Biological and Environmental aspects of GM crop usage

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 • • 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 r. DNA 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 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 : Should be proportional to risk identified • Risk Communication : Nature and Severity Data can be obtained developer, literature, scientists, technical bulletins, regulatory agencies Data should be based on sound science, scientific peer review 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 • • A specific adverse immune reaction to a protein Immediate Ig. E mediated Delayed Cell Mediated Allergy • Most allergic reactions are caused by specific Ig. E antibodies • The mechanism involved is development of Ig. E 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

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 http: //www. Allergen. Online. com (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 nr. NCBI (Gen. Pept, Swiss. Prot, PIR, RPF, PDB) and ex. PASy • 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 Ig. E • 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 Ig. E 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 (Cry 1, cry 3 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 • 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, CP 4 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 r. DNA 1990 Res. in transgenic crop 1998 Ministry of Forests and Environment, Go. I and Dept. of Biotechnology are implementing Agencies r. DNA 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) Approvals IBSC RCGM (DBT) GEAC (Mo. EF)

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

• Rules, 1989 : The Ministry of Environment & Forests, Go. I 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 • r. DNA 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 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 • 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 Mo. A. • Prevention of Food Adulteration Act : Issued by Ministry of health & Family Welfare, Go. I for assuring quality and safety of food and encourage fair trade practices.

The Depts. , Ministries and Institutions… • • Mo. EF : 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 Mo. HFW : Regulates PFA Act ICMR : Advisory body for Mo. HFW Mo. A : 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

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 Allergen. Online (>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 Ig. E 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 • Crop • • • Brinjal Organization • • 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 • Source: Department of Biotechnology, Government of India • • • Gene cry 1 Ab, cry 1 Ac, cry 1 Ab IPCVcp, IPCV replicase, CP 4 EPSPS Cod. A, Osmotin, barnase, barstar Ssu-maize, Psy, Ssu-tp. Crt. I bar, barnase, barstar cry 1 Ac cry 1 Ab + SBTI cry 1 Ac cry 1 Ab Ama-1 Bacterial blight res, Xa-21, cry 1 Ab, gna gene, gna Bt, chitinase, cry 1 Ac and Aa cry 1 Ac chitinase, B-1, 3 -glucanase chitinase cry 1 Ac, OXDC

Thank You