phd thesis dr.ghazanfar
TRANSCRIPT
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)
ii
CERTIFICATE
This is to certify that the research work described in this thesis is the original work
of the author and has been carried out under our direct supervision. We have personally
gone through all the data/results/materials reported in the manuscript and certify their
correctness/authenticity. We further certify that the material included in this thesis have
not been used in part or full in a manuscript already submitted or in the process of
submission in partial/complete fulfillment of the award of any other degree from any
other institution. We also certify that the thesis has been prepared under our supervision
according to the prescribed format and we endorse its evaluation for the award of Ph.D.
degree through the official procedures of the university.
(DR. S. RIAZUDDIN) Co-Supervisor
(DR. TAYYAB HUSNAIN) Supervisor
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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.
iv
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
v
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)
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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
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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%
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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.
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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
1.1 BIOTECHNOLOGY AND THE CONVENTIONAL 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
3.1.2 Agrobacterium Transformation with pKMAB By Heat Shock Method 43
x
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
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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
3.10 COMPARISON OF SOME QUALITATIVE CHARACTERS 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
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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
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4.3.2 Comparative Study of Insecticide Applications on Bt and non-Bt 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
4.4 Bt INHERITANCE STUDIES IN TRANSGENIC 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
4.5 STUDIES ON HETEROSIS AND HETEROBELTIOSIS IN 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
4.6 HERITABILITY AND GENETIC ADVANCE STUDIES IN Bt COTTON 141
4.6.1 Heritability for Bt Resistance 141
4.6.2 Genetic Advance for Bt Resistance 144
4.7 CORRELATION OF Bt TRAIT WITH OTHER ECONOMIC TRAITS 146
4.8 COMPARISON OF SOME QUALITATIVE CHARACTERS 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
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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
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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
xvi
LIST OF ANNEXURES
ANNEXURE-I RECIPES OF VARIOUS MEDIUMS I
ANNEXURE-II RECIPES OF VARIOUS SOLUTIONS III
ANNEXURE-III RECIPES OF VARIOUS BUFFERS V
xvii
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
xviii
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)
xix
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
1
CHAPTER 1
INTRODUCTION
2
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
3
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
4
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).
5
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
6
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.
7
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
8
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
9
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.
10
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
11
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:-
12
13
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
14
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
15
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
16
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.
17
CHAPTER 2
REVIEW OF LITERATURE
18
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.
19
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).
20
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
21
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).
22
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
23
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.
24
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.
25
(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
26
(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
27
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.
28
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
29
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
30
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
31
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
32
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.
33
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
34
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.
35
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
36
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,
37
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
38
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.
39
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
40
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.
41
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.
42
CHAPTER 3
MATERIALS AND METHODS
43
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.
44
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
45
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
46
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
47
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
48
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
49
tubes were incubated at 65°C for 1 hour. The tubes were centrifuged at 12000rpm for 15
minutes. The supernatant was taken and 2.0µl RNAase (10mg ml-1 stock) was added. The
tubes were incubated at 37°C for 15 minutes. 400µl 100% Ethanol (2 volumes) and 20µl
3M Na-Acetate pH5.2 (0.1 volume) were added to each tube. The tubes were incubated at
-20°C for 1 hour. The DNA was spooled out and air-dried. The DNA was finally
resuspended in 100µl low EC water or TE Buffer. The DNA concentration was estimated
by running samples on 0.7% Agarose gel and comparing with the λ-uncut marker or
λ/HindIII bands of known concentrations.
3.3.2 Polymerase Chain Reaction
The PCR was performed to screen positive plants with the following set of
primers:-
Cry1Ab forward primer 5´-GTT ACC CTG ATT GAT AGG C-3´
(nucleotide 1106-1125)
Cry1Ab reverse primer 5´ACA GAA GAC CTT TCA ATA TC-3´
(nucleotide 1636-1656)
The total amplified portion was 550 base pairs. The PCR parameters were initial
denaturation at 94°C for 2 minutes followed by 35 cycles of 94°C for 45 seconds, 52°C
for 45 seconds and 72°C for 45 seconds and a final extension of 10 minutes at 72°C. The
reaction mixture was prepared as described above in section 3.1.4.2.
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3.3.3 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
51
13000rpm for 30 seconds to remove small amounts of silica powder. The supernatant was
transferred to a new tube. The amount of DNA was quantified either with the help of a
spectrophotometer or running an aliquot on 1% Agarose gel. The final concentration of
DNA was maintained at 100ng per µl with the help of DNA concentrator.
3.3.3.1.3 DNA labeling with Biotin-11-dUTP
The labeling was done using Biotin DecaLabel™ DNA Labeling Kit of
Fermentas. 10µl DNA template (100ng per µl), 10µl Decanucleotide in 5X buffer and
44µl water were mixed in an eppendorf by vortexing. The eppendorf was centrifuged at
10000rpm for 3-5 seconds followed by incubation in boiling water for 5-10 minutes. The
eppendorf was cooled down on ice and spun down quickly in microcentrifuge. The 5µl
Biotin Labeling Mix (1mM dGTP, 1mM dATP, 1mM dCTP, 1mM dTTP and 0.35mM
Biotin-11-dUTP aqueous solution) and 1µl (5U) Klenow fragment (exo¯) were added to
the eppendorf. The eppendorf was vortexed briefly and centrifuged at 10000rpm for 3-5
seconds. The eppendorf was incubated at 37°C for 20 hours. The reaction was stopped by
adding 1.0µl 0.5M EDTA pH 8.0.
3.3.3.1.4 Probe Estimation
Four serial dilutions (containing 1.0µl, 0.1µl, 0.01µl and 0.001µl DNA) each of
labeled DNA and control labeled DNA were spotted on Hybond-N membrane. The
membrane was air-dried. The DNA cross-linking was performed by exposing the
membrane to UV light for 3 minutes. The detection was done as given in section 3.3.3.5
below.
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3.3.3.2 Genomic DNA digestion
The DNA (27µg) of transformed plants was digested with EcoRI (160U) and
BamHI (160U) enzymes. The reaction was incubated at 37°C overnight. The samples
were checked for complete digestion on 0.8% Agarose gel.
3.3.3.3 Gel Running for Southern Hybridization
The completely digested samples were loaded on 1cm thick 1% Agarose gel and
run at 30 volts overnight. The gel was immersed in Depurination solution and kept on
shaking for 10 minutes until Bromophenol blue turned yellow. The Depurination solution
was replaced with the Denaturation solution and kept on shaking for 45 minutes until
yellow color turned blue. The Denaturation solution was discarded and Neutralization
solution was added. The gel was kept on agitation for 35 minutes.
3.3.3.4 Gel Transfer Assembly
A 3mm filter paper wick pre-soaked with 2X SSC (Standard Sodium Citrate)
solution was placed on a ceramic tile mounted on a steel tray filled with10X SSC
solution. The sides of the paper wick were immersed in the solution. The three layers of
Whatman Sheets were placed on the wick onto which the gel was placed upside down.
The Hybond-N membrane was placed on the gel above which a pile of blotting papers
and newsprint were placed. The size of the membrane, blotting papers and the newsprint
was exactly same as that of the gel. A 1.5 kg weight was put on top of the assembly. The
assembly was left overnight to facilitate DNA transfer from the gel to the membrane
through capillary action.
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3.3.3.5 Blot Processing
The transfer assembly was disassembled and the membrane air-dried. The
membrane was wrapped in saran wrap and UV cross-linked for 3 minutes. The membrane
was incubated with Pre-hybridization solution at 65°C for 2 hours. The Pre-hybridization
solution was discarded and the membrane was incubated with Biotin-labelled Cry1Ab
Probe solution at 65°C for 12 hours. The Probe solution was denatured in boiling water
for 10 minutes before use. The detection was done using Biotin Chromogenic Detection
Kit (Fermentas). The membrane was kept on shaking in 1X Blocking/Washing Buffer for
5 minutes at room temperature. The Washing Buffer was replaced with 1% Blocking
Solution and kept on shaking for 30 minutes. The Blocking Solution was replaced with
Streptavidin-AP conjugate. The membrane was kept on shaking at room temperature for
30 minutes. The membrane was twice washed with Blocking/Washing Buffer by placing
it on shaking for 15 minutes each time. The membrane was incubated in 1X Detection
Buffer for 10 minutes. The membrane was finally incubated in 1X Substrate solution
(BCIP/NBT) at room temperature in the dark for 30 minutes.
3.3.3.6 Copy Number Estimation
To estimate copy number of the transgene in the transformed plants, 27µg
genomic DNA was digested with 320 units (about 12U/µg) of the enzyme SstI (SacI)
which has a unique site in the plasmid pKMAB. The Southern Hybridization was done as
described above in the sections 3.3.3.3 to 3.3.3.5.
3.3.4 Immunological Assay of Transgenic Plants
The transgenic cotton plants were analyzed through ELISA and Western Dot Blot
for protein expression studies.
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3.3.4.1 Isolation of Protein from Cotton Leaves
About 0.5gm fresh terminal leaves were ground in liquid nitrogen. 1.0ml Protein
Extraction Buffer was added and centrifuged at 14000rpm at 4°C. The supernatant was
taken. The protein concentration was estimated by taking OD value at 595A° and
calculated comparing with the already plotted curve of known concentrations of BSA
(Bovine Serum Albumin).
3.3.4.2 Enzyme Linked Immunosorbent Assay
50µg total protein was loaded on a 96-well ELISA plate. The Carbonate Buffer
(100µl) was added to each well. The plate was incubated at 4°C overnight. The wells
were thrice washed with 1X PBS. 5% skimmed milk (200µl) was put in each well. The
plate was incubated at 37°C for 30 minutes. The wells were thrice washed with 1X PBS.
The primary antibodies (200µl) already diluted in 5% skimmed milk (2µl/ml) were added
to each well. The plate was incubated at 37°C for 2 hours. The wells were thrice washed
with 1X PBS. 200µl of skimmed milk (5%) was added in each well and the plate was
incubated at 37°C for 30 minutes. The wells were thrice washed with 1X PBS. The
secondary antibodies (200µl) already diluted in 5% skimmed milk (0.5µl/ml) were added
to each well. The plate was incubated at 37°C for 1 hour. The wells were thrice washed
with 1X PBS. The color substrate NBT/BCIP (1 tablet dissolved in 10ml water) was
added to each well. The plate was incubated at room temperature for 45 minutes. The
reaction was stopped with 2M Na2CO3.
3.3.4.3 Western Dot Blot
The Hybond-C membrane was used for Western Dot Blot. 10ng protein was
loaded on the membrane. The membrane was completely air-dried and incubated in
Blocking Buffer/Reagent for 30 minutes at room temperature or overnight at 4°C. The
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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
56
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
57
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.
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The plants were again subjected to individual studies and molecular analysis.
Plant selections were made on the basis of the criteria stated above. The plants in this
generation showed 50% to 100% homozygosity, therefore the seed of selected plants
from each progeny row was bulked to have four pure lines.
3.5.1.4 4th & 5th Generation
The four pure lines mentioned above were further analyzed at molecular level, for
two successive generations to eliminate false positive plants and to check for errors in
selection. A sample of 30 plants, at random, was taken from each pure line during every
year and analyzed at molecular level through PCR and Western Dot Blot.
3.5.2 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.
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The data were recorded on the following characteristics:-
1. Bt Protein Percentage
The Bt contents in all genotypes were quantified to have a true picture of variation
in insect resistance of the plants due to Bt concentration. The total protein was isolated
from fresh leaves of the plants. The concentration of total protein was estimated with the
help of spectrophotometer at 595 nm. The reading was compared with the BSA standard
curve. An equal amount of protein of each sample (10ng) was loaded directly onto a
Hybond-C membrane and the procedure of Western Blotting was followed. After
processing, the blot was scanned and the Bt contents quantified using ImageQuant TL
software of the Amersham BioSciences (Pvt) Limited.
2. Natural Infestation of Spotted Bollworm
The pest scouting was done on weekly basis in the cotton trials. The number of
spotted bollworms was counted. Occasionally, a few larvae were found dead on the
transgenic lines due to Bt effect. The dead larvae were not included in the data. The
results were averaged at the end of the season to have a comparison of the occurrence of
the insects.
3. Field Bioassays
Different methods were tried to conduct field bioassays with 1st/2nd instar
American Bollworm (Heliothis armigera) e.g. placing the larvae on leaves of the plants
with the help of camel hair brush; releasing pupae to pupate in the field, grow into adult,
lay eggs and damage the plants at larval stage; and releasing the lab-reared larvae through
glass vials in the field. The method of glass vials was proved to be the better one.
For the purpose of field bioassays, a large number of Heliothis larvae were reared
in the CEMB insectary. Ten larvae of 1st/2nd instar were placed in a small glass vial and
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tied to the middle of the stem of each plant in the experiment. The glass vial was opened
afterwards to let the insects travel to all parts of the plant. The number of live insects per
plant were recorded daily in the field upto seventh day of artificial infestation.
4. Insect Resistance Shown In Lab Bioassays
The laboratory bioassays were conducted as described in section 3.4.
5. Seed Cotton Yield (g)
The seed cotton picked from ten randomly selected plants from each plot during
all pickings was mixed and weighed on an electronic balance. Average yield of seed
cotton per plant for each genotype in each replication was calculated by dividing the total
yield with the number of plants and expressed in grams.
6. Plant Height (cm)
The plant height was recorded in centimeters from base to the apex by using a
meter rod.
7. Number of Monopodial Branches per Plant
At maturity, the number of monopodial branches (indirect fruiting branches) of
ten randomly selected plants from each plot were counted and averaged for the purpose of
statistical analysis.
8. Number of Sympodial Branches
At maturity, the number of sympodial branches (direct fruiting branches) of ten
randomly selected plants from each plot were counted and averaged for the purpose of
statistical analysis.
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9. Number of Bolls per Plant
Ten plants in each plot were randomly selected for data recording. The actual
count of effective bolls on the plant to be sampled was recorded and summed up for all
pickings. The mean was calculated by dividing total number of bolls with the number of
plants.
10. Average Boll Weight (g)
The average boll weight was obtained by dividing the total seed cotton yield of ten
randomly selected plants in each plot by the respective total number of effective bolls.
11. Ginning Outturn (%age)
The produce of each plant was cleaned and dried. A sample of 100 grams from
each plant was taken and ginned separately with a Single Roller Electric Gin. The lint
obtained was weighed and the following formula was used to calculate Ginning Outturn
(GOT):-
GOT %age = (Weight of lint / Weight of seed cotton in the sample) x 100
12. Staple Length (mm)
The Staple Length was measured by Fibrograph Model 530 (electronic).
The “Tuft Method” was also used to measure Staple Length. A representative
sample of lint of each plant was taken and turned into a sliver and run through a draw box
till a uniform band of parallel fibers was obtained. These fibers were then mounted on a
set of metallic comb, fixed parallel to each other on a stand. One end of the processed
sample was aligned using tweezers, and two tufts were drawn from each sample. The tufts
were placed on a velvet covered tuft board and two lines were drawn, one on the even end
of the tuft just beneath the grip mark of the tweezers and second on the opposite end of
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the tuft where the rate of change of visual density of fibers was found to be maximum.
The distance between the two lines was measured with a fine millimeter scale. The
average staple length was calculated by taking the mean of the two tufts of a sample.
13. Fibre Fineness(µg/in)
The Fibre Fineness was measured with the help of “Sheffield 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
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non-Bt lines were regularly sprayed with suitable insecticides to control the Lepidopteran
insects, whereas no insecticide was applied to the Bt lines throughout the season to
control Lepidopteran insects. The seed cotton yield per plot was recorded and the means
were statistically compared by using “t-test assuming unequal variances” as explained in
the statistical analysis section 3.11.
3.5.2.2.2 Insecticide Application Trial, 2005
.In the year 2005, the experiment was conducted using Split Plot Randomized
Complete Block Design with four replications having genotypes in the main plots and
treatments (insecticide applications) in the sub-plots.
The trial comprised of one Bt genotype CEMB-3 and one non-Bt genotype 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
65
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.
66
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
67
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 PARENT1. 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.
68
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
69
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”.
70
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)
71
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.
72
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)
73
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 / s2
2
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
74
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
75
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.
76
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
77
CHAPTER 4
RESULTS AND DISCUSSION
78
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).
79
Figure 1
SCHEMATIC DIAGRAM OF THE CONSTRUCT pKMAB 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
Border Sequence
Pnos Promoter
Neo Gene
3 َOCS
T DNA Gene 7
Border Sequence
CAB 22L Leader Sequence
Bt Gene Cry1Ab
35S Promoter
Bam HI
XbaI TatI
ScaI
SacI
EcoRI NcoI
80
Figure 2
PCR Confirmation of Transformation of Agrobacterium tumefaciens C58C1
1 2 3 4 5 6 7
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.
550bp
81
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.
82
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.
83
Figure 4
PCR OF TRANSFORMED PLANTS
1 2 3 4 5 6 7 8 9 10 11 12 13
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.
550 bp
84
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
85
Figure 5
SOUTHERN BLOT ANALYSIS OF TRANSFORMED PLANTS
(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.
CE
MB
-3
CE
MB
-11
CE
MB
-16
CE
MB
-17
CE
MB
-3
CE
MB
-11
CE
MB
-16
CE
MB
-17
A B
86
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.
87
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).
88
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).
1 2 3 4 5 6 7
89
Figure 8
COMPARATIVE VIEW OF DAMAGED AND HEALTHY COTTON BOLLS
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
A
D C
B
90
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%.
Table 1
Insect Resistance and No. of Bolls of 1st Generation Plants
S.No. NAME
Insect mortality % shown in lab bioassay with Heliothis
No. of Total Bolls (Mature + Immature)
Average Boll Damage %age During the Peak Infestation Period (August-October)
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
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Figure 9
LABORATORY BIOASSAY WITH HELIOTHIS LARVAE
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.
A B
C D
92
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.
93
Figure 10
LAYOUT PLAN OF PROGENY ROWS GROWN DURING KHARIF, 2003 .
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.
CEM
B-3
CEM
B-1
1
CEM
B-2
4
CEM
B-2
2
CEM
B-9
CEM
B-4
CEM
B-1
7
CEM
B-1
0
CEM
B-1
6
Con
trol
Sorghum
Path
Non-Bt Refugia
94
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.
95
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.
96
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.
97
Table 2
CHARACTERISTICS OF SELECTED FIVE PLANTS FROM 2nd GENERATION
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.
S.
No. Plant No.
PCR
Result
Western
Dot Blot
Result
Insect
Mortality
shown in
the Lab
Bioassay
(%)
Boll
Damage
due to
Natural
Infestation
(%age)
No. of
Monopodial
Branches
No. of
Sympodial
Branches
Plant
Height
(cm)
Yield
(gm)
Ginning
Outturn
(%age)
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
98
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).
99
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.
A
B
100
Table-3
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.
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
101
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.
102
Figure 13
COTTON FIELD 2004
COTTON FIELD 2005
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).
A B
C
A
B C
103
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).
104
Figure 14
Figure 15
Bt PROTEIN CONTENT
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
CEMB-3 CEMB-11 CEMB-16 CEMB-17 CEMB-C
PER
CEN
TAG
E
SPOTTED BOLLWORM
0.00
0.20
0.40
0.60
0.80
1.00
1.20
CEMB-3 CEMB-11 CEMB-16 CEMB-17 CEMB-C
NO
.OF
INSE
CTS
PER
PLA
NT
GENOTYPES
The number of live insects per plant were counted on weekly basis during the season and averaged at the end.
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).
105
Table 4
ANALYSIS OF VARIANCE: MEAN SQUARES FOR DIFFERENT CHARACTERS OF THE Bt TRIALS 2004-2005
** 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).
SOURCE OF VARIATION
YIELD PER
PLANT
PLANT HEIGHT
NO. OF MONOPODIAL
BRANCHES
NO. OF SYMPODIAL BRANCHES
NATURAL INFESTATION OF SPOTTED BOLLWORM
FIELD BIOASSAY (Heliothis)
GOT %age
Replication 224.20 *
413.22 *
12.89 * 9.33 * 0.19 ** 0.01 ns 0.10 ns
Genotypes 24.59 ns
653.30 **
6.09 ns 13.11 ** 0.09 ** 0.01 ns 10.04 **
Error 58.96 614.24 2.23 2.59 0.02 0.01 2.11
106
Table 5
ANALYSIS OF VARIANCE: MEAN SQUARES FOR DIFFERENT CHARACTERS OF THE
Bt TRIALS 2004-2005
** 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).
SOURCE OF VARIATION
NO. OF BOLLS PER PLANT
BOLL WEIGHT
STAPLE LENGTH
FIBRE FINENESS
Bt PROTEIN CONTENT
LAB BIOASSAY (Heliothis)
Replication 34.77 ns 0.69 ns 0.65 ns 0.05 ns 0.004070 ns 240.00 *
Genotypes 12.3 ns 1.15 ns 0.84 ns 0.03 ns 0.045343 ** 181.73 *
Error 13.07 0.42 0.54 0.04 0.001435 43.33
107
Table 6
MEAN COMPARISONS FOR DIFFERENT CHARACTERS OF THE Bt TRIALS 2004-2005*
• 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
GENOTYPES
YIELD PER
PLANT (g)
PLANT HEIGHT
(cm)
NO. OF MONOPODIAL
BRANCHES PER PLANT
NO. OF SYMPODIAL BRANCHES PER PLANT
NATURAL INFESTATION OF SPOTTED BOLLWORM (no. of insects
per plant)
FIELD BIOASSAY (Heliothis)
(no. of insects per
plant)
GOT %age
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-CONTROL 19.88 a 131.47 a 6.12 a 9.52 a 0.99 a 0.15 a 31.29 b
108
Table 7
MEAN COMPARISONS FOR DIFFERENT CHARACTERS OF THE Bt TRIALS 2004-2005*
• 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
GENOTYPES NO. OF BOLLS PER PLANT
BOLL WEIGHT
(g)
STAPLE LENGTH
(mm)
FIBRE FINENESS
(µg/in)
Bt PROTEIN (%age of total
protein)
LAB BIOASSAY (Heliothis mortality
%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
109
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
110
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.
111
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
0.00
0.02
0.04
0.06 0.08 0.10 0.12 0.14 0.16
0.18
0.20
CEMB-3 CEMB-11 CEMB-16 CEMB-17 CEMB-C
NO
. OF
SUR
VIVI
NG
HEL
IOTH
IS L
AR
VAE
GENOTYPES
The number of surviving Heliothis larvae after one week of 2nd and 3rd field infestations respectively during both the years was recorded.
112
Figure 18
Figure 19
LABORATORY BIOASSAYS
0
10
20
30
40
50
60
CEMB-3 CEMB-11 CEMB-16 CEMB-17 CEMB-C
MO
RTA
LITY
PER
CEN
TAG
E O
F H
ELIO
THIS
LA
RVA
E
GENOTYPES The data on insect mortality were taken on daily basis upto seventh day.
YIELD PER PLANT
0
5
10
15
20
25
30
CEMB-3 CEMB-11 CEMB-16 CEMB-17 CEMB-C
YIEL
D (g
)
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.
113
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).
114
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.
115
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.
116
Figure 20
Figure 21
PLANT HEIGHT
0
20
40
60
80
100
120
140
160
CEMB-3 CEMB-11 CEMB-16 CEMB-17 CEMB-C
CEN
TIM
ETR
ES
0
1
2
3
4
5
6
7
8
CEMB-3 CEMB-11 CEMB-16 CEMB-17 CEMB-C
NUMBER OF MONOPODIAL BRANCHES PER PLANT
GENOTYPES The plant height was recorded in centimeters from base to the apex by using a meter rod.
NU
MB
ER
GENOTYPES At maturity, the number of monopodial branches of ten randomly selected plants from each plotwere counted and averaged for the purpose of statistical analysis.
117
Figure 22
Figure 23
NUMBER OF SYMPODIAL BRANCHES PER PLANT
0
2
4
6
8
10
12
14
16
CEMB-3 CEMB-11 CEMB-16 CEMB-17 CEMB-C
NU
MB
ER
NUMBER OF BOLLS PER PLANT
0
2
4
6
8
10
12
14
16
18
20
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.
NU
MB
ER
GENOTYPES The actual count of effective bolls on ten randomly selected plants was recorded and summed up forall pickings. The mean was calculated by dividing total number of bolls with the number of plants.
118
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.
119
Figure 24
Figure 25
BOLL WEIGHT
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
CEMB-3 CEMB-11 CEMB-16 CEMB-17 CEMB-C
GINNING OUTTURN PERCENTAGE
29.00
30.00
31.00
32.00
33.00
34.00
35.00
36.00
CEMB-3 CEMB-11 CEMB-16 CEMB-17 CEMB-C
GR
AM
S
GENOTYPES The average boll weight was obtained by dividing the total seed cotton yield of ten randomlyselected plants in each plot by the respective total number of effective bolls.
GO
T %
age
GENOTYPES
A sample of 100 grams from each plant was taken and ginned separately with a Single RollerElectric Gin. The lint obtained was expressed as GOT %age.
120
Figure 26
Figure 27
STAPLE LENGTH
24.00
24.50
25.00
25.50
26.00
26.50
27.00
27.50
CEMB-3 CEMB-11 CEMB-16 CEMB-17 CEMB-C
FIBRE FINENESS
4.40
4.50
4.60
4.70
4.80
4.90
5.00
CEMB-3 CEMB-11 CEMB-16 CEMB-17 CEMB-C
MIL
LIM
ETER
SM
ICR
OG
RA
M P
ER
GENOTYPES
The Staple Length was measured by Fibrograph Model 530 (electronic).
GENOTYPES The Fibre Fineness was measured with the help of “Sheffield Micronaire” and expressed in microgram per inch.
121
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”.
122
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.
123
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
124
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.
125
Table 9
SEED COTTON YIELD COMPARISONS OF Bt AND NON-Bt GENOTYPES UNDER
DIFFERENT INSECTICIDE APPLICATION TREATMENTS
L0 L1 L2 L3
GENOTYPES/ YIELD YIELD (g) YIELD (g)
%AGE INCREASE/ DECREASE
OVER L0
YIELD (g)
%AGE INCREASE/DECREASE
OVER L0
YIELD (g)
%AGE INCREASE/ DECREASE
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
126
Table 10
ANALYSIS OF VARIANCE FOR SEED COTTON YIELD
(COMPARATIVE STUDY OF INSECTICIDE APPLICATIONS, 2005)
SOURCE OF VARIATION df SS MS F.RATIO
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
L X V 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).
127
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.
128
Table 12
COMPARISON OF INSECTICIDE USE AND YIELDS ON Bt
AND NON-Bt COTTON LINES.
Bt (CEMB-3)
NON-Bt (CEMB-C)
No. of sprays against bollworms 1.75 * 3.0
Yield (gm/plant) 49.40 ns 37.52
* 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.
129
Figure 28
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.
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
L0 (No spray)
L1 (3 sprays)
L2 (2.5 sprays)
L3 (1.75 sprays)
CEMB 3 CEMB C
*
*
YIELD COMPARISONS OF Bt AND NON-Bt GENOTYPES
GR
AM
S PE
R P
LAN
T
TREATMENTS
130
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.
131
Table 13 HISTORY SHEET OF TRANSGENIC PURE LINES DEVELOPED AT CEMB
S. NO.
LINE NO.
1ST GENERATION 2ND GENERATION 3RD
GENERATION 4TH GENERATION 5TH GENERATION
1. CEMB-3
Total Plants = 1 PCR positive = 1 ELISA positive = 1 Southern Blot Positive = 1 Western Blot Positive =1
Total Plants Analyzed = 17 PCR positive = 17 Western Dot Blot Positive =17
Total Plants Analyzed = 10 PCR positive = 10 Western Dot Blot Positive =10
Total Plants Analyzed = 30 PCR positive = 30 Western Dot Blot Positive =30
Total Plants Analyzed = 30 PCR positive = 30 Western Dot Blot Positive =30
2. CEMB-11
Total Plants = 1 PCR positive = 1 ELISA positive = 1 Southern Blot Positive = 1 Western Blot Positive =1
Total Plants Analyzed = 18 PCR positive = 12 Western Dot Blot Positive =12
Total Plants Analyzed = 10 PCR positive = 9 Western Dot Blot Positive =9
Total Plants Analyzed = 30 PCR positive = 30 Western Dot Blot Positive =30
Total Plants Analyzed = 30 PCR positive = 30 Western Dot Blot Positive =30
3. CEMB-16
Total Plants = 1 PCR positive = 1 ELISA positive = 1 Southern Blot Positive = 1 Western Blot Positive =1
Total Plants Analyzed = 17 PCR positive = 4 Western Dot Blot Positive =5
Total Plants Analyzed = 10 PCR positive = 9 Western Dot Blot Positive =9
Total Plants Analyzed = 30 PCR positive = 30 Western Dot Blot Positive =30
Total Plants Analyzed = 30 PCR positive = 30 Western Dot Blot Positive =30
4. CEMB-17
Total Plants = 1 PCR positive = 1 ELISA positive = 1 Southern Blot Positive = 1 Western Blot Positive =1
Total Plants Analyzed = 30 PCR positive = 26 Western Dot Blot Positive =26
Total Plants Analyzed = 10 PCR positive = 9 Western Dot Blot Positive =9
Total Plants Analyzed = 30 PCR positive = 30 Western Dot Blot Positive =30
Total Plants Analyzed = 30 PCR positive = 30 Western Dot Blot Positive =30
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.
132
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.
133
Table 14
MOLECULAR ANALYSIS OF F1 PLANTS
S.NO NAME OF CROSS TOTAL PLANTS
ANALYZED
NUMBER OF PLANTS
POSITIVE IN PCR
NUMBER OF PLANTS
POSITIVE IN WESTERN DOT BLOT
1 MNH-93 X CEMB-3 20 20 20
2 CEMB-3 X MNH-93 19 19 19
3 CIM-482 X CEMB-3 11 11 11
4 CEMB-3 X CIM-482 19 19 19
5 MNH-93 X CEMB-11 20 20 20
6 CEMB-11 X MNH-93 19 19 19
All plants of F1 generation were analyzed through PCR and Western Dot Blot to study the transmission and expression of the transgene in F1 generation. It was concluded that the gene was stably integrated in the genome of the transgenic plants; the transgene could be successfully transferred from Bt lines to non-Bt lines; and the gene was dominant
134
Table 15
SEGREGATION OF Bt GENE IN F2 POPULATIONS OF SIX CROSSES
S.No. Combinations Total Plants
No. of Negative
Plants
No. of Positive Plants
Chi-square
value for 3:1 ratio
Ratio
1. MNH-93 X CEMB-3 88 45 43 32.061 Distorted (Non-Mendelian)
2. CEMB-3 X MNH-93 93 23 70 0.004 3:1 (Mendelian)
3. CIM-482 X CEMB-3 60 13 47 0.356 3:1 (Mendelian)
4. CEMB-3 X CIM-482 92 51 41 45.449 Distorted (Non-Mendelian)
5. MNH-93 X CEMB-11 103 56 47 47.382 Distorted (Non-Mendelian)
6. CEMB-11 X MNH-93 67 22 45 2.194 3:1 (Mendelian)
Total 503 210 293
The data were subjected to chi-square goodness of fit test against the Mendelian ratio 3:1.
135
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
136
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.
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Table 16
ANALYSIS OF VARIANCE: MEAN SQUARES OF F1 HYBRIDS FOR DIFFERENT CHARACTERS
Mean Squares Source of Variation df Yield per
Plant No. of Bolls per Plant Boll Weight GOT %age Lab Bioassay
(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 Crosses 1 405.30 * 16.18 * 0.43 ns 260.59 ** 14.08 ns
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).
138
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
139
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
140
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
141
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.
142
Table 17
MEAN PERFORMANCE OF F1 HYBRIDS AND THEIR PARENTS FOR DIFFERENT CHARACTERS
Seed Cotton
Yield per Plant (g)
No. of Bolls per Plant
Boll Weight (g) GOT %age
Lab Bioassay (Mortality
%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
143
Table 18
ESTIMATES OF HETEROSIS AND HETEROBELTIOSIS FOR DIFFERENT
CHARACTERS OF F1 HYBRIDS
GENOTYPES Seed Cotton Yield per plant (g)
No. of Bolls per plant Boll Weight (g) GOT %age Lab Bioassay
(Mortality %age)
Ht(%) Hbt(%) Ht(%) Hbt(%) Ht(%) Hbt(%) Ht(%) Hbt(%) Ht(%) Hbt(%)
MNH-93 X CEMB-3 16.49ns -2.81ns 13.81ns 4.17ns 5.89ns -4.08ns 26.44** 26.17** 36.23** 23.68**
CEMB-3 X MNH-93 50.19* 25.31* 24.58* 14.02ns 21.38** 9.96* 21.43** 21.17** 30.43** 18.42**
CIM-482 X CEMB-3 -15.19ns -18.58ns -2.80ns -4.11ns -6.96ns -9.30ns 16.38** 2.43** 16.22* 19.44*
CEMB-3 X CIM-482 107.07** 98.79** 69.40** 67.12** 12.83* 9.99* 13.02** -0.52** -8.11ns -5.56ns
MNH-93 X CEMB-11 91.35** 73.78** 81.36** 81.36** 6.42ns -3.19ns 22.45** 20.27** 13.43ns 5.56ns
CEMB-11 X MNH-93 -7.19ns -15.71ns -20.34ns -20.34ns 15.52* 5.09* 17.43** 15.33** 10.45ns 2.78ns
** 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).
144
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
S.No. Cross combination F1 Variance (lab bioassay results)
F2 Variance (lab bioassay results)
Broad Sense Heritability (%)
Genetic Advance
Relative Expected
Gain
1 MNH-93 X CEMB-3 462.2222
872.6003
47.03 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.
146
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
Yie
ld (g
)
Plan
t Hei
ght (
cm)
No.
of M
onop
odia
l Bra
nche
s per
Pla
nt
No.
of S
ympo
dial
Bra
nche
s per
Pla
nt
No.
of B
olls
per
Pla
nt
Bol
l Wei
ght (
g)
Gin
ning
Out
turn
(%)
Stap
le L
engt
h (m
m)
Fibr
e Fi
nene
ss (µ
g/in
)
Nat
ural
Infe
stat
ion
of S
pott
ed
Bol
lwor
m (n
umbe
r of
inse
cts p
er p
lant
)
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
Leaf Hairiness Smooth to Sparsely
Hairy Profusely Hairy
Boll Shape Roundish Roundish
Boll Opening Good Fluffy Good Fluffy
Reaction to Virus Susceptible Susceptible
Reaction to Bollworms Tolerant Susceptible
The qualitative characters were also taken into account while screening plants in different generations
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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,
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while evaluating the lines for insect resistance, the data on natural infestation of Spotted
Bollworm was the most reliable criteria because only the spotted bollworm occurs
naturally in Lahore environment. These results were in accordance with the results
obtained after Bt protein quantification. The results obtained after conducting lab
bioassays also supported significant differences among control and Bt lines. The DMR
test clearly indicated the Bt lines to be better in insect resistance than the non-Bt
counterpart.
The data obtained after much hectic and laborious exercise of conducting Field
Bioassays/Artificial Infestation of Heliothis in the field were not so encouraging. This
may be attributed to the reasons already described i.e. the insect moths were collected
from entirely different climatic zones than that of Lahore area, reared artificially in the
labs under controlled conditions of temperature and humidity, and released in the field
directly. Consequently, the mortality rate of the insects had been higher due to abrupt
change in their environment. The method of insect release therefore, needed to be
improved further. Our findings that the control line showed higher survival percentage of
Heliothis than the Bt lines were similar to the results already reported by various research
workers, a few of which are described below:-
The expression of Bt Cry1Ac and Cry1Ab genes have been reported in cotton by
Perlak et al. (1990) and Barton (1989). They reported total protection from insect damage
of leaf tissue from these plants in laboratory assays when tested with Lepidopteran insects
and that Cry1Ab had 5-fold higher unit activity for pink bollworm than for cotton
bollworm and tobacco budworm. Sachs et al. (1996; 1998) reported that cotton plants of
both genetic backgrounds that possessed the Cry1Ab insecticidal protein or high terpenoid
glanding or both were more resistant to Tobacco budworm larvae than plants with other
traits. Estruch et al. (1997); Halcomb et al. (1996); Halcomb et al. (2000) and Van Rie. J.
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(2000) evaluated plant stands of BTK and non-BTK. They found that the attack of
bollworms and tobacco budworm was less on BTK plants as compared to non-BTK
plants and that mortality %age was greater in case of bollworm while no significant
difference was observed in case of Tobacco budworm fed both on BTK and non-BTK
plants. Our findings were also in confirmation of Wu et al. (2003) who reported that the
survival of larvae was significantly reduced in Cry1Ac expressing plants in spite of the
fact that egg densities between transgenic and non-transgenic plants varied 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
157
resistance but keeping the other characters intact/unaltered. Since the seeds used for
transformation were of an approved variety, it was therefore a highly positive aspect of
this transformation that most of the varietal characters remained un-altered.
The most important character in a crop-improvement programme is yield. The
data presented in Table-5 indicated that the genotypes had non-significant differences
among themselves regarding yield. The crop was kept immune to insecticidal sprays
against Lepidopteran insects. Theoretically, the Bt lines should give higher yields than
control. In fact, the Bt lines generally gave higher yields than the control line, in both
years of study; however, the increased yields were non-significant statistically. This
behaviour of the transgenic lines may be attributed to the lower natural infestation of the
Lepidopteran insects during the years of study.
In the comparative studies of insecticide applications on Bt and non-Bt cotton, it
was concluded that a saving of 41% of insecticides (meant for Lepidopteran, only) can be
done by the use of Bt cotton line CEMB-3 in comparison with its non-Bt counterpart
MNH-93. Our findings are in confirmation of many earlier research workers. Field trials
of transgenic cotton (BTK) lines have been studied against Lepidopterans by Benedict et
al (1996) and Altman et al (1996). Their plants were carrying genes that code Cry1Ab and
Cry1Ac delta-endotoxins from Bacillus thuringiensis var. kurstaki. They found that these
insect resistance lines showed a reduction of the insecticide application for tobacco
budworm, Bollworm, Cabbage looper and increased farm profit. Similarly, Stewart et al.
(2001) and Tabashnik et al. (2002) reported that crops genetically engineered to produce
Bacillus thuringiensis toxins for insect control can reduce use of conventional
insecticides, but insect resistance could limit the success of this technology. Our findings
are also in confirmation of Xia et al. (1999) who reported use of Bt cotton in china with a
concomitant reduction in insecticide use. They concluded that Bt cotton required fewer
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chemical insecticides and a potential for higher yields. Carpenter and Gianessi (2001)
reported the primary benefits of increasing yields due to elimination of losses by
European corn borer. Other benefits of modified plants were emphasized by several
authors like reduced environmental impact of insecticides, potential of higher yields and
better food supply in the developing countries, better food safety due to reduced fungal
infections and remediation of polluted soils (Borlaug, 2000; Mackey and Santerre, 2000;
Munkvold and Hellmich, 2000; Mendelsohn et al., 2003; Kasha, 2000). However, the
new modified crops could not be the panacea for solving all the pest problems due to
specific mode of actions of toxins against the target pests (Sharma et al., 2000).
4.9.4 Bt Inheritance
In the selfed-generations studies, the Cry1Ab gene was found to be behaving
differently in different progenies in respect of integration and expression. This may be
attributed to the epistatic and environmental factors besides segregation of the gene.
According to the earlier reports of Sachs et al. (1996; 1998) epistatic and environmental
factors affect the foreign gene expression in cotton (Gossypium hirsutum L); these effects
could influence the stability, breeding, durability and efficacy of foreign genes. They
reported site of gene insertion and cotton background to be the significant sources of
variation for Cry1A gene expression. These effects were heritable and caused similar
effects in several different genetic backgrounds of F2 families.
In the present studies, varietal differences were observed in various characters.
This has been in confirmation of Hoskinson et al. (1964) who observed marked varietal
differences in cotton, some experimental lines being vigorous and tolerant of the early
season disease-insect complex.
159
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
160
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
163
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.
164
CHAPTER 5
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I
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
II
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
III
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
IV
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
V
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
VI
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%