sterile pieces of a whole plant from which cultures are generally initiated explants aerial plant...
TRANSCRIPT
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Sterile pieces of a whole plant from which cultures are generally initiated
Explants
•Aerial plant parts are “cleaner” than underground parts
•The smaller the explant the better the chances to overcome specific phytopathological problems (virus, microplasm, bacteria), but it decreases the survival rate
• Inner tissues are less contaminated than outer ones
•Comparable explants do not always react in a similar way , due to: influence of location on the mother plant,
influence of juvenility status , influence of polarity
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Inoculum
A subculture of plant material which is already in culture
Generally all plant cells can be used as an explant, however young and rapidly growing tissue (or tissue at an early stage of development) are preferred.
Types of explant
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Types of culture (Explant base)
Plant tissue culture
Embryo culture Seed culture Meristem culture
Protoplast cultureCell culture (suspension culture)
Callus cultureBud culture
Organ culture
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Types of In vitro culture (explant based)
Culture of intact plants (seed and seedling culture)
Embryo culture (immature embryo culture)
Organ culture Callus culture Cell suspension culture Protoplast culture
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Seed culture Growing seed
aseptically in vitro on artificial media
Increasing efficiency of germination of seeds that are difficult to germinate in vivo
it is possible to independent on asymbiotic germination. Production of clean seedlings for explants or meristem culture
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Embryo culture Growing embryo
aseptically in vitro on artificial nutrient media
Overcoming seed dormancy and self-sterility of seeds
Study embryo development
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Organ culture Any plant organ can serve as an
explant to initiate cultures
No.
Organ Culture types
1. Shoot Shoot tip culture
2. Root Root culture
3. Leaf Leaf culture
4. Flower Anther/ovary culture
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Shoot apical meristem culture
Production of virus free germplasm
Mass production of desirable genotypes
Facilitation of exchange between locations (production of clean material)
Cryopreservation (cold storage) or in vitro conservation of germplasm
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Root organ culture
1.Production of seedling from crop which multiply through root
2.Production of secondary metabolite
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Ovary or ovule culture Production of haploid plants
A common explant for the initiation of somatic embryogenic cultures
Overcoming abortion of embryos of wide hybrids at very early stages of
development due to incompatibility barriers
In vitro fertilization for the production of distant hybrids avoiding style and
stigmatic incompatibility that inhibits pollen germination and pollen tube
growth
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Anther and microspore culture
Production of haploid plants Production of homozygous diploid lines through chromosome doubling, thus reducing the time required to
produce inbred lines Uncovering mutations or recessive
phenotypes
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SterilizationKilling or excluding microorganisms or their spores with heat, filters, chemicals or other
sterilants
Tissue culture is an aseptic techniqueAseptic technique:
-Sterile-Free of pathogenic microorganisms-Free or freed from pathogenic microorganisms-Free from the living germs of disease and fermentation-Conditions established to exclude contaminants
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Axenic culture
GermfreeUncontaminated Free from germs or pathogenic organisms Free from other microorganism Containing only 1 organismA culture of an organism that is entirely free from all other contaminating organismsNot contaminated by or associated with any other living organism Pure cultures that are completely free of the presence of other organisms
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Sterilization
1. Micro-organism contamination can over
grow the plant culture resulting in
culture death
2. Micro-organism contamination exhaust
the nutrient media
3. Micro-organism can change in secondary
metabolite structure or produce other
compounds .
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Source of contamination
The explant or culture
The vessels
The media
The instruments
The environment where handling is
taking place
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Aseptic Techniques
Chemical treatments • disinfectants, • antibiotics, • sublimatPhysical treatments• heating: the most important disinfection
method • electromagnetic radiation, • filtration• ultrasonic waves.
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Disinfectans
They penetrate into bacteria, They will denature bacterial protein, They decrease the activity of bacterial
enzyme, They inhibit bacterial growth and
metabolism, They damage the structure of cell
membrane, They change membrane permeability.
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Disinfectans – Liquid laundry bleach (NaOCl at 5-6% by vol)• Rinse thoroughly after treatment• Usually diluted 5-20% v/v in water; 10% is most common
– Calcium hypochlorite – Ca(OCl)2
• a powder; must be mixed up fresh each time
– Ethanol (EtOH)• 95% used for disinfesting plant tissues• Kills by dehydration• Usually used at short time intervals (10 sec – 1 min)• 70% used to disinfest work surfaces, worker hands
– Isopropyl alcohol (rubbing alcohol) is sometimes recommended
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Antibiotics
Used only when necessary or when disinfestants are ineffective or impractical
Its use by incorporating in the mediaCommon antibiotics are carbenicillin,
cefotaxime, rifampicin, tetracycline, streptomycin
Problems with antibiotics• tend to be selective• resistance acquisition• may obscure presence of microbes• cell/tissue growth inhibition
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An ideal antibiotics
Broad-spectrum Did not induce resistance Selective toxicity, low side effects Preserve normal microbial flora
BC Yang
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Modes of action Inhibitors of cell wall synthesis.
Penicillins, cephalosporin, bacitracin, carbapenems and vancomycin.
Inhibitors of Cell Membrane.Polyenes - Amphotericin B, nystatin, and condicidin.Imidazole - Miconazole, ketoconazole and clotrimazole.Polymixin E and B.
Inhibitors of Protein Synthesis.Aminoglycosides - Streptomycin, gentamicin, neomycin and kanamycin.Tetracyclines - Chlortetracycline, oxytetracycline, doxycycline and minocycline.Erythromycin, lincomycin, chloramphenicol and clindamycin.
Amphotericin
Tetracyclines
Aminoglycosides
vancomycin
BC Yang
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Modes of action
Inhibitors of metabolites
(Antimetabolites).Sulfonamides - Sulfanilamide,
sulfadiazine silver and
sulfamethoxazole.
Trimethoprim, ethambutol, isoniazid.
Inhibitors of nucleic acids
(DNA/RNA polymerase).Quinolones - Nalidixic acid, norfloxacin
and ciprofloxacin.
Rifamycin and flucytosine.
rifamycin
BC Yang
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Sublimat (0.1 - 1%)
Its activity based on Cl- Heavy metal (Hg) denaturates proteins. Hg is toxic for the environment,
therefore recuperate the Hg-solution after use and collect in a large container.
Hg can be precipitated by adding ammonia to the solution, and siphoning the supernatant
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UV radiation Ultraviolet is light
with very high energy levels and a wavelength of 200-400 nm.
One of the most effective wavelengths for disinfection is that of 254 nm.
BC Yang
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Heating
•Oven (dry heat) Suitable for tools, containers a 160°-180° C for 3 h
•Microwaves (off the shelf)
Useful for melting agar (but not gellan gum types of solidifying agents)Special pressurized containers are required for sterilizing in a microwave
• Flaming or heating of toolsFlaming – e.g., 95% EtOH in an alcohol burner is useful for
sterilizing metal instrumentsBacticinerators – heats metal tools in a hot ceramic coreHeated glass beads
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Heating•Autoclave
Steam heat under pressure (It typically generates 15 lbs/in2 and 250° F (1.1 kg/cm2 and 121° C))It is faster and more effectiveFor liquids (such as water, medium), autoclave time depends on liquid volume Recommended autoclaving times (sterilization time only):
250 ml requires 15 min500 ml requires 20 min1000 ml requires 25 min
Excessive autoclaving can break down organics – a typical symptom is caramelized sucrose
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Heating
• Flaming or heating of toolsFlaming – e.g., 95% EtOH in an alcohol burner is useful for sterilizing metal instrumentsBacticinerators – heats metal tools in a hot ceramic coreHeated glass beads
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Filtration
– Filtration of culture medium•Some medium ingredients are heat labile, e.g., GA, IAA, all proteins, antibiotics
•Most devices use a paper cellulose filter with small pore spaces (0.22 µm)
•Syringes used for small volumes, vacuum filtration for large volumes
– Filtration of air•Transfer hoods usu. generate wind at 27-30 linear m per min (or 90-100 ft per min)
•Too slow and air drops contaminants onto your work surface; too fast causes turbulence and excess filter wear
•air "corridors" must be kept free of barriers to be effective
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Sterilization Equipment
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sterilizing paper: dry heat
sterilizing tools
laminar flow cabinet
Sterilization Equipment
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Sterilization Equipment
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Callus CultureCallus:
An un-organised mass of cells, produced when explants are cultured on the appropriate solid
medium, with both an auxin and a cytokinin and correct conditions.
A tissue that develops in response to injury caused by physical or chemical means
Most cells of which are differentiated although may be and are often highly unorganized within the
tissue
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Explants Callus
Protoplasts Development Suspension cells
Organs
(leaves, roots, shoots, flowers,...)
De-differentiation Re-differentiation
1. Meristems 2. Leaf sections 3. Bulb sections 4. Embryos 5. Anthers 6. Nucellus
Callus formation
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Stimuli :
In vivo : wound, microorganisms, insect feeding
In vitro : Phytohormones 1. Auxin 2. Cytokinin 3. Auxin and cytokinin 4. Complex natural extracts
Callus formation
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Callus
• During callus formation there is some degree of dedifferentiation both in morphology and metabolism, resulting in the lose the ability to photosynthesis.
• Callus cultures may be compact or friable.Compact callus shows densely aggregated cellsFriable callus shows loosely associated cells and
the callus becomes soft and breaks apart easily. • Habituation:
The lose of the requirement for auxin and/or cytokinin by the culture during long-term culture.
•
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When friable callus is placed into the appropriate liquid medium and agitated, single cells and/or small clumps of cells are released into the medium and continue to grow and divide, producing a cell-suspension culture.
The inoculum used to initiate cell suspension culture should neither be too small to affect cells numbers nor too large too allow the build up of toxic products or stressed cells to lethal levels.
When callus pieces are agitated in a liquid medium, they tend to break up.
Cell-suspension cultures
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Cell suspension culture
Suspensions are much easier to bulk up than callus since there is no manual transfer or solid support
Cell suspension culture techniques are very important for plant biotransformation and plant genetic engineering.
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Protoplast culture
The isolation and culture of plant protoplasts in vitro
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Protoplast
The living material of a plant or bacterial cell, including the protoplasm and plasma membrane
after the cell wall has been removed.
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Plant Regeneration Pathways
Existing Meristems (Microcutting)Uses meristematic cells to regenerate whole plant.
Organogenesis
Relies on the production of organs either directly from an explant or callus structure
Somatic Embryogenesis
Embryo-like structures which can develop into whole plants in a way that is similar to zygotic embryos are formed from somatic cells
(Source:Victor. et al., 2004)(Source:Victor. et al., 2004)
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Cell Differentiation
The process by which cells become specialized in form and function. These cells undergo changes
that organize them into tissues and organs.
MorphogenesisAs the dividing cells begin to take form, they are undergoing morphogenesis which means
the “creation of form.”Morphogenetic events lay out the
development very early on in development as cell division, cell differentiation and
morphogenesis overlap
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Morphogenesis
• These morphogenetic events “tell” the organism where the head and tail are, which is the front and back, and what is left and right.
• As time progresses, later morphogenetic events will give instructions as to where certain appendages will be located.
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Morphogenetic Events
• Morphogenetic events, as well as cell division and differentiation, take place in all multicellular organisms.
• In plants, morphogenesis and growth in overall size are not limited to embryonic and juvenile periods, they occur throughout the life of the plant.
• For example, apical meristems of plants are responsible for a plant’s continued growth and development and the formation of new organs throughout the plant’s life. These are perpetually embryonic regions in the tips of shoots and roots.
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Cloning
• Using the somatic cells of a multicellular organism to generate a new organism is
• Each clone is genetically identical to the parent plant.
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Microcutting propagationThe production of shoots from pre-
existing meristems only.
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Organogenesis• The ability of non-
meristematic plant tissues to form various organs de novo.
• The formation of adventitious organs
• The production of roots, shoots or leaves
• These organs may arise out of pre-existing meristems or out of differentiated cells
• This may involve a callus intermediate but often occurs without callus.
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Indirect organogenesis
Explant
Callus
Meristemoid
Primordium
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Direct OrganogenesisDirect shoot/root formation from the
explant
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Somatic Embryogenesis
• The formation of adventitious embryos
• The production of embryos from somatic or “non-germ” cells.
• It usually involves a callus intermediate stage which can result in variation among seedlings
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Types of embryogenic cells
• Pre-embryogenic determined cells, PEDCs– The cells are committed to embryonic
development and need only to be released. Such cells are found in embryonic tissue.
• Induced embryogenic determined cells, IEDCs– In majority of cases embryogenesis is through
indirect method.– Specific growth regulator concentrations and/or
cultural conditions are required for initiation of callus and then redetermination of these cells into the embryogenic pattern of development.
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Various terms for non-zygotic embryos
Adventious embryosSomatic embryos arising directly from other organs or embryos.
Parthenogenetic embryos (apomixis) Somatic embryos are formed by the unfertilized egg.
Androgenetic embryosSomatic embryos are formed by the male gametophyte.
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Somatic Embryogenesis and Organogenesis
• Both of these technologies can be used as methods of micropropagation.
• It is not always desirable because they may not always result in populations of identical plants.
• The most beneficial use of somatic embryogenesis and organogenesis is in the production of whole plants from a single cell (or a few cells).
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Somatic embryogenesis differs from organogenesis
• Bipolar structure with a closed radicular end rather than a monopolar structure.
• The embryo arises from a single cell and has no vascular connection with the mother tissue.
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Two routes to somatic embryogenesis
(Sharp et al., 1980)
• Direct embryogenesis– Embryos initiate directly from explant
in the absence of callus formation.
• Indirect embryogenesis– Callus from explant takes place from
which embryos are developed.
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Direct somatic embryogenesis
Direct embryo formation from an explant
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Indirect Somatic Embryogenesis
Explant → Callus Embryogenic → Maturation → Germination
1.Calus induction2.Callus embryogenic development
3.Multiplication4.Maturation5.Germination
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Induction• Auxins required for induction
–Proembryogenic masses form–2,4-D most used–NAA, dicamba also used
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Development
Auxin must be removed for embryo development
Continued use of auxin inhibits embryogenesis
Stages are similar to those of zygotic embryogenesis– Globular– Heart– Torpedo– Cotyledonary– Germination (conversion)
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Maturation
• Require complete maturation with apical meristem, radicle, and cotyledons
• Often obtain repetitive embryony• Storage protein production necessary• Often require ABA for complete
maturation• ABA often required for normal embryo
morphology – Fasciation– Precocious germination
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Germination
• May only obtain 3-5% germination• Sucrose (10%), mannitol (4%) may be
required• Drying (desiccation)
– ABA levels decrease– Woody plants– Final moisture content 10-40%
• Chilling– Decreases ABA levels– Woody plants
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Somatic embryogenesis as a means of propagation is
seldom usedHigh probability of mutations
The method is usually rather difficult.Losing regenerative capacity become
greater with repeated subculture Induction of embryogenesis is very
difficult with many plant species.A deep dormancy often occurs with
somatic embryogenesis
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Peanut somatic embryogenesis
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Steps of Micropropagation• Stage 0 – Selection & preparation of the mother
plant– sterilization of the plant tissue takes place
• Stage I - Initiation of culture– explant placed into growth media
• Stage II - Multiplication– explant transferred to shoot media; shoots
can be constantly divided• Stage III - Rooting
– explant transferred to root media• Stage IV - Transfer to soil
– explant returned to soil; hardened off