Docstoc

Agrobacterium-mediated transform

Document Sample
Agrobacterium-mediated transform Powered By Docstoc
					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
– 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
Dr. Michael Parkinson 4

07 March 2002

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)

Chop up

Maturation

07 March 2002

Dr. Michael Parkinson

9

Methods of micropropagation
• Axillary branching • >95% of all micropropagation. • Genetically stable • Simple and straightforward • Efficient but prone to genetic instability • Little used. Potentially phenomenally efficient.
10

• Adventitious shoot formation • Somatic embryogenesis
07 March 2002

Dr. Michael Parkinson

Axillary Branching
Shoot tip
Stem 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 • Shoot tips • Axillary buds • Seeds

• Hypocotyl (from germinated seed) • Leaves

07 March 2002

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

Shoot tip - Auxins and Gibberellins

Leaves sugars, GAs

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

07 March 2002

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 2002 special cells in Michael Parkinson bundles. vascular 07 March Dr. 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.
27

Dr. Michael Parkinson

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.
28

Dr. Michael Parkinson

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
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

NaOCl CaOCl H 2O 2 HgCl2 AgNO3

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 Auxin

07 March 2002

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

Adventitious Shoot formation

Meristem culture

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
39

Dr. Michael Parkinson

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 forMichael Parkinson scale-up 07 March 2002 Dr. 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

• 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.
48

07 March 2002

Dr. Michael Parkinson

Introduction into suspension
Initial high density Sieve out lumps 1 2

+
Pick off growing high producers Subculture and sieving

Plate out
07 March 2002 Dr. Michael Parkinson 49

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)
07 March 2002

Plant Cell Suspension typical Growth curve
16 14
Dry w eight (g/l)

12 10 8 6 4 2 0

3

4

2 1
0 2 4 6 8 10 12 14 16 18 20 22 tim e (d)

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) Michael Parkinson Dr.

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.
71

Dr. Michael Parkinson

Overview of molecular farming
• Gene isolation - easy • Vector design
– organ specific promoters – High level expression – Containment

• Regeneration from a crop monocot difficult

• Transformation of maize by Biolistics

• Growth, seed harvesting and downstream processing requires strong agricultural and fermentation expertise. • www.prodigene.com
72

07 March 2002

Dr. Michael Parkinson

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
73

Dr. Michael Parkinson

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 Researchers develop the ability to isolate genes
1st transgenic plant: antibiotic resistant tobacco

1973
1983

1985
1990

GM plants resistant to insects, viruses, and bacteria are field tested for the first time - USEFUL TRAITS
First successful field trial of GM cotton- CROP

1994
1995

Flavr-Savr tomato - 1st FDA approval for a food

Monsanto's Roundup Ready soybeans approved for 07 March 2002 Dr. 76 sale in the United Michael Parkinson States.

Useful single gene traits that have been introduced into plants
• • • • • Herbicide resistance* Insect resistance* Virus resistance Seed protection Fungal resistance • • • • • • • Cold / Frost resistance Drought resistance High starch potatoes Oil production Plastics Digestibility proteins Antibodies

• Delayed ripening

07 March 2002

Dr. Michael Parkinson

77

Genes for pest resistance
• Insects • Protease inhibitors • Bacillus thuringiensis insecticidal proteins** • Lectins • Ribosome-inactivating proteins (RIPs)
07 March 2002

• Fungi • Chitinases and Beta1,3-glucanases • RIPs • Thionins • Antifungal peptides

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).
79

Dr. Michael Parkinson

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.

07 March 2002

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 25% losses post-harvest = 1 tonne/ha

5 tonnes/ha 10% losses post-harvest = 0.5 tonne/ha

07 March 2002

3 tonnes/ha to eat

Dr. Michael Parkinson

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 • • • • • • • Male-sterility Modified fatty acid Flower colour Flower life Delayed fruit ripening Virus resistance Bt

07 March 2002

Dr. Michael Parkinson

84

Plasmid construction
• Useful gene construct • Visible marker • Selectable marker*

07 March 2002

Dr. Michael Parkinson

85

Gene construction
DNA Nucleus

• Plant specific promoter • Plant RBS • Useful gene • Signal peptides* • PolyA-tail

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 • A natural genetic engineer! - causes Crown Galls • Very efficiently transforms most dicotyledonous plants • Problematical with monocots
07 March 2002

Particle guns • Works! • No residual Agrobacterium • Can be used with differing DNAs to probe gene function

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
89

07 March 2002

Dr. Michael Parkinson

Co-integrative and binary vectors
LB RB t-DNA

Bacterial ORI Ampicillin resistance
VIR genes Plasmid DNA Bacterial Chromosome

Co-integrative

Binary vector

07 March 2002

Dr. Michael Parkinson

90

Agrobacterium-mediated transformation
• 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 • A natural genetic engineer • 2 species

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 2002 07 March 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).
93

Dr. Michael Parkinson

„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 Dr. Michael Parkinson

Explosive Charge

Projectile DNA coated pellets Barrel Vent Stop plate

Petri Dish with cultures

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.
95

Dr. Michael Parkinson

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

Adventitious shoot formation • Dividing cells stimulated by high [cytokinin]/[auxin] to form buds which grow to give shoots

07 March 2002

Dr. Michael Parkinson

97

Somatic embryogenesis from Pro-embryonic masses (PEMs)
+ Auxin leads to high [Putrescine] PEM Development and cycling of Pro-embryonic masses

Single cells sloughed off the surface Putrescine to Spermidine
Spermidine to Spermine

E.g. Carrot,
Monocots, some conifers

Remove Auxin Polyamine interconvesions

07 March 2002

Dr. Michael Parkinson

98

Cleavage Polyembryony- conifers
Cleavage lengthways Embryo

Suspensor

07 March 2002

Normal Embyro

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 Researchers develop the ability to isolate genes
1st transgenic plant: antibiotic resistant tobacco

1973
1983

1985
1990

GM plants resistant to insects, viruses, and bacteria are field tested for the first time - USEFUL TRAITS
First successful field trial of GM cotton- CROP

1994
1995

Flavr-Savr tomato - 1st FDA approval for a food

Monsanto's Roundup Ready soybeans approved for 07 March 2002 Dr. 101 sale in the United Michael Parkinson States.

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)

Millions of hectares

• Acreage of transgenic crops has gone from nothing in 1995 to around 135 million acres in 2001.

60 50 40 30 20 10 0 1995 1997 1999 2001
103

07 March 2002

Dr. Michael Parkinson

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
250

M hectares

200 150 100 50 0

07 March 2002

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
Dr. Michael Parkinson 107

W

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)
07 March 2002

40 35 30 25 20 15 10 5 0

area

Soybean

Cotton

Corn

Dr. Michael Parkinson

108

Oil Seed

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
60 50 40 30 20 10 0
an or n yb e Ra p ee O il S
07 March 2002 Dr. Michael Parkinson

% transgenic

So

C

d
109

e

Types of genetic modification
Millions of hectares

• >99% of all transgenic crops are either herbicide or insect resistant • <1% have other traits

25 20 15 10 5 0

Insect resistance

Herbicide

07 March 2002

Dr. Michael Parkinson

110

Others

Herbicide resistant crops

07 March 2002

Dr. Michael Parkinson

111

Approved Transgenic plants
• • • • • • • • Soybean Corn Cotton Oil Seed rape Sugarbeet Squash Tomato Tobacco • • • • • • • Carnations Potato Flax Papaya Chicory Rice Melon

07 March 2002

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
114

Dr. Michael Parkinson

„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
115

Dr. Michael Parkinson

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