Production Of Transgenic Tomato Plant Through Agrobacterium Mediated Transformation Using Green Fluorescent Protein Gene

1. Production Of Transgenic Tomato
Plant Through Agrobacterium
Mediated Transformation Using
Green Fluorescent Protein
DR. PRERNA SONI
ASSISTANT PROFESSOR
BIOTECHNOLOGY
SPCAS COLLEGE NAWAPARA RAIPUR CHHATTISGARH

2. INTRODUCTION
 Tomato History
 How to genetically modify a plant
 Agrobacterium mediated gene transfer methodology
 The process of gene transfer from Agrobacterium to plant cells
implies several essential steps :-
 Bacterial colonization
 Induction of bacterial virulence system
 Generation of T-DNA transfer complex
 T-DNA transfer
 Integration of T-DNA into plant genome.

3. Callus
Growing Plant in Culture Media Modified Plant

4. The Fluorescent Protein
Green Fluorescent Protein (GFP)
 It is discovered by Shimomura in 1962.
 It is derived from Jellyfish Aequorea victoria
 It is a bioluminescent protein
Aequorin
Ca3 – Aequorin - Coelenteramide
OOH
Coelenterazine
3Ca2+
Invitro Green light
λ max = 508 nm
Invitro Blue light
λ max = 470 nm
Ca3 – Aequorin – Coelenteramide +
CO2
GFP

5. EXPLORING THE STRUCTURE
3D GFP FLUORESCENCE IN JELLYFISH 3D IMAGE OF A.victoria’s GFP

6.  The structure of GFP was solved in 1996.
 It has a novel three dimensional structure called  can
 It contains a chromophore which is biosynthetically created between the
amino acid residues of 65-67 modified Ser-Tyr-Gly sequence of the GFP
protein.
 This chromophore absorbs blue light & allows the emission of the green
light

7. Expression of GFP in Tomato and Wheat root
Tomato plant of the variety "Minitom" (left) and colonisation of the root by the gfp-labelled
bacterium Serratia liquefaciens MG1 (middle). The labelled bacterium shows intensive green
fluorescence. Right: Azospirillum brasilense Sp7-cells with activated ipdC-gfp-reporter construc
(arrows) on the surface of wheat roots.

8. Timeline of Plant Biotechnology
1700s -- Naturalists identify hybrid plants.
1860s -- Austrian botanist and monk Gregor Mendel studies pea plants and
recognizes that specific traits are passed from parents to offspring - these traits are
eventually discovered to be genes.
1900 -- European botanists begin to improve plant productivity using genetic
theories based on Mendel's work.
1922 -- Farmers purchase hybrid seed corn created by crossbreeding two corn varieties.
1953 -- Structure of DNA is discovered - marking the beginning of modern genetic research.
1970s -- Hybrid seeds are introduced to developing countries to increase food supplies.
1973 -- Genetic engineering is used to precisely manipulate bacterial DNA.
1983 -- First GM plant is created; a tobacco plant resistant to an antibiotic.
1985 -- GM plants resistant to viruses, bacteria, and insects are field tested.
1986 -- EPA approves the release of the first GM crop (herbicide resistant tobacco).
1990 -- First successful field trial of GM cotton (herbicide resistant).
1992 -- FDA decides GM foods will be regulated as conventional foods.
1994 – FlavrSavr Tomato becomes the first GM food to be approved for sale.
1995 -- Herbicide resistant canola, corn,
2000 -- Cotton, soybeans, sugar beet as well as insect or virus resistant corn,
cotton, papaya, potato, squash, tomato approved in the U.S.
2001 -- "Golden rice" which may help prevent millions of cases of blindness
and death caused by Vitamin A and iron deficiencies undergoes continued testing.

9. MATERIALS AND METHODS
1. Plasmid Isolation (pGPTV, pUC-GFP)
Plasmid isolation involves four steps –
I. Harvesting
II. Alkali lysis
III. Plasmid precipitation
IV. Purification
2. Agarose gel electrophoreusis
I. Preparation of gel
II. Loading of samples
III. Passing of electric current.

10. 3. Restriction Digestion
– Plasmid DNA has digested with the help of
restriction enzymes. EcoRI & Xba1.
– Linearized pGPTV vector & GFP insert were
observed, which were eluted by using
ammonium acetate method.
– Total reaction volume 40 µl
DNA
SAMPLES
DNA (µl) BUFFER(µl) EcoR1(µl) Xbal(µl)
pGPTV 30 6.00 1.00 1.00
pUC-GFP 28 6.00 1.00 1.00

11. 4. Gel Elution
o This was done using 5M, 350 µl ammonium
acetate.
5. Estimating the concentration of DNA in small
volume using Spot Test Method
6. Transfer of GFP gene to pGPTV
o Ligation Reaction
o Total reaction volume – 20 µl
Ligase ADDW
Enzyme (µl)
(µl)
Sl.No. Molar
Ratio
Vector
DNA(µl)
Insert
DNA(µl)
10X
buffer
(µl)
1 1:2 3.5 0.3 2.0 0.5 13.7

12. 7. Transformation
o Competent cell preparation
( E.coli basic strain DH5-  cells were used for
transformation.)
8. Screening
o Isolation of plasmid DNA from Agrobacterium
tumefaciens.
o Restriction digestion
o Agarose gel electrophoresis.
9. Triparental mating
o The transformed recombinant pGPTV plasmid with
gene of interest in E. coli was transferred to
Agrobacterium tumefaciens LBA4404 strain with the
help of E. coli helper strain pRK2013.

13. 10. Agrobacterium mediated plant
transformation
 The GFP gene was transferred from
Agrobacterium to the tomato plant genome to
transform our tomato plant including following
steps :
 Preparation of tomato explants.
 Surface sterilization of tomato leaves
 Leaf disc preparation.
 Cocultivation of tomato leaf discs with Agrobacterium
tumefaciens.
 Selection media transfer.

14. 11. Propagation of transgenic plantlets –
o Callus formation
o Sub culturing of transformed tomato
o Ex vitro establishment.
12. Confirming the presence of transgenes by
isolating genomic DNA
13. Polymerase chain reaction
Total reaction volume – 25 µl

15. TABLE
Sl
No
Sample DNA
(µl)
Taq
MgCl2
(µl)
Taq
buffer
(µl)
dNTP
mixture
(µl)
Forward
Primer I
(µl)
Reverse
Primer
II
(µl)
Enzymes
Taq
polymerase
(µl)
ADDW
(µl)
1. Genomic
DNA
4.0 2.5 2.5 2.25 0.75 0.75 0.75 11.5
2. Genomic
DNA
5.0 2.5 2.5 2.25 0.75 0.75 0.75 10.5
3. Genomic
DNA
5.0 2.5 2.5 2.25 0.75 0.75 0.75 10.5
4.
5.
Genomic
DNA
Genomic
DNA
6.0
6.0
2.5
2.5
2.5
2.5
2.25
2.25
0.75
0.75
0.75
0.75
0.75
0.75
9.5
9.5
6. + Ve
control
6.0 2.5 2.5 2.25 0.75 0.75 0.75 9.5

16. RESULTS

17. RESTRICTION DIGESTION

18. GEL ELUTION

19. SPOT TEST

20. SCREENING

21. TRANSFORMATION

22. TRIPARENTAL MATING

23. CULTURING OF TOMATO PLANT FROM LEAF DISC

24. TRANSGENIC TOMATO

25. GENOMIC DNA ISOLATION

26. POLYMERASE CHAIN REACTION

27. DISCUSSION
&
CONCLUSION

28. THANKYOU