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BE304 Plant Cell culture Dr. Michael Parkinson, School of Biotechnology 07 March 2002 Dr. Michael Parkinson 1 ASSESSMENT • One hour open book exam • 2 experimental protocols in plant cell culture. • You should minutely dissect these and make sense of them. You will get 2 marks for every valid point that you make. • You can also get marks for suggesting alternatives that could have been used. 07 March 2002 Dr. Michael Parkinson 2 Types of points • Seeds were washed overnight under a running tap, rinsed for 10s in 70% ethanol then sterilised in 20% Domestos + 0.1% v/v Tween20 for 10 minutes followed by 3 rinses in sterile distilled water. 07 March 2002 • • • • • • • • Why use seeds? Why wash overnight? Why rinse in 70% EtOH? Why sterilise at all? Why Domestos? Why 20% for 10 mins? Why 0.1% v/v Tween20? Why rinse? Dr. Michael Parkinson 3 Resources • Powerpoint presentation of lectures – [email protected]/~parkinsm/teaching – Partially worked solution to exam question • Text books – – – – Agriculture 631 Plant cell and tissue culture 571 Secondary metabolism 660 Transformation 572 07 March 2002 Dr. Michael Parkinson 4 Lecture outline • Micropropagation • Production of products in cell cultures • Plant transformation – For every item, you will be given an experimental protocol. These will broken down into a number of sections. There will be a series of lectures covering the sections followed by a detailed discussion of another protocol. – We will also try out some of your findings. 07 March 2002 Dr. Michael Parkinson 5 Micropropagation • Advantages and disadvantages of micropropagation • Methods of micropropagation • Choice of explant • Media • Stage I - Sterilisation • Stage II - Multiplication • Stages III and IV- Rooting, hardening off and transfer to greenhouse 07 March 2002 Dr. Michael Parkinson 7 Advantages and disadvantages of micropropagation • Speed - roughly a 10X increase every 2 months (possible to produce 106 plants from a single starting plant in on year). • Axenic - provided that the original explant is free of contaminant, the resulting plants will all be uncontaminated. • Clonal propagation • Cost - 0.15€ per explant 07 March 2002 Dr. Michael Parkinson 8 Historical aspects • First commercially used with orchids conventional propagation rate of 1 per year. • Through protocorms, 1,000,000 per year. Corm (Swollen stem) 07 March 2002 Chop up Maturation Dr. Michael Parkinson 9 Methods of micropropagation • Axillary branching • Adventitious shoot formation • Somatic embryogenesis 07 March 2002 • >95% of all micropropagation. • Genetically stable • Simple and straightforward • Efficient but prone to genetic instability • Little used. Potentially phenomenally efficient. Dr. Michael Parkinson 10 Axillary Branching Stem Shoot tip Leaf petiole Axillary bud in the axil of the leaf 07 March 2002 Dr. Michael Parkinson 11 Choice of explant Desirable properties of an explant • Easily sterilisable • Juvenile • Responsive to culture 07 March 2002 • Shoot tips • Axillary buds • Seeds • Hypocotyl (from germinated seed) • Leaves Dr. Michael Parkinson 12 Media • When you make an explant like an axillary bud, you remove it from the sources of many chemicals and have to re-supply these to the explants to allow them to grow. Shoot tip - Auxins and Gibberellins Leaves sugars, GAs Roots - water, vitamins mineral salts and cytokinins 07 March 2002 Dr. Michael Parkinson 13 Medium constituents • • • • • • • Inorganic salt formulations Source of carbohydrate Vitamins Water Plant hormones - auxins, cytokinins, GA’s Solidifying agents Undefined supplements 07 March 2002 Dr. Michael Parkinson 14 Carbohydrates • Plants in culture usually cannot meet their needs for fixed carbon. Usually added as sucrose at 2-3% w/v. • Glucose or a mixture of glucose and fructose is occasionally used. • For large scale cultures, cheaper sources of sugars (corn syrup) may be used. 07 March 2002 Dr. Michael Parkinson 15 Photoautotrophic culture • Growth without a carbon source. Therefore need to boost photosynthesis. • High light intensities needed (90150mMole/m2/s) compared to normal (30-50). • Usually increase CO2 (1000ppm) compared to normal 369.4ppm. • Much reduced level of contamination and plants are easier to transfer to the greenhouse. 07 March 2002 Dr. Michael Parkinson 16 Inorganic salt formulations • Contain a wide range of Macro-elements (>mg/l) and microelements (<mg/l). • A wide range of media are readily available as spray-dried powders. • Murashige and Skoog Medium (1965) is the most popular for shoot cultures. • Gamborgs B5 medium is widely used for cell suspension cultures (no ammonium). 07 March 2002 Dr. Michael Parkinson 17 Vitamins • A wide range of vitamins are available and may be used. • Generally, the smaller the explant, the more exacting the vitamin requirement. • A vitamin cocktail is often used (Nicotinic acid, glycine, Thiamine, pyridoxine). • Inositol usually has to be supplied at much higher concentration (100mg/l) 07 March 2002 Dr. Michael Parkinson 18 Plant hormones (Growth regulators) • • • • • • Auxins Cytokinins Gibberellic acids Ethylene Abscisic Acid “Plant Growth Regulator-like compounds” 07 March 2002 Dr. Michael Parkinson 19 Auxins • Absolutely essential (no mutants known) • Only one compound, Indole-3-acetic acid. Many synthetic analogues (NAA, IBA, 2,4-D, 2,4,5-T, Pichloram) - cheaper & more stable • Generally growth stimulatory. Promote rooting. • Produced in meristems, especially shoot meristem and transported through the plant in special cells in vascular bundles. 07 March 2002 Dr. Michael Parkinson 20 Cytokinins • Absolutely essential (no mutants known) • Single natural compound, Zeatin. Synthetic analogues Benyzladenine (BA), Kinetin. • Stimulate cell division (with auxins). • Promotes formation of adventitious shoots. • Produced in the root meristem and transported throughout the plant as the Zeatin-riboside in the phloem. 07 March 2002 Dr. Michael Parkinson 21 Gibberellins (GA’s) • A family of over 70 related compounds, all forms of Gibberellic acid. • Commercially, GA3 and GA4+9 available. • Stimulate etiolation of stems. • Help break bud and seed dormancy. • Produced in young leaves. 07 March 2002 Dr. Michael Parkinson 22 Ethylene • Involved in wound responses in plants. • Produced in all cells of the plant and causes thickening of stems and leaf abscission. • Reduces adventitious shoot formation. • Interacts with an ethylene-binding protein (EBP) in the cell membrane. Binding of AgNO3 or norbornadiene to EBP antagonises ethylene effects. 07 March 2002 Dr. Michael Parkinson 23 Abscisic Acid (ABA) • Only one natural compound. • Promotes leaf abscission and seed dormancy. • Plays a dominant role in closing stomata in response to water stress. • Has an important role in embryogenesis in preparing embryos for dessication. Helps ensure ‘normal’ embryos. 07 March 2002 Dr. Michael Parkinson 24 ‘Plant Growth Regulator-like substances’ • Polyamines - have a vital role in embryo development. • Jasmonic acid - involved in plant wound responses. • Salicylic acid. • Not universally acclaimed as plant hormones since they are usually needed at high concentrations. 07 March 2002 Dr. Michael Parkinson 25 Undefined supplements • Sources of hormones, vitamins and polyamines. • e.g. Coconut water, sweetcorn extracts • Not reproducible • Do work. 07 March 2002 Dr. Michael Parkinson 26 Stage I - Sterilisation • Bacteria and fungi will overgrow the explant on the medium unless they are removed. • Pre-treatments to clean up the explant • Detergents • Sterilants and Antibiotics 07 March 2002 Pre-treatments • Transfer plants to a greenhouse to reduce endemic contaminants • Force outgrowth of axillary buds. • Washing removes endemic surface contaminants. Dr. Michael Parkinson 27 Uses of detergents • Air bubbles on the surface of the explant can protect bacteria and fungi from the liquid sterilant. • Mixing should therefore be done in such a way as to reduce air bubble formation 07 March 2002 Air bubble around epidermal hair Leaf surface • Detergents (e.g. Triton, Tween20) reduce the surface tension of the waxy cuticle on the leaf surface and increase wetting. Dr. Michael Parkinson 28 Sterilants • There are 3 principal • There is always a ways to kill off surface trade-off between contaminants. killing the surface contaminants and – oxidant action killing the explant. – Active halogen – Heavy metal poisoning • As far as possible, cut – *Powerful chemicals surfaces should be such as conc. sulphuric protected. acid may be used on seeds. 07 March 2002 Dr. Michael Parkinson 29 Sterilants used NaOCl CaOCl H2O2 HgCl2 AgNO3 Conc 10-20% v/v 10-20% v/v 1% v/v 0.1% w/v 1% w/v time 10-20 mins 10-20 mins 10 mins 10-30 mins 10-30 mins Action oxidant / Halogen oxidant / Halogen oxidant Heavy metal Heavy metal Antibiotics are rarely used since many are bacteriostatic and can cause mass overgrowth of cultures when they are removed. There are no antifungal compounds that are proven to be innocuous. 07 March 2002 Dr. Michael Parkinson 30 Stage II - Multiplication • Nodal cuttings are made. This removes the inhibitory effect of the shoot apex on bud outgrowth (Apical dominance). • GA’s may be added to promote etiolation, especially in species that form rosettes. • Cytokinins may be used to increase bud growth (antogonises auxin effect). • Multiplication is very labour-intensive. 07 March 2002 Dr. Michael Parkinson 31 Stages III and IV Rooting and transfer to the greenhouse • Plants must be rooted by using media containing auxin or by dipping explant bases in auxin solutions. • Progressively, the plants must be hardened by increasing the light intensity, and reducing sugar, inorganic salts and humidity. • Medium must be removed prior to transplantation to prevent contamination. 07 March 2002 Dr. Michael Parkinson 32 Micropropagation by adventitious shoot formation • Adventitious shoot formation is the de-novo development of shoots from cell clusters in the absence of pre-existing meristems. • In some species (e.g. Saintpaulia), many shoots can be induced (3000 from one leaf). • In other species (e.g. coffee), it may be necessary to induce an unorganised mass proliferation of cells (callus) prior to adventitious shoot formation. 07 March 2002 Dr. Michael Parkinson 33 Control of organogenesis Cytokinin Leaf strip Adventitious Shoot Root Callus 07 March 2002 Auxin Dr. Michael Parkinson 34 Plant Hygiene • Pathogens affect yield (average 30% reduction) • There are strict plant sanitation requirements for import of plants. • Viruses and bacteria will be multiplied along with the explants and need to be removed prior to plant multiplication. 07 March 2002 Dr. Michael Parkinson 35 Ways to eliminate viruses • 1 Heat treatment. Plants grow faster than viruses at high temperatures. • 2 Meristemming. Viruses are transported from cell to cell through plasmodesmata and through the vascular tissue. Apical meristem often free of viruses. Trade off between infection and survival. • 3. Not all cells in the plant are infected Adventitious shoots formed from single cells can give virus-free shoots. 07 March 2002 Dr. Michael Parkinson 36 Elimination of viruses Plant from the field Pre-growth in the greenhouse Active growth Heat treatment 35oC / months ‘Virus-free’ Plants Meristem culture Adventitious Shoot formation Virus testing Micropropagation cycle 07 March 2002 Dr. Michael Parkinson 37 PRODUCTION OF PRODUCTS • • • • • Advantages and disadvantages Cost of production Plant cell culture systems Ways to increase product formation Commercial production 07 March 2002 Dr. Michael Parkinson 38 Advantages and disadvantages Advantages • Can manipulate environment • Can feed precursors • Possible to select in culture • Possible to get all cells in a culture producing. 07 March 2002 • Can continuously extract. • Can retain biomass • • • • Disadvantages High cost Contamination Low intrinsic production Dr. Michael Parkinson 39 Cost of production • • • • Plant cells are slow growing. Full of water (90% - 95%). Easily contaminated. Shear-sensitivity means specially modified fermenters necessary • All this puts the cost of production of dry mass to $25 per kilo. Product only a fraction of this. 07 March 2002 Dr. Michael Parkinson 40 Plant cell culture systems Organised Unorganised • Shoot cultures. • Callus • ‘Hairy root’ cultures • Cell suspension culture • Embryo fermentations. 07 March 2002 Dr. Michael Parkinson 41 Shoot cultures • Under conditions of high cytokinin, a culture producing a mass of shoots may be produced by adventitious shoot formation. • For light-associated products, may be much more high yielding. • Sensitive to shear • Illumination a problem for scale up 07 March 2002 Dr. Michael Parkinson 42 ‘Hairy root’ cultures • ‘Hairy roots’ are produced by infecting sterile plants with a natural genetic engineer, Agrobacterium rhizogenes. • Genes for auxin synthesis and sensitivity are engineered into plant cells leading to gravity-insensitive mass root production. • Very useful for products produced in roots. • Aggregration and shear sensitivity are a major problem for scale-up 07 March 2002 Dr. Michael Parkinson 43 Embryo Fermentations • Somatic Embryos may be produced profusely from leaves or zygotic embryos. • For micropropagation, potentially phenomenally productive. • Shear sensitivity is a problem. • Maturation in liquid is a problem. 07 March 2002 Dr. Michael Parkinson 44 Shikonin production in culture • Shikonin production in the intact plant • Introduction into culture • Optimisation of production through medium manipulations • Fermentation 07 March 2002 Dr. Michael Parkinson 45 Callus • Equimolar amounts of auxin and cytokinin stimulate cell division. Leads to a mass proliferation of an unorganised mass of cells called a callus. • Requirement for support ensures that scaleup is limited (Ginseng saponins successfully produced in this way). 07 March 2002 Dr. Michael Parkinson 46 Cell suspension culture • When callus pieces are agitated in a liquid medium, they tend to break up. • Suspensions are much easier to bulk up than callus since there is no manual transfer or solid support. • Large scale (50,000l) commercial fermentations for Shikonin and Berberine. 07 March 2002 Dr. Michael Parkinson 47 Introduction of callus into suspension • ‘Friable’ callus goes easily into suspension. – – – – 2,4-D Low cytokinin semi-solid medium enzymic digestion with pectinase – blending 07 March 2002 • Removal of large cell aggregates by sieving. • Plating of single cells and small cell aggregates - only viable cells will grow and can be reintroduced into suspension. Dr. Michael Parkinson 48 Introduction into suspension Sieve out lumps 1 2 Initial high density + Pick off growing high producers Subculture and sieving Plate out 07 March 2002 Dr. Michael Parkinson 49 Growth kinetics Plant Cell Suspension typical Growth curve 16 14 Dry w eight (g/l) • 1. Initial lag dependent on dilution • 2. Exponential phase (dt 1-30 d) • 3. Linear/deceleration phase (declining nutrients) • 4. Stationary (nutrients exhausted) 3 12 10 4 8 6 4 2 0 2 1 0 2 4 6 8 10 12 14 16 18 20 22 tim e (d) 07 March 2002 Dr. Michael Parkinson 50 Characteristics of plant cells • Large (10-100mM long) • Tend to occur in aggregates • Shear-sensitive • Slow growing • Easily contaminated • Low oxygen demand (kla of 5-20) 07 March 2002 • Will not tolerate anaerobic conditions • Can grow to high cell densities (>300g/l fresh weight). • Can form very viscous solutions Dr. Michael Parkinson 51 Shear and plant cells • Oxygen demand proportional to cell density. • Shear rate proportional to viscosity • shear rate proportional to **power of viscosity 07 March 2002 Dr. Michael Parkinson 52 Special reactors for plant cell suspension cultures • • • • • Modified stirred tank Air-lift Air loop Bubble column Rotating drum reactor 07 March 2002 Dr. Michael Parkinson 53 Modified Stirred Tank Standard Rushton turbine 07 March 2002 Dr. Michael Parkinson Wing-Vane impeller 54 Airlift systems Poor mixing Bubble column 07 March 2002 Airlift (draught tube)Dr. Michael Parkinson Airloop (External Downtube) 55 Rotating Drum reactor • Like a washing machine • Low shear • Easy to scale-up 07 March 2002 Dr. Michael Parkinson 56 Ways to increase product formation • Select • Start off with a producing part • Modify media for growth and product formation. • Feed precursors or feed intermediates (bioconversion) 07 March 2002 • Produce ‘plant-like’ conditions (immobilisation) Dr. Michael Parkinson 57 Selection • Select at the level of the intact plant • Select in culture – single cell is selection unit – possible to plate up to 1,000,000 cells on a Petri-dish. – Progressive selection over a number of phases 07 March 2002 Dr. Michael Parkinson 58 Selection Strategies • • • • Positive Negative Visual Analytical Screening 07 March 2002 Dr. Michael Parkinson 59 Positive selection • Add into medium a toxic compound e.g. hydroxy proline, kanamycin • Only those cells able to grow in the presence of the selective agent give colonies • Plate out and pick off growing colonies. • Possible to select one colony from millions of plated cells in a days work. • Need a strong selection pressure - get escapes 07 March 2002 Dr. Michael Parkinson 60 Negative selection • Add in an agent that kills dividing cells e.g. chlorate / BUdR. • Plate out leave for a suitable time, wash out agent then put on growth medium. • All cells growing on selective agent will die leaving only non-growing cells to now grow. • Useful for selecting auxotrophs. 07 March 2002 Dr. Michael Parkinson 61 Visual selection • Only useful for coloured or fluorescent compounds e.g. shikonin/Berberine/ some alkaloids. • Plate out at about 50,000 cells per plate. • Pick off coloured / fluorescent compounds • Possible to screen about 1,000,000 cells in a days work. 07 March 2002 Dr. Michael Parkinson 62 Analytical Screening • Cut each piece of callus in 2. • One half subcultured. • Other half extracted and amount of compound determined analytically (HPLC/ GCMS/ ELISA). • Extraction V. laborious and limits number of callus pieces that can be assayed to 200/d (Zenk by Radioimmunoassay). 07 March 2002 Dr. Michael Parkinson 63 Media manipulations 07 March 2002 Dr. Michael Parkinson 64 Immobilisation 07 March 2002 Dr. Michael Parkinson 65 Plant Genetic Transformation Dr Michael Parkinson 07 March 2002 Dr. Michael Parkinson 66 Overview • • • • Introduction Plant genetic transformation Current status of GM crops Future trends & ‘Problems’ 07 March 2002 Dr. Michael Parkinson 67 Introduction • Potential of Plant Biotechnology • Uses of introduced novel genes • Traits that plant breeders would like in plants 07 March 2002 Dr. Michael Parkinson 68 Potential of Plant Biotechnology • • • • • Micropropagation Somatic hybrids / Cybrids Haploid plants Fermentations Introduction of novel genes into plants 07 March 2002 Dr. Michael Parkinson 69 Uses of introduced novel genes • Research into gene functions • ‘Molecular farming’ • Crop improvement in a single step 07 March 2002 Dr. Michael Parkinson 70 Molecular farming • Polyhydroxy butyrate (PHB) is a renewable source of plastics. • Monoclonal antibodies* • Human Serum Albumin • Interleukins. • Vaccines (virus coat protein genes) 07 March 2002 • Neurotransmitters e.g. 50mg/kg Leu-enkaphalin produced in Oil seed rape. • Modification of oils to improve Biodiesel. • Prodigene now producing enzymes, oral vaccines & antibodies from Maize seeds. Dr. Michael Parkinson 71 Overview of molecular farming • Gene isolation - easy • Vector design – organ specific promoters – High level expression – Containment • Transformation of maize by Biolistics 07 March 2002 • Regeneration from a crop monocot difficult • Growth, seed harvesting and downstream processing requires strong agricultural and fermentation expertise. • www.prodigene.com Dr. Michael Parkinson 72 Traits that plant breeders would like in plants • High primary productivity • High crop yield • High nutritional quality • Adaptation to intercropping • Nitrogen Fixation 07 March 2002 • Drought resistance • Pest resistance • Adaptation to mechanised farming • Insensitivity to photoperiod • Elimination of toxic compounds Dr. Michael Parkinson 73 Plant genetic transformation • Overview of requirements for plant genetic transformation • Development of GM foods • Genes for crops • Benefits of GM crops, especially in developing countries 07 March 2002 • How to get genes into cells to give transformed cells • How to get a plant back from a single transformed cell Dr. Michael Parkinson 74 Overview of requirements for plant genetic transformation • Trait that is encoded by a single gene • A means of driving expression of the gene in plant cells (Promoters and terminators) • Means of putting the gene into a cell (Vector) • A means of selecting for transformants • Means of getting a whole plant back from the single transformed cell (Regeneration) 07 March 2002 Dr. Michael Parkinson 75 Development of GM foods 1950 First regeneration of entire plants from an in vitro culture 1973 Researchers develop the ability to isolate genes 1983 1st transgenic plant: antibiotic resistant tobacco 1985 GM plants resistant to insects, viruses, and bacteria are field tested for the first time - USEFUL TRAITS 1990 First successful field trial of GM cotton- CROP 1994 Flavr-Savr tomato - 1st FDA approval for a food Monsanto's Roundup Ready soybeans approved for 07 March 2002 Dr. Michael Parkinson 76 sale in the United States. 1995 Useful single gene traits that have been introduced into plants • • • • • Herbicide resistance* Insect resistance* Virus resistance Seed protection Fungal resistance • Delayed ripening 07 March 2002 • • • • • • • Cold / Frost resistance Drought resistance High starch potatoes Oil production Plastics Digestibility proteins Antibodies Dr. Michael Parkinson 77 Genes for pest resistance • Insects • Fungi • Protease inhibitors • Bacillus thuringiensis insecticidal proteins** • Lectins • Ribosome-inactivating proteins (RIPs) • Chitinases and Beta1,3-glucanases • RIPs • Thionins • Antifungal peptides 07 March 2002 Dr. Michael Parkinson 78 Improved post-harvest properties Up to 50% of harvested food is lost post-harvest in Africa. • Any poisonous protein can be detoxified by heating and rendered safe e.g. lectins; inhibitors. • Ripening control 07 March 2002 • Wheat germ agglutinin • Cowpea trypsin inhibitor • Flavrsavr tomatoes contain antisense to polygalacturonase (softens tomatoes by dissolving the cell wall). Dr. Michael Parkinson 79 Other useful traits • Improved Agronomic properties • Improved plant breeding • Improved nutritional properties 07 March 2002 • High starch potatoes • Pollen-specific promoter plus RNAse • Golden rice (gene from Chrysanthemum giving - converted to vitamin A. Dr. Michael Parkinson 80 Potential of GM crops in low input, sustainable agriculture Traditional GM crop with pest resistance plus post-harvest qualities 4 tonnes/ha produced 5 tonnes/ha 25% losses post-harvest = 1 tonne/ha 3 tonnes/ha to eat 07 March 2002 Dr. Michael Parkinson 10% losses post-harvest = 0.5 tonne/ha 4.5 tonnes/ha to eat 81 • Cassava is a very important crop in Africa • Viral infection of the crop is increasing • Possible to engineer Cassava Mosaic virus resistance by using coat protein genes 07 March 2002 Dr. Michael Parkinson 82 Perceived benefits of GM crops 07 March 2002 Dr. Michael Parkinson 83 Approved Traits • • • • • Glufosinater herbicide Sethoxydimr herbicide Bromoxynilr herbicide Glyphosater herbicide Sulfonylurear herbicide 07 March 2002 • • • • • • • Male-sterility Modified fatty acid Flower colour Flower life Delayed fruit ripening Virus resistance Bt Dr. Michael Parkinson 84 Plasmid construction • Useful gene construct • Visible marker • Selectable marker* 07 March 2002 Dr. Michael Parkinson 85 Gene construction DNA • Plant specific promoter • Plant RBS • Useful gene • Signal peptides* • PolyA-tail Nucleus transcription mRNA Cytoplasm translation Polypeptide chain Post-translational modification 07 March 2002 Dr. Michael Parkinson 86 2 Types of delivery systems Naked DNA Cell wall is the primary resistance to DNA uptake • Biolistics • SiC fibres • Protoplasts • Electroporation • Pollen 07 March 2002 Vectored • Agrobacterium • Viruses Dr. Michael Parkinson 87 Getting genes into cells (Vectors) Agrobacterium Particle guns • A natural genetic engineer! - causes Crown Galls • Very efficiently transforms most dicotyledonous plants • Problematical with monocots • Works! • No residual Agrobacterium • Can be used with differing DNAs to probe gene function 07 March 2002 Dr. Michael Parkinson 88 Transformation with Agrobacterium • Agrobacterium contains a circle of DNA (Ti plasmid) that carries the desired genes • Co-cultivation of the Agrobacterium with plant pieces transfers the DNA Bacterial Ti Plasmid chromosome Petri dish with leaf pieces plus Agrobacterium 07 March 2002 Dr. Michael Parkinson 89 Co-integrative and binary vectors LB t-DNA RB Bacterial ORI Ampicillin resistance VIR genes Plasmid DNA Co-integrative 07 March 2002 Binary vector Dr. Michael Parkinson Bacterial Chromosome 90 Agrobacterium-mediated transformation • A natural genetic engineer • 2 species • In the presence of exudates (e.g. acetosyringone) from wounded plants, – A.tumefaciens Virulence (VIR) genes (produces a gall) are activated and cause – A. rhizogenes (produces roots) the t-DNA to be transferred to plants. • Oncogenes (for auxin Everything between and cytokinin the left and right synthesis) + Opines border is transferred. 91 07 March 2002 Dr. Michael Parkinson General transformation protocol Transformation O/N A.r culture Sterile explants with dividing cells Wash Inoculate (mins-hrs) (bacterial attachment) Co-cultivate (days) Transfer of t-DNA Recovery of transgenic plants Transfer to regeneration medium plus selective antibiotics Regeneration of transgenic plants 07 March 2002 Transfer to medium with bactericidal antibiotics plus selective antibiotics (months) Kill off Agrobacterium and select transgenic cells Dr. Michael Parkinson Transfer to medium with bactericidal antibiotics (days) Kill off Agrobacterium 92 Naked DNA • Biolistics now used routinely. DNA coated particles are literally blasted into cells by an explosive discharge. • SiC fibres 1mm * 70mm are strong and will penetrate cell wall. Vortex cells with medium, SiC fibres and plasmid DNA. 07 March 2002 • Protoplasts are cells without a cell wall. Produced by enzymic degradation of the cell wall. DNA uptake enhanced by electroporation or treatments to change plasmalemma charge (Polyethylene Glycol). Dr. Michael Parkinson 93 ‘Particle Gun’ • DNA coated on pellets is forced down the barrel of a ‘Particle Gun’ by an explosive charge • The particles are forced through the cell wall where the DNA is released 07 March 2002 Petri Dish with cultures Dr. Michael Parkinson Explosive Charge Projectile DNA coated pellets Barrel Vent Stop plate 94 Visible markers B-glucuronidase (GUS) • The UidA gene encoding activity is commonly used. Gives a blue colour from a colourless substrate (X-glu) for a qualitative assay. Also causes fluorescence from Methyl Umbelliferyl Glucuronide (MUG) for a quantitative assay. 07 March 2002 • • • • Green Fluorescent Protein (GFP) Fluoresces green under UV illumination Non-destructive Problems with a cryptic intron now resolved. Has been used for selection on its own. Dr. Michael Parkinson 95 Selection • Transformation frequency is low (Max 3% of all cells) and unless there is a selective advantage for transformed cells, these will be overgrown by non-transformed. • Usual to use a positive selective agent like antibiotic resistance. The NptII gene encoding Neomycin phospho-transferase II phosphorylates kanamycin group antibiotics and is commonly used. 07 March 2002 Dr. Michael Parkinson 96 Regeneration of whole plants back from single cells - 2 means Somatic embryogenesis • Multiple embryos are formed. • 3 types – Pro-embryonic masses – Cleavage polyembryony – Secondary embryo formation 07 March 2002 Adventitious shoot formation • Dividing cells stimulated by high [cytokinin]/[auxin] to form buds which grow to give shoots Dr. Michael Parkinson 97 Somatic embryogenesis from Pro-embryonic masses (PEMs) + Auxin leads to high [Putrescine] PEM Development and cycling of Pro-embryonic masses E.g. Carrot, Monocots, some conifers 07 March 2002 Remove Auxin Polyamine interconvesions Dr. Michael Parkinson Single cells sloughed off the surface Putrescine to Spermidine Spermidine to Spermine 98 Cleavage Polyembryony- conifers Cleavage lengthways Embryo Suspensor Normal Embyro 07 March 2002 Lateral division Dr. Michael Parkinson New embryos 99 Secondary embryo formation - Most dicots Abundant Secondary Embryos +Cytokinin +Charcoal +ABA -Cytokinin Early embryo 07 March 2002 Dr. Michael Parkinson 100 Development of GM foods 1950 First regeneration of entire plants from an in vitro culture 1973 Researchers develop the ability to isolate genes 1983 1st transgenic plant: antibiotic resistant tobacco 1985 GM plants resistant to insects, viruses, and bacteria are field tested for the first time - USEFUL TRAITS 1990 First successful field trial of GM cotton- CROP 1994 Flavr-Savr tomato - 1st FDA approval for a food Monsanto's Roundup Ready soybeans approved for 07 March 2002 Dr. Michael Parkinson 101 sale in the United States. 1995 Current status of GM crops • The worlds most important crops • GM crops • Traits 07 March 2002 Dr. Michael Parkinson 102 Global area of transgenic crops (ISAA Brief. Global Review of Commercialised Transgenic crops: 1998 & 2001) 60 Millions of hectares • Acreage of transgenic crops has gone from nothing in 1995 to around 135 million acres in 2001. 50 40 30 20 10 0 1995 07 March 2002 Dr. Michael Parkinson 1997 1999 2001 103 The worlds most important crops 07 March 2002 Dr. Michael Parkinson 104 Root Crops 07 March 2002 Dr. Michael Parkinson 105 Pulses 07 March 2002 Dr. Michael Parkinson 106 The worlds most important crops M hectares 250 200 150 100 50 W he at R ic e C or Ba n So rle rg y So hu yb m ea ns M ille C t an ol Po a C tato as sa va 0 07 March 2002 Dr. Michael Parkinson 107 07 March 2002 Dr. Michael Parkinson Oil Seed Cotton Corn 40 35 30 25 20 15 10 5 0 Soybean • Soybean and corn are the major GM crops * Large acreage * Grown in the USA * Can be regenerated • Acreage of potatoes is small (<0.1 million hectares) area Types of GM crops (1998) 108 e Ra p or n O il S ee d C yb e an 60 50 40 30 20 10 0 So • Almost 1/3rd of the Soybean crop in the US is GM (60% of crop in Argentina) • Almost 1/4 of US corn • 50% of Canadian oil seed rape % transgenic GM crop areas in North America 07 March 2002 Dr. Michael Parkinson 109 07 March 2002 20 15 10 5 Dr. Michael Parkinson Others 0 Insect resistance • <1% have other traits 25 Herbicide • >99% of all transgenic crops are either herbicide or insect resistant Millions of hectares Types of genetic modification 110 Herbicide resistant crops 07 March 2002 Dr. Michael Parkinson 111 Approved Transgenic plants • • • • • • • • Soybean Corn Cotton Oil Seed rape Sugarbeet Squash Tomato Tobacco 07 March 2002 • • • • • • • Carnations Potato Flax Papaya Chicory Rice Melon Dr. Michael Parkinson 112 Problems and potential 07 March 2002 Dr. Michael Parkinson 113 Future traits and methodology • Environmental stress resistance • Edible vaccines • Post-harvest quality • ‘Plantibodies’ • Biodegradeable plastics • Fungal resistance 07 March 2002 • Targetting to the chloroplast • Organ specific expression • Antibiotic-free selection • Greater gene stability • More crop species Dr. Michael Parkinson 114 ‘Problems’ with GM foods • Unethical to meddle with nature • ‘Contamination’ of non-GM crops • Lack of public choice • Allergic reactions • Generation of ‘Superweeds’ 07 March 2002 • Transfer of antibiotic resistance genes • Re-activation of latent viruses • Toxins • Loss of diversity • Poisoning / reduction of beneficial insects Dr. Michael Parkinson 115 Summary • There are several ways that plant biotechnology can be beneficial • A wide range of useful traits can be put into plants • The benefits of GM crops are such that the technology has been taken up very quickly • We have to balance the potential benefits with potential risks and assess release on a case by case basis 07 March 2002 Dr. Michael Parkinson 116