Transgenic crops and application

Transgenic crops and application in plant biotechnology

1. TRANSGENIC Crops & its APPLICATION Submitted to: Dr. K. P. Pachchigar Submitted by: Gami Pankajkumar B. M.Sc.(Agri.)GPB 1 st sem. MBB501: Principles of Biotechnology

2. What Are Transgenic Plants? A transgenic crop plant contains a gene or genes which have been artificially inserted instead of the plant acquiring them through pollination. The inserted gene sequence (known as the  transgene ) may come from another unrelated plant, or from a completely different species : for example transgenic Bt corn, which produces its own insecticide, contains a gene from a bacterium. Plants containing transgenes are often called  genetically modified or GM crops , although in reality all crops have been genetically modified from their original wild state by domestication, selection and controlled breeding over long periods of time.

3. What is the need of transgenic plants? A plant breeder tries to assemble a combination of genes in a crop plant which will make it as useful and productive as possible. The desirable genes may provide features such as higher yield or improved quality, pest or disease resistance, or tolerance to heat, cold and drought . This powerful tool enables plant breeders to do what they have always done - generate more useful and productive crop varieties containing new combinations of genes - but this approach expands the possibilities beyond the limitations imposed by traditional cross-pollination and selection techniques.

4. Procedure in production of Transgenic crops The novel transgene is identified ,isolated and clonned Designing of gene The target plants are transformed (by vector mediated or direct method) Selection of transgenic plant tissue/ cells Regeneration of the transgenic plant by the process of plant tissue culture

5. TRANSFORMATION Gene transfer or uptake of DNA refers to the process that moves a specific piece of DNA into cell. The directed desirable gene transfer from one organism to another and the subsequent stable integration & expression of foreign gene into the genome is referred as genetic transformation.

6. TRANSFORMATION METHODS:

7. Applications: Crop Improvement The following traits are potentially useful to plant genetic engineering for abiotic and biotic stress resistance. Genetically Engineered Traits: The Big Six. Herbicide Resistance Insect Resistance Virus Resistance Altered Oil Content Delayed Fruit Ripening Drought tolerence

8. 1. Herbicide Resistance Over 63% of transgenic crops grown globally have herbicide resistance traits. Herbicide resistance is achieved through the introduction of a gene from a bacterium conveying resistance to some herbicides . In modern agriculture, the herbicides have been taken the major role in weed control. Though the uses of herbicide offers several advantages, i.e., permitting economic weed control, increasing the efficiency of crop production resulting in higher crop yield and biodegradability etc., they are endowed with several limitations as well, i.e., lack of selectivity is one of the most important limitation.

9. Genetic engineering offers the scope of modifying plants through integration of genes providing resistance to broad spectrum herbicides. Three approaches have been followed in the production of herbicide resistant plants: i ) over production of herbicide sensitive biochemical compounds; ii) structural alteration of a biochemical target compound resulting in reduced herbicide affinity, and iii) detoxification or degradation of the herbicide before it reaches the biochemical target inside the plant cell.

10. A) Glyphosate Resistance One of the most famous kinds of GM crops are " Roundup Ready ", or  glyphosate -resistant. Glyphosate, (the active ingredient in Roundup) kills plants by interfering with the  shikimate  pathway in plants , which is essential for the synthesis of the aromatic amino acids like a  phenylalanine, tyrosine and tryptophan. More specifically, glyphosate inhibits the enzyme  5-enolpyruvylshikimate-3-phosphate synthase  ( EPSPS ). The gene encoding EPSPS has been transferred from glyphosate -resistant E. coli into plants, which allow plants to be resistant.

11. Also some micro-organisms have a version of EPSPS that is resistant to glyphosate inhibition. One of these was isolated from an Agrobacterium  strain CP4 (CP4 EPSPS) that was resistant to glyphosate . It produce an enzyme that inactivates glyphosate . Glyphosate is rapidly metabolized by Glyphosate oxidoreductase (GOX)

12. Glyphosate Sensitive Plants Without amino acids, plant dies X Phenylalanine

13. Glyphosate Resistant Plants With amino acids, plant lives Glyphosate oxidoreductase Phenylalanine

14. b) Glufosinate Resistance Glufosinate (the active ingredient in phosphinothricin ) mimics the structure of the amino acid glutamine , which blocks the enzyme glutamate synthase. Plants receive a gene from the bacterium Streptomyces that produce a protein that inactivates the herbicide. c) Bromoxynil Resistance A gene encoding the enzyme bromoxynil nitrilase (BXN) is transferred from Klebsiella pneumoniae bacteria to plants. Nitrilase inactivates the Bromoxynil before it kills the plant . d) Sulfonylurea . Kills plants by blocking an enzyme needed for synthesis of the amino acids valine , leucine , and isoleucine . Resistance generated by mutating a gene in tobacco plants, and transferring the mutated gene into crop plants.

15. 2 . Insect Resistance Progress in engineering insect resistance in transgenic plants has been achieved through the use of insect control protein genes of Bacillus thuringiensis . Insect resistance was first reported in tobacco ( Vaeck et al., 1987) and tomato ( Fischhoff et al., 1987). Today insect resistant transgenes , whether of plant, bacterial or other origin, can be introduced in to plants to increase the level of insect resistance. More than 400 genes encoding toxins from wide range of B. thuringiensis have been identified so far and approximately 40 different genes conferring insect resistance have been incorporated into crops.

16. Genes conferring insect resistance to plants have been obtained from microorganisms: Bt gene from Bacillus thuringiensis ; ipt gene ( isopentyl transferase ) from Agrobacterium tumefaciens , cholesterol oxidase gene from a streptomyces fungus and Pht gene from Photorhabdus luminescens . Bt toxin gene: Bacillus thuringiensis (Bt) is an entomocidal bacterium that produces an insect control protein. Bt genes code for the Bt toxin. Most Bt toxins are active against Lepidopteran larvae but some are specific for Dipteran and Coleopteran insects. The insect toxicity of Bt resides in a large protein.

17. The toxins accumulate as crystal proteins (CS- endotoxins ) inside the bacteria during sporulation . They are converted to active form upon infection by susceptible insect, thereby killing the insect by disruption of ion transport across the brush borders/ membranes of susceptible insect. Based on their host range Hofte and Whiteley classified Bt toxins into 14 distinct groups and 4 classes ( Hofte and Whiteley 1989) viz. - CryI (active against Lepidoptera) - CryII (Lepidoptera and Diptera ) CryIII ( Coleoptera ) and - CryIV ( Diptera ).

18. Cry proteins are toxic to insects (mainly against lepidopteran , coleopteran, dipteran , and nematodes), but non-toxic to human and animals (BANR, 2000).

19. Toxic Action of Cry Proteins When Cry protein ingested by lepidopteran insect larvae , a protoxin is solubilized by the high pH of the gut lumen and solubilization of the protoxin is activated through cleavage by digestive enzymes into a smaller (~60kDa) fragment ( Hofte and Whiteley , 1989; OECD, 2007). Then, activated toxic fragment can binds to receptors on the membrane of the insect’s midgut epithelial cells (Bravo et al., 1992) and follows the activation of an apoptotic signal cascade pathway (Zhang et al., 2006) , causing formation of pores . This leads to osmotic shock, cell lysis and insect death ( Lorence et al., 1995).

20. Mode of action of Cry toxin Mode of action of Bacillus thuringiensis in lepidopteran caterpillar: 1. ingestion by bacteria; 2. solubilization of the crystals; 3. activation protein; 4. binding of proteins to the receptors 5. membrane pore formation and cell disruption (Modified from: Schünemann et al., 2014).

21. Credit:Transgenic plants: resistance to abiotic and biotic stresses : Akila Wijerathna-Yapa ;Journal of Agriculture and Environment for International Development - JAEID 2017, 111 (1): 245-275DOI: 10.12895/jaeid.20171.643

22. 3. Virus Resistance Chemicals are used to control the insect vectors of viruses, but controlling the disease itself is difficult because the disease spreads quickly. Plants may be engineered with genes for resistance to viruses, bacteria, and fungi. Virus diseases of cultivated plants cause substantial loss in food, forage and fiber crops throughout the world. No large scale methods exist for curing plants once they have become virus infected. The development of molecular strategies for the control of virus diseases has been especially successful owing to small genomic size of plant viruses which make them particularly amenable to molecular techniques for cloning.

23. The approach is to identify those viral genes or gene products which was present at an improper time or in wrong amount ,will interfere with the normal functions of infection process and prevent disease development. Virus Coat Protein Mediated Cross Protection The concept of cross protection is the ability of one virus to prevent or inhibit the effect of a second challenge virus. Transgenic tobacco expressing tobacco mosaic virus (TMV) coat protein which resistance similar to that occurs in viral mediated cross protection (Powell-Abel et al., 1986). The resistance reduce numbers of infection sites on inoculated leaves, suggesting that an initial step in the virus life cycle has been disrupted . It has been demonstrated that TMV cross protection may result from the coat protein of the protecting virus preventing un-coating of the challenge virus RNA.

24. This approach has been used in several crops like tobacco, tomato, potato, rice, maize, melons, alfalfa, sugar beet etc. Courtesy:Transgenic plants: resistance to abiotic and biotic stresses : Akila Wijerathna-Yapa ;Journal of Agriculture and Environment for International Development - JAEID 2017, 111 (1): 245-275DOI: 10.12895/jaeid.20171.643

25. Altered Oil Content Oil quality can be changed by genetic modification of crops or by chemical modification of the fatty acids. Conventional breeding has been remarkably successful in modifying oil quality. Genetic engineering now offers unique opportunities to produce novel fatty acids. The various strategies used for modifying fatty acids are as follows: ( i ) introduction of a novel enzyme, e.g., an acyl transferase or an acyl -ACP thioesterase , acyl -ACP desaturase , etc., (ii) suppression of an enzyme activity, e.g., α acyl -ACP desaturase ), (iii) site-directed mutagenesis to alter the specificity, etc. of an enzyme, e.g., acyl -ACP desaturase , etc., and (iv) creation of hybrid genes to generate novel enzyme activities.

26. Perhaps the most successful example is the increased lauric acid content of B. napus ; a transgenic line name ' Laurical ' has been released for commercial cultivation. Lauric acid does not occur naturally in Brassica sp. oil. But seeds of undomesticated California bay ( Ullibellularia californica ) accumulate laurate (12:0). The gene encoding lauroyl -ACP thioesterase was isolated from U. californica and transferred into B. napus . Some of the transgenic lines showed upto 60% lauric acid in their oils. Thereis some evidence that a part of the lauric acid in transgenic B. napus is subjected to ß-oxidation. Varieties of canola and soybean plants have been genetically engineered to produce oils with better cooking and nutritional properties. Genetically engineered plants may also be able to produce oils that are used in detergents, soaps, cosmetics, lubricants, and paints.

27. Delayed Fruit Ripening Allow for crops, such as tomatoes, to have a higher shelf life. Tomatoes generally ripen and become soft during shipment to a store. Tomatoes are usually picked and sprayed with the plant hormone ethylene to induce ripening, although this does not improve taste. Tomatoes have been engineered to produce less ethylene so they can develop more taste before ripening, and shipment to markets.

28. Transgenic tomato with delayed ripening:lower level of ethylene production Reduced activity of the cell wall degrading enzymes, e.g. polygalacturonases

29. Produced by calgene by blocking the polygalacturonase (PG) gene, which is involved in spoilage. PG is an enzyme that breaks down pectin, which is found in plant cell walls. Plants were transformed with the anti-sense PG gene, which is mRNA that base pair with mRNA which plant produces, essentially blocking the gene from translation. First genetically modified organism to be approved by the FDA, in 1994.

30. The Flavr Savr Tomato (First transgenic Plant Product) Cited by: https://www.google.com/search?q=flavour+saver+tomato&source=lnms&tbm=isch&sa=X&ved=0ahUKEwjqlduvbThAhW57nMBHephCCMQ_AUIDigB&biw=1500&bih=711&dpr=0.9#imgrc=oSDrw5FBxewfTM:

31. Use of gene for 1-aminocyclopropane-1-carboxylic acid(ACC) deaminase : s- adenosyl methionine (SAM) 1-aminocyclopropane-1-carboxylic acid(ACC) ethylene Other metabolites SAM hydrolase ( bacteriophage T3)

32. 6.Drought resistance: A number of such genes have been identified, isolated, cloned and expressed in plants, which are potential sources of resistance to abiotic stresses. These genes include Rab (responsive to abscisic acid) and SalT (induced in response to salt stress) genes of rice; genes for enzymes involved in proline biosynthesis in bacteria ( proBA and proC in E. coli) and plants. In plants, proline is preferentially produced from ornithine under normal conditions. However, under stress it is made directly from glutamate, the first two reactions of the pathway being catalyzed by a single enzyme ∆ 1 -pyrroline-5-carboxylate synthetase (P5CS) .

33. The gene encoding P5CS (as well as that encoding P5CR, A1-pyrroline-5-carboxylate reductase ; a non-rate limiting enzy me of the pathway) has been isolated from soybean and mothbean , and cloned. The mothbean PSCS gene has been transferred and overexpressed in tobacco. The transgenic plants produced 10- to 15-fold more proline than the control plants. The leaves of transgenic plants retained a higher osmotic potential and showed a greater root biomass under water stress than did the control plants. Thesefindings indicates that overexpression of P5CS in plants enhances their tolerance to osmotic stress.

34. Genetically Engineered Foods 1. More than 60% of processed foods in the United States contain ingredients from genetically engineered organisms. 2. 12 different genetically engineered plants have been approved in the United States, with many variations of each plant, some approved and some not. 3. Soybeans. Soybean has been modified to be resistant to broad-spectrum herbicides. Scientists in 2003 removed an antigen from soybean called P34 that can cause a severe allergic response.

35. 4. Corn Bt insect resistance is the most common use of engineered corn, but herbicide resistance is also a desired trait. Products include corn oil, corn syrup, corn flour, baking powder, and alcohol. By 2002 about 32% of field corn in the United States was engineered. 5. Canola. More than 60% of the crop in 2002 was genetically engineered; it is found in many processed foods, and is also a common cooking oil.

36. 6. Cotton. More than 71% of the cotton crop in 2002 was engineered. Engineered cottonseed oil is found in pastries, snack foods, fried foods, and peanut butter. 7.Nutritionally Enhanced Plants—Golden Rice: More than one third of the world’s population relies on rice as a food staple, so rice is an attractive target for enhancement. Golden Rice was genetically engineered to produce high levels of beta-carotene, which is a precursor to vitamin A. Vitamin A is needed for proper eyesight.

37. Golden Rice was developed by Ingo Potrykus and Peter Beyer, and several agencies are attempting to distribute the rice worldwide Other enhanced crops include iron-enriched rice and tomatoes with three times the normal amount of beta-carotene Rice endosperm synthesize geranyl-geranyl pyrophosphate which can be converted into ß-carotene by 3 enzymes. Phytoene desaturase and Lycopene ß Cyclase derived from daffodil( Narcissus pseudonarcissus ) and Carotene desaturase from Erwinia uredova .

38. The transgenes providing these enzyme activities were transferred into rice through Agrobacterium mediated transformation. The resulting rice looks yellowish orange in colour and contain ß-carotene kwnon as ‘Golden Rice’.

39. Golden rice

40. Advantages of Transgenic crops: Resistance to biotic stress( insects,diseases,viruses ) Resistance to abiotic stress Herbicide resistance Improved quality Male sterile crops High yield Cheap to maintain disease resistant species High adaptability

41. Disadvantages Allergic reactions Reducing the numbers of pest insects in farmland and impacting biodiversity. Significant negative impact on wildlife(wider effects on food chains ) Chance of developing resistance in weeds (uncontrollable weeds)

42. References books: 1.Introduction to plant biotechnology- H.S. Chawla 2.Biotechnology, expanding horizons - B.D.Singh  

43. Thank you