Download to read offline
Development of biotech crops, biosafety and regulatory system in India
- 1. Development of biotechcrops, Biosafety and Regulatory system in India Presentation by Sugandh chauhan
- 2. Terms to know •Genetic modification involves altering the genes of an organism, be it a plant, animal or microorganism. It allows the transfer of genes for specific traits between species using laboratory techniques. • Transgene is a foreign gene that has been transferred from one organism to another • Transgenic crops are those whose genome is altered by adding transgene • Transgenesis is the phenomenon of introduction of exogenous DNA into the genome to create and maintain a stable and heritable character.
- 3. Genetically modified crops(GM crops) • With the rapid advances in biotechnology, a number of genetically modified (GM) crops carrying novel traits have been developed. • GM crops are developed by a process of genetic modification by which selected individual genes are inserted from one organism into another to enhance desirable characteristics or to suppress undesirable ones. • GM crops are also known as genetically engineered or transgenic or biotech crops. • The first transgenic plant was developed in 1983 in tobacco in U.S.A. • Commercial cultivation of transgenic crops started in the early 1990s.
- 5. Need of GMcrops • To improve the yield of crops • To reduce dependance on pesticides • To enhance resistance to weeds, pests and disease • To reduce environmental stress • To improve nutritional quality • To improve taste and texture • To provide additional health constituents
- 6. Area under GMcrops is on the rise globally • Globally, area under GM crops has increased manifold from 17 lakh hectares in 1996 to 1917 lakh hectares in 2018. • India has the world’s fifth largest cultivated area under GM crops. • In India, the area under Bt-Cotton has increased from 0.29 lakh hectares in 2002-03 to 117.47 lakh hectares in 2019-20, according to Directorate of Economics and Statistics. • Major companies interested in GM crops in India include Monsanto India, Mahyco.
- 7. History of Bacillusthuringiensis (Bt) • Discovered in 1901 by Ishiwataria a cause of sotto disease that was killing silkworms and named it Bacillus sotto. • In 1911, Berliner isolated this bacterium from dead Mediterranean flour moth in Thuringia, Germany, and named it Bt. • In 1915, Berliner reported the existence of a parasporal body, or crystalline inclusion (called crystal) close to the endospore within Bt spore. • In 1956, it was found that the main insecticidal activity against lepidopteran insects was due to the parasporal crystal. • In 1956, Bt was used commercially in the USA. • In the 1980s, the use of Bt increased worldwide when insects became increasingly resistant to the chemical insecticides.
- 8. Bacillus thuringiensis (Bt) •Bacillus thuringiensis (Bt) is a naturally occurring soilborne gram-positive, rod-shaped, sporulating bacterium found worldwide. • Bt strains produce three types of insecticidal toxins • Crystal (cry) toxins • Cytolytic (cyt) toxins • Vegetative insecticidal proteins (Vip)
- 9. Crystal Toxins (δ-endotoxins) •The most widely known δ- endotoxins, include Cry and Cyt toxins. • The Cry toxin (cry from crystal), insecticidal crystal protein is a protein formed during sporulation in Bt strains and aggregate to form crystals. • Some strains of Bt, produce another toxic crystal, named cytolytic protein or Cyt toxin.
- 10. Mode of actionof Bt 1. Ingestion 2. Solubilization and proteolytic activation 3. Binding to target site 4. Formation of toxic lesions
- 12. Development of transgeniccrops • Isolation of the gene of interest • Insertion of the gene into a transfer vector and plant transformation • Selection and regeneration of the modified plant cells into whole plants • Lab analysis and safety testing • Approval by Government agencies • Commercialization
- 13. Methods of genetransfer • The methods of foreign gene (DNA) transfer in crop plants are- • Electroporation • Gene transfer by polyethylene glycol (PEG) • Gene transfer through microinjection • Agrobacterium mediated gene transfer • Biolistics
- 14. Electroporation • Short pulseof high voltage are applied to protoplasts which make temporary pores in plasma membrane to increase their permeability and facilitate the uptake of foreign gene. Gene transfer by PEG • It is applicable for protoplast only. The chemical used is polyethylene glycol. It stimulates endocytosis and thereby causing the uptake of DNA.
- 15. Microinjection • DNA isintroduced into the cytoplasm or nucleus by using microinjection. • Mainly used for transformation of large animal cells.
- 16. Agrobacterium mediated genetransfer • Agrobacterium tumefaciens is a soil borne gram negative bacterium which infects wounded plant tissues and induces the formation of a plant tumor called crown gall disease. • The ability to cause crown gall disease is associated with the presence of Ti plasmid within the bacterial cell. • A segment of Ti plasmid known as T-DNA is transferred from bacteria into host where it gets integrated into the host genome. • T-DNA carries the gene that involve in the biosynthesis of hormones auxin and cytokinin and plant metabolites opines and agropines. • Agrobacterium has proved to be an incredible useful tool for the integration of genes into plants.
- 18. Biolistics • It isa physical method first described by Stanford and co- workers in 1984. • Also known as Gene gun method/particle bombardment/microprojectiles. • Gene gun is a device that fires DNA into target cells. • DNA to be transformed is coated onto microscopic beads made of either gold or tungsten.
- 20. Bt-cotton • Bt cottonis genetically modified cotton crop that expresses an insecticidal protein whose gene has been derived from a soil bacterium called Bacillus thuringiensis. • The transgenic cotton is of two types (1) bollgard (confers resistance to bollworms). (2) Roundup ready cotton (confers resistance to herbicides). • Bt cotton is the only genetically modified (GM) crop that has been approved for commercial cultivation in 2002 by the Government of India.
- 21. Bt cotton Cryprotein Year Company Bollgard cotton Cry 1 Ac 1996 Monsanto Bollgard II cotton Cry 2 Ab 2003 Monsanto Wide strike cotton Cry 1 Ac and Cry 1 F 2004 Dow Agro- sciences
- 22. Need of Bt-cotton •Cotton is a long duration crop and is attacked by large number of insect pests throughout its growth and development. • The three bollworms, American bollworm , Pink bollworm and the Spotted bollworms, are major pests and cause serious threat to cotton production. • About 9400 M tonnes of insecticides worth Rs 747 crores were used only for bollworm control in 2001. • Cotton bolls are highly vulnerable to insects.
- 24. Commercial Release ofDifferent Bt-Cotton Events in India, 2002-2014
- 25. Benefits of Bt-cotton •Resistance to bollworms • Yield improvement • Reduce use of pesticides • Reduce cost of cultivation • More profit
- 26. Bt-brinjal • Bt Brinjalis the first genetically modified vegetable crop in India, developed by the Maharashtra Hybrid Seed Company Ltd., (Mahyco) in 2000. • Bt brinjal incorporates the cry1Ac gene from the soil bacterium Bacillus thuringiensis (Bt) expressing insecticidal protein to confer resistance against FSB. • The company used a DNA construct containing the cry1Ac gene, a CaMV 35S promoter and the selectable marker genes nptII and aad, to transform young cotyledons of brinjal plants.
- 27. Cont. • In India,the Genetic Engineering Appraisal Committee (GEAC) approved its commercialization in 2009 following field trials and safety evaluations. However, the seeds have not yet been released to farmers. • The National Center on Plant Biotechnology (NRCPB) has developed Bt brinjal varieties expressing the cryFa1 gene. • The Indian Institute of Horticultural Research (IIHR) is also developing Bt brinjal using the cry1Ab gene.
- 28. Need of Bt-brinjal •Brinjal or eggplant (Solanum melongena) is vulnerable to many diseases caused by insects, pests, fungi, bacteria and viruses. • Production is affected by the brinjal fruit and shoot borer (FSB, Leucinodes orbonalis) and other fruit borer insects Helicoverpa arimegera.
- 29.
- 30. Bt-corn • Bt cornis a GM crop developed in 1996 that helps reduce pesticide use against the European Corn Borer, a pesky caterpillar that eats the crop. • Bt corn incorporates the cry1Ab gene from Bt expressing insecticidal protein to confer resistance against corn borers.
- 32. GM-Mustard • Dhara MustardHybrid-11 or DMH-11 is a genetically modified variety of mustard developed by the Delhi University’s Centre for Genetic Manipulation of Crop Plants in 2002. • Hybridised mustard DMH-11 developed using “barnase / barstar” technology. It is Herbicide Tolerant (HT) crop. • 2016: GEAC gave green signal to GM Mustard for field trial, but SC stayed the order. • GM mustard Dhara is pending for commercial release
- 33. Golden Rice • GoldenRice was engineered from normal rice by Ingo Potrykus and Peter Beyer in 1990 to help improve human health. • Rice in its milled form is without vitamin A and its carotenoid precursors. • Millions of rice consumers who depend on rice for a large proportion of their calories suffer from vitamin A deficiency. • Golden Rice has an engineered multi-gene β-carotene pathway in its genome. This pathway produces beta-carotene, precursor of vitamin A • The genes phytoene synthase (psy) from Narcissus pseudonarcissus, phytoene desaturase (crt1) from Erwinia uredovora and lycopene cyclase (lcy) have been introduced into the rice, driven by the endosperm specific glutelin promoter (Gt1).
- 34.
- 36. How to makeGolden Rice
- 37. Transgenic Tomato “FlavrSavr” • The FLAVR SAVR tomato was the first GM food crop to be marketed commercially following FDA approval in 1994, • It was developed by Calgene company of California in 1992 through the use of antisense RNA technology to regulate the expression of the enzyme polygalacturonase (PG) in ripening tomato fruit. • Flavr Savr tomato has longer shelf life.
- 38. Antisense RNA Technology •Antisense RNA is a single stranded RNA that is complementary to mRNA strand transcribed within a cell. • Antisense RNA introduced into a cell to inhibit translation of a complementary mRNA by base pairing to it and creating barrier to the translation machinery.
- 39. Genes involved 1. pTOM5encodes for phytoene synthase that promote lycopene synthesis that gives red coloration 2. pTOM6 encodes for polygalacturanase (PG) that degrades the cell wall resulting in fruit softening 3. pTOM encodes for ACC oxidase that catalyze ethylene formation which triggers fruit ripening • PG breakdown the polygalacturonic acid component of the cell wall of fruit pericarp of tomato resulting in softening of fruit. It also makes it more prone to bruises and decreases its shelf life.
- 40. Mechanism 1. Isolation ofgene from tomato plant that encodes the enzyme PG 2. Synthesis of complementary DNA (cDNA) 3. Introduction of cDNA into a fresh tomato plant to produce transgenic plant. GM tomato plant has two type of genes- • Gene for PG that produces sense mRNA • Synthetic antisense PG gene that produces antisense mRNA
- 42. Stacking of Transgenes •Introduction of more than one gene into crop plants simultaneously or sequentially, called transgene stacking. • It is an effective strategy for conferring higher and durable insect and disease resistance in transgenic plants than single-gene technology. • The first stack that gained regulatory approval in 1995 was a hybrid Cotton stack produced by crossing Bt-cotton expressing cry1Ab gene and Roundup ready cotton producing EPSPS enzyme conferring resistance to herbicide glyphosate.
- 43. Methods for transgenestacking The methods used can be separated into two main groups: (1) simultaneous introduction methods (2) sequential introduction methods Simultaneous introduction methods This method refer to the introduction of several genes in the same process of genetic transformation. This technique is described as co-transformation and can be of two types- • co-transformation with single plasmid • co-transformation with multiple plasmids
- 44. Cont. • Co-transformation witha single plasmid - introducing a single genetic construction with all genes, each with its own promoter and terminator, in the same plasmid. • In Co-transformation with multiple plasmids, several constructions are made, each with one or a few genes, and each construct is inserted into a separate plasmid.
- 45. Sequential introduction methods Re-transformationinvolves the transformation of an already transgenic plant to insert a new characteristic. Sexual crossing – plants containing several transgenes produced by crossing parents with different transgenes.
- 47. Biosafety • As moreand more transgenic crops are released for field-testing and commercialization, concerns have been expressed regarding potential risks to both human health and environment. • Biosafety describes the principles, procedures and policies to be adopted to ensure the environmental and personal safety. The major biosafety concerns falls into following categories: • Bio-safety of human and animal health • Ecological concerns • Public attitude
- 48. Bio-safety of humanand animal health • Risk of toxicity, due to the nature of the product or the changes in the metabolism and the composition of the organisms resulting from gene transfer. • Newer proteins in transgenic crops from the organisms, which have not been consumed as foods, sometimes has the risk of these proteins becoming allergens. • Genes used for antibiotic resistance as selectable markers have also raised concerns regarding the transfer of such genes to microorganisms and thereby aggravate the health problems.
- 49. Ecological concerns • Geneflow due to cross pollination for the traits involving resistance can result in development of tolerant or resistant weeds that are difficult to eradicate. • GM crops could lead to erosion of biodiversity and pollute gene pools of endangered plant species. • Genetic erosion has occurred as the farmers have replaces the use of traditional varieties with monocultures. Public attitude • Consumer response depends on perceptions about risks and benefits of genetically modified foods. The media, individuals, scientists and administrator and NGO have the responsibility to educate the people about the benefits of GM foods.
- 50. Regulatory system inIndia Regulatory approval of GM crops • The rules governing the handling of genetically modified organisms (GMOs) and products thereof were notified in 1989 under Environment Protection Act 1986. • Two government agencies, the Ministry of Environment and Forests (MoEF) and the Department of Biotechnology (DBT), Ministry of Science and Technology, are responsible for implementation of the regulations.
- 51. Cont. • There arebasically 6 authorities to handle different aspects of the regulation. 1. Recombinant DNA Advisory Committee (RDAC) 2. Review Committee on Genetic Manipulation (RCGM) 3. Genetic Engineering Approval Committee (GEAC) 4. Institutional Biosafety Committees (IBSC) 5. State Biosafety Coordination Committees (SBCC) 6. District Level Committees (DLC).
- 52. Cont. • RDAC isconstituted by DBT to monitor the developments in biotechnology at national and international levels. RDAC submits recommendations from time to time that are suitable for implementation for upholding the safety regulations in research and applications of GMOs and products thereof. • IBSC - set-up at each institution for monitoring institute level research in genetically modified organisms. • RCGM - committee is constituted by DBT to review all ongoing projects involving high risk and controlled field experiments. • GEAC - set-up in the Ministry of Environment, Forests and Climate Change to authorize large-scale trials and environmental release of GMO.
- 53. Cont. • SBCC -set up in each state where research and application of GMOs are contemplated, coordinate the activities related to GMOs in the state with the central ministry. SBCCs have powers to inspect, investigate and to take action in case of violations. • DLC is constituted at district level to monitor the safety regulations in installations engaged in the use of GMOs in research and application.
- 54. References • H. Khan,Gene transfer technologies in plants: Roles in improving crops. Recent Research in Science and Technology 2009, 1(3): 116–123 ISSN: 2076-5061 • S Kumar, A Misra et al. Bt Brinjal in India: A long way to go. GM crops, 2011 • CC Ceccon, A Caverzan, R Margis, JR Salvadori. Gene stacking as a strategy to confer characteristics of agronomic importance in plants by genetic engineering. Ciência Rural, Santa Maria, v.50:6, e20190207, 2020 • L Palma, D Muñoz, C Berry, J Murillo, P Caballero. Bacillus thuringiensis toxins: an overview of their biocidal activity. Toxins 2014, 6, 3296-3325
- 55.