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) (DR. TAYYAB HUSNAIN)

Co-Supervisor 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 re-

confirmed 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 non-

Bt 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 ii
ACKNOWLEDGEMENTS iv
SUMMARY vi
LIST OF FIGURES xiv
LIST OF TABLES xv
LIST OF ANNEXURES xvi
ABBREVIATIONS xvii
1 INTRODUCTION 1
BIOTECHNOLOGY AND THE CONVENTIONAL
1.1 TECHNOLOGY 2
1.2 AGRICULTURE IN PAKISTAN 3
1.3 COTTON SITUATION IN PAKISTAN 4
1.4 PLANT PROTECTION 5
1.5 ADVANCES IN COTTON RESEARCH 5
1.6 INSECT RESISTANCE THROUGH Bt GENES 7
1.7 Bt SAFETY STUDIES 8
1.8 WORLDWIDE ADOPTION OF Bt CROPS 11
1.9 BENEFITS OF Bt CROPS 13
1.10 NEED OF THE DAY 15
1.11 OBJECTIVES 16
2 REVIEW OF LITERATURE 17
2.1 BACILLUS THURINGIENSIS 18
2.2 PLANT TRANSFORMATION 22
2.3 FIELD STUDIES 28
2.4 INHERITANCE 33
3 MATERIALS AND METHODS 42
3.1 AGROBACTERIUM TRANSFORMATION 43
3.1.1 Agrobacterium tumefaciens Competent Cells Preparation 43
Agrobacterium Transformation with pKMAB By Heat Shock
3.1.2 Method 43

ix
3.1.3 Long and Short Term Storage of Bacterial Strain 44
3.1.4 Confirmation of Agrobacterium Transformation 44
3.1.4.1 Plasmid Isolation 44
3.1.4.2 Confirmation of Transformation Through PCR 45
3.2 COTTON TRANSFORMATION 45
3.2.1 Selection of a Suitable Variety 45
3.2.2 Seed Delinting 46
3.2.3 Seed Sterilization 46
3.2.4 Bombardment with Tungsten Particles 46
3.2.5 Agrobacterium Mediated Transformation 47
3.3 MOLECULAR ANALYSES OF TRANSGENIC PLANTS 48
3.3.1 Genomic DNA Isolation 48
3.3.2 Polymerase Chain Reaction 49
3.3.3 Southern Hybridization 50
3.3.3.1 Probe Making/DNA Labelling 50
3.3.3.1.1 Plasmid Digestion 50
3.3.3.1.2 Gel Elution 50
3.3.3.1.3 DNA Labeling with Biotin-11-dUTP 51
3.3.3.1.4 Probe Estimation 51
3.3.3.2 Genomic DNA Digestion 52
3.3.3.3 Gel Running for Southern Hybridization 52
3.3.3.4 Gel Transfer Assembly 52
3.3.3.5 Blot Processing 53
3.3.3.6 Copy Number Estimation 53
3.3.4 Immunological Assay of Transgenic Plants 53
3.3.4.1 Isolation of Protein from Cotton Leaves 54
3.3.4.2 Enzyme Linked Immunosorbent Assay 54
3.3.4.3 Western Dot Blot 54
3.4 INSECT BIOASSAYS 55
3.5 FIELD STUDIES 55
3.5.1 Development of Transgenic Pure Lines 56
3.5.1.1 1st Generation 56

x
3.5.1.2 2nd Generation 57
3.5.1.2.1 Selection Criteria 58
3.5.1.3 3rd Generation 58
3.5.1.4 4th & 5th Generations 59
3.5.2 Field Trials 59
3.5.2.1 Bt trials 2004-2005 59
3.5.2.2 Comparative Study of Insecticide Applications on Bt and non-Bt
Cotton Lines 2004-2005. 63
3.5.2.2.1 Insecticide Application Trial, 2004 63
3.5.2.2.2 Insecticide Application Trial, 2005 64
3.6 Bt INHERITANCE STUDIES 65
3.6.1 Bt Inheritance in Transgenic Selfed Generations 65
3.6.2 Bt Inheritance in Filial Generations 65
3.6.2.1 Crossing among Bt and non-Bt Lines 66
3.6.2.1.1 Emasculation 66
3.6.2.1.2 Pollination 66
3.6.2.1.3 Combinations of Crosses 67
3.6.2.2 Inheritance Studies in F1 Generation 67
3.6.2.3 Inheritance Studies in F2 Generation 67
3.7 HETEROSIS AND HETEROBELTIOSIS STUDIES 68
3.8 HERITABILITY AND GENETIC ADVANCE STUDIES 69
3.9 CORRELATION STUDIES 70
COMPARISON OF SOME QUALITATIVE CHARACTERS
3.10 OF Bt AND NON-Bt COTTON 71
3.11 STATISTICAL ANALYSES 72
3.11.1 Analysis of Variance 72
3.11.2 t-test Assuming Unequal Variances 72
3.11.3 Chi Square Test 73
3.11.4 Estimation of Heterosis and Heterobeltiosis 73
3.11.5 t-Test For Heterosis 74
3.11.6 t-test for Heterobeltiosis 74
3.11.7 Heritability Estimates 74
3.11.8 Genetic Advance Estimates 75

xi
3.11.9 Estimation of Correlation 75
3.11.10 t-test For Correlation 76
4 RESULTS AND DISCUSSION 77
4.1 COTTON TRANSFORMATION 78
4.1.1 Selection of a Suitable Variety 78
4.1.2 Agrobacterium Transformation with pKMAB 78
4.1.3 Agrobacterium Mediated Transformation of Cotton with pKMAB 81
4.1.4 Cotton Genomic DNA Isolation 81
4.1.5 Polymerase Chain Reaction 81
4.1.6 Southern Blot Analysis 84
4.1.7 Enzyme Linked Immunosorbent Assay 84
4.1.8 Western Blot Analysis 84
4.2 DEVELOPMENT OF TRANSGENIC PURE LINES 87
4.2.1 1st Generation 87
4.2.2 2nd Generation 92
4.2.3 3rd Generation 98
4.2.4 4th & 5th Generations 101
4.3 FIELD STUDIES ON Bt COTTON 101
4.3.1 Bt Trials 2004-05 101
4.3.1.1 Bt Protein %age 103
4.3.1.2 Natural Infestation of Spotted Bollworm 103
4.3.1.3 Field Bioassay with Heliothis 109
4.3.1.4 Lab Bioassay 113
4.3.1.5 Seed Cotton Yield per Plant 113
4.3.1.6 Plant Height 114
4.3.1.7 Number of Monopodial Branches per Plant 115
4.3.1.8 Number of Sympodial Branches per Plant 115
4.3.1.9 Number of Bolls per Plant 115
4.3.1.10 Boll Weight 118
4.3.1.11 Ginning Outturn Percentage 118
4.3.1.12 Staple Length 118
4.3.1.13 Fibre Fineness 121

xii
 Comparative Study of Insecticide Applications on Bt and non-Bt
4.3.2 Cotton Lines 2004-2005. 121
4.3.2.1 Studies During the Year, 2004 121
4.3.2.2 Studies During the Year, 2005 123
Bt INHERITANCE STUDIES IN TRANSGENIC
4.4 GENERATIONS 130
4.4.1 Bt Inheritance Studies in Selfed Generations 130
4.4.2 Bt Inheritance Studies in Filial Generations 130
4.4.2.1 Inheritance of Transgene in F1 Generation 130
4.4.2.2 Mendelian Inheritance Studies in F2 Generation 132
STUDIES ON HETEROSIS AND HETEROBELTIOSIS IN
4.5 F1 GENERATION 135
4.5.1 Seed Cotton Yield per Plant 135
4.5.2 Number of Bolls per Plant 138
4.5.3 Boll Weight 138
4.5.4 Ginning Outturn Percentage 139
4.5.5 Lab Bioassay Results (Mortality %age of Heliothis Larvae) 140
HERITABILITY AND GENETIC ADVANCE STUDIES IN
4.6 Bt COTTON 141
4.6.1 Heritability for Bt Resistance 141
4.6.2 Genetic Advance for Bt Resistance 144
CORRELATION OF Bt TRAIT WITH OTHER
4.7 ECONOMIC TRAITS 146
COMPARISON OF SOME QUALITATIVE CHARACTERS
4.8 OF Bt AND NON-Bt COTTON 148
4.9 DISCUSSION 150
4.9.1 Transformation 150
4.9.2 Development of Transgenic Pure Lines 152
4.9.3 Field Studies 154
4.9.4 Bt Inheritance 158
4.9.5 Heterosis and Heterobeltiosis 160
4.9.6 Heritability and Genetic Advance 161
4.9.7 Correlation 162
5 LITERATURE CITED 164

xiii
 LIST OF FIGURES

Figure 1 Schematic Diagram of the construct pKMAB 79

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

xiv
 LIST OF TABLES

Table 1 Insect Resistance and Number of Bolls of 1st Generation Plants 90

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

xv
 LIST OF ANNEXURES

ANNEXURE-I RECIPES OF VARIOUS MEDIUMS I

ANNEXURE-II RECIPES OF VARIOUS SOLUTIONS III

ANNEXURE-III RECIPES OF VARIOUS BUFFERS V

xvi
 ABBREVIATIONS

µl micro litre

µg micro gram

°C degree centigrade

% Percent

A° Angstrom

ATP Adenosine Triphosphate

BSA Bovine Serum Albumin

Bt Bacillus thuringiensis

BTK Bacillus thuringiensis var. kurstaki

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

cm Centimeter

Cry Crystal

DNA Deoxy Ribonucleic Acid

dNTPs Dinucleotide Triphosphate

EC Electric Conductivity

EDTA Ethylene diamine tetra acetic acid

ELISA Enzyme Linked Immunosorbent Assay

ems error mean square

et al. (et alii) and others

ETL Economic Threshold Level

exo Exonuclease

F1 First Filial Generation

F2 Second Filial Generation

g Gram

xvii
GCA General Combining Ability

GOT Ginning Outturn

GUS Glucuronidase

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

HgCl2 Mercuric Chloride

Kb Kilobase

KCl Potassium Chloride

KDa kilo Dalton

kg Kilogram

L Litre

lbs/in2 pounds per square inch

Na2CO3 Sodium Carbonate

NaCl Sodium Chloride

NaOH Sodium Hydroxide

No. Number

M Molar

mg Milligram

mgL-1 Milligram per litre

ml milli litre

mM milli molar

MS Murashige and Skoog

N Normal

N2 Nitrogen

Na Sodium

NADP Nicotinamide adenine dinucleotide phosphate

ng nano gram

nm nano meter (wavelength)

xviii
NPT Neomycin phosphotransferase

OD Optical Density

PCR Polymerase Chain Reaction

PBS Phosphate Saline Buffer

pH power of Hydrogen

p.mol pico moles

q/ha quintals per hectare

RNA Ribonucleic Acid

rpm rounds per minute

SCA Specific Combining Ability

SDS Sodium Dodecyl Sulphate

SSC Standard Sodium Citrate

TE Tris Ethylene diamine tetra acetic acid

Ti Tumor inducing

U Units

UV ultra violet

viz. Namely

YEP 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 RESISTANCE THROUGH Bt

GENES

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, soil-

dwelling 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 cotton-

producing 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 Bt-

protected 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 Bt-

protected 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 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. 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 Agrobacterium-

mediated 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 kanamycin-

resistant 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 T-

DNA 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. They selected the

26
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 dual-

toxin 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 cross-

resistance 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 Cry1Ac-

resistant strains (2-10%) than for susceptible strains (0%) (Chitkowski et al., 2003).

Shelton et al., (2000) conducted field tests on managing resistance to Bt-

engineered 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.

32
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.

34
 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.

40
 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.

41
 CHAPTER 3

MATERIALS AND METHODS

42
3.1 AGROBACTERIUM TRANSFORMATION

3.1.1 Agrobacterium tumefaciens Competent Cells

Preparation

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

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.

43
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 Confirmation of Agrobacterium Transformation

3.1.4.1 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 COTTON TRANSFORMATION

3.2.1 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

45
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 MS-

kanamycin 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.

49
3.3.3 Southern Hybridization

3.3.3.1 Probe Making/DNA Labelling

3.3.3.1.1 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.

51
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.

52
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.

53
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 40% or more insect mortality

Susceptible plants 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

56
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. Boll Damage Percentage due to natural infestation of bollworms

2. Insect Mortality Percentage in lab bioassay with Heliothis larvae

3. Seed Cotton Yield (g)

4. Plant Height (cm)

5. Number of Monopodial Branches per Plant

6. Number of Sympodial Branches per Plant

7. Confirmation of transgene presence through PCR, and

8. 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. The plant must be higher yielding in terms of seed cotton;

3. 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 25-

40ºC and 30-70%, respectively.

58
 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 Field Trials

3.5.2.1 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.

59
 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.

61
 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

62
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 Micronaire-

complete 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

63
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 MNH-

93. There were four insecticide treatments as follows:-

L0 No insecticide application during the entire season

L1 Insecticide Application at an ETL (economic threshold level) of 3 larvae

per 25 plants – general recommendation by the Punjab Agriculture

Department

L2 Insecticide Application at an incidence of 5 larvae per 25 plants

L3 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 0 sprays

Class 2 1.75 spray

Class 3 2.5 sprays

Class 4 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. Inheritance of Bt gene in selfed transgenic generations, and

2. 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.

65
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 high-

powered 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 (un-

opened 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

66
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. FEMALE PARENT MALE PARENT

1. MNH-93 CEMB-3
2. CEMB-3- MNH-93
3. CIM-482 CEMB-3
4. CEMB-3 CIM-482
5. MNH-93 CEMB-11
6. 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.

67
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. Seed Cotton Yield (g)

2. Number of bolls per Plant

3. Boll Weight (g)

4. Ginning Outturn Percentage (GOT %age)

5. 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

68
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”.

69
 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. Seed Cotton Yield (g)

2. Plant Height (cm)

3. Number of Monopodial Branches per Plant

4. Number of Sympodial Branches per Plant

5. Number of Bolls per Plant

6. Boll Weight (g)

7. Ginning Outturn Percentage

8. Staple Length (mm)

9. 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 OF Bt AND NON-Bt

COTTON

The observations on the following qualitative characters of Bt and non-Bt cotton

were also taken during the present studies:-

1. Plant Shape

2. Leaf Hairiness

3. Boll Shape

4. Boll Opening

5. Reaction to Virus

6. 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 Estimation of Correlation

The 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 COTTON TRANSFORMATION

4.1.1 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 EcoRI

TatI SacI XbaI

Border Pnos Neo 3َ 35S CAB 22L Bt Gene T DNA Border

Sequence Promoter Gene OCS Promoter Leader Sequence Cry1Ab Gene 7 Sequence

ScaI
Bam HI

Pnos Promoter Nopaline Synthase Promoter

Neo Gene Neomycin Phosphotransferase (nptII) gene
OCS Octapine Synthase Terminator Gene
35S Promoter Cauliflower Mosaic Virus 35S Promoter
CAB 22L 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

CEMB-17
CEMB-3

CEMB-3

A B

(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.

85
 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 B C

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
No. of Total Average Boll Damage
mortality %
Bolls %age During the Peak
S.No. NAME shown in lab
(Mature + Infestation Period
bioassay with
Immature) (August-October)
Heliothis
1 CEMB-1 90 72 24
2 CEMB-2 50 63 14
3 CEMB-3 60 71 24
4 CEMB-4 80 79 8
5 CEMB-5 80 51 37
6 CEMB-6 20 19 26
7 CEMB-7 50 11 64
8 CEMB-8 90 2 100
9 CEMB-9 50 10 40
10 CEMB-10 70 22 86
11 CEMB-11 60 60 18
12 CEMB-12 80 52 12
13 CEMB-13 90 65 34
14 CEMB-14 70 22 50
15 CEMB-15 60 13 92
16 CEMB-16 70 7 29
17 CEMB-17 100 46 17
18 CEMB-18 100 40 15
19 CEMB-19 80 39 46
20 CEMB-20 100 29 24
21 CEMB-21 80 24 21
22 CEMB-22 100 37 19
23 CEMB-23 70 41 27
24 CEMB-24 80 57 18
25 CEMB-25 70 30 37
26 CEMB-26 100 36 25
27 Control 20 8 63

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, pre-
fasted for 4-6 hours, was released in the each plate and allowed to feed on the leaf. After 48-
72 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

CEMB-16
CEMB-3

CEMB-9

CEMB-4
Control

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
 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 Boll
Mortality Damage
Western No. of No. of Plant Ginning
S. PCR shown in due to Yield
Plant No. Dot Blot Monopodial Sympodial Height Outturn
No. Result the Lab Natural (gm)
Result Branches Branches (cm) (%age)
Bioassay Infestation
(%) (%age)

1 CEMB 3-2 + + 60 3.33 4 17 195 87.8 43.96

2 CEMB 11-2 + + 40 2.86 3 33 105 102.3 39.6

3 CEMB 16-10 + + 50 2.63 1 12 172 109.3 41.0

4 CEMB 16-15 + + 60 0.00 4 22 117 88.6 42.0

5 CEMB 17-25 + + 50 3.45 5 19 170 95.3 38.5

6 Control - - 20 20 2 18 190 46.8 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 16-

10 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 night-time view of the green house

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 30-
70%, respectively.

99
 Table-3

Bt Protein %age in 3rd Generation Plants 2003-2004

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

Plant No.1 0.56 1.07 0.53 0.40 0.76

Plant No.2 0.88 1.02 0.32 0.44 0.54

Plant No.3 0.55 0.16 1.35 0.40 0.56

Plant No.4 0.40 1.18 0.26 0.39 0.58

Plant No.5 0.70 0.94 0.27 0.62 0.00

Plant No.6 0.09 0.85 0.32 0.62 0.39

Plant No.7 0.11 0.61 0.49 0.26 0.63

Plant No.8 0.20 0.20 0.39 0.00 0.40

Plant No.9 0.60 1.06 0.33 0.83 0.52

Plant No.10 0.46 0.00 0.30 0.68 0.55

Standard Deviation 0.26 0.44 0.33 0.24 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 FIELD STUDIES ON Bt COTTON

4.3.1 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

B A

COTTON FIELD 2005

B
C

The field trials comprising of four Bt genotypes/pure lines viz. CEMB-3, CEMB-
11, 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

0.30
PERCENTAGE

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

NATURAL
YIELD NO. OF NO. OF FIELD
SOURCE OF PLANT INFESTATION GOT
PER MONOPODIAL SYMPODIAL BIOASSAY
VARIATION HEIGHT OF SPOTTED %age
PLANT BRANCHES BRANCHES (Heliothis)
BOLLWORM

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 NO. OF BOLLS BOLL STAPLE FIBRE Bt PROTEIN LAB BIOASSAY

VARIATION PER PLANT WEIGHT LENGTH FINENESS CONTENT (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*

NATURAL FIELD
YIELD NO. OF NO. OF INFESTATION BIOASSAY
PLANT
PER MONOPODIAL SYMPODIAL OF SPOTTED (Heliothis) GOT
GENOTYPES HEIGHT
PLANT BRANCHES PER BRANCHES BOLLWORM (no. of %age
(cm)
(g) PLANT PER PLANT (no. of insects insects per
per plant) plant)

CEMB-3 24.44 a 110.00 b 4.08 a 12.32 b 0.69 b 0.10 a 34.23 a

CEMB-11 21.70 a 95.17 b 3.98 a 12.47 b 0.76 b 0.05 a 34.28 a

CEMB-16 19.31 a 102.50 b 5.82 a 11.82 b 0.76 b 0.07 a 34.15 a

CEMB-17 22.04 a 96.83 b 4.42 a 13.50 b 0.72 b 0.12 a 34.04 a

CEMB-
19.88 a 131.47 a 6.12 a 9.52 a 0.99 a 0.15 a 31.29 b
CONTROL

• 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*

BOLL STAPLE FIBRE Bt PROTEIN LAB BIOASSAY

NO. OF BOLLS
GENOTYPES WEIGHT LENGTH FINENESS (%age of total (Heliothis mortality
PER PLANT
(g) (mm) (µg/in) protein) %age)

CEMB-3 15.00 a 2.93 a 24.96 a 4.63 a 0.215987 b 41.333 a

CEMB-11 12.50 a 2.91 a 25.91 a 4.59 a 0.287177 ab 33.333 ab

CEMB-16 11.03 a 4.02 a 26.13 a 4.80 a 0.260833 ab 40.667 a

CEMB-17 13.37 a 2.70 a 25.87 a 4.64 a 0.298383 a 34.667 ab

CONTROL 16.17 a 2.38 a 26.35 a 4.82 a 0.000000 c 22.000 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

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
50
HELIOTHIS LARVAE

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

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 2004-

2005’ 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 un-

alteration 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

5
NUMBER

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

10
NUMBER

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

12
NUMBER

10

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

3.0
GRAMS

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 R1 R2 R3 TOTAL MEAN

CEMB-3 494.10 337.70 639.90 1471.70 490.56

CEMB-C 1296.10 1051.50 1517.00 3864.60 1288.20

t-TEST: TWO-SAMPLE ASSUMING UNEQUAL VARIANCES

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

† 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 non-

significantly, 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 L1 L2 L3

%AGE %AGE %AGE

GENOTYPES/ INCREASE/ INCREASE/ INCREASE/
YIELD (g) YIELD (g) YIELD (g) YIELD (g)
YIELD DECREASE DECREASE DECREASE
OVER L0 OVER L0 OVER L0

CEMB-3 41.19 58.49 42.00 45.68 10.90 49.40 19.93

CEMB-C 29.36 38.70 31.81 37.52 27.79 34.39 17.13

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
df SS MS F.RATIO
VARIATION

TREATMENTS 7.00 2357.55 336.79 0.62 ns

GENOTYPES 1.00 1501.41 1501.41 2.75 ns

LEVELS 3.00 710.18 236.73 0.43 ns

LXV 3.00 145.96 48.65 0.09 ns

ERROR 24.00 13122.38 546.77

TOTAL 31.00 15479.94

ns non-significant

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) Class-2 (L1) Class-3 (L2) Class-4 (L3)

CEMB-3 CEMB-C CEMB-3 CEMB-C CEMB-3 CEMB-C CEMB-3 CEMB-C

R1 0 0 4 4 3 4 2 2

R2 0 0 4 3 3 3 2 3

R3 0 0 2 3 2 2 2 1

R4 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 NON-Bt
(CEMB-3) (CEMB-C)

No. of sprays against bollworms 1.75 * 3.0

Yield (gm/plant) 49.40 ns 37.52

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

ns 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 CEMB C
60.00

50.00
GRAMS PER PLANT

*
40.00

*
30.00

20.00

10.00

0.00
L0 L1 L2 L3
(No spray) (3 sprays) (2.5 sprays) (1.75 sprays)

TREATMENTS

Economic Threshold Levels (ETLs) / Treatments

L0 No Spray (Control)
L1 3 insects per 25 plants
L2 5 insects per 25 plants
L3 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 Bt Inheritance Studies in Filial Generations

4.4.2.1 Inheritance of Transgene in F1 Generation

Two transgenic lines viz. CEMB-3 and CEMB-11 were crossed to two non-

transgenic 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. LINE 1ST 3RD

2ND GENERATION 4TH GENERATION 5TH GENERATION
NO. NO. GENERATION GENERATION
Total Plants = 1 Total Plants Total Plants Total Plants Total Plants
PCR positive = 1 Analyzed = 17 Analyzed = 10 Analyzed = 30 Analyzed = 30
ELISA positive = 1 PCR positive = 17 PCR positive = 10 PCR positive = 30 PCR positive = 30
1. CEMB-3 Southern Blot Western Dot Blot Western Dot Blot Western Dot Blot Western Dot Blot
Positive = 1 Positive =17 Positive =10 Positive =30 Positive =30
Western Blot
Positive =1
Total Plants = 1 Total Plants Total Plants Total Plants Total Plants
PCR positive = 1 Analyzed = 18 Analyzed = 10 Analyzed = 30 Analyzed = 30
ELISA positive = 1 PCR positive = 12 PCR positive = 9 PCR positive = 30 PCR positive = 30
2. CEMB-11 Southern Blot Western Dot Blot Western Dot Blot Western Dot Blot Western Dot Blot
Positive = 1 Positive =12 Positive =9 Positive =30 Positive =30
Western Blot
Positive =1
Total Plants = 1 Total Plants Total Plants Total Plants Total Plants
PCR positive = 1 Analyzed = 17 Analyzed = 10 Analyzed = 30 Analyzed = 30
ELISA positive = 1 PCR positive = 4 PCR positive = 9 PCR positive = 30 PCR positive = 30
3. CEMB-16 Southern Blot Western Dot Blot Western Dot Blot Western Dot Blot Western Dot Blot
Positive = 1 Positive =5 Positive =9 Positive =30 Positive =30
Western Blot
Positive =1
Total Plants = 1 Total Plants Total Plants Total Plants Total Plants
PCR positive = 1 Analyzed = 30 Analyzed = 10 Analyzed = 30 Analyzed = 30
ELISA positive = 1 PCR positive = 26 PCR positive = 9 PCR positive = 30 PCR positive = 30
4. CEMB-17 Southern Blot Western Dot Blot Western Dot Blot Western Dot Blot Western Dot Blot
Positive = 1 Positive =26 Positive =9 Positive =30 Positive =30
Western Blot
Positive =1
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 chi-

square 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

NUMBER OF
NUMBER OF
TOTAL PLANTS
PLANTS
S.NO NAME OF CROSS PLANTS POSITIVE IN
POSITIVE IN
ANALYZED WESTERN
PCR
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

Chi-
No. of No. of
Total square
S.No. Combinations Negative Positive Ratio
Plants value for
Plants Plants
3:1 ratio

Distorted
1. MNH-93 X CEMB-3 88 45 43 32.061
(Non-Mendelian)
3:1
2. CEMB-3 X MNH-93 93 23 70 0.004
(Mendelian)
3:1
3. CIM-482 X CEMB-3 60 13 47 0.356
(Mendelian)
Distorted
4. CEMB-3 X CIM-482 92 51 41 45.449
(Non-Mendelian)
Distorted
5. MNH-93 X CEMB-11 103 56 47 47.382
(Non-Mendelian)
3:1
6. CEMB-11 X MNH-93 67 22 45 2.194
(Mendelian)

Total 503 210 293

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 non-

Mendelian 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 CIM-

482 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 CEMB-

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

482 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 non-

significantly 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 MNH-

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

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
Seed Cotton Yield No. of Bolls per Lab Bioassay
GENOTYPES Boll Weight (g) GOT %age
per plant (g) plant (Mortality %age)

Ht(%) Hbt(%) Ht(%) Hbt(%) Ht(%) Hbt(%) Ht(%) Hbt(%) Ht(%) Hbt(%)

MNH-93 X
16.49ns -2.81ns 13.81ns 4.17ns 5.89ns -4.08ns 26.44** 26.17** 36.23** 23.68**
CEMB-3
CEMB-3 X
50.19* 25.31* 24.58* 14.02ns 21.38** 9.96* 21.43** 21.17** 30.43** 18.42**
MNH-93
CIM-482 X
-15.19ns -18.58ns -2.80ns -4.11ns -6.96ns -9.30ns 16.38** 2.43** 16.22* 19.44*
CEMB-3
CEMB-3 X
107.07** 98.79** 69.40** 67.12** 12.83* 9.99* 13.02** -0.52** -8.11ns -5.56ns
CIM-482
MNH-93 X
91.35** 73.78** 81.36** 81.36** 6.42ns -3.19ns 22.45** 20.27** 13.43ns 5.56ns
CEMB-11
CEMB-11 X
-7.19ns -15.71ns -20.34ns -20.34ns 15.52* 5.09* 17.43** 15.33** 10.45ns 2.78ns
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).

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 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 CIM-

482 (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 CEMB-

3 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.

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 Table 19

HERITABILITY AND GENETIC ADVANCE FOR Bt RESISTANCE IN THE CROSSES

BETWEEN Bt AND NON-Bt COTTON LINES
Relative
F1 Variance F2 Variance Broad Sense Genetic
S.No. Cross combination Expected
(lab bioassay results) (lab bioassay results) Heritability (%) Advance
Gain

462.2222 872.6003 47.03

1 MNH-93 X CEMB-3 28.62 50.50

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.

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 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).

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 Table 20

CORRELATION OF Bt INSECT RESISTANCE TRAIT WITH THE ECONOMINC TRAITS OF COTTON

Bollworm (number of insects per plant)

No. of Monopodial Branches per Plant

No. of Sympodial Branches per Plant

Natural Infestation of Spotted

Fibre Fineness (µg/in)
Ginning Outturn (%)
No. of Bolls per Plant

Staple Length (mm)

Plant Height (cm)

Boll Weight (g)

Yield (g)

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 -0.83 *

* 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.

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4.8 COMPARISON OF SOME QUALITATIVE

CHARACTERS OF Bt AND NON-Bt

COTTON

The observations on some important qualitative characters of cotton were also

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.

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 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 Profusely Hairy
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

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4.9 DISCUSSION

4.9.1 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 Agrobacterium-

mediated. 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

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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)

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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 Bt-

engineered 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,

154
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.

155
(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 non-

significantly. 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

156
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

157
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 non-

Mendelian. 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

159
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

162
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.

163
 CHAPTER 5

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 ANNEXURE-I

RECIPES OF VARIOUS MEDIUMS

YEP MEDIUM

Yeast Extract 10.0 g/l

Bactopeptone 10.0 g/l

NaCl 5.0 g/l

Agar 14.0 g/l

pH 7.5

MS MEDIUM

Ms Basal Medium 4.43 g/l

Sucrose 30.0 g/l

Phytagel 3.0 g/l

pH 5.7

LB MEDIUM

NaCl 10.0 g/l

Tryptone 10.0 g/l

Yeast Extract 5.0 g/l

Bacto Agar 15.0 g/l

I
T3 MEDIUM

Tryptone 3.12 g/l

Tryptose 2.08 g/l

Yeast Extract 1.56 g/l

MnCl2 0.0052 g/l

1M K2PO4(pH 6.8) 2.6ml

Agar 15.76 g/l

Glucose 3.0 g/l

II
 ANNEXURE-II

RECIPES OF VARIOUS SOLUTIONS

20X PBS SOLUTION

NaCl 160 g/l

KCl 4.0 g/l

Na2HPO4 28.4 g/l

SOB SOLUTION

Tryptone 20 g/l

Yeast Extract 5 g/l

NaCl 0.584 g/l

KCl 0.186 g/l

pH 7.0

SOC SOLUTION

SOB solution As required

Mg++ 20 mM

Glucose 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 100ml

Skimmed Milk 5g

Tween 20 0.05%

VI