GENETICALLY MODIFIED ORGANISMS (GMOs) AND ENVIRONMENT

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APPLICATIONS OF BIOTECHNOLOGY FOR CROP IMPROVEMENT Agricultural Biotechnology rank second only next to medicine Potential market: $ 123 billion/year Do we really need crop improvement?

* Despite improvements over last 50 years, some 300 million people

have insufficient food and over a billion people have (women & children) are affected by specific nutrient deficiencies.

* Starvation is still a big problem- 28 out of 97 newborn children die due to starvation

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* A billion more people could starve by 2010; we have to triple our food production * World population - 6 billion (1999) expected to reach 8 billion (by 2020) * Plants are best source of food for man * Plants directly supply 90% of human calorie intake and 80% of protein intake * With 30,000 plant species occurring on earth, only around 40 crop species provide food source * Organic farming-Scientifically not possible-Green House Effect * High input farming-costly, Environmental risk (pesticides, fungicides, fertilizers www.biotecnika.org

A brief chronology of important developments of genetic engineering

1953 1972-73 Discovery of double helix structure of DNA by Watson and Crick DNA cloning techniques involving recombinant DNA developed by Herbert Boyer, Stanley Cohen, Paul Berg and others in the USA. A bacterial gene was the first gene to be cloned in 1973 First expression in bacteria of a gene from a different species Bacteria produced human somatostatin from a chemically synthesized gene; later that year human insulin was also produced in bacteria from a chemically synthesized gene US supreme court ruled that genetically modified microorganisms can be patented Insulin (Eli Lillys Humulin) was the first pharmaceutical made by genetically engineered bacteria to be approved for use www.biotecnika.org in Britain and USA

1974 1978 -

1980 1982 -

1982 1983 -

First transgenic animal (mice) produced

First transgenic plant expressing a gene of another plan species produced

1985 1986 1989 -

First transgenic farm animals produced (rabbits, pigs and sheep)

First controlled experimental releases of genetically engineered organisms into the environment US patent office announced that it would accept patent applications for genetically engineered plants and animals. First patented transgenic animal produced for pharmaceutical research- DuPonts Oncomouse Genetically engineered tomato marketed in USA Cloning of transgenic sheep in UK

1995 1997 -

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1998 -

Regulatory and biosafety issues became globally very important. Regulation for transboundary movement of GMOs are under preparation by the Convention on Biological Diversity Cloned human embryo developed into 4 cell stage

2001 -

2002 onwards - Recent developments in cloning, transgenics, GMOs, GMFs.

2004- Global status of biotech crops (over all more than 50,000 m hect) USA- 47.6, Argentina-16.2, Canada-5.4, Brazil-5.0, China-3.7 Paraguay- 1.2, India, South Africa-0.5, Uruguay-0.3, Australia0.2, Romania, Mexico, Spain, Philippines- 0.1 m hectares

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Production of GMOs involves knowledge of:

Gene Structure Gene Expression Gene Cloning * Enzymes for DNA manipulation * Vectors * Expression hosts * Selection of clone/identification of the gene Production of transgenic plants and animals

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The ability to introduce foreign genes into a wide range of plants has created a revolution in plant biology Recent research has demonstrated that delivering DNA to a plant cell can be achieved by innovative methods.

Methods of genetic engineering

a) Conventional method for DNA delivery i) Agrobacterium mediated transformation ii) Microprojectile bombardment-mediated transformation iii) Electroporation System iv) Protoplast mediated transformation

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Manipulating yield:

Photosynthesis:
Ribulose bipohsphate carboxylase-oxygenase (RuBisCo) is the most abundant in the world. Photorespiration, which leads to loss of CO2, therefore appears to reduce the efficiency of carbon fixation. An improvement in carbohydrate yield may be achieved by increasing the relative rate of carboxylase to the oxygenase activity of RuBisCo The Rubisco -composed of eight identical large and eight identical small unit, encoded in the chloroplast and nuclear genomes respectively. The genes, present as small multigene families, have been cloned and characterized, and are amenable to manipulation. It may be possible to delete the oxygenase activity of the enzyme by protein engineering, or to select for mutant enzymes which possess no or reduced oxygenase activity and transfer the encoding genes to crop plants.

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Nitrogen Fixation:
Approaches to genetically manipulate nitrogen-fixing symbiosis require an understanding of recognition, infection and nodule development. The main genes involved are the nodulation genes (nod), polysaccharide biosynthesis genes (exo and lps), nodule development genes (ndv), and nitrogen fixing genes (nif and fix). On the plant side, plant genes specifically expressed during the symbiosis are termed nodulins (e.g., leghaemoglobin, Uricase II, glutamine synthase, choline kinase, sucrose synthase).

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Fruit Ripening:

A considerable proportion of agricultural produce is lost after harvesting.

Molecular techniques may be used to reduce this loss. For example, in tomato, two enzymes involved, ACC synthase and EFE (Ethylene forming Enzyme), have been cloned.

Ethylene production in transgenic plants expressing antisense to constructs to both ACC bacterial enzyme that degrades ACC.
Transgenic tomatoes from suchplants can last without decaying for much longer than control plants, and they can be transported with less damage. A similar approach can be used to prolong transport and vase of life of some flowers.

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Quality of food:

A great deal of study carried on nutritional and technological qualities of the seed storage products of the cereals, legumes, and Brassicas.
The storage proteins of a number of species are low in one or more essential amino acids. Wheat, barley, maize, and sorghum are low in lysine, while legume storage proteins are different in sulfur-containing amino acids. Barley and sorghum are further lacking threonine, and maize in tryptophan. Many storage protein genes have been cloned, and have been transferred successfully across species barriers.

Golden rice rich in Vitamin A and Fe

Completely artificial proteins, high in limiting amino acids, can also be synthesized. Cystein, Ferritin rich cereals

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Technological Quality:

The biochemical properties of crops can also, it is hoped, be modified to improve quality in relation to technological requirements

Bread making quality of wheat Beer Production- Malting and Brewing Genetic engineering for specialty oil production by making
mutants of Arabiodopsis thaliana

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RECENT DEVELOPMENTS IN TRANSGENIC PLANTS

Strawberries
Identified genes that govern the sweetness, tasteness and smell of strawberries Sucrose Transporter protein (Horticulture Research International, U.K Caffeine-free coffee plants are on the horizon (NCL, Pune) Wild gene has been transferred, which blocks the synthesis of caffeine Super-grain, high iron content, more yield and famine resistant (IRRI, Philippines) Modified for higher starch content lot of saving to processed tomato products (Univ. of Dayton)
Modified leaf veins-by selecting group of genes- leaf shape (Cambridgeshire, U.K.)
The genetically modified nector of Petunia will contain Vaccine (CPBR, Wageningen) Potato plant is modified if required foliage is produced if required the tubers (USA) Tobacco plants have been produced which synthesizes human blood factors (USA)

Coffee

Rice Beefier Tomato Lettuce Bioactive nector Dual use crops Plant produce blood factors

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Strategies for expression of antigens in plants

Gene Antigen DNA for epitope

Transformation vector Genetic Transformation and regeneration

Virus with chimeric coat protein Infection of plants

Transgenic plant

Infected plant Isolation of chimeric virus

Extraction of Antigen Feeding of edible organs

Immunogenicity www.biotecnika.org

Antigen produced in transgenic Plants

Protein
Hepatitis B surface antigen Rabies virus glycoprotein Norwalk virus capsid protein E. Coli heat-labile enteroprotein B subunit Cholera toxin B subunit Mouse glutamate decarboxylase VP I protein of foot-and-mouth disease virus Insulin Glycoprotein of Swine-transmissible gasteroenteritis coronavirus

Plant
Tobacco Tomato Tobacco Potato Potato, Tobacco Potato Arabidopsis Potato Arabidopsis

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Some of the value added crops

Some of the value added crops: Golden rice: containing betacarotene to overcome Vitamin A deficiencies in regions where rice is a major staple food. Canola containing high levels of oleic acids and laurate Barley containing feed enzymes Other vegetables and fruits with delayed ripening as well as modified flavor characteristics

Ability to grow in harsh environment

Plant based pharmaceuticals

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It has been reported that about 60 recombinant products have been approved till 2003 and are being used as therapeutics in various forms across the world. The annual global market for biopharmaceutical is estimated at more than US$ 30 billion in 2002, as compared to US$ 12 billion in 1999 and US$ 5 billion in 1989. Notable target indications for approved therapetics include diabetes, heamophilia, hepatitis, myocardial infarction and various cancers.

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Some of approved recombinant therapeutics

Products
Blood factors Factor VIII Blood clot dissolvers Tissue plasminogen activator Growth factors and hormones Insulin Cytokines INTERFERONS IFN-2a Vaccines Hepatitis B surface antigen Additional products TNFR-IgG fragment Fusion protein

Therapeutic indication
Hemophilia Type A

Produced in host cells

Recombinate

Company
Baxter Healthcare/Genetics Institute Genentech Eli Lilly Hoffman La-Roche

Acute myocardial infarction Diabetes mellitus Hairy cell leukemia

CHO cells E.coli E.coli

Hepatits B prevention

S.cereviviae

Merck

Rheumatoid arthritis

CHO cells

Immunex (US)

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List of recombinant therapeutics approved for marketing in India through imports

S.No. 1 2 3 4 5 6 7 8 9 10 11 12 13 Molecules Human Insulin Erythrropoeitin Hepatitis B vaccine (recombinant surface antigen based) Human growth hormone Interleukin 2 Interleukin 11 Granulocyte colony stimulating factor Granulocyte macrophage colony stimulating factor Interferon -2a Interferon -2b Interferons Blood factor VIII Follicle stimulating hormone Therapeutics applications Diabetes Treatment of anemia Immunization against Hepatitis B Deficiency of growth hormone in children Renal cell carcinoma Thrombocytopenia Chemotherapy induced Neutropenia Chemotherapy induced Neutropenia Chronic myeloid leukemia Chronic myeloid leukemia, Hepatitis B and Hepatitis C Chronic granulomatous disease and severe malignant osteopetrosis Heamophilia Type A Reproductive disorder

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Companies producing rDNA therapeutics in India and their products

S.No.
1 2 3 4 5 6

Name of the industry

Shantha Biotechnics Bharath Biotech Panacea Biotech Wockhardt Dr. Reddys Laboratory Serum institute of India

rDNA products
Hepatitis B Vaccine, Interferon Hepatitis B Vaccine Hepatitis B Vaccine Hepatitis B Vaccine, Erythropoietin, Insulin Granulocyte Colony stimulating factor Hepatitis B Vaccine

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Biosafety
Biosafety regulations have been developed by many countries involved in transgenic research and commercialization.

The most ambitious global attempt has been under the Convention on Biological Diversity (CBD).
Article 19 of the CBD committed members to a protocol on biosafety specifically addressing transboundary movement of GMOs. The Cartagena Protocol adopted on January 29, 2000. under the aegis of CBD. It seeks to protect biological diversity from the potential risks posed by living modified organisms.

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India is a party to the CBD and a signatory to Cartagena Protocol on Biosafety.

India has a well defined regulatory mechanism for development and evaluation of GMOs and the products thereof.

Rules notified in 1989 under Environmental Protection Act, 1986 (EPA) define the competitive authorities and composition of such authorities for the handling of all aspects of GMOs and products thereof.

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Five new criteria of risks to be considered on the basis of the model proposed by the Office of the Technology Assessment (OTA) Formation: deliberate or accidental creation of genetically modified microorganisms;

Liberation: deliberate or accidental liberation of these microorganisms at the work site and/or in the environment
Proliferation: multiplication, genetic reconstruction, growth, transport, modification and disappearance of these microorganisms in the environment and possible transfer of genetic material to other microorganisms; Installation: installation of these microorganisms in an ecological niche and possible colonization of human beings or other living organisms; Effect: subsequent appearance of effects on human beings or on the ecosystem due to interaction between the organism and a host or an experimental factor.

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The differences between rDNA technologies and conventional breeding methods

1.

derived form disease-causing viruses, plasmids and mobile genetic elements-parasitic DNA that have the ability to invade cells and insert themselves into the cells genome designed to breakdown species barriers so that they can shuttle genes between a wide range of species. Have wide host range-can infect many animals and in the process pick up genes from viruses of all these species to create new pathogens. carry genes for antibiotic resistance which will speed up the evolution of antibiotic resistance which is already a big public health problem

2.

3.

3.

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The short time-scale- allows little time for testing or for ecological adjustments

The great variety of genes being shuffled, whose ecological effects could be hard to predict from past experience The degree to which genes introduced into one species could eventually move into another species, by natural, if very rare, means of interspecies gene transfer, are so far quite unpredictable.

The ownership of the technologies is largely private and oriented to short-term profit. Industries are only just becoming aware of the need for environmental protection The risks, however, small, must be taken seriously

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These guidelines cover:

Genetically engineered organisms,

Genetic transformation of green plants, animals

rDNA technology in vaccine development; and

large scale production and deliberate/accidental release of organisms, plants, animals & products derived by rDNA technology.

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REGULATORY FRAMEWORK IN INDIA:

Rules for GMOs: Environment and Protection Act (1986) by The Ministry of Environment and Forests the rules and procedures for the manufacture, import, use, research and release of GMOs as well as products made by the use of such organisms were notified by the MoEF through their Notification No. 621 in Official Gazette of Govt. of India on December 5, 1989. Following statuary bodies are involved in decision making:
i. iii. iv. v.

The recombinant DNA Advisory Committee (RDAC) Review Committee on Genetic Manipulation (RCGM)

Genetic Engineering Approval Committee (GEAC) State Biotechnology Coordination Committee (SBCC)

vi. Institutional Biosafety Committee (IBSC)

vi.

District Level Committee (DLC) www.biotecnika.org

These committees work in the framework of the following rules

1.

Recombinant DNA guidelines, 1990 Guidelines for research in transgenic plants, 1998

2.

3.
4.

Drugs and Cosmetics Rules (8th amendment), 1998

Guidelines for generating preclinical and clinical data for rDNA therapeutics, 1999 Drug Policy, 2002 Seed Policy, 2002

5. 6.

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Status of Biosafety regulations for GMOs:

The two main areas of concern are the impact environmental/ecosystem and the effects on human health. on

These risks can be broadly classified as follows:

Risks for animal and human health: toxicity & food quality/safety, allergies, resistance of pathogen to drugs (antibiotic resistance)

Risks for the environment: persistence of the gene or transgene (volunteers, increased fitness, invasiveness) or of the transgene products (accumulative effects); susceptibility of non target organisms; increased use of the chemicals in agriculture; unpredictable gene expression or transgene instability; genetic pollution through pollen or seed dispersal.

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Risks for agriculture: Resistance/tolerance or target organisms; weeds or superweeds; alteration of nutritional value (attractiveness of the organisms to pests); reduction of cultivars (increase of susceptibility and loss of biodiversity)

General Concerns: Loss of familiarity; higher cost of agriculture; field trials not planned for risk management ethical issues.

Risks of interaction with non target organisms: Horizontal gene transfer (Transgene or promoter dispersion); transfer of foreign gene to microorganisms (DNA uptake); generation of new live viruses by recombination (transcapsidation, complementation, etc)

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Biosafety guidelines for rDNA research activities:

Category

I: which are exempt for the purpose of intimation and approval of competent authority Category II: Those requiring prior intimation of competent authority
Category

III: those requiring review and approval of competent authority before commencement

Toxin gene clonings of genes for vaccine production of mosquito and tick DNA experiments

Cloning Cloning Genes

coding for antibiotic resistance into cultured human cells of recombinant DNA

Introduction

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 Introduction Experiments

into animal cells of unidentified DNA molecules

involving the use of infectious animal and plant viruses in tissue culture systems involving gene transfer involving gene transfer

Experiments Experiments Cell

fusion experiments in animal experiments

Transgenasis

All

experiments involving the genetic manipulation of plant pathogens and the use of such genetically manipulated plant pathogens would require approval of competent authority (IBSC).

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Bioethical issues

Transgenic crops themselves become weeds Plants engineered to contain viral genes may aid the creation of new viruses Plants engineered to express drugs and pesticides may present risks to other organisms that are not the intended targets of new chemicals Terminator gene concept

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Biosafety issues

Issues of food toxicity or allergenicity from GMO GMO with antibiotic resistance (marker gene) may reduce the effectiveness of the same Transfer or spread of resistant genes to bacteria in the environment is the main concern

Genetic material including the resistant gene can survive the passage through gut, enter blood stream and finally may integrate with the genetic material of consumer
Present genes inadventantly entering food chain by cross pollination or mixing crops during harvesting Loss of biodiversity, increased cancer risk, changes in nutritional quality, genetic pollution, damage to beneficial organisms and creation of superweeds.

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Monsanto & Food for alarm:

Human explore to Monsantos herbicide Roundup has resulted in nausea, skin and eye inflammation, bronchial constriction and nervous system disorders

AP asks Monsanto to atop trials in cotton fields:

The Andhra Pradesh Govt. has asked the Mahyco - Monsanto Biotech (India) Pvt. Ltd. to stop all its field trials being conducted in the 11 places in seven district of the state

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Transgenic Trials attacked in Brisbane:

In March 2000, a small plot of transgenic pineapples was vandalized at a field research site in Brisbane. As a result of that damage to the plants, the research has been set back by several months

Call to curb adverse impact of Genetic Technologies in crops:

Dr. M. S. Swaminathan said India should take advantage of recombinant DNA technologies without associated harm to human or ecological health. A recent statement issued by Royal Society of London on Genetically modified plants for food use provides guidelines for the safe handling of Biotechnological application and those could be adopted in India too with suitable modifications

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Problems with GM crop is political

The problems should be left to technological to solve It is mainly concerned with industrialization of agriculture by multinationals This powerful technology is being used to further commercial aims at the expense of public fund

The first green revolution was achieved by the non-profit sector but this gene revolution is being achieved by industry
No technology is perfect, the benefits and drawbacks need to be judged against the conventional technology they replace Developing countries: It is not appropriate for well-fed and rich west to push its capitalist, socialist or environmentalist, philosophies on to developing world

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Challenges ahead for young crop biotechnologists:

Public debate on various aspects of gene technology Genome research- rice genome project Improvement in the gene technology Rigorous bio-safety related trials

Research mainly by public-funded institutions on selected crops.

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THANK YOU

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