8Batch cultivation: extract products from time to time with cleaning & sterilization to begin the whole process againContinuous cultivation: used medium and products are continuously removed, raw materials are added throughout the processA stirred tank fermenter
9Immobilization of cells and enzymes One problem with the fermentation processes so far is that at some point the cell culture is removed and discarded.Any mechanism for immobilizing the microorganism and/or the enzymes they produce, improves the economics of the process.1 Entrapment – cells or enzyme molecules are trapped in a suitable meshwork of inert material, e.g. agar, cellulose, etc2 Binding – cells or enzyme become physically attached to the surface of a suitable material, e.g. sand or gravel
103 Cross-linking – cells or enzymes are chemically bonded to a suitable chemical matrix However immobilized, the cells or enzymes are made into small beads which are then either packed into column, or kept in the nutrient medium.The nutrient can be continually added and the product removed without frequent removal of the microorganisms/enzymes.The process cannot be continued indefinitely because impurities may accumulate.
11Single Cell Protein (SCP) Single cell protein comprises the cells, or their products, of microorganisms which are grown for animal and human consumption.The product also contains fats, carbohydrates, vitamins and minerals.Raw materials: petroleum chemicals, alcohols, sugars, agricultural & industrial wastes.Microorganisms: bacteria, filamentous fungi, algae, yeast.
12Cell, tissue and organ culture Plant and animal cells can also be grown in vitro to make a variety of products.In vitro: by artificial means outside the bodyThe plant meristems retain the growing ability of plant cells.If a tissue containing meristematic cells, e.g. a bud, root tip, etc., is removed from the plant and grown aseptically on a nutrient medium, an undifferentiated mass (callus) develops in the presence of hormones and growth regulators.
13Plant Tissue CultureMicropropagation is the production of whole plants from small sections of plant such as a stem tip, node, meristem, embryo, or even a seedPlant tissue culture is basically the same thing, except that it implies the use of callus tissue generated from plant cells cultured in-vitro.
14Plant Tissue CultureMicropropagation and plant tissue culture are used to produce large numbers of plants from small pieces of the stock plant in relatively short periods of time.
15Why does micropropagation work? Plant Tissue CultureWhy does micropropagation work?Plant cells have the ability to reproduce the whole plant from single cells. This is called totipotency.Totipotency is the ability of a single cell to express the full genome in the cells to which it gives rise by cell division.
16Plant Tissue CultureTotipotency in reference to fertilized eggs (zygotes) are totipotent because they produce a population of differentiated cells forming an entire organism, whereas for example human skin cells are not totipotent since in culture they divide to produce only more skin cells (not nerve, muscle etc.).
17Plant Tissue CulturePlants have the ability to reproduce asexuallyIt is this natural ability that is the basis of micropropagation
18Virtually any plant cell Plant Tissue CultureWhere does the new growth come from in plants?Meristematic TissueParenchyma TissueAdventitious growthVirtually any plant cell
19Plant Tissue CultureMeristematic tissue - which are undifferentiated cells from shoot and root tips that have not been programmed for their ultimate developmentParenchyma cells – the most common type of plant cell, which can regenerate and differentiate to initiate the growth of new and varied tissue and organsAdventitious growth is the development of new shoots, buds, roots, or leaves from atypical or unusual locations
20Plant Tissue Culture The genetic basis of micropropagation There are two types of plant cell divisions which include somatic cells and sex cellsMitosis – somatic cellsMeiosis – sex cells
21Plant Tissue CultureMitosis – every somatic cell is diploid (2n) with 2 sets of chromosomesThe chromosomes duplicate and then segregateFrom this 2 new cells form, each with an identical set of chromosomes to the original cell
222 Manufacture of useful chemicals by plant culture – atropine (dilation of pupil),codeine (pain killer),digoxin (treatment of cardiovascular problems),jasmine (perfume),menthol (flavouring).
23Types of Cultures Seed culture Organ culture Callus culture Seed cultureOrgan cultureCallus cultureCell cultureProtoplast culture
24Different Techniques of Plant Tissue Culture: Callus and Cell cultureSomatic embryogenesisHaploid cultureProtoplast cultureMicropropagationOrganogenesisProduction of virus-free plantsSomaclonal variationIn vitro Mutagenesis
25For obtaining virus-free plants. Callus CulturesProduction of plantlets through somatic embryogenesis or organogenesis.For obtaining virus-free plants.For generation of useful somaclonal and gametoclonal variants.As a source of protoplasts and suspension cultures.Production of useful secondary metabolites.For biotransformation studies.Selection of cell lines with valuable properties such as resistance to disease, herbicides, overproduction of secondary metabolites etc.For mutagenetic studies.
26Somatic Embryogenesis Production of plantlets through somatic embryogenesis or organogenesis.For obtaining virus-free plants.As a source of protoplasts and suspension cultures.Production of useful secondary metabolites.Selection of cell lines with valuable properties such as resistance to disease, herbicides, overproduction of secondary metabolites etc.For mutagenetic studies.Somatic Embryogenesis fromGrape Callus
27Somatic Embryogenesis Stimulation of callus or suspension cells to undergo a developmental pathway that mimics the development of the zygotic embryo.
28Anther and Microspore Culture Haploid CultureAnther and Microspore Culture-Production of haploid plants.-Production of homozygous diploid lines through chromosome doubling, thus reducing the breeding cycle.Ovary or Ovule Culture-Achievement of In vitro fertillization.
29Protoplast Isolation,Culture and FusionCombining distant genomes to produce somatic hybrids, asymmetric hybrids.Production of organelle recombinants.Transfer of CMS (cytoplasmic male sterility) in elite lines.Source material for genetic transformation.
30Introduction of foreign DNA to generate novel genetic combinations. Genetic TransformationIntroduction of foreign DNA to generate novel genetic combinations.Transfer of desirable genes for disease and pest resistance from related or unrelated plant species into high yielding susceptible cultivars.Study of structure and function of genes.Over-production of secondary metabolites, naturally present in mother plant.Production of novel secondary metabolites absent in parent plant.
31Mass multiplication of elite germplasm. Organognesis and Enhanced Axillary BuddingMass multiplication of elite germplasm.As source material for protoplast work, genetic transformation and mirografting.Conservation of endangered genotypes either at normal or at sub-zero temperatures.Organogenesis may not produce clones!
32Somaclonal Variations (Genetic or Epigenetic)Isolation of useful variants in well-adapted, high yielding genotypes lacking in a few desirable traits.Isolation of useful variants overproducing primary or secondary metabolites.Isolation of useful variants with better disease resistance, stress tolerance capacities.Creation of additional genetic variation without hybridization in useful cultivars.
33Plant Tissue Culture - Definition The growth and development of plant seeds, organs, explants, tissues, cells or protoplasts on nutrient media under sterile (axenic) conditions.
34Explant - Definition This means to simply cut-out a very small piece of leaf or stem tissue, or even isolate individual cells, and place them in a tissue culture container.The tissue has to be surface-sterilized so it will not have any contaminating bacteria or fungus. It is then placed inside the tissue culture vessel (dish, jar, etc.)containing a gel called agar. In the agar is dissolved all the sugar, nutrients and plant growth regulators the explant needs.
35Characteristic of Plant Tissue Culture Techniques Environmental condition optimized (nutrition, light, temperature).Ability to give rise to callus, embryos,adventitious roots and shoots.Ability to grow as single cells (protoplasts, microspores, suspension cultures).Plant cells are totipotent, able to regenerate a whole plant.
36Totipotency or Totipotent: The capacity of a cell (or a group of cells) to give rise to an entire organism.Differentiation (De-):The physiological and morphological changes that occur in a cell, tissue, or organ during development.Organogenesis:The development of tissues and/or organs from individual cells not from pre-existing meristems.
38WATERGlass Distilled – Heat & CondensationCartridge System -- Filtered
39Major Mineral Nutrients (mM) Nitrogen as Either Nitrate (NO3) and Ammonium (NH4)KNO3, NH4NO3, Ca(NO3)2 etc.Calcium as CaCl2 or Ca(NO3)2Magnesium as MgSO4Potassium as KCl or K2HPO4Phosphorus as K2HPO4 or KH2PO4 or Na SaltsSulfur as Many SO420-50% of Osmotic Potential
40Minor Mineral Elements (uM) Boron (B)Cobalt (Co)Iron (Fe --Usually Chelated with NaEDTA)Manganese (Mn not Mg)Molybdenum (Mo)Copper (Cu)Zinc (Zn)Iodine (I)
41Typically Added Between 20 and 40 g/l Carbon SourcesCane Sugar = Sucrose (Fructose and Glucose)Corn Sugar = FructoseMaltose, Glucose Sorbitol, Raffinose and Other SugarsTypically Added Between 20 and 40 g/l20-50% of the Osmotic potential of the medium
42Vitamins usually added to medium at 0.2-1.0 mg/L Vitamins and Other Organic CompoundsVitamins usually added to medium at mg/LVitamin B1 or Thiamine is considered essential -- CarbohydratesVitamin C – AntioxidantYeast extract – Source of Many B Vitamins – Rarely UsedInositol or myo-inositol – Really a Sugar Alcohol -- Membranes
43Casein Hydrolysate or Peptone (Amino Acids), Ammonium, etc. Vitamins and Other Organic CompoundsCoconut Milk (Really the Water) – Source of PGRs (Kinetin and/or Zeatin) Varies GreatlyCasein Hydrolysate or Peptone (Amino Acids), Ammonium, etc.Polyamines – Somatic Embryogenesis,Root FormationActivated Charcoal, PPVP,Ascorbic and Citric Acid -- Polyphenols
44Plant Growth Regulators Auxins -- IAA, IBA, NAA, 2,4-D, TDZ, Dicamba, etc.Cytokinins – Kinetin, BA, 2iP, Zeatin, Thidiazuron, etc.Gibberellic Acids -- More Than 60 Forms GA 4 & 7 Most Commonly UsedAbscisic Acid -- Cis and Trans FormsEthylene – The Only Gaseous PGR
45Function in Plant Tissue Culture HormoneProduct NameFunction in Plant Tissue CultureAuxinsIndole-3-Acetic AcidIndole-3-Butyric AcidIndole-3-Butyric Acid, Potassium Salt-Naphthaleneacetic Acid2,4-Dichlorophenoxyacetic Acidp-Chlorophenoxyacetic acidPicloramDicambaAdventitous root formation (high concen)Adventitious shoot formation (low concen)Induction of somatic embryosCell DivisionCallus formation and growthInhibition of axillary budsInhibition of root elongationCytokinins6-Benzylaminopurine6-,-Dimethylallylaminopurine (2iP)KinetinThidiazuron (TDZ)N-(2-chloro-4-pyridyl)-N’PhenylureaZeatinZeatin RibosideAdventitious shoot formationInhibition of root formationPromotes cell divisionModulates callus initiation and growthStimulation of axillary’s bud breaking and growthInhibition of shoot elongationInhibition of leaf senescenceGibberellinsGibberellic AcidStimulates shoot elongationRelease seeds, embryos, and apical buds from dormancyInhibits adventitious root formationPaclobutrazol and ancymidol inhibit gibberellin synthesis thus resulting in shorter shoots, and promoting tuber, corm, and bulb formation.Abscisic AcidStimulates bulb and tuber formationStimulates the maturation of embryosPromotes the start of dormancyPolyaminesPutrescineSpermidinePromotes adventitious root formationPromotes somatic embryogenesisPromotes shoot formation
46Plant Growth Regulators Used in Concentrations of – 10 uMMany Can Be Autoclaved (Especially Synthetic Such as 2,4-D, Dicamba, TDZ, BA), But Others Degrade With Heat and Should Be Filter-Sterilized (IAA, Kinetin, Zeatin, etc).Most Have Interactions With Each Other -- Can Cause a Multitude of EffectsCan be Prepared in Water, KOH, Ethanol, DMSO
47Preparation of Plant Growth Regulators Example: Benzyladenine or BADissolve 100 mg of Either BA (Benzyladenine) in 5 ml of 95% Ethanol or 1.0N KOHBring Volume to 100 ml with WaterYields 1mg/mlStore at 4C (Lasts Over a Year if Not Contaminated)
48Agar and Alternatives (Support) Crude Agar Contains Lots of Impurities – Minerals, Organic Compounds, which may interfere with tissue culturePhytoagar is Purified (Lacking Most Impurities) and Has a Melting Point of About 65C and a Gelling Point Between 40-50CAgarose is A Purified Fraction of Agar and Typically Has Low Melting and Gelling Points. More Expensive and Use for Protoplasts
49Agar and AlternativesGellan Gums – Gelrite and Phytagel Require Additional Ca Ions to Gel. Care Must be Taken to Assure That Tissues Have Sufficient Calcium for GrowthMechanical Supports – Filter Paper Bridges, Rafts, Rock Wool (Fiberglass), Foam and Glass or Polyurethane Beads
50MetabolitesPrimary metabolites: Molecules that are essential for growth and development of an organism.Secondary metabolites: molecules that are not essential for growth and development of an organism.Primary – mostly present in most of the cells – important for function of cells; ex: proteins, AA, nucleotides, lipidsSecondary – if knock out synthesis of it, development will be mostly fine – small defect; produced by specialized cell during certain development stages and diff. organisms may produce different 2nd metabolites – all of them derived from primary ones
51Secondary metabolites are derived from primary metabolites
52Why secondary metabolites? Chemical warfare to protect plants from the attacks by predators, pathogens, or competitorsAttract pollinators or seed dispersal agentsImportant for abiotic stressesMedicineIndustrial additives2nd metabolites – important role to make plant more adaptive to the environment-used as repellant to deal w/ biological stresses-food and other additives
53Secondary metabolites Possibly over 250,000 secondary metabolites in plantsClassified based on common biosynthetic pathways where a chemical is derived.Four major classes:Alkaloids, glycosides, phenolics, terpenoids-only 10% have been chemically identified and even less used in daily life-each of the 4 major classes can be subdivided to subclasses
54AlkaloidsMost are derived from a few common amino acids (i.e., tyrosine, tryptophan, ornithine or argenine, and lysine)Compounds have a ring structure and a nitrogen residue.Indole alkaloids is the largest group in this family, derived from tryptophanWidely used as medicineAlkaloids--have nitrogen atom in their structure – most also have ring structure-Indole (largest group) – derived from tryptophan-pathways to produce secondary metabolites can also produce primary metabolites
55PhenolicsDerived from aromatic amino acids, such as phenylalanine, tyrosin, and trytophan.All contain structures derived from phenolSome examples:Coumarins: antimicrobial agents, feeding deterrents, and germination inhibitors.Lignin: abundant in secondary cell wall, rigid and resistant to extraction or many degradation reagents.All derived from phenol precursor-three aromatic acid – phenylalanine, tyrosine, and tryptophan-Coumarins – used to make perfumes; toxic to animals and humans-Lignin – highly branched polysaccharide; oxidized and cross linked to other cell wall component
56TerpenoidsTerpenes are generally polymers of 5-carbon unit called isopreneGive scent, flavors, colors, medicine...Three plant hormones are derived from the terpenoid pathway.Terpenoids-Three plant hormones – GA, brassinosteroids, and abscissic acid
57GlycosidesCompounds that contain a carbonhydrate and a noncarbohydrateGlucosinolates: found primarily in the mustard family to give the pungent taste.Glycosides –Found in mustard familyMany oils produced by plants in mustard family(Canola plants produce less glucosinolates)
58Taxol Taxol is a terpenoid Taxus brevifolia Nutt.Taxol is a terpenoid"the best anti-cancer agent” by National Cancer InstituteHas remarkable activity against advanced ovarian and breast cancer, and has been approved for clinical use.
59Camptothecin is an indole alkaloid, derived from tryptophan. Has anticancer and antiviral activityTwo CPT analogues have been used in cancer chemotherapy, topotecan and irinotecan.Used to treat AIDS
60class of alkaloids, the vinca alkaloids from Vinca rosea, the Madagascar periwinkle, can also bind to tubulin and inhibit microtubule polymerization. Vinblastine and vincristine are used as potent agents for cancer chemotherapy,The alkaloid colchicine, a constituent of the swollen, underground stems of the autumn crocus (Colchicum autumnale) and meadow saffron, inhibits the polymerization of tubulin into microtubules.
61Genetic EngineeringThe simple addition, deletion, or manipulation of a single trait in an organism to create a desired change.
62Genetic Engineering Genetic Engineering -major tool is recombinant DNA.-Recombinant- DNA joined to other unrelated foreign DNA.-also called gene splicing.-tiny segments of a gene are taken out and replaced.
63Genetic Engineering-gene splicing, gene cloning, molecular cloning -process cutting a gene out of a DNA strand and inserting the gene into another DNA strand.
64DNA IsolationInside all plants cells is a nucleus, the "brain" of the cell. The nucleus contains all of the information the cell needs. This information is stored on chromosomes, made up of tightly spiraled DNA.
65DNA is composed of four different nucleotides: adenine (A), guanine (G), thymine (T), and cytosine (C). These nucleotides make up the genetic language of life. The order of the nucleotides encodes all of the cell's information.A set of nucleotides that code for a particular protein is called a gene, and each chromosome contains thousands of genes. Since the proteins a cell produces are responsible for its specific traits, by changing the genes of an organism you can change its proteins, and therefore its traits.
66Cloning GenesGene cloning is used to locate and copy a specific gene from the entire DNA of an organism. For example, suppose the red gene in this bacterium needs to be extracted from the rest of the DNA in order to be added to a plant.
67The DNA is removed from bacteria cells and isolated in a test tube The DNA is removed from bacteria cells and isolated in a test tube. A restriction enzyme is added to the isolated DNA, and cuts the extracted bacterial DNA into gene-sized pieces.
68In another test tube, extracted bacterial plasmids are cut using the same restriction enzyme. The cut plasmids are mixed with the gene-sized pieces of DNA.
69The two combine to form recombinant plasmids The two combine to form recombinant plasmids. Some of the plasmids will recombine with themselves without picking up the bacterial DNA. These will be useless. Other plasmids will contain the gene of interest
70Designing GenesEach gene has three distinct regions:Promoter - Signals how much protein to produce and when it should be made.Coding Region - Encodes which protein to produce. In order, codons (sets of three nucleotides) are read by the cell, specifying the next amino acid that must be made and added to the chain.Termination Sequence - Signals the end of a gene, preventing the cell from combining two or more coding regions.
72Genetic engineers can alter or replace one or more of the three regions to design a gene so that it will be expressed in a specific way in a plant cell. Here is an example of different genetic modifications that can be achieved by manipulating the gene sequence.
74Combining 35S with Bt. CRYIA and inserting this gene in corn will make all parts of the plant poisonous to the corn borer. Combining PEP Carboxylase with Bt. CRYIA will produce a plant that is poisonous to the corn borer only if the pest eats the green parts of the plant. Corn stalks, silks, and late season plants that have slowed their photosynthesis will remain edible to the borer.The same holds for the Round Up Resistance coding region. Combining it with 35S will produce a plant that is completely immune to Roundup.
75Designing GenesEach gene has three distinct regions:Promoter - Signals how much protein to produce and when it should be made.Coding Region - Encodes which protein to produce. In order, codons (sets of three nucleotides) are read by the cell, specifying the next amino acid that must be made and added to the chain.Termination Sequence - Signals the end of a gene, preventing the cell from combining two or more coding regions.
76Endonucleases-type of enzyme in DNA strand. -produced nucleic acid strand breaks interior of nucleic acid strand. -restriction endonucleases-enzyme produced by bacteria that is used in recombinant DNA. -cuts open bacterial plasmid. -gene construct engineered to plasmid with ligasees. Plasmids back to bacterium.
77Cloning Vectors-carrier for DNA during the recombinant DNA process. -plasmid-piece of free-floating DNA in the cytoplasm of bacteria. -double-stranded, circular molecules that replicate independently of the chromosome.
78TransformationTransformation- process of introducing free DNA into bacteriaCompetent cell- a cell that is capable of taking up DNA.Electroporation- The use of an electric shock to momentarily open or disrupt cell walls.
79TransformationConjugation- the contact of bacteria that involves the exchange of DNA with a mating tube.Transformed cell- cell with new DNAMarker gene- a gene that identifies which organisms have been successfully transformed
80Other ProcessesTotipotent- means that an organism has the ability to grow from a single cell-especially important with plants, also called regeneration.
81AgroBacterium Transformation Agro bacterium tumefacians is a bacterium that causes a disease known as crown gall in plants.Infects plants by transferring its genetic material into plant cell.Agrobacterium transformation is the most common technique for genetically engineered plants
82Ballistic Gene Transfer Ballistic Gene Transfer- the use of tiny DNA-coated projectiles as carriers. It is important to transport DNA through the walls of intended recipient cells.Projectiles are often known as micro projectilesBallaistic transformation is done by using a ‘gene gun’ the gene gun has been useful in creating agricultural crops.
83The Role of Tissue Culture Callus- a mass of undifferentiated plant cells.By making a callus the number of transformed cells is increased
84Genetic engineering – change DNA composition of an organism Genetic engineering: using molecular biology methods to modify the genetic information of an organism.To learn about the biology of an organismTo generate a new or improved commercial productPlant biotechnology: manipulating or modifying plants to improve agriculture or to generate a “new” or improved commercial product.Genetic engineering – change DNA composition of an organismGenetically modified organisms (GMOs):Organisms are modified by genetic engineering to express desirable traits.
85Essential components of genetic engineering Methods of introducing the DNA into the host plantsA source of DNA fragment containing desirable traits
86Methods to introduce DNA fragments to plants AgrobacteriumVirusChemically inducedPhysically assisted
87AgrobacteriumThe bacterium can transfer its own DNA into plants and modulates plant growth and development (causes crown gall disease)Efficiently transforms many dicotyledonous plantsProblematical with monocotsAgrobacterium – soil bacteria; isolate from any kind of soil-natural genetic engineer-can transfer own piece of DNA into host which can integrate into CH of host cell; integrated DNA then expressed in host cell – can modulate development of plant growthForm tumor like growth (crown gall disease)Efficiently transform dicots (not monocots) – problem b/c many grains such as wheat, rice, etc. are monocots – if want to improve crop quality – can’t use agrobacterium – best way to introduce DNA into host
89Ti Plasmid 1. T-DNA (Transferred DNA) transferred and expressed into plant causingtumor (crown gall) formation2. Virulence Genesessential for the transfer and integration of the T-DNA3. ori4. Noc (Opine Catabolism) genes (other microbes do NOT have)
90Genes within T-DNA 1. Enzymes to produce Auxin (iaa) 2. Enzyme to produce cytokinins3. Genes for synthesis of Opines - carbon source for bacteriano use in plants4. Tum -genes responsible for tumor formation
92Engineered Ti plasmid 1. Clone foreign gene into T-DNA 2. Delete genes responsible for tumor formation3. Add selectable marker
93Infection of Plant Cell Continued 1. Bacteria attaches to host2. Virulence gene expression activated by compounds secreted by wounds3. T-DNA is transferred4. T-DNA integrates into host (plant) cellT-DNA is cut out of plasmid at left and right borderRight border- responsible for integration5. Other T-DNA genes are activated causing tumor formation
94Viruses as a tool in genetic engineering Enter plant cells via insect carriers, wounding sites, and seeds.Use host transcription, translation, and replication machinery to express viral genes.mostly transient, occasionally DNA can integrate into the host genome to become stable transformationWidely used in research laboratories to study gene function but less applicable in plant biotechnology-Viruses can use insects, enter thru wounding sites, or by seeds-use host cell’s machinery-can hijack virus genome and put desired traits (allow virus carry out gene of interest into host cell)(usually DNA don’t get integrated – transient until get integrated0
95Chemically induced gene transformation Protoplasts are cells without a cell wall.DNA (any piece of DNA) uptake enhanced by chemical treatmentsCan be transient or stable. Stable transgenic plants can be obtained via regeneration.Produced by enzymatic degradation of the cell wall.of the cell wall and induction of callus which will further differentiate to form plants.Protoplasts – plant cells w/o cell wall-enzymatic treatment to degrade cell wall and leave round, green plant cells-only have outer plasma membrane – can use chemical to induce permeability in plasma membrane-can uptake any kind of DNA into pore (so more transient; occasionally get integration to form stable)Protoplasts
96Physically assisted gene transformation The particle gunPhysically assisted gene transformation – mediated by gene gunTiny gold particles bind to DNA and load into gene gunShoot that into plant tissue – can get transformed (more transient, occasionally get integrated into CH)
97Crop improvement increase crop yield nutritional improvement improved timber: faster growing trees, increased quality (harder, stronger wood)increased shelf lifeimproved taste and texturestress resistance: drought, heat, cold, salt tolerancepest resistanceherbicide resistancerenewable biofuelFor crop improvement, need to find traits that can be useful
98Immobilized Plant Cells Plant cell cultures can also be used for the production of metabolites such as pharmaceuticals, chemicals, flavors, and fragrances. The first product obtained from mass plant cell cultures was shikonin [ ], a red pigment composed of eight naphthoquinone molecules. Shikonin is produced by a two-stage fermentation process and is a high-value chemical ($ 4000/kg) with a limited annual market capacity of ca. 15 kg. Immobilized plant cell systems will be used mainly for products of cells in the stationary growth phase. The release of intracellularly stored products by intermittent permeabilization of immobilized cells can be a great economic advantage, allowing reutilization of the biomass. The continuous immobilized plant cell process in combination with strain selection and improved product leakage allows production of plant-derived chemicals in the range of $ 20 – 25/kg. However, in the present industrial state of technology for plant cell cultures, a relatively small number of products have both high value per weight and sufficient market size.
99Herbicides and herbicide-resistant plants Herbicides are generally non-selective (killing both weeds and crop plants) and must be applied before the crop plants germinateFour potential ways to engineer herbicide resistant plantsInhibit uptake of the herbicideOverproduce the herbicide-sensitive target proteinReduce the ability of the herbicide-sensitive target to bind to the herbicideGive plants the ability to inactivate the herbicide
100Herbicide-resistant plants: Giving plants the ability to inactivate the herbicide Herbicide: BromoxynilResistance to bromoxynil (a photosytem II inhibitor) was obtained by expressing a bacterial (Klebsiella ozaenae) nitrilase gene that encodes an enzyme that degrades this herbicide
101Herbicide-resistant plants: Reducing the ability of the herbicide-sensitive target to bind to the herbicideHerbicide: Glyphosate (better known as Roundup)Resistance to Roundup (an inhibitor of the enzyme EPSP involved in aromatic amino acid biosynthesis) was obtained by finding a mutant version of EPSP from E. coli that does not bind Roundup and expressing it in plants (soybean, tobacco, petunia, tomato, potato, and cotton)5-enolpyruvylshikimate-3-phosphate synthase (EPSP) is a chloroplast enzyme in the shikimate pathway and plays a key role in the synthesis of aromatic amino acids such as tyrosine and phenylalanineThis is a big money maker for Monsanto!
102Fungus- and bacterium-resistant plants Genetic engineering here is more challenging; however, some strategies are possible:Individually or in combination express pathogenesis-related (PR) proteins, which include b1,3-glucanases, chitinases, thaumatin-like proteins, and protease inhibitorsOverexpression of the NPR1 gene which encodes the “master” regulatory protein for turning on the PR protein genesOverproducing salicylic acid in plants by the addition of two bacterial genes; SA activates the NPR1 gene and thus results in production of PR proteins
103Development of stress- and senescence-tolerant plants: genetic engineering of salt-resistant plants Overexpression of the gene encoding a Na+/H+ antiport protein which transports Na+ into the plant cell vacuoleThis has been done in Arabidopsis and tomato plants allowing them to survive on 200 mM salt (NaCl)
113ANDROGENSprototype is testosterone (produced by interstitial cells of testis)main function: development and maintenance of primary and secondary sex characteristics in males (androgenic)protein retention (anabolic action)other naturally occuring androgens: androsterone, isoandrosterone, dehydroandrosterone, dehydroisoandrosterone
119Phytoandrogens (Plant Chemicals that Mimic or Boost Testosterone) - from tree bark (cortex) of the Gutta-Percha tree, Eucommia ulmoides-male hormone-like effects that interact with the human androgen receptor-androgen receptor (AR) plays a pivotal role in skeletal muscle development, bone density, fertility and sex drive
124AROMATASE INHIBITORSaromatase is a cytochrome P450 enzyme that catalyzes the conversion of adrenal androgen androstenedione to estrone in both pre- amd post menopausal womenreaction occurs in the liver, muscle, adipose and breast tissuein post-menopausal women, aromatization is responsible for the majority of circulating estrogenaminoglutethimide was used but has now been replaced by more selective drugsdrugs may be steroidal (testolactone, emestane) or non-steroidal (anastrozole, letrozole)estrogen deprivation through aromatase inhibition is an effective and selective treatment for some post-menopausal patients with hormone-dependent breast cancer
125AROMATASE INHIBITORSboth of these drugs are used in the treatment of advancedbreast cancer in post-menopausal women with diseaseprogression following tamoxifen therapy