BE304

BE304 Plant Cell culture
Dr. Michael Parkinson,

School of Biotechnology
07 March 2002
Dr. Michael Parkinson
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
Types of points
Seeds were washed
overnight under a
running tap, rinsed for
10s in 70% ethanol
then sterilised in 20%
Domestos + 0.1% v/v
Tween20 for 10
minutes followed by 3
rinses in sterile
distilled water.
07 March 2002
Why use seeds?

Why wash overnight?
Why rinse in 70% EtOH?
Why sterilise at all?
Why Domestos?
Why 20% for 10 mins?
Why 0.1% v/v Tween20?
Why rinse?
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
07 March 2002
Lecture outline
Micropropagation
Production of products in cell cultures
Plant transformation
For every item, you will be given an
experimental protocol. These will broken down
into a number of sections. There will be a series
of lectures covering the sections followed by a
detailed discussion of another protocol.
We will also try out some of your findings.
07 March 2002
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
Advantages and disadvantages of

micropropagation
Speed - roughly a 10X increase every 2
months (possible to produce 106 plants from
a single starting plant in on year).
Axenic - provided that the original explant
is free of contaminant, the resulting plants
will all be uncontaminated.
Clonal propagation
Cost - 0.15 per explant
07 March 2002
Historical aspects
First commercially used with orchids conventional propagation rate of 1 per year.
Through protocorms, 1,000,000 per year.
Corm
(Swollen stem)
07 March 2002
Chop up
Maturation
Methods of micropropagation
Axillary branching
Adventitious shoot
formation
Somatic
embryogenesis
07 March 2002
>95% of all
micropropagation.
Genetically stable
Simple and
straightforward
Efficient but prone to
genetic instability
Little used. Potentially
phenomenally efficient.
10
Axillary Branching
Stem
Shoot tip
Leaf petiole
Axillary bud in the

axil of the leaf
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11
Choice of explant
Desirable properties of
an explant
Easily sterilisable
Juvenile
Responsive to culture
07 March 2002
Shoot tips
Axillary buds
Seeds
Hypocotyl (from
germinated seed)
Leaves
12
Media
When you make an
explant like an axillary
bud, you remove it
from the sources of
many chemicals and
have to re-supply
these to the explants to
allow them to grow.
Shoot tip - Auxins

and Gibberellins
Leaves sugars, GAs
Roots - water, vitamins

mineral salts and cytokinins
07 March 2002
13
Medium constituents
Inorganic salt formulations

Source of carbohydrate
Vitamins
Water
Plant hormones - auxins, cytokinins, GAs
Solidifying agents
Undefined supplements
07 March 2002
14
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
15
Photoautotrophic culture
Growth without a carbon source. Therefore
need to boost photosynthesis.
High light intensities needed (90150mMole/m2/s) compared to normal (30-50).
Usually increase CO2 (1000ppm) compared to
normal 369.4ppm.
Much reduced level of contamination and
plants are easier to transfer to the greenhouse.
07 March 2002
16
Inorganic salt formulations

Contain a wide range of Macro-elements
(>mg/l) and microelements (<mg/l).
A wide range of media are readily available
as spray-dried powders.
Murashige and Skoog Medium (1965) is the
most popular for shoot cultures.
Gamborgs B5 medium is widely used for
cell suspension cultures (no ammonium).
07 March 2002
17
Vitamins
A wide range of vitamins are available and
may be used.
Generally, the smaller the explant, the more
exacting the vitamin requirement.
A vitamin cocktail is often used (Nicotinic
acid, glycine, Thiamine, pyridoxine).
Inositol usually has to be supplied at much
higher concentration (100mg/l)
07 March 2002
18
Plant hormones (Growth regulators)
Auxins
Cytokinins
Gibberellic acids
Ethylene
Abscisic Acid
Plant Growth Regulator-like compounds
07 March 2002
19
Auxins
Absolutely essential (no mutants known)
Only one compound, Indole-3-acetic acid.
Many synthetic analogues (NAA, IBA,
2,4-D, 2,4,5-T, Pichloram) - cheaper &
more stable
Generally growth stimulatory. Promote
rooting.
Produced in meristems, especially shoot
meristem and transported through the plant
in
special
cells
in
vascular
bundles.
07 March 2002
20
Cytokinins
Absolutely essential (no mutants known)
Single natural compound, Zeatin. Synthetic
analogues Benyzladenine (BA), Kinetin.
Stimulate cell division (with auxins).
Promotes formation of adventitious shoots.
Produced in the root meristem and
transported throughout the plant as the
Zeatin-riboside in the phloem.
07 March 2002
21
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.
07 March 2002
22
Ethylene
Involved in wound responses in plants.
Produced in all cells of the plant and causes
thickening of stems and leaf abscission.
Reduces adventitious shoot formation.
Interacts with an ethylene-binding protein
(EBP) in the cell membrane. Binding of
AgNO3 or norbornadiene to EBP
antagonises ethylene effects.
07 March 2002
23
Abscisic Acid (ABA)

Only one natural compound.
Promotes leaf abscission and seed
dormancy.
Plays a dominant role in closing stomata in
response to water stress.
Has an important role in embryogenesis in
preparing embryos for dessication. Helps
ensure normal embryos.
07 March 2002
24
Plant Growth Regulator-like

substances
Polyamines - have a vital role in embryo
development.
Jasmonic acid - involved in plant wound
responses.
Salicylic acid.
Not universally acclaimed as plant
hormones since they are usually needed at
high concentrations.
07 March 2002
25
Undefined supplements
Sources of hormones, vitamins and
polyamines.
e.g. Coconut water, sweetcorn extracts
Not reproducible
Do work.
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26
Stage I - Sterilisation
Bacteria and fungi will
overgrow the explant
on the medium unless
they are removed.
Pre-treatments to clean
up the explant
Detergents
Sterilants and
Antibiotics
07 March 2002
Pre-treatments
Transfer plants to a
greenhouse to reduce
endemic contaminants
Force outgrowth of
axillary buds.
Washing removes
endemic surface
contaminants.
27
Uses of detergents
Air bubbles on the
surface of the explant
can protect bacteria
and fungi from the
liquid sterilant.
Mixing should
therefore be done in
such a way as to
reduce air bubble
formation
07 March 2002
Air bubble
around epidermal hair
Leaf surface
Detergents (e.g.
Triton, Tween20)
reduce the surface
tension of the waxy
cuticle on the leaf
surface and increase
wetting.
28
Sterilants
There are 3 principal
There is always a
ways to kill off surface
trade-off between
contaminants.
killing the surface
contaminants and
oxidant action
killing the explant.
Active halogen
Heavy metal poisoning As far as possible, cut
*Powerful chemicals
surfaces should be
such as conc. sulphuric
protected.
acid may be used on
seeds.
07 March 2002
29
Sterilants used
NaOCl
CaOCl
H 2O 2
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.
07 March 2002
30
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.
07 March 2002
31
Stages III and IV Rooting and

transfer to the greenhouse
Plants must be rooted by using media
containing auxin or by dipping explant
bases in auxin solutions.
Progressively, the plants must be hardened
by increasing the light intensity, and
reducing sugar, inorganic salts and
humidity.
Medium must be removed prior to
transplantation to prevent contamination.
07 March 2002
32
Micropropagation by
adventitious shoot formation
Adventitious shoot formation is the de-novo
development of shoots from cell clusters in
the absence of pre-existing meristems.
In some species (e.g. Saintpaulia), many
shoots can be induced (3000 from one leaf).
In other species (e.g. coffee), it may be
necessary to induce an unorganised mass
proliferation of cells (callus) prior to
adventitious shoot formation.
07 March 2002
33
Control of organogenesis
Cytokinin
Leaf strip
Adventitious
Shoot
Root
Callus
07 March 2002
Auxin
34
Plant Hygiene
Pathogens affect yield (average 30%
reduction)
There are strict plant sanitation
requirements for import of plants.
Viruses and bacteria will be multiplied
along with the explants and need to be
removed prior to plant multiplication.
07 March 2002
35
Ways to eliminate viruses

1 Heat treatment. Plants grow faster than
viruses at high temperatures.
2 Meristemming. Viruses are transported
from cell to cell through plasmodesmata
and through the vascular tissue. Apical
meristem often free of viruses. Trade off
between infection and survival.
3. Not all cells in the plant are infected
Adventitious shoots formed from single
cells can give virus-free shoots.
07 March 2002
36
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
07 March 2002
37
PRODUCTION OF PRODUCTS
Advantages and disadvantages

Cost of production
Plant cell culture systems
Ways to increase product formation
Commercial production
07 March 2002
38
Advantages and disadvantages

Advantages
Can manipulate
environment
Can feed precursors
Possible to select in
culture
Possible to get all cells
in a culture producing.
07 March 2002
Can continuously
extract.
Can retain biomass
Disadvantages
High cost
Contamination
Low intrinsic
production
39
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
40
Plant cell culture systems

Organised
Unorganised
Shoot cultures.
Callus
Hairy root cultures
Cell suspension
culture
Embryo fermentations.
07 March 2002
41
Shoot cultures
Under conditions of high cytokinin, a
culture producing a mass of shoots may be
produced by adventitious shoot formation.
For light-associated products, may be much
more high yielding.
Sensitive to shear
Illumination a problem for scale up
07 March 2002
42
Hairy root cultures

Hairy roots are produced by infecting
sterile plants with a natural genetic
engineer, Agrobacterium rhizogenes.
Genes for auxin synthesis and sensitivity
are engineered into plant cells leading to
gravity-insensitive mass root production.
Very useful for products produced in roots.
Aggregration and shear sensitivity are a
major problem for
scale-up
07 March 2002
43
Embryo Fermentations
Somatic Embryos may be produced
profusely from leaves or zygotic embryos.
For micropropagation, potentially
phenomenally productive.
Shear sensitivity is a problem.
Maturation in liquid is a problem.
07 March 2002
44
Shikonin production in culture

Shikonin production in the intact plant
Introduction into culture
Optimisation of production through medium
manipulations
Fermentation
07 March 2002
45
Callus
Equimolar amounts of auxin and cytokinin
stimulate cell division. Leads to a mass
proliferation of an unorganised mass of
cells called a callus.
Requirement for support ensures that scaleup is limited (Ginseng saponins successfully
produced in this way).
07 March 2002
46
Cell suspension culture

When callus pieces are agitated in a liquid
medium, they tend to break up.
Suspensions are much easier to bulk up than
callus since there is no manual transfer or
solid support.
Large scale (50,000l) commercial
fermentations for Shikonin and Berberine.
07 March 2002
47
Introduction of callus into

suspension
Friable callus goes
easily into suspension.
2,4-D
Low cytokinin
semi-solid medium
enzymic digestion with
pectinase
blending
07 March 2002
Removal of large cell

aggregates by sieving.
Plating of single cells
and small cell
aggregates - only
viable cells will grow
and can be reintroduced into
suspension.
48
Introduction into suspension

Sieve out lumps
1
2
Initial high
density
+
Pick off
growing
high
producers
Subculture
and sieving
Plate out
07 March 2002
49
Growth kinetics
Plant Cell Suspension typical Growth
curve
16
14
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)
12
10
8
6
4
2
0
2
1
0
2 4
6 8 10 12 14 16 18 20 22
tim e (d)
07 March 2002
50
Characteristics of plant cells

Large (10-100mM
long)
Tend to occur in
aggregates
Shear-sensitive
Slow growing
Easily contaminated
Low oxygen demand
(kla of 5-20)
07 March 2002
Will not tolerate

anaerobic conditions
Can grow to high cell
densities (>300g/l
fresh weight).
Can form very viscous
solutions
51
Shear and plant cells

Oxygen demand
proportional to cell
density.
Shear rate proportional
to viscosity
shear rate proportional
to **power of
viscosity
07 March 2002
52
Special reactors for plant cell

suspension cultures
Modified stirred tank

Air-lift
Air loop
Bubble column
Rotating drum reactor
07 March 2002
53
Modified Stirred Tank
Standard Rushton turbine

07 March 2002
Wing-Vane impeller
54
Airlift systems
Poor mixing
Bubble column
07 March 2002
Airlift (draught
tube)Dr. Michael Parkinson
Airloop (External
Downtube)
55
Rotating Drum reactor

Like a washing
machine
Low shear
Easy to scale-up
07 March 2002
56
Ways to increase product

formation
Select
Start off with a
producing part
Modify media for
growth and product
formation.
Feed precursors or
feed intermediates
(bioconversion)
07 March 2002
Produce plant-like
conditions
(immobilisation)
57
Selection
Select at the level of the intact plant
Select in culture
single cell is selection unit
possible to plate up to 1,000,000 cells on a
Petri-dish.
Progressive selection over a number of phases
07 March 2002
58
Selection Strategies
Positive
Negative
Visual
Analytical Screening
07 March 2002
59
Positive selection
Add into medium a toxic compound e.g.
hydroxy proline, kanamycin
Only those cells able to grow in the
presence of the selective agent give colonies
Plate out and pick off growing colonies.
Possible to select one colony from millions
of plated cells in a days work.
Need a strong selection pressure - get
escapes
07 March
2002
60
Negative selection
Add in an agent that kills dividing cells e.g.
chlorate / BUdR.
Plate out leave for a suitable time, wash out
agent then put on growth medium.
All cells growing on selective agent will die
leaving only non-growing cells to now
grow.
Useful for selecting auxotrophs.
07 March 2002
61
Visual selection
Only useful for coloured or fluorescent
compounds e.g. shikonin/Berberine/ some
alkaloids.
Plate out at about 50,000 cells per plate.
Pick off coloured / fluorescent compounds
Possible to screen about 1,000,000 cells in a
days work.
07 March 2002
62
Analytical Screening
Cut each piece of callus in 2.
One half subcultured.
Other half extracted and amount of
compound determined analytically (HPLC/
GCMS/ ELISA).
Extraction V. laborious and limits number
of callus pieces that can be assayed to 200/d
(Zenk by Radioimmunoassay).
07 March 2002
63
Media manipulations
07 March 2002
64
Immobilisation
07 March 2002
65
Plant Genetic
Transformation
Dr Michael Parkinson
07 March 2002
66
Overview
Introduction
Plant genetic transformation
Current status of GM crops
Future trends & Problems
07 March 2002
67
Introduction
Potential of Plant Biotechnology

Uses of introduced novel genes
Traits that plant breeders would like in
plants
07 March 2002
68
Potential of Plant Biotechnology
Micropropagation
Somatic hybrids / Cybrids
Haploid plants
Fermentations
Introduction of novel genes into plants
07 March 2002
69
Uses of introduced novel genes

Research into gene functions
Molecular farming
Crop improvement in a single step
07 March 2002
70
Molecular farming
Polyhydroxy butyrate
(PHB) is a renewable
source of plastics.
Monoclonal
antibodies*
Human Serum Albumin
Interleukins.
Vaccines (virus coat
protein genes)
07 March 2002
Neurotransmitters e.g.
50mg/kg Leu-enkaphalin
produced in Oil seed
rape.
Modification of oils to
improve Biodiesel.
Prodigene now
producing enzymes, oral
vaccines & antibodies
from Maize seeds.
71
Overview of molecular farming

Gene isolation - easy
Vector design
organ specific
promoters
High level expression
Containment
Transformation of
maize by Biolistics
07 March 2002
Regeneration from a crop

monocot difficult
Growth, seed harvesting

and downstream
processing requires
strong agricultural and
fermentation expertise.
www.prodigene.com
72
Traits that plant breeders would

like in plants
High primary
productivity
High crop yield
High nutritional
quality
Adaptation to intercropping
Nitrogen Fixation
07 March 2002
Drought resistance
Pest resistance
Adaptation to
mechanised farming
Insensitivity to photoperiod
Elimination of toxic
compounds
73
Plant genetic transformation

Overview of
requirements for plant
genetic transformation
Development of GM
foods
Genes for crops
Benefits of GM crops,
especially in
developing countries
07 March 2002
How to get genes into

cells to give
transformed cells
How to get a plant
back from a single
transformed cell
74
Overview of requirements for

plant genetic transformation
Trait that is encoded by a single gene
A means of driving expression of the gene in
plant cells (Promoters and terminators)
Means of putting the gene into a cell (Vector)
A means of selecting for transformants
Means of getting a whole plant back from the
single transformed cell (Regeneration)
07 March 2002
75
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
76
sale in the United
States.
1995
Useful single gene traits that

have been introduced into plants
Herbicide resistance*
Insect resistance*
Virus resistance
Seed protection
Fungal resistance
Delayed ripening
07 March 2002
Cold / Frost resistance

Drought resistance
High starch potatoes
Oil production
Plastics
Digestibility proteins
Antibodies
77
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
07 March 2002
78
Improved post-harvest properties

Up to 50% of
harvested food is lost
post-harvest in Africa.
Any poisonous protein
can be detoxified by
heating and rendered
safe e.g. lectins;
inhibitors.
Ripening control
07 March 2002
Wheat germ agglutinin

Cowpea trypsin
inhibitor
Flavrsavr tomatoes
contain antisense to
polygalacturonase
(softens tomatoes by
dissolving the cell
wall).
79
Other useful traits

Improved Agronomic
properties
Improved plant
breeding
Improved nutritional
properties
07 March 2002
High starch potatoes

Pollen-specific
promoter plus RNAse
Golden rice (gene
from Chrysanthemum
giving - converted to
vitamin A.
80
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
07 March 2002
10% losses
post-harvest
= 0.5 tonne/ha
4.5 tonnes/ha to eat

81
Cassava is a
very important
crop in Africa
Viral infection
of the crop is
increasing
Possible to
engineer
Cassava
Mosaic virus
resistance by
using coat
protein genes
07 March 2002
82
Perceived benefits of GM crops
07 March 2002
83
Approved Traits
Glufosinater herbicide
Sethoxydimr herbicide
Bromoxynilr herbicide
Glyphosater herbicide
Sulfonylurear
herbicide
07 March 2002
Male-sterility
Modified fatty acid
Flower colour
Flower life
Delayed fruit ripening
Virus resistance
Bt
84
Plasmid construction
Useful gene construct
Visible marker
Selectable marker*
07 March 2002
85
Gene construction
DNA
Plant specific
promoter
Plant RBS
Useful gene
Signal peptides*
PolyA-tail
Nucleus
transcription
mRNA
Cytoplasm
translation
Polypeptide chain
Post-translational
modification
07 March 2002
86
2 Types of delivery systems

Naked DNA
Cell wall is the
primary resistance to
DNA uptake
Biolistics
SiC fibres
Protoplasts
Electroporation
Pollen
07 March 2002
Vectored
Agrobacterium
Viruses
87
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
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
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
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
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).
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
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.
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
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
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
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
New embryos
99
Secondary embryo formation

- Most dicots
Abundant
Secondary
Embryos
+Cytokinin
+Charcoal
+ABA
-Cytokinin
Early embryo
07 March 2002
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
101
sale in the United
States.
1995
Current status of GM crops

The worlds most important crops
GM crops
Traits
07 March 2002
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
1997
1999
2001
103
07 March 2002
104
Root Crops
07 March 2002
105
Pulses
07 March 2002
106

M hectares
250
200
150
100
50
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
07 March 2002
107
07 March 2002
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
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
109
07 March 2002
20
15
10
5
Others
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
111
Approved Transgenic plants
Soybean
Corn
Cotton
Oil Seed rape
Sugarbeet
Squash
Tomato
Tobacco
07 March 2002
Carnations
Potato
Flax
Papaya
Chicory
Rice
Melon
112
Problems and potential
07 March 2002
113
Future traits and methodology

Environmental stress
resistance
Edible vaccines
Post-harvest quality
Plantibodies
Biodegradeable
plastics
Fungal resistance
07 March 2002
Targetting to the
chloroplast
Organ specific
expression
Antibiotic-free
selection
Greater gene stability
More crop species
114
Problems with
GM foods
Unethical to meddle
with nature
Contamination of
non-GM crops
Lack of public choice
Allergic reactions
Generation of Superweeds
07 March 2002
Transfer of antibiotic
resistance genes
Re-activation of latent
viruses
Toxins
Loss of diversity
Poisoning / reduction
of beneficial insects
115
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
116

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BE304 Plant Cell culture

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