01/01/2014 SAMI 1
PLANT
BREEDING
SELECTION
HYBRIDIZATION
INTRODUCTION
MUTATION
TISSUE
CULTURE
GENETIC
ENGINEERING
transgen...
UNIVERSITY OF AGRICULTURAL AND HORTICULTURAL SCIENCES, SHIMOGA.
COLLEGE OF HORTICULTURE, MUDIGERE.
 Introduction
 Transformation methods
 Practical application
 Herbicidal, Pest, Disease,
Drought – Tolerance, and
Qual...
Introduction
01/01/2014 SAMI 4
Biotechnology is one of the tools , but a
potentially important one, in the struggle to
red...
 Modification of an organism’s DNA in order
to achieve a desired trait.
01/01/2014
SAMI
5
What are genetically modified o...
01/01/2014 SAMI 6
Brief history of transgenics
ISAAA, 2011
1985
1st
transgenic
plants
produced.
1994
Flavr-Savr
tomato is
...
01/01/2014 SAMI 7
How transgenic breeding
differs from conventional
breeding?
Conventional Breeding Transgenesis
Current cultivar Wild relative
GOI
Unwanted
genes
Back cross
F1 generation
F2 generatio...
TOMATO
• Most common vegetable
• Indian meals incomplete without tomato.
• Botanical name Solanum lycopersicum L.
• Chromo...
Source
– NBPGR, NCBI etc.,
Vector Construction
– Plasmid, cosmid, bacteriphage, virus etc.,
Transformations
– Microinjecti...
01/01/2014 SAMI 11
Transformation Methods
Microinjection
Biolistics - gene gun/
particle bombardment
Electroporation
Silicon carbide fibers
PEG
DEAE-dextran
Calcium...
Agrobacterium mediated transformation
• Most extensively used method
• Natural ability of Agrobacterium to transfer plants...
Tomato transformation mediated by
Agrobacterium
01/01/2014 SAMI 14
01/01/2014 SAMI 15
Simple and efficient Agrobacterium mediated
procedure for transformation of Tomato
01/01/2014 SAMI 16
Bacterial strain use...
Figure 1 : Vectors used for transformation of Tomato using A. tumefaciens
strain AGL 1
(Sharma et al., 2009)
01/01/2014 SA...
Plate 1: Different stages of Solanum lycopersicum var. Pusa Ruby
transformation
01/01/2014 SAMI 18
(Sharma et al., 2009)
Table 1: Effect of bacterial concentration and co-cultivation
period on transformation of Pusa Ruby
(Sharma et al., 2009)
...
Table 2: Transformation efficiency of different Tomato varieties
using optimized protocol
(Sharma et al., 2009)
01/01/2014...
Biolistic gun
• Most successfully applied
• Also known as ‘Particle bumbardment’
• Principle: High velocity discharge of D...
Plate 2 : Gene gun
01/01/2014 SAMI 22
01/01/2014 SAMI 23
01/01/2014 SAMI 24
Tomato transformation using biolistic gun
• Intoduced gene : β glucuronidase (gusA)
• Genotype : IPA-3
• Explants : Shoot ...
Source df
Mean sum of squares
Target
distance
DNA
quantity
Pre-
bumbardment
culture
Post-
bumbardment
culture
Explant 2 21...
Source df
Mean sum of squares
Target
distance
DNA
quantity
Pre-
bumbardment
culture
Post-
bumbardment
culture
Explant 2 21...
Source df
Mean sum of squares
Target
distance
DNA
quantity
Pre-
bumbardment
culture
Post-
bumbardment
culture
Explant 2 21...
Source df
Mean sum of squares
Target
distance
DNA
quantity
Pre-
bumbardment
culture
Post-
bumbardment
culture
Explant 2 21...
Plate 2: Different explants showing GUS expresion
a. Cotyledons, b. Shoot tips, c. Hypocotyls, d. Leaf, e. Callus, f. Root...
Pollen mediated transformation of
Tomato
01/01/2014 SAMI 31
01/01/2014 SAMI 32
Does not require tissue culture and plant
regeneration
Produces genetically uniform progeny
Not expe...
01/01/2014 SAMI 33
Bacterial strain used: A. tumefaciens strain
LBA4404
Growth conditions:
Growth room temperature : 28 ± ...
(Satish, 2009)
01/01/2014 SAMI 34
Plate 3: Vector used for transformation
of pollen of Tomato
01/01/2014 SAMI 35
Table 7: In vitro pollen germination medium for Tomato (Pusa Ruby)
(Satish, 2009)
Medium Germination (%...
01/01/2014 SAMI 36
Herbicidal tolerance
Pest tolerance
Disease tolerance
Abiotic stress tolerance
Quality improvement
01/01/2014 SAMI 37
Engineering herbicidal resistance in plants by
expression of detoxifying enzyme
01/01/2014 SAMI 38
Block et al., 2007, Eng...
Figure 6: Ammonia determination (% basal NH4
+ content) after
spraying Basta® (20 l/ha)
(Block et al., 2007)
01/01/2014 SA...
Class of
herbicide
Compound/
Herbicide
Transgene/
Mechanism
Company
Phosphonic
acid
Phoshanothricin
(Basta®
, Liberty®
)
b...
Genetic manipulation for pest tolerance
01/01/2014 SAMI 41
table 9: Different versions of the Cry genes effective
against different orders of insects
Cry gene designation
Toxic to t...
Experimental details:
Bacterial strain used : A. tumefaciens strain LBA 4404
Variety used : Pusa Ruby
Explant : Cotyledona...
01/01/2014 SAMI 44
Mandaokar et al., 2000, New Delhi
Table 10: Expression of Cry 1 Ac protein in transformed Pusa
Ruby
Pla...
Experimental details:
Bacterial strain used : A. tumefaciens LBA 4402
Variety used : Italian tomato genotype LEPA
Explant ...
01/01/2014 SAMI 46
Plate 4: Damage caused by H. armigera to the fruit of non-transgenic and
transgenic tomato plants in th...
01/01/2014 SAMI 47
Harish Kumar*, Vinod Kumar, 2003, Gurgaon
Plate 5: Extent of damage caused by larvae of H. armigera to ...
Genetic manipulation for disease tolerance
01/01/2014 SAMI 48
01/01/2014 SAMI 49
Experimental details-
Bacterial strain used : A. tumefaciens SRC 1102
Explant : young leaf discs
Gene :...
Figure 7: Physical map of binary vector Pn-AMP2643
used for transformation
01/01/2014 SAMI 50
Lee et al., 2003
Plate 6: Transgenic tomato conforming resistance against F. oxysporum
transformed with pnAMPs gene.
01/01/2014 SAMI 51
Lee...
Evaluation of transgenic Tomato plants against CMV strain
WL under field conditions
01/01/2014 SAMI 52
Fuchs et al., 1996,...
Table 11: Evaluation of transgenic tomato lines for resistance
under field conditions
(Fuchs et al., 1996)
01/01/2014 SAMI...
Plate 7: Disease reactions TT5-007-11 and G-80
(Fuchs et al., 1996)
01/01/2014 SAMI 54
Table 12: Performance of transformed and non-
transformed lines
(Fuchs et al., 1996)
01/01/2014 SAMI 55
Line No. of
plants...
Virus Method Workers
Spotted wilt
virus
RNA mediated Goldbatch et al. (2003)
Yellow leaf curl Rep Protein
Brunetti et al. ...
Genetic manipulation for abiotic stress
tolerance
01/01/2014 SAMI 57
LeERF1 improves tolerance to drought in
Tomato
01/01/2014 SAMI 58
Lu et al., 2010, Beijing
Experimental details:
Variety u...
Plate 8: Evaluation of drought tolerance in 4 week old seedlings
by withholding water for 10 days
Control
Drought
stress
N...
(Lu et al., 2010)
Figure 9: proline content on water stress
01/01/2014 SAMI 60
01/01/2014 SAMI 61
Experimental details:
Bacterial strain used : A. tumefaciens
Variety used : Micro tom
Explant : 7 day o...
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 ...
Genetic manipulation for quality
improvement
01/01/2014 SAMI 63
Suppression of ACC oxidase expression in
Tomato using heterologus gene from Banana
01/01/2014 SAMI 64
Batra et al., 2010, ...
Plate 10: On vine (a) and post harvest (b) developmental stages of non-
transformed and transformed tomatoes
a
b
(Batra et...
The ‘FLAVR SAVR
TM
’ affair
 First GM whole food to be sold in public market
 FDA approved on 18 May, 1994 & sale begun ...
‘FLAVR SAVR
TM
’ Contd..
 Stay ‘ripe’ but ‘not rot’
 For 10 days
 No refrigeration needed
01/01/2014 SAMI 67
Table 14: Comparison between FLAVR SAVR and control
line for nutritional values
01/01/2014 SAMI 68
Transgenic Tomato Researches in India
01/01/2014 SAMI 69
Trait Gene Stage
Year of
completion
Salinity, Drought, Cold
ect.
Mannitol-1-phospate
dehydrogenase
T4 2014
Shelf life Argi...
Regulatory bodies in India
1. Institutional Biosafety Committees (IBSC)
2. Recombinant DNA Advisory Committee (RDAC)
3. Re...
Some news clippings
01/01/2014 SAMI 72
Take home message
01/01/2014 SAMI 73
SAFETY: GM foods and crops are as safe
as conventional ones
REGULATION: Highly regula...
01/01/2014 SAMI 74
ENVIRONMENTAL BENEFITS: Require
less pesticides, less tillage.
BETTER NUTRITION: futuristic GM crops
FA...
Conventional breeding for traits
is unpredictable
Use of biotech tools will allow us
to go from unpredictable to
predictab...
01/01/2014 SAMI 76
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Transgenics - a biotech approach for improvement of tomato (tomato breeding)

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Transgenics - a biotech approach for improvement of tomato (tomato breeding)

  1. 1. 01/01/2014 SAMI 1 PLANT BREEDING SELECTION HYBRIDIZATION INTRODUCTION MUTATION TISSUE CULTURE GENETIC ENGINEERING transgenics
  2. 2. UNIVERSITY OF AGRICULTURAL AND HORTICULTURAL SCIENCES, SHIMOGA. COLLEGE OF HORTICULTURE, MUDIGERE.
  3. 3.  Introduction  Transformation methods  Practical application  Herbicidal, Pest, Disease, Drought – Tolerance, and Quality  Indian research  Conclusion 01/01/2014 SAMI 3
  4. 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. 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. 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
  7. 7. 01/01/2014 SAMI 7 How transgenic breeding differs from conventional breeding?
  8. 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. 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. 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
  11. 11. 01/01/2014 SAMI 11 Transformation Methods
  12. 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. 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
  14. 14. Tomato transformation mediated by Agrobacterium 01/01/2014 SAMI 14
  15. 15. 01/01/2014 SAMI 15
  16. 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. 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. 18. Plate 1: Different stages of Solanum lycopersicum var. Pusa Ruby transformation 01/01/2014 SAMI 18 (Sharma et al., 2009)
  19. 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. 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. 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
  22. 22. Plate 2 : Gene gun 01/01/2014 SAMI 22
  23. 23. 01/01/2014 SAMI 23
  24. 24. 01/01/2014 SAMI 24
  25. 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. 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. 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. 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. 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. 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
  31. 31. Pollen mediated transformation of Tomato 01/01/2014 SAMI 31
  32. 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. 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
  34. 34. (Satish, 2009) 01/01/2014 SAMI 34 Plate 3: Vector used for transformation of pollen of Tomato
  35. 35. 01/01/2014 SAMI 35 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)
  36. 36. 01/01/2014 SAMI 36
  37. 37. Herbicidal tolerance Pest tolerance Disease tolerance Abiotic stress tolerance Quality improvement 01/01/2014 SAMI 37
  38. 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. 39. Figure 6: Ammonia determination (% basal NH4 + content) after spraying Basta® (20 l/ha) (Block et al., 2007) 01/01/2014 SAMI 39
  40. 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) 01/01/2014 SAMI 40
  41. 41. Genetic manipulation for pest tolerance 01/01/2014 SAMI 41
  42. 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. 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. 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. 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. 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. 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
  48. 48. Genetic manipulation for disease tolerance 01/01/2014 SAMI 48
  49. 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. 50. Figure 7: Physical map of binary vector Pn-AMP2643 used for transformation 01/01/2014 SAMI 50 Lee et al., 2003
  51. 51. Plate 6: Transgenic tomato conforming resistance against F. oxysporum transformed with pnAMPs gene. 01/01/2014 SAMI 51 Lee et al., 2003
  52. 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. 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. 54. Plate 7: Disease reactions TT5-007-11 and G-80 (Fuchs et al., 1996) 01/01/2014 SAMI 54
  55. 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. 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
  57. 57. Genetic manipulation for abiotic stress tolerance 01/01/2014 SAMI 57
  58. 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. 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. 60. (Lu et al., 2010) Figure 9: proline content on water stress 01/01/2014 SAMI 60
  61. 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. 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
  63. 63. Genetic manipulation for quality improvement 01/01/2014 SAMI 63
  64. 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. 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. 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. 67. ‘FLAVR SAVR TM ’ Contd..  Stay ‘ripe’ but ‘not rot’  For 10 days  No refrigeration needed 01/01/2014 SAMI 67
  68. 68. Table 14: Comparison between FLAVR SAVR and control line for nutritional values 01/01/2014 SAMI 68
  69. 69. Transgenic Tomato Researches in India 01/01/2014 SAMI 69
  70. 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. 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
  72. 72. Some news clippings 01/01/2014 SAMI 72
  73. 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. 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. 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
  76. 76. 01/01/2014 SAMI 76

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