Definition: 2 met. - - / 3 1) (OH+ring bd B. Single B) 2) ( C,N , steroid terpine Sex H. plant sterol, beta-sitosterol deri. steroid padinax steroidal glycoside) 3) ( N . ring chain N ) 2 met. -shikonin,Alkannin: Shikon organic solvent Indigo indole alkaloid vary -Hyoscyamine,scopolamine atropine 2 met. Tropane alkaloid alkaloid , Hyoscyamus niger Hyos. deri. Hyos.HBr (Buscopan) Atropine -codeine Alkaloid Morphenan alkaloid Morphine,codeine,heroine opium= papaver somniferum morphenan MethylG. codeine methylG. OH G. morphine . -Sanguinarine alkaloid -Berberine alkaloid . . toxicity -L-DOPA dopamine Parkinson precursor L DOPA -Vinca alkaloid dimeric alkaloid Vincristine, vinblastine (Madagascar periwinkle) Leukemia -Camptothecin -Homoharringtonine alkaloid -Digitoxin cardiac glycoside 3 primer 3, steroid part, unsat.lactone ring -Valepotriate terpenoid -Podophyllotoxin podophyllum -Etoposide ovarian cancer -Taxol Taxus terpenoid -Rosmarinic acid anti-ox terpenoid curcumin -Arbutin Whittening enz.tyrosinase phenolic -Carthamin

-Crocin tetraterpenoid Bixin , Bixin Crocin (Crosud sativus) -Hernandulcin Paper1: indole alkaloid Paper2:

1): ginseng 1964 2)suspension culture condition ginsenoside Medium susp.cul Sucrose(Carbon source) , Nitrate , , Growth H. ginsenoside 2,4-D, ,temp:24-28 , ginsenoside K, Cu 3)Bioreactor culture 2 stir Air-lift 2 met. Bioreactor stir flat /Angle-disc /anchor 4)Hairy root culture ginsenoside rg1 2 met. HPLC peak 5)Shoot culture ginseng embryo cul. petiole petriole Bioreactor cell ., stress cell subcul. - -1 - - -(species) - = cell chlorophyll Anthocyanin . = UV , Fluor. fluor Taxol - immune Taxol cell 2 met. screen cell Ab taxol Ab ginsenoside screen cell Ab cell . Ab ginsenoside (G) G Ab G Ab cell cell ELISA technique, Immuno assay Ab Ab Microplate reader screen * . TLC Ab run Ag TLC -= vary vary Carbon source sucrose salvia officinalis rosmarinic acid auxin 2,4-D dedifferentiation secondary metabolites

L-DOPA Mucuna pruriens, Ubiquinone 10 N. tabacum diosgenin Dioscorea deltoidea 2,4-D Taxus cuspidata gibberellic acid

2 met. media cell cell media gel bead gel Medium medium medium cell cell susp. 2.Entrapment in net or foam=biomass support particle constructed, block of polyurethane foam, reticulate polyurethane at differences pore sizes= susp.cul./Biorea. foam cell medium POP! 3.Immobilization in hollow-fiber membrane=Tubular fiber from cellulose acetate silicone polycarbonate organized in parallel bundle= cell cell wall mb cell mb Bioreactor configurations for use with immobilized cell paper Sanguinarine -Fluidized-bed bioreactor -Fixed bed media inoculation - morphinan alkaloid Papaver somniferum morphine codeine sanguinarine -Botrytis sp. sanguinarine Papaver somniferum 26% - codeine immobilized cells Papaver somniferum biotransformation codeinone codeine codeine 88% Berberine isoquinoline alkaloid coptis japonica berberine gibberellic acid 10-8 M berberine 1.66g/L high-density cell culture cell mass 70g/L berberine 0.45 g Tropane alkaloid - tropic acid Scopolia japonica suspension culture 15 -high tropane alkaloid producing strain Hyoscyamus niger hyoscyamine 7 - Cardinolides Digitalis purpurea cardiac glycosides digitoxin digitoxin digoxin/ - b-methyldigitoxin substrate immobilized cell Digitalis lanata 17 bioreactor 20 L b methyl digoxin 1.4% b-methyldigitoxin

- = 2,4-D, NAA - = ginseng - 24 -= Air-lift Bioreactor Bioreactor feed Flow rate, N2,O2, %gas Taxol - = 250 rpm 1000 rpm - = light/dark . Ex. G 2 met. MVA(Mevalanic acid), farnesol. G terpenoid G. Isoprene unit (C5) -Phenyl alanine= Aromatic ring phenolic, flavonid, alkaloid -Tropic acid= Tropane alkaloid Atropine, Hyos -Farnesyl 3 medium enz. -Biotic elicitor=, bact. autoclave elicitor ? 2 met. infect -Abiotic elicitor=, , UV, ., methyl jusmonate Ex. Ginseng precursor MVA bio.elicitor Oligosac. carbohydrate component of ginseng cell wall Abiotic Ex. Taxol Methyl jusmonate Taxol Elicitor and mode of action - elicitor / / / -elicitor elicitation -elicitor= Nigeran, Glucan, chitosan , Phytophtera

megasperma, Phaseolus vulgaris hypocotyls extract,HgCl2-Coculture= elicitor cell cul detect - elicitor complex preparation Immobilization 1.Entrapment in gel=Calcium alginate agar polyacrylamide agarose gelatin carageenan paper Solanum gel bead Ca2+alginate cell susp. Alginate(-) CaCl2 sol (+) gel media polysac (elicitor) 2 / eli. 2 met. biomass susp.cul.

L-DOPA L-3,4-dihydroxyphenylalanine betalain, melanine cathecolamine Parkinson/ Mucana pruriens 2, 4 D L-DOPA 25 mg/L/ immobilized cell M. pruriens alginate tyrosine DOPA 2% Valepotriates Valepotriates * cell line valerianaceae 9 MS media Fedia cornucopiae

immobilized cell glass fiber mat 6 taxol 0.012% Ginseng 4 7 Panax ginseng callus crown gall calli redifferentiated root 21.1%,19.3% 27.4%/ 30L cell mass 2KL cell mass 700 mg/L/day Rosmarinic acid a-O-caffeoyl-3,4-dihydroxyphenyllactic acid curcurmin 10 / Coleus blumei Anchusa officinalis Gamborg and Eveleighs B5 medium 16 g dry weight/L 30-40 medium 7.5% 3.7g/L / Coleus blumei spiral module stirrer fed batch fermentor sucrose production medium rosmarinic acid 5.5g/L 910 mg/L/day 200 g 14 32 L Arbutin ericaceae Arctostaphylos uva-ursi / Datura innoxia hydroquinone arbutin 10 / Catharanthus roseus 5 L hydroquinone 34 arbutin 9.2g/L medium 1% medium 2 high density cell culture 3-4 biotransformation hydroquinone Plant virus inhibitor Mirabilis jalapa 95mg/L 7 24KD sequence 24% homology ricinD-A chain Shikonin Lithospermum erythrorhizon shikonin / Protoplast shikonin two stage culture high cell density culture shikonin / shikonin root culture medium 2 L XAD-2 shikonin 5 mg 220 Anthocyanins Euphorbia millii MS medium 2,4-D, NAA, malt extract, yeast extract cyaniding-3arabinoside / cell aggregate 28 1.32% dry weight ( 0.28% ) Safflower yellow Carthamus tinctorius saffron carthamin 30%

Valeriana locusta - trifluoroleucine nystatin treatment colchicines - two phase culture media bioregulator dimethylmorpholinium-bromide , dimethyl-piperidinium-bromide, dimethyl piperidinium chloride, 2-(3,4-dichloro-phenoxy)-triethylamine 2-(3,5 diisopropylphenoxy)-triethylamine Valepotriates Camptothecin Camptotheca

acuminata Camptotheca acuminata MSmedium 4mg/L NAA camtothecin 0.998 mg/L Homoharringtonine Cephalotaxus harringtonia

colon tumor, melanoma leukemia 3-6 5-10 mg/kg dry weight 1-3% Podophyllotoxin Podophyllum peltatum KB cell etoposide (V16) Hodgkinsdisease 2,4DKinetin -embryogenic root liquid MS medium 1mg/L NAA, 0.2 mg/L kinetin 500 mg/L casein hydrolysate growth regulator podophyllotoxin 1.6% 6 / precursor coniferyl alcohol b cyclodextrin cell suspension culture Podophyllum peltatum podophyllotoxin precursor 4 Vinca alkaloid dimeric indole alkaloid vincristine vinblastine leukemia, Hodgkinss disease / abscisic acid 7 catharanthine 85 mg/L 30 / ferric ion anhydrovinblastine vinblastine 52.8% 12.3% 30 C pH 7.0 3 / catharanthine MS medium with 1mg/L NAA 0.1mg/L kinetin NaCl KCl high-cell density culture catharanthine 230 mg/L/week / ferric chloride oxalate maleate sodium borohydrate vinblastine anhydrovinblastine 50% Taxol diterpene amide depolymerization stabilized microtubule / Taxus

brevifolia 26 taxol 3.9 mg/L

/ chitin, cellulose chitosan 1mM D-phynylalanine Mg Mg Ca medium / stigma-like structure crocin picrocrocin Madder colorant alizarin, purpurine glycosides / LS agar medium 2,4-D gellan gum kinetin sucrose 100 21 1.5 g Hernandulcin sesquiterpenes Lippia

metabolic, morphological and physiological modulation seemed to be generated by the gel bead networks and by the cell-cell interactions inherent to the microenvironmental constraints. An accumulation of starch grains was observed through amyloplasts. Immobilised S. chrysotrichum cells accumulated 5 mgg1 dry weight of SC1. Although this was four times less than the free cells, the desired metabolite was excreted into the surrounding medium (around 40 % of the total production).

dulcia(verbenaceae) MS medium 16 0.25mg/g dry weight / chitosan 5 shoot culture MS solid medium hernandulcin 2.9% dry weight

(secondary metabolites) (totipotency) (callusculture) (undifferentiatedcells) (regenerate) (subculture) 25 2C ( 1 x 1 ) 2 /

Production of sanguinarine by elicited plant cell culture III. Immobilized bioreactor culturesPapaver somniferum cells were surface-immobilized and grown efficiently andmaintained highly viable in 6-1 modified airlift bioreactors. These immobilized cultures were elicited with chitin in the presence of XAD-7 adsorbent polymeric resin, in parallel with control suspension cultures carried out in flasks, for sanguinarine production. Results showed production levels of the larger cultures operated under apparently non limiting conditions of up to 80 mg l1 or 0.52% dw after 600 h, of which 7080% was found in the resin. These productions were 3- to 14-times lower than obtained from control cultures. This poor performance of immobilized cultures is explained by dissolved oxygen limitations within the biofilm as well as by various other restrictive factors associated to the culture system used in relation to the elicitation process, including the inherent physiological heterogeneity of the plant cell biofilm and the insoluble nature of the elicitor used which may have hindered its penetration of the immobilized biomass. Immobilisation of Solanum chrysotrichum plant cells withinCa-alginate gel beads to produce an antimycotic spirostanol saponin Plant cell cultures of the Mexican species Solanum chrysotrichum can be established from friable calli. These cells produce an antifungal spirostanol saponin designated SC1. Plant cell immobilisation within Ca-alginate gel beads can lead to an enhancement in secondary metabolite production. Different matrices, obtained by varying calcium (0.1 to 0.8 molL1) and alginate (1 to 1.5 % w/v) concentrations were used to study the influence of immobilisation parameters on the behaviour of S.

chrysotrichum cells. Throughout the growth cycle and in the various immobilisationconditions, fresh and dry weight, SC1 yield and viability were measured. Important

2 - (pH) 5.0 6.0 5 auxins, cytokinins, gibberllins, ethylene absicic acid auxins cytokinins auxins cytokinins Lithospermum

tumefaciens transformed roots (hairy roots) transformed shoots saikosaponins

Bupleurum falcatum L. adventitious roots 0.02-0..1% (NH4 : NO3 = 1 : 11) adventitious roots 2% 10 hairy roots Hyssopus officinalis rosmarinic acid 8.03% 8 11 3. Biotransformation of precursors (biotransformation) (Rauwolfia serpentina) hydroquinone glycosylation arbutine arbutine 18 g/l 7 codeine (Papaver

erythrorhizon (shikonin), Panax ginseng(ginsenoside), Coleus blumeii(rosmarinic acid) Coptis japonica(berberine) undifferentiated cells undifferentiatedcells (biological technology) 1. Cell immobilization degree of cell-to-cell contact differentiated cells undifferentiated cells gel entrapment, biofilms, adsorption, film immobilization membranes Gel entrapment calcium alginate (Catharanthus roseus) calcium alginate ajmalicine 3 7 Lithospermum erythrorhizon calcium alginate shikonin 2.5 8 2. Morphological modification differentiated cells 9 organized plant culture organogenesis Agrobacterium rhizogenes A.

somniferum) undifferentiated cells codeine specialized latex cells thebaine precursor codeine thebaine codeine tetrahydrothebaine thebainone (cell-free) biotransformation (C.roseus) precursor catharanthine vindoline vinblastine 4. Elicitation phytoalexin elicitor 2 biotic elicitors abiotic elicitors Ruta graveolens phenylalanine ammonia lyase (PAL) 8-12 Rhootorula rubra acridone epoxide, furoquinolines furano coumarins 13 (Papaver somniferum) tyrosine/dopa decarboxylase sanguinarine 80 5. Permeabilisation (electroporation) 100% 15 dimethylsulfoxide

(DMSO)16 polyoxyethylenesorbitanemonolaurate (Tween-20)17 6. Two-phase system 2 two-phase n-hexadecane Miglyol(triglyceride) lipophillic carrier ion exchanger neutral resin XAD18 Miglyol non-toxic triglyceride 19 Miglyol monoterpenes Thuja occidentalis 0.8 mg/g.l 3.0 mg/g.l (precursor) Vitis vinifera 5 21 amberlite resin XAD-4 XAD-7 XAD-7 anthraquinones Cinchona ledgeriana 15 (large scale production) 40-60 g/l 2% rosmarinic acid (21.4%) Coleus blubeii, anthraquinone (18%) Morinda citrifolia, benzylisoquinolines (15%) Coptis japonica, shikonin (12%) Lithospermum erythrorhizon, berberine (10%) Berberis wilsonae, shikimic acid (10%) Gallium mollugo, diosgenin (8%) Dioscorea deltoidea, nicotine(5%) Nicotiana tabacum

Nicotiana

tabaccum Secondary metabolites from plant cell cultures Suggested reading Vasil ch 11, Bhojwani ch17 Types of secondary metabolites (SMs) pharmaceuticals from plant products (e.g., alkaloids, analgesics, cancer-fighting chemicals) plant-derived flavors and colorings (where natural compounds are necessary/desirable

SMs made in: batches (small to medium-scale) bioreactors (large-scale production)

Secondary metabolites from plant cell cultures Features of these compounds low molecular wt synthesis (in plant cell) is tightly regulated

A few products are commercially produced examples shikonin, ginseng compounds, berberine more are not produced because of two main problems low yields in in vitro culture feedback inhibition from SMs stored intracellularly

Secondary metabolites from plant cell cultures Transport mechanisms in cultivated plant cells cells that transport SMs to vacuolar space for storage final yield limited to storage capacity of medium cells that secrete SMs into the medium final yield limited to properties of the medium production is more easily manipulated, but separation of culture medium from cultures is required SMs originating from cells in the medium, secreted into the medium, taken up by other cells and stored

Secondary metabolites from plant cell cultures example sanguinarine from California poppy cells sanguinarine is an alkaloid that acts as an agent against dental caries cells are grown in a 2-liter air-lift bioreactor this bioreactor uses 2 phases: an aqueous culture phase and a silicone oil extraction phase bioreactor is sparged thru a central draft tube; gas bubbles are disengaged thru a top layer of silicone fluid

Secondary metabolites from plant cell cultures example sanguinarine from California poppy cells difference in specific gravity between aqueous and extraction phases, as well as gentle mixing in the airlift system maintains a clean separation between phases a small contact area between phases is sufficient for secretion into the extraction phase advantages (of this two-phase system) silicone extraction phase doesn't affect cell viability

Secondary metabolites from plant cell cultures example sanguinarine from California poppy cells advantages (of this two-phase system) SM product is isolated rapidly SM product can be localized in a reduced volume elicitation can be incorporated biosynthetic enzymes induced (usu. with an oligosaccharide) increase in SM production results

Secondary metabolites from plant cell culturesSecondary metabolites from plant cell cultures

Other systems immobilized cell culture (2 approaches) entrapment in alginate beads adherence of cells to a porous membrane in both cases, the objectives: control of growth (high nutrient levels first, then elicitation or starvation) release of SM into the medium where it can be harvested

Other systems hairy root culture culture of fast-growing roots by: inoculation with Agrobacterium rhizogenes (esp. useful where differentiated cells are required for SM production) transfer of T-DNA causing hairy roots, then elimination of Agrobacterium by antibiotic (transformed) hairy roots are first subcultured to solid, then liquid medium

Secondary metabolites from plant cell cultures Other systems hairy root culture (contin) SM is released into the medium, then harvested primary advantage a simple system w/o problems inherent with cell suspensions limited to spp. susceptible to A. rhizogenes biotransformation