Biological and Environmental aspects of
GM crop usage
Prof. Parthadeb Ghosh
UGC Emeritus Fellow
Plant Biotechnology Research Unit
Department of Botany
University of Kalyani
History of crop improvement
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By trial and error for almost 9900 years
By scientific principles of breeding for last 110 years
By chemical induced mutation for last 85 years
By rDNA technology last 34 years
Green revolution
Dr. MS Swaminathan
Dr. NE Borlaug
Irrigation facilities
Improved/hybrid seeds
Chemical fertilizers
Pest management
Farm credit
Political will
CHALLENGES AHEAD
-Population in 2050 AD: 1.5 B
-Shrinking area of cultivated land
-Diminishing water resources
-Malnutrition and undernourishment
-Deterioration in soil quality
-Climate change (global warming)
The Role of Biotechnology
• Raise the yield ceiling and provide sustainable production
systems
Agriculture
Resource Based
Science Based Industry
Food Security ??
Genetically Modified (GM) Crops
What is a GM crop?
GM crops are genetically improved and contain a
gene or genes from the same or a different species
artificially inserted in its genome.
Tissue Culture & Transformation – gives the
maximum flexibility for moving genes within or
between species.
Building the Transgenes
ON/OFF Switch
PROMOTER INTRON
Makes Protein
CODING SEQUENCE
Plant Transgene
Plant Selectable
Marker Gene
Plasmid DNA
Construct
bacterial genes
•antibiotic marker
•replication origin
stop sign
poly A signal
Leaf Disk Method for A. t. Mediated Transformation
Leaf Disk Preparation
Co-cultivation with Agrobacterium
Regeneration of Shoots
Selection for Transformation
22 countries, 117.7 M ha
21 crops, 107 events, 539 approvals,
29 countries
Biotech crop countries and mega-countries
Total plantation in India = 8.0 M Ha
0.80 M Ha
0.80 M Ha
3.0 M Ha
1.40 M Ha
Others
1.6 M Ha
Bt- COTTON
Bt-cotton - First GM crop – 2002
Second in global cotton production
Area – 8.0 million hectares – 2008
Yield gain - 31%
Reduction in pesticide sprays – 39%
TARGET CROPS AND TRAITS
IN THE ICAR NETWORK
Insect pest resistance
• Stem borer
 Rice
 Sorghum
 Maize
• Pod Borer
 Pigeon pea
 Chick pea
• Boll worm
 Cotton
• Fruit borer
 Tomato
 Brinjal
• Aphid
 Brassica
Drought stress
Brassica
TRANSGENICS
Delayed ripening
 Tomato
Virus
resistance
Cotton
Soybean
Tomato
Potato
Banana
Papaya
Cassava
Fungal resistance
Rice
Banana
NUTRITIONAL QUALITY
“Golden Rice”
*Expression of enzymes
of β-carotene
pathway in rice
endosperm
*Amelioration of
Vitamin A deficiency
Bt Brinjal: Putting Science into Agriculture
Bt-Brinjal
Food consisting of living organisms, e.g.
soybean, maize
Food derived from GMO e.g. soy oil, corn
flour
Foods containing ingredients produced by
GMO, e.g. Vitamins or essential amino acids
Foods containing ingredients processed by
enzymes produced by GMO, e.g. high
fructose corn syrup produced using
recombinant glucose isomerase
The Risks…..
Human Health
Environment
Human Health…
• Alteration in nutrition profile of the food
• Introduction of toxins
• Introduction of existing or new allergens
Environmental concerns…
• Horizontal gene transfer
• Effect on non target organisms
• Development of resistance by pests
Societal concerns…
• Unfamiliarity with the technology
• Lack of reliable information
• Negative media opinion
• Opposition by activists group
• Mistrust of the industry
Traditionally…….
• Hardly anything what we eat today has been
assessed for food safety
• Even food known to be toxic or allergic or contain
anti nutrients are being used based on our
experience and history
(Potatoes, tomatoes, eggs, milk, peanuts, fish, wheat
etc)
Is this food safe ?
Is the food safe… ???
Some known allergenic food sources
What is safety ?
• OECD defined it as “ the one which , as far as we know,
and with the exception of some individual, who me be
sensitive or allergic, when consumed in moderation over a
period of time does not result in identifiable harm to the
consumer”
• Absolute safety is difficult to prove
• One can show the absence of evidence of any harm at the
most
Codex Alimentarius Commission
TO PROVIDE A SUITABLE FRAMEWORK FOR
UNDERTAKING RISK ANALYSIS ON THE SAFETY AND
NUTRITIONAL ASPECTS OF FOOD DERIVED FROM
MODERN BIOTECHNOLOGY
The Principles
• Risk assessment :
Identification of hazard
Intended and unintended effects
New and altered hazards
Changes in nutrients relevant to human health
• Risk Management :
Nature and Severity
Data can be obtained
developer, literature,
scientists, technical bulletins,
regulatory agencies
Data should be based on
sound science, scientific
peer review
Should be proportional to risk identified
• Risk Communication :
Food labeling,
conditional marketing approvals,
post-marketing monitoring
Should involve all stake holders, should be transparent, all stages
documented
The Framework
Core considerations
Gene (s)
•Source (s)
•Molecular characterization
•Insert/copy no./integrity/
stability
Food/Feed Composition
•Proximate analysis
•Key nutrients/anti nutrients
•Animal performance
Protein
•History of safe use &
Consumption
•Function/specificity/
mode of action
•Levels
•Toxicology & allergenicity
Environmental
The Molecular Characterization
Molecular Characterization
• Rigorous molecular characterization of each transgenic plant must be
completed
The following should be considered
The transformation system
(i) Agrobacterium mediated
(ii) Microparticle bombardment
Molecular characterization of the inserted DNA
(i) Insert number
(ii) Insert composition
Genetic stability of the introduced trait
(i) Segregation analysis
(ii) Integron stability
Transformation system
• A. tumefaciens mediated transformation is characterised by
– Low transgene copy number
– Limited molecular rearrangements in the
insert
– Higher transformation efficiency
However it may show species specificity
Microparticle bombardment is
characterised by
• Introduction of full length of transgene
• Transgene rearrangements
• Transgene copy number can vary between 120.
• Multiple copies within an insert generally cosegregate as a transgenic locus
• No species specificity
The information required…
• All the genetic elements (promoter, leader,
terminator, marker etc) transferred along with
citation
• Detailed map of plasmid used as a vector indicating
location, orientation, size etc of genetic elements
• Relevant restriction enzyme sites, location of
primers used in PCR, regions used as a probe
Allergenecity
• One of the components of overall risk analysis of
GM Food.
• Potential or life threatening allergies are relatively
rare
• It is important that a food allergen does not enter
the food supply
Allergy : Some background
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A specific adverse immune reaction to a protein
Immediate
IgE mediated
Delayed
Cell Mediated
Allergy
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Most allergic reactions are caused by specific IgE antibodies
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The mechanism involved is development of IgE antibodies which upon re-exposure bind to
mast cells and release histamines
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Occurrence ranges between 2-4% in adults and 4-8 % in children (US, Europe)
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Peanuts, milk, wheat, eggs, fish, soybeans, crustacean, tree nuts together accounts for over
90% cases (EU adds celery roots, mustard and sesame seeds)
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Disease management by avoidance
The two steps….
The causative agents
• Food borne
Peanut, tree nut, milk, eggs, crustaceans, (wheat,
soybean), celery, sesame, kiwi, mustard
• Air borne
Pollen, weeds, molds, dust mites, latex
• Other
Bee and ant venom
Three Questions
• Is the novel protein an existing allergen ?
• Is the newly expressed protein going to cause
allergic cross reactivity ??
• Is the new protein likely to sensitize and
become an allergen ???
The Tests
• Bioinformatics
• Specific serum testing
• Searchable specific allergen databases
(1313 in version 8.0 of known or putative
allergens)
NCBI (all sequences)
• Review scientific literature for evidence of
allergenicity
Guidelines for Allergenicity Assessment
• International Food Biotechnology Council and
International Life Sciences Institute (IFBC-ILSI),
1996
• Food and Agriculture Organization and World
Health Organization (FAO/WHO), 2001
• Codex Alimetarius Commission (CAC), 2003
Databases
• Comprehensive databases like nrNCBI (GenPept,
SwissProt, PIR, RPF, PDB) and exPASy
• Swiss Prot is a highly annotated database with a lot
of valuable biological information
• Several specialized databases are also available
Some points to ponder…
• IFBS-ILSI and FAO/WHO guidelines follow a decision tree approach for
evaluating risk of allergenicity whereas CAC follows a weight of evidence
approach
(Decision tree approach appears rigid as no single criteria is sufficiently predictive)
• IFBC-ILSI recommends in vivo clinical testing (SPT) and DBPCFC if a
single 8 aa match is found but no cross reactivity in vitro with IgE
• FAO/WHO found in vivo clinical testing “impractical” or even
“unethical”. Recommends 6 aa match rather than 8 aa match and
targeted serum* and animal model testing
(*even when the transgenic protein does not show sequence similarity and cross reactivity
in specific serum test)
• CAC recommends a 35% identity over an 80 aa window to be a sufficient
conservative prediction for potential cross reactivity.
Interpretation of results
• Evaluate the matches : E score is more useful than bit
score or % identity
• A low E score with alignment over the entire sequence
length is significant
• Review literature extensively
Issues…. ?????
• Animal Model tests : No validated models as yet
• Targeted serum IgE tests : most probably will lead to false
positive results ???
• Heat Stability ???
(CAC, 2003 guidelines and weight of evidence approach
appears practical??)
GM Product Classification
Insert
Does not code
For protein
Anti viral
Codes for simple
Functional/storage
protein
Insecticidal (Cry1, cry3
etc.), ug/g
Anti-fungal, ug/g
Storage protein (high
protein potato/high
met corn), mg/g
Codes for functional
enzyme
Herbicide tolerance
(EPSPS roundup, PAT
Soybean, rice or maize)
Nutri. Enhancement
(golden rice, high lys corn)
Altered FA Synthesis
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No significant sequence match with the aforementioned GM proteins as per data of
major biotech companies around the world based on bio-informatics (no > 50%
overall or > 35% identity in 80 aa match)
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Cry 1, 2, 3, CP4 EPSPS, NPT II and cry 1 F (except one 6 - mer match)
Toxicological Studies
• Food Ingredients
Food additives, contaminants, pesticide
• Grains from GM crops
Protein from the GM plant
Whole grain
residues etc.
Acute Toxicity
Protein
Characterization
Source, HOSU,
Mechanism of action
Specificity
Expression levels
Bioinformatics
Digestion/Heat Stability
Toxicology
Q’tative safety
Acute oral toxicity
(mice)
Limit dose
(2000 mg/kg, OECD)
Mortality, body wt.,
behavior, necropsy
Toxic
Yes
Equivalence
SDS-PAGE, AA Composition, peptide finger
Printing, N terminal sequencing, glycosylation,
MALDI-TOF, Enzymatic/Biological activity
No
ILSI Guidelines, 2008
Grains from GM crops
• Codex approach needs to be slightly modified
• Foods are generally considered safe but absolute
safety is difficult to establish
• Take off point is ‘substantial equivalence’
• Objective is to establish that food from GM plant is
‘as safe as….’ the conventional counterpart
Food from GM plant
Food
Characterization
Toxicology
HOSU
Comparison with
non GM isogenic
parental line
Subchronic rodent
dietary feeding
studies
(rats, 90 days)
Compositional
Analyses
Agronomic
characters
Biochemistry,
Haematology
Histopathology
Organ wt etc
Reference non GM
Tolerance limit
Nutritional Eq… Q’litative C’tion
Broiler chicken
(42 days)
GM Food
As Safe As
Rapid growing sp
Sensitive to
changed nutrition
Yes
No
Animal tests may not be warranted….
• Source not known to synthesize toxin protein (s)
• The protein has a history of safe use
• Amino acid sequence analysis lacks identity with known
toxins
• Protein is easily digested/degraded
• Protein is unstable to heat and other processing
The choice should be based on objective
science based decision
Indian Scenario
The regulatory framework for transgenic crop in India comprises
following rules and guidelines
Rules & Policies
Rule 1989 under EPA of 1986
Seed policy of 2002
Guidelines
rDNA 1990
Res. in transgenic crop 1998
Ministry of Forests and Environment, GoI and Dept. of Biotechnology
are implementing Agencies
rDNA Advisory Committee (RDAC)
Rev Com on Gen Manipulations (RCGM)
Gen. Engg. Approval Committee (GEAC)
Approvals
Institutional Biosafety Committee (IBSC)
State Biosafety coord committee (SBCC)
District Level Committee (DLC)
IBSC
RCGM (DBT)
GEAC (MoEF)
Biotechnology Policy of India, 2005
Some excerpts……
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A comprehensive and integrated view should be developed of r-DNA and non
r-DNA
based applications of biotechnology with other technological components required for
agriculture as a whole
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Regulatory requirement in compliance with Cartagena Protocol, and other international
treaties and protocol for biosafety, germplasm exchange and access and the guiding
principles of Codex Alimentarius will be implemented through inter ministerial consultative
process
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availability, access, release and efficient system for biosafety assessment of GMOs and
products thereof; safe use of approved technologies and prevention of unauthorized ones;
building public trust
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India : a signatory and has ratified, along with 138 countries, the Cartagena Protocol
which provide guidelines for safe handling and trans-boundary movement of LMOs
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Rules, 1989 : The Ministry of Environment & Forests, GoI notified the rules
and procedures under EPA of 1986 covering areas of research as well as large
scale applications of GMOs and products made there from throughout India
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rDNA Guidelines : Formulated by DBT, revised in 1994, cover R & D activities
on GMOs, transgenic crops, large scale production, deliberate release of GMOS
in environment, shipment and import for lab. research
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Guidelines for research in transgenic plants : Separate guidelines formulated by
DBT in 1998. Covers R & D in plants, development of transgenics, their growth
in soil for molecular and field evaluation, also include guidelines for toxicity and
allergenicity evaluation of transgenic seeds, plants or plant parts
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Seed Policy : Issued by Ministry of Agriculture in 2002 contains a separate
section (no. 6) on transgenic plants. It states that all transgenics will be tested for
environment and biosafety before commercial release, agronomic traits to
evaluated for at least 2 seasons under the all India coordinated project trials
(AICPT) by ICAR. Seeds to be registered as per the provisions of seed act. Post
market monitoring for 3-5 years by MoA.
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Prevention of Food Adulteration Act : Issued by Ministry of health & Family
Welfare, GoI for assuring quality and safety of food and encourage fair trade
practices.
The Depts., Ministries and Institutions…
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MoEF : Holds GEAC, apex body that gives approval for manufacture, sale, import
and export of all GMOs and products thereof
DBT : Holds RCGM, approves research and small scale trials etc of GMOs
MoHFW : Regulates PFA Act
ICMR : Advisory body for MoHFW
MoA : Nodal Ministry for Agriculture growth. Implements Seed Policy, 2002
Ministry of Commerce & Industry : Formulates EXIM policy
Ministry of Food Processing Industries
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National Institute of Nutrition, Hyderabad
Central Food Technology Research Institute, Mysore
Defense Food Research Laboratory, Mysore
Industrial Toxicology Research Institute, Lucknow
National Bureau of Plant Genetic Resources, New Delhi
Centre for DNA Finger Printing and Diagnostics, Hyderabad
Indian Regulatory Framework
Part A
•Rationale and benefits
•Biology of plant system
•Molecular biology and
transformation method
Part B
•Field trial plans
•Phenotype of plant,
fruits/seeds
Part C
Consequence to the
environment
Part D
Food Safety Evaluations
Source of gene, cloning strategy,
Sequence details of inserted gene
Vector and method of t’formation
Genetic analysis and segregation stability
Biochemistry of expressed gene product
by chemical, immunological methods
Competitive toxicant analysis
Potential for weediness
Risk during processing/handling
Gene and pollen transfer
Effect of diseases & pests
Nutritional studies,
acute and chronic toxicity
Allergenecity testing
Classical animal feeding trials
Immunotoxicological studies
Gut toxicological studies
Effect of transgene product on gene
integration, regulation & expression
Bioinformatics….
• Learnt and practiced FASTA and BLASTP local sequence alignment
tools for matching sequences of interest with online databases
• Result analyses based on E score, % identity, similarity and literature
review
• E-score is inversely related to similarity of two proteins & depends on
overall length of joined sequence alignments, quality (similarity, identity)
of overlap & database size.
• Codex (2003) recommendations for bioinformatics search using
FASTA/BLASTP algorithms for allergenecity assessment
35% identity over 80 aa sliding window
50% identity over entire sequence length
Purpose
• To have an in depth understanding of the
framework for biosafety analysis of GM food
– Molecular characterization
– Toxicology studies
– Allergenicity assessment
– Bioinformatics
The outcome
• Pretty good understanding of different frameworks for analysis (ILSI,
FAO/WHO, Codex)
• Understanding from risk assessor, academician and developers points of
view
• Personal interactions for studies wrt toxicology, allergenecity, genetic
characterization and bioinformatics
• Industrial visits and interactions
• A well informed University Professor
• Transfer to students of Masters Program (MAP)
• Participation in capacity building programs (HRD &T)
• Strengthening technical capacity to assess, manage and monitor risks
associated with GMO ?
• Assist the regulatory agency in the home country ??
Biosafety
Protecting human and animal health and environment from
possible side effects of the products of modern biotechnology
such as genetically modified plants
• Many food considered safe based on history of safe
consumption
• Hazards associated are analyzed as per guidelines of
CAC and CWPRA
• Being used for long time for chemical, microbial and
nutritional factors
• May need suitable modification for whole food
Limitations….
• Disproportionate attention relative to information it imparts
in terms of food safety
• In the absence of phenotypic data, unlikely to predict
unforeseen effects on nutrient profile
• No correlation between copy number and safety
• May provide information on positional and pleiotropic
effects and gene silencing
• Ensures appropriate characterization of the genetic
modification
The Mechanism …..
From Nagai et al, 2006
Choice of Algorithm
• It is based on desired comparison
computational resources available and goal
type,
• FASTA, BLAST P and Ssearch are the algorithm of
choice for protein sequence alignments
• Low specificity filter help avoid false positive
statistically significant scores
• Scoring matrix and Gap penalties : use default
Amino acid sequence comparison
• Overall FASTA vs AllergenOnline
(>50% overall identity or E score <1 e-7 )
(Most predictive, allergic cross reactivity most likely)
• FASTA scanning over 80 aa window
(>35% identity indicates some chances of reactivity)
• Scanning 6 or 8 aa segments : ???
• If identity detected above the specified limit, go for specific
serum testing
Serum IgE test
• Serum donors must have relevant and proven allergy
• The test must be specific and validated (should be able to detect
binding to conformational or linear epitopes)
• Must include positive (who react to gene source or sequence matched
allergen) and negative control allergic sera and
proteins/extracts
• Use protein free from N linked glycosylation, if possible
• No established claims of allergenecity of new proteins in GM
crops (upto Dec., 2007)
Some more considerations………
• Many potent food allergen are stable in pepsin
• Many are abundant protein, up to 1% of total proteins in food
• If the transgene is transferred to known allergen source, then changes in
endogenous allergenicity needs to be monitored
• However, it is necessary to establish natural variability of allergenicity
among the non GM varieties.
•
Special attention must be given when a transcription activator is
transferred or transgene is transferred in the coding region for an
allergen
Transgenic crop under development and field trials in India
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Crop
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Brinjal
Organization
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IARI, New Delhi
MAHYCO, Mumbai
Cauliflower
MAHYCO, Mumbai
Sungrow Seeds Ltd., New Delhi
Cabbage
Sungrow Seeds Ltd., New Delhi
Chickpea
ICRISAT, Hyderabad
Groundnut
ICRISAT, Hyderabad
Maize
Monsanto, Mumbia
Mustard
IARI, New Delhi
NRCWS, Jabalpur
TERI, New Delhi
UDSC, New Delhi
Okra
MAHYCO, Mumbai
Pigeonpea
ICRISAT, Hyderabad
MAHYCO, Mumbai
Potato
CPRI, Simla
NCPGR, New Delhi
Rice
Directorate of Rice Research,
Hyderabad
Osmania University, Hyderabad
IARI, New Delhi
MAHYCO, Mumbai
MKU, Madurai
MSSRF, Chennai
TNAU, Coimbatore
Sorghum
MAHYCO, Mumbai
Tomato MAHYCO, Mumbai
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Source: Department of Biotechnology, Government of India
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Gene
cry1Ab, cry1Ac
cry1Ac
cry1Ac
cry1Ac, cry1Ab
IPCVcp, IPCV replicase,
CP4 EPSPS
CodA, Osmotin,
bar, barnase, barstar
Ssu-maize, Psy, Ssu-tpCrtI
bar, barnase, barstar
cry1Ac
cry1Ab + SBTI
cry1Ac
cry1Ab
Ama-1
Bacterial blight res, Xa-21,
cry1Ab, gna gene,
gna
Bt, chitinase, cry1Ac and Aa
cry1Ac
chitinase, B-1,3-glucanase
chitinase
cry1Ac
cry1Ac, OXDC
Thank You