TRANSGENIC CROPS CHALLENGES AND PROSPECTS Transgenic Technology : Transform gene from any source. Eg: animals, bacteria, virus etc Traditional Breeding : Move genes only between members of a particular genus of plants. Take multiple growing seasons to develop and test a new variety. Lot of man power Limited possibility of improved traits.

1. TRANsGENIC CROPS
CHALLENGES
AND
PROSPECTS
R.S. ARCHANA

2. Key Highlights
• INTRODUCTION
• PRODUCTION OF GENETICALLY MODIFIED ORGANISMS
• APPLICATIONS
• CASE STUDIES
• REGULATORY FRAMEWORK
• POTENTIAL RISKS
• CHALLENGES
• FUTURE PROSPECTS
• CONCLUSION

3. • Transgene: Genetic material that has been transferred naturally or by
using any genetic engineering techniques from one organism to
another.
• Transgenesis : The process of introducing an exogenous gene called
a transgene into a living organisms so that the organism will exhibit a
new property and transmit that property to its offspring.

4. Transgenic Plants : The plants which expresses the characters
coded by the transgene are called Transgenic plants.

5. • Despite the success of the Green Revolution, the battle to ensure food
security for hundreds of millions miserably poor people is far from
won.
• Boosts agricultural production and fight global hunger
• Scientists and food producers believe that GM food crops could help
to solve many problems in agriculture.
Need of Transgenics

6. • Allows innovations that are impossible to achieve with
conventional breeding
• Precursor to the modern concept of genetic modification.

7. 1972
1974
1982
1987
1987
Paul Berg created the 1st r DNA molecule
Rudolf Jaenisch created the 1st GM mouse
The 1st GM crop, an
antibiotic-resistant tobacco
plant, was produced.
Gene gun was invented
Ice-minus strain of Pseudomonas
syringae became the 1st GM organism released
into the environment when a strawberry field and
a potato field in California were sprayed with it.
1992
Calgene attained approval to
commercially release
the Flavr Savr tomato, the
first GM food 1994
China was the first
country to
commercialize
transgenic plants,
introducing a virus-
resistant tobacco
HISTORY OF
GENETIC
MODIFICATION

8. 1994
1995
1996
2010
2017
An insect resistant Potato was approved for release
and GMO corn hits the market in the USA.
scientists at the J. Craig Venter Institute announced that they
had created the 1st synthetic bacterial genome. They named
it as Synthia and it was the world's first synthetic life form.
2000
1st GM flower moondust bluish
colour carnation was introduced
European Union approved tobacco to be resistant to the
herbicide bromoxynil, making it 1st genetically engineered crop
commercialized in Europe.
Researchers genetically modified a virus to express
spinach defensin proteins injected into orange trees for citrus
greening disease
Vitamin A enriched Golden rice, was
the 1st plant developed with
increased nutrient value.

9. Increased
yields
Protection against
pests and pathogens Reduced losses due to
disease and infestation
Tolerance towards
abiotic stresses
Improving
shelf life
Plant based
Pharmaceuticals
Manipulation of
plant development
Eliminate
anti-nutritional
factors
Herbicide
Tolerance
Higher
nutritional
value
Remove constraints Increase potential
HUNGER/MALNUTRITION
Product quality
improvement
Tolerance of
extreme weather
WHY TRANSGENIC PLANTS

10. Difference between Transgenic technology and
Traditional breeding
Transgenic Technology : Transform gene from any source.
Eg: animals, bacteria, virus etc
Traditional Breeding : Move genes only between members of a
particular genus of plants.
• Take multiple growing seasons to develop and test a new variety.
• Lot of man power
• Limited possibility of improved traits.

13. Global GM area by Country

14. Worlds agricultural land
under GM
Four GM crops account for 99%
of worldwide
GM crop hectarage

15. Production of transgenic plants
Isolate and clone gene of interest
Add DNA segments to initiate or enhance gene
expression
Add selectable markers
Introduce gene construct into plant cells
(transformation)
Select transformed cells or tissues
Regenerate whole plants
karthikumarbt@kcetvnr.org 15

16. Plant Transformation Methods
Physical Chemical Biological
• Microinjection
• Biolistics - gene gun/
particle
bombardment
• protoplast
Electroporatio
n
•Liposome fusion
• PEG mediated
• DEAE-dextran
mediated
• Calcium
phosphate
• Agrobacterium-
mediated
• Virus-
mediated
16

17. Agrobacterium - mediated Gene
Transfer
• Pioneered by J. Schell (Max-Planck Inst., Cologne)
• A. tumefaciens is a gram-negative soil bacterium which naturally
transforms plant cells, resulting in crown gall (cancer) tumors
• Related to Rhizobia species:
tumefaciens- causes crown galls on many dicots
rubi- causes small galls on a few dicots
rhizogenes- hairy root disease
radiobacter- avirulent

18. • Tumor formation is the result of the transfer, integration and expression of genes on
a specific segment of A. tumefaciens plasmid DNA called the T-DNA (transferred
DNA)
• The T-DNA resides on a large plasmid called the Ti (tumor inducing) plasmid found
in A.tumefaciens
Crown Gall on
Tobacco
Infection of a plant with
A. tumefaciens and formation of
crown galls

19. Ti Plasmid :
• Large ( 200-kb)
• Conjugative
Ti plasmid has 3 important regions:
(i) T-DNA region
(ii) Virulence region
(iii) Opine catabolism region
 There is ori region that is
responsible for the origin of DNA
replication.

20. Agrobacterium Transfers Plasmid DNA into Infected Plants
Agrobacterium carrying a Ti plasmid is attracted by acetosyringone to a wounded plant stem. The Ti plasmid
is cut by endonucleases to release single-stranded T-DNA, which is covered with protective proteins, and
transported into the plant cell through a conjugation-like mechanism. The T-DNA enters the plant nucleus
where it integrates into plant chromosomal DNA.
FIGURE 14.4

21. Leaf Disk Method for A. t. Mediated Transformation
Leaf Disk Preparation Co-cultivation with Agrobacterium Selection for Transformation
Regeneration of Shoots 21

22. Advantages :
• Technically simple
• Transfers large fragments of foreign DNA
• Capable of infecting intact Plant cells,tissues and organs.
• Introduces 1 to several copies of T DNA
• Transferred traits have been found to be stable over many generations
Dis advantages:
• Host range is limited
• Sometimes, the cells in a tissue are difficult to transform because of deep layers
to be reached by agrobacterium.

23. Virus mediated transformation
• Efficient gene transfer agents
• Amplify the transferred genes through viral genome replication
• Spread systematically with in a plant
Plant viruses as vectors:
• Caulimo virus- ds DNA : CaMV
• Gemini virus- 2 ss DNA : Maize streak virus
• RNA Plant viruses : TMV

24. Limitations
• Vast majority of plant viruses have genome not of DNA but of
RNA
• Limited capacity for insertion of foreign genes.
• Infective capacity of CaMV is lost if more than a few hundred
nucleotides are introduced
• In most cases, viral genome has to be modified to accommodate
the insertion of foreign sequences.
• Helper viruses cannot be used since the foreign DNA gets expelled
and wild-type viruses are produced.

25. The biolistic method (particle gun method)
• Discovered by klein and colleagues
(1987)
PRINCIPLE:
• DNA is bound to the tiny particles of
Gold or Tungsten, which is subsequently
shot into plant tissue or single plant cells,
under high helium pressure using gun.
• Also known as the “micro-projectile
bombardment”

26. 5/29/2020
26
DNA separates from the coated metal after penetrating into the
cell and integrates into the plant genome inside the nucleus

27. Method
• DNA coated on microscopic gold beads are placed
at the end of a plastic bullet.
• Blast of helium or strong electrical discharge used
to project them.
• Plastic meshwork stop is used to stop the bullet
• Beads enter the cytoplasm or nucleus of the cell
• DNA is free and recombine with chromosomal
DNA
• Leaf transferred to selection media for cell to grow
• Only cells with selectable marker grow other die
• Transformed plants are regenerated.
• Screened for gene of interest.

28. Advantages :
 This technique is clean and safe.
 Work with all types of plants.
 Specially in species like corn, wheat and rice, for which
transformation using agrobacterium tumefaciens has been less
successful
Dis advantages :
o Serious damage can be happened to the cellular tissue.
o Shallow penetration of particles
o Equipment itself is very expensive

29. Microinjection
• Direct physical method involving the mechanical insertion of the
desirable DNA into a target cell
• Transfer of the gene through a micropipette into the cytoplasm or
nucleus of a plant cell

30. Limitations:
• Slow and expensive
• To be performed by trained and skilled persons.
• In order to manipulate the protoplasts without damage, they have to
be cultured for 1 to 5 days before injection.
Protoplast culture

31. Electroporation
PRINCIPLE:
Creation pores in plant cell membrane by electric impulse
through which protoplasts take up desired molecules from their
surrounding fluid.
 More useful for animal cells

32.  Transformed cells can then regenerate their cell walls and grow to
whole, fertile transgenic plants.
Protoplast colony derived Mass of callus Regenerated plant
Fig: (A) Diagram showing formation of transient pores in cell
membrane on applying electrical pulse, entry of DNA inside the cell
(B) Sealing of pores afterwards

33. Advantages:
• Fast, convenient, simple, and inexpensive method.
• Low cell toxicity.
Drawback
o Regeneration is difficult.
o Wrong length impulses causes large pores, fail to closure
of membranes causing cell damage
o Non specific transport creates ion imbalance lead to cell
death

34. Liposome mediated transformation
• Lipofection is a very efficient technique and is used for the transfer of
genes to bacterial, animal and plant cells.
• Liposomes are circular lipid molecules, which have an aqueous
interior that carry nucleic acids.
• The positively charged liposomes very efficiently complex with DNA

35. • On treatment of DNA fragment with liposomes, the DNA pieces get
encapsulated inside liposomes.
• liposomes adher to cell membranes and
fuse with them to transfer DNA fragments.
• Thus, the DNA enters the cell and then to
the nucleus.
Advantages:
• Simplicity
• Long term stability
• Low toxicity
• Protection of nucleic acid from degradation

36. CHEMICAL GENE MEDIATED
TRANSFER
Polyethylene glycol- mediated transformation
PEG, in the presence of divalent cations, destabilizes the
plasma membrane of protoplasts and renders it permeable to naked
DNA.

37. Advantages:
A large number of protoplasts can be simultaneously
transformed.
This technique can be successfully used for a wide variety of
plant species.
limitations:
i) The DNA is susceptible for degradation and rearrangement.
ii) Random integration of foreign DNA into genome may result
in undesirable traits.
iii) Regeneration of plants from transformed protoplasts is a
difficult task.

38. DEAE-dextran mediated transfer
The desirable DNA can be complexed with a high
molecular weight polymer diethyl amino ethyl (DEAE) dextran
and transferred.
limitation
Does not yield stable transformants.

39. Calcium phosphate co-precipitation
• DNA is mixed with CaCl2 solution and isotonoic buffer to form DNA CaPo4
precipitate.
• This precipitate is allowed to react with actively dividing cells
• Efficiency is low but can be increased by giving physiological shock with
DMSO

40. Screening of transgene
• Genes that facilitates the detection of genetically modified plant tissue during
development referred to as marker genes.
• Marker genes present along with transgene in the T-DNA segment.
• These are resistance genes useful for monitoring successfull transformation
Two major types of genes
• Conferring resistance to antibiotics
• Conferring tolerance to herbicides

41. Drug (Antibiotic) resistance Marker Substrate for selection
nptII (Neomycin Phosphotransferase) G418, Kanamycin, Neomycin
hpt (Hygromycin Phosphotransferase) Hygromycin
dhfr( Dihydro folate reductase) Methotrexate, Trimethoprim
Herbicide resistance Marker Substrate for Selection
bar&pat(Phosphinothricinacetyl
transferase)
Phosphenothricin(Bialaphos, Basta)
Epsps(5-enolpyruvylshikhimate-3-
phosphate synthase)
Glyphosate

42. APPLICATIONS
RESISTANCE TO BIOTIC STRESS
1) Insect resistance
2) Virus resistance
3) Fungal and bacterial resistance
RESISTANCE TO ABIOTIC STRESS
1) Herbicide resistance
2) Glyphosate resistance
IMPROVEMENT OF CROP YIELD & QUALITY
1) Extended shelf life of fruits
2) Improved nutrition
3) Improved colouration
PRODUCTION OF LOW-COST PHARMACEUTICALS
1) Edible vaccines
2) Essential proteins

43. More Specific examples of GM foods
FLAVR SAVR
• The first approved GM food in 1994 by FDA.
• Licensed in may 17, 1994.
Before the Flavr Savr
• Picked before ripe and gassed with ethylene to give red color –
keeps them from becoming bruised and spoiled.
• Tomatoes lose their taste and taste
more like “cardboard.”

44. What is the Flavr Savr?
• A tomato implanted with a gene from E. coli
• A tomato that will not soften while ripening on the vine.
• The transgenic tomato would allow tomatoes to be shipped
safely, keep their color, and have their natural flavors.
• Increased shelf life in Canada, Japan, Mexico, and the US

45. Flavr Savr Gene
• A strand of RNA that is reverse of the RNA that occurs in the plant
• Original RNA- Produces enzyme Polygalacturonase
• Flavr Savr RNA- binds to Polygalacturonase so the enzyme can’t
break down pectin (causes tomato to become soft).

46. Golden rice
• Transgenic technology produced a type of rice that accumulates β-
carotene in rice grains.
• It is consumed, when β-carotene is converted into vitamin-A.
• It contains 37 mg/g of carotenoid of which 84% is β-carotene
Normal rice Golden rice

47. Who Began the Golden Rice Project?
• Started in 1982 by Ingo Potrykus-Professor emeritus of the
Institute for Plant Sciences
• 1st plant developed with increased nutrient value in 2000,
Purpose:
To provide pro vitamin A to
world mainly developing countries where
malnutrition and vitamin A deficiency are
common

48. How Does It Work?
• Addition of 2 genes in rice genome will complete the biosynthetic
pathway
1. Phytoene synthase (psy) – derived from daffodils
2. Lycopene cyclase (crt1) – from soil bacteria Erwinia uredovora
Produces enzymes and catalysts for the biosynthesis of carotenoids (β-
carotene) in the endosperm.
• Presence of pro-vitamin A
gives rice grains a yellowish-
orange color, thus, the name
‘Golden Rice’

49. Controversy Against “Fool’s Gold”
Supply does not provide a
substantial quantity as the
recommended daily intake
Genetic contamination of natural,
global staple foods Culture
Some people prefer to cultivate
and eat only white rice based on
traditional values and spiritual
beliefs

50. Insect resistant plants
• Bacillus thuringiensis (Bt) is a common gram positive, spore-forming,
soil bacterium.
• Japanese biologist, Ishiwatari was investigating the cause of the sotto
disease (sudden-collapse disease) that was killing large populations of
silkworms when he first isolated the bacterium Bacillus
thuringiensis (Bt) as the cause of the disease in 1901.
• Farmers started to use Bt as a pesticide in 1920.

51. • In the 1980's use of Bt increased when insects became increasingly resistant
to the synthetic insecticides.
• Scientists and environmentalists became aware that the chemicals were
harming the environment.
• Because of this, governments and private industries started to fund research
on Bt.
Different Cry proteins produced by Bacillus :
• Cry I : kills Butterflies and moths
• Cry II : kills Butterflies and flies
• Cry III : kills beetles
• Cry IV : kills only flies.

52. • These toxins is used to prevent
 1. cotton boll worm ---- destroy cotton
 2. European corn borer ---- destroy corn
• The first genetically engineered plant, corn, was registered with the
EPA in 1995.
• Today, GM crops including, potato and cotton are planted throughout
the world

53. 53
1. Insect eats Bt crystals and spores.
2. The toxin binds to specific receptors in
the gut and the insects stops eating.
3. The crystals cause the gut wall to break
down, allowing spores and normal gut
bacteria to enter the body.
4. The insect dies as spores and gut
bacteria proliferate in the body.
How Bt works ?

54. 54
Govt. of India approved Mahyco’s Bt-cotton
for control of bollworms
India’s first transgenic crop

55. • Development and commercialization of insect-resistant transgenic Bt
crops expressing Cry toxins revolutionized the history of agriculture.

56. Herbicide Tolerance
• Over 63% of Gm crops grown globally have herbicide tolerance traits.
• Achieved through the introduction of a gene from a bacterium or virus conveying
resistance to some herbicides.
• Eg : Glyphosate

57. • As of 1999 the most prevalent GM trait was glyphosate-resistance.
Glyphosate Inhibits EPSPS in the Aromatic Pathway

58. What scientists did ?
• A plasmid which was transferred to the soybean cells through the
cauliflower mosaic virus was soon developed to provide
immunity to glyphosate-containing herbicides.
• HOW?
 Inserting constitutive EPSP gene
– (5-enolpyruvylshikimate-3-phosphate synthase)

59. • The 1st crops to be engineered for glyphosate resistance were
produced by Monsanto Co. and called “Roundup Ready” in 1996.
• Corn, cotton have also been genetically engineered to be
herbicide resistant.
• About 75% of all soybeans and 65% of all cotton grown in
America is GM for herbicide resistance.

60. • Tobacco plants have been engineered to be resistant to the
herbicide bromoxynil.
• In October 2014 the US EPA registered Duo maize, which is
genetically modified to be resistant to both glyphosate and 2,4-D, in
six states.
• Inserting a bacterial aryloxyalkanoate dioxygenase
gene, aad1 makes the corn resistant to 2,4-D.

61. CASE
STUDIES

62. • Continuous practice of old methods in agriculture cannot reduce the
food scarcity.
• Over use of chemicals, drought and infertile soil are a severe issues
where the investment is more and the yielding is less.
• To overcome these issues concentrating on the new technology by
combining the specific genes in-vivo for the crops which is called
transgenic crops.

63. • A novel synthetic cry2A* gene was introduced into the elite indica rice
restorer line Minghui 63 by Agrobacterium-mediated transformation.
• Insect bioassays were conducted in both the laboratory and field.
• A total of 102 independent transformants were obtained. Among them, 71
transformants were positive cry2A*
• They were significantly resistant to lepidopteran rice pests.
• These cry2A* transgenic lines can be used to produce insect-resistant hybrids
and serve as a resistant source for the development of Bt rice.

64. Enhanced provitamin A content in maize kernels by
Overexpression of the bacterial genes crtB (for phytoene synthase)
and crtI (for the four desaturation steps of the carotenoid pathway in
plants), under the control of a ‘super γ-zein promoter’ for endosperm-
specific expression, resulted in an increase of total carotenoids of up to
34-fold with a preferential accumulation of β-carotene in the maize
endosperm.

65. M37W
 White endosperm
 Total carotenoids: 1.1µg/g
 Lutein : Zeaxanthin 0.5 : 0.27 µg/g DW
A632
 Yellow endosperm
 Total carotenoids: 28 µg/g
 Lutein : Zeaxanthin 15.61 : 7.77 µg/g DW
WHITE vs YELLOW MAIZE
The high β-carotene trait was found to be reproducible over at least
four generations.

67. Cold tolerant crops
• Scientists have created a frost resistant crops plant by adding an
antifreeze gene from a cold water fish to it.
• The antifreeze genes come from the cold water flounder, a fish that
can survive in very cold conditions.

68. Pathway for cold tolerant tomatoes using
antifreeze gene

69. Virus-resistant crops
Biotech squash Biotech papaya
These new varieties contain the coat protein genes resist
watermelon mosaic virus and zucchini yellow mosaic
virus and papaya ring spot virus

70. Biopharmaceuticals
• The genes for proteins to be used in human medicine can be inserted
into plants and expressed by them.
• Glycoprotein gene gp 120 of the AIDS virus HIV-1 incorporated into
GM maize as a cheap edible oral vaccine

71. • In April 2012, the only protein received approval for human use
is glucocerebrosidase, an enzyme lacking in Gaucher's disease.
• It is synthesized by transgenic carrot cells grown in tissue culture.

74. REGULATORY FRAMEWORK
• India is a signatory to the Cartagena Protocol on Biosafety
(CPB) since 2003
• Genetic Engineering Approval Committee (GEAC) that
functions as a statutory body under the Environment
Protection Act 1986 of the Ministry of Environment &
Forests (MoEF), has been changed to Genetic Engineering
Appraisal Committee on July 22, 2010

75. Milestones
• GEAC’s decided to permit field trials of 21 new varieties out
of 28 genetically modified (GM) crops such as rice, wheat,
maize and cotton crops – February 27, 2014
• GEAC bio-technology supporters rejected just one out of the
28 proposals.
• Six proposals were rejected for want of more information.

76. .
•In 2007, GEAC recommended the commercial release of Bt Brinjal,
which was developed by Mahyco (Maharashtra Hybrid Seeds Company)
in collaboration with the Dharward University of Agricultural sciences
and the Tamil Nadu Agricultural University.
•But the initiative was blocked in 2010.

77. POTENTIAL RISKS OF GM PLANTS
Harmful Effects on Crops:
• The 'Bt' genes present in GM crops kill beneficial insects
• Creation of “super weeds”
• crop-to-crop gene flow
• Disruption of traditional practices and economies in less
developed countries.

78. Damage to human health
• Allergies
• Antibiotic resistance
• Eating foreign DNA
• Changed nutrient levels

79. Economic concerns
• Lengthy and costly process
• May be patented
 Monsanto, Novartis, Dow, DuPont hold patents for GM crops Make substantial
profit by exporting it to Ems’.
• More gap between rich and poor
Other invention
• Discouraged/stopped
• Suicide gene technology
• Only one growing per season
• Next time would produce sterile seeds that do not germinate.

80. Challenges
• Inability, incapability and unpreparedness of the Indian GM
research establishment to deal with this unpredictable and
uncontrollable technology.
• Need of genotype independent protocols for regeneration and
transformation
• International organizations should promote networking of
interested regional countries and labs to work together towards
novel goal

81. • Companies advanced in transgenic technologies could
participate in providing solutions and expertise assistance to
overcome difficulties
• considerable time is needed for productiveness
• Much still needs to be done to improve our knowledge of
specific genes and their actions, potential side effects of
foreign DNA and of manipulating genes within an organism.
• Although these and other GM foods show promise for
increasing agricultural productivity and decreasing disease, the
political pressure from anti-GM critics remains a powerful
force.

82. Future prospects
• Current research focusing on understanding and developing
useful promoter sequences to control transgenes
• Establishing precise methods to insert and place the transgene at
specific locations in the recipient chromosomes.
• Developing Various products including vaccines, vitamin
enrichments, hormones that result in faster maturation and
disease resistance.
• Major solution to meet food requirements for 920 crores
population of world by 2050.

83. Conclusion
Improved Nutrition
Resistance to disease
Reduced use of
chemicals
Environment
al risks
Health risks
Economic
risks
• GMO’s present both positive and
negative aspects to society.
• GMO’s are still lies in its embryonic
stage and in the developmental
process Only.
•We cannot ignore a technology that
has such enormous potential benefits.
• Time will reveal their ultimate
effects.
• Leave the decision making to the
scientists.

84. FOR YOUR KIND ATTENTION