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In Vitro Developmental
Pathways
Mother or donor
plant
drip irrigated mother plants
•is a plant used as a source of explants and selected for its
genotype, hygienic or physiological status
•see also stage O : improving hygienic and physiological
status and preconditioning of mother plant
Explant
Explant
Inoculum
a piece of plant material isolated from mother plant to be
cultured
a subculture of plant material which is already in culture
For 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
•influence of location on the mother plant (topophysis, cyclophysis and
periphysis)
•influence of juvenility status
•influence of polarity
Location
Regeneration can be influenced by the position of
the explant on the plant or by different physical
exposure:
Juvenile is different from young. Young refers to the age, and is the opposite of old.
Juvenile is the opposite of adult, and can refer to:
•the period in the lifetime that the organ was initiated (sleeping buds at the base
of a stem were initiated on the seedling);
•the initiation period of an organ on which new structures develop (root sprouts
develop on roots which were originally initiated in the seedling stage);
•the proximity to the root system.
Juvenility in several hard-woods:
•Density of crosshatching
indicates degree of juvenility.
•In the juvenile zone, note the
single trunk, retention of
leaves
close to the trunk in winter,
and
obtuse branch angle. In the
mature
zone, note the forked trunk
(Bonga and Durzan,
and
1982)
acute branch angles.
Juvenility gradients in trees
Juvenility in a regulary shaped
conifer:
the degree of juvenility of an apical
meristem is inversely proportional to
the distance (along trunk and
branches)
between the root-shoot junction (A)
and the meristem. The distance
AB>AC>
AD>AE>AF, and therefore, meristem
B is
the most mature, and meristem F is
the
most juvenile.
(Bonga and Durzan, 1982)
Juvenility and propagation
In vitro propagation of conifers is generally only possible with
sections of embryos (A) or young germinants (B). Thus in vitro
propagation capability is already lost in young seedlings (C),
but these can still be propagated by conventional rooting or
cutting techniques. Cuttings taken from these seedling's root
readily and generally produce true-to-type propagules.
Cuttings from a somewhat older tree (D) have a reduced
rooting capacity, and the propagules often show varying
degrees of plagiotropic growth. Cuttings from mature trees (E)
do not root.
Terminology
Tissue culture is an aseptic technique
asepsis
avoiding contaminations using sterilization procedures
axenic
free from association with other living organisms
sterilization
killing or excluding microorganisms or their spores with heat,
filters, chemicals or other sterilants
An elaborated example of surface sterilization
•Cut the plant material to an appropriate size to fit the
container
which will be used during the sterilsation procedure
•Rinse plant material under running tap water
•Shake for a few seconds in alcohol
•HgCl2 (0.1 - 1%) + Teepol 2 drops/100ml: 3-5 min
•Rinse in autoclaved distilled water
•Commercial bleach 7-15% + Teepol 2 drops/100ml:
10-30 min
•Rinse several times in autoclaved distilled water
sterilization of orchid flower
stalks
Notes:
•Tap water: to remove large debris, detergent can be used to shrub the surface
•Ethanol: 70% is most effective to denaturate proteins. Sometimes 95% is used to
dissolve the surface wax layer
•NaOCl (commercial bleach) or Ca(OCl)2 : activity is based on both
Cl- and other oxidation reactions
•HgCl2* activity based on Cl- and heavy metal denaturation of proteins.
•wetting agent (e.g. “Teepol”) can be added to the forementioned agents
* 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
Sterilization of culture media
Thermostabile ingredients are autoclaved; thermolabile ones are filter
sterilized
•Autoclaving: adequate autoclaving requires that the medium is maintained at a
choosen temperature and at the corresponding pressure for a given time; e.g. 50
kPa corresponds to 112°C, and 100 kPa to 120°C
•Filter sterilization: disposable filter membranes are first autoclaved; the filter pore
size is not greater than 0.22µm; sterilized solutions are filtered a sterile container.
Autoclaves
Bench top model
(pressure cooker
type)
Vertical
autoclave,
also used as
medium
preparation
device
Top view of left
figure
Vertical
autocalve,
laboratory
model
Horizontal
autoclave
Sterilization of tissue culture containers, working tools and
environment
•glassware: autoclaving, dry heat (180°C a few hours), radiation
•disposable containers: radiation
•paper: dry heat
•working tools: flaming or heat (flame, glass beads)
•bench surface: swapping with alcohol
•environment: laminar flow cabinet (HEPA-filtered)
sterilizing paper: dry heat
sterilizing tools
laminar flow cabinet
Explants
• Pieces of organs
– Leaves
– Stems
– Roots
• Specific cell types
– Pollen
– Endosperm
– Nucellus
Callus
• Unorganized, growing mass of cells
• Dedifferentiation of explant
– Loosely arranged thinned walled, outgrowths from
explant
– No predictable site of organization or differentiation
• Auxin + cytokinin
• Often can be maintained indefinitely by
subculture, but may lose ability to redifferentiate
• Compact vs friable
• Habituation
Three stages of callus culture
• Induction: Cells in explant dedifferentiate
and begin to divide
• Proliferative Stage: Rapid cell division
• Differentiation stage (sometimes):
metabolic pathway or organogenesis
Callus
© 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS
Induction
© 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS
Division
E. Sutton, UC Davis
Differentiation
• Organogenesis
• Somatic embryogenesis
Cell and Suspension Culture
• Cell Cultures?
• Suspension Cultures
Suspension cultures
• Can be initiated
from any part of the
plant.
• Usually initiated
from friable callus
already growing in
culture.
• Transferred into
liquid medium.
Agitation
• Breakdown of cell aggregates into
smaller clumps of cells
• Maintains a uniform distribution of
cells and cell clumps in the medium
• Provides gas exchange
Medium
• Same as for
callus culture?
• Gamborg B5
• Conditioning
Growth Curve
E. Sutton, UC Davis
Batch Cultures
• A certain number of cells is used to
inoculate the culture, in a given
volume
• Erlenmeyer flask: volume should be
about 20% of flask capacity for
aeration.
• Roller cultures
Continuous Culture
• Bioreactors
• Closed continuous cultures: Remove
some of the media and replace with fresh.
Continuous removal or periodic.
Terminate growth at harvest. Start over.
• Open continuous culture: Not only remove
some of media, but cells too. Maintain cell
density at optimal level. Can be grown for
years.
Why is it possible to regenerate in vitro?
• Totipotency
– Initial state
– Competence
– Determination
– Differentiation
Only occurs in a few cells in culture.
Why?
• Pre-determination prior to culture
• Newly formed meristems may act as sinks
 Meristematic centers might actually
produce compounds that inhibit
neighboring cells.
Organogenesis
The formation of organs (such as
leaves, shoots, roots) on a plant
organ, usually of a different kind.
Organogenesis
• Rule of thumb: Auxin/cytokinin 10:1100:1 induces roots.
• 1:10-1:100 induces shoots
• Intermediate ratios around 1:1 favor
callus growth.
Indirect organogenesis
Explant → Callus → Meristemoid → Primordium
Indirect Organogenesis
• Dedifferentiation
– Less committed, more plastic
developmental state
• Induction
– Cells become organogenically
competent and fully determined for
primordia production
– Change in culture conditions?
• Differentiation
© 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS
© 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS
© 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS
Direct Organogenesis
Collin & Edwards,1998, Plant Cell Culture
http://www.liv.ac.uk/~sd21/tisscult/publications
.htm
Somatic Embryogenesis
 Parthenocarpy
 Apomixis
 In vitro somatic embryogenesis
Soybean – Wayne Parrot, UGA
Somatic Embryos
• Bipolar
• Not connected to explant or callus
cells by vascular tissue
• In most woody plants, tissue must be
juvenile or reproductive
Indirect Somatic
Embryogenesis
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
–
–
–
–
–
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
Rubber tree from somatic
embryo
CIRAD
Factors that Influence SE
•
•
•
•
Genotype
Growth regulators
Carbon source
Nitrogen
Maturation and Germination
(Conversion)