BE304

BE304 Plant Cell culture : 

07 March 2002 Dr. Michael Parkinson 1 BE304 Plant Cell culture Dr. Michael Parkinson, School of Biotechnology

ASSESSMENT : 

07 March 2002 Dr. Michael Parkinson 2 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.

Types of points : 

07 March 2002 Dr. Michael Parkinson 3 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. 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?

Resources : 

07 March 2002 Dr. Michael Parkinson 4 Resources Powerpoint presentation of lectures Webpages@dcu.ie/~parkinsm/teaching Partially worked solution to exam question Text books Agriculture 631 Plant cell and tissue culture 571 Secondary metabolism 660 Transformation 572

Lecture outline : 

07 March 2002 Dr. Michael Parkinson 5 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.

Grand Overview : 

07 March 2002 Dr. Michael Parkinson 6 Grand Overview Explant Sterile explant Sterilisation Shoot cultures Media Adventitious shoots / Somatic embryos Micro- propagation Callus Product formation Transformation regeneration

Micropropagation : 

07 March 2002 Dr. Michael Parkinson 7 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

Advantages and disadvantages of micropropagation : 

07 March 2002 Dr. Michael Parkinson 8 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

Historical aspects : 

07 March 2002 Dr. Michael Parkinson 9 Historical aspects First commercially used with orchids - conventional propagation rate of 1 per year. Through protocorms, 1,000,000 per year. Corm (Swollen stem) Chop up Maturation

Methods of micropropagation : 

07 March 2002 Dr. Michael Parkinson 10 Methods of micropropagation Axillary branching Adventitious shoot formation Somatic embryogenesis >95% of all micropropagation. Genetically stable Simple and straightforward Efficient but prone to genetic instability Little used. Potentially phenomenally efficient.

Axillary Branching : 

07 March 2002 Dr. Michael Parkinson 11 Axillary Branching Shoot tip Axillary bud in the axil of the leaf Stem Leaf petiole

Choice of explant : 

07 March 2002 Dr. Michael Parkinson 12 Choice of explant Desirable properties of an explant Easily sterilisable Juvenile Responsive to culture Shoot tips Axillary buds Seeds Hypocotyl (from germinated seed) Leaves

Media : 

07 March 2002 Dr. Michael Parkinson 13 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 Roots - water, vitamins mineral salts and cytokinins Leaves - sugars, GAs

Medium constituents : 

07 March 2002 Dr. Michael Parkinson 14 Medium constituents Inorganic salt formulations Source of carbohydrate Vitamins Water Plant hormones - auxins, cytokinins, GA’s Solidifying agents Undefined supplements

Carbohydrates : 

07 March 2002 Dr. Michael Parkinson 15 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.

Photoautotrophic culture : 

07 March 2002 Dr. Michael Parkinson 16 Photoautotrophic culture Growth without a carbon source. Therefore need to boost photosynthesis. High light intensities needed (90-150mMole/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.

Inorganic salt formulations : 

07 March 2002 Dr. Michael Parkinson 17 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).

Vitamins : 

07 March 2002 Dr. Michael Parkinson 18 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)

Plant hormones (Growth regulators) : 

07 March 2002 Dr. Michael Parkinson 19 Plant hormones (Growth regulators) Auxins Cytokinins Gibberellic acids Ethylene Abscisic Acid “Plant Growth Regulator-like compounds”

Auxins : 

07 March 2002 Dr. Michael Parkinson 20 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.

Cytokinins : 

07 March 2002 Dr. Michael Parkinson 21 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.

Gibberellins (GA’s) : 

07 March 2002 Dr. Michael Parkinson 22 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.

Ethylene : 

07 March 2002 Dr. Michael Parkinson 23 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.

Abscisic Acid (ABA) : 

07 March 2002 Dr. Michael Parkinson 24 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.

‘Plant Growth Regulator-like substances’ : 

07 March 2002 Dr. Michael Parkinson 25 ‘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.

Undefined supplements : 

07 March 2002 Dr. Michael Parkinson 26 Undefined supplements Sources of hormones, vitamins and polyamines. e.g. Coconut water, sweetcorn extracts Not reproducible Do work.

Stage I - Sterilisation : 

07 March 2002 Dr. Michael Parkinson 27 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 Pre-treatments Transfer plants to a greenhouse to reduce endemic contaminants Force outgrowth of axillary buds. Washing removes endemic surface contaminants.

Uses of detergents : 

07 March 2002 Dr. Michael Parkinson 28 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 Leaf surface Air bubble around epidermal hair Detergents (e.g. Triton, Tween20) reduce the surface tension of the waxy cuticle on the leaf surface and increase wetting.

Sterilants : 

07 March 2002 Dr. Michael Parkinson 29 Sterilants There are 3 principal ways to kill off surface contaminants. oxidant action Active halogen Heavy metal poisoning *Powerful chemicals such as conc. sulphuric acid may be used on seeds. There is always a trade-off between killing the surface contaminants and killing the explant. As far as possible, cut surfaces should be protected.

Sterilants used : 

07 March 2002 Dr. Michael Parkinson 30 Sterilants used 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.

Stage II - Multiplication : 

07 March 2002 Dr. Michael Parkinson 31 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.

Stages III and IV Rooting and transfer to the greenhouse : 

07 March 2002 Dr. Michael Parkinson 32 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.

Micropropagation by adventitious shoot formation : 

07 March 2002 Dr. Michael Parkinson 33 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.

Control of organogenesis : 

07 March 2002 Dr. Michael Parkinson 34 Control of organogenesis Cytokinin Auxin Leaf strip Adventitious Shoot Root Callus

Plant Hygiene : 

07 March 2002 Dr. Michael Parkinson 35 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.

Ways to eliminate viruses : 

07 March 2002 Dr. Michael Parkinson 36 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.

Elimination of viruses : 

07 March 2002 Dr. Michael Parkinson 37 Elimination of viruses Plant from the field Pre-growth in the greenhouse ‘Virus-free’ Plants Heat treatment 35oC / months Active growth Meristem culture Micropropagation cycle Virus testing Adventitious Shoot formation

PRODUCTION OF PRODUCTS : 

07 March 2002 Dr. Michael Parkinson 38 PRODUCTION OF PRODUCTS Advantages and disadvantages Cost of production Plant cell culture systems Ways to increase product formation Commercial production

Advantages and disadvantages : 

07 March 2002 Dr. Michael Parkinson 39 Advantages and disadvantages Advantages Can manipulate environment Can feed precursors Possible to select in culture Possible to get all cells in a culture producing. Can continuously extract. Can retain biomass Disadvantages High cost Contamination Low intrinsic production

Cost of production : 

07 March 2002 Dr. Michael Parkinson 40 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.

Plant cell culture systems : 

07 March 2002 Dr. Michael Parkinson 41 Plant cell culture systems Organised Shoot cultures. ‘Hairy root’ cultures Embryo fermentations. Unorganised Callus Cell suspension culture

Shoot cultures : 

07 March 2002 Dr. Michael Parkinson 42 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

‘Hairy root’ cultures : 

07 March 2002 Dr. Michael Parkinson 43 ‘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

Embryo Fermentations : 

07 March 2002 Dr. Michael Parkinson 44 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.

Shikonin production in culture : 

07 March 2002 Dr. Michael Parkinson 45 Shikonin production in culture Shikonin production in the intact plant Introduction into culture Optimisation of production through medium manipulations Fermentation

Callus : 

07 March 2002 Dr. Michael Parkinson 46 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 scale-up is limited (Ginseng saponins successfully produced in this way).

Cell suspension culture : 

07 March 2002 Dr. Michael Parkinson 47 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.

Introduction of callus into suspension : 

07 March 2002 Dr. Michael Parkinson 48 Introduction of callus into suspension ‘Friable’ callus goes easily into suspension. 2,4-D Low cytokinin semi-solid medium enzymic digestion with pectinase blending Removal of large cell aggregates by sieving. Plating of single cells and small cell aggregates - only viable cells will grow and can be re-introduced into suspension.

Introduction into suspension : 

07 March 2002 Dr. Michael Parkinson 49 Introduction into suspension + Plate out Sieve out lumps 1 2 Pick off growing high producers Initial high density Subculture and sieving

Growth kinetics : 

07 March 2002 Dr. Michael Parkinson 50 Growth kinetics 1. Initial lag dependent on dilution 2. Exponential phase (dt 1-30 d) 3. Linear/deceleration phase (declining nutrients) 4. Stationary (nutrients exhausted) 1 2 3 4

Characteristics of plant cells : 

07 March 2002 Dr. Michael Parkinson 51 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) Will not tolerate anaerobic conditions Can grow to high cell densities (>300g/l fresh weight). Can form very viscous solutions

Shear and plant cells : 

07 March 2002 Dr. Michael Parkinson 52 Shear and plant cells Oxygen demand proportional to cell density. Shear rate proportional to viscosity shear rate proportional to **power of viscosity

Special reactors for plant cell suspension cultures : 

07 March 2002 Dr. Michael Parkinson 53 Special reactors for plant cell suspension cultures Modified stirred tank Air-lift Air loop Bubble column Rotating drum reactor

Modified Stirred Tank : 

07 March 2002 Dr. Michael Parkinson 54 Modified Stirred Tank Standard Rushton turbine Wing-Vane impeller

Airlift systems : 

07 March 2002 Dr. Michael Parkinson 55 Airlift systems Bubble column Airlift (draught tube) Poor mixing Airloop (External Downtube)

Rotating Drum reactor : 

07 March 2002 Dr. Michael Parkinson 56 Rotating Drum reactor Like a washing machine Low shear Easy to scale-up

Ways to increase product formation : 

07 March 2002 Dr. Michael Parkinson 57 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) Produce ‘plant-like’ conditions (immobilisation)

Selection : 

07 March 2002 Dr. Michael Parkinson 58 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

Selection Strategies : 

07 March 2002 Dr. Michael Parkinson 59 Selection Strategies Positive Negative Visual Analytical Screening

Positive selection : 

07 March 2002 Dr. Michael Parkinson 60 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

Negative selection : 

07 March 2002 Dr. Michael Parkinson 61 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.

Visual selection : 

07 March 2002 Dr. Michael Parkinson 62 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.

Analytical Screening : 

07 March 2002 Dr. Michael Parkinson 63 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).

Media manipulations : 

07 March 2002 Dr. Michael Parkinson 64 Media manipulations

Immobilisation : 

07 March 2002 Dr. Michael Parkinson 65 Immobilisation

Plant GeneticTransformation : 

07 March 2002 Dr. Michael Parkinson 66 Plant GeneticTransformation Dr Michael Parkinson

Overview : 

07 March 2002 Dr. Michael Parkinson 67 Overview Introduction Plant genetic transformation Current status of GM crops Future trends & ‘Problems’

Introduction : 

07 March 2002 Dr. Michael Parkinson 68 Introduction Potential of Plant Biotechnology Uses of introduced novel genes Traits that plant breeders would like in plants

Potential of Plant Biotechnology : 

07 March 2002 Dr. Michael Parkinson 69 Potential of Plant Biotechnology Micropropagation Somatic hybrids / Cybrids Haploid plants Fermentations Introduction of novel genes into plants

Uses of introduced novel genes : 

07 March 2002 Dr. Michael Parkinson 70 Uses of introduced novel genes Research into gene functions ‘Molecular farming’ Crop improvement in a single step

Molecular farming : 

07 March 2002 Dr. Michael Parkinson 71 Molecular farming Polyhydroxy butyrate (PHB) is a renewable source of plastics. Monoclonal antibodies* Human Serum Albumin Interleukins. Vaccines (virus coat protein genes) 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.

Overview of molecular farming : 

07 March 2002 Dr. Michael Parkinson 72 Gene isolation - easy Vector design organ specific promoters High level expression Containment Transformation of maize by Biolistics Overview of molecular farming Regeneration from a crop monocot difficult Growth, seed harvesting and downstream processing requires strong agricultural and fermentation expertise. www.prodigene.com

Traits that plant breeders would like in plants : 

07 March 2002 Dr. Michael Parkinson 73 Traits that plant breeders would like in plants High primary productivity High crop yield High nutritional quality Adaptation to inter-cropping Nitrogen Fixation Drought resistance Pest resistance Adaptation to mechanised farming Insensitivity to photo-period Elimination of toxic compounds

Plant genetic transformation : 

07 March 2002 Dr. Michael Parkinson 74 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 How to get genes into cells to give transformed cells How to get a plant back from a single transformed cell

Overview of requirements for plant genetic transformation : 

07 March 2002 Dr. Michael Parkinson 75 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)

Development of GM foods : 

07 March 2002 Dr. Michael Parkinson 76 Development of GM foods Flavr-Savr tomato - 1st FDA approval for a food 1995 Monsanto's Roundup Ready soybeans approved for sale in the United States. 1994 First successful field trial of GM cotton- CROP 1990 GM plants resistant to insects, viruses, and bacteria are field tested for the first time - USEFUL TRAITS 1985 1st transgenic plant: antibiotic resistant tobacco 1983 Researchers develop the ability to isolate genes 1973 First regeneration of entire plants from an in vitro culture 1950

Useful single gene traits that have been introduced into plants : 

07 March 2002 Dr. Michael Parkinson 77 Useful single gene traits that have been introduced into plants Herbicide resistance* Insect resistance* Virus resistance Seed protection Fungal resistance Delayed ripening Cold / Frost resistance Drought resistance High starch potatoes Oil production Plastics Digestibility proteins Antibodies

Genes for pest resistance : 

07 March 2002 Dr. Michael Parkinson 78 Genes for pest resistance Insects Protease inhibitors Bacillus thuringiensis insecticidal proteins** Lectins Ribosome-inactivating proteins (RIPs) Fungi Chitinases and Beta-1,3-glucanases RIPs Thionins Antifungal peptides

Improved post-harvest properties : 

07 March 2002 Dr. Michael Parkinson 79 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 Wheat germ agglutinin Cowpea trypsin inhibitor Flavrsavr tomatoes contain antisense to polygalacturonase (softens tomatoes by dissolving the cell wall).

Other useful traits : 

07 March 2002 Dr. Michael Parkinson 80 Other useful traits Improved Agronomic properties Improved plant breeding Improved nutritional properties High starch potatoes Pollen-specific promoter plus RNAse Golden rice (gene from Chrysanthemum giving - converted to vitamin A.

Potential of GM crops in low input, sustainable agriculture : 

07 March 2002 Dr. Michael Parkinson 81 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 10% losses post-harvest = 0.5 tonne/ha 4.5 tonnes/ha to eat

Slide 82: 

07 March 2002 Dr. Michael Parkinson 82 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

Perceived benefits of GM crops : 

07 March 2002 Dr. Michael Parkinson 83 Perceived benefits of GM crops

Approved Traits : 

07 March 2002 Dr. Michael Parkinson 84 Approved Traits Glufosinater herbicide Sethoxydimr herbicide Bromoxynilr herbicide Glyphosater herbicide Sulfonylurear herbicide Male-sterility Modified fatty acid Flower colour Flower life Delayed fruit ripening Virus resistance Bt

Plasmid construction : 

07 March 2002 Dr. Michael Parkinson 85 Plasmid construction Useful gene construct Visible marker Selectable marker*

Gene construction : 

07 March 2002 Dr. Michael Parkinson 86 Gene construction Plant specific promoter Plant RBS Useful gene Signal peptides* PolyA-tail DNA mRNA Polypeptide chain transcription translation Post-translational modification Nucleus Cytoplasm

2 Types of delivery systems : 

07 March 2002 Dr. Michael Parkinson 87 2 Types of delivery systems Naked DNA Cell wall is the primary resistance to DNA uptake Biolistics SiC fibres Protoplasts Electroporation Pollen Vectored Agrobacterium Viruses

Getting genes into cells (Vectors) : 

07 March 2002 Dr. Michael Parkinson 88 Getting genes into cells (Vectors) Agrobacterium A natural genetic engineer! - causes Crown Galls Very efficiently transforms most dicotyledonous plants Problematical with monocots Particle guns Works! No residual Agrobacterium Can be used with differing DNAs to probe gene function

Transformation with Agrobacterium : 

07 March 2002 Dr. Michael Parkinson 89 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 chromosome Ti Plasmid Petri dish with leaf pieces plus Agrobacterium

Co-integrative and binary vectors : 

07 March 2002 Dr. Michael Parkinson 90 Co-integrative and binary vectors Binary vector t-DNA VIR genes Plasmid DNA Bacterial Chromosome Bacterial ORI Ampicillin resistance LB RB Co-integrative

Agrobacterium-mediated transformation : 

07 March 2002 Dr. Michael Parkinson 91 Agrobacterium-mediated transformation A natural genetic engineer 2 species A.tumefaciens (produces a gall) A. rhizogenes (produces roots) Oncogenes (for auxin and cytokinin synthesis) + Opines In the presence of exudates (e.g. acetosyringone) from wounded plants, Virulence (VIR) genes are activated and cause the t-DNA to be transferred to plants. Everything between the left and right border is transferred.

General transformation protocol : 

07 March 2002 Dr. Michael Parkinson 92 General transformation protocol O/N A.r culture Sterile explants with dividing cells Inoculate (mins-hrs) (bacterial attachment) Co-cultivate (days) Transfer of t-DNA Wash Transfer to medium with bactericidal antibiotics (days) Kill off Agrobacterium Transfer to medium with bactericidal antibiotics plus selective antibiotics (months) Kill off Agrobacterium and select transgenic cells Transfer to regeneration medium plus selective antibiotics Regeneration of transgenic plants Transformation Recovery of transgenic plants

Naked DNA : 

07 March 2002 Dr. Michael Parkinson 93 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. 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).

‘Particle Gun’ : 

07 March 2002 Dr. Michael Parkinson 94 ‘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 Barrel Explosive Charge Vent Stop plate Petri Dish with cultures Projectile DNA coated pellets

Visible markers : 

07 March 2002 Dr. Michael Parkinson 95 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. 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.

Selection : 

07 March 2002 Dr. Michael Parkinson 96 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.

Regeneration of whole plants back from single cells - 2 means : 

07 March 2002 Dr. Michael Parkinson 97 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 Adventitious shoot formation Dividing cells stimulated by high [cytokinin]/[auxin] to form buds which grow to give shoots

Somatic embryogenesis from Pro-embryonic masses (PEMs) : 

07 March 2002 Dr. Michael Parkinson 98 Somatic embryogenesis from Pro-embryonic masses (PEMs) PEM Development and cycling of Pro-embryonic masses + Auxin leads to high [Putrescine] Putrescine to Spermidine Spermidine to Spermine Single cells sloughed off the surface Remove Auxin Polyamine interconvesions E.g. Carrot, Monocots, some conifers

Cleavage Polyembryony- conifers : 

07 March 2002 Dr. Michael Parkinson 99 Cleavage Polyembryony- conifers Embryo Suspensor Normal Embyro Lateral division Cleavage lengthways New embryos

Secondary embryo formation - Most dicots : 

07 March 2002 Dr. Michael Parkinson 100 Secondary embryo formation - Most dicots Early embryo +Cytokinin Abundant Secondary Embryos -Cytokinin +Charcoal +ABA

Development of GM foods : 

07 March 2002 Dr. Michael Parkinson 101 Development of GM foods Flavr-Savr tomato - 1st FDA approval for a food 1995 Monsanto's Roundup Ready soybeans approved for sale in the United States. 1994 First successful field trial of GM cotton- CROP 1990 GM plants resistant to insects, viruses, and bacteria are field tested for the first time - USEFUL TRAITS 1985 1st transgenic plant: antibiotic resistant tobacco 1983 Researchers develop the ability to isolate genes 1973 First regeneration of entire plants from an in vitro culture 1950

Current status of GM crops : 

07 March 2002 Dr. Michael Parkinson 102 Current status of GM crops The worlds most important crops GM crops Traits

Global area of transgenic crops(ISAA Brief. Global Review of Commercialised Transgenic crops: 1998 & 2001) : 

07 March 2002 Dr. Michael Parkinson 103 Global area of transgenic crops(ISAA Brief. Global Review of Commercialised Transgenic crops: 1998 & 2001) Acreage of transgenic crops has gone from nothing in 1995 to around 135 million acres in 2001.

The worlds most important crops : 

07 March 2002 Dr. Michael Parkinson 104 The worlds most important crops

Root Crops : 

07 March 2002 Dr. Michael Parkinson 105 Root Crops

Pulses : 

07 March 2002 Dr. Michael Parkinson 106 Pulses

The worlds most important crops : 

07 March 2002 Dr. Michael Parkinson 107 The worlds most important crops

Types of GM crops (1998) : 

07 March 2002 Dr. Michael Parkinson 108 Types of GM crops (1998) 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)

GM crop areas in North America : 

07 March 2002 Dr. Michael Parkinson 109 GM crop areas in North America 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

Types of genetic modification : 

07 March 2002 Dr. Michael Parkinson 110 Types of genetic modification >99% of all transgenic crops are either herbicide or insect resistant <1% have other traits

Herbicide resistant crops : 

07 March 2002 Dr. Michael Parkinson 111 Herbicide resistant crops

Approved Transgenic plants : 

07 March 2002 Dr. Michael Parkinson 112 Approved Transgenic plants Soybean Corn Cotton Oil Seed rape Sugarbeet Squash Tomato Tobacco Carnations Potato Flax Papaya Chicory Rice Melon

Problems and potential : 

07 March 2002 Dr. Michael Parkinson 113 Problems and potential

Future traits and methodology : 

07 March 2002 Dr. Michael Parkinson 114 Future traits and methodology Environmental stress resistance Edible vaccines Post-harvest quality ‘Plantibodies’ Biodegradeable plastics Fungal resistance Targetting to the chloroplast Organ specific expression Antibiotic-free selection Greater gene stability More crop species

‘Problems’ with GM foods : 

07 March 2002 Dr. Michael Parkinson 115 ‘Problems’ with GM foods Unethical to meddle with nature ‘Contamination’ of non-GM crops Lack of public choice Allergic reactions Generation of ‘Super-weeds’ Transfer of antibiotic resistance genes Re-activation of latent viruses Toxins Loss of diversity Poisoning / reduction of beneficial insects

Summary : 

07 March 2002 Dr. Michael Parkinson 116 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