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PLANT BIOTECHNOLOGY TRANSGENIC PLANTS USES and PROFITS DEFINITIONS • Genetic transformation (transgenesis) – procedures leading to creation of a transgenic organism • Transgenic organism – an organism in which a new gene (transgene) has been added to a set of genes created during evolution: • transgene originates from another cell or organism • transgene is integrated into the recipient genome and inherited by next generations – synonym of a genetically modified organism (GMO), usually used for plants and animals • Transgene expression/transformation: – transient – temporal expression of a transgene, not necessary resulting from its integration into the recipient genome – stabile – long lasting expression of a transgene integrated into the recipient genome For successful production of transgenic plants, • • • DNA delivery methods are used, Selection markers or reporter genes are useful, Regeneration from single cells is required PLANT TRANSFORMATION METHODS • Direct transfer of DNA: – PEG – polyethylene glycol – electroporation • Transfer of DNA via a carrier: – microinjection – bombardment • Transfer of DNA via a vector: – Agrobacterium – viral vectors DNA Delivery Agrobacterium Particle gun Agrobacterium Agrobacterium is a soil borne gram-negative bacterium, that has a unique ability to introduce part of its DNA into plant cells. Agrobacterium Most of the native transferred bacterial DNA is replaced with genes of interest. In the laboratory, bacteria are co-cultured or inoculated with plant tissue and the bacteria transfer part of their DNA into plant cells. Particle gun For particle bombardment, tungsten or gold particles are coated with DNA and accelerated towards target plant tissues. Most devices use compressed helium as the force used to accelerate the particles. PDS-1000/He Gene gun Particle gun The particles punch holes in the plant cell wall and usually penetrate only 1-2 cell layers. Particle bombardment is a physical method for DNA introduction and the biological incompatibilities associated with Agrobacterium are avoided. The DNA-coated particles can end up either near or in the nucleus, where the DNA comes off the particles and integrates into plant chromosomal DNA. MARKERS USED FOR PLANT TRANSFORMATION • SELECTION MARKERS: – resistance to antibiotics, antimetabolites, herbicides and toxic levels of amino acids and their analogs • REPORTER GENES: – – – – – – cat – chloramphenicol acetyl transferase (CAT) lacZ - b-galactosidase (LacZ) uidA - b-glucuronidase (GUS) gfp – green fluorescent protein (GFP) luxA/B – bacterial luciferase (Lux) luc – firefly luciferase (Luc) GFP Jellyfish Green Fluorescent Protein GFP - Jellyfish Green Fluorescent Protein GFP is a reporter gene used in DNA transfer studies. The jellyfish green fluorescent protein gene has been modified for optimum expression in plants. The protein from the gene will fluoresce green when illuminated with high intensity UV/blue light. 398 475 510 Chalfie et al., Science, 1994 GFP - Jellyfish Green Fluorescent Protein Chlorophyll can fluoresce red under the same conditions which cause GFP to appear green. GFP fluorescence occurs as spots if individual cells are targeted or the whole tissue can be green if all of the cells within the tissue contain the gfp gene. GFP is very useful as a marker or indicator of successful gene transfer. Introduction of the gfp gene into different target tissues Petunia petal cells Soybean seed – whole seedling (on right) Wheat callus cells GFP expression in wheat seeds (left seed, on left) and roots (below) GFP expression in soybean tissues No GFP – red chlorophyll fluorescence GFP – green fluorescence gif animation of GFP expression in soybean tissue Shows variability in expression pattern standard illumination on left – GFP illumination on right PLANT REGENERATION • Plants can be generated from single cells using “tissue culture”, where parts of the plants are placed in Petri dishes. The specific response of the plant tissue depends on the starting plant materials and the medium in the Petri dish. • Under the right conditions, plant tissues regenerate into whole plants via two distinct processes: – somatic embryogenesis – shoot morphogenesis. PLANT REGENERATION For somatic embryogenesis, embryos or artificial seeds form, which can germinate into a whole plant. Plant Recovery Starting Material Immature seeds Germination Soybean Embryogenesis Development Induction Proliferation PLANT REGENERATION For shoot morphogenesis, shoots are formed first, which must generate roots before they can be transferred to the soil. Production of Transgenic Plants When DNA delivery, plant regeneration and selection for transgenic cells are merged, transgenic plants can be produced. The idea is to introduce DNA into cells, which can be selected and generated into whole transgenic organisms. transformation plant explants selection medium with antibiotics regeneration transgenic callus transgenic plant TRANSGENIC PLANTS USES and PROFITS Transgenic plants with new features • Tolerance to herbicides • Resistance to diseases and negative environmental factors • Improved hybrid breeding • Improved plant architecture and photosynthetic effectiveness • Phytoremediation • Improved productive/industrial features • Production of biopharmaceuticals and edible vaccines Engineering herbicide tolerance • Non-selective herbicides used for weeds: – – – – glufosinate (phosphinotrycin) - amino acid analogue glycophosphates sulfonylureas imidazolinones • Examples: transgenic plants resistant to glyphosate herbicide due to expression of the glyphosateinsensitive EPSPS gene of E. coli and/or GOX (glyphosate oxidoreductase) of a soil bacterium (Ochrobactrum anthropi strain LBAA) - soybean, corn, cotton, canola, alfalfa, sugarbeet , wheat • Njiti et al., Agron. J., 2003 Round-up herbicide (Monsanto) Glyphosate (N-(phosphonomethyl) glycine) RESISTANCE TO DISEASES • Plant pathogens: – – – – – bacteria viruses fungi nematodes insects Sources of resistance to viruses • Production of the components of virus particles: – – – – virus capsid proteins virus coat proteins cDNA of satellite RNA (RNA viruses) anti-sense RNA Transgenic tobacco resistant to TMV (tobacco mosaic virus) A B A B A B Nicotiana tobaccum infected with TMV: A – nontransgenic plants B – transgenic plants producing viral coat protein or antisense RNA Abel et al., Science 1986 Nelson et al., Gene, 1993 TRANSGENIC PLANTS RESISTANT TO INSECTS (http://butterflywebsite.com/) INSECTICIDES • Worldwide expenditure on insecticides (in millions of USD): • Worldwide crop losses caused by insect pests, in spite of the use of chemical insecticides (w millions of USD): 1965 25000 2465 45000 620 20000 1190 1870 Fruit and Vegetables Cotton Rice Maize Other 8000 Vegetables Fruit Maize Rice Maagd et al., Trends in plant science, 1999 Sources of resistance to insect pests • Production of toxins and inhibitors: – – – – – ”Crystal” proteins (Cry proteins from Bacillus thuringiensis) protease inhibitors a-amylase inhibitors lectins chitinases Schuler et al., TIBTECH, 1998 Commercial transgenic Bt crop plants • Bt-potato, 1995: – NewLeaf™, Monsanto, USA; countries: Canada, Japan, Mexico – Production of Cry3A protein – protection against Leptinotarsa decemlineata (Colorado potato beetle); 40% reduction in insecticide usage • Bt-cotton, 1996: – Bollgard™, Monsanto, USA; countries: Australia, China, Mexico, South Africa and USA – Production of Cry1Ac protein – protection against Helicoverpa virescens (tobacco budworm), H. zea (cotton bollworm, corn earworm, tomato fruitworm) and Pectinophera gossypiella (pink bollworm); reduction in insecticide usage by 750 000 L, yield increase by 14% • Bt-maize, 1996: – Maximizer, Novartis, Switzerland; YieldGard™, Monsanto, USA; countries: Argentina, Canada, Japan, USA, European Union – Production of Cry1Ab protein, protection against Ostrinia nubilalis (corn borer); 99% efficiency in field tests Ostrinia nubilalis (European Corn Borer ) Losses in maize cultivation: 7% (40 million of tons per year), in some regions of Europe and North America – 20% http://www.ent.iastate.edu/pest/cornborer/ Bt-maize Zea mays plants infected with Ostrinia nubilalis larvae nontransgenic plant transgenic plant http://www.american.edu/TED/maize.htm MON810 Maize • accepted for cultivation by the European Committee in September 2004 Losses in Europe – up to 50% http://www.monsanto.com TRANSGENIC PLANTS RESISTANT TO NEMATODES Defense systems against root-parasitic nematodes Effectors inhibitors, toxins specific binding degrading enzymes proteinase inhibitors Bt toxins, cytotoxins lectins monoclonal antibodies collagenases chitinases epidermis cortex vascular cylinder cortex epidermis feeding migration and/or feeding migration egg development Targets Jung et al., Trends in plant science, 1998 Nematode resistance genes from crop species Plant species Nematode potato Globodera pallida G. rostochiensis Gpa2 Gro1, H1 soybean Heterodera glycines Rhg4, rhg1 barley H. avenae Ha2, Ha3, Ha4 wheat H. avenae Cre1, Cre3 peanut Meloidogyne arenaria Mae, Mag sugar beet H. schachtii Hs1pro-1, Hs2 tomato M. incognita G. rostochiensis Mi-1, Mi-3 Hero Jung et al., Trends in plant science, 1998 Resistance gene Sugar beet plants expressing the Hs1pro-1 gene The life cycle of the nematode Heretodera schachtii in roots of susceptible (a) and resistant (b) plants Jung et al., Trends in plant science, 1998 TRANSGENIC PLANTS RESISTANT TO NEGATIVE ENVIRONMENTAL FACTORS TRANSGENIC PLANTS RESISTANT TO ABIOTIC STRESS • Protection from oxidative stress: – production of enzymes involved in neutralization of reactive oxygen species (ROS: oxygen ions and peroxides): – superoxide dismutases, catalases, lactoperoxidases, glutathione peroxidases and peroxiredoxins. • Salt tolerance: – accumulation of salt in the vacuoles of tomato leaf cells, but not in fruit: • overexpression of AtNHX1 – vacuolar Na+/H+ antiporter from Arabidopsis thaliana – transgenic tobacco plants with increased salt tolerance: • overexpression of SbGST - Tau class glutathione transferase from an extreme halophyte Salicornia brachiata (glasswort, succulent) Blumwald., Biotechnol. Gen. Eng. Rev., 2003; Jha et al., Mol. Biol. Rep., 2011 IMPROVED HYBRID BREEDING IMPROVEMENT OF HYBRID BREEDING • Transgenic plants deficient in pollen production: – expression of the bacterial rybonuclease gene (barnase) under the control of anther-specific promoter • Restoration of the fertility: – induced expression of the gene for bacterial rybonuclease inhibitor (barstar) The tightly bound complex between barnase and its inhibitor barstar (blue). Kuvshinov et al., Environ. Biosafety Res., 2005; Kobayashi et al., Plant Cell Rep. 2006 IMPROVEMENT OF PLANT ARCHITECTURE AND PHOTOSYNTHETIC EFFECTIVENESS Modulation of plant reaction to light by engineering phytochrome (PHY) gene family • Growth of plants in a high density – shadow avoidance syndrome: • elongation of plant shoots on the costs of leaves, storage organs and fertility – low yields • Overexpression of PHYA gene – suppression of the shadow avoidance syndrome – yield increase Rice line PBNT PA26 PA41 PA53 PHYA transgene copy number 0 2 1 1 Plant height (cm) 92±4 76±6 73±5 68±5 Grain yield per plant (gm) 39.2 ± 8.1 47.4 ± 7.0 41.7 ± 8.3 43.4 ± 9.5 Relative grain yield (%) 100 121 106 111 Garg et al., Planta, 2005 Acceleration in formation of multiple floral buds • Stimulation of scion bud release by rol gene transformed rootstocks of Rosa hybrida L Salm et al., J. Exp. Botany, 1998 TRANSGENIC PLANTS AND PHYTOREMEDIATION Phytodetoxification of hazardous organomercurials • Expression of the bacterial merA (mercuric reductase) and merB (organomercurial lyase) genes in Arabidopsis thaliana: – conversion of highly toxic organomerucrials to less toxic elemental mercury Hg(0) 0 mM 1 mM 5 mM 10 mM CH3HgCl concentration accumulation of Hg(0) in plants Bizily et al., Nature Biotechnol., 2000 Biodegradation of explosives • expression of the PETN reductase gene in Nicotiana tobaccum: – degradation of TNT (2, 4, 6 – trinitrotoluene) or GTN (glycerol trinitrate) with concomitant release of nitrogen GTN GTN 0 mM 1 mM 1mM GMN GDN TNT 0 mM 50 mM nontransgenic plants 50mM transgenic plants GTN degradation French et al., Nature Biotechnol., 1999 TRANSGENIC PLANTS WITH IMPROVED PRODUCTIVE/INDUSTRIAL FEATURES Prolonged storage time • Transgenic tomato with delayed ripping: – lower level of ethylene production – reduced activity of the cell wall degrading enzymes, e.g. polygalacturonases – suppression of N-glycan processing enzymes Sitrit & Bennett, Plant Physiol., 1998 Meli et al., PNAS USA, 2010 „Flavr Savr” tomato In 1994, the first “genetically modified” food was approved by the FDA to go to market. The tomato, Flavr Savr, was modified by Calgene (a biotechnology company) using antisense technology resulting in altered ripening. In an attempt to slow the softening process, the Flavr Savr employs antisense technology to block PG enzyme production. PG enzyme is responsible for the breakdown of pectin. Pectin is a building block in cell walls, and is what gives tomatoes their firmness. http://www.biotechnolog.pl/gmo-13.htm A reversed, “antisense” copy of the PG (polygalacturonase) DNA was added to the tomato genome. When this gene is transcribed, it produces an RNA, that has the complementary bases of the actual PG RNA sequence. These two RNA strands base pair, in effect, disabling the RNA that would produce the PG enzyme protein. This disabling is enough to slow down softening of the tomatoes, allowing them to be transported to their consumer destinations ripe. http://dragon.zoo.utoronto.ca/~jlm2000/T0501D/methods_index.html „Flavr Savr” vs traditional tomato The Flavr Savr tomato ripens on the vine – resulting in fuller flavour. It is modified so that it remains firm after harvesting Flavr Savr Traditional The traditional tomato must be harvested while it is still green and firm so that it is not crushed on the way to the supermarket. The traditional tomato is sprayed with ethylene after shipping to induce ripening. Ripe and Increased Flavour. Supermarket Supermarket http://dragon.zoo.utoronto.ca/~jlm2000/T0501D/introduction.html „Flavr Savr” – commercial use • Influence on human health- studies on safety of Flavr Savr tomato have not reveald any adverse effects on health …BUT… • In 1996 Flavr Savr tomatoes have been taken off the market shelves because manipupation of the ripening gene caused unintended consequences such as: * soft skin * strange taste http://www.btinternet.com/~nlpWESSEX/images/gentomss.jpg * compositional changes in tomato • Favr Savr tomatoes were more expensive than non-modified tomatoes http://make-money-fast.50webs.com/images/money.jpg • Flavr Savr tomatoes are still used in processes tomato products http://www.ncbe.reading.ac.uk/NCBE/GMFOOD/menu.html Engineering plant metabolism for improved nutrition • Increased level of non-saturated fatty acids in the seeds of oil plants – modulation of stearyl-ACP desaturase expression • Modification of the amino acid content in plants by: Grayburn et al., Biotechnology, 1992 – manipulating methionine biosynthesis and expressing Tabe & Higgins, Trends in Plant Science, 1998 methionine-rich proteins in legume plant seeds • Vitamin precursors in transgenic plants: – „golden rice”: beta-carotene - vitamin A precursor in rice grains (genes from daffodil plants) Ye et al., Science, 2000; Beyer et al., J. Nutr., 2003 „Golden Rice” Golden rice was created by Ingo Potrykus of the Institute of Plant Sciences at the Swiss Federal Institute of Technology Golden rice was designed to produce Vitamin A precursor beta-carotene in the part of rice that people eat, the endosperm. The rice plant can naturally produce beta-carotene, which is a carotenoid pigment that occurs in the leaves and is involved in photosynthesis. http://en.wikipedia.org/wiki/Golden_rice However, the plant does not normally produce the pigment in the endosperm since photosynthesis does not occur in the endosperm. Golden rice was created by transforming rice with three beta-carotene biosynthesis genes: • • • psy (phytoene synthase) both from daffodil lyc (lycopene cyclase) (Narcissus pseudonarcissus) crt1 from the soil bacterium Erwinia uredovora http://en.wikipedia.org/wiki/Golden_rice „Golden Rice” - what for? The research that led to golden rice was conducted with the goal of helping children who suffer from Vitamin A deficiency (VAD). At the beginning of the 21st century, 124 million people, in 118 countries in Africa and South East Asia, were estimated to be affected by VAD. VAD is responsible for 1-2 million deaths, 500,000 cases of irreversible blindness and millions of cases of xerophthalmia annually. Children and pregnant women are at highest risk. http://en.wikipedia.org/wiki/Golden_rice http://maitri.diecezja.gda.pl/gazetka/my_14/images/my14-1.jpg Controversial „Golden Rice” www.jewishworldreview.com/ 0601/saunders062701.asp • Influence on human health • Lack of experience in tillage of the Golden Rice, it is not known what proprieties it will show in different ecosystems • Lack of information about the influence on environment, different organisms Production of new biopolymers • Polyhydroxy butyrate – thermolabile, biodegradable plastic produced in Arabidopsis thaliana transgenic plants Poirier et al., Int J Biol Macromol, 1995 TRANSGENIC PLANTS AS SOURCES OF MEDICAL PRODUCTS Medical molecular farming • Transgenic plants as bioreactors for production of recombinant antibodies (plantibodies) • Transgenic plants as vaccine production systems (edible vaccines = plantigens) • Transgenic plants as pharmaceutical proteins factories Daniell, Streatfield & Wycoff, Trends in Plant Science, 2001 PLANTIBODIES Costs per gram for purified IgA produced by different expression systems Daniell, Streatfield & Wycoff, Trends in Plant Science, 2001 Therapeutic and diagnostic plantibodies application specificity plant dental caries streptococcal antigen I i II N. tabaccum diagnostic anti-human IgG alfalfa cancer treatment carcinoembryonic antigen wheat, rice, N. tabaccum B-cell lymphoma treatment idiotype vaccine N. benthamiana colon cancer surface antigen N. benthamiana Herpes simplex 2 HSV2 soybean Daniell, Streatfield & Wycoff, Trends in Plant Science, 2001 PLANTIGENES – edible vaccines FOODS UNDER STUDY as alternatives to injectable vaccines include bananas, potatoes and tomatoes, as well as lettuce, rice, wheat, soybeans and corn. Langridge, Scientific American, 2000 How to make an edible vaccine? Langridge, Scientific American, 2000 How edible vaccines provide protection? Langridge, Scientific American, 2000 Stages of an edible vaccine production a – identification and isolation of the antigen gene b – gene cloning into an expression vector c – stable transformation of plant tissue d – selection of transformants e – regeneration of transgenic plants f – antigen expression in edible plant organs g – detection of antigen in protein extracts from plant tissues h – immunogenecity and protection tests on animals i – immunization of humans Proteins with applications for human vaccines, expressed in plants • Heat-labile toxin B subunit - against enterotoxigenic E. coli: – tobacco, potato, maize – immunogenic and protective when administered orally • Cholera toxin B subunit - against Vibrio cholerae – potato - immunogenic and protective when administered orally • Envelope surface protein - against Hepatitis virus B (HBV): – tobacco, potato, lupin, lettuce - immunogenic when administered orally • Capsid protein - against Norwalk virus: – tobacco, potato - immunogenic when administered orally • Glycoprotein – against rabies virus: – tomato – intact protein • Glycoprotein B – against human cytomegalovirus: – tobacco – immunologically related protein Daniell, Streatfield & Wycoff, Trends in Plant Science, 2001 In preparation • transgenic bananas providing resistance to: – cholera – typhoid fever – hepatitis • transgenic tomatoes – HBV – vaccine against AIDS (HIV) Proteins with applications for animal vaccines, expressed in plants • VP60 - against rabbit hemorrhagic disease virus: – potato – immunogenic and protective when administered by injection • VP1 - against foot-and-mouth disease virus: – Arabidopsis, alfalfa - immunogenic and protective when administered by injection or orally • Glycoprotein S - against transmissible gastroenteritis coronavirus – Arabidopsis, tobacco - immunogenic when administered by injection – maize – protective when administrated orally Daniell, Streatfield & Wycoff, Trends in Plant Science, 2001 Biopharmaceuticals Production of biopharmaceuticals for human health in transgenic plants (1) human proteins plant application protein C tobacco anticoagulant hirudin canola thrombin inhibitor granulocyte-macrophage colony-stimulating factor tobacco neutropenia somatotropin tobacco growth hormone erythropoietin tobacco anemia encephalins Arabidopsis antihyperanalgesic EGF tobacco wound repair and control of cell prolif. interferon-a rice, turnip Hepatitis B and C interferon-b tobacco Hepatitis B and C Production of biopharmaceuticals for human health in transgenic plants (2) human proteins plant application HSA tobacco liver cirrhosis, burns, surgery hemoglobin a, b tobacco blood substitute homotrimeric collagen tobacco collagen synthesis a-1-antitrypsin rice cistic fibrosis, liver disease, hemorrhage aprotinin maize trypsin inhibitor for transplantation surgery lactoferrin potato antimicrobial Daniell, Streatfield & Wycoff, Trends in Plant Science, 2001 Literature • H.S. Chawla, Introduction to plant biotechnology, Chapter 24 (Transgenics in crop improvement), 2002.
