1. Biological and Environmental aspects of GM crop usage
Prof. Parthadeb Ghosh
UGC Emeritus Fellow
Plant Biotechnology Research Unit
Department of Botany
University of Kalyani

2. History of crop improvement
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

3. Green revolution Irrigation facilities Improved/hybrid seeds
Dr. MS Swaminathan
Dr. NE Borlaug
Irrigation facilities
Improved/hybrid seeds
Chemical fertilizers
Pest management
Farm credit
Political will

4. 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)

5. The Role of Biotechnology
Raise the yield ceiling and provide sustainable production systems
Agriculture
Resource Based Science Based Industry
Food Security ??

6. 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.

7. Building the Transgenes
ON/OFF Switch
Makes Protein
stop sign
CODING SEQUENCE
INTRON
poly A signal
PROMOTER
Plant Selectable
Marker Gene
Plasmid DNA
Construct
Plant Transgene
bacterial genes
antibiotic marker
replication origin

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

9. 21 crops, 107 events, 539 approvals,
22 countries, M ha
21 crops, 107 events, 539 approvals,
29 countries

10. Biotech crop countries and mega-countries

11. Total plantation in India = 8.0 M Ha
Others
1.6 M Ha
3.0 M Ha
1.40 M Ha

12. 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%

13. 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
Virus resistance
Cotton
Soybean
Tomato
Potato
Banana
Papaya
Cassava
TRANSGENICS
Delayed ripening
Tomato
Fungal resistance
Rice
Banana

14. *Expression of enzymes of β-carotene pathway in rice endosperm
NUTRITIONAL QUALITY
“Golden Rice”
                      
*Expression of enzymes of β-carotene pathway in rice endosperm
*Amelioration of Vitamin A deficiency

15. Bt Brinjal: Putting Science into Agriculture

16. Bt-Brinjal

17. 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

18. The Risks…..
Human Health
Environment

19. Human Health… Alteration in nutrition profile of the food
Introduction of toxins
Introduction of existing or new allergens

20. Environmental concerns…
Horizontal gene transfer
Effect on non target organisms
Development of resistance by pests

21. Societal concerns… Unfamiliarity with the technology
Lack of reliable information
Negative media opinion
Opposition by activists group
Mistrust of the industry

22. 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)

23. Is this food safe ?

24. Is the food safe… ???

25. Some known allergenic food sources

26. 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

27. Codex Alimentarius Commission
TO PROVIDE A SUITABLE FRAMEWORK FOR UNDERTAKING RISK ANALYSIS ON THE SAFETY AND NUTRITIONAL ASPECTS OF FOOD DERIVED FROM MODERN BIOTECHNOLOGY

28. The Principles Risk assessment : Risk Management :
Identification of hazard Nature and Severity
Risk Management :
Should be proportional to risk identified
Risk Communication :
Should involve all stake holders, should be transparent, all stages documented
Data can be obtained
developer, literature,
scientists, technical bulletins,
regulatory agencies
Intended and unintended effects
New and altered hazards
Changes in nutrients relevant to human health
Data should be based on
sound science, scientific
peer review
Food labeling,
conditional marketing approvals,
post-marketing monitoring

29. The Framework Core considerations Gene (s) Food/Feed Composition
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

30. The Molecular Characterization

31. 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

32. 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

33. Microparticle bombardment is characterised by
Introduction of full length of transgene
Transgene rearrangements
Transgene copy number can vary between
Multiple copies within an insert generally co- segregate as a transgenic locus
No species specificity

34. 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

35. 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

36. Allergy : Some background
A specific adverse immune reaction to a protein
Immediate IgE mediated
Allergy
Delayed Cell Mediated
Most allergic reactions are caused by specific IgE antibodies
The mechanism involved is development of IgE antibodies which upon re-exposure bind to mast cells and release histamines
Occurrence ranges between 2-4% in adults and 4-8 % in children (US, Europe)
Peanuts, milk, wheat, eggs, fish, soybeans, crustacean, tree nuts together accounts for over 90% cases (EU adds celery roots, mustard and sesame seeds)
Disease management by avoidance

37. The two steps….

38. The causative agents Food borne Air borne Other
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

39. 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 ???

40. 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

41. 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

42. 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

43. 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.

44. 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

45. 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??)

46. GM Product Classification (golden rice, high lys corn)
Insert
Codes for simple
Functional/storage
protein
Does not code
For protein
Codes for functional
enzyme
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)
Cry 1, 2, 3, CP4 EPSPS, NPT II and cry 1 F (except one 6 - mer match)
Insecticidal (Cry1, cry3
etc.), ug/g
Anti-fungal, ug/g
Storage protein (high
protein potato/high
met corn), mg/g
Herbicide tolerance
(EPSPS roundup, PAT
Soybean, rice or maize)
Nutri. Enhancement
(golden rice, high lys corn)
Altered FA Synthesis
Anti viral

47. Toxicological Studies
Food Ingredients
Food additives, contaminants, pesticide residues etc.
Grains from GM crops
Protein from the GM plant
Whole grain

48. Acute Toxicity Protein Characterization Toxicology Q’tative safety
Acute oral toxicity
(mice)
Limit dose
(2000 mg/kg, OECD)
Mortality, body wt.,
behavior, necropsy
Source, HOSU,
Mechanism of action
Specificity
Expression levels
Bioinformatics
Digestion/Heat Stability
Toxic
Yes No
Equivalence
SDS-PAGE, AA Composition, peptide finger
Printing, N terminal sequencing, glycosylation,
MALDI-TOF, Enzymatic/Biological activity
ILSI Guidelines, 2008

49. 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

50. Food from GM plant Food Characterization Toxicology Nutritional Eq…
Q’litative C’tion
HOSU
Comparison with
non GM isogenic
parental line
Subchronic rodent
dietary feeding
studies
(rats, 90 days)
Broiler chicken
(42 days)
GM Food
As Safe As
Rapid growing sp
Sensitive to
changed nutrition
Biochemistry,
Haematology
Histopathology
Organ wt etc
Compositional
Analyses
Agronomic
characters
Yes No
Reference non GM
Tolerance limit

51. 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

52. The choice should be based on objective science based decision

53. Indian Scenario
The regulatory framework for transgenic crop in India comprises following rules and guidelines
Rule 1989 under EPA of rDNA 1990
Seed policy of Res. in transgenic crop 1998
Ministry of Forests and Environment, GoI and Dept. of Biotechnology are implementing Agencies
Approvals
Rules & Policies
Guidelines
rDNA Advisory Committee (RDAC)
Rev Com on Gen Manipulations (RCGM)
Gen. Engg. Approval Committee (GEAC)
Institutional Biosafety Committee (IBSC)
State Biosafety coord committee (SBCC)
District Level Committee (DLC)
IBSC
RCGM (DBT)
GEAC (MoEF)

54. Biotechnology Policy of India, 2005 Some excerpts……
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
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
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
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

55. 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
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
Guidelines for research in transgenic plants : Separate guidelines formulated by DBT in 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
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.
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.

56. The Depts., Ministries and Institutions…
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
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

57. Indian Regulatory Framework
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
Rationale and benefits
Biology of plant system
Molecular biology and
transformation method
Part A
Competitive toxicant analysis
Potential for weediness
Risk during processing/handling
Gene and pollen transfer
Effect of diseases & pests
Field trial plans
Phenotype of plant,
fruits/seeds
Part B
Consequence to the
environment
Part C
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
Part D
Food Safety Evaluations

59. 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

60. Purpose
To have an in depth understanding of the framework for biosafety analysis of GM food
Molecular characterization
Toxicology studies
Allergenicity assessment
Bioinformatics

61. 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 ??

62. Biosafety
Protecting human and animal health and environment from possible side effects of the products of modern biotechnology such as genetically modified plants

63. 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

64. 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

65. The Mechanism …..
From Nagai et al, 2006

66. Choice of Algorithm
It is based on desired comparison type, computational resources available and goal
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

67. 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

68. 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)

69. 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

70. Transgenic crop under development and field trials in India
Crop Organization Gene
Brinjal IARI, New Delhi cry1Ab, cry1Ac
MAHYCO, Mumbai
Cauliflower MAHYCO, Mumbai cry1Ac
Sungrow Seeds Ltd., New Delhi
Cabbage Sungrow Seeds Ltd., New Delhi cry1Ac
Chickpea ICRISAT, Hyderabad cry1Ac, cry1Ab
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-tpCrtI
UDSC, New Delhi bar, barnase, barstar
Okra MAHYCO, Mumbai cry1Ac
Pigeonpea ICRISAT, Hyderabad cry1Ab + SBTI
MAHYCO, Mumbai cry1Ac
Potato CPRI, Simla cry1Ab
NCPGR, New Delhi Ama-1
Rice Directorate of Rice Research, Bacterial blight res, Xa-21,
Hyderabad
Osmania University, Hyderabad cry1Ab, gna gene,
IARI, New Delhi gna
MAHYCO, Mumbai Bt, chitinase, cry1Ac and Aa
MKU, Madurai cry1Ac
MSSRF, Chennai chitinase, B-1,3-glucanase
TNAU, Coimbatore chitinase
Sorghum MAHYCO, Mumbai cry1Ac
Tomato MAHYCO, Mumbai cry1Ac, OXDC
Source: Department of Biotechnology, Government of India

71. Thank You