Cell suspension culture

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.

Aggregates of cells suspended in a liquid medium.

Transfer of callus to liquid media. Then they are agitated.

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.

FRIABILITY

Characteristics of plant cells• Large (10-100M

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

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.

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.

Small portion is withdrawn and cell density will be checked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– 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 lumps1 2Initial high

density

Subcultureand sieving

Growth kinetics1. Initial lag dependent

on dilution2. Exponential phase

(dt 1-30 d)3. Linear/deceleration

phase (declining nutrients)

4. Stationary (nutrients exhausted)

12

3 4

Reactors for plant 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)

Synchronization• Cold treatment: 4oC • Starvation: deprivation of an essential growth

compound, e.g. N →accumulation in G1• Use of DNA synthesis inhibitors: thymidine, 5-

fluorodeoxyuridine, 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

• 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 PreservationExtension 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: • Undifferentiated plant cells• Embryonic suspension• Callus• Pollen• Seeds• Somatic embryos• Shoot apices

Preparation• 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

CryopreservationRequirements:

•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