Plant Tissue Culture - Definition

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

sequential stages of somatic embryo development

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

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

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.

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.

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.

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.

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.

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.

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.).

Plant Tissue Culture

• Plants have the ability to reproduce asexually• It is this natural ability that is the basis of

micropropagation

Plant Tissue Culture

• Where does the new growth come from in plants?

– Meristematic Tissue

– Parenchyma Tissue

– Adventitious growth

– Virtually any plant cell

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

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

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

2 Manufacture of useful chemicals by plant culture –

atropine (dilation of pupil),

codeine (pain killer),

digoxin (treatment of cardiovascular problems),

jasmine (perfume),

menthol (flavouring).

 

•Seed culture

•Organ culture

•Callus culture

•Cell culture

•Protoplast culture

Types of Cultures

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

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.

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

Somatic Embryogenesis

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

Somatic Embryogenesis

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

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

-Production of haploid plants.-Achievement of In vitro fertillization.

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.

 

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.

 

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!

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.

Plant Tissue Culture - Definition

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

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.

 

Characteristic of Plant Tissue Culture Techniques

1. Environmental condition optimized (nutrition, light, temperature).2. Ability to give rise to callus, embryos,

adventitious roots and shoots.3. Ability to grow as single cells (protoplasts, microspores, suspension

cultures).4. Plant cells are totipotent, able to regenerate a whole plant.

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.

What’s in Tissue Culture Medium?What’s in Tissue Culture Medium?

•Water

• Mineral Salts

• Carbon Source(s)

• Vitamins

• Other Complex Addenda

• Plant Growth Regulators

WATERWATER

Glass Distilled – Heat & CondensationGlass Distilled – Heat & Condensation

Cartridge System -- FilteredCartridge System -- Filtered

Major Mineral Nutrients (mM)Major Mineral Nutrients (mM)

•Nitrogen Nitrogen as Either Nitrate (NO as Either Nitrate (NO33) and Ammonium (NH) and Ammonium (NH44))

KNOKNO33, NH, NH44NONO33, Ca(NO, Ca(NO33))2 2 etc. etc.

•CalciumCalcium as CaCl as CaCl22 or Ca(NO or Ca(NO33))2 2

•MagnesiumMagnesium as MgSO as MgSO4 4

•Potassium Potassium as KCl or Kas KCl or K22HPOHPO44

•Phosphorus Phosphorus as Kas K22HPOHPO4 4 or KH or KH22POPO44 or Na Salts or Na Salts

•SulfurSulfur as Many SO as Many SO44

20-50% of Osmotic Potential20-50% of Osmotic Potential

Minor Mineral Elements (uM)Minor Mineral Elements (uM)

•Boron (B)Boron (B)

•Cobalt (Co)Cobalt (Co)

•Iron (Fe --Usually Chelated with NaEDTA)Iron (Fe --Usually Chelated with NaEDTA)

•Manganese (Manganese (Mn Mn not Mg)not Mg)

•Molybdenum (Mo)Molybdenum (Mo)

•Copper (Cu)Copper (Cu)

•Zinc (Zn)Zinc (Zn)

•Iodine (I)Iodine (I)

Carbon SourcesCarbon Sources

•Cane Sugar = Sucrose (Fructose and Glucose)Cane Sugar = Sucrose (Fructose and Glucose)

•Corn Sugar = FructoseCorn Sugar = Fructose

•Maltose, Glucose Sorbitol, Raffinose and Other SugarsMaltose, Glucose Sorbitol, Raffinose and Other Sugars

20-50% of the Osmotic potential of the medium20-50% of the Osmotic potential of the medium

Typically Added Between 20 and 40 g/lTypically Added Between 20 and 40 g/l

Vitamins and Other Organic CompoundsVitamins and Other Organic Compounds

Vitamins usually added to medium at 0.2-1.0 mg/LVitamins usually added to medium at 0.2-1.0 mg/L

Vitamin B1 or Thiamine is considered essential -- CarbohydratesVitamin B1 or Thiamine is considered essential -- Carbohydrates

Vitamin C – AntioxidantVitamin C – Antioxidant

Inositol or myo-inositol – Really a Sugar Alcohol -- MembranesInositol or myo-inositol – Really a Sugar Alcohol -- Membranes

Yeast extract – Source of Many B Vitamins – Rarely UsedYeast extract – Source of Many B Vitamins – Rarely Used

Vitamins and Other Organic CompoundsVitamins and Other Organic Compounds

Coconut Milk (Really the Water) – Source of PGRs Coconut Milk (Really the Water) – Source of PGRs (Kinetin and/or Zeatin) Varies Greatly (Kinetin and/or Zeatin) Varies Greatly

Casein Hydrolysate or Peptone (Amino Acids), Ammonium, etc.Casein Hydrolysate or Peptone (Amino Acids), Ammonium, etc.

Polyamines – Somatic Embryogenesis,Polyamines – Somatic Embryogenesis,

Root FormationRoot Formation

Activated Charcoal, PPVP, Activated Charcoal, PPVP,

Ascorbic and Citric Acid -- PolyphenolsAscorbic and Citric Acid -- Polyphenols

Plant Growth RegulatorsPlant Growth Regulators

•AuxinsAuxins -- IAA, IBA, NAA, 2,4-D, TDZ, Dicamba, etc. -- IAA, IBA, NAA, 2,4-D, TDZ, Dicamba, etc.

•Cytokinins Cytokinins – Kinetin, BA, 2iP, Zeatin, Thidiazuron, etc.– Kinetin, BA, 2iP, Zeatin, Thidiazuron, etc.

•Gibberellic AcidsGibberellic Acids -- More Than 60 Forms GA 4 & 7 Most Commonly Used -- More Than 60 Forms GA 4 & 7 Most Commonly Used

•Abscisic AcidAbscisic Acid -- Cis and Trans Forms -- Cis and Trans Forms

•EthyleneEthylene – The Only Gaseous PGR – The Only Gaseous PGR

Hormone Product Name Function in Plant Tissue Culture

Auxins Indole-3-Acetic AcidIndole-3-Butyric AcidIndole-3-Butyric Acid, Potassium Salt-Naphthaleneacetic Acid2,4-Dichlorophenoxyacetic Acidp-Chlorophenoxyacetic acidPicloramDicamba

Adventitous root formation (high concen)Adventitious shoot formation (low concen)Induction of somatic embryosCell DivisionCallus formation and growthInhibition of axillary budsInhibition of root elongation

Cytokinins 6-Benzylaminopurine6-,-Dimethylallylaminopurine (2iP)KinetinThidiazuron (TDZ)N-(2-chloro-4-pyridyl)-N’PhenylureaZeatinZeatin Riboside

Adventitious shoot formationInhibition of root formationPromotes cell divisionModulates callus initiation and growthStimulation of axillary’s bud breaking and growthInhibition of shoot elongationInhibition of leaf senescence

Gibberellins Gibberellic Acid Stimulates 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 Acid Abscisic Acid Stimulates bulb and tuber formationStimulates the maturation of embryosPromotes the start of dormancy

Polyamines PutrescineSpermidine

Promotes adventitious root formationPromotes somatic embryogenesisPromotes shoot formation

Plant Growth RegulatorsPlant Growth Regulators

• Used in Concentrations of 0.001 – 10 uMUsed in Concentrations of 0.001 – 10 uM

•Many Can Be Autoclaved (Especially Synthetic Such as 2,4-D, Many Can Be Autoclaved (Especially Synthetic Such as 2,4-D, Dicamba, TDZ, BA), But Others Degrade With Heat and Should Be Dicamba, TDZ, BA), But Others Degrade With Heat and Should Be Filter-Sterilized (IAA, Kinetin, Zeatin, etc).Filter-Sterilized (IAA, Kinetin, Zeatin, etc).

•Most Have Interactions With Each Other -- Can Cause a Multitude of EffectsMost Have Interactions With Each Other -- Can Cause a Multitude of Effects

•Can be Prepared in Water, KOH, Ethanol, DMSOCan be Prepared in Water, KOH, Ethanol, DMSO

Preparation of Plant Growth RegulatorsPreparation of Plant Growth Regulators

Dissolve 100 mg of Either BA (Benzyladenine) in 5 ml Dissolve 100 mg of Either BA (Benzyladenine) in 5 ml of 95% of 95% EthanolEthanol or 1.0N or 1.0N KOH KOH

Bring Volume to 100 ml with WaterBring Volume to 100 ml with Water

Yields 1mg/mlYields 1mg/ml

Store at 4C (Lasts Over a Year if Not Contaminated)Store at 4C (Lasts Over a Year if Not Contaminated)

Example:Example: Benzyladenine or BA Benzyladenine or BA

Agar and Alternatives (Support)Agar and Alternatives (Support)

•Crude Agar Contains Lots of Impurities – Minerals, Organic Crude Agar Contains Lots of Impurities – Minerals, Organic Compounds, which may interfere with tissue cultureCompounds, which may interfere with tissue culture

•Phytoagar is Purified (Lacking Most Impurities) and Has a Melting Point of Phytoagar is Purified (Lacking Most Impurities) and Has a Melting Point of About 65C and a Gelling Point Between 40-50CAbout 65C and a Gelling Point Between 40-50C

•Agarose is A Purified Fraction of Agar and Typically Has Low Agarose is A Purified Fraction of Agar and Typically Has Low Melting and Gelling Points. More Expensive and Use for Melting and Gelling Points. More Expensive and Use for ProtoplastsProtoplasts

Agar and AlternativesAgar and Alternatives

•Gellan Gums – Gelrite and Phytagel Require Additional Ca Ions to Gel. Gellan Gums – Gelrite and Phytagel Require Additional Ca Ions to Gel. Care Must be Taken to Assure That Tissues Have Sufficient Calcium for Care Must be Taken to Assure That Tissues Have Sufficient Calcium for GrowthGrowth

•Mechanical Supports – Filter Paper Bridges, Rafts, Rock Wool Mechanical Supports – Filter Paper Bridges, Rafts, Rock Wool (Fiberglass), Foam and Glass or Polyurethane Beads(Fiberglass), Foam and Glass or Polyurethane Beads

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• 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.

Metabolites

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Secondary metabolites are derived from primary metabolites

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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

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• 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

Secondary metabolites

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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

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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.

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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.

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Glycosides

• Compounds that contain a carbonhydrate and a noncarbohydrate

• Glucosinolates: found primarily in the mustard family to give the pungent taste.

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• 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.

Taxol

Taxus brevifolia Nutt.

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• 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.

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.

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

-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.

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.

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.

• 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.

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.

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.

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.

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

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.

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.

• 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.

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.

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.

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.

• 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.

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

Totipotent- means that an organism has the ability to grow from a single cell

-especially important with plants, also called regeneration.

• 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

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.

Callus- a mass of undifferentiated plant cells.By making a callus the number of transformed

cells is increased

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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.

Genetically modified organisms (GMOs):Organisms are modified by genetic engineering to express desirable traits.

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Essential components of genetic engineering

• Methods of introducing the DNA into the host plants

• A source of DNA fragment containing desirable traits

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Methods to introduce DNA fragments to plants

• Agrobacterium

• Virus

• Chemically induced

• Physically assisted

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• 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

Structure of the nopaline Ti plasmid pTi C58, showing selected components.

© 2003 John Wiley and Sons Publishers

Ti Plasmid

1. T-DNA (Transferred DNA)transferred and expressed into plant causing tumor (crown gall) formation

2. Virulence Genesessential for the transfer and integration of the T-DNA

3. ori

4. Noc (Opine Catabolism) genes (other microbes do NOT have)

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

Engineered Ti plasmid

1. Clone foreign gene into T-DNA

2. Delete genes responsible for tumor formation

3. Add selectable marker

Infection 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) cell

T-DNA is cut out of plasmid at left and right borderRight border- responsible for integration

5. Other T-DNA genes are activated causing tumor formation

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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

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• 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.

Chemically induced gene transformation

Protoplasts

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The particle gun

Physically assisted gene transformation

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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

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 [517-89-5], 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.

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

1. Inhibit uptake of the herbicide

2. Overproduce the herbicide-sensitive target protein

3. Reduce the ability of the herbicide-sensitive target to bind to the herbicide

4. Give plants the ability to inactivate the herbicide

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

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!

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 1,3-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

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)

CH3OH

H

H

H

HO

ESTRADIOL

CH3

H

H

H

HO

O

ESTRONE

CH3OH

H

H

H

HO

OH

ESTRIOL

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

HO

HH

H

HO

HH

H

O

HO

HH

H

O

OH

HO

HH

H

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CHOLESTEROLPREGNENOLONE

17-HYDROXYPREGNENOLONEDEHYDROEPIANDROSTERONE

HO

HH

H

O

O

HH

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DEHYDROEPIANDROSTERONE

ANDROSTENEDIONE

OH

HH

H

O

TESTOSTERONE

OH

HH

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HO

ESTRADIOL

HO

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H

O

CHOLESTEROLPREGNENOLONE

17-HYDROXYPREGNENOLONEDEHYDROEPIANDROSTERONE

ANDROSTENEDIONE

OH

HH

H

O

TESTOSTERONE

OH

HH

H

HO

ESTRADIOL

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

OOH

OH OOH

OOH

OH O

GENISTEIN BIOCHANIN A

O

OOCH3

FORMONONETIN

OOH

OOH

DAIDZIN

OOH

HO O

COUMESTROL

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

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

Natural androgens

O

OH

HH

H

TESTOSTERONE

O

OH

HH

H

H

DIHYDROTESTOSTERONE

HO

BA

O

Steroid Aromatase

Estrogens

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

AROMATASE INHIBITORS

N

NN

NC CN

LETROZOLE (FEMARA)

both of these drugs are used in the treatment of advancedbreast cancer in post-menopausal women with diseaseprogression following tamoxifen therapy

NN

N

C

CCH3

NC

H3C CN

CH3

CH3

ANASTROZOLE (ARIMIDEX)