genne transfer and transgenic cotton soybean corn mustard

agrobacterium, and production of transgenic plants in cotton, corn,soybean,mustard, recent status, problems in india and worldwide statistics.

1. THE ROLE OF VECTOR MEDIATED GENE TRANSFER AND PRODUCTION OF TRANSGENIC PLANTS IN COTTON,MAIZE,SOYBEAN AND OILSEED Submitted To : Submitted By : Dr. A. K. Sharma Smrutishree S ahoo Professor M.Sc.(Ag.) PBG Previous Department of PLANT BREEDING AND GENETICS Collage of Agriculture Swami Keshwanand Rajasthan Agricultural University, Bikaner

2. GENE TRANSFER IN PLANTS Transfer a gene from one DNA molecule to another DNA molecule. GENETIC TRANSFORMATION desirable gene transfer from one organism to another stable integration expression of foreign gene into the genome Transient transformation - DNA is not integrated into host genome Stable transformation - DNA is integrated into host genome and is inherited in subsequent generations. The transferred gene is known as TRANSGENE and the organism that develop after a successful gene transfer is known as TRANSGENIC.

3. METHODS OF GENE TRANSFER NATURAL METHODS 1 . Conjugation 2. Bacterial transformation 3. Retroviral transduction 4. Agrobacterium mediated transfer( Ti vector) ARTIFICIAL METHODS Physical methods 1. Microinjection 2. Biolistics transformation/particle gun Chemical methods 1. DNA transfer by calcium phosphate method 2. Liposome mediated transfer Electrical methods 1. Electroporation Ti vectors mostly used followed by paricle gun and electroporation .

4. VECTORS FOR THE PRODUCTION OF TRANSGENIC PLANTS A vector is a DNA molecule capable of independent existence and replication. Properties of a good vectors 1) replicate autonomously 2) easy to isolate and purify 3) easily introduced into the host cells. 4)suitable marker genes that allow easy detection of the transformed host cell. Eg . Genes for amphicillin and Tetracycline resistance. 5) unique target sites for restriction enzymes 6) vector should contain suitable regulatory elements like promoter, operator, ribosome binding sites.

5. Types of plant vectors PLANT VECTORS PLASMID VECTORS 1-pTi(tumour inducing plasmid) 2-pRi (root inducing plasmid) PLANT VIRUS VECTORS 1-CaMV(cauliflower mosaic virus) 2-Gemini virus 3-TMV(tobacco mosaic virus) 4-BMV(brome mosaic virus )

6. Agrobacterium - mediated Gene Transfer Most common method of engineering dicots , but also used for monocots Pioneered by J. Schell (Max-Planck Inst., Cologne) Agrobacteria soil bacteria, gram-negative, related to Rhizobia species: tumefaciens - causes crown galls on many dicots rubi - causes small galls on a few dicots rhizogenes - hairy root disease radiobacter - avirulent

7. Infection and tumorigenesis Infection occurs at wound sites Involves recognition and chemotaxis of the bacterium toward wounded cells galls are “real tumors”, can be removed and will grow indefinitely without hormones genetic information must be transferred to plant cells

8. Why Agrobacterium ? Agrobacterium tumefaciens or Agrobacterium rhizogenes mediated transformation is to date the most commonly used method for obtaining transgenic plants. host plant species range A. tumefaciens include : Large number of dicots and some monocots and Gymnosperms . Agrobacteria are naturally occurring, ubiquitous soil borne pathogens. 1- A. tumefaciens causes crown gall disease ( tumors ) 2- A. rhizogenes causes root hair disease (hairy root) Other bacterial groups also contain species capable of interkingdom genetic exchange ( Gelvin 2005).

9. Gene Transfer using Agrobacterium Ti-plasmid(200kb) Ti plasmid produce octopine and nopaline . Ri plasmid produce agropine and mannopine . Regions in Ti plasmid- 1-T-DNA contains oncogene and opine synthesis genes 2-vir region-regulates transfer of T-DNA to plant cells 3-opine catabolism region 4-conjugative transfer region( oriT or tra ) region 5-origin of replication Right and left border (RB, LB) sequences are the only parts of T-DNA needed to enable transfer into plants.

10. LB RB LB, RB – left and right borders (direct repeat) auxA + auxB – enzymes that produce auxin cyt – enzyme that produces cytokinin Ocs – octopine synthase , produces octopine T-DNA These genes have typical eukaryotic expression signals! On the Ti plasmid Transfer the T-DNA to plant cell Acetosyringone (AS) (a flavonoid ) released by wounded plant cells activates vir genes. virA,B,C,D,E,F,G (7 complementation groups), span about 30 kb of Ti plasmid. auxA auxB cyt ocs Vir (virulent) genes

11. Overview of the Infection Process

12. A. tumefaciens & Crown Gall Disease

13. Agrobacterium tumefaciens for TRANGENIC PLANTS DRAWBACKS: Auxine/ Cytochine made by T-DNA do not allow proper plant regeneration Opine is not usefull for plant Ti plasmids are big (200-800Kb) Monocots don't produce AS in response to wounding. couldn't regenerate plants from tumors

14. 1) BINARY VECTOR SYSTEM Strategy: 1. Move T-DNA onto a separate, small plasmid .(mi-Ti) 2. Remove aux and cyt genes. 3. Insert selectable marker (kanamycin resistance) gene in T-DNA. 4. Vir genes are retained on a separate plasmid .(helper Ti) 5. Put foreign gene between T-DNA borders. 6. Co-transform Agrobacterium with both plasmids. Infect plant with the transformed bacteria. 2) CO-INTEGRATE pTi VECTORS Produced by integrating the modified E. coli plasmid(pBR322)in to disarmed pTi . VECTORS DERIVED FROM pTi- Disarmed pTi - the deletion of genes governing auxin and cytokinin production from T-DNA of a Ti-plasmid.

15. Tearless Onion Colorful Cauliflowers Purple tomatoes Blue Roses TRANSGENICS

16. TRANSGENIC PLANTS “ Transgenics ” or GMOs are defined as those organisms with a gene or genetic construct of interest that has been introduced by molecular or recombinant DNA techniques. The power of this technique lies in its ability to move genes from one organism to crop plants to impart novel characteristics. It is possible to transfer genetic material from algae, bacteria, viruses or animals to plants or to move genes between sexually incompatible species.

17. TARGET TRAITS Disease resistance Improving the quantity of the protein Increasing vitamin content Stress tolerance Herbicide resistance Delayed ripening Edible vaccine

18. VEGETABLES Tomato, Potato, Eggplant Lettuce, Celery, Cauliflower Cabbage, Sugarbeet , Carrot, Cucumbers, Sweetpotato , Cassava FRUITS Apple, Strawberry, Walnut, Muskmelon, Papaya, Grape Transgenic Crops for Food EDIBLE OILS Mustard Oilseed rape Canola Sunflower CEREALS Wheat, Rice Maize, Rye LEGUMES Soybean, Pigeon pea, Chick pea

19. TRANSGENIC COTTON HERBICIDE RESISTANCE Glyphosate Resistance Glyphosate = “Roundup”, “Tumbleweed” = Systemic herbicide Marketed under the name Roundup, glyphosate inhibits the enzyme EPSPS (S-enol p yruvl s hikimate-3 p hosphate – involved in chloroplast amino acid synthesis) , makes aromatic amino acids. The gene encoding EPSPS has been transferred from glyphosate -resistant E. coli into plants, allowing plants to be resistant.

20. EPSP SYNTHASE Phosphoenol pyruvate Tryptophan Tyrosine Phenylalaline ROUNDUP ( Glyphosate ) Glucose 3 phosphoglycerate Glycolysis EPSP SYNTHASE ROUNDUP (Glyphosate ) In transgenic plant, herbicide cannot bind the mutant of EPSPS (Roundup resistant cotton and soybean) ROUNDUP ( Glyphosate ) RESISTANCE ( HERBICIDE TOLERANCE IN CROPS)

21. Bt COTTON-INSECT RESISTANCE Genetically modified to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis . Toxins are crystalline proteins (Cry-proteins) that target specific pests. DISCOVERY OF Bt- COTTON Ernst Berliner isolated  a bacteria that had killed a Mediterranean flour moth in 1911, and rediscovered Bt. He named it Bacillus thuringiensis , after the German town Thuringia where the moth was found. INTRODUCTION TO INDIA? In 2002, a joint venture between  Monsanto  and Mahyco introduced Bt cotton to India. In 2011, India grew the largest GM cotton crop at 10.6 million hectares.

22. ( Agrobacterium mediated)

23. Bacillus thuringiensis  (or  Bt ) a  Gram-positive , soil-dwelling  bacterium , commonly used as a  biological pesticide Cry gene- Upon sporulation , B. thuringiensis forms crystals of proteinaceous insecticidal δ- endotoxins (called crystal proteins or  Cry  proteins), which are encoded by  cry genes . How Bt works? 1. Insect eats  Bt  crystals and spores. 2. The toxin binds to specific receptors in the gut and the insects stops eating. 3. The crystals cause the gut wall to break down, allowing spores and normal gut bacteria to enter the body. 4. The insect dies as spores and gut bacteria proliferate in the body. alkaline digestive tracts denature the insoluble crystals soluble and being cut with  proteases  found in the insect gut, which liberate the toxin from the crystal. The Cry toxin paralyzing the digestive tract and forming a pore.The insect stops eating and starves to death. 

24. ADVANTAGE AND DISADVANTAGE Advantages High insect specificity Control crop damage and disease vectors Nontoxic to non-target species Biodegradable Reduction of other insecticides 94.5 million kg (19.4%) from 1996 to 2005 for cotton Yield increases Limitations Susceptible to resistance High seed cost

25. Bt-COTTON INDIA At present, 96% of India cotton cultivation area is under Bt cotton crops but it wasn’t always so. Cotton production rose from 14 million bales in the pre-Bt year of 2001-'02 to 39 million bales in 2014-'15, a rise of almost 180%. India’s cotton imports fell, exports grew and as of 2015-16 India is expected to have overtaken China as the biggest cotton producer it the world.

26. PROBLEMS FACED the adoption of Bt cotton is that the seeds are more expensive than local, non-genetically modified varieties. the seeds cannot be reused and farmers need to buy new stock for every growing season. This, along with licencing agreements with local seed companies, has given Monsanto a near monopoly on cotton seeds in India that has been the biggest worry for activists. the diffusion of illegal Bt hybrids that hadn't been cleared for biosafety standards, leading to fears of environmental toxicity. Bt hybrids were unsuitable for rain-fed cotton lands so yield stagnation of more than 1000 varieties cause farmer suicide. Bt provides protection only against one type of cotton pest, use of insecticides has risen again close to the levels of the pre-Bt years.

27. PRESENT ISSUES Despite Monsanto's warning the government cut royalty by more than 70% on March 9, 2017and followed that up by capping the price of seeds at Rs 800. They were earlier sold at between Rs 830 and Rs 1,000. Monsanto faces a big challenge from the Central Institute of Cotton Research, which has introduced Bt genes into 21 cotton seed varieties and is offering to provide these seeds to farmers at 10% the cost of Monsanto’s products. if Monsanto leaves then India will lose access to the new iterations of its Bollgard seed that farmers might need in the next three to five years

28. TRANSGENIC CORN(MAIZE) HERBICIDE RESISTANT MAIZE Corn varieties resistant to  glyphosate  herbicides were first commercialized in 1996 by  Monsanto , and are known as "Roundup Ready Corn".They tolerate the use of  Roundup . Bayer CropScience  developed "Liberty Link Corn" that is resistant to  glufosinate .   Pioneer Hi-Bred  has developed and markets corn hybrids with tolerance to  imidazoline  herbicides under the trademark "Clearfield "

29. INSECTICIDE-PRODUCING CORN Bt-CORN The  European corn borer ,  Ostrinia nubilalis , destroys corn crops by burrowing into the stem, causing the plant to fall over. The  European corn borer  causes about a billion dollars in damage to corn crops each year. In recent years, traits have been added to ward off  corn ear worms  and  root worms , the latter of which annually causes about a billion dollars in damages. Bt-corn is poisonous to these insects.

30. Bt-corn Non Bt-corn Bt-corn reduces the need for conventional pesticides:

31. DROUGHT RESISTANCE In 2013  Monsanto  launched the first transgenic drought tolerance trait in a line of corn hybrids called DroughtGard . The MON 87460 trait is provided by the insertion of the cspB gene from the soil microbe  Bacillus subtilis ; it was approved by the USDA in 2011 and by China in 2013 SWEET CORN GM sweet corn varieties include "Attribute", the brand name for insect-resistant sweet corn developed by  Syngenta and Performance Series™ insect-resistant sweet corn developed by Monsanto

32. PRESENT SCENARIO(INDIA) India does not currently allow the growing of GM food crops but the government of Prime Minister Narendra Modi , keen to improve farms' productivity, has encouraged open field trials after a five-year de facto ban. Monsanto's ( MON.N ) Indian subsidiary expects to submit final trial results for its genetically modified (GM) corn to lawmakers within a year for the government to then decide on a commercial launch. the resulting corn will be insect- and herbicide-tolerant, helping raise yields by 15-20 percent . The current average corn yield in India is 2-2.25 tonnes per hectare, compared with 10 tonnes in the top producer United States

33. TRANSGENIC SOYBEAN  In 1994 the first genetically modified soybean was introduced to the U.S. market, by  Monsanto . In 2014, 90.7 million hectares of GM soy were planted worldwide, 82% of the total soy cultivation area ROUNDUP READY SOYBEAN Roundup Ready Soybeans (The first variety was also known as GTS 40-3-2 (OECD UI: MON-04032-6)) are a series of  genetically engineered varieties of  glyphosate -resistant  soybeans  produced by  Monsanto .

34. TRANSGENIC OILSEEDS

35. ß-carotene rich Mustard oil

36. GM HYBRIDISED MUSTARD , As it is claimed, gives up to 30% more yield than the present best varieties. Researchers have used “ barnase / barstar ” technology for genetic modification. A barnase gene is isolated from a soil bacterium called Bacillus amyloliquefaciens . The gene can code for a protein that impairs the pollen production in a plant making it male-sterile . This male-sterile variety is crossed with a parent variety having a gene called ‘ barstar ’ to block the action of barnase gene. The resulting variety, having both foreign genes , is a fertile plant and it can increase yield of the crop. This canola crop is cultivated 10% in Canada.

37. STATISTICS Global overview Values Acreage of genetically modified crops worldwide 179.7m ha Acreage of genetically modified soybean crops worldwide 92m ha Leading GMO crops producing country, based on acreage United States Acreage of genetically modified crops in the U.S. 70.9m ha Adoption of GMO technology for soybeans worldwide 83% Adoption of GMO technology for cotton worldwide 75%

38. Global Adoption of Biotech Soybean, Maize, Cotton, and Canola The most planted biotech crops in 2016 were soybean, maize, cotton, and canola. Although there was only 1% increase in the planting of biotech soybean, it maintained its high adoption rate of 50% of the global biotech crops or 91.4 million hectares. This area is 78% of the total soybean production worldwide.

39. Global Area of Biotech Crops in 2015 and 2016: by Country (million hectares) rank Country 2015 2016 1 USA* 70.9 72.9 2 Brazil* 44.2 49.1 3 Argentina* 24.5 23.8 4 Canada* 11.0 11.6 5 India* 11.6 10.8

40. Canada overview Values Genetically modified soybeans seeded in Ontario 718.3k ha Genetically modified corn for grain seeded in Ontario 679.9k ha Genetically modified soybeans seeded in Quebec 210k ha Genetically modified corn for grain seeded in Ontario 303k ha U.S. overview Values Share of GMO corn in the U.S., based on acreage 92% Share of GMO cotton in the U.S., based on acreage 80%

41. Attitudes of farmers and consumers Values U.S. Millennials who think that GMOs should never be used in food and beverages 34% U.S. Millennials who think that GMOs need more testing 32% U.S. consumers who drink less coffee because of GMO concerns 22% GMO is the most important food safety issue for U.S. consumers 24% U.S. consumers strongly agree that GMOs should be labeled as such 48%

42. crop variety Maize MON 810, MON 863, StarLink Soybean Roundup ready soybean, Vistive gold mustard DMH-11, Varuna   , the east European EH-2 Cotton PAU Bt 1, F1861 and RS 2013

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