Genetically Modified Crops SMG

Genetically Modified Crops : Techniques , some examples

1. GENETICALLY MODIFIED CROPS Dr. Saji Mariam George Associate Professor, Dept. of Botany, Assumption College Autonomous, Changanacherry

2. GENETICALLY MODIFIED CROPS (GMCs) Genetically manipulated or genetically engineered or Transgenic crops or Transgenics . Produced by introducing a gene from an unrelated organism, also known as a foreign gene or transgene (= trans ferred gene ) by suitable genetic engineering techniques. Transgene on expression give new character which is absent in the population of the species . GMCs can be exploited in Agriculture, Medicine, Horticulture, Floriculture etc.

3. TECHNIQUES FOR PRODUCTION OF GMCs I. Recombinant DNA( rDNA ) Technology (Herbert W.Boyer , Stanley N. Cohen & Paul Berg , 1975) Isolation of the desired gene or DNA fragment to be cloned from the donor organism by suitable biochemical methods. Cutting or slicing the DNA with suitable restriction endonuclease enzyme. Insertion of the desired gene/s (foreign DNA) into a suitable vector (Splicing of DNA into vectors) to form recombinant DNA ( rDNA ) Introduction of rDNA to a suitable organism for transformation (uptake of DNA into cells) Selection of transformed cells with the rDNA with the help of a selectable marker gene (Antibiotic resistance gene) inserted in the vector. Multiplication/ expression/ integration followed by expression of the introduced gene in the host .

4. Image: http://www.biologydiscussion.com/dna/recombinant-dna-technology/recombinant-dna-technology-with-diagram/17785

5. TECHNIQUES… II. Direct gene transfer techniques Introduction of DNA into plant cells without the involvement of a biological agent which results in transformation. Methods : a) Chemically stimulated DNA uptake by plant protoplasts Polyethylene glycol (PEG) is the most commonly used chemical. Enhance DNA uptake by plant protoplasts (Protoplasts- cell without cell wall). Protoplasts are incubated in a mixture of 15 - 20 % PEG dissolved in a buffer containing Mg ++ or Ca ++ washed and plated in Petriplates for growth. PEG causes precipitation of DNA and stimulates their uptake by endocytosis (taking in by invagination of plasma membrane)and transforms 0.1 to 0.4% of the total protoplasts treated. Transformed protoplasts are cultured to regenerate plants .

6. TECHNIQUES… b) Electroporation The cells are exposed to high voltage electricity for a very brief period with an instrument, Electroporator . This will induce temporary pores in the plasma membrane through which the foreign DNA can enter into the protoplast and may produce transformation . The optimal voltage and time will depend on plant species, the source of protoplasts and the resistance of the medium.

7. Electroporation ….. Images: https://bio-equip.cn http://www.biologydiscussion.com/gene/gene-delivery-systems-steps-types/17737

8. TECHNIQUES… c) Shot Gun Method ( Particle gun method, Microprojectiles , Biolistic process, Particle acceleration etc.) Shooting DNA coated 1-2 µm tungsten or gold particles into plant cells with a device. This method accelerates particles either by using pressurised Helium gas or by the electrostatic energy released by a drop let of water exposed to high voltage.

9. Shot Gun Method….. Images : http://www.nwcreation.net/images/genegun2.gif http://nepad-abne.net/biotechnology/process-of-developing-genetically-modified-gm-crops/plant-transformation-using-particle-bombardment/

10. Indirect gene transfer technique – Agrobacterium mediated gene transfer This involves the use of a biological agent, a gram negative soil bacterium , Agrobacterium tumefaciens that causes crown gall, a tumorous growth in many dicots . After entry into a plant cell through wounds, the Agrobacterium transfers a small part of its Ti plasmid( Tumour inducing plasmid ) known as T- DNA (Transferred DNA) into the nucleus of the host plant cell and integrates some of its DNA into the chromosomes of the host plant cell. Hence Agrobacterium is known as Natural Plant Genetic Engineer. The Ti plasmid of A. tumefaciens is an ideal vector (vehicle DNA or carrier DNA ) for Plant Genetic Engineering.

11. Agrobacterium tumefaciens , Crown gall Images https://www.labroots.com/trending/microbiology/1518/better-know-a-microbe-agrobacterium-tumefaciens https://www.google.com/imgres?imgurl=https://apps.rhs.org.uk/Advice/ACEImages/SCN0000013_931579. : http://www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/Agrotrans.GIF

12. Agrobacterium mediated gene transfer… Steps : Isolation of the desired foreign gene from a plant or micro organism by suitable biochemical methods. Isolation of Ti plasmid from Agrobacterium tumefaciens and the separation of T- DNA portion of Ti plasmid . Insertion of the T-DNA in to pBR322 plasmid ( pBR 322 - An intermediate vector plasmid , p for plasmid, B for Bolivar & R for Rodriguez, the scientists who constructed the vector , 322 is the strain number , constructed using naturally occuring Col E1 plasmid of E. coli. ) . to form a shuttle vector ( capable of replicating in two organisms) Construction of Recombinant DNA or rDNA or recombinant plasmid or Chimeric DNA (Chimera – contain sequences from at least two different sets of DNA) – by cutting the T-DNA with a suitable restriction endonuclease enzyme (an enzyme that cut DNA at specific sites, known as molecular scalpels ,isolated from bacteria ) and the insertion of the foreign DNA into it.

13. Insertion of the rDNA into the E.coli cells in presence of Calcium chloride solution for Transformation . Selection of transformed E.coli cells using selectable marker genes (Antibiotic resistance genes ). Coculture of transformed E.coli cells with a culture of Agrobacterium tumefaciens may result in Conjugation and the E.coli cells may transfer the recombinant plasmid to the cells of Agrobacterium . In Agrobacterium , the rDNA may undergo genetic recombination with the Ti plasmid of that cell. As a result, the inserted foreign DNA is transferred to the unmanipulated Ti plasmid of Agrobacterium tumefaciens .

14. Selection of transformed A. tumefaciens Transformed A. tumefaciens are then allowed to infect the cultured plant cells whose genomes are to be improved and they insert the DNA into the plant cells which get integrated with the homologous sequence of plant genome. Thus the plant cells become genetically transformed to synthesize the product of the desired gene. Many transgenic plants were produced by Agrobacterium mediated gene transfer technique.

15. Agrobacterium mediated gene transfer… Image:https :// www.facebook.com / LBMBdz /posts/1290027324435746

16. GENETICALLY MODIFIED CROPS Flavr Savr Tomato Produced by Calgene Co. California (1992) The gene for the enzyme Polygalacturonase (PG) which promotes fruit softening by degrading Pectin is blocked – prevents softening Slow ripening & improved shelf life. – retain natural colour & flavour – nutritional content unchanged . No health risks First commercially grown genetically engineered food - given license for human consumption.

17. GOLDEN RICE The Creators : Dr. Ingo Potrykus & Dr. Peter Beyer (2000) Image : http://www.goldenrice.org/

18. GOLDEN RICE… Created by transforming rice by the addition of three beta carotene biosynthesis genes - two Daffodil( Narcissus pseudonarcissus ) genes – PSY ( Phytoene synthase ) & LCY ( Lycopene cyclase ) and one bacterial gene , CRT I ( Phytoene desaturase ) from the soil bacterium Erwinia uredovora , which get expressed in the edible part , endosperm and hence the rice is golden yellow in colour . Image :https://www.slideshare.net/varshiny2711/golden-rice-9703634

19. GOLDEN RICE Golden rice has great potential to reduce the occurrence of Vitamin A deficiency diseases. Image : http://www.goldenrice.org/Content2-How/how1_sci.php

20. Bt CROPS These are insect resistant genetically modified crops, produced by inserting the Bt gene( cry genes) from Bacillus thuringiensis by genetic engineering techniques & get the gene expressed. Bt crops can produce the toxic crystal protein & protect themselves from insects (Lepidoptera : Moths, Butterflies etc. ; Diptera : Flies, Mosquitoes etc. ) without any external application of pesticides. Bacillus thuringiensis Images https://twitter.com/sfamtweets/status/929077958102327296 http://web.utk.edu/~jurat/Btresearchtable.html

21. EDIBLE VACCINES Crop plants can be genetically engineered to produced Edible Vaccines. The genes encoding antigenic proteins from pathogens are isolated and inserted into plants by genetic engineering techniques and get them expressed. Crops under study : Banana - Vaccine against AIDS, Rabies, Hepatitis B Tomato - Rabies Potato - Hepatitis B Lettuce - Hepatitis B Images https://www.mcdb.ucla.edu/Research/Goldberg/HC70A_W12/pdf/EdibleVaccines. Copyright 2000 Scientific American, Inc. https://www.taringa.net/+info/el-origen-y-significado-del-nombre-de-los-paises_gyr33 ( Stockphoto ) https://link.springer.com/article/10.1007/s00299-016-2056-1

22. GENETIC ENGINEERING IN FLORICULTURE GENETICALLY MODIFIED CARNATIONS Pigment Profiling: Transgenic Carnations( Dianthus caryophyllus  ) - Blue to Violet. FLORIGENE Moon series of genetically modified carnations – have delphinidin - based anthocyanins by expressing heterologous F3 0 5 0 H genes. The first genetically modified flowers that were available on sale in Japan in 1997.

23. Image : uploaded by  Filippa Brugliera Tanaka and Brugliera 2013 FLORIGENE Moon series of genetically modified carnations

24. GENETICALLY MODIFIED CHRYSANTHEMUM Florigene ( Australia ), Suntory, and NARO Institute of Floricultural Science (NIFS), Japan developed transgenic blue Chrysanthemums by genetic engineering techniques. Generated by overexpression of the gene encoding flavonoid 3′,5′-hydroxylase (F3′5′H), the key enzyme for delphinidin biosynthesis. 

25. A blue transgenic Taihei Chrysanthemum, created by Japanese scientists by modifying two genes . CreditNaonobu Noda/NARO Source : Noda et.al., Plant and Cell Physiology 2013 (10) https://www.ncbi.nlm.nih.gov/pubmed/23926063

26. GENETICALLY MODIFIED ROSES Introduced the blue genes of Pansies ( Viola sps .) into calluses of Rose and get them expressed. https://www.suntory.com/sic/research/s_bluerose/story

27. Transgenic Blue Rose was developed by Suntory Flowers  in collaboration with an Australian venture company Florigene Ltd. (2002) The first blue roses in the world were named "SUNTORY blue rose  APPLAUSE ’’ 

28. GENETICALLY MODIFIED CROPS… POTENTIAL BENEFITS Foods with enhanced nutritional quality Production of Drugs & Edible Vaccines Crops with built-in resistance Fruits with improved shelf life POTENTIAL RISKS Production of toxic or allergenic products Spread of transgenes to related species Reduction in Biodiversity Economic losses due to international boycotts of GM products

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