Transgenic approaches to disease resistance (1).pptx

Transgenic approaches to disease resistance

1. TRANSGENIC APPROACHES TO DISEASE RESISTANCE Submitted to, Dr. Aghil Soorya. A Assistant Professor Dept of Botany St Teresa’s college Ernakulam Submitted by, Silpa Selvaraj Roll no:14 I Msc Botany St Teresa’s college Ernakulam ‹#›

2. TRANSGENIC PLANTS Transgenic plants  are the those plants whose genome is integrated with a desired foreign DNA using genetic engineering techniques. The aim is to introduce a new trait to the plant which does not occur naturally . The inserted gene sequence is known as the transgene. The purpose of inserting a combination of genes in a plant, is to make it as useful and productive as possible. This process provides advantages like improving shelf life, higher yield, improved quality, pest resistance, tolerance to heat, cold and drought resistance, against a variety of biotic and abiotic stresses. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

3. HISTORY The first transgenic plants were reported in 1983 . It was developed through the insertion of  nptII   bacterial antibiotic resistance gene into tobacco. Since then, many  recombinant proteins have been expressed in several important agronomic species of plants including corn, tomato, potato, banana, alfalfa and canola. Tobacco plants were generally used, however potatoes and bananas are also considered, for the purpose of vaccines for human beings. Alfalfa seeds and sprouts Canola ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

4. DEVELOPMENT OF TRANSGENIC PLANTS Most transgenic plants are generated by ; ☆ B iolistic method (Particle gun method) ☆ Agrobacterium   tumefaciens  mediated transformation method. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

5. 1. BIOLISTIC METHOD In this method, DNA of interest is coated with Gold or Tungsten, which is subsequently shot into the target cells, under high pressure using a gene gun . The DNA separates from the coated metal and it integrates into the plant genome inside the nucleus. This method has been applied successfully for many crops, especially monocots, like wheat,maize etc for which transformation using  Agrobacterium tumefaciens  has been less successful. The major limitation of this technique is that the DNA is randomly introduced into the target cell. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

6. Particle gun method ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

7. 2. Agrobacterium tumefaciens MEDIATED TRANSFORMATION METHOD It involves the use of soil-dwelling, gram negative bacteria, known as Agrobacterium tumefaciens . It cause crown gall disease . In addition to the bacterial genome, it contains an extra chromosomal DNA called Ti plasmid which can replicate on its own . The Ti plasmid contains major three regions; T DNA region, Virulence region and Opine catabolism region. Crown gall disease ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

8. Ti PLASMID 1. T-DNA region: This region has the genes for the biosynthesis of auxin (aux), cytokinin (cyt) and opine (ocs) and is flanked by left and right borders . These three genes-aux, cyt and ocs are referred to as oncogenes , as they are the determinants of the tumor phenotype. 2.Virulence region or vir region Vir region codes for proteins involved in T-DNA transfer . At least nine vir-gene operons have been identified. These include vir A, vir G, vir B1, vir C1, vir D1, D2, D4, and vir E1 and E2. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

9. 3. Opine catabolism region: The opine catabolism region is the region from where the bacteria sources its nutrients for the whole process. Opines are derivatives of amino acid or sugar phosphates that can be catabolized to use in the form of nutrients. The types of opines found in Ti-plasmid are nopaline and octopine types. Besides the above three, there is ori region that is responsible for the origin of DNA replication. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

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11. The importance of this plasmid is that, it contain T-DNA region where the genes that cause crown gall formation are removed and replaced with the genes of interest, which can be transferred to a plant cell. In addition, Agrobacterium is capable of transferring large fragments of DNA very efficiently. It also helps in the transfer of DNA pieces with defined ends. One of the biggest limitations of Agrobacterium is that, it has narrow host range This method works well for the dicotyledonous plants like potatoes, tomatoes and tobacco . ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

12. T-DNA transfer and integration: 1. Signal induction to Agrobacterium : The wounded plant cells release certain chemicals- phenolic compounds and sugars which are recognized as signals by Agrobacterium . 2. Attachment of Agrobacterium to plant cells : The Agrobacterium attaches to plant cells through polysaccharides, particularly cellulose fibres produced by the bacterium. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

13. 3. Production of virulence proteins: To start with, signal induction by phenolics stimulates vir A which in turn activates vir C. This induces expression of virulence genes of Ti plasmid to produce the corresponding virulence proteins (D1, D2, E2, B, etc.). Certain sugars (e.g. glucose, galactose, xylose) that induce virulence genes have been identified. 4. Production of T-DNA strand: The right and left borders of T-DNA are recognized by vir D1/vir D2 proteins. These proteins are involved in the production of single-stranded T-DNA , its protection and export to plant cells. The ss T-DNA gets attached to vir D2. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

14. 5. Transfer of T-DNA out of Agrobacterium: The ss T-DNA-vir D2 complex in association with vir G is exported from the bacterial cell. Vir B products form the transport apparatus. 6. Transfer of T-DNA into plant cells and integration : The T-DNA-vir D2 complex crosses the plant plasma membrane. In the plant cells, T-DNA gets covered with vir E2. This covering protects the T-DNA from degradation by nucleases. T-DNA-vir D2-vir E2-plant protein complex enters the nucleus through nuclear pore complex. Within the nucleus, the T-DNA gets integrated into the plant chromosome . ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

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16. Agrobacterium tumefaciens mediated transformation method ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

17. HERBICIDE RESISTANT PLANTS Weeds are unwanted & useless plants that grow along with the crop plants . They compete with the crops for light & nutrients. It is estimated that the worlds crop yield is reduced by 10 – 15 % due to the presence of weeds. To tackle the problem of weeds , modern agriculture has developed a wide range of weed killers called herbicides . The plants which develop resistance against herbicides are called herbicide resistant plants . Eg : Avena fatua (W ild oat), Conyza canadensis (Horseweed) ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

18. An ideal herbicide is to posses the following characters : - Capable of killing weeds with out affecting crop plants . Not toxic to animals & microorganisms . Rapidly degraded in the soil . Commercially available herbicides cannot discriminate weeds from the crop plants . For this reason , crops are also affected by herbicides hence there is need to develop herbicide resistance plants ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

19. Glyphosate It is a broad spectrum herbicide . Ef fective against 76 of world’s worst 78 weeds . Less toxic to animals . Easily degradable. The A merican company (Monsanto) market it as round up . Mechanism of Glyphosate action :-  Capable of killing the plants in low concentrations  Rapidly transported to growing tissues .  Glyphosate is a competitive inhibitor of EPSPS( 5-enolpyruvylshikimate-3-phosphate synthase ) ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

20. EPSPS is blocked by the herbicide glyphosate. This prevents the plant from producing the amino acids necessary for its growth thereby blocking the growth of the plant. ‹#› Enol

21. STRATEGIES FOR ENGINEERING HERBICIDE RESISTANCE O ver expression of EPSPS gene Use of mutant variety of EPSPS gene . D etoxification of herbicide by a foreign gene. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

22. 1.Over expression of EPSPS gene :- An over expression of EPSPS gene was detected in Petunia . Gene from Petunia was isolated & introduced into other plants . The transgenic plants can tolerate glyphosate 2 -4 times higher than that required to kill wild type weed plants . ‹#› Petunia Presented by Silpa Selvaraj Transgenic approaches to disease resistance

23. 2.Use of mutant EPSPS gene ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

24. 3.Detoxification of glyphosate :- The soil microorganisms possess an enzyme glyphosate oxidase reductase that converts glyphosate to glyoxylate and aminomethyl phosphonic acid . Th e gene was isolated from ochrobactrum anthropy & was introduced in to crop plants. e.g: oil seed rape ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

25. PEST RESISTANT PLANTS There is clearly a benefit to farmers, if transgenic plants are developing resistance to a specific pest. There may also be a benefit to the environment, if the use of pesticides is reduced. Transgenic crops, containing insect resistance genes from Bacillus thuringiensis , have made it possible to reduce significantly the amount of insecticide, applied on cotton in the USA. Eg ; Bt cotton, Bt brinjal etc. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

26. INSECT RESISTANT PLANTS Bacillus thuringiensis is a bacterium that is pathogenic for a number of insect pests. Its lethal effect is mediated by a protein toxin it produces. Through recombinant DNA methods, the toxin gene can be introduced directly into the genome of the plant, where it is expressed and provides protection against insect pests of the plant. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

27. VIRUS RESISTANT PLANTS TMV resistant tobacco and tomato plants are produced by introducing viral coat proteins. Other viral resistant transgenic plants are (a) Potato virus resistant potato plants (b) RSV resistant rice (c) YMV resistant black gram (d) YMV resistant green gram. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

28. FUNGAL & BACTERIAL RESISTANT PLANTS The human lysozyme gene, was introduced into tobacco ( Nicotiana tabacum) by the Agrobacterium-mediated method. The introduced human lysozyme gene was highly expressed and the gene product accumulated in the tobacco plants. They showed enhanced resistance against the fungus Erysiphe cichoracearum. Growth of the bacterium Pseudomonas syringae pv. Tabaci was also strongly retarded in the transgenic tobacco, and the disease symptoms were reduced to 17% of that observed in the wild-type tobacco. Thus, the introduction of a human lysozyme gene is an effective approach to protect crops against both fungal and bacterial diseases. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

29. GOLDEN RICE Rice is an important crop for nearly 4 billion people all around the world. At the same time it is deficient in vitamin A . Deficiency of vitamin A leads to eye disorders. The most effective solution for this problem is to genetically engineer rice in such way that beta carotene ,the precursor of vitamin A will be produced in rice endosperm. Such beta carotene rich rice grains will have a characteristic yellow or orange colour. It is called as golden rice . It was developed by two research teams, one headed by Dr.Ingo Potrykus and the other by Dr. Peter Beyer. They genetically engineered a Japanica rice variety by incorporating three genes , two from daffodil ( Narcissus pseudonarcissus) and other one from bacterium Erwinia uredovora . ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

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31. Advantages of golden rice ; It primarily benefits the economically poor and the disadvantaged section of the society. It can be sown every year. Satisfies urgent nutritional needs. Sustainable and cost free solution for vitamin A deficiency. No side effects. Neither adversely affects natural biodiversity nor does it reduce agricultural biodiversity. No negative environmental impact or any conceivable risk to consumer’s health. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

32. Objections against golden rice Although golden rice is claimed to be potentially effective in solving the problems related to vitamin A deficiency, there are strong apprehensions about its acceptability. Environmental groups like Greenpeace, describe it as a “ Trogen horse ” which would open the door for other GM crops, gradually displacing the traditional crops. It is argued that golden rice is quite insufficient to solve the problems of vitamin A deficiency all by itself. Only a mixed diet consisting of several beta carotene containing items would solve the problem of vitamin A deficiency. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

33. FLAVR SAVR TOMATO Among the first GM crops to reach the market in 1994 were the Flavr Savr tomatoes. Flavr Savr tomatoes were launched by the biotechnological company, Calgene (California). It is a transgenic variety of tomato, characterized by reduced and delayed fruit softening. They are very tasty, remain attached to the plant for much longer time and retain their flavour and freshness for a long time even after harvesting. They are developed by applying the antisense RNA technology which minimizes the production of fruit ripening enzyme polygalacturonase . ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

34. Decrease in the synthesis of this enzyme reduces and delays fruit softening. This enables the marketing of fresh tomatoes. Flavr Savr tomatoes were first launched and marketed under the brand name “ Macgreger tomatoes ”. Even though no health risks were found, it was not widely accepted by the consumer sector . The major reasons for this are high price, unattractive texture of skin, undesirable compositional change, objection from environmentalists etc Gradually it was withdrawn from the market. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

35. Flavr Savr tomatoes ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

36. Bt CROPS Bt crops are pest resistant transgenic crops which contain the bacterial Bt toxin gene integrated to their genome. Bt toxin is a toxin produced by Bacillus thuringiensis bacterium. This bacterium was discovered by Japanese bacteriologist Shigetane Ishiwata in 1901 in diseased silkworms. It has adverse effect on insect larvae . The bacterium produces an insecticidal crystal protein ( ICP ) called as delta endotoxin . The gene which encodes this endotoxin is called cry gene . ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

37. When the ICP reach the midgut of the larvae, it gets proteolytically cleaved, releasing active fragments. This fragments interact with the receptors in the midgut membrane. This results in the flow of cations into the cells which leads to the lysis and destruction of the midgut membrane. The endotoxin even at very low concentrations are lethal to the insect larvae. But they are non toxic to other animals like mammals. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

38. Bt COTTON Bt cotton is a genetically engineered form of natural cotton. Bt toxin has to be ingested by the insect larvae to cause death. The toxin becomes active at the high pH of the insect gut. It then attacks the gut cells, punching holes in the lining. Bt spores spill out of the gut and germinate inside the insect causing the death of the insect within a couple of days. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

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40. Why Bt cotton is a good choice over conventional cotton? Cotton is the most important commercial crop of India, It is called as white gold . In India it is grown in 5 percent of the total crop area but it uses upto 55 percentage of all pesticides. Bt cotton has high resistance to pest attacks, gives high yield, environment safe and cost effective. Since the plants produce toxins in their tissues, there is no need to spray synthetic pesticides. Bt toxins do not destroy beneficial insects. Exposure of farm workers and non target organisms to pesticides also get very much reduced. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

41. Bt BRINJAL Bt Brinjal is the first Genetically Modified food crop in India. It is developed by inserting a gene called cry1Ac from a soil bacterium called Bacillus thuringiensis through Agrobacterium- mediated gene transfer. Bt Brinjal was developed by Maharashtra Hybrid Seeds Company (Mahyco). It gives resistance against insects such as the Brinjal Fruit and Shoot Borer ( Leucinodes orbonalis ) Once a fruit and shoot borer larva feed on Bt brinjal plants, the cry1Ac protein reacts with the alkaline insect gut, It disrupts the insect’s digestive process, causing paralysis and eventually the death of the larvae. Fruit and shoot borer ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

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43. Advantages of Bt Brinjal Bt brinjal has been found to be safe for human consumption and safe for the environment. The Cry1Ac endo-toxin is a protein that breaks down when cooked. It is active only in an alkaline medium, and since humans consume brinjal only when cooked, it will not interfere with digestion. Additionally, as the stomach is acidic, the digestive process will not be affected by the introduction of the Cry1Ac toxin. The toxin breaks down into common amino acids in the digestive system, which are part of the normal diet and are neither toxic nor allergic. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

44. The Cry1Ac endo-toxin would only damage the fruit and shoot borer gut which is alkaline. Bt brinjal increases marketable yields, thus resulting in higher incomes for farmers. Farmers will be able to continue to save and re-use their seeds for the hybrids varieties. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

45. CONCERNS ABOUT Bt Brinjal Environmental Impact: The use of pest-resistant Bt Brinjal may lead to the extinction of species considered important for farm ecology. Bt Brinjals can become costly: Companies that own Bt Brinjal seeds can charge a high price for their procurement, as it is predicted that its high yield and increased shelf life will fuel demand. If that is the case, it might lead to a scenario where small and marginal farmers can ill afford Bt brinjal seeds. Scientists are of the opinion that Bt brinjal poses a serious health risk, as its resistance to antibiotics can make medicines ineffective. There also might be adverse effects of forming allergens and other anti-nutritions in foods. M.S Swaminathan, the father of India’s Green Revolution , had asked for an independent study on the long toxicity of Bt Brinjal before approving it for commercial use. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

46. Some interesting transgenic plants ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

47. Transgenic colorful corns ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

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49. Are blue java bananas transgenic??? Blue Java Banana ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

50. Blue java bananas are transgenic Blue java bananas are not transgenic ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

51. Blue Java which owes its name to the characteristic blue colour of the peel when it is unripe. However, no genetic modification is involved – it’s all work of mother nature. Its peculiarity is not so much in the colour, but precisely in its sweet taste that is said to have the consistency of an ice cream and the flavour that recalls vanilla. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

52. REFERENCE https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.frontiersin.org/articles . https://www.google.com/url?sa=t&source=web&rct=j&url=http://www.igntu.ac.in/eContent/IGNTU-eContent-317364151984-M.Sc-EnvironmentalScience-2-ManojkumarRai-Environmentalmicrobiologyandbiotechnology . Kikkert, J. R., Vidal, J. R., & Reisch, B. I. (2005). Stable transformation of plant cells by particle bombardment/biolistics.  Methods in molecular biology (Clifton, N.J.) ,  286 , 61–78. Nakajima, H., Muranaka, T., Ishige, F., Akutsu, K., & Oeda, K. (1997). Fungal and bacterial disease resistance in transgenic plants expressing human lysozyme. Plant cell reports, 16(10), 674–679. ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance

53. THANK YOU ‹#› Presented by Silpa Selvaraj Transgenic approaches to disease resistance