BE304 Plant Cell Culture: Dr. Michael Parkinson, School of Biotechnology

07 March 2002 Dr.
Michael Parkinson 1
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.
Types of points
Seeds were washed
overnight under a
running tap, rinsed for
10s in 70% ethanol
then sterilised in 20%
Domestos + 0.1% v/v
Tween20 for 10
minutes followed by 3
rinses in sterile
distilled water.
Why use seeds?
Why wash overnight?
Why rinse in 70% EtOH?
Why sterilise at all?
Why Domestos?
Why 20% for 10 mins?
Why 0.1% v/v Tween20?
Why rinse?
Resources
Powerpoint presentation of lectures
Webpages@dcu.ie/~parkinsm/teaching
Partially worked solution to exam question
Text books
Agriculture 631
Plant cell and tissue culture 571
Secondary metabolism 660
Transformation 572

Lecture outline
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.
Micropropagation
Advantages and disadvantages of
micropropagation
Methods of micropropagation
Choice of explant
Media
Stage I - Sterilisation
Stage II - Multiplication
Stages III and IV- Rooting, hardening off
and transfer to greenhouse
Advantages and disadvantages of
micropropagation
Speed - roughly a 10X increase every 2
months (possible to produce 10
6
plants from
a single starting plant in on year).
Axenic - provided that the original explant
is free of contaminant, the resulting plants
will all be uncontaminated.
Clonal propagation
Cost - 0.15 per explant
Historical aspects
First commercially used with orchids -
conventional propagation rate of 1 per year.
Through protocorms, 1,000,000 per year.
Corm
(Swollen stem)
Chop up Maturation
Methods of micropropagation
Axillary branching

Adventitious shoot
formation

Somatic
embryogenesis
>95% of all
micropropagation.
Genetically stable
Simple and
straightforward
Efficient but prone to
genetic instability
Little used. Potentially
phenomenally efficient.
Axillary Branching
Shoot tip
Axillary bud in the
axil of the leaf
Stem Leaf petiole
Choice of explant
Desirable properties of
an explant
Easily sterilisable
Juvenile
Responsive to culture
Shoot tips
Axillary buds
Seeds

Hypocotyl (from
germinated seed)
Leaves
Media
When you make an
explant like an axillary
bud, you remove it
from the sources of
many chemicals and
have to re-supply
these to the explants to
allow them to grow.
Shoot tip - Auxins
and Gibberellins
Roots - water, vitamins
mineral salts and cytokinins
Leaves -
sugars, GAs
Medium constituents
Inorganic salt formulations
Source of carbohydrate
Vitamins
Water
Plant hormones - auxins, cytokinins, GAs
Solidifying agents
Undefined supplements
Carbohydrates
Plants in culture usually cannot meet their
needs for fixed carbon. Usually added as
sucrose at 2-3% w/v.
Glucose or a mixture of glucose and
fructose is occasionally used.
For large scale cultures, cheaper sources of
sugars (corn syrup) may be used.
Photoautotrophic culture
Growth without a carbon source. Therefore
need to boost photosynthesis.
High light intensities needed (90-
150mMole/m
2
/s) compared to normal (30-50).
Usually increase CO
2
(1000ppm) compared to
normal 369.4ppm.
Much reduced level of contamination and
plants are easier to transfer to the greenhouse.
Inorganic salt formulations
Contain a wide range of Macro-elements
(>mg/l) and microelements (<mg/l).
A wide range of media are readily available
as spray-dried powders.
Murashige and Skoog Medium (1965) is the
most popular for shoot cultures.
Gamborgs B5 medium is widely used for
cell suspension cultures (no ammonium).
Vitamins
A wide range of vitamins are available and
may be used.
Generally, the smaller the explant, the more
exacting the vitamin requirement.
A vitamin cocktail is often used (Nicotinic
acid, glycine, Thiamine, pyridoxine).
Inositol usually has to be supplied at much
higher concentration (100mg/l)
Plant hormones (Growth regulators)
Auxins
Cytokinins
Gibberellic acids
Ethylene
Abscisic Acid
Plant Growth Regulator-like compounds
Auxins
Absolutely essential (no mutants known)
Only one compound, Indole-3-acetic acid.
Many synthetic analogues (NAA, IBA,
2,4-D, 2,4,5-T, Pichloram) - cheaper &
more stable
Generally growth stimulatory. Promote
rooting.
Produced in meristems, especially shoot
meristem and transported through the plant
in special cells in vascular bundles.
Cytokinins
Absolutely essential (no mutants known)
Single natural compound, Zeatin. Synthetic
analogues Benyzladenine (BA), Kinetin.
Stimulate cell division (with auxins).
Promotes formation of adventitious shoots.
Produced in the root meristem and
transported throughout the plant as the
Zeatin-riboside in the phloem.
Gibberellins (GAs)
A family of over 70 related compounds, all
forms of Gibberellic acid.
Commercially, GA3 and GA4+9 available.
Stimulate etiolation of stems.
Help break bud and seed dormancy.
Produced in young leaves.
Ethylene
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
AgNO
3
or norbornadiene to EBP
antagonises ethylene effects.
Abscisic Acid (ABA)
Only one natural compound.
Promotes leaf abscission and seed
dormancy.
Plays a dominant role in closing stomata in
response to water stress.
Has an important role in embryogenesis in
preparing embryos for dessication. Helps
ensure normal embryos.
Plant Growth Regulator-like
substances
Polyamines - have a vital role in embryo
development.
Jasmonic acid - involved in plant wound
responses.
Salicylic acid.
Not universally acclaimed as plant
hormones since they are usually needed at
high concentrations.
Undefined supplements
Sources of hormones, vitamins and
polyamines.
e.g. Coconut water, sweetcorn extracts
Not reproducible
Do work.
Stage I - Sterilisation
Bacteria and fungi will
overgrow the explant
on the medium unless
they are removed.
Pre-treatments to clean
up the explant
Detergents
Sterilants and
Antibiotics
Pre-treatments
Transfer plants to a
greenhouse to reduce
endemic contaminants
Force outgrowth of
axillary buds.
Washing removes
endemic surface
contaminants.
Uses of detergents
Air bubbles on the
surface of the explant
can protect bacteria
and fungi from the
liquid sterilant.
Mixing should
therefore be done in
such a way as to
reduce air bubble
formation
Leaf surface
Air bubble
around epidermal hair
Detergents (e.g.
Triton, Tween20)
reduce the surface
tension of the waxy
cuticle on the leaf
surface and increase
wetting.
Sterilants
There are 3 principal
ways to kill off surface
contaminants.
oxidant action
Active halogen
Heavy metal poisoning
*Powerful chemicals
such as conc. sulphuric
acid may be used on
seeds.
There is always a
trade-off between
killing the surface
contaminants and
killing the explant.
As far as possible, cut
surfaces should be
protected.
Sterilants used
Conc time Action
NaOCl 10-20% v/v 10-20 mins oxidant / Halogen
CaOCl 10-20% v/v 10-20 mins oxidant / Halogen
H
2
O
2
1% v/v 10 mins oxidant
HgCl
2
0.1% w/v 10-30 mins Heavy metal
AgNO
3
1% w/v 10-30 mins 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.
Stage II - Multiplication
Nodal cuttings are made. This removes the
inhibitory effect of the shoot apex on bud
outgrowth (Apical dominance).
GAs may be added to promote etiolation,
especially in species that form rosettes.
Cytokinins may be used to increase bud
growth (antogonises auxin effect).
Multiplication is very labour-intensive.
Stages III and IV Rooting and
transfer to the greenhouse
Plants must be rooted by using media
containing auxin or by dipping explant
bases in auxin solutions.
Progressively, the plants must be hardened
by increasing the light intensity, and
reducing sugar, inorganic salts and
humidity.
Medium must be removed prior to
transplantation to prevent contamination.
Micropropagation by
adventitious shoot formation
Adventitious shoot formation is the de-novo
development of shoots from cell clusters in
the absence of pre-existing meristems.
In some species (e.g. Saintpaulia), many
shoots can be induced (3000 from one leaf).
In other species (e.g. coffee), it may be
necessary to induce an unorganised mass
proliferation of cells (callus) prior to
adventitious shoot formation.
Control of organogenesis
Cytokinin
Auxin
Leaf strip
Adventitious
Shoot
Root
Callus
Plant Hygiene
Pathogens affect yield (average 30%
reduction)
There are strict plant sanitation
requirements for import of plants.
Viruses and bacteria will be multiplied
along with the explants and need to be
removed prior to plant multiplication.
Ways to eliminate viruses
1 Heat treatment. Plants grow faster than
viruses at high temperatures.
2 Meristemming. Viruses are transported
from cell to cell through plasmodesmata
and through the vascular tissue. Apical
meristem often free of viruses. Trade off
between infection and survival.
3. Not all cells in the plant are infected
Adventitious shoots formed from single
cells can give virus-free shoots.
Elimination of viruses
Plant from the field
Pre-growth in the greenhouse
Virus-free Plants
Heat treatment
35
o
C / months
Active
growth
Meristem culture
Micropropagation cycle
Virus testing
Adventitious
Shoot
formation
PRODUCTION OF PRODUCTS
Advantages and disadvantages
Cost of production
Plant cell culture systems
Ways to increase product formation
Commercial production
Advantages and disadvantages
Advantages
Can manipulate
environment
Can feed precursors
Possible to select in
culture
Possible to get all cells
in a culture producing.
Can continuously
extract.
Can retain biomass

Disadvantages
High cost
Contamination
Low intrinsic
production
Cost of production
Plant cells are slow growing.
Full of water (90% - 95%).
Easily contaminated.
Shear-sensitivity means specially modified
fermenters necessary
All this puts the cost of production of dry
mass to $25 per kilo. Product only a fraction
of this.
Plant cell culture systems
Organised
Shoot cultures.
Hairy root cultures
Embryo fermentations.
Unorganised
Callus
Cell suspension
culture
Shoot cultures
Under conditions of high cytokinin, a
culture producing a mass of shoots may be
produced by adventitious shoot formation.
For light-associated products, may be much
more high yielding.
Sensitive to shear
Illumination a problem for scale up
Hairy root cultures
Hairy roots are produced by infecting
sterile plants with a natural genetic
engineer, Agrobacterium rhizogenes.
Genes for auxin synthesis and sensitivity
are engineered into plant cells leading to
gravity-insensitive mass root production.
Very useful for products produced in roots.
Aggregration and shear sensitivity are a
major problem for scale-up
Embryo Fermentations
Somatic Embryos may be produced
profusely from leaves or zygotic embryos.
For micropropagation, potentially
phenomenally productive.
Shear sensitivity is a problem.
Maturation in liquid is a problem.
Shikonin production in culture
Shikonin production in the intact plant
Introduction into culture
Optimisation of production through medium
manipulations
Fermentation
Callus
Equimolar amounts of auxin and cytokinin
stimulate cell division. Leads to a mass
proliferation of an unorganised mass of
cells called a callus.
Requirement for support ensures that scale-
up is limited (Ginseng saponins successfully
produced in this way).
Cell suspension culture
When callus pieces are agitated in a liquid
medium, they tend to break up.
Suspensions are much easier to bulk up than
callus since there is no manual transfer or
solid support.
Large scale (50,000l) commercial
fermentations for Shikonin and Berberine.
Introduction of callus into
suspension
Friable callus goes
easily into suspension.
2,4-D
Low cytokinin
semi-solid medium
enzymic digestion with
pectinase
blending
Removal of large cell
aggregates by sieving.
Plating of single cells
and small cell
aggregates - only
viable cells will grow
and can be re-
introduced into
suspension.
Introduction into suspension
+

Plate out

Sieve out lumps
1 2

Pick off
growing
high
producers
Initial high
density
Subculture
and sieving
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)
Plant Cell Suspension typical Growth
curve
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12 14 16 18 20 22
time (d)
D
r
y

w
e
i
g
h
t

(
g
/
l
)
1
2
3
4
Characteristics of plant cells
Large (10-100mM
long)
Tend to occur in
aggregates
Shear-sensitive
Slow growing
Easily contaminated
Low oxygen demand
(kla of 5-20)
Will not tolerate
anaerobic conditions
Can grow to high cell
densities (>300g/l
fresh weight).
Can form very viscous
solutions
Shear and plant cells
Oxygen demand
proportional to cell
density.
Shear rate proportional
to viscosity
shear rate proportional
to **power of
viscosity

Special reactors for plant cell
suspension cultures
Modified stirred tank
Air-lift
Air loop
Bubble column
Rotating drum reactor
Modified Stirred Tank
Standard Rushton turbine
Wing-Vane impeller
Airlift systems
Bubble column Airlift (draught
tube)
Poor mixing
Airloop (External
Downtube)
Rotating Drum reactor
Like a washing
machine
Low shear
Easy to scale-up

Ways to increase product
formation
Select
Start off with a
producing part
Modify media for
growth and product
formation.
Feed precursors or
feed intermediates
(bioconversion)
Produce plant-like
conditions
(immobilisation)
Selection
Select at the level of the intact plant
Select in culture
single cell is selection unit
possible to plate up to 1,000,000 cells on a
Petri-dish.
Progressive selection over a number of phases
Selection Strategies
Positive
Negative
Visual
Analytical Screening
Positive selection
Add into medium a toxic compound e.g.
hydroxy proline, kanamycin
Only those cells able to grow in the
presence of the selective agent give colonies
Plate out and pick off growing colonies.
Possible to select one colony from millions
of plated cells in a days work.
Need a strong selection pressure - get
escapes
Negative selection
Add in an agent that kills dividing cells e.g.
chlorate / BUdR.
Plate out leave for a suitable time, wash out
agent then put on growth medium.
All cells growing on selective agent will die
leaving only non-growing cells to now
grow.
Useful for selecting auxotrophs.
Visual selection
Only useful for coloured or fluorescent
compounds e.g. shikonin/Berberine/ some
alkaloids.
Plate out at about 50,000 cells per plate.
Pick off coloured / fluorescent compounds
Possible to screen about 1,000,000 cells in a
days work.
Analytical Screening
Cut each piece of callus in 2.
One half subcultured.
Other half extracted and amount of
compound determined analytically (HPLC/
GCMS/ ELISA).
Extraction V. laborious and limits number
of callus pieces that can be assayed to 200/d
(Zenk by Radioimmunoassay).
Media manipulations

Immobilisation

Plant Genetic
Transformation
Dr Michael Parkinson
Overview
Introduction
Plant genetic transformation
Current status of GM crops
Future trends & Problems
Introduction
Potential of Plant Biotechnology
Uses of introduced novel genes
Traits that plant breeders would like in
plants
Potential of Plant Biotechnology
Micropropagation
Somatic hybrids / Cybrids
Haploid plants
Fermentations
Introduction of novel genes into plants
Uses of introduced novel genes
Research into gene functions
Molecular farming
Crop improvement in a single step
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.
Gene isolation - easy
Vector design
organ specific
promoters
High level expression
Containment
Transformation of
maize by Biolistics
Overview of molecular farming
Regeneration from a crop
monocot difficult

Growth, seed harvesting
and downstream
processing requires
strong agricultural and
fermentation expertise.
www.prodigene.com
Traits that plant breeders would
like in plants
High primary
productivity
High crop yield
High nutritional
quality
Adaptation to inter-
cropping
Nitrogen Fixation
Drought resistance
Pest resistance
Adaptation to
mechanised farming
Insensitivity to photo-
period
Elimination of toxic
compounds
Plant genetic transformation
Overview of
requirements for plant
genetic transformation
Development of GM
foods
Genes for crops
Benefits of GM crops,
especially in
developing countries

How to get genes into
cells to give
transformed cells
How to get a plant
back from a single
transformed cell
Overview of requirements for
plant genetic transformation
Trait that is encoded by a single gene
A means of driving expression of the gene in
plant cells (Promoters and terminators)
Means of putting the gene into a cell (Vector)
A means of selecting for transformants
Means of getting a whole plant back from the
single transformed cell (Regeneration)
Development of GM foods
Flavr-Savr tomato - 1st FDA approval for a food
1995
Monsanto's Roundup Ready soybeans approved for
sale in the United States.
1994
First successful field trial of GM cotton- CROP 1990
GM plants resistant to insects, viruses, and bacteria are
field tested for the first time - USEFUL TRAITS
1985
1st transgenic plant: antibiotic resistant tobacco
1983
Researchers develop the ability to isolate genes 1973
First regeneration of entire plants from an in vitro culture
1950
Useful single gene traits that
have been introduced into plants
Herbicide resistance*
Insect resistance*
Virus resistance
Seed protection
Fungal resistance

Delayed ripening
Cold / Frost resistance
Drought resistance
High starch potatoes
Oil production
Plastics
Digestibility proteins
Antibodies
Genes for pest resistance
Insects

Protease inhibitors
Bacillus thuringiensis
insecticidal proteins**
Lectins
Ribosome-inactivating
proteins (RIPs)
Fungi

Chitinases and Beta-
1,3-glucanases
RIPs
Thionins
Antifungal peptides

Improved post-harvest properties
Up to 50% of
harvested food is lost
post-harvest in Africa.
Any poisonous protein
can be detoxified by
heating and rendered
safe e.g. lectins;
inhibitors.
Ripening control

Wheat germ agglutinin
Cowpea trypsin
inhibitor
Flavrsavr tomatoes
contain antisense to
polygalacturonase
(softens tomatoes by
dissolving the cell
wall).
Other useful traits
Improved Agronomic
properties
Improved plant
breeding
Improved nutritional
properties

High starch potatoes

Pollen-specific
promoter plus RNAse
Golden rice (gene
from Chrysanthemum
giving - converted to
vitamin A.

Potential of GM crops in low
input, sustainable agriculture
Traditional GM crop with pest resistance
plus post-harvest qualities
4 tonnes/ha produced
5 tonnes/ha
25% losses
post-harvest
= 1 tonne/ha
3 tonnes/ha to eat
10% losses
post-harvest
= 0.5 tonne/ha
4.5 tonnes/ha to eat
Cassava is a
very important
crop in Africa
Viral infection
of the crop is
increasing
Possible to
engineer
Cassava
Mosaic virus
resistance by
using coat
protein genes
Perceived benefits of GM crops
Approved Traits
Glufosinate
r
herbicide
Sethoxydim
r
herbicide
Bromoxynil
r
herbicide
Glyphosate
r
herbicide
Sulfonylurea
r

herbicide

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

Plasmid construction
Useful gene construct
Visible marker
Selectable marker*
Gene construction
Plant specific
promoter
Plant RBS
Useful gene
Signal peptides*
PolyA-tail
DNA
mRNA
Polypeptide chain
transcription
translation
Post-translational
modification
Nucleus
Cytoplasm
2 Types of delivery systems
Naked DNA
Cell wall is the
primary resistance to
DNA uptake
Biolistics
SiC fibres
Protoplasts
Electroporation
Pollen
Vectored
Agrobacterium
Viruses
Getting genes into cells (Vectors)
Agrobacterium

A natural genetic
engineer! - causes
Crown Galls
Very efficiently
transforms most
dicotyledonous plants
Problematical with
monocots
Particle guns

Works!
No residual
Agrobacterium
Can be used with
differing DNAs to
probe gene function
Transformation with Agrobacterium
Agrobacterium
contains a circle of
DNA (Ti plasmid) that
carries the desired
genes
Co-cultivation of the
Agrobacterium with
plant pieces transfers
the DNA
Bacterial
chromosome
Ti Plasmid
Petri dish
with leaf pieces
plus Agrobacterium
Co-integrative and binary vectors
Binary vector
t-DNA
VIR genes
Plasmid DNA
Bacterial
Chromosome
Bacterial ORI
Ampicillin
resistance
LB RB
Co-integrative
Agrobacterium-mediated
transformation
A natural genetic
engineer
2 species
A.tumefaciens
(produces a gall)
A. rhizogenes
(produces roots)
Oncogenes (for auxin
and cytokinin
synthesis) + Opines
In the presence of
exudates (e.g.
acetosyringone) from
wounded plants,
Virulence (VIR) genes
are activated and cause
the t-DNA to be
transferred to plants.
Everything between
the left and right
border is transferred.
General transformation protocol
O/N A.r culture
Sterile explants
with dividing cells
Inoculate (mins-hrs)
(bacterial attachment)
Co-cultivate (days)
Transfer of t-DNA
Wash
Transfer to medium
with bactericidal
antibiotics (days)
Kill off Agrobacterium
Transfer to medium
with bactericidal
antibiotics plus
selective antibiotics
(months)
Kill off Agrobacterium
and select transgenic
cells
Transfer to
regeneration
medium plus
selective
antibiotics
Regeneration
of transgenic
plants
Transformation
Recovery of transgenic plants
Naked DNA
Biolistics now used
routinely. DNA coated
particles are literally
blasted into cells by an
explosive discharge.
SiC fibres 1mm *
70mm are strong and
will penetrate cell
wall. Vortex cells with
medium, SiC fibres
and plasmid DNA.
Protoplasts are cells
without a cell wall.
Produced by enzymic
degradation of the cell
wall. DNA uptake
enhanced by
electroporation or
treatments to change
plasmalemma charge
(Polyethylene Glycol).
Particle Gun
DNA coated on pellets
is forced down the
barrel of a Particle
Gun by an explosive
charge
The particles are
forced through the cell
wall where the DNA is
released
Barrel
Explosive
Charge
Vent
Stop plate
Petri Dish
with cultures
Projectile
DNA coated
pellets
Visible markers
B-glucuronidase (GUS)
The UidA gene encoding
activity is commonly
used. Gives a blue colour
from a colourless
substrate (X-glu) for a
qualitative assay. Also
causes fluorescence from
Methyl Umbelliferyl
Glucuronide (MUG) for
a quantitative assay.
Green Fluorescent
Protein (GFP)
Fluoresces green
under UV illumination
Non-destructive
Problems with a
cryptic intron now
resolved.
Has been used for
selection on its own.
Selection
Transformation frequency is low (Max 3%
of all cells) and unless there is a selective
advantage for transformed cells, these will
be overgrown by non-transformed.
Usual to use a positive selective agent like
antibiotic resistance. The NptII gene
encoding Neomycin phospho-transferase II
phosphorylates kanamycin group antibiotics
and is commonly used.
Regeneration of whole plants
back from single cells - 2 means
Somatic embryogenesis
Multiple embryos are
formed.
3 types
Pro-embryonic masses
Cleavage polyembryony
Secondary embryo
formation
Adventitious shoot
formation
Dividing cells
stimulated by high
[cytokinin]/[auxin] to
form buds which grow
to give shoots
Somatic embryogenesis from
Pro-embryonic masses (PEMs)
PEM
Development and cycling
of Pro-embryonic masses
+ Auxin leads to high [Putrescine]
Putrescine
to Spermidine
Spermidine
to Spermine
Single cells sloughed
off the surface
Remove
Auxin
Polyamine
interconvesions
E.g. Carrot,
Monocots,
some
conifers
Cleavage Polyembryony- conifers
Embryo
Suspensor
Normal
Embyro
Lateral division
Cleavage lengthways
New embryos
Secondary embryo formation
- Most dicots
Early embryo
+Cytokinin
Abundant
Secondary
Embryos
-Cytokinin
+Charcoal
+ABA
Development of GM foods
Flavr-Savr tomato - 1st FDA approval for a food
1995
Monsanto's Roundup Ready soybeans approved for
sale in the United States.
1994
First successful field trial of GM cotton- CROP 1990
GM plants resistant to insects, viruses, and bacteria are
field tested for the first time - USEFUL TRAITS
1985
1st transgenic plant: antibiotic resistant tobacco
1983
Researchers develop the ability to isolate genes 1973
First regeneration of entire plants from an in vitro culture
1950
Current status of GM crops
The worlds most important crops
GM crops
Traits
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.

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20
30
40
50
60
1995 1997 1999 2001
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a
r
e
s
Root Crops
Pulses
0
50
100
150
200
250
W
h
e
a
t
R
i
c
e
C
o
r
n
B
a
r
l
e
y
S
o
r
g
h
u
m
S
o
y
b
e
a
n
s
M
i
l
l
e
t
C
a
n
o
l
a
P
o
t
a
t
o
C
a
s
s
a
v
a
M

h
e
c
t
a
r
e
s

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)
0
5
10
15
20
25
30
35
40
S
o
y
b
e
a
n
C
o
r
n
C
o
t
t
o
n
O
i
l

S
e
e
d
a
r
e
a
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
0
10
20
30
40
50
60
S
o
y
b
e
a
n
C
o
r
n
O
i
l

S
e
e
d

R
a
p
e
%

t
r
a
n
s
g
e
n
i
c
Types of genetic modification
>99% of all
transgenic crops
are either
herbicide or
insect resistant

<1% have other
traits

0
5
10
15
20
25
H
e
r
b
i
c
i
d
e
I
n
s
e
c
t
r
e
s
i
s
t
a
n
c
e
O
t
h
e
r
s
M
i
l
l
i
o
n
s

o
f

h
e
c
t
a
r
e
s
Herbicide resistant crops
Approved Transgenic plants
Soybean
Corn
Cotton
Oil Seed rape
Sugarbeet
Squash
Tomato
Tobacco
Carnations
Potato
Flax
Papaya
Chicory
Rice
Melon
Problems and potential
Future traits and methodology
Environmental stress
resistance
Edible vaccines
Post-harvest quality
Plantibodies
Biodegradeable
plastics
Fungal resistance

Targetting to the
chloroplast
Organ specific
expression
Antibiotic-free
selection
Greater gene stability
More crop species
Problems with
GM foods
Unethical to meddle
with nature
Contamination of
non-GM crops
Lack of public choice
Allergic reactions
Generation of Super-
weeds
Transfer of antibiotic
resistance genes
Re-activation of latent
viruses
Toxins
Loss of diversity
Poisoning / reduction
of beneficial insects
Summary
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

BE304 Plant Cell Culture: Dr. Michael Parkinson, School of Biotechnology

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BE304 Plant Cell Culture: Dr. Michael Parkinson, School of Biotechnology

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07 March 2002 Dr.

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