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RIDDHI KARNIK
 Was under trial and error for almost 9900 years.
 The first genetically modified plant was produced in 1982,
using an antibiotic-resistant tobacco plant.
 The first genetically modified crop approved for sale in the
U.S., in 1994, was the FlavrSavr tomato, which had a
longer shelf life, as it took longer to soften after ripening.
 As of mid-1996, a total of 35 approvals had been granted
to commercially grow 8 transgenic crops and one flower
crop of carnations, with 8 different traits in 6 countries
plus the EU. In 2000, with the production of golden rice,
scientists genetically modified food to increase its
nutrient value for the first time.
HISTORY
• To improve the agricultural, horticultural or ornamental
value of a crop plant
• To serve as a bioreactor for the production of economically
important proteins or metabolites
• To provide a powerful means for studying the action of
genes (and gene products) during development and other
biological processes
WHY GENETICALLY ENGINEER PLANTS?
Applications of Plant Genetic
Engineering
A.Crop Improvement
B.Genetically Engineered Traits: The Big Six
1.Herbicide Resistance
2.Insect Resistance
3.Virus Resistance
4.Altered Oil Content
5.Delayed Fruit Ripening
6.Pollen Control
C.Biotech Revolution: Cold and Drought
Tolerance and Weather-Gaurd Genes
D.Genetically Engineered Foods
1.Soybeans
2.Corn
3.Cotton
4.Other Crops
An Overview of the Crop Genetic Engineering cycle
Leaf Disc Method for A. t. Mediated Transformation
Leaf Disk Preparation Co-cultivation with Agrobacterium Selection for Transformation
Regeneration of Shoots
6
Genetic engineering techniques
applied to plants
METHOD SALIENT FEATURES
1.VECTOR MEDIATED GENE
TRANSFER
a. Agrobacterium mediated
gene transfer
b. Plant viral vectors
Very efficient but limited to a selected group of plants
Ineffective, hence not widely used
2.DIRECT OR VECTORLESS
DNA TRANSFER
a. Electroporation
b. Microprojectile
c. Liposome fusion
d. Silicon carbide fibres
Mostly confined to protoplasts that can be regenerated to
viable plants
Limited use only one cell can be microinjected at a time
Confined to protoplasts that can be regenerated into viable
whole plants
Requires regenerable cell suspensions
3 CHEMICAL METHODS
a. Polyethylene glycol
mediated
b.Diethylaminoethyl(DEAE)dext
ran- mediated
Confined to protoplasts. Regeneration of fertile plants is
frequently problematical
Very less results
 Herbicides are generally non-selective (killing both
weeds and crop plants) and must be applied before
the crop plants germinate
 Four potential ways to engineer herbicide resistant
plants
1. Inhibit uptake of the herbicide
2. Overproduce the herbicide-sensitive target protein
3. Reduce the ability of the herbicide-sensitive target
to bind to the herbicide
4. Give plants the ability to inactivate the herbicide
HERBICIDES AND HERBICIDE-RESISTANT
PLANTS
HERBICIDE-RESISTANT PLANTS:
REDUCING THE ABILITY OF THE HERBICIDE-SENSITIVE
TARGET TO BIND TO THE HERBICIDE
 Herbicide: Glyphosate (better known as Roundup)
 Resistance to Roundup (an inhibitor of the enzyme
EPSP involved in aromatic amino acid biosynthesis)
was obtained by finding a mutant version of EPSP
from E. coli that does not bind Roundup and
expressing it in plants (soybean, tobacco, petunia,
tomato, potato, and cotton)
 5-enolpyruvylshikimate-3-phosphate synthase
(EPSP) is a chloroplast enzyme in the shikimate
pathway and plays a key role in the synthesis of
aromatic amino acids such as tyrosine and
phenylalanine
 Genetic engineering here is more challenging;
however, some strategies are possible:
 Individually or in combination express pathogenesis-
related (PR) proteins, which include b1,3-glucanases,
chitinases, thaumatin-like proteins, and protease
inhibitors
 Overexpression of the NPR1 gene which encodes the
“master” regulatory protein for turning on the PR
protein genes
 Overproducing salicylic acid in plants by the addition
of two bacterial genes; SA activates the NPR1 gene
and thus results in production of PR proteins
FUNGUS- AND BACTERIUM-RESISTANT
PLANTS
Modification of plant nutritional
content: increasing the vitamin A
content of plants
• 124 million children
worldwide are deficient in
vitamin A, which leads to
death and blindness
• Mammals make vitamin A
from b-carotene, a common
carotenoid pigment normally
found in plant photosynthetic
membranes
• Here, the idea was to
engineer the b-carotene
pathway into rice
• The transgenic rice is yellow
or golden in color and is
called “golden rice”
*Expression of enzymes
of β-carotene pathway
in rice endosperm
*Amelioration of Vitamin
A deficiency
Edible Vaccines – Ongoing
Research Areas
Hepatitis B
Dental caries - Anti-tooth decay Ab
Autoimmune diabetes
Cholera
Rabies
HIV
Rhinovirus
Foot and Mouth
Enteritis virus
Malaria
Influenza
Cancer
EDIBLE VACCINES FROM PLANTS
Two strategies for production
1) Expression of foreign antigens in plant via stable
transformation
2) Delivery of vaccine epitopes via plant virus
(Mason and Arntzen, 1995)
Strategy for the production of candidate vaccine
antigens in plant tissues
e
RABIES VIRUS G PROTEIN IN TOMATO
• Gene linked to CaMV35S
promoter
• Introduced to tomato plants by
Agrobacterium- mediated
transformation
• Expression of recombinant
glycoprotein in leaves and fruits
• Protein localized in Golgi bodies,
vesicles and plasma lemma
Norwalk virus (cold virus) capsid protein in potato
and tobacco
• Causative agent for acute epidemic
gastroenteritis
• NVCP was fused to CaMV35S promoter
• Transformation by Agrobacterium
• Expression level: varies with plant
(
DEVELOPMENT OF STRESS- AND
SENESCENCE-TOLERANT PLANTS: GENETIC
ENGINEERING OF SALT-RESISTANT PLANTS
 Overexpression of
the gene encoding
a Na+/H+ antiport
protein which
transports Na+ into
the plant cell
vacuole
 This has been done
in Arabidopsis and
tomato plants
allowing them to
survive on 200 mM
salt (NaCl)
Frost Resistance
• Ice-minus bacteria
• Ice nucleation on plant surfaces caused by
bacteria that aid in protein-water coalescence 
forming ice crystals @ 0oC (320F)
• Ice-minus Pseudomonas syringae
• Modified by removing genes responsible for
crystal formation
• Sprayed onto plants
• Displaces wild type strains
• Protected to 23oF
• Dew freezes beyond this point
• Extends growth season
• First deliberate release experiment – Steven
Lindow – 1987- sprayed potatoes
Development of stress- and senescence-tolerant
plants: genetic engineering of flavorful tomatoes
Fruit ripening is a natural aging or senescence process that involves two
independent pathways, flavor development and fruit softening.
Typically, tomatoes are picked when they are not very ripe (i.e., hard and
green) to allow for safe shipping of the fruit.
Polygalacturonase is a plant enzyme that degrades pectins in plant cell
walls and contribute to fruit softening.
In order to allow tomatoes to ripen on the vine and still be hard enough
for safe shipping of the fruit, polygalacturonase gene expression
was inhibited by introduction of an antisense polygalacturonase
gene and created the first commercial genetically engineered
plant called the FLAVR SAVR tomato.
Flavor development pathway
Fruit softening pathway
Green Red
Hard Soft
polygalacturonaseantisense polygalacturonase
 Crop Organization Gene
 Brinjal IARI, New Delhi cr y1Ab, cr y1Ac
 MAHYCO, Mumbai
 Cauliflower MAHYCO, Mumbai cr y1Ac
Sungrow Seeds Ltd., New Delhi
 Cabbage Sungrow Seeds Ltd., New Delhi cr y1Ac
 Chickpea ICRISAT, Hyderabad cr y1Ac, cr y1Ab
 Groundnut ICRISAT, Hyderabad IPCVcp, IPCV replicase,
 Maize Monsanto, Mumbia CP4 EPSPS
 Mustard IARI, New Delhi CodA, Osmotin,
NRCWS, Jabalpur bar, barnase, barstar
TERI, New Delhi Ssu-maize, Psy, Ssu-tpCr tI
UDSC, New Delhi bar, barnase, barstar
 Okra MAHYCO, Mumbai cr y1Ac
 Pigeonpea ICRISAT, Hyderabad cr y1Ab + SBTI
MAHYCO, Mumbai cr y1Ac
 Potato CPRI, Simla cr y1Ab
NCPGR, New Delhi Ama-1
 Rice Directorate of Rice Research, Bacterial blight res, Xa -21,
Hyderabad
Osmania University, Hyderabad cr y1Ab, gna gene,
IARI, New Delhi gna
MAHYCO, Mumbai Bt, chitinase, cr y1Ac and Aa
MKU, Madurai cr y1Ac
MSSRF, Chennai chitinase, B -1,3-glucanase
TNAU, Coimbatore chitinase
 Sorghum MAHYCO, Mumbai cr y1Ac
Transgenic crop under development and field trials in India
• improved nutritional quality
• increased crop yield
• insect resistance
• disease resistance
• herbicide resistance
• salt tolerance
• biopharmaceuticals
• saving valuable topsoil
• ability to grow plants in harsh environments
ADVANTAGES OF GM CROPS
• Damage to human health
•allergies
•horizontal transfer and antibiotic resistance
•eating foreign DNA
•changed nutrient levels
• Damage to the natural environment
•crop-to-weed gene flow
•leakage of GM proteins into soil
•reductions in pesticide spraying: are they real?
• Disruption of current practices of farming and food
production in developed countries
•crop-to-crop gene flow
• Disruption of traditional practices and economies in
less developed countries.
• Lack of research on consequences of transgenic
crops.
DISADVANTAGES OF GM CROPS
 Foods produced using biotechnology has not been
established as safe and are not adequately
regulated.
 Crops produced using biotechnology will negatively
impact the environment.
 The long term
effects of foods developed using biotechnology are
unknown.
MYTHS RELATED TO GENETIC MODIFICATION
 Genetically-modified foods have the potential
to solve many of the world's hunger and
malnutrition problems, and to help protect and
preserve the environment by increasing yield
and reducing reliance upon chemical pesticides
and herbicides. Yet there are many challenges
ahead for governments, especially in the areas
of safety testing, regulation, international
policy and food labeling. Many people feel that
genetic engineering is the inevitable wave of
the future and that we cannot afford to ignore
a technology that has such enormous potential
benefits. However, we must proceed with
caution to avoid causing unintended harm to
human health and the environment as a result
of our enthusiasm for this powerful technology.
CONCLUSION
 Principles of genetic manipulations.
PRIMROSE 5th EDITION
 INTERNET
 MOLECULAR BIOTECHNOLOGY by GLICK
 http://en.wikipedia.org/wiki/Genetically_modified_crops
REFERENCES

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Transgenic plants

  • 2.  Was under trial and error for almost 9900 years.  The first genetically modified plant was produced in 1982, using an antibiotic-resistant tobacco plant.  The first genetically modified crop approved for sale in the U.S., in 1994, was the FlavrSavr tomato, which had a longer shelf life, as it took longer to soften after ripening.  As of mid-1996, a total of 35 approvals had been granted to commercially grow 8 transgenic crops and one flower crop of carnations, with 8 different traits in 6 countries plus the EU. In 2000, with the production of golden rice, scientists genetically modified food to increase its nutrient value for the first time. HISTORY
  • 3. • To improve the agricultural, horticultural or ornamental value of a crop plant • To serve as a bioreactor for the production of economically important proteins or metabolites • To provide a powerful means for studying the action of genes (and gene products) during development and other biological processes WHY GENETICALLY ENGINEER PLANTS?
  • 4. Applications of Plant Genetic Engineering A.Crop Improvement B.Genetically Engineered Traits: The Big Six 1.Herbicide Resistance 2.Insect Resistance 3.Virus Resistance 4.Altered Oil Content 5.Delayed Fruit Ripening 6.Pollen Control C.Biotech Revolution: Cold and Drought Tolerance and Weather-Gaurd Genes D.Genetically Engineered Foods 1.Soybeans 2.Corn 3.Cotton 4.Other Crops
  • 5. An Overview of the Crop Genetic Engineering cycle
  • 6. Leaf Disc Method for A. t. Mediated Transformation Leaf Disk Preparation Co-cultivation with Agrobacterium Selection for Transformation Regeneration of Shoots 6
  • 7. Genetic engineering techniques applied to plants METHOD SALIENT FEATURES 1.VECTOR MEDIATED GENE TRANSFER a. Agrobacterium mediated gene transfer b. Plant viral vectors Very efficient but limited to a selected group of plants Ineffective, hence not widely used 2.DIRECT OR VECTORLESS DNA TRANSFER a. Electroporation b. Microprojectile c. Liposome fusion d. Silicon carbide fibres Mostly confined to protoplasts that can be regenerated to viable plants Limited use only one cell can be microinjected at a time Confined to protoplasts that can be regenerated into viable whole plants Requires regenerable cell suspensions 3 CHEMICAL METHODS a. Polyethylene glycol mediated b.Diethylaminoethyl(DEAE)dext ran- mediated Confined to protoplasts. Regeneration of fertile plants is frequently problematical Very less results
  • 8.  Herbicides are generally non-selective (killing both weeds and crop plants) and must be applied before the crop plants germinate  Four potential ways to engineer herbicide resistant plants 1. Inhibit uptake of the herbicide 2. Overproduce the herbicide-sensitive target protein 3. Reduce the ability of the herbicide-sensitive target to bind to the herbicide 4. Give plants the ability to inactivate the herbicide HERBICIDES AND HERBICIDE-RESISTANT PLANTS
  • 9. HERBICIDE-RESISTANT PLANTS: REDUCING THE ABILITY OF THE HERBICIDE-SENSITIVE TARGET TO BIND TO THE HERBICIDE  Herbicide: Glyphosate (better known as Roundup)  Resistance to Roundup (an inhibitor of the enzyme EPSP involved in aromatic amino acid biosynthesis) was obtained by finding a mutant version of EPSP from E. coli that does not bind Roundup and expressing it in plants (soybean, tobacco, petunia, tomato, potato, and cotton)  5-enolpyruvylshikimate-3-phosphate synthase (EPSP) is a chloroplast enzyme in the shikimate pathway and plays a key role in the synthesis of aromatic amino acids such as tyrosine and phenylalanine
  • 10.  Genetic engineering here is more challenging; however, some strategies are possible:  Individually or in combination express pathogenesis- related (PR) proteins, which include b1,3-glucanases, chitinases, thaumatin-like proteins, and protease inhibitors  Overexpression of the NPR1 gene which encodes the “master” regulatory protein for turning on the PR protein genes  Overproducing salicylic acid in plants by the addition of two bacterial genes; SA activates the NPR1 gene and thus results in production of PR proteins FUNGUS- AND BACTERIUM-RESISTANT PLANTS
  • 11. Modification of plant nutritional content: increasing the vitamin A content of plants • 124 million children worldwide are deficient in vitamin A, which leads to death and blindness • Mammals make vitamin A from b-carotene, a common carotenoid pigment normally found in plant photosynthetic membranes • Here, the idea was to engineer the b-carotene pathway into rice • The transgenic rice is yellow or golden in color and is called “golden rice” *Expression of enzymes of β-carotene pathway in rice endosperm *Amelioration of Vitamin A deficiency
  • 12. Edible Vaccines – Ongoing Research Areas Hepatitis B Dental caries - Anti-tooth decay Ab Autoimmune diabetes Cholera Rabies HIV Rhinovirus Foot and Mouth Enteritis virus Malaria Influenza Cancer
  • 13. EDIBLE VACCINES FROM PLANTS Two strategies for production 1) Expression of foreign antigens in plant via stable transformation 2) Delivery of vaccine epitopes via plant virus (Mason and Arntzen, 1995)
  • 14. Strategy for the production of candidate vaccine antigens in plant tissues
  • 15. e RABIES VIRUS G PROTEIN IN TOMATO • Gene linked to CaMV35S promoter • Introduced to tomato plants by Agrobacterium- mediated transformation • Expression of recombinant glycoprotein in leaves and fruits • Protein localized in Golgi bodies, vesicles and plasma lemma
  • 16. Norwalk virus (cold virus) capsid protein in potato and tobacco • Causative agent for acute epidemic gastroenteritis • NVCP was fused to CaMV35S promoter • Transformation by Agrobacterium • Expression level: varies with plant (
  • 17. DEVELOPMENT OF STRESS- AND SENESCENCE-TOLERANT PLANTS: GENETIC ENGINEERING OF SALT-RESISTANT PLANTS  Overexpression of the gene encoding a Na+/H+ antiport protein which transports Na+ into the plant cell vacuole  This has been done in Arabidopsis and tomato plants allowing them to survive on 200 mM salt (NaCl)
  • 18. Frost Resistance • Ice-minus bacteria • Ice nucleation on plant surfaces caused by bacteria that aid in protein-water coalescence  forming ice crystals @ 0oC (320F) • Ice-minus Pseudomonas syringae • Modified by removing genes responsible for crystal formation • Sprayed onto plants • Displaces wild type strains • Protected to 23oF • Dew freezes beyond this point • Extends growth season • First deliberate release experiment – Steven Lindow – 1987- sprayed potatoes
  • 19. Development of stress- and senescence-tolerant plants: genetic engineering of flavorful tomatoes Fruit ripening is a natural aging or senescence process that involves two independent pathways, flavor development and fruit softening. Typically, tomatoes are picked when they are not very ripe (i.e., hard and green) to allow for safe shipping of the fruit. Polygalacturonase is a plant enzyme that degrades pectins in plant cell walls and contribute to fruit softening. In order to allow tomatoes to ripen on the vine and still be hard enough for safe shipping of the fruit, polygalacturonase gene expression was inhibited by introduction of an antisense polygalacturonase gene and created the first commercial genetically engineered plant called the FLAVR SAVR tomato. Flavor development pathway Fruit softening pathway Green Red Hard Soft polygalacturonaseantisense polygalacturonase
  • 20.  Crop Organization Gene  Brinjal IARI, New Delhi cr y1Ab, cr y1Ac  MAHYCO, Mumbai  Cauliflower MAHYCO, Mumbai cr y1Ac Sungrow Seeds Ltd., New Delhi  Cabbage Sungrow Seeds Ltd., New Delhi cr y1Ac  Chickpea ICRISAT, Hyderabad cr y1Ac, cr y1Ab  Groundnut ICRISAT, Hyderabad IPCVcp, IPCV replicase,  Maize Monsanto, Mumbia CP4 EPSPS  Mustard IARI, New Delhi CodA, Osmotin, NRCWS, Jabalpur bar, barnase, barstar TERI, New Delhi Ssu-maize, Psy, Ssu-tpCr tI UDSC, New Delhi bar, barnase, barstar  Okra MAHYCO, Mumbai cr y1Ac  Pigeonpea ICRISAT, Hyderabad cr y1Ab + SBTI MAHYCO, Mumbai cr y1Ac  Potato CPRI, Simla cr y1Ab NCPGR, New Delhi Ama-1  Rice Directorate of Rice Research, Bacterial blight res, Xa -21, Hyderabad Osmania University, Hyderabad cr y1Ab, gna gene, IARI, New Delhi gna MAHYCO, Mumbai Bt, chitinase, cr y1Ac and Aa MKU, Madurai cr y1Ac MSSRF, Chennai chitinase, B -1,3-glucanase TNAU, Coimbatore chitinase  Sorghum MAHYCO, Mumbai cr y1Ac Transgenic crop under development and field trials in India
  • 21. • improved nutritional quality • increased crop yield • insect resistance • disease resistance • herbicide resistance • salt tolerance • biopharmaceuticals • saving valuable topsoil • ability to grow plants in harsh environments ADVANTAGES OF GM CROPS
  • 22. • Damage to human health •allergies •horizontal transfer and antibiotic resistance •eating foreign DNA •changed nutrient levels • Damage to the natural environment •crop-to-weed gene flow •leakage of GM proteins into soil •reductions in pesticide spraying: are they real? • Disruption of current practices of farming and food production in developed countries •crop-to-crop gene flow • Disruption of traditional practices and economies in less developed countries. • Lack of research on consequences of transgenic crops. DISADVANTAGES OF GM CROPS
  • 23.  Foods produced using biotechnology has not been established as safe and are not adequately regulated.  Crops produced using biotechnology will negatively impact the environment.  The long term effects of foods developed using biotechnology are unknown. MYTHS RELATED TO GENETIC MODIFICATION
  • 24.  Genetically-modified foods have the potential to solve many of the world's hunger and malnutrition problems, and to help protect and preserve the environment by increasing yield and reducing reliance upon chemical pesticides and herbicides. Yet there are many challenges ahead for governments, especially in the areas of safety testing, regulation, international policy and food labeling. Many people feel that genetic engineering is the inevitable wave of the future and that we cannot afford to ignore a technology that has such enormous potential benefits. However, we must proceed with caution to avoid causing unintended harm to human health and the environment as a result of our enthusiasm for this powerful technology. CONCLUSION
  • 25.  Principles of genetic manipulations. PRIMROSE 5th EDITION  INTERNET  MOLECULAR BIOTECHNOLOGY by GLICK  http://en.wikipedia.org/wiki/Genetically_modified_crops REFERENCES