Genetic engineering and development of transgenic plants

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1. Genetic Engineering & Development of Transgenic plants Presented by, L.N.Nisha I-PhD-Entomology 1

2. Genetic Engineering/ Genetic manipulation It is a process where the gene for a particular character is introduced inside the chromosome of a cell When the gene for a particular character is introduced in a plant cell a transgenic plant is produced. These transgenic plants exhibit characters governed by the newly introduced gene It helps in improving crop variety It ensures food security and insect resistant crops It also improves the quality and yield of crops 2

3. Genetic Engineering/ Genetic Manipulation Is the direct manipulation of an organisms genome using biotechnology. It is a set of technologies used to change the genetic makeup of cells, including the transfer of genes within & across species boundaries to produce improved (or) novel organisms 3

4. Genetically modified crops (GMCs, GM crops or biotech crops) are used in agriculture, the DNA of which has been modified using genetic engineering techniques. In most cases the aim is to introduce a new trait to plant which does not occur naturally in species 4

5. Why genetically engineered plants??? To improve the agricultural, horticultural (or) ornamental value of a crop plant Resistance to certain pests, diseases and environmental conditions Reduction of spoilage Resistance to chemical treatments ( Eg - Resistance to herbicide) Improving the nutrient profile of the crop 5

6. Transgenic Plants It is a foreign gene/genetic material that has been transferred naturally (or) by any of a number of genetic engineering techniques from one organism to another Transgene 6

7. Transgenesis The phenomenon of introduction of exogenous DNA into the genome to create and maintain a stable and heritable character 7

8. Transgenic Plants The plants whose genome is altered by adding one or more transgenes are known as transgenic plants 8

9. History of GMO’s 1982- 1 st transgenic plant produced which is an antibiotic resistance tobacco plant 9

10. 1984- 1 st successful plant genetic engineering experiments was done by using Caulimovirus vector 10

11. 1994- 1 st genetically modified crop approved for sale in US was Flavr - Savr tomato 11

12. 1994- The European Union approved genetically engineered tobacco resistant to the herbicide ( Bromoxynil ). First commercially engineered crop marketed in Europe 12

13. 1995- Bt - potato was approved by the US- Environmental Protection Agency making it the country’s first pesticide producing crop One of the most consequential potato plant pest is CPB which often becomes resistant to chemical insecticides. Modified potatoes carrying gene Cry3A originating from the bacteria Bt. 13

14. 1996- 1 st genetically modified flower ( Moondust - bluish colored carnation was introduced) 14

15. 2000- Golden rice with ß- carotene developed with increased nutrient value 15

16. Development of Transgenic plants The basic requirements of transformation are, 1. A target genome 2. A vector to carry the gene 3. Modification of the foreign DNA to increase the level of gene expression 4. Method to deliver the plasmid DNA into the cell 5. Methodology to identify the transformed cell 6. Tissue culture to recover the viable plants from the transformed cells 16

17. Transgenic plants have been produced by addition of one or more following genes Endo-toxin gene from Bt Protease inhibitors Alpha amylase inhibitors Lectins Enzymes Pyramiding genes 17

18. Endotoxin gene from Bt 18 The gene responsible for producing endotoxin is isolated from Bt and cloned into plants to develop resistance to insects

19. 19 Protease inhibitors Insects have protease in their gut which are the enzymes helping in digestion of protein PI inhibit the proteases and affect digestion in insects The PI are isolated from one plant and cloned into another to produce transgenic plants Four classes- Serine, Thiol , Metallo and Aspartyl Serine PI is the most important Eg - Cowpea trypsin inhibitor (CpTi) gene derived from cowpea and cloned into tobacco against Helicoverpa

20. 20 Alpha amylase inhibitors- Alpha amylase is a digestive enzyme present in insects for digestion of carbohydrates. AAI affects the digestion of carbohydrates in insects. Transgenic tobacco & tomato expressing AAI which are resistant to Lepidopteran pests Lectin genes- Lectin are a group of plant proteins that bind to carbohydrates, including chitin. The deleterious effect of chitin binding lectins on insect development is mediated by binding to chitin in the peritrophic membrane that lines the midgut of insect thus interfering with the uptake of nutrients Eg - Gene encoding the pea lectin (P- Lec ) has been expressed in transgenic tobacco against Helicoverpa virescens

21. 21 Enzymes- Chitinase enzyme gene, cholesterol oxidase and lipo - oxygenase genes have been cloned into plants and these transgenic plants show insecticidal properties. Eg - Expression of bean chitinase (BCH) in transgenic tobacco against aphid Pyramiding genes- Engineering transgenic crops with more than one gene to get multi mechanistic resistance is called pyramiding of genes Eg - CpTi gene and pea lectin gene were cloned to produce transgenic tobacco. Lectins and bean chitinase (BHC) genes were cloned to produce transgenic potato

22. Bt insect resistant plants development Bacillus thuringiensis – a soil bacterium whose spores contain a crystalline protein ( Cry ) In the insect gut the protein breaks down to release a toxin known as delta- endotoxin This toxin binds to and creates pores in the intestinal lining Resulting in ion imbalance Paralysis of the digestive system After few days the insect died Different versions of Cry genes also known as “ Bt genes” have been identified 22

23. Bt - Mode of entry 23

24. More than 100 different variations of Bt toxin have been identified in diverse strains of Bt . The different variations have different target specificity Cry gene designation Toxic to the insect orders Cry 1A(a), Cry1A(b), Cry1A(c) Lepidoptera Cry1B, Cry1C, Cry1D Lepidoptera Cry II Lepidoptera, Diptera Cry III Coleoptera Cry IV Diptera Cry V Lepidoptera, Coleoptera 24

25. Steps in development of transgenic plants 25 3 1 2

26. Genetic engineering techniques applied to plants Vector mediated gene transfer Agrobacterium mediated gene transfer Direct (or) vectorless DNA transfer Gene gun Electroporation Microinjection 26

27. Agrobacterium mediated gene transfer Natural soil borne bacterium – Crown gall disease in plants It is an efficient vector of DNA because it has the ability to transmit a fragment of its large plasmid into the nuclear genome of an infected cell The fragment called T-DNA, transfer contained genes- Oncogenes, that induce tumors in the plant tissues The transgenic Agrobacterium is then used to transfer the delta endotoxin protein into the plant cell chromosomes Plant cell is cultured and grown into a whole plant whose ells contains the toxic protein These transgenic resistant plants then produce seed expressing the insect resistant trait, which can be commercialized This procedure has been used to transfer Bt delta endotoxin gene to many plants including (Cotton, tobacco, tomato and potato) 27

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29. List of transgenic plants carrying Bt genes for insect resistance S.N0. Crop Genes Target insect 1 Brinjal Cry IIIb Leptinotarsa 2 Cotton Cry 1A (b) H.zea, PBW 3 Maize Cry 1A (b) O. nubilalis 4 Potato Cry 1A (b) PTM 5 Rice Cry 1A (b) YSB, LF 6 Sugarcane Cry 1A (b) D. saccharalis 7 Tobacco Cry 1A (b) H.virescens 8 Tomato Cry 1A (c) Manduca sexta 29

30. Biolistic method (Particle gun) Also called as “Gene gun or Micro-projectile bombardment” method DNA is bound to tiny particles of gold or tungsten which are shot into plant tissue The particles penetrate both the cell wall and membranes DNA separates from the metal and is integrated into the plant genome inside the nucleus Successfully for monocots- Wheat, maize Disadvantage- Serious damage to the cellular tissue 30

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32. Electroporation Short pulse 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 32

33. Microinjection 33 DNA of interest is taken in microinjector and then delivered inside the cell Micromanipulator Successful- Tobacco & Alfalfa Maximum of 40-50 protoplasts can be microinjected in one hr

34. Success of transgenic plants/ GM plants 34 Insect resistant cotton- Bt toxin kills the cotton boll worm. Transgene- Bt gene from Bt

35. Insect resistant corn- Bt toxin kills European corn borer Transgene- Bt protein 35

36. Herbicide resistant crop- EPSP synthase 36

37. 37 Virus resistant papaya- Resistant to papaya ring-spot virus Transgene- Virus coat protein

38. Advantage & Disadvantage of transgenic plants 38

39. Advantages of Transgenic plants Improvement in nutritional value of food Increase in farmers income Increase in food supply More convenient and flexible to use Safer environment through decreased use of pesticides Improved the quality of ground and surface water with less pesticide residues Safe to non-target organisms and human beings 39

40. Disadvantages of transgenic plants Secondary pest incidence Disruption of pollinators and plant communities would occur if the toxin is expressed in plant nectar and pollen CCD- Is affecting bee hives and it is supposed to be the use of Bt transgenic crops GM ingredients cause cancer- Histopathologist (Dr. Stanley Ewan) “food and water contaminated with GE material could increase the growth of Malignant tumor GM food could raise new allergy outbreak in humans – GM soybean containing “Brazilian protein” was allergic to humans and was withdrawn from production 40

41. Conclusion Transgenic plants have the potential to solve many of the worlds hunger and malnutrition problems Help to protect and preserve the environment by increasing yield and reducing reliance upon chemical pesticides and herbicides 41

42. THANK YOU……….. 42