Transgenic crop production and Biosafety concerns

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The development of transgenic plants is the result of integrated application of rDNA technology, Gene transfer methods and Tissue culture techniques. The major biosafety concerns are divided into different categories, namely, health, nutritional, environmental, ecological, socio-economic, and ethical concerns. These concerns include those arising due to properties of transgenic plants themselves, those resulting from the spread of the transgenes to other organisms, and also those resulting from their release into the environment. Biosafety regulations are required to assess the safety of transgenic crops before its release in to environment. The controversy about the health safety of transgenic foods is complex and good science and its communication are required in order to find solutions.

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Transgenics production and Bio-Safety concerns Credit seminar on 19 th April, 2014 Department of Genetics and Plant Breeding

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contents…. Top 10 facts : IASSS Report 2 013 Transgenic organisms/GMOs/GM Crops-definitions Transgenic crops- Types Building of Transgene and T ransgenic plant Techniques for gene transfer- Direct and indirect methods Transgenic crops – Applications Story of Bt Cotton, Golden rice, F lavr savr tomato Biosafety regulation agencies in World and India Major biosafety concerns, issues, risks and recommendations Action Plan for the Development and Utilization of GM Crops Tests for assessing GMO safety Supporting articles

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2013 was the 18th year of successful commercialization of biotech crops . Biotech crops were first commercialized in 1996 . Remarkably , Hectarage of biotech crops increased every single year between 1996 to 2013, with 12 years of double-digit growth rates, reflecting the confidence and trust of millions of risk-averse farmers around the world. Fact # 01 2013 Report

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Biotech crop hectares increased by more than 100-fold from 1.7 mha in 1996, to over 175 mha in 2013 . In 2013, hectarage of biotech crops grew by 5 million hectares, at an annual growth rate of 3%. It is important to note that more modest annual gains, and continued plateauing, are predicted for the next few years due to the already optimal (between 90% and 100%) adoption rates for the principal biotech crops, leaving little or no room for expansion. Fact # 02

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Number of countries growing biotech crops and stacked traits Of the 27 countries which planted biotech crops in 2013, 19 were developing and 8 were industrial countries. Stacked traits occupied 47.1 million hectares, or 27%. Fact # 03

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For the second consecutive year, in 2013, developing countries planted more hectares than industrial countries. Notably , developing countries grew more, 54% (94 million hectares) of global biotech crops in 2013 than industrial countries at 46% (81 million hectares). Fact # 04

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Number of farmers growing biotech crops. In 2013, a record 18 million farmers, up 0.7 million from 2012, grew biotech crops – remarkably over 90%, or over 16.5 million. In 2013, a record 7.5 million small farmers in China and another 7.3 million in India, elected to plant more than 15 million hectares of Bt cotton, because of the significant benefits it offers. In 2013, almost 400,000 small farmers in the Philippines benefited from biotech maize. Fact # 05

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The top 5 countries planting biotech crops – deployment of the first drought tolerant maize and stacked HT/IR soybean . Fact # 06

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Status of biotech crops in Africa. The continent continued to make progress with South Africa benefiting from biotech crops for more than a decade. Both Burkina Faso and Sudan increased their Bt cotton hectarage by an impressive 50% and 300%, respectively, in 2013 . Status of biotech crops in the EU. Five EU countries planted a record 148,013 hectares of biotech Bt maize, up 15% from 2012. Spain led the EU with 136,962 hectares of Bt maize, up 18% from 2012 with a record 31% adoption rate in 2013. Fact # 07 Fact # 08

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Diagrammatic illustration representing the area (in million hectares) covered by biotech crops in major countries of the world. 10

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Benefits offered by biotech crops. From 1996 to 2012, biotech crops contributed to Food Security, Sustainability and the Environment/Climate Change Future Prospects. Cautiously optimistic with more modest annual gains expected due to the already high rates of adoption (90% or more) in the principal biotech crops in mature markets in both developing and industrial countries. Bangladesh, Indonesia and Panama approved biotech crop planting in 2013 with plans for commercialization in 2014. Fact # 09 Fact # 10 (James, 2013 )

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Only one crop approved 14 crops under various stages of contained field trials Include brinjal , cotton, cabbage, groundnut, pigeon pea, mustard, potato, sorghum, tomato, tobacco, rice, okra and cauliflower Traits include insect resistance, herbicide tolerance, virus resistance, nutritional enhancement, salt tolerance, fungal resistance Indian status of Transgenic crops

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Traits Crops Insect resistance Blackgram , brinjal , cabbage, cauliflower, chickpea, cotton, maize, pigeon pea, potato, rice, tobacco, tomato, wheat Disease resistance Blackgram , brinjal , chickpea, coffee, rice, tomato Herbicide tolerance Blackgram , cotton, mustard, rice   Stress tolerance Mustard, rice   Edible vaccines Muskmelon, tomato   Fruit ripening Banana, potato, tomato   Nutrition enhancement Mustard, potato Transgenic research on other crops in I ndia

What are transgenic organisms ??:

What are transgenic organisms ?? An organism whose genome has been altered by the inclusion of foreign genetic material. This foreign genetic material may be derived from other individuals of the same species., from wholly different species or synthetic sources ( Verma , 2013). Normal plant Transgenic plant transgene

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GMOs - Genetically modified organisms GMO - an organism that expresses traits that result from the introduction of foreign DNA Originally a term equivalent to transgenic organism TRANASGENIC CROPS Generally a transgenic crop contains one or more genes that have been inserted artificially either from an unrelated plant or from different species altogether. GMOs and TRANSGENIC ORGANISMS

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Second generation of GM crops Increased levels of protein Modified and healthier fats Modified carbohydrates Improved flavor characteristics Increased levels of micronutrients Third generation of GM crops Resistance to abiotic stress “ Pharmaplants ” First-generation GM crops Herbicide resistance:- Corn, Soybean, rice, and Sugar beet Insect Pest resistance:- Corn, rice tomato and potato Viral resistance:- Papaya, Squash and potato Slow ripening and softening- Tomato and melon Improved oil quality -Canola and soybean Male sterility - Canola and corn 16 Types of GM/Transgenic crops

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

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How to make Transgenic Plants Overview of the process for creating transgenic plants/ Crop Genetic Engineering cycle 1.Locating gene for plant trait. 2.Designing gene for insertion. 3.Transformation. 4.Selection and Regeneration. 5.Plant Breeding and Testing.

Techniques for plant genetic transformation:

Techniques for plant genetic transformation Indirect method - Agrobacterium mediated gene transfer Direct methods - Particle bombardment ( biolistics ) Microprojectile gun method Electroporation Silicon carbide fibres Polyethylene glycol (PEG)/protoplast fusion Liposome mediated gene transfer

Why Agrobacterium ??:

Why Agrobacterium ?? The tumorigenic host plant species for range A.tumefaciens include: Large number of dicots and Gymnosperms. Agrobacteria are naturally occurring, ubiquitous soil borne pathogens. A. tumefaciens causes crown gall disease ( tumors ) A. rhizogenes causes root hair disease (hairy root) Agrobacterium genetically transforms its host by transferring a well-defined DNA segment from its tumour inducing plasmid to the host cell genome Tumor Agrobacterium tumefaciens 1907: Smith and Townsend demonstrated that Agrobacterium tumefaciens causes crown gall tumors. Later, causative agent of hairy root disease was determined to be Agrobacterium rhizogenes .

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T-DNA Contains oncogenes and opine synthesis genes & is transferred into host plant genome. vir region Regulates the transfer of T-DNA into plant nucleus. Opine catabolism regions Produce enzymes necessary for the utilization of opines by Agrobacterium. Conjugative transfer ( oriT or tra ) region Functions in conjugative transfer of the plasmid. Origin of replication For propagation in Agrobacterium . Functional regions of Ti plasmid

The basic protocol used for any Agrobacteruim mediated transformation experiments :

The basic protocol used for any Agrobacteruim mediated transformation experiments 1. Identify a suitable explants 2. Co-cultivate with the Agrobacterium 3. Kill the Agrobacterium with a suitable antibiotic 4 . Select for transformed plant cells 5. Regeneration of whole plant Agrobacterium mediated transformation methods are thought to induce less rearrangement of the transgene. Lower transgene copy number that direct DNA delivery methods. Successful production of transgenic plants depends on the suitable transformation protocols. Practical application of Agrobacterium-mediated plant transformation

Gene gun method:

“ Biolistics ” – shotgun cloning Coat gold or tungsten particles with Plasmid DNA DNA is driven by helium blast (old days gunpowder driven pistons) Particles fired into plant tissue at 430 m/sec Can be used against cells or whole plants (mono or dicots) Good for plants that cannot be transformed by Agrobacterium, e.g. most monocot Can be used with all plants or cells or organelles Drawbacks include tissue damage and low efficiency Lots of rearrangement of integrated DNA – can lead to instable gene transfer Gene gun method

Electroporation:

Electroporation In this technique,short pulses of high voltage are applied to protoplasts which make temporary pores in the plasma membrane to increase their permeability and facilitate the uptake of foreign gene. Advantages and disadvantages: Both intact cells and tissue can be transformed. The efficiency of transformation depends upon the plant materials, electroporation and tissue treatment conditions used for transformation. ~40 to 50% incubated cells receive DNA ~50% of the transformed cells can survive

Microinjection method:

Microinjection method Microinjection involves direct physical approach in depositing DNA into specific target cells. The protoplasts are immobilised in agarose or on glass slides coated with polylysine or by holding them under suction by a micropipette. The exogenous DNA of interest is taken in microinjector and then directly delivered inside the cell. Micromanipulator is used for microinjection of the DNA. A maximum of 40-50 protoplasts can be microinjected in one hour. Successful transformation by microinjection of cells has been achieved in tobacco , alfalfa etc. Holding peppite Nucleus Host cells Micro injection needle

Polyethylene glycol (PEG)/Protoplast fusion method:

Polyethylene glycol (PEG)/Protoplast fusion method Plant protoplast can be transformed with naked DNA by treatment with PEG in the presence of divalent cations e. g., Calcium. PEG and divalent cations destabilize the plasma membrane of the plant protoplast and rendered it permeable to naked DNA. DNA enters the nucleus and integrates into the host genome. Disadvantage and advantages: Regeneration of fertile plants from protoplasts is a problematic for some species. The DNA used for transformation is also susceptible to degradation and rearrangement. Despite the limitations, the technique have the advantages and protoplast can isolated and transformed in number of plants species.

Silicon carbide fibres-Whiskers:

Silicon carbide fibres -Whiskers Plant materials (Cells in suspension culture, embryos and embryo-derived callus) is introduced into a buffer containing DNA and the silicon fibers which is then vortexed . The fibers (0.3-0.6 μm in diameter and 10-100μm long) penetrate the cell wall and plasma membrane, allowing the DNA to gain access to the inside of the cells. Disadvantages and advantages The drawbacks of this technique relate to the availability of suitable plant material and the inherent dangers of the fibers, which require careful handing. Many cereals, produce embryonic callus that is hard and compact and not easily transformed with this technique. Despite the some disadvantages, this method is recently used for successful transformation of wheat, baerly , and maize without the need to cell suspension.

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Liposome mediated gene transfer/LIPOFECTION It is a technique used to inject genetic material into a cell by means of liposomes, which are  vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer. Lipofection generally uses a positively charged (cationic) lipid to form an aggregate with the negatively charged (anionic) genetic material .   The main advantages Its high efficiency, its ability to transfect all types of nucleic acids in a wide range of cell types, its ease of use, reproducibility, and low toxicity. In addition, this method is suitable for all transfection applications. S creening assay has also shown good efficiency in some in vivo models.

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The ultimate goal of transgenics is to improve the crops, with desired traits Resistance to biotic stresses i.e. resistance to diseases caused by insects, virus, fungi and bacteria. Resistance to abiotic stresses i.e. herbicides, temperature (heat, chilling, freezing), drought, salinity, ozone, intense light. Improvement of crop yield, and quality e.g. longer shelf life of fruits and flowers. Transgenic plants with improved nutrition. Transgenic plants as bioreactors for the manufacture of commercial products e.g. proteins, vaccines and biodegradable plastics. Applications of Transgenic crops/GM crops

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Gene Crystal shape Protein size (KD) Insect actively Cry I A(a), A(b), A(c), B, C, D, E, F, G Bipyramidal 130 – 138 Lepidopteran larva Cry II (Sub group) A, B, C Cuboidal 69 - 71 Lepidoptera, diptera Cry III (Sub group) A, B, C Flat irregular 73 – 74 Coleoptara Cry IV (Sub group) A, B, C, D Biopyrimidal 73 – 134 Diptera Cry V – IX Various 35 – 129 Various The organism Bacillus thuringiensis was discovered in 1911 as a pathogen in flour moth, Thuringia, Germany. Later it was commercially utilized as bio pesticides in France in 1938 and then in USA in 1950 for the toxin produced by this bacterium. From 1950 onwards the bio pesticides containing this soil bacterium were popular. In 1992 the gene which is responsible for the toxin production was introduced to the cotton crop was grown in six locations in USA. Development and Genetics of Bt – Cotton

Story of Golden Rice Synthesis:

Story of Golden Rice Synthesis Two Daffodil genes and one bacterial gene Erwinia uredovora were cloned into agrobacterium T DNA and inserted into rice genome to generate needed enzymes Phytoene synthase & Lycopene- b -cyclase Carotene desaturase T DNA Germ-line transformation with agrobacterium X Cross T-formed rice with genes T-formed rice with gene Progeny rice plant with complete b carotene pathway

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 -Carotene Pathway in Rice Geranylgeranyl diphosphate Phytoene Lycopene  -carotene (vitamin A precursor) Phytoene synthase Phytoene desaturase Lycopene -beta- cyclase ξ-carotene desaturase Problem: Rice lacks these enzymes Normal Vitamin A “Deficient” Rice Daffodil gene Single bacterial gene; performs both functions Daffodil gene Vitamin A Pathway is complete and functional Golden Rice

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Shikimic acid + Phosphoenol pyruvate 3-Enolpyruvyl shikimic acid-5-phosphate (EPSP) Plant EPSP synthase Aromatic amino acids + Glyphosate Without amino acids, plant dies RoundUp has no effect; enzyme is resistant to herbicide Bacterial EPSP synthase With amino acids, plant lives The Roundup Ready Story

Story of Flavr savr tomato:

Story of Flavr savr tomato Production of the Flavr-Savr tomatoes started in 1994. Enzyme polygalacturonase breaks down structural polysaccharide pectin in wall of a plant. This is part of the natural decay process in a plant Monsanto identified the gene than encodes the enzyme and made another gene that blocked the production of the enzyme. Antisense technology

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What are the differences between a hybrid and a GMO ? sown Sown ?

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Biosafety concerns

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Biosafety “It may be defined as the policies and procedures invariably adopted to ensure the environmentally safe applications of biotechnology” Biosafety levels (BSL) It usually refers to a classification system used to indicate the safety precautions required for those investigating microorganisms ,especially viruses known to be dangerous or lethal to those exposed to them.

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The European Community (EC) Organization for Economic Cooperation and Development (OECD) WHO/FAO Working Group on Biosafety United Nations Conference on the Environment and Development (UNCED) Codex Alimentarius Commission Bio safety regulating agencies of world Bio safety regulating agencies of India Nodal agencies Ministry of Environment and Forestry ( MoEf ) Dept of Biotechnology- Ministry of Science and Technology Competent authorities RDAC- Recombinant DNA Advisory Committee IBSC-Institutional biosafety committee RCGM-Review Committee on Genetic Manipulation GEAC- Genetic Engineering Appraisal Committee SBCC-State Biotechnology Coordination Committee DLC- district level committee

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Rules for Manufacture, Use, Import, Export and Storage of Hazardous Microorganisms (Genetically Engineered Organisms or Cells, 1989 under the EPA (1986) known as ‘ Rules 1989’ by MoEF The Biological Diversity Act, 2002 by MoEF Plant Quarantine Order, 2004 by NBPGR under MoA Seed Policy, 2002 by MoA DGFT Notification Relating to Inclusion of GM Policy in Foreign Trade Policy (2006-09) by MoC&I Food Standards and Safety Act, 2006 by MoH&FW Drugs and Cosmetics Amendment Act, 1972 by MoH&FW Acts/Rules - Biosafety Regulation

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The Protocol is an international legally binding treaty which sets procedures and mechanisms to be applied in the transboundary movements of Living Modified Organisms (LMOs)- living organisms that possesses a novel combination of genetic material obtained through the use of modern biotechnology (genetic modification). The Protocol does not apply to other products of biotechnology. Cartagena Protocol Major elements of the protocol: Advance informed Agreement procedure Simplified system for agricultural commodities Risk assessments Risk management and emergency procedures Export documentation Bio-safety clearing House Capacity-building and finance Public awareness and participation Issue of non-parties.

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The major Biosafety concerns…

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Biosafety Issues Following are some of the biosafety issues, which have been widely discussed at the national and international levels and continue to receive attention of scientists and the society at large: Pollen flow and horizontal gene transfer of transgene to wild relatives and bacteria Allergenicity and toxicity Invasiveness Development of resistance in insects Development of resistance in weeds against herbicides giving rise to superweeds Adverse effects on non-target organisms Loss of biodiversity Selectable and scorable markers involving antibiotic resistance Disruption of ecosystem S ocial , ethical and economic issues These issues have been discussed in some detail in a recent document entitled “Agricultural Biotechnology-A Lot More than Just GM Crops” brought out by International Service for the Acquisition of Agri -Biotech Applications (ISAAA)

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Some important studies suggesting possible risks include the following: ( i ) adverse effect of Bt -corn on monarch butterfly ( ii) contamination of land races due to Bt -corn; ( iii) adverse effect of lectin in GM potato on rats ( iv) Aventis’s Star-Link corn with Bt gene Cry9C, which had to be withdrawn from the market, although there was no evidence of any harmful effect (v) Adverse effect of herbicide resistant transgenic soybeans on the fertility of rats, as reported by Irina Ermakova from Moscow

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Recommendations R esearch on transgenics must be continued with the aim of developing safer, more productive and nutritious food crops This concept of ‘ Cisgenic ’ technology, which has negligible food safety risk, and therefore may face less opposition/criticism GM crop events must be actually evaluated by the experts before their commercial release to the farmers The bio-safety is to be evaluated on a case-by-case basis The process of in situ conservation of biodiversity of crop varieties. The consequences of gene transfer via pollen should be evaluated on a case-to-case basis and due precaution must be ensured Reorganisation of resistance developed in the insects against insecticides and that in the weeds against herbicides. Contd.,

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Access to seed of approved GM crops .,the government should make a policy for procuring the seeds of useful GM food crops A “National Institute of Bio-safety and Bio-Security” should be created with state of the art infrastructure, human resource and research programs for conducting frontier research, providing policy support and technical advice to the government. The public needs to be educated properly about the facts regarding both food safety and economic benefits of the GM food technology. Need to establish PPP (Public-Private P artnership ) for joint development and ownership of the GM food crop products. The monopolistic control of seed business by MNCs ( multi-national companies )

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Action Plan for the Development and Utilization of GM Crops In view of the long gestation period for the development of useful GM crop events, and the high cost of research and development, there seems to be a need to have a sound biosafety evaluation and regulatory infrastructure. Following plan of action is suggested for this purpose: Bio-safety Evaluation and Regulatory Mechanism Pre- and Post-release Monitoring and Evaluation of GM Crops Accessibility of Approved GM Seeds to the Farmers Research and Development including PPP Education and Public Awareness

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Risk Assessment and Management Risk is defined as the probability of harm. Risk analysis consists of three steps

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Green peace Navdanya Friends of earth GMO awareness GMO free new jersey GMO free Utah Millions against Monsanto Texas Gene watch site Organic consumer association Gene Campaign Popular anti- GMO organizations

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S ubstantial E quivalence A functional part of the current risk assessment T he concept of substantial equivalence is that novel crops or foods, such as those made using genetic engineering, can be compared with the same kinds of conventional crops or foods to know the risk factors. A number of properties of the novel and conventional products, including the levels of nutrients, toxic substances, and potential allergens, may be compared taking into account established patterns of processing and consumption. If the comparison reveals that there are no significant differences between the two kinds of food, the novel food is presumed to be no less safe than the conventional food.

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Acute tests Sub chronic tests Chronic tests Single generations studies Multi generation studies

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Adverse effects of a substance that result either from a single exposure Acute testing on mice Helps in determining … The nature and duration of any acute toxic response. Provide the maximum non-lethal dose Preliminary information relevant to single exposure or over-dosage in humans. No observed effect concentration Lowest observed effect concentration Maximum allowable toxicant concentration Acute tests…. Alterations in metabolic pathways due to GM. Potential impacts on nutritional status. Minimum test to assess safety of long-term consumption of GM foods. Effects of repeated exposure over prolonged period of time Information on, immunological, histological and neurological effects Chronic tests Sub chronic tests Proliferative changes in tissues during the 90-day study Possible nervous, reproductive and hormonal disorders

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Indian journal of biotechnology vol. 5, January 2006, pp. 26-31 Stable integration, expression and inheritance of the ferritin gene in transgenic elite indica rice cultivar BR29 with enhanced iron level in the endosperm M K halekuzzaman 1,2 , K Datta 1 , S K Datta 1,4 , M F Alam 2 , I O Joarder 3 Genetics and Biotechnology Division, International Rice Research Institute, Manila, Philippines . The ferritin gene driven by an endosperm-specific glutelin promoter inserted into a Bangladeshi rice cultivar, BRRI Dhan 29 (BR29), using the biolistic method. Analysis demonstrated integration, inheritance and expression of the ferritin gene up to the T 3 generation. All transgenic plants accumulated higher levels of iron in the grain, with as much as 9.2 mg/kg versus the control as 3.8 mg/kg.

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Partial map of the plasmid pGPTV -bar/ fer containing 0.8 kb fragment of the ferritin gene . bar gene as selectable marker. PPT ( P hosphinothricin ) at 2.5 mg/L. Plasmid Constructs and Rice Transformation Materials and Methods

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PCR (Polymerase Chain Reaction) Analysis: fer F: 5'-GACGCACAATCCCACTATCCTT-3' (forward primer) fer R: 5'-CATTGTTGCGATCTGCCACACT-3' (reverse primer) bar F: 5'-GTCTGAACCATCGTCAACC-3' (forward primer) bar R: 5'-GAAGTCCAGCTGCCAGAAAC-3' (reverse primer) Southern Blot Analysis: Restriction Enzyme - Bam Hl and Sst l Probe - Rediprime labeling kit Estimation of Iron Content in Transgenic Seeds: ICP (Inductively Coupled Argon Plasma) Spectrometer Histochemical Localization of Iron in Rice Endosperm: Pearl's Prussian blue technique

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Results A total of 109 putative transformed (T 0 ) plants were obtained after bombardment and calli selection. Insertion of the transgene in the genome was primarily checked by PCR analysis. M = 1 kb DNA molecular marker N = non-transformed control lanes1-6= transformants P = positive control. Inheritance and Segregation Studies in T 1 , T 2 and T 3 Progenies Southern blot analysis showing the integration of ferritin gene in T 3 progeny of cultivar BR29. N = non-transformed control P = positive control.

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ICP Spectrometer T1 Brown Seeds Control-9.7 Transformed – 10.5 to 15.0 T2 Polished Seeds Control-3.3 to 3.8 Transformed- 4.9 to 11.0 T3 Polished Seeds Control- 3.8 Transformed- 6.3 to 9.2 UNIT - mg/Kg Iron content in different transformed lines of BR29 , polished T 3 seeds with control Iron Content in Rice Seeds Expressing ferritin Gene Newly expressed gene did not affect morphology, growth and fertility

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A display of immuno -strip: (a)GM sample having protein of interest(2bands-positive control); (b)non-GM sample having no protein(single band-negative control of interest))

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The fate of transgenes in the human gut Netherland T, John Heritage Division of Microbiology, University of Leeds, Woodhouse Lane, Leeds ,LS2 9JT, UK. e-mail; j.heritage@leeds.ac.uk Gut microbes that cannot be recovered in artificial culture may acquire and harbor genes from genetically modified plants. Volume 22 number 2 february 2004 nature biotechnology

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“ The controversy about the health safety of Transgenic foods is complex and good science and its communication are required in order to find solutions” Biosafety regulations required to assess the safety of transgenic crops before its release in to environment. The development of Transgenic plants is the result of integrated application of rDNA technology, G ene transfer methods and Tissue culture techniques. T ransgenic plants are designed to acquire useful quality attributes such as insect resistance, herbicide tolerance, abiotic stress tolerance, disease resistance, high nutritional quality, high yield potential , delayed ripening, enhanced ornamental value, male sterility, and production of edible vaccines. Conclusion

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Queries & Discussions

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_Presented by_ Ashok Singamsetti BAM-13-09 Department of Genetics and Plant Breeding

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