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

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.

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.

Methodology

Agrobacterium Mediated Transformation

Putative Transgenic Cotton Plants

PCR ELISA ELISA Insect

Bioassay

Spray

Assay

Morphological

Characteristics

Copy No. of Gene in

Transgenic Cotton

Inheritance Studies

Putative Transgenic Cotton Plants

OBJECTIVE 1

Transformation of CEMB synthetic GTGene

and Cry1Ac+Cry2A genes in cotton Variety

VH-289

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.)

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)

OBJECTIVE 2

Molecular confirmation of putative

cotton plants of Variety VH-289

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

0

0,5

1

1,5

2

2,5

3

GTG ProteinConc. ng/g

Cry2A ProteinConc. ng/g

Quantification of GTG protein of transgenic cotton

plants in T1 generation

(ug/g)

(ug/g)

Quantification of Cry2A protein of transgenic cotton

plants in T1 generation

(ug/g)

(ug/g)

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

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

OBJECTIVE 3

Evaluation of transgenic cotton plants

through insect assay and Glyphosate spray

assay

• 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

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.

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

14 Control 1600 ml/acre Plant Died

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 93a Least 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)

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

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

OBJECTIVE 4

Determination of field performance and

morphological characteristics of transgenic

cotton plants

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.

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

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

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

OBJECTIVE 5

Expression and Inheritance studies of

transgenic plants in advanced generation

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

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

Cotton

Lines

(O-E) (O-E)^2/E F.

Value

F.

Table Conclusion Mendelian

Ratio Resistant 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……

OBJECTIVE 6

Determination of the copy no. of gene in

transgenic cotton plants

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.

Transgenic plant line VH 289 (55-4) have shown signal in

nucleus at chromosome no: 6 for Cry2A

Transgenic plant line VH 289 (55-4) have shown

signal in nucleus at chromosome no: 3 for GTGene

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.

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)