9Commercialization Shikonin Ginseng Dye Medicine – to treat fungal, bacterial or viral infectionsProduced via continuous-flow reactorcells retained by membrane / periodic harvesting4000 L systemGinsengHealth foodL systemSanguinarineProduced from Papaver somniferumDental care – treatment for gum disease (gingivitis)
10Plant CellEukaryotesProkaryotes do not have nucleus, no internal organsAnimal cell – do not have chloroplasts, may or may not have cilia, no cell wallPlant cell – have LARGE vacuoles, cell wall, chloroplast, lack of lysosomes, centrioles, pseudopods and flagella and cillia.
121. The Quest for Commercial Production of Biochemicals from Plant Cell Culture. 1.1 WHY PLANT? WHY PLANT CELL CULTURE?AphrodisiacAnxietyMalariaCancerFeverParasitic infectionTestosterone increaseUlcersMale infertilityAnalgesicHypertensionCUTEurycoma longifoliaEXTINCT!!
13Industrial Chemical Sectors to Which Plant Products Contribute 1) Medicinals- 25% of prescribed drugs include compoundsfrom plants.The Ten Most Prescribed Medicinals from Plant SourcesMedicinal agent Activity Plant SourceSteroids from diosgenin Anti-fertility agents Dioscorea deltoideaCodeine Analgesic Papaver somniferumAtropine Anticholinergic Atropa belladonna L.Reserpine Antihypertensive Rauwolfia serpentina L.Hycoscyamine Anticholinergic Hyoscyamus niger L.Digoxin Cardiatonic Digitalis lanata L.Scopolamine Anticholinergic Datura metel L.Digitoxin Cardiovascular Digitalis purpurea L.Pilocarpine Cholinergic Pilocarpus jabonandiQuinidine Antimalarial Chinchona ledgerianafrom M.W.Fowler (1982)p.3 In PlantBiotech.(Mantel &Smith, ed.)
142) Agrochemicalseg: Insect repellence from Cymbopogun nardus3) Fine Chemicals- including perfumes, flavours, aromas, colorants and food materials, for example quinine alkaloid (bittering agent), chalcone (non-nutritive sweetener), Jasmine (perfume)
15Preparation and composition of nutrient media Factors that determine the in-vitro growth and development of plant are:NutrientsPhysical factorsSome organic substances
16NutrientsWaterNecessary constituent of all living plant cell and tissueAs biochemical medium and solventA chemical reactant or product in many metabolic processes, including photosynthesisResponsible for cell turgorResponsible for the function of the stomataActs as a coolant and temperature buffer
17Nutrients cont…… Macro-elements Used in large amounts Carbon, hydrogen, oxygen, nitrogen, phosphorus, potassiumEg; deficiency of nitrogen cause stunted growth, yellow lower leaves, spindly stalk and pale green colorDeficiency of phosphorus cause purplish color in lower leaves and stems, dead spots on leaves and fruitsSecondary nutrient – calcium ,magnesium and sulfur
18Nutrients cont…… Micro-elements Sugar Required in small quantities Boron, chlorine, copper, iron, manganese, molybdenum, zincSugarSince plants or parts of plants in tissue culture condition are not completely autotrophic carbon source is needed
19Physical growth factor LightPlant contain pigment chlorophyll that traps light energy and change it to chemical energy called ATP (adenosine triphosphate),a compound used by cells for energy storage.This chemical is made of the nucleotide adenine bonded to a ribose sugar, and that is bonded to three phosphate groups.The dark reaction converts CO2 to sugar and ATPLight stimulated secondary metabolite
21Temperature Vary in their ability to tolerate Eg: Snapdragon grow best at 12oC but Nicotiana sp at 22oCEffects many essential plant growth processes (biological reaction in plant)17-25oC is normally used for induction of callusCallus Nicotiana tabacum
22pH and oxygen Normally adjusted to between 5 to 6 before autoclaving pH control equipment if use bioreactorWhy need oxygen?Remember that they are not Autotrophic
23Organic substances Plant growth regulators (PGRs) Hormones are produced naturally by plants, while plant growth regulators are applied to plants by humans.PGR may be synthetic compounds (e.g., IBA and Cycocel) that mimic naturally occurring plant hormones, or they may be natural hormones that were extracted from plant tissue (e.g., IAA).(PGRs are chemicals that are designed to affect plant growth and/or development.They are applied for specific purposes to affect specific plant responses.
24Plant growth regulators cont…. Eg: auxin, gibberellin (GA), cytokinin, ethylene, and abscisic acid (ABA)Auxin - is the active ingredient in most rooting compounds in which cuttings are dipped during vegetative propagationEx: Auxin (e.g IBA): mg/lCytokinin (e.g BA): mg/lCytokinins - stimulate cell division and often are included in the sterile media used for growing plants from tissue culture.If a medium's mix of growth-regulating compounds is high in cytokinins and low in auxin, the tissue culture explant (small plant part) will produce numerous shoots.
25Plant growth regulators cont…. Ethylene - found only in the gaseous formIt induces ripening, causes leaves to droop (epinasty) and drop (abscission), and promotes senescence.Plants often increase ethylene production in response to stress, and ethylene often is found in high concentrations within cells at the end of a plant's lifeThe increased ethylene in leaf tissue in the fall is part of the reason leaves fall off trees. Ethylene also is used to ripen fruit (e.g., green bananas).
26Plant growth inhibitor Abscisic acid (ABA) - induces dormancy and prevents seeds from germinating; causes abscission of leaves, fruits, and flowers; and causes stomata to closeEg; High concentrations of ABA in guard cells during periods of drought stress probably play a role in stomatal closure.
27Culture Conditions Affecting Growth and Product Accumulation in Plant Cell Cultures The internal culture conditionsmedia componentsprecursors and elicitorsAerationculture pHThe external culture conditionsLightTemperatureCulture agitation
28Type of cultures Suspension culture Root culture Shoot culture Seed cultureEg: orchid seed (because during in vivo seeds do not germinate well)Sugar is extremely important as energy sourceEmbryo cultureIs the sterile isolation and growth of an immature or mature embryo in vitro, with the goal of obtaining a viable plant.
29Type of culture cont… Callus culture Culture of non-organized tumor tissue, which arise on wounds of differentiated tissues and organsTakes place under the influence of exogenously supplied growth regulators present in the nutrient medium. Auxin alone or cytokinin alone or both auxin and cytokinin
30Type of culture cont… Organ culture Cell culture Protoplast culture It is an isolated organ grown in vitroCell cultureThe growing of individual cells that have been obtained from an explant tissue or callus or it is refer to as cell suspension cultureProtoplast cultureCulture of cells without cell wall
31Cell Suspension Culture A cell suspension culture consists of cell aggregates dispersed and growing in moving liquid media.Initiated by transferring pieces of undifferentiated and friable calli to a liquid mediumPlatform (orbital) shakers are widely used for the initiation and serial propagation of plant cell suspension culture. With variable speed control ( rpm).Agitation serves 2 purposes: 1. it exerts a mild pressure on cell aggregates, breaking them in to smaller clumps and single cell. 2. maintains uniform distribution of cell and cell clumps in the medium.Movement of the medium provides good gaseous exchange between the culture medium and air.Volume of the culture should be 20 ml for 100ml flask or 70 ml for 250 ml flask.
32Types of suspension culture Batch cultureA cell suspension culture grown in a fixed volume of nutrient culture medium.Cell suspension increases in biomass by cell division and cell growth until a factor in the culture environment (nutrient or oxygen availability) becomes limiting and the growth ceases.
33Types of suspension culture cont… The cells exhibit the following five phases of a growth cycle1. Lag phase, where cells prepare to divide2. Exponential phase, where the rate of cell division is highest3. Linear phase, where cell division slows but the rate of cells expansion increases4. Deceleration phase, where the rates of cell division and elongation decreases5. Stationary phase, where the number and size of cells remain constantThe cell generation time (doubling time) in suspension culture varies from 24 to 48 h in well established cell cultures.Doubling time (td) is the time required for the concentration of biomass of a population of cell to double.
34Types of suspension culture cont… Continuous cultureA culture is continuously supplied with nutrient by the inflow of fresh medium but the culture volume is normally constant.2 types:Open continuous cultureThe inflow of fresh medium is balanced by outflow of corresponding volumes of culture including harvest of cellsThe rate of inflow of medium and culture harvest are adjusted so that the cultures are maintained indefinitely at a steady state, the rate of cells washout equals the rate of formation of new cells in the system.A situation of balanced growth is achieved; i.e. majority of cells in the culture are in a similar metabolic stateThe growth rate and cell density are held constant by a fixed rate of input of growth limiting nutrients and removal of cells and spend medium.
35Continuous culture cont… 1. Chemostates – growth rate and cell density are held constant by a fixed rate of input of a growth limiting nutrient medium (nitrogen, phosphorus or glucose). In such a medium, all the constituents other than growth limiting nutrients are present at concentrations higher than that required to maintain the desired rate of cell growth. The growth limiting substances is so adjusted that its increase or decrease is reflected by a corresponding increase or decrease in the growth rate of cells.2. Turbidostates- fresh medium flows in response to increase in turbidity so as to maintain the culture at a fixed optical density of suspension. A pre-selected biomass density is maintained by the washout of cells.
36Closed continuous culture Cells are retained and inflow of fresh medium is balanced by outflow of corresponding volumes of spent medium only.The cells from the out-flowing medium are separated mechanically and added back to the culture. So cell biomass continues to increase as the growth proceedsIt has potential value in studies on cytodifferentiation, where it may be important to grow cells under a particular regulated environment and then maintain them for a considerable period in a non-dividing but viable state.It can also be used in cases where secondary products produced by cell suspension cultures have been shown to be released in significant amounts into their culture medium. In such cases, a maintenance culture in a closed continuous system should enable the chemical product to be continuously harvested from a fixed culture biomass.
37Semi-continuous culture The inflow of fresh medium is manually controlled at infrequent intervals by a “drain and refill” process, such that the volume of culture removed is always replaced by an equivalent volume of fresh medium.Although the number of cell increases exponentially, the cell density is maintained within fixed limits by the periodic replacement of harvested culture by fresh medium
38Cell suspension culture- DisadvantagesProductivity decrease – genetic alterationSlow growth, have to maintain for several weekShear effect size of plant aggregate than effect performanceSeparate media for growth and secondary metaboliteAdvantagesBetter controlAble to reproduce condition in large scale
39Important factor for bioreactor Gas-liquid mass transferPlant cell have lower respiration rate-oxygen transfer requirements are lessPlant cell bioreactor typically operated at KLa (oxygen transfer coefficient) values of 10 – 30 hr-1High KLa results in poor plant cell growthIncrease shearCO2 stripping from the liquidBioreactor is equipped with a dissolved oxygen probe and KLa is characteristic of the bioreactor system
40Important factor for bioreactor cont… ShearPlant cells are shear sensitiveShear refer to forces exerted on the surface of a body in a directional parallel to the surfaceTurbulent Eddy TheoryAgitation system –impart energy into the liquid-transfer from larger to smaller eddies-so the power input can be related to the size of the smallest eddies-the greater the power added the smaller is the size of the smallest eddies-when the size of aggregate is small relative to the size of the smallest eddy –
41Shear cont….then it is carried around with the fluid in the eddy and is probably undamaged-however, when the size of aggregate is larger or same size of the smallest eddy-then it can be caught between eddies such that the dissipation of energy occurs at the surface of the cell.
42Shear cont…. Important Implication of theory Shear forces may act on aggregates such that the aggregates size is reduced at increased turbulentPlant cell enlarge as they age, they will also become more shear sensitive (eg secondary metabolite produce during stationary phase, reduce productivity)Other report suggested that cell damage may caused by cell-cell and cell-impeller collisions,Also by gas sparging (even in the absent of mechanical agitation)
43Important factor for bioreactor cont… Mixing – refer to the convective transport of matter (eg: the transfer of solute associated with bulk fluid motion)ProblemLarge size of plant cell or cell aggregate-lead to settling at the bottom of bioreactor (then settle into dead zone or unmixed region of the bioreactor)-dead zone can depleted of nutrient (eg dissolved oxygen)Attachment of cells onto surface above the level of the liquid- cell not bathed in liquid media-deprived of nutrient
44Type of Bioreactor Mechanically Agitated Bioreactor Employ impellers and mechanical energy for gas-liquid mass transfer and mixingEg: flat-blade turbine impeller- provide radial fluid mixing-but plant cell can not tolerate high-shear condition- so marine propellor has been used (low-shear mixing)-axial fluid mixingDisadvantage- Because of shear sensitivity-agitation speeds appropriate for plant cell cultivation are insufficient to break the incoming gas stream into small bubbles ----Obtaining sufficient oxygen transfer require that the incorporating gas stream be dispersed as fine bubbles by using appropriate gas distributor.
45Type of Bioreactor cont… Pneumatically Agitated BioreactorMotion of the rising gas stream should capable of providing the energy for fluid mixingDifferent byNo impellerTall and thin (high-to-diameter ratio is 10)Advantage- no moving parts
46Type of Bioreactor cont… Bubble column-eg (picture from text books)AirliftProvide for liquid circulationPromote better top-to-bottom mixingBaffle use to separate the two sectionsAdvantage in suspending cells and clumpsDisadvantage – little oxygen transfer occurs in the downcomer section
47Type of Bioreactor cont… Comparison of BioreactorMechanically agitatedBubble columnAirliftOxygen transfer++++++Low shearMixing
48Operating modeRefer to how the nutrient and product streams are supplied or removed with respect to the timeDetermination of suitable operating mode needIdentification of timing of product synthesis to growthGrowth-associated productionNon-growth-associated productionWhere does the product have been secretedExtracellularIntracellular
49Operating mode cont… Type of operating mode Batch-when all the nutrients the culture requires are supplied initiallyFed BatchRepeated Fed BatchTwo-stage Batch operationContinuous Cultivation – chemostatContinuous Cultivation-perfusion Operating mode