1. The objectives of the study were to transform cotton variety VH-289 with Cry1Ac+Cry2A and GTGene using Agrobacterium mediated transformation. 2. Putative transgenic plants were confirmed by PCR and ELISA. Transgenic plants showed resistance against lepidopteran insects and tolerance to glyphosate. 3. Evaluation of transgenic plants in advanced generations showed increased yield components such as plant height, boll number, boll weight and yield compared to control plants. 4. Gene expression and inheritance studies showed that the transgenes followed a 3:1 Mendelian ratio of inheritance in subsequent generations.

2. objectives
2.Molecular
confirmation of
putative cotton
plants of Variety
VH-289
1.Transformationof
CEMB synthetic
GTGene and
Cry1Ac+Cry2A
genes in cotton
Variety VH-289
3. Evaluation of
transgenic cotton
plants through
insect assay and
Glyphosate spray
assay
4. Determination of
field performance and
morphological
characteristics of
transgenic cotton
plants
5. Expression and
Inheritance studies
of transgenic plants
in advanced
generation
6. Determination
of the copy no. of
gene in transgenic
cotton plants

4. Introduction.. Cotton is one of the most important economic crops in
the world, which provides more than 90% of the total
cotton production.
Beside importance of cotton it has many problems like
insect, pest, weeds and viruses.
Pakistan and Indonesia will get more attention to
insect, pest and weed problems because both countries
have obstacles in production costs of the pest or weed
maintenance which requires huge cost.
A number of Cry and GTGene, which are used to
produce resistance in cotton plants because they are
differentially expressed during different stages of
cotton development.
On the basis of their specific expression, these genes
may offer potential for improvement of cotton yield.
Genetic transformation is a powerful tool for plant
breeding and genetical, physiological or biochemical
research; consequently it is an extremely dynamic
field.

5. Pakistan is the fifth largest producer of cotton in the
world, the third largest exporter of raw cotton, the fourth
largest consumer of cotton, and the largest exporter of
cotton yarn.
The export of Indonesian cotton yarn 138.000 MT during
2013 and Textile export of Indonesia in 2012 esteemed
at $ 12.5 billion and $ 6.4 billion from January to June
2013.
In 2012-13, 16,495.00 tons of pesticides and weedicides
were consumed which cost 66 Millions US Dollars in
Indonesia same like Pakistan.
Biotechnology has the potential to create new plants, new
genes and new products such as GM cotton that have
resistance against weeds because Weeds compete with
crop plants and bollworms reduce the yield.

7. Methodology
Agrobacterium Mediated Transformation
Putative Transgenic Cotton Plants
PCR ELISAELISA Insect
Bioassay
Spray
Assay
Morphological
Characteristics
Copy No. of Gene in
Transgenic Cotton
Inheritance Studies
Putative Transgenic Cotton Plants

8. OBJECTIVE 1
Transformation of CEMB synthetic GTGene
and Cry1Ac+Cry2A genes in cotton Variety
VH-289

9. Fig: Physical maps of Cry1Ac+Cry2A and GTG constructs.
A) Plasmid construct drawing of Cry1Ac +Cry2A.
B) Plasmid construct drawing of GTGene.
(Maps show only those restriction sites, used for cloning.)

11. A: Germinated
Seeds of VH-289
E: embryos shifted to
selection free MS
medium plate
D: co-culturing
injured embryos in
MS (Cry2A & GTG)
C: injuring cotton
embryos with blade
B: embryo isolation
F: embryos shifted to
test tubes with
selection drug (Kana)

12. OBJECTIVE 2
Molecular confirmation of putative
cotton plants of Variety VH-289

13. Figure A: PCR amplification of Cry2A gene
Lane 1: 1kb ladder, Lane 2 to 14: transgenic cotton plants with Cry2Ac gene, Lane 15: negative control,
Lane 16: positive control cotton plants
Figure B: PCR amplification of GTG
Lane 1: 1kbladder, Lane 2-14: transgenic cotton plants with GTGene, Lane 15: negative control, Lane
16: positive control

14. 0
0,5
1
1,5
2
2,5
3
GTG Protein
Conc. ng/g
Cry2A Protein
Conc. ng/g

15. Quantification of GTG protein of transgenic cotton
plants in T1 generation
(ug/g)

16. (ug/g)
Quantification of Cry2A protein of transgenic cotton
plants in T1 generation

17. (ug/g)
(ug/g)

18. Fig: PCR Amplification of Cry2A gene
Lane 2-16: transgenic cotton plants, Lane 17: control cotton
plant, Lane 18: positive control, Lane 4, 12 and 13: negative
amplification
585 bp

19. Fig: PCR Amplification of Cry2A gene
Lane 2-16: transgenic cotton plants, Lane 17: control cotton plant,
Lane 18: positive control, Lane 4, 12 and 13: negative amplification
230 bp

20. OBJECTIVE 3
Evaluation of transgenic cotton plants
through insect assay and Glyphosate spray
assay

21. • Plate A: Transgenic plant leaf showing killed larvae after
eating a small portion of leaf
• Plate B: Control non-transgenic plant. Larva remained
active and alive after damage

23. A: transgenic cotton plant after spray
B: control cotton plant after spray, after 5-7 days of spray, results
showed the death of the control plant while the transformed plant
remains alive.

24. Serial No. Plant Name Glyphosate spray concentration
(ml/acre)
Results
1 VH 289 (2) 1600 ml/acre Plant survived and showed Resistance
2 VH 289 (18) 1600 ml/acre Plant survived and showed Resistance
3 VH 289 (25) 1600 ml/acre Plant survived and showed Resistance
4 VH 289 (34) 1600 ml/acre Plant Died
5 VH 289 (52) 1600 ml/acre Plant survived and showed Resistance
6 VH 289 (53) 1600 ml/acre Plant survived and showed Resistance
7 VH 289 (55) 1600 ml/acre Plant survived and showed Resistance
8 VH 289 (66) 1600 ml/acre Plant Resistant
9 VH 289 (57) 1600 ml/acre Plant Died
10 VH 289 (64) 1600 ml/acre Plant Died
11 VH 289 (69) 1600 ml/acre Plant survived and showed Resistance
12 VH 289 (72) 1600 ml/acre Plant survived and showed Resistance
13 VH 289 (73) 1600 ml/acre Plant survived and showed Resistance

25. Cotton Lines
Bio-toxic Leaf Assay (Mortality %)
20 days 40 days 60 days
Control 0e 0e 0e
2 78b 79b 81b
18 87a 88a 89a
25 65cd 67d 71d
52 70c 72cd 75cd
53 66cd 70cd 74d
55 82ab 85a 89a
66 69cd 73c 80bc
69 64d 67d 72d
72 85a 87a 93a
73 85a 86a 93aLeast significant difference test (LSD) of results determined that there are only four line
of transgenic plant which significantly differ from control and other transgenic crops i.e.
line VH 289 (18), VH 289 (55), VH 289 (72), and VH 289 (73)

26. Serial No. Plant Name Glyphosate spray
conc. (ml/acre)
Survival %age
1 Controls 1600 ml/acre 0%
2 VH 289(2) 1600 ml/acre 75% survive, 10% died, 15% undeveloped seed
3 VH 289(18) 1600 ml/acre 75% survived, 20% died, 5% undeveloped seed
4 VH 289(25) 1600 ml/acre 75% survived, 20% died, 5% undeveloped seed
5 VH 289(52) 1600 ml/acre 75% survived, 20% died, 5% undeveloped seed
6 VH 289(53) 1600 ml/acre 75% survived, 10% died, 15% undeveloped seed
7 VH 289(55) 1600 ml/acre 75% survived, 25% undeveloped seed
8 VH 289(66) 1600 ml/acre 75% survived, 25% dead
9 VH 289(69) 1600 ml/acre 75% survived, 20% dead, 5% undeveloped seed
10 VH 289(72) 1600 ml/acre 75% survived, 10% dead, 15% undevelopedseed
11 VH 289(73) 1600 ml/acre 75% survived, 15% died, 10% undeveloped seed

27. Line
Bio-toxic Leaf Assay (Mortality %)
20 days 40 days 60 days
Controls 0.000b 0.000b 0.000b
18-6
84.167a 85.833a 88.333a
55-4
86.455a 95.455a 98.182a
72-2
80.636a 94.545a 97.273a
73-10 80.231a 94.615a 96.154a

28. OBJECTIVE 4
Determination of field performance and
morphological characteristics of transgenic
cotton plants

29. Data of morphological characteristics of T1 progeny
transgenic cotton plants was collected in comparison
to control.
Variation in all these characteristics i.e no. of bolls,
yield and height was obtained in all transgenic cotton
line in comparison to control.

30. Line Plant Height Number of Boll Boll weight Yield
Control 147.858a 31.15a 3.60b 100.201b
2 47.869e 16.00f 2.73e 41.532e
18 66.206c 25.10d 4.16a 98.141b
25 40.360g 17.40f 3.051d 51.145d
52 42.703f 21.10e 3.28c 67.537c
53 42.369f 21.30e 3.25c 66.398c
55 68.266b 27.00c 4.25a 110.989a
66 46.371e 20.20e 3.30c 63.906c
69 54.760d 20.10e 3.29c 61.802c
72 68.154b 28.30bc 4.20a 117.472a
73 67.719b 28.70b 4.18a 112.663a

31.  Plant Height: Agronomic characteristics of transgenic plant is of great
value. The data of T2 generation revealed that plant height in T2
generation was greater than control
 Total Number of Bolls: The data of T2 generation of transgenic cotton
plants namely VH 289 (18-6), VH 289 (55-4), VH 289 (72-2) and VH 289
(73-10) have showed that transgenic plants have significantly higher no.
of bolls than control
 Boll Weight: Boll weight per plant in T2 generation transgenic cotton
was calculated and then compared with the average value controls On
ANOVA, LSD and Dunnett’s test showed significant difference of boll
weight at the level of 5% and 1% than control plant
 Yield: Similarly, showed significant difference of Yield weight at the level
of 5% and 1% than control plant

32. Line Plant Height Number of Boll Boll Weight Yield
Controls
148.054d 33.115d 3.633d 102.908d
18-6 166.383c 53.600c 4.188a 191.263c
55-4
179.664a 60.400ab 4.163a 219.317ab
72-2 173.882ab 65.764a 4.004c 230.349a
73-10
168.715bc 59.231b 4.028bc 203.592bc

33. OBJECTIVE 5
Expression and Inheritance studies of
transgenic plants in advanced generation

34.  The plants selected in T1 generation namely VH 289 (18-6),
VH 289 (55-4), VH 289 (72-2), VH 289 (73-10) on the basis
of transgene expression, insect bioassay, spray assay and yield
were sown to raise T2 generation.
 Molecular analysis like PCR, ELISA, insect assay and
herbicides assay was done to evaluate the transgenic cotton
plants in T2 generation. Entire T2 progeny plants is comprised
of sixty plants

35. Cotton
Lines
No. of
Plants
Observation Prob. Expected
Value
Prob
E
No. of
Resistant
Plants
No. of
Dead
Plants
Survival
rate (3/4)
Death
rate
(1/4)
Resistant Dead
Line of VH
289 (18-6)
15 12 3 0.75 0.25 1 11.25 3.75
Line of VH
289 (55-4)
15 10 5 0.75 0.25 1 11.25 3.75
Line of VH
289 (72-2)
15 11 4 0.75 0.25 1 11.25 3.75
Line of VH
289 (73-10)
15 13 2 0.75 0.25 1 11.25 3.75

36. Cotton
Lines
(O-E) (O-E)^2/E F.
Value
F.
Table
Conclusion Mendelian
RatioResistant Dead Resistant Dead
Line of VH
289 (18-6)
0.75 -0.75 0.05 0.15 0.2 3.84 H0 reject 3:1
Line of VH
289 (55-4)
-1.25 1.25 0.138889
0.41666
7
0.5555
56
3.84 H0 reject 3:1
Line of VH
289 (72-2)
-0.25 0.25 0.005556
0.01666
7
0.0222
22
3.84 H0 reject 3:1
Line of VH
289 (73-10)
1.75 1.75 0.272222
0.81666
7
1.0888
89
3.84 H0 reject 3:1
Continued……

37. OBJECTIVE 6
Determination of the copy no. of gene in
transgenic cotton plants

38.  The transgenic plant line VH 289 (55-4) which was showing
good expression of Cry2A and GTGene and resulting in
higher yield was subjected for determination of its transgene
copy no. and location by using gene specific probe.
 All transgenic plant line VH 289 (55-4) have shown signal in
nucleus at chromosome no: 6 for Cry2A and chromosome
no: 3 for GTGene but no signal was observed in control plant.

39. Transgenic plant line VH 289 (55-4) have shown signal in
nucleus at chromosome no: 6 for Cry2A

40. Transgenic plant line VH 289 (55-4) have shown
signal in nucleus at chromosome no: 3 for GTGene

41. CEMB transgenic cotton lines harboring Cry1Ac+Cry2A
showed good potential against lepidopteron insects by showing
100% mortality and codon optimized cp4EPSPS gene
(GTGene) against broad spectrum herbicide with tolerance
limit of 1600ml/acre which ultimately leads towards increase
of cotton yield.
These selected transgenic cotton lines after trials in advance
generations can be raised as variety for poor farmers of
Pakistan because it holds good potential. This study will help
me to get the idea to implement in my country for solution of
such problems.

42. PUBLICATION
Transformation and Evaluation of Cry1Ac+Cry2A And GTGene in
Gossypium Hirsutum L. Front. Plant Sci. 6:708.
doi:10.3389/fpls.2015.00708.
Variation in Expression of Phytochrome B Gene in Cotton (Gossypium
hirsutum L.) Journal of Agricultural Science & Technology;2013, Vol. 15
Issue 5, p1033. AJCS 7(2):206-212 (2013)
Genetic diversity assessment of cotton (Gossypium hirsutum L.)
genotypes from Pakistan using simple sequence repeat markers.
AJCScience Vol. 7 Issue 2 (Feb 2013)
Expression of Calotropis procera expansin gene CpEXPA3 enhances
cotton fibre strength (2014)