3. Introduction
Transformation methods
Practical application
Herbicidal, Pest, Disease,
Drought – Tolerance, and
Quality
Indian research
Conclusion
01/01/2014 SAMI 3
4. Introduction
01/01/2014 SAMI 4
Biotechnology is one of the tools , but a
potentially important one, in the struggle to
reduce poverty, improve food security, reduce
malnutrition and improve livelihood of the rural
and the urban poor.
- Persley, 1999
5. Modification of an organism’s DNA in order
to achieve a desired trait.
01/01/2014
SAMI
5
What are genetically modified organisms (GMO) ?
+ =
Arctic fish DNA Tomato
A tomato resistant to
frost
What is transgenic?
http://en.wikipedia.org/wiki/Fish_tomato
6. 01/01/2014 SAMI 6
Brief history of transgenics
ISAAA, 2011
1985
1st
transgenic
plants
produced.
1994
Flavr-Savr
tomato is
released
1996
Herbicide-
and insect-
resistant
crops
approved
for
cultivation
2000
Golden rice
with
ß-carotene
developed
2011
160 million
ha. of GM
crops
planted.
GM crops
considered
substantially
equivalent to
hybrid
varieties
1992
8. Conventional Breeding Transgenesis
Current cultivar Wild relative
GOI
Unwanted
genes
Back cross
F1 generation
F2 generation
Many backcrosses
New Cultivar
Unwanted
Genes
GOI
Current
Cultivar
Transfer in to
plasmid
agrobacterium
GOI
Genes
required
for transfer
& marker
genes
New Cultivar
Unrelated organism
Genes
required
for transfer
& marker
genes
GOI
01/01/2014 SAMI 8
9. TOMATO
• Most common vegetable
• Indian meals incomplete without tomato.
• Botanical name Solanum lycopersicum L.
• Chromosome number 2n = 24
• Well distributed throughout the world
• One among the top five vegetables
01/01/2014 SAMI 9
Area Production Productivity(t/ha)
INDIA 8.65 lakh ha. 16.82 lakh tons 19.5
KARNATAKA 51.2 thousand
ha.
1756.7 thousand
tons
19.5
Anon 2012
10. Source
– NBPGR, NCBI etc.,
Vector Construction
– Plasmid, cosmid, bacteriphage, virus etc.,
Transformations
– Microinjection, Gene gun, Electrophoretion, Agrobacterium, etc.,
Selection of transgenic plant (Antibiotic containing media)
Elimination of marker and backbone (through DEX media)
Confirmation (through PCR)
Expression
– Southern blotting, RT PCR, ELISA
01/01/2014 SAMI 10
12. Microinjection
Biolistics - gene gun/
particle bombardment
Electroporation
Silicon carbide fibers
PEG
DEAE-dextran
Calcium
phosphate
A. tumefaciens
A. rhizogenes
Virus-mediated
Pollen
transformation
Meristem
transformation
Singh B. D., 2009
01/01/2014 SAMI 12
Methods of plant transformation
PHYSICAL CHEMICAL BIOLOGICAL IN PLANTA
13. Agrobacterium mediated transformation
• Most extensively used method
• Natural ability of Agrobacterium to transfer plants
• First report: McCormick et al.(1986)
• Cotyledons/leaves are used for transformation
• A. tumefaciens and A. rhizogenes
01/01/2014 SAMI 13
16. Simple and efficient Agrobacterium mediated
procedure for transformation of Tomato
01/01/2014 SAMI 16
Bacterial strain used : A. tumefaciens strain AGL 1
Growth conditions-
Growth room temperature : 28 10C
Culturing of Agrobacterium : Rotary shaker (280C) for 72hrs at 200 rpm
Explants : Cotyledons from 10 day old plants
Size of explant : 0.7 cm x 1.0 cm
Varieties studied : Pusa Ruby, Arka Vikas and Sioux
Sharma et al., 2009, New Delhi
17. Figure 1 : Vectors used for transformation of Tomato using A. tumefaciens
strain AGL 1
(Sharma et al., 2009)
01/01/2014 SAMI 17
18. Plate 1: Different stages of Solanum lycopersicum var. Pusa Ruby
transformation
01/01/2014 SAMI 18
(Sharma et al., 2009)
19. Table 1: Effect of bacterial concentration and co-cultivation
period on transformation of Pusa Ruby
(Sharma et al., 2009)
01/01/2014 SAMI 19
Bacterial
concentration
No. of
explants
Averaga percent transformation
efficeincy + SE
72 h
cocultivation
96 h
cocultivation
0.5 X 108 70 20.7 + 1.01 22.1 + 1.01
1.0 X 108 70 41.4 + 2.02 24.3 + 2.02
2.0 X 108 70 38.6 + 2.02 19.3 + 3.03
5.0 X 108 70 36.4 + 1.01 17.9 + 1.01
10 X 108 70 12.9 + 2.02 -
20. Table 2: Transformation efficiency of different Tomato varieties
using optimized protocol
(Sharma et al., 2009)
01/01/2014 SAMI 20
Vector used Tomato
variety
Average No. of
explants co-
cultivated
Average transformation
efficiency (% + SE)
pCTBE2L Pusa Ruby 174 40.5 + 0.80
pRINASE2L Pusa Ruby 163 41.1 + 0.89
pCTBE2L Sioux 150 41.0 + 1.00
pCTBE2L Arka Vikas 150 22.0 + 1.50
21. Biolistic gun
• Most successfully applied
• Also known as ‘Particle bumbardment’
• Principle: High velocity discharge of DNA
‘bullets’ coated with gold or nickel particles
• Non specificity of species
• Gene integration is random
• Low rate of stable transformation (10-5)
01/01/2014 SAMI 21
25. Tomato transformation using biolistic gun
• Intoduced gene : β glucuronidase (gusA)
• Genotype : IPA-3
• Explants : Shoot tips, hypocotyls and
cotyledon
• Particle gun used : Bio-Rad Gun
• TC media : MS + BAP (2.0 mg/l) + Kinetin
(1.0 mg/l)
01/01/2014 SAMI 25
Ruma et al., 2009, Ludhiana
26. Source df
Mean sum of squares
Target
distance
DNA
quantity
Pre-
bumbardment
culture
Post-
bumbardment
culture
Explant 2 210.71* 9.66 45.29* 8.24*
Parameters 3 1457.75* 1508.98* 772.85* 548.94*
Explant x
Parameters
6 37.12* 52.72* 1207.10* 3.21
Error 24 7.04 14.78 12.25 26.42
Table 3 : ANOVA for GUS expression as affected by different
parameters of particle gun
(Ruma et al., 2009)
Figure 2: Effect of target
distance on
transformation
01/01/2014 SAMI 26
27. Source df
Mean sum of squares
Target
distance
DNA
quantity
Pre-
bumbardment
culture
Post-
bumbardment
culture
Explant 2 210.71* 9.66 45.29* 8.24*
Parameters 3 1457.75* 1508.98* 772.85* 548.94*
Explant x
Parameters
6 37.12* 52.72* 1207.10* 3.21
Error 24 7.04 14.78 12.25 26.42
Table 4: ANOVA for GUS expression as affected by different parameters of
particle gun
(Ruma et al., 2009)
Figure 3: Effect of DNA quantity
on transformation
01/01/2014 27SAMI
28. Source df
Mean sum of squares
Target
distance
DNA
quantity
Pre-
bumbardment
culture
Post-
bumbardment
culture
Explant 2 210.71* 9.66 45.29* 8.24*
Parameters 3 1457.75* 1508.98* 772.85* 548.94*
Explant x
Parameters
6 37.12* 52.72* 1207.10* 3.21
Error 24 7.04 14.78 12.25 26.42
Table 5: ANOVA for GUS expression as affected by different parameters
of particle gun
(Ruma et al., 2009)
Figure 4: Effect of pre-bumbardment
culture on transformation
01/01/2014 SAMI 28
29. Source df
Mean sum of squares
Target
distance
DNA
quantity
Pre-
bumbardment
culture
Post-
bumbardment
culture
Explant 2 210.71* 9.66 45.29* 8.24*
Parameters 3 1457.75* 1508.98* 772.85* 548.94*
Explant x
Parameters
6 37.12* 52.72* 1207.10* 3.21
Error 24 7.04 14.78 12.25 26.42
Table 6: ANOVA for GUS expression as affected by different
parameters of particle gun
(Ruma et al., 2009)
Figure 5: Effect of post bumbardment
culture on transformation
01/01/2014 SAMI 29
30. Plate 2: Different explants showing GUS expresion
a. Cotyledons, b. Shoot tips, c. Hypocotyls, d. Leaf, e. Callus, f. Root like
structures
01/01/2014 SAMI 30
32. 01/01/2014 SAMI 32
Does not require tissue culture and plant
regeneration
Produces genetically uniform progeny
Not expensive
Genotype independent – monocots/dicots
Utilizes normal fertilization process
Benefits of pollen transformation
33. 01/01/2014 SAMI 33
Bacterial strain used: A. tumefaciens strain
LBA4404
Growth conditions:
Growth room temperature : 28 ± 10C
Culturing medium : Yeast Extract Mannitol Agar
+ Streptomycin (100 µg/ml) +
Rifampicin (25 µg/ml) +
Kanamycin (50 µg/ml)
In vitro manipulation of pollen for
transformation in Tomato
Satish, 2009, Dharwad
38. Engineering herbicidal resistance in plants by
expression of detoxifying enzyme
01/01/2014 SAMI 38
Block et al., 2007, England
Experimental details:
Bacterial strain used : A. tumefaciens strain C58C1 Rif
Variety used : Petit Havana
Explant : Leaf disc
Media (5.7 pH) : ½ MS + Sucrose (1%) + Agar
(0.8%)
Gene : bar gene
Mechanism : PAT (Phosphinothricin
acetyltransferace)
detoxification
39. Figure 6: Ammonia determination (% basal NH4
+ content) after
spraying Basta® (20 l/ha)
(Block et al., 2007)
01/01/2014 SAMI 39
42. table 9: Different versions of the Cry genes effective
against different orders of insects
Cry gene designation
Toxic to these insect
orders
CryIA(a), CryIA(b), CryIA(c) Lepidoptera
Cry1B, Cry1C, Cry1D Lepidoptera
CryII Lepidoptera, Diptera
CryIII Coleoptera
CryIV Diptera
CryV Lepidoptera, Coleoptera
01/01/2014 SAMI 42
Byrne et al., 2002
43. Experimental details:
Bacterial strain used : A. tumefaciens strain LBA 4404
Variety used : Pusa Ruby
Explant : Cotyledonary leaves from 10-day old
seedlings
Media (5.7 pH) : ½ MS + Sucrose (1%) + Agar (0.8%)
Gene : cry1Ac gene
01/01/2014 SAMI 43
Transgenic tomato plants resistant to fruit borer
(Helicoverpa armigera Hubner)
Mandaokar et al., 2000, New Delhi
44. 01/01/2014 SAMI 44
Mandaokar et al., 2000, New Delhi
Table 10: Expression of Cry 1 Ac protein in transformed Pusa
Ruby
Plant no. Cry1Ac content(% of total
soluble protien)
Insect mortality (%)
Leaf Fruit
Control 0.00 0 0
Bt1 0.26 + 0.07 80 50
Bt2 0.10 + 0.03 100 100
Bt3 0.11 + 0.03 100 100
Bt4 0.04 + 0.005 100 100
Bt5 0.11 + 0.04 60 80
Bt6 0.42 + 0.09 100 100
Bt7 0.60 + 0.05 100 100
45. Experimental details:
Bacterial strain used : A. tumefaciens LBA 4402
Variety used : Italian tomato genotype LEPA
Explant : Cotyledonary leaves from 10-day old seedlings
Media (5.7 pH) : ½ MS + Sucrose (1%) + Agar (0.8%)
Gene : cry1Ab gene
01/01/2014 SAMI 45
Tomato expressing Cry1A(b) insecticidal protein protected
against tomato fruit borer, damage in the laboratory and
green house
Harish Kumar*, Vinod Kumar, 2003, Gurgaon
46. 01/01/2014 SAMI 46
Plate 4: Damage caused by H. armigera to the fruit of non-transgenic and
transgenic tomato plants in the laboratory.
Harish Kumar*, Vinod Kumar, 2003, Gurgaon
47. 01/01/2014 SAMI 47
Harish Kumar*, Vinod Kumar, 2003, Gurgaon
Plate 5: Extent of damage caused by larvae of H. armigera to transgenic Bt and
non transgenic tomato plants in the green house
49. 01/01/2014 SAMI 49
Experimental details-
Bacterial strain used : A. tumefaciens SRC 1102
Explant : young leaf discs
Gene : Pn-AMPs
Source : Pharbitis nil
Hevein-like proteins from pharbitis nil confers disease
resistance against phytopathogenic fungi in tomato
Lee et al, 2003
50. Figure 7: Physical map of binary vector Pn-AMP2643
used for transformation
01/01/2014 SAMI 50
Lee et al., 2003
51. Plate 6: Transgenic tomato conforming resistance against F. oxysporum
transformed with pnAMPs gene.
01/01/2014 SAMI 51
Lee et al., 2003
52. Evaluation of transgenic Tomato plants against CMV strain
WL under field conditions
01/01/2014 SAMI 52
Fuchs et al., 1996, Geneva
Experimental details-
Bacterial strain used : A. tumefaciens strain C58Z707
Variety used : TT5-007-11, TT5-007-11 x Solarset (Hy)
Explant : 7 day old cotyledon sections (0.2 x 0.5 cm)
Media (5.7 pH) : MS + B5 Vit + BA (2.5 mg/l) + IAA (1.0 mg/l) +
Agar (0.8 %)
Gene : CMV-WL CP gene
53. Table 11: Evaluation of transgenic tomato lines for resistance
under field conditions
(Fuchs et al., 1996)
01/01/2014 SAMI 53
Lines/hybrids Infected/tested Infection (%)
Transgenic homozygous
TT5-007-11
0/176 0
Control G80 65/176 37
Transgenic TT5-007-11 X
Solarset hybrid
0/22 0
Control solarset 17/22 77
54. Plate 7: Disease reactions TT5-007-11 and G-80
(Fuchs et al., 1996)
01/01/2014 SAMI 54
55. Table 12: Performance of transformed and non-
transformed lines
(Fuchs et al., 1996)
01/01/2014 SAMI 55
Line No. of
plants
tested
Average
Plant
height
Plants
with
fruits
(%)
Average
No. of
fruits/plant
Average
Yield
(g)/plant
Average
Fruit
wieght
(g)
Transgenic
TT5-007-11
176 64 + 12 92 19 +11 1840 97
Non transformed
G-80
65 47 + 11 56 2 + 3 108 54
56. Virus Method Workers
Spotted wilt
virus
RNA mediated Goldbatch et al. (2003)
Yellow leaf curl Rep Protein
Brunetti et al. (1997); Antignus
et al. (2007)
Goldenn
mosaic virus
Sense and
antisense RNA
Day et al. (1991)
TMV
N gene from
Tobacco
Whitham et al. (1996)
Bushy Stunt
Virus
ScFvs Boonrod et al. (2000)
Table 13: Reports on development of transgenic tomato
plants for virus resistance
(Prins et al., 2007)
01/01/2014 SAMI 56
58. LeERF1 improves tolerance to drought in
Tomato
01/01/2014 SAMI 58
Lu et al., 2010, Beijing
Experimental details:
Variety used : Zhongshu No.4
Explant : 7 day old cotyledon sections (0.2 x 0.5 cm)
Gene : LeERF1 gene
59. Plate 8: Evaluation of drought tolerance in 4 week old seedlings
by withholding water for 10 days
Control
Drought
stress
Non-
transfered
Sense
LeERF1
Antisense
LeERF1
(Lu et al., 2010)01/01/2014 SAMI 59
60. (Lu et al., 2010)
Figure 9: proline content on water stress
01/01/2014 SAMI 60
61. 01/01/2014 SAMI 61
Experimental details:
Bacterial strain used : A. tumefaciens
Variety used : Micro tom
Explant : 7 day old cotyledon
Gene : betaine aldehyde dehydrogenase
(BADH) gene,
Salt-inducible expression of SIBADH gene in transgenic
tomato enhances salt tolerance
Wang et al, 2013
62. 01/01/2014 SAMI 62
(A) Under the non-stress condition (B) After 7 days of salt
stress by treatment with 200 mM NaCl,
Wang et al, 2013
64. Suppression of ACC oxidase expression in
Tomato using heterologus gene from Banana
01/01/2014 SAMI 64
Batra et al., 2010, Lucknow
Experimental details:
Variety used : Ailsa craig
Explants : Cotyledons
Source of gene : Banana
Gene : MaCO gene
Method : Particle bombardment
65. Plate 10: On vine (a) and post harvest (b) developmental stages of non-
transformed and transformed tomatoes
a
b
(Batra et al., 2010)01/01/2014 SAMI 65
66. The ‘FLAVR SAVR
TM
’ affair
First GM whole food to be sold in public market
FDA approved on 18 May, 1994 & sale begun on 21st
May
Technology: Antisense RNA to regulate the
expression of polygalacturunase (PG)
Developed by Calgene company, Davis, California
01/01/2014 SAMI 66
67. ‘FLAVR SAVR
TM
’ Contd..
Stay ‘ripe’ but ‘not rot’
For 10 days
No refrigeration needed
01/01/2014 SAMI 67
68. Table 14: Comparison between FLAVR SAVR and control
line for nutritional values
01/01/2014 SAMI 68
70. Trait Gene Stage
Year of
completion
Salinity, Drought, Cold
ect.
Mannitol-1-phospate
dehydrogenase
T4 2014
Shelf life Arginine decarboxylase T3 2014
Male sterility i-Ornithine decarboxylase T3 2015
Salinity Glyoxalsae I & II T3 2015
Fungus, Nematode i-Ornithine decarboxylase T3 2015
Fungus, Nematode i-Chitin synthase T3 2015
Fungus Afp-ca T3 2015
Rakesh, 2012
Table 15: List of on-going transgenic work in India through
ICAR
01/01/2014 SAMI 70
71. Regulatory bodies in India
1. Institutional Biosafety Committees (IBSC)
2. Recombinant DNA Advisory Committee (RDAC)
3. Review Committee on Genetic Manipulation (RCGM)
4. Genetic Engineering Approval Committee (GEAC)
5. State Biosafety Coordination Committees (SBCC)
01/01/2014 SAMI 71
73. Take home message
01/01/2014 SAMI 73
SAFETY: GM foods and crops are as safe
as conventional ones
REGULATION: Highly regulated. Approval
process requires many tests and years
ENVIRONMENT: Their is no evidence
74. 01/01/2014 SAMI 74
ENVIRONMENTAL BENEFITS: Require
less pesticides, less tillage.
BETTER NUTRITION: futuristic GM crops
FARMERS: want GM crops because crop
production is cheaper.
OPPONENTS OF GM CROPS: have not
brought forth scientific evidence to backup
their claims.
75. Conventional breeding for traits
is unpredictable
Use of biotech tools will allow us
to go from unpredictable to
predictable engineering
01/01/2014 SAMI 75