, T. R. (1985). A simple and general method for transferring genes into plants. Sci. 12291239. Hoskinson, P. E., Pate J.B. and Duncan, E. N. (1964). The influence of early season diseases and insects on seedling vigour in cotton. Plant. Dis. Reptr. 48: 499-602. Huang, J., Rozelle, S., Pray, C. and Wang, Q. (2002). Plant biotechnology in China. Science (Washington). 295: 674–677. Husnain, T., Asad, J., Maqbool, B.S., Datta, S. K. and Riazuddin, S. (2002). Variability in expression of insecticidal Cry1Ab gene in Indica Basmati rice. Euphytica 128: 121-128. Hussain, B., Amin, M. A. and Khan, M. A. (1998). Assessment of genetic mechanism in seed and lint indices in upland cotton. J. Agic. Res. 36(2): 103-110. Hussain, S. S. (2002). Genetic Transformation of Cotton with Galanthus Nivalis Agglutinin (GNA) gene. PhD Thesis, CEMB, Univ. of the Punjab, Lahore. p:83.

170

IPCS (2000). International Programme on Chemical Safety—Environmental Health Criteria 217: Bacillus thuringiensis. WHO. http://www.who.int/pcs/docs/ehc_217.html. Iqbal, M. Z. and Nadeem, M. A. (2003). Generation Mean Analysis for seed cotton yield and number of sympodial branches per plant in cotton (Gossypium hirsutum L.). Asian J. Plant. Sci., 2(4): 395-399. James, C. (1997). Global Status of Transgenic Crops in 1997. ISAAA Briefs. No.5. ISAAA. Ithaca. NY. James, C. (1998). Global Review of Commercialized Transgenic Crops: 1998, ISAAA Briefs. No.8. ISAAA. Ithaca. NY. James, C. (1999). Preview—Global Review of Commercialized Transgenic Crops: 1999, ISAAA Briefs. No.12. ISAAA, Ithaca, NY. James, C. and Krattiger, A. F. (1996). Global review of the field testing and commercialization of transgenic plants, 1986 to 1995: The first decade of crop biotechnology. ISAAA Briefs. 1: 1–38. Jeon, G. A., Eum, J. S. and Sim, W. S. (1998). The role of inverted repeat (IR) sequence of the virE gene expression in Agrobacterium tumefaciens pTiA6. Mol. Cells. 8: 49-53. Kasha, K. J. (2000). Biotechnology and world food supply. Genome 42: 642-645. Khan, M.A., Bashir, E. and Bantel, R. (2001). Plant Breeding. 2nd edition, National Book Foundation, Islamabad. Pakistan. Klein, T. M., Gradziel, T., Fromm, M. E. and Sangord, J. C. (1987). Factors influencing gene delivery into maize (Zea mays) cell by high velocity microprojectiles. Bio/Tech. 6: 559-563. Klotz-Ingram, C., Jans, S., Fernandez-Cornejo, J. and McBride, W. (1999). Farm-level production effects related to the adoption of genetically modified cotton for pest management. AgBioForum. 2(2): 73–84. Knowles, B. H. and Ellar, D. J. (1987). Colloid-osmotic lysis is a general feature of the mechanisms of action of Bacillus thuringiensis (delta)-endotoxins with different insect specificity. Biochem. Biophys. Acta. 924: 509–518.

171

Knowles, B. H. and Dow, J. A. T. (1993). The crystal delta-endotoxins of Bt models for their mechanism of action of the insect midgut. Bioessays. 15: 469-476. Kolganova, T. V., Shrivastava, D. K., Mett, V. L. and Piruzyan, E. S. (1991). Morphogenetic potential of transformed cotton callus tissues. Bio/Tech. 3: 469474. Kronstad, W. E. (1977). Correlation of field emergence rate and seed vigour criteria in barley cultivars. Crop. Sci. 17(2): 312-314. Lee, M. K., Milne, R. E., Ge, A. Z. and Dean, D. H. (1992). Location of Bombyx mori receptor binding regime on Bacillus thuringiensis δ-endotoxin. App. Environ. Microbiol. 267: 3115-3121. Liu, G., Chen, N., Kaji, A., Bode, A.M., Ryan, C. A. and Dong, Z. (2001). Potato inhibitor I and II from potatoes block the UVB-induced AP-1 protein compositional pattern in JB6 cells. Proc. Natl. Acad. Sci. USA. 8: 98 (10): 57865791. Mackey, M. A. and Santerre, C. R. (2000). Biotechnology and our food supply. Nutrition Today. 35: 120-127. Magoja, J. L. and Palacios, I. G. (1987). Early expression of heterosis in diploperennial teosinte-maize hybrids. Maize Genet. Coop. Newsl. No. 61: 63-64. Majeed, A., Husnain, T. and Riazuddin, S. (2000). Transformation of virus-resistant Gossypium hirsutum L. genotype CIM-443 with pesticidal gene. Plant Biotech. 17(2): 105-110. Maluf, W. R., Azevedo, S. M., Gomes, L. A. A. and Olivera, A. C. B. (2002). Inheritance of resistance to the root-knot nematode Meloidogyne javanica in lettuce. Genet. Mol. Res. 1 (1): 64-71. Marra, M., Carlson, G. and Hubbell, B. (1998). Economic impacts of the first crop biotechnologies. Available on the World Wide Web at http://www.ag.econ.ncsu.edu/faculty/marra/online.html. Martin, P. A. and Travers, R. S. (1989). Worldwide abundance and distribution of Bacillus thuringiensis isolates. App. Environ. Microbiol. 55: 2437-2442.

172

McCabe, D. E. and Martinell, B. J. (1993). Particle gun transformation applied to cotton. International Congress of Plant Mol. Biol. Tucson AZ. McClintock, J. T., Schaffer, C. R. and Sjoblad, R. D. (1995). A comparative review of the mammalian toxicity of Bacillus thuringiensis-based pesticides. Pestic. Sci. 45: 95– 105. Mendelsohn, M., Kough, J., Vaituzis, Z. and Matthews, K. (2003). Are Bt crops safe? Nature Biotechnol. 21: 1003-1009. Meredith, W. R. Jr. and Brown, J. S. (1998). Heterosis and combining ability of cottons originating from different regions of the United States. The Journal of Cotton Science. 2:77-84(1998). Milicia, C. I. and Juncu, A. M. (1966). Cold resistance of maize hybrids. Probl. Agric. Bucuresti. 19(12): 10-18. Munkvold, G. P. and Hellmich, R. L. (2000). Genetically modified, insect resistant maize: implications for management of ear and stalk disease. Plant Health Progress.12. September 2000. Available from: http://www.plantmanagementnetwork.org/sub/php/review/maize. Noteborn, H. P. J. M., Rienenmann-Ploum, M. E., Van den Berg, J. H. J., Alink, G. M., Zolla, L. and Kuiper, H. A. (1993). Food safety of transgenic tomatoes expressing the insecticidal crystal protein Cry1Ab from Bacillus thuringiensis and the marker enzyme APH (39) II. Med. Fac. Landbouw. Univ. Gent. 58/4b. Olsen, K. M. and Daly, J. C. (2000). Plant toxin interactions in transgenic Bt cotton and their effect on mortality of Helicoverpa armigera (Lepidoptera: Noctuidae). J. Econ. Entomol. 93(4): 1293-1299. Panhwar, G. N., Kalhoro, A. D., Soomro, A. H., Tunio, G. H. and Chang, M. S. (2002). Heterosis studies in varietal crosses of Gossypium hirsutum L. for certain economic characters. Asian Journal of Plant Sciences. 1(1): 44-47. 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. Biotechnology. 8: 939– 943. Poehlman, J. M. (1978). Breeding Field Crops. 2nd edition. The AVI publishing company, Inc. Westport, Connecticut. 173

Potrykus, I. (1991). Gene transfer to plants - assessment of published approaches and results. Ann. Rev. Plant Physiol. and Plant Mol. Biol. 42: 205-225. Rajanna, B. and de la Cruz, A. A. (1975). Growth analysis–an aid in determining seedling vigour of field crops. Agron. Abst. P. 95. Amer. Soc. of Agron. Madison, Wisconsin, U.S.A. Reed, G. L., Puls, K., Jensen, A. S., Feldman, J. and Berry, R. E. (1993). The effect of Colorado Potato Beetle control measures on non-target arthropods. In Proceedings of the 1993 Washington State Potato Conference and Trade Fair, pp. 125–140. Riva, G. A., Cabrera, G. J., Pardon, R. V. and Pardo, C. A. (1998). Agrobacterium tumefaciens: a natural tool for plant transformation. Elec. J. Biotech. 1(3): 117133. Royal Society. (1998). Genetically modified plants for food use. Available at. www.royalsoc.ac.uk/files/statfiles/document-56.pdf. Royal Society. (2000). Transgenic plants and world agriculture.

<http://www.royalsoc.ac.uk/policy/index.html> Sachs, S. E., Benedict, H. J., Taylor, F. J., Stelly, M. D., Davis, K. S. and Altman, W. D. (1996). Pyramiding Cry1Ab insecticidal protein and terpenoids in cotton to resist tobacco budworm (Lepidoptera: Noctuidae). Environ. Entomol. 25(6): 1257-1261. Sachs, S. E., Benedict, H. J., Taylor, F. J., Stelly, M. D., Altman, W. D., Berberich, A. S., and Davis, K. S. (1998). Expression and segregation of genes encoding Cry1A insecticidal proteins in cotton. Crop Sci. 38: 1-11. Saha, S., Callahan, F.E., Douglas, A.D. and Creech, J.B. (1997). Lyophilization of cotton tissue on quality of extractable DNA, RNA and protein. The J. Cotton Sci. 1:1014. Singh, B.D. (2005). A Textbook of Plant Breeding. 2nd edition. Kalyani Publishers, New Delhi, India. Schnepf, H. E. and Whiteley, H. R. (1981). Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coli. Proc. Natl. Acad. Sci. USA 78: 2893–2897.

174

Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D. R. and Dean, D. H. (1998). Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and Molecular Biology Reviews. 62(3): 775-806. Schrammeijer, B., Sijmons, P. C., Van den Elzen, P. J. M. and Heokema, A. (1990). Meristem transformation of sunflower via Agrobacterium. Plant. Cell Report. 5560. Sharma, H. C., Sharma, K. K., Seetharama, N. and Ortiz, R. (2000). Prospects for using transgenic resistance to insects in crop improvement. Electronic Journal of Biotechnology [online]. 15 August 2000. Vol. 3. No. 2. Available from: http://www.ejbiotechnology.info/content/vol3/issue2/full/3/index.html. Shelton, A. M., Tang, J. D., Roush, R. T., Metz, T. D. and Earle, E. D. (2000). Field tests on managing resistance to Bt-engineered plants. Nature Biotechnology. 18: 339342 Shrivastava, D. K., Kolganova, T. V., Mett, V. L. and Piruzian, E. S. (1991). Genetic transformation of Gossypium hirsutum L. Plant Biotech. 289: 263-264. Shwartz, J. M., Tabashnik, B. E. and Johnson, M. W. (1991). Early response of cultured Lepidopteran cells to expose to δ-endotoxin from Bacillus thuringiensisinvolvement of calcium and anionic channels. Entomologia experimentalis et. Applicata. 61:179-187. Sjoblad, R. D., McClintock, J. T. and Engler, R. (1992). Toxicological considerations for protein components of biological pesticide products. J. Econ. Entomol. 80: 717– 723. Snedecor, G. W. and Cochran, W. G. (1989). Statistical Methods. Eighth Edition. Iowa State University Press. Sonsgtad, D. D., Somers, D. A. and Greisback, R. J. (1995). Advances in alternative DNA delivery techniques. Plant Cell Tiss. Organ Cult. 40: 1-15. Smith, E. F. and Townsend, C.O. (1907). A plant tumor of bacterial origin. Sci. 25: 671673. Smith, R. H. (1997). An extension entomologist’s 1996 observations of Bollgard Bt technology. In 1997 Proceedings Beltwide Cotton Conferences.

175

Smith, R. H. (1999). Alabama entomologist believes genetic engineering and eradication will usher in a new era of cotton pests. Cotton Grower Plus. March, 1999. Stamp, P. (1987). Seedling growth and photosynthetic traits of a maize cross and its parental lines at constant and fluctuating temperatures. Angewandte Boptanik. 61(3-4): 193-201. Steel, R. G. D. and Torrie, J. H. (1980). Principles and Procedures of Statistics. A Biometrical Approach. McGraw Hill Book Inc. New York. Stewart, S. D., Adamczyk, J. J., Knighten, K. S. and Davis, F. M. (2001). Impact of Bt cottons expressing one or two insecticidal proteins of Bacillus thuringiensis Berliner on growth and survival of noctuid (Lepidoptera) larvae. J. Econ. Entomol. 94(3): 752-760. Tabashnik, B. E., Liu, Y. B., Dennehy, J. T., Sims, A. M., Sisterson, S. M., Biggs, W. R. and Carriere, Y. (2002). Inheritance of resistance to Bt toxin Cry1Ac in a fieldderived strains of pink bollworm (Lepidoptera; Gelechiidae). J. Econ. Entomol. 59(5): 1018-1026. Thomas, W. E. and Ellar, D. J. (1983). Mechanism of action of Bt. var. israelensis insecticidal delta endotoxins. FEBS Lett. 154: 362-368. Taylor, S. L. and Lehrer, S. B. (1996). Principles and characteristics of food allergens. Crit. Rev. Food Sci. Nutr. 36 (S): S91–S118. Umbeck, P., Jahnson, G., Barton, K. and Swain, W. (1987). Genetically transformed cotton (Gossypium hirsutum L.). Plant. Biotech. 5: 263-266. USDA (1975). Cooperative Economic Insect Report. APHIS 25. 32. USDA (1998). Pest Management Practices: 1997 Summary. National Agricultural Statistics Service, SPCRI (98). Van Frankenhuyzen, K. V. 1993. The challenge of Bacillus thuringiensis. P. 35. In P. F. Entwistle, J. S. Van Rie, J. (2000). Bacillus thuringiensis and its use in transgenic insect control technology. Int. J. Med. Microbiol. 290 (4-5): 463-469.

176

Weinzierl, R., Pierce, C. and Steffey, K. (1997). Preliminary results of 1997 summer survey for Bt-resistant European corn borers. Pest Manage. Crop Dev. Bull. 22: 183–184. Williams, M. R. (1997). Cotton insect losses 1979–1996. In 1997 Proceedings Beltwide Cotton Conferences. Wolfersberger, M. G. (1992). V-ATPase energized epithelia and biological insect control. J. Exp. Biol. 172: 377-386. Williams, M. R. (1999). Cotton Crop Losses. Retrieved June 1999 from the World Wide Web: http://www.msstate.edu/Entomology/ CTNLOSS/1998loss.html. Wilson L. J., Mensah, R. K. and Fitt, G. P. (2004). Implementing integrated pest management in Australian cotton. In Insect pest management: field and protected crops. Ed. by Horowitz AR and Ishaaya I, Springer, New York. pp 97–118. Wu, K., Guo, Y., Lv, N., Greenplate, J. T. and Deaton, R. (2003). Efficacy of transgenic cotton containing a Cry1Ac gene from Bacillus thuringiensis against Helicoverpa armigera (Lepidoptera: Noctuidae.) in Northern China. J. Econ. Entomol. 96(4): 1322-1328. Wu, G., Cui, H., Ye, G., Xia, Y., Sardana, R., Cheng, X., Li, Y., Altosaar, I and Shu, R. (2002). Inheritance and expression of the Cry1Ab gene in Bt (Bacillus thuringiensis) transgenic rice. Thoer. appl. Genet. 104: 727-734. Wynne, J. C., Every, D. A. and Rice, P. H. (1970). Combining ability in Arachis hypogea L. II Field Performance of F1 Hybrids. Crop Sci. 10 (6): 713-715. Xia, J. Y., Cui, J. J., Ma, L. H., Dong, S. X. and Cui, X. F. (1999). The role of transgenic Bt cotton in integrated insect pest management. Acta Gossypii Sim. 11: 57–64. Zapata, C., Park, S. H., El-Zik, K. M. and Smith, R. H. (1999). Transformation of a Texas cotton cultivar by using Agrobacterium and shoot apex. Theor. Appl.Genet. 80: 250-255. Zapata, C., Srivatanakul, M., Park, S. H., Lee, B. M., Salas, M. G. and Smith, R. H. (1999). Improvements in shoot apex regeneration of two fibre crops: cotton and kenaf. Plant Cell Tiss. Cult. 56: 185-191.

177

Zeng, Q. C., Wu, Q., Zhou, K. D., Feng, D. J., Wang, F., Su, J., Altosaar, I. and Zhu, Z. (2002). Obtaining stem borer resistant homozygous transgenic lines of Minghui 81 harboring novel Cry1Ac gene via particle bombardment. Yi Chuan Xue Bao, 29(6): 519-524. Zhang, J. and Stewart, J. M. (2000). Economical and rapid method for extracting cotton genomic DNA. The J. Cotton Sci. 4:193-201. Zhang, Z. J., Yang, G. H., Li, G. H., Jin, S. L., and Yang, X. B. (2001). Transgressive segregation, heritability, and number of genes controlling durable resistance to stripe rust in one Chinese and two Italian wheat cultivars. Phytopathology. 91:680-686. Zupan, J. R. and Zambryski, P. C. (1995). Transfer of T-DNA from Agrobacterium to the plant cell. Plant Physiol. 107: 1041-1047. 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.

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ANNEXURE-I

RECIPES OF VARIOUS MEDIUMS

YEP MEDIUM Yeast Extract Bactopeptone NaCl Agar pH 10.0 g/l 10.0 g/l 5.0 g/l 14.0 g/l 7.5

MS MEDIUM Ms Basal Medium Sucrose Phytagel pH 4.43 g/l 30.0 g/l 3.0 g/l 5.7

LB MEDIUM NaCl Tryptone Yeast Extract Bacto Agar 10.0 g/l 10.0 g/l 5.0 g/l 15.0 g/l

I

T3 MEDIUM Tryptone Tryptose Yeast Extract MnCl2 1M K2PO4(pH 6.8) Agar Glucose 3.12 g/l 2.08 g/l 1.56 g/l 0.0052 g/l 2.6ml 15.76 g/l 3.0 g/l

II

ANNEXURE-II

RECIPES OF VARIOUS SOLUTIONS

20X PBS SOLUTION NaCl KCl Na2HPO4 160 g/l 4.0 g/l 28.4 g/l

SOB SOLUTION Tryptone Yeast Extract NaCl KCl pH 20 g/l 5 g/l 0.584 g/l 0.186 g/l 7.0

SOC SOLUTION SOB solution Mg++ Glucose As required 20 mM 20 mM

DEPURINATION SOLUTION 0.25N HCl

III

DENATURATION SOLUTION 5N NaOH 5M NaCl

NEUTRALIZATION SOLUTION 2M Tris-HCl pH 8.0 5M NaCl

20X SSC SOLUTION 3M NaCl 0.3M Na-Citrate

PRE-HYBRIDIZATION SOLUTION 5X SSC 0.1% Sarkosine 0.02% SDS 1% Blocking Reagent

2X WASH SOLUTION 2X SSC 0.1% SDS

IV

ANNEXURE-III

RECIPES OF VARIOUS BUFFERS

PCR BUFFER 10X 100 mM Tris-HCl pH 8.3 500 mM KCl 30 mM MgCl2

TE BUFFER 1mM Tris-HCl pH 8.0 1mM EDTA pH 8.0

DNA EXTRACTION BUFFER 0.5M Glucose 0.2M Tris-HCl pH 8.0 5mM EDTA pH 8.0 2% PVP 0.2% Mercaptoethanol

DNA LYSIS BUFFER 0.2M Tris-HCl pH 8.0 1.4M NaCl 25mM EDTA 2% CTAB 2% PVP

V

COLOR SUBSTRATE 1 tablet of NBT/BCIP + 10ml Genius Buffer-III/ Water

PROTEIN EXTRACTION BUFFER 0.04M EDTA 10% Glycerol 0.15M NaCl 0.01M Tris-HCl pH 7.5 0.1M NH4Cl 0.02M DTT 3.5mM PMSF CARBONATE BUFFER 0.06M NaHCO3 0.04M Na2CO3

BLOCKING BUFFER 1X PBS Skimmed Milk Tween 20 100ml 5g 0.05%

VI

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