Transcript Lecture 2: Applications of Tissue Culture to Plant

Explants
Sterile pieces of a whole plant from which cultures
are generally initiated
• 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
Types of explant
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.
Inoculum
A subculture of plant material which is already in culture
Types of culture
(Explant base)
Embryo culture
Cell culture
(suspension culture)
Callus culture
Seed culture
Plant tissue culture
Bud culture
Meristem culture
Protoplast culture
Organ culture
Types of In vitro culture
(explant based)
 Culture of intact plants (seed and seedling culture)
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Embryo culture (immature embryo culture)
Organ culture
Callus culture
Cell suspension culture
Protoplast culture
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
Embryo culture
 Growing embryo aseptically in
vitro on artificial nutrient media
 Overcoming seed dormancy and
self-sterility of seeds
 Study embryo development
Organ culture
Any plant organ can serve as an explant to initiate
cultures
No.
1.
2.
3.
4.
Organ
Shoot
Root
Leaf
Flower
Culture types
Shoot tip culture
Root culture
Leaf culture
Anther/ovary culture
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
Root organ culture
1. Production of
seedling from crop
which multiply
through root
2. Production of
secondary metabolite
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
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
Sterilization
Killing or excluding microorganisms or their spores with
heat, filters, chemicals or other sterilants
Tissue culture is an aseptic technique
Aseptic 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
Axenic culture
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Germfree
Uncontaminated
Free from germs or pathogenic organisms
Free from other microorganism
Containing only 1 organism
A culture of an organism that is entirely free from all
other contaminating organisms
 Not contaminated by or associated with any other
living organism
 Pure cultures that are completely free of the presence
of other organisms
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 .
Source of contamination
 The explant or culture
 The vessels
 The media
 The instruments
 The environment where handling is taking place
Aseptic Techniques
Chemical treatments
• disinfectants,
• antibiotics,
• sublimat
Physical treatments
• heating: the most important disinfection method
• electromagnetic radiation,
• filtration
• ultrasonic waves.
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.
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)
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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
Antibiotics
Used only when necessary or when disinfestants are
ineffective or impractical
Its use by incorporating in the media
Common antibiotics are carbenicillin, cefotaxime,
rifampicin, tetracycline, streptomycin
Problems with antibiotics
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tend to be selective
resistance acquisition
may obscure presence of microbes
cell/tissue growth inhibition
An ideal antibiotics
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Broad-spectrum
Did not induce resistance
Selective toxicity, low side effects
Preserve normal microbial flora
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Modes of action
 Inhibitors of cell wall synthesis.
Penicillins, cephalosporin, bacitracin,
carbapenems and vancomycin.
vancomycin
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Amphotericin
Inhibitors of Cell Membrane.
Polyenes - Amphotericin B, nystatin, and
condicidin.
Imidazole - Miconazole, ketoconazole and
clotrimazole.
Polymixin E and B.
 Inhibitors of Protein Synthesis.
Aminoglycosides
Tetracyclines
Aminoglycosides - Streptomycin, gentamicin,
neomycin and kanamycin.
Tetracyclines - Chlortetracycline, oxytetracycline,
doxycycline and minocycline.
Erythromycin, lincomycin, chloramphenicol and
clindamycin.
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Modes of action
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Inhibitors of metabolites
(Antimetabolites).
Sulfonamides - Sulfanilamide, sulfadiazine silver and
sulfamethoxazole.
Trimethoprim, ethambutol, isoniazid.
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Inhibitors of nucleic acids
(DNA/RNA polymerase).
Quinolones - Nalidixic acid, norfloxacin and
rifamycin
ciprofloxacin.
Rifamycin and flucytosine.
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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
UV radiation
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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.
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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 tools
Flaming – e.g., 95% EtOH in an alcohol burner is useful for sterilizing
metal instruments
Bacticinerators – heats metal tools in a hot ceramic core
Heated glass beads
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 effective
For liquids (such as water, medium), autoclave time depends on
liquid volume
Recommended autoclaving times (sterilization time only):
250 ml requires 15 min
500 ml requires 20 min
1000 ml requires 25 min
Excessive autoclaving can break down organics – a typical symptom
is caramelized sucrose
Heating
• Flaming or heating of tools
Flaming – e.g., 95% EtOH in an alcohol burner is useful for
sterilizing metal instruments
Bacticinerators – heats metal tools in a hot ceramic core
Heated glass beads
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 90100 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
Sterilization Equipment
Sterilization Equipment
sterilizing paper: dry heat
sterilizing tools
laminar flow cabinet
Sterilization Equipment
Callus Culture
Callus:
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
Callus formation
1. Meristems
2. Leaf sections
De-differentiation
3. Bulb sections
4. Embryos
Explants
Re-differentiation
Callus
5. Anthers
6. Nucellus
Protoplasts
Development
Suspension cells
Organs
(leaves, roots, shoots, flowers,...)
Callus formation
Stimuli :
In vivo : wound, microorganisms, insect feeding
In vitro : Phytohormones
1. Auxin
2. Cytokinin
3. Auxin and cytokinin
4. Complex natural extracts
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 cells
Friable 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.
•
Cell-suspension cultures
 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 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.
Protoplast culture
The isolation and culture of plant protoplasts in vitro
Protoplast
The living material of a plant or bacterial cell, including the
protoplasm and plasma membrane after the cell wall has been
removed.
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)
Cell Differentiation
The process by which cells become specialized in form
and function. These cells undergo changes that organize
them into tissues and organs.
Morphogenesis
As 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
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.
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.
Cloning
• Using the somatic cells of a multicellular
organism to generate a new organism is
• Each clone is genetically identical to the parent
plant.
Microcutting propagation
The production of shoots from pre-existing
meristems only.
Organogenesis
• The ability of nonmeristematic 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.
Indirect organogenesis
Explant
Callus
Meristemoid
Primordium
Direct Organogenesis
Direct shoot/root formation from the explant
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
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.
Various terms for nonzygotic embryos
 Adventious embryos
Somatic embryos arising directly from other organs or
embryos.
 Parthenogenetic embryos (apomixis)
Somatic embryos are formed by the unfertilized egg.
 Androgenetic embryos
Somatic embryos are formed by the male gametophyte.
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).
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.
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.
Direct somatic embryogenesis
Direct embryo formation from an explant
Indirect Somatic Embryogenesis
Explant → Callus Embryogenic → Maturation → Germination
1. Calus induction
2. Callus embryogenic development
3. Multiplication
4. Maturation
5. Germination
Induction
• Auxins required for induction
– Proembryogenic masses form
– 2,4-D most used
– NAA, dicamba also used
Development
 Auxin must be removed for embryo development
 Continued use of auxin inhibits embryogenesis
 Stages are similar to those of zygotic embryogenesis
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Globular
Heart
Torpedo
Cotyledonary
Germination (conversion)
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
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
Somatic embryogenesis as a
means of propagation is
seldom used
 High 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
Peanut somatic embryogenesis
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