blue2 LL_logo_neg_RGB Bi7430 Molecular Biotechnology 11. Molecular Biotechnology in Agriculture blue3 Outline qDefinition of green biotechnology qGenetic engineering of plants qGenetic engineering of animals qBiopharming qGMO benefits and controversies blue3 Green (agricultural) biotechnology qgreen biotechnology applied to agricultural processes qenvironmentally-friendly solutions as alternative to traditional agriculture, horticulture, and animal breeding qmodification of plants and animals increasing value in agriculture §traditional agriculture – selective crossbreeding and hybridization §modern molecular biotechnology – transgenesis (rDNA) qtransgenic organism - altered by addition of exogenous DNA qtransgene – DNA that is introduced blue3 Genetic engineering of plants q> 150 different plant species in 50 countries worldwide qDNA sequence of A. thaliana (2000), rice (2005), cotton (2006), corn (2009), potato (2011), tomato (2012), etc. qtransgenic plants engineered to §overcome biotic and abiotic stress opesticides (herbicides) opests and diseases (insects, viruses, bacteria, fungi) oenvironmental stress (salt, temperature, cold and drought) §improved crop quality oimproved nutritional quality oenhance taste, appearance and fragrance oincrease shelf-life §biopharming oplants as bioreactors for production of useful compounds (e.g., therapeutics, vaccines, antibodies) §phytoremediation blue3 Genetic engineering of plants qplant transgenesis procedure 1.construction of vector/plasmid (restriction digests, ligation) 2.propagation in E.coli 3.transformation 4.culture and selection qtotipotency - entire plant generated from a single, non-reproductive cell File:Protoplasts Petunia sp.jpg blue3 Methods of plant transformation qdirect methods §protoplast polyethylene glycol (PEG) method ofirst technique for plant transgenesis oPEG induces reversible permeabilization of the plasma membrane §protoplast electroporation ointensive electrical field leads to pores on plasma membrane §silicon carbide fibers ofibers punch holes through plant cells during vortexing §protoplast microinjection File:Protoplasts Petunia sp.jpg https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcRKtXQgTGyKFn5wC1l5ls6LDxohfq7reo4r54QTTTVPSwF w13e9BA blue3 Methods of plant transformation qdirect methods §particle bombardment omost common technique for direct transformation o„particle gun” or „gene gun” oDNA precipitated onto tungsten or gold particles oparticles shot into the plant tissue/cells https://encrypted-tbn2.gstatic.com/images?q=tbn:ANd9GcQLtEIzWHVNn_KHGxuvFSLSWlAkIqWGXEMKGuq5NDZLjov mcuLI http://upload.wikimedia.org/wikipedia/commons/thumb/d/d3/Genegun.jpg/220px-Genegun.jpg http://physics.ucsd.edu/~groisman/Gene%20guns_files/image004.jpg http://4.bp.blogspot.com/_CDIj-5Jol4w/R8MUUMS-8GI/AAAAAAAAAA8/WDyL19q6xiw/s200/genegun+2.jpg blue3 Methods of plant transformation qindirect methods (vectored) §Agrobacterium-mediated transformation oA. tumefaciens plant pathogenic bacteria causes Crown gall (tumors) otumor inducing (Ti) plasmid oT-DNA transferred and integrated into plant cell http://upload.wikimedia.org/wikipedia/commons/thumb/d/d1/Ti_plasmid.svg/350px-Ti_plasmid.svg.png Crown Gall Disease blue3 Markers and selection qtransformation frequency is low (less than 3%) qwithout selective advantage transformed cells overgrown by non-transformed qselection markers §antibiotics resistance (Kanamycin, Geneticin) §herbicides resistance (Phosphinothricin) qreporter genes §GUS (β-glucuronidase) §GFP (green fluorescent protein) §LUC (luciferase) http://images-mediawiki-sites.thefullwiki.org/01/7/0/1/9413573470085496.png http://www.jic.ac.uk/staff/wendy-harwood/images/AHC12(B)%20luc%20in%20seedling%20cropped.72%20ppi.j pg http://ars.els-cdn.com/content/image/1-s2.0-S0167779910000181-gr1.jpg blue3 Application of transgenic plants qpest and disease resistance §toxin gene from Bacillus thuringiensis oBt-corn resistant to European corn borer oBt-cotton resistant to cotton bollworm oBt-peanut resistant to cornstalk borer § §Papaya ringspot virus resistance inserting gene from pathogen into crop affords the crop plant resistance http://www.meta-helix.com/images/pbt_cotton_imgc1.jpg http://upload.wikimedia.org/wikipedia/commons/thumb/1/1d/Bt_plants.png/220px-Bt_plants.png http://www.apsnet.org/edcenter/intropp/lessons/viruses/Article%20Images/PapayaRingspotVirus01.jpg blue3 Application of transgenic plants qherbicide resistance §herbicide target modification §herbicide target overproduction §herbicide detoxification (enzymatic) qEXAMPLES §sulfonylurea resistance blocking the enzyme for synthesis Val, Leu, isoLeu mutated gene transferred from resistant tabaco §bromoxynil resistance transgene encoding enzyme bromoxynil nitrilase §glyphosinate resistance bacterial transgene protein inactivating herbicide http://www.scielo.br/img/revistas/bjpp/v18n1/a07fig01.jpg http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/bromoxynil2.jpg Fig. 3. blue3 Application of transgenic plants qresistance to environmental stress qmarginal land or climate change induced drought qcrucial ways of securing the world's food supply §drought tolerance - gene from Xerophyta viscosa - unique protein in cell membrane - gene for production of protective waxy cuticle on leaves - gene for expression of trehalose (stabilization of biomolecules) §salt tolerance - gene for enhanced glycinebetaine production Xerophyta retinervis00.jpg Drought tolerance Water is crucial for all living things. Plants use water as a solvent, a transport medium, an evaporative coolant, physical support, and as a major ingredient for photosynthesis. Without sufficient water, agriculture is impossible. Therefore, drought tolerance is an extremely important agricultural trait. One way of engineering drought tolerance is by taking genes from plants that are naturally drought tolerant and introducing them to crops. The ressurection plant (Xerophyta viscosa), a native of dry regions of southernmost Africa, possesses a gene for a unique protein in its cell membrane. Experiments have shown that plants given this gene are less prone to stress from drought and excess salinity. Some genes have been found that control the production of the thin, protective cuticle found on leaves. If crops can be grown with a thickened waxy cuticle, they could be better equipped for dealing with dryness. Salt tolerance Irrigation has enabled the transformation of arid regions into some of the world's most productive agricultural areas. Excess salinity, however, is becoming a major problem for agriculture in dry parts of the world. In several cases, scientists have used biotechnology to develop plants with enhanced tolerance to salty conditions. Researchers have noticed that plants with high tolerance to salt stress possess naturally high levels of a substance called glycinebetaine. Further, plants with intermediate levels of salinity tolerance have intermediate levels, and plants with poor tolerance to salinity have little or none at all. Genetically modified tomatoes with enhanced glycinebetaine production have increased tolerance to salty conditions. Another approach to engineering salt tolerance uses a protein that takes excess sodium and diverts it into a cellular compartment where it does not harm the cell. In the lab, this strategy was used to create test plants that were able to flower and produce seeds under extreme salt levels. Commercially available crops with such a modification are still several years away. blue3 Application of transgenic plants qimproved crop quality §higher nutrition value ogolden rice (beta – carotene genes) 120 million children suffers from vitamin A deficiency healthy vision and prevents night blindness oblack tomato (anthocyanin antioxidant gene) prevent heart disease, diabetes and cancer §improve shelf life odelayed fruit ripening (FlavrSavr tomato) antisense gene blocking pectinase §improved appearance odelphinine gene from pansy cloned to rose §biopharming http://plantsinaction.science.uq.edu.au/edition1/?q=files/imagecache/figure-large/Fig%2011.22.jpg http://www.gatesfoundation.org/agriculturaldevelopment/PublishingImages/golden-rice-hero.jpg http://s.ecrater.com/stores/59305/4fa15a8da5a3c_59305b.jpg http://img.ehowcdn.com/article-new/ehow/images/a07/40/m9/transgenic-ornamental-plants-1.1-800x800.j pg blue3 Break 5 min http://3.bp.blogspot.com/-z3V88N6why0/TuX93jdb30I/AAAAAAAABUw/20uX6GmuiHk/s1600/coffee_break_ahead_ sign.jpg blue3 Genetic engineering of animals qselective breeding §time consuming and costly §limited number of properties available §difficult to introduce new genetic traits / lines qtransgenic animals §fast generation lines carrying desired properties oincreased growth oimproved disease resistance oimproved nutritional quality oincreased wool quality §model animals for human disease research §biopharming - production of useful molecules §biosensors for environmental pollution § http://scitechdaily.com/images/aquabounty-ge-salmon.jpg fatmouse.jpg http://www.practicalfishkeeping.co.uk/custom/images/medium/4c2c6378706af.jpg blue3 Genome targeting technologies qdirect microinjection (pronucleus method) §injection of desired DNA to male pronucleus §most popular, commercial available §success range from 10 to 30% §transfer of large genes possible §no theoretical limit for gene construct size §random insertion of the transgene (affecting other genes and expression patterns) blue3 qretrovirus mediated gene transfer §retroviruses used as vectors (gene therapy) §virus gene is replaced by transgene §replication defective virus infect host cells (e.g., ES cells, embryo cells) §efficient mechanism of transgene integration §transfer of genes < 8 kb only possible §random insertion of the transgene http://firstivf.net/pictures/icsi3/8cell_embryo.jpg https://encrypted-tbn2.gstatic.com/images?q=tbn:ANd9GcTFxJCDoecfv7AVDlyuyJx8WoXIjTT8cA8KAls1w9poZLb zCi8cwA Genome targeting technologies blue3 qembryonic stem cell method §transfection of gene construct into in vitro culture of embryonic stem (ES) cells §ES recombinant cells incorporated into embryo at blastocyst stage §1 in a million incorporated at desired position §ES cell lines not available in farm animals §random insertion of the transgene http://www.medicine.virginia.edu/research/cores/transgenic/images/blackwhitechimera.jpg Genome targeting technologies blue3 Genome targeting technologies qengineered nucleases, „molecular scissors" §site-specific double stranded breaks §Zinc finger nucleases (ZFNs) §transcription-activator like effector nucleases (TALEN) §RNA-guided DNA endonuclease (CRISPR-Cas9) blue3 Genome targeting technologies qCRISPR-Cas9 §synthetic guide RNA (gRNA) §delivering Cas9 nuclease complexed with gRNA into a cell §in vivo (nucleus), stem cells, fertilized egg §can target several genes at once blue3 Application of transgenic animals qdisease-resistant livestock §in vivo immunization - overexpress genes encoding monoclonal antibodies §eliminate production of host cell components interacting with infectious agent qimproving milk quality §increase casein contents let to increase cheese production §decrease lactose content by overexpress lactase §abolish lacto globulin expression (for milk allergic consumer) qimproving animal production traits §transgenic fish - enhanced growth 3-5 times (growth hormone) §transgenic pig - production of omega-3-fatty acids (roundworm gene) §transgenic poultry - lower cholesterol and fat in eggs qbiopharming blue3 Biopharming quse of plants or animals for the production of useful molecules qindustrial products §proteins (enzymes) §fats and oils §polymers and waxes qpharmaceuticals §recombinant human proteins §therapeutic proteins and pharmaceuticals §vaccines and antibodies blue3 Biopharming qindustrial products from plants §cheap and easy to produce §free of animal viruses §risk of food supply contamination §environmental contamination qEXAMPLES (transgenic corn, Sigma): §trypsin otraditionally isolated from bovine pancreas ofirst large scale transgenic plant product oworldwide market = US$120 million §avidin omedical diagnostics §b-glucuronidase ovisual marker in research labs http://americanpreppersnetwork.com/wp-content/uploads/2012/06/corn.jpg avidin-biotin (Vitamin B[7], vitamin H) system as a powerful tool in biological sciences, high degree of affinity and specificity, KD ≈ 10−15 M, making it one of the strongest known non-covalent bonds trypsin blue3 Biopharming qedible vaccines from plants §no purification required §no hazards associated with injections §may be grown locally where needed §no transportation costs §no need for refrigeration or special storage qEXAMPLES: §HIV-suppressing protein in spinach §rabies virus G protein in tomato §vaccine for rotavirus or hepatitis in potato http://www.infonet-biovision.org/res/res/files/3965.300x200.jpeg http://indianapublicmedia.org/amomentofscience/files/2009/08/tomat-940x626.jpg http://www.indepthinfo.com/potato/gifs/potatoes.jpg blue3 Biopharming qplant-made antibodies §plantibodies - monoclonal antibodies produced in plants §free from potential contamination of mammalian viruses §plants used include tobacco, corn, potatoes, soya and rice §EXAMPLES: cancer, herpes simplex virus qplant-made pharmaceuticals §therapeutic proteins and intermediates §EXAMPLES: proteins to treat cystic fibrosis, HIV, hypertension blue3 Biopharming qproduction of pharmaceuticals in milk §easy to purify - few other proteins in milk §dairy cattle produce 10,000 liters of milk/year (35 g protein/liter) §only few transgenic cows can meet worldwide demand §risk of food supply contamination qEXAMPLES: §COW: human serum albumin, human lactoferrin §SHEEP: alpha-1-antitrypsin §GOAT: human antithrombin III (FDA approved), tissue plasminogen activator, malaria antigen q qproduction of materials in milk §BioSteel from spider silk (Nexia Biotech) http://3.bp.blogspot.com/-g5DE4gse7W0/UE3yBYuvD6I/AAAAAAAAAjE/y1ljqxRQmrc/s200/article-new-ehow-ima ges-a08-1k-34-goats-produce-spider-silk-800x800.jpg http://4.bp.blogspot.com/_ZNi61PTNovg/TD7QzJEvzpI/AAAAAAAAAK4/h3o9LXelaZA/s320/spiderweb_herotech.j pg blue3 GMO benefits qcrops §increased stress tolerance §improved resistance to disease, pests and herbicides §increased nutrients, yields, enhanced taste and quality qanimals §improved animal health, resistance, productivity and feed efficiency §better yields of meat, eggs, and milk qenvironment §more efficient processing §conservation of soil, water, and energy §better natural waste management qsociety §increased food security for growing populations §climate change induced drought blue3 GMO controversies qsafety §human health – toxicity, allergens, antibiotic resistance, unknown effects §environment - unintended transfer through cross-pollination, unknown effects on other organisms, loss of biodiversity qethics §tampering with nature by mixing genes among species / cloning §violation of natural organism´s intrinsic values §stress for animals qaccess and intellectual property §domination of world food production by few companies §increasing dependence on industrialized nations by developing countries blue3 GMO future qGMO crop first commercialized in 1996 q17.3 million farmers grew biotech crops on 170 million hectares q90% of new users are small resource-poor farmers in developing countries qEU research on risk of GMOs over the past two decades unable to detect any risks that have not yet been known from conventional agriculture* http://www.decodedscience.com/wp-content/uploads/2013/01/World_map_GMO_production_2005.png * EU Commission (2012): A Decade of EU-funded GMO Impacts Research squash – tykev canola – olejovita rostlina blue3 Questions http://www.avanceon.com/Portals/31626/images/C--Users-sschlegel-Pictures-Question-Mark-Man.jpg blue3 Reading qU.S. Agency for International Development, Agricultural Biotechnology Support Project II, and the Program for Biosafety Systems qHow are Biotech Crops & Foods Assessed for Safety?, Developing a Biosafety System (BRIEF #5 and #6)