1. Sterile pieces of a whole plant from which cultures
are generally initiated
Explants
• The smaller the explant the better the
chances to overcome specific
phytopathological problems (virus,
microplasm, bacteria), but it decreases the
survival rate
2. 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
4. 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
5. 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
6. Embryo culture
Growing embryo aseptically in
vitro on artificial nutrient media
Overcoming seed dormancy and
self-sterility of seeds
Study embryo development
7. 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
8. 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
10. 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
11. 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
12. 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 from the living germs of disease and fermentation
-Conditions established to exclude contaminants
13. Axenic culture
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
Pure cultures that are completely free of the presence of
other organisms
14. Source of contamination
The explant or culture
The vessels
The media
The instruments
The environment where handling is taking place
15. Aseptic Techniques
Chemical treatments
• disinfectants,
• antibiotics,
• sublimat
Physical treatments
• heating: the most important disinfection method
• electromagnetic radiation,
• filtration
• ultrasonic waves.
16. 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.
17. 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
18. 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
• tend to be selective
• resistance acquisition
• Make unclear, the presence of microbes
• cell/tissue growth inhibition
19. An ideal antibiotics
Broad-spectrum
Did not induce resistance
Selective toxicity, low side effects
Preserve normal microbial flora
19
BC Yang
20. 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.
20
Amphotericin
Tetracyclines
Aminoglycosides
vancomycin
BC Yang
21. 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.
21
BC Yang
22. 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
23. 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
24. 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
25. 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 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
29. 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
31. Stimuli :
In vivo : wound, microorganisms, insect feeding
In vitro : Phytohormones
1. Auxin
2. Cytokinin
3. Auxin and cytokinin
4. Complex natural extracts
Callus formation
32. 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.
•
33. 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
34. 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.
36. Protoplast
The living material of a plant or bacterial cell, including the
protoplasm and plasma membrane after the cell wall has been
removed.
37. 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
38. 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 development as cell division, cell differentiation and
morphogenesis overlap
39. 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.
41. 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.
45. 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
46. Various terms for non-zygotic
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.
47. 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.
50. 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
52. 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
