The ppt provide information about basic knowledge about plant genetic transformation & crop improvement in tomato by use of biotechnology.

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PLANT
BREEDING
SELECTION
HYBRIDIZATION
INTRODUCTION
MUTATION
TISSUE
CULTURE
GENETIC
ENGINEERING
transgenics

2. UNIVERSITY OF AGRICULTURAL AND HORTICULTURAL SCIENCES, SHIMOGA.
COLLEGE OF HORTICULTURE, MUDIGERE.

3.  Introduction
 Transformation methods
 Practical application
 Herbicidal, Pest, Disease,
Drought – Tolerance, and
Quality
 Indian research
 Conclusion
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4. Introduction
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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.
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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

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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

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How transgenic breeding
differs from conventional
breeding?

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
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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
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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
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Transformation Methods

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
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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
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14. Tomato transformation mediated by
Agrobacterium
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16. Simple and efficient Agrobacterium mediated
procedure for transformation of Tomato
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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)
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18. Plate 1: Different stages of Solanum lycopersicum var. Pusa Ruby
transformation
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(Sharma et al., 2009)

19. Table 1: Effect of bacterial concentration and co-cultivation
period on transformation of Pusa Ruby
(Sharma et al., 2009)
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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)
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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)
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22. Plate 2 : Gene gun
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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)
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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
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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
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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
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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
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30. Plate 2: Different explants showing GUS expresion
a. Cotyledons, b. Shoot tips, c. Hypocotyls, d. Leaf, e. Callus, f. Root like
structures
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31. Pollen mediated transformation of
Tomato
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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

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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

34. (Satish, 2009)
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Plate 3: Vector used for transformation
of pollen of Tomato

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Table 7: In vitro pollen germination medium for Tomato (Pusa Ruby)
(Satish, 2009)
Medium Germination (%) Tube length (m)
PGM-1 32.00c 190c
PGM-2 46.80b 213b
PGM-3 92.96a 281a
CD @1% 1.62 1.00
Media constitution:
PGM-1: Sucrose (18%) + Agarose (1%) + Boric acid (0.015%)
PGM-2: Sucrose (16%) + Boric acid (0.6mM) + CaNO3 (0.8mM) +
MgSO4 (0.5mM)
PGM-3: Sucrose (10%) + Boric acid (100mg/l) +GA (125µM)

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37. Herbicidal tolerance
Pest tolerance
Disease tolerance
Abiotic stress tolerance
Quality improvement
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38. Engineering herbicidal resistance in plants by
expression of detoxifying enzyme
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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)
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40. Class of
herbicide
Compound/
Herbicide
Transgene/
Mechanism
Company
Phosphonic
acid
Phoshanothricin
(Basta®
, Liberty®
)
bar gene/ PAT
detoxification
Syngenta
Sulphonylurea
Chlorosulphuron
(Glean®
)
Mutant plant/
acetolactate
synthase
Dupont-
Poineer-Hi-
Bred
Nitriles
Bromoxynil
(Buctril®
)
Nitrilase
detoxification
Calgene/
Bayer crop
science
(Slater, 2010)
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41. Genetic manipulation for pest tolerance
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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
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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
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Transgenic tomato plants resistant to fruit borer
(Helicoverpa armigera Hubner)
Mandaokar et al., 2000, New Delhi

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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
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Tomato expressing Cry1A(b) insecticidal protein protected
against tomato fruit borer, damage in the laboratory and
green house
Harish Kumar*, Vinod Kumar, 2003, Gurgaon

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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

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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

48. Genetic manipulation for disease tolerance
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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
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Lee et al., 2003

51. Plate 6: Transgenic tomato conforming resistance against F. oxysporum
transformed with pnAMPs gene.
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Lee et al., 2003

52. Evaluation of transgenic Tomato plants against CMV strain
WL under field conditions
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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)
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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)
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55. Table 12: Performance of transformed and non-
transformed lines
(Fuchs et al., 1996)
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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)
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57. Genetic manipulation for abiotic stress
tolerance
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58. LeERF1 improves tolerance to drought in
Tomato
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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
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60. (Lu et al., 2010)
Figure 9: proline content on water stress
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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

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(A) Under the non-stress condition (B) After 7 days of salt
stress by treatment with 200 mM NaCl,
Wang et al, 2013

63. Genetic manipulation for quality
improvement
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64. Suppression of ACC oxidase expression in
Tomato using heterologus gene from Banana
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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
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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
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67. ‘FLAVR SAVR
TM
’ Contd..
 Stay ‘ripe’ but ‘not rot’
 For 10 days
 No refrigeration needed
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68. Table 14: Comparison between FLAVR SAVR and control
line for nutritional values
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69. Transgenic Tomato Researches in India
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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
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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)
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72. Some news clippings
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73. Take home message
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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

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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
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