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Plant Tissue Culture - Definition

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1 Plant Tissue Culture - Definition
The growth and development of plant seeds, organs, explants, tissues, cells or protoplasts on nutrient media under sterile (axenic) conditions.




5 sequential stages of somatic embryo development



8 Batch cultivation: extract products from time to time with cleaning & sterilization to begin the whole process again Continuous cultivation: used medium and products are continuously removed, raw materials are added throughout the process A stirred tank fermenter

9 Immobilization 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, etc 2 Binding – cells or enzyme become physically attached to the surface of a suitable material, e.g. sand or gravel

10 3 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.

11 Single 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.

12 Cell, 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 body The 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.

13 Plant Tissue Culture Micropropagation is the production of whole plants from small sections of plant such as a stem tip, node, meristem, embryo, or even a seed Plant tissue culture is basically the same thing, except that it implies the use of callus tissue generated from plant cells cultured in-vitro.

14 Plant Tissue Culture Micropropagation 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.

15 Why does micropropagation work?
Plant Tissue Culture Why 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.

16 Plant Tissue Culture Totipotency 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.).

17 Plant Tissue Culture Plants have the ability to reproduce asexually It is this natural ability that is the basis of micropropagation

18 Virtually any plant cell
Plant Tissue Culture Where does the new growth come from in plants? Meristematic Tissue Parenchyma Tissue Adventitious growth Virtually any plant cell

19 Plant Tissue Culture Meristematic tissue - which are undifferentiated cells from shoot and root tips that have not been programmed for their ultimate development Parenchyma cells – the most common type of plant cell, which can regenerate and differentiate to initiate the growth of new and varied tissue and organs Adventitious growth is the development of new shoots, buds, roots, or leaves from atypical or unusual locations

20 Plant Tissue Culture The genetic basis of micropropagation
There are two types of plant cell divisions which include somatic cells and sex cells Mitosis – somatic cells Meiosis – sex cells

21 Plant Tissue Culture Mitosis – every somatic cell is diploid (2n) with 2 sets of chromosomes The chromosomes duplicate and then segregate From this 2 new cells form, each with an identical set of chromosomes to the original cell

22 2 Manufacture of useful chemicals by plant culture –
atropine (dilation of pupil), codeine (pain killer), digoxin (treatment of cardiovascular problems), jasmine (perfume), menthol (flavouring).

23 Types of Cultures Seed culture Organ culture Callus culture
Seed culture Organ culture Callus culture Cell culture Protoplast culture

24 Different Techniques of Plant Tissue Culture:
Callus and Cell culture Somatic embryogenesis Haploid culture Protoplast culture Micropropagation Organogenesis Production of virus-free plants Somaclonal variation In vitro Mutagenesis

25 For obtaining virus-free plants.
Callus Cultures Production 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.

26 Somatic 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 from Grape Callus

27 Somatic Embryogenesis
Stimulation of callus or suspension cells to undergo a developmental pathway that mimics the development of the zygotic embryo.

28 Anther and Microspore Culture
Haploid Culture Anther 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.

29 Protoplast Isolation, Culture and Fusion Combining 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.

30 Introduction of foreign DNA to generate novel genetic combinations.
Genetic Transformation Introduction 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.

31 Mass multiplication of elite germplasm.
Organognesis and Enhanced Axillary Budding Mass 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!

32 Somaclonal 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.

33 Plant Tissue Culture - Definition
The growth and development of plant seeds, organs, explants, tissues, cells or protoplasts on nutrient media under sterile (axenic) conditions.

34 Explant - 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.

35 Characteristic 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.

36 Totipotency 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.

37 What’s in Tissue Culture Medium?
Water Mineral Salts Carbon Source(s) Vitamins Other Complex Addenda Plant Growth Regulators

38 WATER Glass Distilled – Heat & Condensation Cartridge System -- Filtered

39 Major Mineral Nutrients (mM)
Nitrogen as Either Nitrate (NO3) and Ammonium (NH4) KNO3, NH4NO3, Ca(NO3)2 etc. Calcium as CaCl2 or Ca(NO3)2 Magnesium as MgSO4 Potassium as KCl or K2HPO4 Phosphorus as K2HPO4 or KH2PO4 or Na Salts Sulfur as Many SO4 20-50% of Osmotic Potential

40 Minor 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)

41 Typically Added Between 20 and 40 g/l
Carbon Sources Cane Sugar = Sucrose (Fructose and Glucose) Corn Sugar = Fructose Maltose, Glucose Sorbitol, Raffinose and Other Sugars Typically Added Between 20 and 40 g/l 20-50% of the Osmotic potential of the medium

42 Vitamins usually added to medium at 0.2-1.0 mg/L
Vitamins and Other Organic Compounds Vitamins usually added to medium at mg/L Vitamin B1 or Thiamine is considered essential -- Carbohydrates Vitamin C – Antioxidant Yeast extract – Source of Many B Vitamins – Rarely Used Inositol or myo-inositol – Really a Sugar Alcohol -- Membranes

43 Casein Hydrolysate or Peptone (Amino Acids), Ammonium, etc.
Vitamins and Other Organic Compounds Coconut Milk (Really the Water) – Source of PGRs (Kinetin and/or Zeatin) Varies Greatly Casein Hydrolysate or Peptone (Amino Acids), Ammonium, etc. Polyamines – Somatic Embryogenesis, Root Formation Activated Charcoal, PPVP, Ascorbic and Citric Acid -- Polyphenols

44 Plant 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 Used Abscisic Acid -- Cis and Trans Forms Ethylene – The Only Gaseous PGR

45 Function in Plant Tissue Culture
Hormone Product Name Function in Plant Tissue Culture Auxins Indole-3-Acetic Acid Indole-3-Butyric Acid Indole-3-Butyric Acid, Potassium Salt -Naphthaleneacetic Acid 2,4-Dichlorophenoxyacetic Acid p-Chlorophenoxyacetic acid Picloram Dicamba Adventitous root formation (high concen) Adventitious shoot formation (low concen) Induction of somatic embryos Cell Division Callus formation and growth Inhibition of axillary buds Inhibition of root elongation Cytokinins 6-Benzylaminopurine 6-,-Dimethylallylaminopurine (2iP) Kinetin Thidiazuron (TDZ) N-(2-chloro-4-pyridyl)-N’Phenylurea Zeatin Zeatin Riboside Adventitious shoot formation Inhibition of root formation Promotes cell division Modulates callus initiation and growth Stimulation of axillary’s bud breaking and growth Inhibition of shoot elongation Inhibition of leaf senescence Gibberellins Gibberellic Acid Stimulates shoot elongation Release seeds, embryos, and apical buds from dormancy Inhibits adventitious root formation Paclobutrazol and ancymidol inhibit gibberellin synthesis thus resulting in shorter shoots, and promoting tuber, corm, and bulb formation. Abscisic Acid Stimulates bulb and tuber formation Stimulates the maturation of embryos Promotes the start of dormancy Polyamines Putrescine Spermidine Promotes adventitious root formation Promotes somatic embryogenesis Promotes shoot formation

46 Plant Growth Regulators
Used in Concentrations of – 10 uM Many 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 Effects Can be Prepared in Water, KOH, Ethanol, DMSO

47 Preparation of Plant Growth Regulators
Example: Benzyladenine or BA Dissolve 100 mg of Either BA (Benzyladenine) in 5 ml of 95% Ethanol or 1.0N KOH Bring Volume to 100 ml with Water Yields 1mg/ml Store at 4C (Lasts Over a Year if Not Contaminated)

48 Agar and Alternatives (Support)
Crude Agar Contains Lots of Impurities – Minerals, Organic Compounds, which may interfere with tissue culture Phytoagar is Purified (Lacking Most Impurities) and Has a Melting Point of About 65C and a Gelling Point Between 40-50C Agarose is A Purified Fraction of Agar and Typically Has Low Melting and Gelling Points. More Expensive and Use for Protoplasts

49 Agar and Alternatives Gellan Gums – Gelrite and Phytagel Require Additional Ca Ions to Gel. Care Must be Taken to Assure That Tissues Have Sufficient Calcium for Growth Mechanical Supports – Filter Paper Bridges, Rafts, Rock Wool (Fiberglass), Foam and Glass or Polyurethane Beads

50 Metabolites Primary 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, lipids Secondary – 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

51 Secondary metabolites are derived from primary metabolites

52 Why secondary metabolites?
Chemical warfare to protect plants from the attacks by predators, pathogens, or competitors Attract pollinators or seed dispersal agents Important for abiotic stresses Medicine Industrial additives 2nd metabolites – important role to make plant more adaptive to the environment -used as repellant to deal w/ biological stresses -food and other additives

53 Secondary metabolites
Possibly over 250,000 secondary metabolites in plants Classified 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

54 Alkaloids Most 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 tryptophan Widely used as medicine Alkaloids- -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

55 Phenolics Derived from aromatic amino acids, such as phenylalanine, tyrosin, and trytophan. All contain structures derived from phenol Some 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

56 Terpenoids Terpenes are generally polymers of 5-carbon unit called isoprene Give scent, flavors, colors, medicine... Three plant hormones are derived from the terpenoid pathway. Terpenoids -Three plant hormones – GA, brassinosteroids, and abscissic acid

57 Glycosides Compounds that contain a carbonhydrate and a noncarbohydrate Glucosinolates: found primarily in the mustard family to give the pungent taste. Glycosides – Found in mustard family Many oils produced by plants in mustard family (Canola plants produce less glucosinolates)

58 Taxol Taxol is a terpenoid
Taxus brevifolia Nutt. Taxol is a terpenoid "the best anti-cancer agent” by National Cancer Institute Has remarkable activity against advanced ovarian and breast cancer, and has been approved for clinical use.

59 Camptothecin is an indole alkaloid, derived from tryptophan.
Has anticancer and antiviral activity Two CPT analogues have been used in cancer chemotherapy, topotecan and irinotecan. Used to treat AIDS

60 class 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.

61 Genetic Engineering The simple addition, deletion, or manipulation of a single trait in an organism to create a desired change.

62 Genetic 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.

63 Genetic Engineering -gene splicing, gene cloning, molecular cloning -process cutting a gene out of a DNA strand and inserting the gene into another DNA strand.

64 DNA Isolation Inside 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.

65 DNA 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.

66 Cloning Genes Gene 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.

67 The 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.

68 In 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.

69 The 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

70 Designing Genes Each 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.


72 Genetic 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.


74 Combining 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.

75 Designing Genes Each 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.

76 Endonucleases -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.

77 Cloning 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.

78 Transformation Transformation- process of introducing free DNA into bacteria Competent cell- a cell that is capable of taking up DNA. Electroporation- The use of an electric shock to momentarily open or disrupt cell walls.

79 Transformation Conjugation- the contact of bacteria that involves the exchange of DNA with a mating tube. Transformed cell- cell with new DNA Marker gene- a gene that identifies which organisms have been successfully transformed

80 Other Processes Totipotent- means that an organism has the ability to grow from a single cell -especially important with plants, also called regeneration.

81 AgroBacterium 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

82 Ballistic 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 projectiles Ballaistic transformation is done by using a ‘gene gun’ the gene gun has been useful in creating agricultural crops.

83 The Role of Tissue Culture
Callus- a mass of undifferentiated plant cells. By making a callus the number of transformed cells is increased

84 Genetic 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 organism To generate a new or improved commercial product Plant biotechnology: manipulating or modifying plants to improve agriculture or to generate a “new” or improved commercial product. Genetic engineering – change DNA composition of an organism Genetically modified organisms (GMOs): Organisms are modified by genetic engineering to express desirable traits.

85 Essential components of genetic engineering
Methods of introducing the DNA into the host plants A source of DNA fragment containing desirable traits

86 Methods to introduce DNA fragments to plants
Agrobacterium Virus Chemically induced Physically assisted

87 Agrobacterium The bacterium can transfer its own DNA into plants and modulates plant growth and development (causes crown gall disease) Efficiently transforms many dicotyledonous plants Problematical with monocots Agrobacterium – 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 growth Form 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

88 Fig Structure of the nopaline Ti plasmid pTi C58, showing selected components. The Ti plasmid is 210 kb in size. Symbols used are ori, origin of replication; Tum, genes responsible for tumor formation; Nos, genes involved in nopaline bioynthesis; Noc, genes involved in the catabolism of nopaline; vir, virulence genes required for T-DNA transfer. The nucleotide-pair sequence of the left and right terminal repeats are shown at the top; the asterisks mark the four base pairs that differ in the two border sequences. Structure of the nopaline Ti plasmid pTi C58, showing selected components. © 2003 John Wiley and Sons Publishers

89 Ti Plasmid 1. T-DNA (Transferred DNA)
transferred and expressed into plant causing tumor (crown gall) formation 2. Virulence Genes essential for the transfer and integration of the T-DNA 3. ori 4. Noc (Opine Catabolism) genes (other microbes do NOT have)

90 Genes within T-DNA 1. Enzymes to produce Auxin (iaa)
2. Enzyme to produce cytokinins 3. Genes for synthesis of Opines - carbon source for bacteria no use in plants 4. Tum -genes responsible for tumor formation


92 Engineered Ti plasmid 1. Clone foreign gene into T-DNA
2. Delete genes responsible for tumor formation 3. Add selectable marker

93 Infection of Plant Cell Continued
1. Bacteria attaches to host 2. Virulence gene expression activated by compounds secreted by wounds 3. T-DNA is transferred 4. T-DNA integrates into host (plant) cell T-DNA is cut out of plasmid at left and right border Right border- responsible for integration 5. Other T-DNA genes are activated causing tumor formation

94 Viruses 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 transformation Widely 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

95 Chemically induced gene transformation
Protoplasts are cells without a cell wall. DNA (any piece of DNA) uptake enhanced by chemical treatments Can 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

96 Physically assisted gene transformation
The particle gun Physically assisted gene transformation – mediated by gene gun Tiny gold particles bind to DNA and load into gene gun Shoot that into plant tissue – can get transformed (more transient, occasionally get integrated into CH)

97 Crop improvement increase crop yield nutritional improvement
improved timber: faster growing trees, increased quality (harder, stronger wood) increased shelf life improved taste and texture stress resistance: drought, heat, cold, salt tolerance pest resistance herbicide resistance renewable biofuel For crop improvement, need to find traits that can be useful

98 Immobilized 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.

99 Herbicides and herbicide-resistant plants
Herbicides are generally non-selective (killing both weeds and crop plants) and must be applied before the crop plants germinate Four potential ways to engineer herbicide resistant plants Inhibit uptake of the herbicide Overproduce the herbicide-sensitive target protein Reduce the ability of the herbicide-sensitive target to bind to the herbicide Give plants the ability to inactivate the herbicide

100 Herbicide-resistant plants: Giving plants the ability to inactivate the herbicide
Herbicide: Bromoxynil Resistance to bromoxynil (a photosytem II inhibitor) was obtained by expressing a bacterial (Klebsiella ozaenae) nitrilase gene that encodes an enzyme that degrades this herbicide

101 Herbicide-resistant plants: Reducing the ability of the herbicide-sensitive target to bind to the herbicide Herbicide: 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 phenylalanine This is a big money maker for Monsanto!

102 Fungus- 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 inhibitors Overexpression of the NPR1 gene which encodes the “master” regulatory protein for turning on the PR protein genes Overproducing salicylic acid in plants by the addition of two bacterial genes; SA activates the NPR1 gene and thus results in production of PR proteins

103 Development 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 vacuole This has been done in Arabidopsis and tomato plants allowing them to survive on 200 mM salt (NaCl)


105 natural mamalian estrogens; plants also produce estrogenic substances (isoflavones)






111 Plant estrogens: phytoestrogens
A variety of compounds exist in plant which possess estrogenic activity Soy products contain flavonoids and lignin derived compounds which have estrogenic activity


113 ANDROGENS prototype 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






119 Phytoandrogens (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



122 Natural androgens

123 Steroid Aromatase Estrogens

124 AROMATASE INHIBITORS aromatase is a cytochrome P450 enzyme that catalyzes the conversion of adrenal androgen androstenedione to estrone in both pre- amd post menopausal women reaction occurs in the liver, muscle, adipose and breast tissue in post-menopausal women, aromatization is responsible for the majority of circulating estrogen aminoglutethimide was used but has now been replaced by more selective drugs drugs 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

125 AROMATASE INHIBITORS both of these drugs are used in the treatment of advanced breast cancer in post-menopausal women with disease progression following tamoxifen therapy

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