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.

Characteristics of plant cells Large (  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

 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 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 Small portion is withdrawn and cell density will be checked  9-15×103 cells / cm3 for sycamore.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 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 lumps 12 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)

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 columnAirlift (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: 4 o C 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 F 1 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: 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

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 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