Download Organogenesis

Problems in tissue culture
Culture contamination
Vitrification
Acclimatization
Post culture behavior
Culture Contamination
 Two sources:
1. Carry over of microorganism on the surface
or in tissue of explants
2. Through faulty procedures in the laboratory
Culture Contamination
 Cause economic losses, by overrunning the
culture either killing the explant or rendering
it for the subculture
 Affect the productivity both in vitro and of
the progeny plant
Organisms associated with plant surface
 Fungi including yeast
 Bacteria
 Mollicutes (mycoplasmas, spiroplasmas and related
organism)
Endophytic microorganisms
 Intercellular endophytic microorganism
Virus
Viroid
Fastidius prokaryotes
 Intracellular endophytic microorganism
L-forms of common plant associated bacteria
Fastidius bacteria associated with plant vascular tissue
vector transmitted and may be spread by contact between infected
and healthy plant
they are capable of clonal propagation
Aspect of quality control
 Awareness of the range and natural history of possible
contaminant of the crop, including specific pathogen
 Adequate preparation of the donor plant including
treatment to reduce or eliminate pathogens
 Confirmation of the status of culture in stage I following
employment of strategies to obtain healthy cultures and
again based on reliable screening methods
Aspect of quality control
 Rigorous monitoring of production to confirm the status
of the cultures. In large scale production this will
necessitate sampling production and is dependent on an
appropriate sampling protocol
 An awareness that the spectrum of contaminating
microorganism may alter with time in culture
 Monitoring of progeny based on sampling of production
Detection and identification methods
Test
Non specific tests:
Application
Culture indexing
DNA staining
Leaf dip electron Microscope
Gel electrophoresis
Cultivable bacteria
Mycoplasmas and related prokaryotes
Viruses
Viroids
Specific tests:
ELISA
DNA probes
Rapid diagnostic kit
Fatty acid profiling
Viruses and bacteria
All organisms
Bacteria
Bacteria
Vitrification
Translucency
Hyperhydration
Succulency
Glassines
Change into a glassy appearance
Produce fragile plants which have a glassy and
hyperhydrous appearance
Vitrification
 The cells were surrounded by thin walls and contained a relatively
poor and largely vacuolated cytoplasm
 Increased cellular space due to extra protoplastic water
 Many of the chloroplasts lacked of the normal organization into grana
and stroma
 Chloroplasts contained large starch grains whereas the chlorophyl
content was lower
 Defective epidermal tissue
 Faulty deposition if epicuticular waxes
 The guard cell did not function properly
 Abnormal stomata
 Reduced lignifications
Factors associated to vitrification
Physical and chemical state of the medium
1. Type of culture media
2. Gelling agents
3. Organic component
4. Inorganic component
5. Growth regulators
6. Relative humidity
7. Environmental condition
8. Various addition
Acclimatization
Process during which plants or other organisms
become adjusted or accustomed to a new climate
or situation as a result of natural processes
Hardening-off
Moving the complete plants to greenhouse or field
Not unique to micro-propagation
Why acclimatization is important?
The greenhouse and field have substantially
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lower RH
higher light levels
autotrophic growth
septic environment
 stressful to micro-propagated plants compared
to in vitro condition
Common issue
Agar has to be thoroughly washed from the
root
Pesticide may be phytotoxic to some species
of micro-propagated plantlet
Gradually reduce the RH or amount of mist
Maintain light level in the greenhouse at 50%
shade before plants are transplanted to the
field
Acclimatization
 Control environment acclimatization
Specific place that all environmental conditions
can be control either automatically or manually
 Direct field acclimatization
Transferring the rooted plantlet directly to the
field
Important aspect in the
control environment
acclimatization
Humidity
Light
Soil and container
Diseases
Temperature
Nutrient
Control humidity
 Avoid the use of an automatic mist system due to
mist leaches nutrients, causes the medium to
become too wet, allows the plantlets to dry, creates
an environment favorable for the growth of algae
and some fungi and bacteria
 Fogging
 The use of a humidifier
 Placing plantlets in an enclosed area that will water
vapor
 The use of anti-transpirants to reduce water losses
Light
 While in vitro, plantlet has been exposed to relatively low
level of light and their leaves are thin and thus resemble
shade leaves
 Leaves of plantlet place under too high a light level will
become chlorotic and necrotic
 Shading up to four weeks under up to 90% will reduce
transpirational demand and excessive light that can destroy
chlorophyll molecules
 Following a period of shading, plantlet should be gradually
moved to the light level under which they will be grown
 Control of photoperiod is also important to prevent
dormancy or to control plant development
Soil and containers
Requirement
A uniform medium that adequately supports the plants,
has suitable pH, well buffered and sufficiently porous
Inhibitors or dramatic shift in pH in medium can
adversely affect root growth
 Larger container is better
 Peat plugs or small foam blocks are recommended
Diseases
Very essential
Plantlet is generally suitable to diseases- causing
organisms
High humidity is conducive to the growth of many
plant diseases causing fungi and bacteria
An integrated approach of sanitation and
application of pesticides is generally used:
Disinfested medium, new or disinfested container and
benches
New poly-ethylene covers
Clean hand
Clean and disinfested instruments
Temperature
 The temperature of the air and growing medium are
generally controlled
 Adjusting the amount of shading and humidity can aid in
temperature control
 Ventilation and fan system
 Fog and air condition
 The temperature of the root zone is important to
encourage root growth
Nutrient
Nutrient can be originally from the
Media, if the media consists of soil, sand
and compost
Fertilizer may be incorporated or top
dressed in slow release form
A soluble complete fertilizer diluted to
¼ to ½ the recommended rate is
recommended
Direct field acclimatization
 It is possible in some species
 Vanilla, Teak, Potato
 A covering of 40 mesh screen
 Only 6 – 14% survived
 Survival and yield varied among clones
Rooting and acclimatization
1. In vitro rooting
2. Ex vitro rooting
In vitro rooting
Disadvantage:
Lack root hair
Died and collapsed after plantlet was removed from culture,
however new lateral and adventitious root formed during
acclimatization
The transition zone between root and shoot was abnormal
The vascular connection were poorly form
Restricted water uptake
Labor intensive and expensive
Ex-vitro rooting
Direct rooting during acclimatization
Attention must be paid to humidity, light and
temperature
Treatment with root inducing growth
regulators may be required prior to
acclimatization
No agar adheres to the base of the cutting
Post Culture Behavior
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Dwarfs
Color changes or mosaic pattern (Chimera)
Growth habit changes
Change in productivity
Cause:
 When shoots are derived from dedifferentiated cell
 Rapid proliferation of single cells or multi-cellular primordia through
organogenesis or embryogenesis
 In vitro process or by added biochemical and stress agent
 Temporary or heritable deformities
Cause
Variation that existed in the source plant
 Chimera
 Non chimeric chromosomal variation
Genetic changes
 Mitotic abnormality
 Somatic crossing over
 polyploidy
Epigenetic or physiological effect
 A non heritable change in phenotype that occurs in a substantial
percentage of the propagated population through an inducible
directed and reversible process
Chimera
Plant or plant part composed of genetically
different sort of cells as a result of mutation
or grafting
Plant with two or more distinct genotypes
Chimera
Cell origin
Mericlinal chimera
a section of one or two of the histogenic layers are
different
Sectorial chimera
all histogens in a sector are different
Periclinal chimera
one histogen is different from the others
Mimicked by variegated or mosaic forms
Due to en-even distribution of viruses in plant
tissue
Histogen
 Cell layers in all higher plant tissue that trace back to distinct
layers in the apical meristem
 3 layers in angiosperm
 LI an outer epidermal layer
 LII an internal tunica layer
 LIII a cortical layer
 LII layer produce gametic tissue and some surrounding
maternal tissue
 The remaining maternal tissue is also formed by LIII and LI
 Root derived from LII and LIII layers
GROWING POINT (APICAL MERISTEM)
Layer Gives rise to:
L-I
Epidermis of all organs;
Monocot leaves - L-I contributes to the outermost region of
the leaf mesophyll giving rise to a strip along the leaf
margin.
Dicot leaves - L-I usually gives rise to only the colorless
epidermis, thus cannot be seen; sometimes L-I gives rise to
small islands of tissue along the margin
L-II Stem and roots:
Outer and inner cortex and some of vascular cylinder leaves:
mesophyll in outer region of leaf
L-III
Stem and roots:
inner cortex, vascular cylinder and pith leaves: mesophyll in
central region of leaf
Non chimeric
chromosomal variation
Breakage in heterochromatic region
Somatic crossing over (mitotic exchange
between homologous chromosomes)
Gene amplification due to mutagenic
agent
Permanent genetic change
 Somaclonal variation
 Genetic change
 Polyploidy
 Aneuploidy and breakage
 Micronucleus formation
 Bi- or multi nucleate cells
 Duplication
 Recombination
 Inversion
 Amplification
 Simple base pair change
 Organelle genome variation
 Isozyme differences
 Expression of cryptic transposable element
 Change in chromosome structure
Definition
• Euploidy
An even increase in number of genomes (entire
chromosome sets)
• Aneuploidy
An increase in number of chromosomes within a
genome
Euploid
Euploid
Symbol
Somatic (2n)
monoploid
x
(ABC)
diploid
2x
(ABC)(ABC)
triploid
3x
(ABC)(ABC)(ABC)
autotetraploid
4x
(ABC)(ABC)(ABC)(ABC)
allotetraploid
2x+2x'
(ABC)(ABC)(DEF)(DEF)
Aneuploid
Aneuploids
Symbol
Somatic (2n)
Description
nullisomic
2x-2
(AB)(AB)
(missing a chromosome set)
monosomic
2x-1
(ABC)(AB)
(missing a chromosome)
double monosomic
2x-1-1
(AB)(AC)
(missing 2 different
chromosomes)
trisomic
2x+1
(ABC)(ABC)(A)
(additional chromosome)
double trisomic
2x+1+1
(ABC)(ABC)(A)(B)
(2 additional different
chromosomes)
tetrasomic
2x+2
(ABC)(ABC)(A)(A)
(2 additional chromosomes same)
trisomic-monosomic
2x+1-1
(ABC)(AB)(A)
(missing a chromosome +
additional chromosome)
Plant variation from
dedifferentiated cell
Mitotic asynchrony caused by growth regulator
effect on DNA biochemistry (2,4,5-T; 2,4-D;
antibiotic; alkaloid; physical mutagen)
Disorientation or dysfunction of the mitotic
apparatus (spindle fiber)
Selection pressure due to the change in plant’s
environment
2,4-D
Increase growth and reduced cell cycle time
Stimulate DNA synthesis
Endo-reduplication lead to nuclear fragmentation
Increased mitotic crossing over
Increase poly-ploid
Temporary alterations
 Altered flowering, sex expression, fertility and yield
 Increased vigor and root-ability
 Increased branching
 Expression of off-type and off-color phenotypes
 Alter susceptibility to diseases and biochemical including
herbicide
 Rejuvenation
Rejuvenation
Bring back to youthful appearance (juvenile)
Juvenility:
The condition of a seedling plant that prevents
flowering or sexual gameto-genesis