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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
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 invitro.
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).
Types of Cultures
•Seed culture
•Organ culture
•Callus culture
•Cell culture
•Protoplast culture
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
Somatic Embryogenesis from
Grape Callus
•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
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 welladapted, 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.
2.
3.
4.
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.
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?
•Water
• Mineral Salts
• Carbon Source(s)
• Vitamins
• Other Complex Addenda
• Plant Growth Regulators
WATER
Glass Distilled – Heat & Condensation
Cartridge System -- Filtered
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
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)
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
Vitamins and Other Organic Compounds
Vitamins usually added to medium at 0.2-1.0 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
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
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
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
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
Plant Growth Regulators
• Used in Concentrations of 0.001 – 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
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)
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
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
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.
50
Secondary metabolites are derived from
primary metabolites
51
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
52
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
53
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
54
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.
55
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.
56
Glycosides
•
Compounds that contain a carbonhydrate and a noncarbohydrate
•
Glucosinolates: found primarily in the mustard family to give the pungent
taste.
57
Taxol
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.
58
• 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.
59
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 genesized 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 DNAcoated 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
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.
84
Essential components of genetic
engineering
• Methods of introducing the DNA into
the host plants
• A source of DNA fragment containing
desirable traits
85
Methods to introduce DNA
fragments to plants
• Agrobacterium
• Virus
• Chemically induced
• Physically assisted
86
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
87
© 2003 John Wiley and Sons Publishers
Structure of the nopaline Ti plasmid pTi C58, showing
selected components.
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)
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 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
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
94
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.
Protoplasts
95
Physically assisted gene transformation
The particle
gun
96
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
97
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 plantderived 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
•
•
1.
2.
3.
4.
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
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 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
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)
CH 3
OH
CH 3
H
H
H
H
H
HO
H
HO
ESTRONE
ESTRADIOL
CH3
H
H
OH
OH
H
HO
ESTRIOL
natural mamalian estrogens; plants also produce estrogenic substances
(isoflavones)
O
O
H
H
H
H
H
HO
H
HO
CHOLESTEROL
PREGNENOLONE
O
O
OH
H
H
H
H
H
HO
H
HO
DEHYDROEPIANDROSTERONE
17-HYDROXYPREGNENOLONE
O
H
H
H
HO
DEHYDROEPIANDROS TERONE
O
OH
H
H
H
H
H
O
H
O
ANDROS TENEDIONE
TES TOS TERONE
OH
H
H
HO
ES TRADIOL
H
O
H
H
H
H
H
HO
H
HO
CHOLES TEROL
PREGNENOLONE
O
O
OH
H
H
H
H
H
H
HO
HO
DEHYDROEPIANDROS TERONE
17-HYDROXYPREGNENOLONE
O
OH
H
H
H
H
H
O
H
O
ANDROS TENEDIONE
TES TOS TERONE
OH
H
H
HO
ES TRADIOL
H
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
OH
OH
O
OH
O
OH
O
O
OH
BIOCHANIN A
GENISTEIN
O
OH
O
OCH 3
OH
O
O
COUMESTROL
O
OH
DAIDZIN
FORMONONETIN
HO
O
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
OH
OH
H
H
H
H
O
H
H
O
H
TESTOSTERONE
DIHYDROTESTOSTERONE
Steroid Aromatase
A
O
B
HO
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 nonsteroidal (anastrozole, letrozole)
• estrogen deprivation through aromatase inhibition is an
effective and selective treatment for some postmenopausal patients with hormone-dependent breast
cancer
AROMATASE INHIBITORS
N
N
N
N
CN
H3 C
N
C
N
CH3
CH3
C
NC
NC
CH3
CN
LETROZOLE (FEMARA)
ANASTROZOLE (ARIMIDEX)
both of these drugs are used in the treatment of advanced
breast cancer in post-menopausal women with disease
progression following tamoxifen therapy