This slides of highlights the methods for development of transgenic plants, their application and biosafety issues

1. GM Crops: an alternative model
to address the food security
SOUMITRA PAUL, Ph.D.
Assistant Professor
Molecular Plant Physiology laboratory
Department of Botany
University of Calcutta
Email:psoumitra@ymail.com,
sopbot@caluniv.ac.in

2.  Gene Modification: Overview
 Why GM Crops?
 Classical Breeding vs. Molecular breeding
 Food security aspects
 Development of different events of GM Crops
 Abiotic stress tolerant GM Crops
 Nutritionally improved GM Crops
 Bt-cotton and myth
 Biosafety analysis of GM Crops
 Substancial equivalence analysis
 Genome editing technology for gene manipulation
Points of Discussion

3. Genetic Modification/Gene Manipulation/Genetic Engineering
The use of modern biotechnology techniques to alter the DNA of an
organism, such as a plant or animal, for the purpose of beneficial product
development or improvement.
A genetically modified organism (GMO) is a plant, animal or other organism
that has been changed using genetic modification.
1. Transgene technology: Genes from one organism and inserting them
into another.
GM does not necessarily mean that a gene from another organism has to be used
to create the GMO. GM can mean that the organism’s own genes are changed.
Modern Biotechnology Techniques:
2. Silencing Technology: Antisense RNA, RNAi, Artificial miRNA
3. Genome Editing/ targeting : TALEN, CRISPR/Cas

4. Gene cloning
(through PCR)Identification of a
gene (functional
genomics
approaches)
Vector
Construction
Promoter cloning
(Desirable
expression)
Genetic Transformation
of plants (Different
methods)
Selection of
regenerated
plants
Analysis of
transgenic plants
Gene transfer in plants: An overview

7. Transgenic events: Categorizing GM Crops
Biotic stress tolerance
Insect , bacterial, fungus, viral
resistance, Herbicide resistance
Abiotic stress tolerance
Drought, salinity
Nutritional enrichment/
Biofortification
Glden rice, ferritin rice
Metabolic engineering
Flavr-savr tomato,
Low phytate rice

8. Abiotic Stress:
Drought, Cold, Heat, Salinity
Transgenic plants transformed with
AtDREB1A gene plants during vegetative
stages (A) Wild type (B) Transgenic plant
[adopted from Datta et al.,2012]
 Drought: Overexpressing dreb1A gene in rice
 Cold: Engineering with COR15a Tf, role in freezing
tolerance.
 Plants engineered with Choline oxidase (codA) soil
tolerated saline and cold
 Homeodomain-leucine zipper (HD-Zip) transcription
factors respond to H2O & osmotic stress, exogenous
abscisic acid
 Transport protein. Grow and fruit even in irrigation
water that is > 50X saltier than normal. > 1/3 salty as
seawater (Blumwald and Zhang 2010)

9. Major abiotic stress
modulation to
increase abiotic
stress tolerance

10. Why we need Bt crops?
• No natural resistance in germplasm (Cotton, Brinjal, Corn, etc)
• Limited success with traditional management strategies
• We can not incorporate cry gene by crossing with B. thuringiensis
• Farmers have no option except to apply huge insecticide at early stage before
FSB bore
• Huge use of pesticides results in
– development of resistance in insects
– Harmful effects on environment, health of farmer and consumers
Insect bioassays of leaves
from Bt cotton. (A)
Consumed surface area of leaf
in (a) the control plant (b)-(f)
different transgenic lines; (B).
Percent mortality of larvae fed
on leaf tissues of different
transgenic lines (adopted from
Singh et al., 2016)

11. Safety issues of the most controversial- Bt
technology
• Since 1938, used extensively as a spray all
over the world to control a variety of
lepidopteron pests.
Bt
62%
1. Acidic stomach
2. Very low pH (∽1.5 in humans)
3. Absence of required receptors
Safe to human and other mammals
Selective action of CRY protein

12. Virus resistance
1. Cross protection by virus
2. coat protein of TMV expressing tobacco,
AC2 gene expressing, nucleocapsid protein
gene of tomato spotted wilt virus expressing
tobacco, SAHH gene down regulation by
antisense technology

13. Virus resistance
Viral infections
Major crop losses
tobacco mosaic virus
(TMV) causes
losses of over $50
million per annum in
the tomato
industry.cross-protection
a plant
Infected by one
strain of virus
protects against superinfection
with a second related strain.

14. Gene Transgenic plants Resistance
Cowpea protease
inhibitor (CpTI)
Tobacco Insect (bruchid beetle)
Cystatin, cysteine
protease inhibitor from
rice, corn
Rice insect
Cross protection by virus Tobacco, potato TMV, PSV
Virus capsid protein or
coat protein gene
Tobacco, potato, tomato TMV, PSV
Nucleocapsid protein
from tomatto spotted
wilt virus (TSWV)
Tomato, tobacco, potato TSWV
S-sdenosyl homocysteine
hydrolase (SAHH) by
antisense
Tobacco Broad spectrum virus
Other insecticidal and resistance against virus

15. Resistance against bacteria and fungi
Gene Transgenic plants Resistance
Acetyl transferase Tobacco Pseudomonas syringe
(wild fire)
Chitinase gene from
Serratia marcescens
Tobacco Alternaria longipes
Chitinase gene Tobacco, Brassica, rice Sheath blight, damping
off (Rhizoctonia solani)
Osmotin gene Potato Late blight
(Phytophthora
infestans)
Xa-21 Rice Bacterial blight
(Xanthomonas oryzae)

16. Xa21 gene has been cloned
from an African rice variety and
introduced into modern
cultivars to confer resistance to
rice leaf blight. Pam Ronald,
Plant pathology, UC
Pathology, UC Davis
Bacterial disease resistant:
Bacterial blight resistance rice (xa-21 overexpressing)

17. – Transgenic breeding lines preferred because resistance can be
introduced into commercial lines with greater speed. The
BASF GM potatoes involves the use of two broad spectrum
resistance genes, Rpi-blb1 and Rpi-blb2. These two genes
have a structure associated with regulatory genes called
nucleotide binding site-leucine rich repeat (NBS-LRR) class of
regulatory proteins. Many disease resistance genes code for
proteins of that class. Numerous plant NBS-LRR genes are
present in the typical plant genome, each protein is specific for
a particular pathogen signaling a defense response, frequently a
localized plant cell death called hypersensitive response. The
C terminus of the protein containing LRR recognizes a ligand
feature of a pathogen activating the NBS signaling module to
initiate the defense response. The blight fungus suppresses the
potato defense genes in sensitive plants but is thwarted by
successful defense genes.
– Blight-resistant potato (BASF -Rpi-blb1 and Rpi-blb2 NBS-
LRR) -UI study concluded for the major potato-producing
regions of the world would be $4.3 billion.
Blight-resistant potato

18. Herbicide tolerant transgenic plants
Active principle Pathway blocked Target product Mechanism of
resistance
Glyphosate
(Roundup)
Aromatic amino
acid
EPSPS (5-enol—
pyruvyl-shikimat-
3 phospahte
synthase)
Overexpression of
EPSPS gene
Phosphinothricin
(Basta)
Glutamine
biosyntheis
GS (glutamyl
synthase)
Bar gene
Sulphonyl urea Branched chain
amino acid
ALS (acetolactate
synthase)
Mutant als gene
Atrazine Photosystem II QB Mutant psbA
gene, GST gene
Bromoxynil photosyntheis bxn (nitrillase)
gene

19. Host plants release factors required by parasitic plants:
control strategy to inactivate host recognition factors
DMBQ
sorgoleone
Host plants Parasitic plants
maize
cowpea
Arabidopsis
Striga
Orobanche
Triphysaria
Seed germination
0 h 12 h 24 h
Haustorium development
Weed Resistant

20. Male sterility
Disrupt pollen development by expressing a lethal gene in pollen.
Promoter of a tapetum specific gene (TA29) was used to drive the
expression of RNase T1 gene, which disrupted pollen formation.
Barstar Barnase system
Barnase (110 amino acids) is a secreted ribonuclease from Bacillus
amyloliquefaciens. Barstar (89 amino acids) is a cytoplasmic barnase inhibitor
with which the host protects itself. RNase is linked with bar gene (glufosinate
tolerant), so glufosinate tolerant plant will be male sterile.
GM canola containing barstar/ barnase system composes about 10% of
commercially cultivated crops in Canada and is one of the few GMO cleared for
agricultural use in Europe.

21. Induction of male sterility in plants by a chimaeric ribonuclease gene
Nature 347, 737-741 (25 October 1990)
Chimaeric ribonuclease genes that are expressed in the anthers of transformed
tobacco and oilseed rape plants were constructed. Chimaeric ribonuclease gene
expression within the anther selectively destroys the tapetal cell layer that
surrounds the pollen sac, prevents pollen formation, and leads to male sterility.
These nuclear male sterility genes should facilitate the production of hybrid seed in
various crop plants.
Isoltaion of a tapetum-specific gene, TA29, facilitated tapetum specific
expression of RNase gene
Tetrad stage: maturation of microspores depends on tapetal cells
SMC= spore mother cells
V= vascular bundle
Fi= filament
w= anther inner wall
t= tapetum

23. A Novel Cell Ablation Strategy Blocks Tobacco Anther Dehiscence
•TA56 gene is stomium specific
•Lectin gene is active in many vegetative and floral tissues.
Ablation of the Stomium (St) Region Leads to Anthers That
Fail to Dehisce

24. Cell Ablation Strategy Using Chimeric Barnase and Barstar Genes with Overlapping Cell
Specificities.
Blocks represent cross-sections through a hypothetical organ system that has four different cell
types. The circular cells in the lower right quadrants are the targets of the ablation experiment.
(A) Blue represents transcriptional activity of the promoter (lectin gene) fused with the anti-cytotoxic
barstar gene.
(B) Red represents transcriptional activity of the promoter (TA56 gene) fused with the cytotoxic
barnase gene.
(C) Combined transcriptional activities of the chimeric barnase and barstar genes. Both chimeric
genes are active within the dark gray cells in the upper right quadrant. Only the chimeric barnase
gene is active in the target cells present in the lower right quadrant.
(D) Selective ablation of the target cells. Barnase/barstar complexes are formed within the dark
gray cells in the upper right quadrant protecting them from the cytotoxic effects of barnase. The
target cells in the lower right quadrant have been ablated selectively due to the cell-specific activity
A Novel Cell Ablation Strategy Blocks Tobacco Anther Dehiscence

25. Seed sterility
Genes involved in seed development may be used to deliver toxicity to seeds
A. Terminator technology:
Not published in scientific literature. Patented by USDA and licensed by Monsanto.
Seeds are treated with tetracycline before sale.
Following components have been described:
1. Ribosomal inhibitor protein (RIP) gene under the control
of LEA promoter. LEA (Late Embryonic Abundance) is a seed-specific gene.
2. A loxP flanked spacer between LEA promoter and the RIP ORF.
3. Cre expression controlled by a “Tet-on” system.
B. Hormone manipulation
LEA promoter controls iaaH gene.

26. High iron/zinc biofortified Rice
Low Phytate Rice
Golden Rice
b-carotene + Vit E rice
Hyperfortified α linolenic acid
Rice (ALA) for PUFA
Insulin promoting rice
Improved protein-potato (Ama1)
Carotenoids enriched potato
Canola with b-carotene
Vitamin C food crop
High iron rice
Vitamin E + b-carotene maize
Biofortified Food Crops
Major GM Rice for
nutritional improvement
Needs to focus
Folate, Lysine, Vitamin E enrichment
and more……….

27. Development of high iron rice

28. Development of Low phytate rice

29. Lutein Zeaxanthin
b-carotene biosynthesis
-carotene b-carotene (3)
LC (lyc)
PDS (crt1)
PS (psy)
(1)
(2)
Phytoene
Lycopene
Vitamin E
Gibberellins
Chlorophyll
IPP
GGPP
Metabolic engineering for development of Golden Rice
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31. To Address the Food Security……
Quantity as well as Quality both are important
Increasing the productivity and decreasing flowering time without
hampering so much physiological activities..by gene manipulation
Rice gene SPIKE (NAL1 allele) can boost yield by 36% (Fujita et
al., 2013), OsFIE2 gene can regulate grain filling in rice (Nallamilli-
Reddy et al., 2013)
Protect the plants from different biotic and abiotic stresses
Why GM Crops: for improvement of
single trait/multiple traits?
Sufficient Production ≠ Good Nutrition
Production + Nutrition = Food Security

32. Major Cereals production in India

33. Still, 42% malnourished and 58% underweight children have been reported
recently in India. In addition, 12 states are considered as high alert regions for
malnutrition!!!
2040, 2050…..?????
India will overtake China as the world's most populous country by 2050, according to
a new French study which predicts the global population to surge to 9.7 billion people
by the middle of the century.
The report by the French Institute of Demographic Studies (INED), predicts India will
take the top spot with a staggering population of 1.6 billion to leave behind current
world leader in population, China, at the second place with 1.3 billion people.

34. Malnutrition in India
HUNgAMA, 2011

35. Biotech growers in the world

36. Beyond Promises : Current status GM Crops
2017 was the 21st year of commercialization of biotech crops (1996-2012)
Biotech crop plantings increased to over 185 million hectares in 2017
30 developing and industrial countries planted biotech crops in 2017
A record 18 million farmers grew GM Crops in 2017
(Source: ISAAA, 2016)
Kenya, Malwai, Nigeria, Ghana, Ethiopia
confirmed their participation for commercial
cultivation of wide varieties of GM Crops in 2016
FACT 1

37. Economic benefit- 98.2 billion US dollar
Reduction in pesticide usage- 473 million Kg(Source: ISAAA, 2016)
In 2011 alone fewer insecticide and pesticide spray
results in reduced CO2 emission by 23.1 billion
kilograms = 10.2 million cars taking off from the
road/year

38. Protocols for Food and Feed Safety
Assessment of GE crops
Test
I. Acute Oral Safety Limit Study In Rats
and Mice- -a limit dose of 2000 mg/kg
of bodyweight of test animal. In case of
protein solution (maximum limit 10
ml/kg).
II. Sub-chronic Feeding Study In
Rodents -- 90-day study in al least 20
animals (ten female and ten male)
III. Protein Thermal Stability- to test
allergenicity. Resistance to heat
denaturation has been observed in
several food allergens; thus a
correlation exists between heat stability
and allergenic potential. incubated at a
range of temperatures from 25°C to
95°C for up to 30 minutes.
IV. Pepsin Digestibility Assay – for
allergenic potential determination. 10
units of pepsin/µg of test protein at
37°C, pH-1.2 & 2
V. Livestock Feeding Study- provided as
feed to an appropriate livestock
(Ruminant and nonruminant) species
for a period of time approximating a
normal production cycle
Observation
1. visual, Body Weight and Feed
Consumption and Pathology.
2. visual, Body Weight and Feed
Consumption and Pathology.
3. Proteins with more than 50%
biological activity remaining are
considered stable. 50- 10% = partially
Stable. < 10% biological activity =
labile at the relevant temperature.
4. most of the non-allergenic food
proteins that have been tested are
digested by 30 seconds, while major
food allergens are stable, or produce
pepsin-stable fragments that are
detectable for from eight to 60
minutes.
5. Visual, Body Weight and Feed
Consumption, Carcass
Measurements, milk yield, milk
composition (e.g., fat, protein, and
lactose)

39. Analysis of transgenic high iron rice with Wild type

41. Regulatory authorities of India for
GM crops
One of the most robust regulatory systems in the world
Efforts of Ministry of Environment and forests (MoEF) & Department of
Biotechnology (DBT)
There are seven competent authorities to handle various issues viz.,
• Genetic Engineering Approval Committee (GEAC)
• Review Committee on Genetic Manipulation (RCGM)
• Recombinant DNAAdvisory Committee (RDAC)
• Monitoring and Evaluation committee (MEC)
• Institutional Biosafety Committee (IBSC)
• State Biotechnology Coordination Committee (SBCC)
• District Level Committee (DLC)
Central Government
undertaking
State Government
undertaking
“Rules for the manufacture, use, import, export and storage of hazardous
microorganism, genetically engineered organism or cells, 1989” notified under the
environment (Protection) Act, 1986

42. Procedure of approval of GM crops for
commercial release (DBT)
Source: Karihaloo & Kumar, 2009

43. Genome editing: Opening New Door of gene modification

44. Development of GM Crops might become a sustainable
solution to the people of developing countries
GENE MANIPULATION : One of the way to address
FOOD SECURITY
For next generation, One of the alternative for better nutrition