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

4. Resources Powerpoint presentation of lectures Text books
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

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.
07 March 2002
Dr. Michael Parkinson

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

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

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

13. Media
Shoot tip - Auxins
and Gibberellins
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.
Leaves -
sugars, GAs
Roots - water, vitamins
mineral salts and cytokinins
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
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.
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.
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).
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)
07 March 2002
Dr. Michael Parkinson

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

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

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

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

39. Advantages and disadvantages
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
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.
07 March 2002
Dr. Michael Parkinson

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

49. Introduction into suspension
Sieve out lumps
1 2
Initial high
density +
Subculture
and sieving
Pick off
growing
high
producers
Plate out
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) 3 4 2 1
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
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
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
07 March 2002
Dr. Michael Parkinson

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

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

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

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

64. Media manipulations
07 March 2002
Dr. Michael Parkinson

65. Immobilisation
07 March 2002
Dr. Michael Parkinson

66. Plant Genetic Transformation
Dr Michael Parkinson
07 March 2002
Dr. Michael Parkinson

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

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

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

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

76. Development of GM foods
1950
First regeneration of entire plants from an in vitro culture
1973
Researchers develop the ability to isolate genes
1st transgenic plant: antibiotic resistant tobacco
1983
GM plants resistant to insects, viruses, and bacteria are field tested for the first time - USEFUL TRAITS
1985
1990
First successful field trial of GM cotton- CROP
Flavr-Savr tomato - 1st FDA approval for a food
1994
Monsanto's Roundup Ready soybeans approved for sale in the United States.
1995
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
Many of the traits that we can currently put into crops are highly applicable to less intensive agriculture (emboldened) which is practised in the 3rd world.
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
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).
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.
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
10% losses
post-harvest
= 0.5 tonne/ha
3 tonnes/ha to eat
4.5 tonnes/ha to eat
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
07 March 2002
Dr. Michael Parkinson

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

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

86. Gene construction Plant specific promoter Plant RBS Useful gene
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

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
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
07 March 2002
Dr. Michael Parkinson

89. Transformation with Agrobacterium
Bacterial
chromosome
Agrobacterium contains a circle of DNA (Ti plasmid) that carries the desired genes
Co-cultivation of the Agrobacterium with plant pieces transfers the DNA
Ti Plasmid
Petri dish
with leaf pieces
plus Agrobacterium
07 March 2002
Dr. Michael Parkinson

90. Co-integrative and binary vectorsLB RB
t-DNA
Bacterial ORI
Ampicillin
resistance
VIR genes
Plasmid DNA
Bacterial
Chromosome
Co-integrative vectors require the genes that are transferred from bacteria to go into the plasmid DNA by homologous recombination. For binary vectors, the plasmid containing the t-DNA is able to replicate in E.coli and can be mobilised into Agrobacterium by a triparental mating with a helper strain of E.coli. This greatly simplifies the process of plasmid construction.
Co-integrative
Binary vector
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.
07 March 2002
Dr. Michael Parkinson

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

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

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

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

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

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

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

103. Global area of transgenic crops (ISAA Brief
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.
07 March 2002
Dr. Michael Parkinson

104. The worlds most important crops
From top left: MHa
Wheat 232 Rice146 Corn/Maize 140
Barley 77 Sorghum 55 Millet 42
Wheat & Rice (not transformed or only just starting to be transformed) acreage greater than other major crops put together.
07 March 2002
Dr. Michael Parkinson

105. Root Crops
Potato 20 Cassava 9.8
07 March 2002
Dr. Michael Parkinson

106. Pulses
Soybean 55Mha
07 March 2002
Dr. Michael Parkinson

107. The worlds most important crops
Of the major crops, only Corn and soybeans have been released as GM crops in any large extent. Reasons:
Regeneration from grasses/cereals difficult, especially for crop varieties.
Small acreage of Millet / potato/ cassava in the US.
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)
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
Several factors have combined to give such high uptake rates for a new technology:
They work!
Pushed by the Agrochemical companies.
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
07 March 2002
Dr. Michael Parkinson

111. Herbicide resistant crops
Examples of transgenic crop plants expressing herbicide resistance.
a. Soybean (Bialaphos); b. Cotton (Bialaphos);
c. phaseolus (Bialaphos); d. soybean field trial for resistance to the herbicides Glyphosate and Bialaphos; and
e. rice (Bialaphos).
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
07 March 2002
Dr. Michael Parkinson

113. Problems and potential
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
07 March 2002
Dr. Michael Parkinson

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