Transcript Agrobacterium-mediated transformation

BE304 Plant Cell culture
Dr. Michael Parkinson,
School of Biotechnology
07 March 2002
Dr. Michael Parkinson
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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.
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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.
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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
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Resources
• Powerpoint presentation of lectures
– [email protected]/~parkinsm/teaching
– Partially worked solution to exam question
• Text books
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Agriculture 631
Plant cell and tissue culture 571
Secondary metabolism 660
Transformation 572
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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.
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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
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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
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Historical aspects
• First commercially used with orchids conventional propagation rate of 1 per year.
• Through protocorms, 1,000,000 per year.
Corm
(Swollen stem)
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Chop up
Maturation
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Methods of micropropagation
• Axillary branching
• Adventitious shoot
formation
• Somatic
embryogenesis
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• >95% of all
micropropagation.
• Genetically stable
• Simple and
straightforward
• Efficient but prone to
genetic instability
• Little used. Potentially
phenomenally efficient.
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Axillary Branching
Stem
Shoot tip
Leaf petiole
Axillary bud in the
axil of the leaf
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Choice of explant
Desirable properties of
an explant
• Easily sterilisable
• Juvenile
• Responsive to culture
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• Shoot tips
• Axillary buds
• Seeds
• Hypocotyl (from
germinated seed)
• Leaves
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Media
• When you make an
explant like an axillary
bud, you remove it
from the sources of
many chemicals and
have to re-supply
these to the explants to
allow them to grow.
Shoot tip - Auxins
and Gibberellins
Leaves sugars, GAs
Roots - water, vitamins
mineral salts and cytokinins
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Medium constituents
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Inorganic salt formulations
Source of carbohydrate
Vitamins
Water
Plant hormones - auxins, cytokinins, GA’s
Solidifying agents
Undefined supplements
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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.
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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.
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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).
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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)
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Plant hormones (Growth regulators)
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Auxins
Cytokinins
Gibberellic acids
Ethylene
Abscisic Acid
“Plant Growth Regulator-like compounds”
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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.
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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.
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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.
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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.
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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.
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‘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.
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Undefined supplements
• Sources of hormones, vitamins and
polyamines.
• e.g. Coconut water, sweetcorn extracts
• Not reproducible
• Do work.
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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
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Pre-treatments
• Transfer plants to a
greenhouse to reduce
endemic contaminants
• Force outgrowth of
axillary buds.
• Washing removes
endemic surface
contaminants.
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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
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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.
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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.
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Sterilants used
NaOCl
CaOCl
H2O2
HgCl2
AgNO3
Conc
10-20% v/v
10-20% v/v
1% v/v
0.1% w/v
1% w/v
time
10-20 mins
10-20 mins
10 mins
10-30 mins
10-30 mins
Action
oxidant / Halogen
oxidant / Halogen
oxidant
Heavy metal
Heavy metal
Antibiotics are rarely used since many are bacteriostatic and can
cause mass overgrowth of cultures when they are removed.
There are no antifungal compounds that are proven to be innocuous.
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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.
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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.
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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.
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Control of organogenesis
Cytokinin
Leaf strip
Adventitious
Shoot
Root
Callus
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Auxin
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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.
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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.
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Elimination of viruses
Plant from the field
Pre-growth in the greenhouse
Active
growth
Heat treatment
35oC / months
‘Virus-free’ Plants
Meristem culture
Adventitious
Shoot
formation
Virus testing
Micropropagation cycle
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PRODUCTION OF PRODUCTS
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Advantages and disadvantages
Cost of production
Plant cell culture systems
Ways to increase product formation
Commercial production
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Advantages and disadvantages
Advantages
• Can manipulate
environment
• Can feed precursors
• Possible to select in
culture
• Possible to get all cells
in a culture producing.
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• Can continuously
extract.
• Can retain biomass
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Disadvantages
High cost
Contamination
Low intrinsic
production
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Cost of production
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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.
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Plant cell culture systems
Organised
Unorganised
• Shoot cultures.
• Callus
• ‘Hairy root’ cultures
• Cell suspension
culture
• Embryo fermentations.
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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
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‘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
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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.
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Shikonin production in culture
• Shikonin production in the intact plant
• Introduction into culture
• Optimisation of production through medium
manipulations
• Fermentation
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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).
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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.
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Introduction of callus into
suspension
• ‘Friable’ callus goes
easily into suspension.
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2,4-D
Low cytokinin
semi-solid medium
enzymic digestion with
pectinase
– blending
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• 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.
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Introduction into suspension
Sieve out lumps
1
2
Initial high
density
+
Pick off
growing
high
producers
Subculture
and sieving
Plate out
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Growth kinetics
Plant Cell Suspension typical Growth
curve
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Dry w eight (g/l)
• 1. Initial lag dependent
on dilution
• 2. Exponential phase
(dt 1-30 d)
• 3. Linear/deceleration
phase (declining
nutrients)
• 4. Stationary (nutrients
exhausted)
3
12
10
4
8
6
4
2
0
2
1
0
2 4
6 8 10 12 14 16 18 20 22
tim e (d)
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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)
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• Will not tolerate
anaerobic conditions
• Can grow to high cell
densities (>300g/l
fresh weight).
• Can form very viscous
solutions
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Shear and plant cells
• Oxygen demand
proportional to cell
density.
• Shear rate proportional
to viscosity
• shear rate proportional
to **power of
viscosity
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Special reactors for plant cell
suspension cultures
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Modified stirred tank
Air-lift
Air loop
Bubble column
Rotating drum reactor
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Modified Stirred Tank
Standard Rushton turbine
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Wing-Vane impeller
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Airlift systems
Poor mixing
Bubble column
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Airlift (draught
tube)Dr. Michael Parkinson
Airloop (External
Downtube)
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Rotating Drum reactor
• Like a washing
machine
• Low shear
• Easy to scale-up
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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)
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• Produce ‘plant-like’
conditions
(immobilisation)
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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
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Selection Strategies
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Positive
Negative
Visual
Analytical Screening
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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
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2002
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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.
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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.
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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).
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Media manipulations
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Immobilisation
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Plant Genetic
Transformation
Dr Michael Parkinson
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Overview
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Introduction
Plant genetic transformation
Current status of GM crops
Future trends & ‘Problems’
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Introduction
• Potential of Plant Biotechnology
• Uses of introduced novel genes
• Traits that plant breeders would like in
plants
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Potential of Plant Biotechnology
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Micropropagation
Somatic hybrids / Cybrids
Haploid plants
Fermentations
Introduction of novel genes into plants
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Uses of introduced novel genes
• Research into gene functions
• ‘Molecular farming’
• Crop improvement in a single step
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Molecular farming
• Polyhydroxy butyrate
(PHB) is a renewable
source of plastics.
• Monoclonal
antibodies*
• Human Serum Albumin
• Interleukins.
• Vaccines (virus coat
protein genes)
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• 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.
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Overview of molecular farming
• Gene isolation - easy
• Vector design
– organ specific
promoters
– High level expression
– Containment
• Transformation of
maize by Biolistics
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• Regeneration from a crop
monocot difficult
• Growth, seed harvesting
and downstream
processing requires
strong agricultural and
fermentation expertise.
• www.prodigene.com
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Traits that plant breeders would
like in plants
• High primary
productivity
• High crop yield
• High nutritional
quality
• Adaptation to intercropping
• Nitrogen Fixation
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• Drought resistance
• Pest resistance
• Adaptation to
mechanised farming
• Insensitivity to photoperiod
• Elimination of toxic
compounds
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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
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• How to get genes into
cells to give
transformed cells
• How to get a plant
back from a single
transformed cell
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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)
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Development of GM foods
1950
First regeneration of entire plants from an in vitro culture
1973
Researchers develop the ability to isolate genes
1983
1st transgenic plant: antibiotic resistant tobacco
1985
GM plants resistant to insects, viruses, and bacteria are
field tested for the first time - USEFUL TRAITS
1990
First successful field trial of GM cotton- CROP
1994
Flavr-Savr tomato - 1st FDA approval for a food
Monsanto's Roundup Ready soybeans approved for
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sale in the United
States.
1995
Useful single gene traits that
have been introduced into plants
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Herbicide resistance*
Insect resistance*
Virus resistance
Seed protection
Fungal resistance
• Delayed ripening
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Cold / Frost resistance
Drought resistance
High starch potatoes
Oil production
Plastics
Digestibility proteins
Antibodies
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Genes for pest resistance
• Insects
• Fungi
• Protease inhibitors
• Bacillus thuringiensis
insecticidal proteins**
• Lectins
• Ribosome-inactivating
proteins (RIPs)
• Chitinases and Beta1,3-glucanases
• RIPs
• Thionins
• Antifungal peptides
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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
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• Wheat germ agglutinin
• Cowpea trypsin
inhibitor
• Flavrsavr tomatoes
contain antisense to
polygalacturonase
(softens tomatoes by
dissolving the cell
wall).
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Other useful traits
• Improved Agronomic
properties
• Improved plant
breeding
• Improved nutritional
properties
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• High starch potatoes
• Pollen-specific
promoter plus RNAse
• Golden rice (gene
from Chrysanthemum
giving - converted to
vitamin A.
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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
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Dr. Michael Parkinson
10% losses
post-harvest
= 0.5 tonne/ha
4.5 tonnes/ha to eat
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• 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
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Perceived benefits of GM crops
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Approved Traits
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Glufosinater herbicide
Sethoxydimr herbicide
Bromoxynilr herbicide
Glyphosater herbicide
Sulfonylurear
herbicide
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Male-sterility
Modified fatty acid
Flower colour
Flower life
Delayed fruit ripening
Virus resistance
Bt
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Plasmid construction
• Useful gene construct
• Visible marker
• Selectable marker*
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Gene construction
DNA
• Plant specific
promoter
• Plant RBS
• Useful gene
• Signal peptides*
• PolyA-tail
Nucleus
transcription
mRNA
Cytoplasm
translation
Polypeptide chain
Post-translational
modification
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2 Types of delivery systems
Naked DNA
Cell wall is the
primary resistance to
DNA uptake
• Biolistics
• SiC fibres
• Protoplasts
• Electroporation
• Pollen
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Vectored
• Agrobacterium
• Viruses
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Getting genes into cells (Vectors)
Agrobacterium
Particle guns
• A natural genetic
engineer! - causes
Crown Galls
• Very efficiently
transforms most
dicotyledonous plants
• Problematical with
monocots
• Works!
• No residual
Agrobacterium
• Can be used with
differing DNAs to
probe gene function
07 March 2002
Dr. Michael Parkinson
88
Transformation with Agrobacterium
• Agrobacterium
contains a circle of
DNA (Ti plasmid) that
carries the desired
genes
• Co-cultivation of the
Agrobacterium with
plant pieces transfers
the DNA
Bacterial
Ti Plasmid chromosome
Petri dish
with leaf pieces
plus Agrobacterium
07 March 2002
Dr. Michael Parkinson
89
Co-integrative and binary vectors
LB
t-DNA
RB
Bacterial ORI
Ampicillin
resistance
VIR genes
Plasmid DNA
Co-integrative
07 March 2002
Binary vector
Dr. Michael Parkinson
Bacterial
Chromosome
90
Agrobacterium-mediated
transformation
• A natural genetic
engineer
• 2 species
• In the presence of
exudates (e.g.
acetosyringone) from
wounded plants,
– A.tumefaciens
Virulence (VIR) genes
(produces a gall)
are activated and cause
– A. rhizogenes
(produces roots)
the t-DNA to be
transferred to plants.
• Oncogenes (for auxin
Everything between
and cytokinin
the left and right
synthesis) + Opines
border is transferred. 91
07 March 2002
Dr. Michael Parkinson
General transformation protocol
Transformation
O/N A.r culture
Sterile explants
with dividing cells
Wash
Inoculate (mins-hrs)
(bacterial attachment)
Co-cultivate (days)
Transfer of t-DNA
Recovery of transgenic plants
Transfer to
regeneration
medium plus
selective
antibiotics
Regeneration
of transgenic
plants
07 March 2002
Transfer to medium
with bactericidal
antibiotics plus
selective antibiotics
(months)
Kill off Agrobacterium
and select transgenic
cells Dr. Michael Parkinson
Transfer to medium
with bactericidal
antibiotics (days)
Kill off Agrobacterium
92
Naked DNA
• Biolistics now used
routinely. DNA coated
particles are literally
blasted into cells by an
explosive discharge.
• SiC fibres 1mm *
70mm are strong and
will penetrate cell
wall. Vortex cells with
medium, SiC fibres
and plasmid DNA.
07 March 2002
• Protoplasts are cells
without a cell wall.
Produced by enzymic
degradation of the cell
wall. DNA uptake
enhanced by
electroporation or
treatments to change
plasmalemma charge
(Polyethylene Glycol).
Dr. Michael Parkinson
93
‘Particle Gun’
• DNA coated on pellets
is forced down the
barrel of a ‘Particle
Gun’ by an explosive
charge
• The particles are
forced through the cell
wall where the DNA is
released
07 March 2002
Petri Dish
with cultures
Dr. Michael Parkinson
Explosive
Charge
Projectile
DNA coated
pellets
Barrel
Vent
Stop plate
94
Visible markers
B-glucuronidase (GUS)
• The UidA gene encoding
activity is commonly
used. Gives a blue colour
from a colourless
substrate (X-glu) for a
qualitative assay. Also
causes fluorescence from
Methyl Umbelliferyl
Glucuronide (MUG) for
a quantitative assay.
07 March 2002
•
•
•
•
Green Fluorescent
Protein (GFP)
Fluoresces green
under UV illumination
Non-destructive
Problems with a
cryptic intron now
resolved.
Has been used for
selection on its own.
Dr. Michael Parkinson
95
Selection
• Transformation frequency is low (Max 3%
of all cells) and unless there is a selective
advantage for transformed cells, these will
be overgrown by non-transformed.
• Usual to use a positive selective agent like
antibiotic resistance. The NptII gene
encoding Neomycin phospho-transferase II
phosphorylates kanamycin group antibiotics
and is commonly used.
07 March 2002
Dr. Michael Parkinson
96
Regeneration of whole plants
back from single cells - 2 means
Somatic embryogenesis
• Multiple embryos are
formed.
• 3 types
– Pro-embryonic masses
– Cleavage polyembryony
– Secondary embryo
formation
07 March 2002
Adventitious shoot
formation
• Dividing cells
stimulated by high
[cytokinin]/[auxin] to
form buds which grow
to give shoots
Dr. Michael Parkinson
97
Somatic embryogenesis from
Pro-embryonic masses (PEMs)
+ Auxin leads to high [Putrescine]
PEM
Development and cycling
of Pro-embryonic masses
E.g. Carrot,
Monocots,
some
conifers
07 March 2002
Remove
Auxin
Polyamine
interconvesions
Dr. Michael Parkinson
Single cells sloughed
off the surface
Putrescine
to Spermidine
Spermidine
to Spermine
98
Cleavage Polyembryony- conifers
Cleavage lengthways
Embryo
Suspensor
Normal
Embyro
07 March 2002
Lateral division
Dr. Michael Parkinson
New embryos
99
Secondary embryo formation
- Most dicots
Abundant
Secondary
Embryos
+Cytokinin
+Charcoal
+ABA
-Cytokinin
Early embryo
07 March 2002
Dr. Michael Parkinson
100
Development of GM foods
1950
First regeneration of entire plants from an in vitro culture
1973
Researchers develop the ability to isolate genes
1983
1st transgenic plant: antibiotic resistant tobacco
1985
GM plants resistant to insects, viruses, and bacteria are
field tested for the first time - USEFUL TRAITS
1990
First successful field trial of GM cotton- CROP
1994
Flavr-Savr tomato - 1st FDA approval for a food
Monsanto's Roundup Ready soybeans approved for
07 March 2002
Dr. Michael Parkinson
101
sale in the United
States.
1995
Current status of GM crops
• The worlds most important crops
• GM crops
• Traits
07 March 2002
Dr. Michael Parkinson
102
Global area of transgenic crops
(ISAA Brief. Global Review of Commercialised Transgenic crops: 1998 & 2001)
60
Millions of hectares
• Acreage of transgenic
crops has gone from
nothing in 1995 to
around 135 million
acres in 2001.
50
40
30
20
10
0
1995
07 March 2002
Dr. Michael Parkinson
1997
1999
2001
103
The worlds most important crops
07 March 2002
Dr. Michael Parkinson
104
Root Crops
07 March 2002
Dr. Michael Parkinson
105
Pulses
07 March 2002
Dr. Michael Parkinson
106
The worlds most important crops
M hectares
250
200
150
100
50
W
he
at
R
ic
e
C
or
Ba n
So rle
rg y
So hu
yb m
ea
ns
M
ille
C t
an
ol
Po a
C tato
as
sa
va
0
07 March 2002
Dr. Michael Parkinson
107
07 March 2002
Dr. Michael Parkinson
Oil Seed
Cotton
Corn
40
35
30
25
20
15
10
5
0
Soybean
• Soybean and corn are
the major GM crops
* Large acreage
* Grown in the USA
* Can be regenerated
• Acreage of potatoes is
small (<0.1 million
hectares)
area
Types of GM crops (1998)
108
e
Ra
p
or
n
O
il S
ee
d
C
yb
e
an
60
50
40
30
20
10
0
So
• Almost 1/3rd of the
Soybean crop in the
US is GM (60% of
crop in Argentina)
• Almost 1/4 of US corn
• 50% of Canadian oil
seed rape
% transgenic
GM crop areas in North America
07 March 2002
Dr. Michael Parkinson
109
07 March 2002
20
15
10
5
Dr. Michael Parkinson
Others
0
Insect
resistance
• <1% have other
traits
25
Herbicide
• >99% of all
transgenic crops
are either
herbicide or
insect resistant
Millions of hectares
Types of genetic modification
110
Herbicide resistant crops
07 March 2002
Dr. Michael Parkinson
111
Approved Transgenic plants
•
•
•
•
•
•
•
•
Soybean
Corn
Cotton
Oil Seed rape
Sugarbeet
Squash
Tomato
Tobacco
07 March 2002
•
•
•
•
•
•
•
Carnations
Potato
Flax
Papaya
Chicory
Rice
Melon
Dr. Michael Parkinson
112
Problems and potential
07 March 2002
Dr. Michael Parkinson
113
Future traits and methodology
• Environmental stress
resistance
• Edible vaccines
• Post-harvest quality
• ‘Plantibodies’
• Biodegradeable
plastics
• Fungal resistance
07 March 2002
• Targetting to the
chloroplast
• Organ specific
expression
• Antibiotic-free
selection
• Greater gene stability
• More crop species
Dr. Michael Parkinson
114
‘Problems’ with
GM foods
• Unethical to meddle
with nature
• ‘Contamination’ of
non-GM crops
• Lack of public choice
• Allergic reactions
• Generation of ‘Superweeds’
07 March 2002
• Transfer of antibiotic
resistance genes
• Re-activation of latent
viruses
• Toxins
• Loss of diversity
• Poisoning / reduction
of beneficial insects
Dr. Michael Parkinson
115
Summary
• There are several ways that plant
biotechnology can be beneficial
• A wide range of useful traits can be put into
plants
• The benefits of GM crops are such that the
technology has been taken up very quickly
• We have to balance the potential benefits
with potential risks and assess release on a
case by case basis
07 March 2002
Dr. Michael Parkinson
116