Transcript Cell suspension

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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Aggregates of cells suspended in a liquid
medium.
Transfer of callus to liquid media.
Then they are agitated.
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Separation of cells following cell division.
Good suspension :
Culture consisting of a high percentage of
single cells and small clusters of cells.
Different requirements for different cases.
The choice of Suitable conditions is largely
determined by trial and error.
Characteristics of plant cells
• Large (10-100M
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
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Large amount of callus is required.
2-3 gm for 100 cm3
Three phases will be observed
Lag phase
Cell division
Stationary phase
The cells should be subcultured early during
the stationary phase.
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Different time periods for subculturing
Some plants max. cell density is reached
within about 18-25 days.
In some plants as short as 6-9 days
Nylon net or stainless steel filter is used to
remove larger cell aggregates.
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Small portion is withdrawn and cell density
will be checked
9-15×103 cells / cm3 for sycamore.
The best speed for 100-120 rpm
Liquid should be filled 20% of the size of the
flask for adequate aeration.
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
• 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
Introduction into suspension
Sieve out lumps
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Initial high
density
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Subculture
and sieving
Plate out
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)
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Reactors for plant suspension cultures
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Modified stirred tank
Air-lift
Air loop
Bubble column
Rotating drum reactor
Modified Stirred Tank
Standard Rushton turbine
Wing-Vane impeller
Airlift systems
Poor mixing
Bubble column
Airlift (draught tube)
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)
Synchronization
• Cold treatment: 4oC
• Starvation: deprivation of an essential growth
compound, e.g. N →accumulation in G1
• Use of DNA synthesis inhibitors: thymidine, 5fluorodeoxyuridine, hydroxyurea
• Colchicine method: arresting the cells in
metaphase stage, measured in terms of mitotic
index (% cells in the mitotic bphase)
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
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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 colored or fluorescent
compounds e.g. shikonin, berberine, some
alkaloids
• Plate out at about 50,000 cells per plate
• Pick off colored / fluorescent-expressing
compounds (cell compounds?)
• Possible to screen about 1,000,000 cells
in a days work.
Analytical Screening
• Cut each piece of callus in half
• One half subcultured
• Other half extracted and amount of
compound determined analytically (HPLC/
GCMS/ ELISA)
Embryo Culture
Embryo Culture Uses
• Rescue F1 hybrids from wide crosses
• Overcome seed dormancy, usually with
addition of hormone (GA) to medium
• To overcome immaturity in seeds
– To speed generations in a breeding program
– To rescue a cross or self (valuable genotype)
Haploid Plant Production
• Embryo rescue of interspecific crosses
– Bulbosum method
• Anther culture/Microspore culture
– Culturing of anthers or pollen grains
(microspores)
– Derive a mature plant from a single
microspore
• Ovule culture
– Culturing of unfertilized ovules (macrospores)
Anther/Microspore Culture Factors
• Genotype
• Optimum growth of mother plant
• Correct stage of pollen development
– Need to be able to switch pollen development from
gametogenesis to embryogenesis
• Pretreatment of anthers
– Cold and heat have been effective
• Culture medium
– Additives
– Agar vs. ‘Floating’
Ovule Culture for Haploid Production
• Essentially the same as embryo culture –
difference is an unfertilized ovule instead
of a fertilized embryo
• Effective for crops that do not yet have an
efficient microspore culture system – e.g.:
melon, onion
Haploids
• Weak, sterile plant
• Usually want to double the chromosomes,
creating a dihaploidbplant with normal
growth & fertility
– Chromosomes can be doubled by
– Colchicine treatment
– Spontaneous doubling
Germplasm Preservation
Extension of micropropagation techniques:
Two methods:
• 1.Slow growth techniques
– ↓Temp., ↓Light, media supplements (osmotic
inhibitors, growth retardants), tissue dehydration, etc
– Medium-term storage (1 to 4 years)
• 2.Cryopreservation
– Ultra low temperatures. Stops cell division &
metabolic processes
– Very long-term (indefinite?)
Most economical germplasm storage –
Why not seeds?
• Some crops do not produce viable seeds
• Some seeds remain viable for a limited duration
only and are recalcitrant to storage
• Seeds of certain species deteriorate rapidly due
to seed borne pathogen
• Some seeds are very heterozygous not suitable
for maintaining true to type genotypes
• Effective approach to circumvent the above
problems may be application of cryopreservation
technology
Cryogenic explants:
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Undifferentiated plant cells
Embryonic suspension
Callus
Pollen
Seeds
Somatic embryos
Shoot apices
Preparation
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Pretreatment
Cryopreservation method
Thawing method
Recovery method is critical
Cryobiology
• Is the study of the effects of extremely low
temperatures on biological systems,
such as cells or organisms.
• Cryopreservation
– an applied aspect of cryobiology
– has resulted in methods that permit low
temperature maintenance of a diversity of
cells
Cryopreservation
Requirements:
•Preculturing–Usually a rapid growth rate to create cells
with small vacuoles and low water content
•Cryoprotection–Glycerol, DMSO, PEG, etc…, to protect
against ice damage and alter the form of ice crystals
•Freezing–The most critical phase; one of two methods:
•Slow freezing allows for cytoplasmic dehydration
•Quick freezing results in fast intercellular freezing with
little dehydration
Cryopreservation Requirements
• Storage–Usually in liquid nitrogen (-96 C) to
avoid changes in ice crystals that occur above 100 C
• Thawing–Usually rapid thawing to avoid damage
from ice crystal growth
• Recovery –
– Thawed cells must be washed of cryoprotectants and
nursed back to normal growth–
– Callus production avoided to maintain genetic stability