PROBLEMS IN TISSUE
CULTURE
 There are problems that can disrupt
and cause tissue culture activity goals
not reached
 In General, disorders comes from:
- materials planting (explant)
- environmental culture
- Human
1
• Problems relating to the material planting 
usually appears at the beginning of the
growth phase :
˜ The quality of material planting/ explant is low
(less well)
˜ The stagnation of growth
˜ Uncontrolled growth
˜ Contamination
˜ Browning
˜ Vitrification
˜ Genetic variability (can be viewed also as
potential
2
 Problems related to environmental factors:
- The demise of the flow of electricity
- Damage of Air Conditioner
- Contamination
 Problems related to human beings:
- Carelessness
- Negligence
- Low level of skills
3
CONTAMINATION
 Sterile condition is essential for success in the
tissue culture procedures  The aseptic
condition are necessary to bottle culture, media,
and planting equipment and explant
 Sterilization is one of the procedures used for
eliminating microorganisms or efforts for
freeing environmental from contamination by
microorganisms
4
Contamination  occurs as a consequence of use
of the media –enriched  the enriched a media,
then the level of contamination is also increasingly
and simple media components, the possibility of
contamination is getting low
VW
Kudson
MS
5
• Contamination is very diverse, as seen from:
- Types of contaminants (bacteria, fungi, etc.)
- Time of occurrence (fast  can emerge within
hours; medium  can emerge within days; slow
 can emerge within month)
- What is contaminated (media or explant)
Need specific methode to each
contamination
6
 Based on differences in objects, sterilization can
be classified in three categories, namely:
1. Sterilization of work environment.
2. Sterilisation of tools and media.
3. Sterilization of plant material (explant)
 The kinds of sterilization forms are distinguished:
a. Sterilization with the heating (dry and wet)
b. Sterilization with ultrafiltrasi
c. Sterilization with chemicals
 Sterilization with moist heating:
- Use steam heated  autoclave
- Almost all microbes die at temperatures of 120 0C
- Time  depend on volume
 Sterilization with dry heating:
- Using the oven  the tools are not flammable
(material glass)  long time warming up ± 45
minutes, the temperature of 160 0C)
• Sterilization using flame:
- Tools dipped in alcohol, then burned
- Used for inoculation activities, planting of
explant, etc.
 Sterilization with chemicals:
- Used to sterilize the surface
eg: explant, instruments, hand, room,
or LAF
- Chemicals that are commonly used 
alcohol, calcium hypochlorida, sodium
hypochlorida, sublimat and chlorox
• Sterilization using light:
- Used on space/room and LAF
- Using ultra violet rays
Sterilization of Media and Tools



Glass tools, equipment of metal or other materials
not easily damaged by high temperature 
generally, sterilization with dry heating or wet
warming
Dissecting set and glass ware  autoclave 121 0C
about 20 – 30 minutes, then save in the oven at a
temperature of 106 0 C for a few minutes
Dissecting set  dipped in alcohol 96% then
burned before use
10
The kinds of Autoclave
 Media sterilization should not be too long because it can
cause:
a. The decomposition of sugar.
b. The degradation of vitamins and amino acids
c. Inactivation of sitocinin (zeatin riboside)
d. Change in pH  cause depolymerization of compactor
.
• The suggestion of a minimum time for media sterilization
Media Volume (ml)
Long time (121 0C)
20 – 50
15 minutes
75
20 minutes
250 – 500
25 minutes
1000
30 minutes
1500
35 minutes
2000
40 minutes
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Sterilization of Culture Equipment
1. Clean bottles given some drops of aquadest
and cover with paper or aluminum foil . To
have the bottles lids autoclaveable, don't
close it too tight, because during the
heating expansion occurs.
2. Tools need to be sterilized before
planting are: scissors, tweezers, scalpel
handles, filter paper, petridish, empty
bottles, and needles.
3. Alat-alat dan kertas saring dibungkus rapi dengan kertas
tebal atau ditaruh dalam baki stainless steel dan bakinya
dibungkus dengan kain tebal sebelum dimasukkan
dalam autoklaf. Alumunium foil tidak direkomendasikan
sebagai pembungkus, karena uap tidak dapat masuk ke
dalam bungkusan. Alat-alat sektio seperti pinset,
gunting, gagang skalpel, dan jarum, dibungkus dengan
kertas kopi atau kertas merang. Hindarkan penggunaan
Al-foil karena uap sukar masuk kedalam bungkusan
sehingga sterilisasi kurang efektif.
4. Petri-dish akan disterilkan, juga dibungkus dengan
kertas kopi atau kertas merang.
3. Tools and filter paper wrapped with a thick paper or put
tray in stainless steel and the rest were wrapped with a
thick cloth before you put in an autoclave. Tools such as
tweezers, scissors, and a needle, scalpel, wrapped in paper
copies. Avoid using of Al-foil because steam is difficult to
enter the parcel, that the sterilization less effective.
4. Petri-dish would be sterilized, also wrapped in paper
copies or paper volvacea.
5. Temperature used for empty bottle sterilization and the
tools that will be used to explant planting is 121oC at 15
psi pressure (pounds per square inch) or 1 atm for 30-60
minutes. Sterilization time calculation begins after the
desired pressure and temperature has been reached.
Sterilization using the oven
• Bottles/reaction tubes/erlemeyer which is used
as a container, usually are sterilized in the
oven.
• The bottles were washed, put in the oven and
heated for 4 hours at a temperature of 160 oC.
After sterilized can be directly used.
• When the bottles are stored for some time, then
during the sterilization, the mouth of the bottle
should be covered with aluminum foil.
Explant Sterilization
Tissue culture/culture in vitro include:
planting cells/ cells aggregate, tissues, and
plants organs in the media.
Growth media  very profitable for fungi and
bacteria growth  so in the initiation of culture
should be arsenic cultures (culture consisting only
of one kinds of organisms  plants tissue )
 Factors that affect to surface explant
contaminants :
Type of vegetation.
The partt of plant is used.
Surfaces morphology (e.g.: hairy or not).
Growth environmental (green house/field)
The time of taken (rainy/dry).
The age of the plant (seedling or plant
maturity).
• Plant conditions (sick or in good health).
•
•
•
•
•
•
 It is difficult to determine a standard
sterilization methods for a type of plants
 in different places need a preliminary
experiment
• In tropical countries, this surface
contamination is usually serious thing 
so some stages of sterilization must be done.
Explant Sterilization Techniques
Some materials for surface explant sterilization:
No
1
Material
Kalsium hipoklorid
Concentration
Long Soaking
1 – 10 %
5 – 30 minutes
2
Natrium hipklorid
1–2%
7 – 15 minutes
3
Hidrogen peroksida
3 – 10 %
5 – 15 minutes
4
Gas klorin
-
1 – 4 hours
5
Perak nitrat
1%
5,30 minutes
6
Merkuri klorid
0,1 – 0,2 %
10 – 20 minutes
7
Betadine
2,5 – 10 %
5 – 10 minutes
8
Benlate
2 gram/lt
20 – 30 minutes
9
Antibiotik
50 mg/lt
0,5 – 1 hours
10
Alkohol
70 %
0,5 – 1 minutes
Materials sterilization, in general are toxic to the
plant tissues  need flushing numerous times after
soaking in solution sterilization to eliminate remain
of the active ingredient that is stuck to the surface of
the explant.
In the sterilization, sometimes used two or more
materials sterilization, for example: soaking in the
alcohol first, then the sodium hipoklorid and rinsed.
It can also soaking solution starts with fungicides or
antibiotics, then Mercury klorid, and rinsed with
sterile water.
Modifications in the implementation of
sterilization can be performed using:
a. Fungicides/antibiotics – mercury klorid – rinse
with sterile aquadest
b. Alcohol-sodium hipoklorid-mercury kloridaquadest sterile.
c. Alcohol-sodium hipoklorid – betadine –
aquadest sterile.
d. and so on, depending on the materials used.
Sterilisasi pada daun africa violet ( Saintpaulia ionantha), sterilisasi dimulai
dengan mencelupkan daun pada alkohol 70%, dipindah ke larutan bleach 0,5%,
dipindah ke larutan bleach 1 %, dipindah ke alkohol 70%, selanjutnya di bilas
menggunakan aquades steril 4 kali (sumber: JA Negrón, UIPR Barranquitas, 2006)
Browning
• Browning  a character appearance of brown or
black often make no occurrence of the growth and
development of explant
• Browning  can be caused by the media and
variety of media suplemen, the use of sterilization,
injure, use of fire, etc.)
• Browning is considered as disrupt due to
symptoms of browning is generally a sign of the
decline of explant physiology and often end up the
death
 Based on the process, browning or blackning
are grouped into two, namely:
1. Browning by enzymatic (Fenolase)
2. Browning by non enzymatic:
- Maillard - Browning
- Caramelization Browning
- Oxidation of Ascorbic Acid Browning
1. Browning by enzymatic (Fenolase)
 occure by enzymatic, usually acting of enzyme
polyphenol oxidases (e.g.: phenol hydroxylase,
kresolase, katekolase)
.
• For occuring of browning reaction  beside
substrate, need prosthetic group of Cu 2+ and
oxygen as hydrogen acceptors
• The basic reaction: the formation of melamine are
brown
hidroksiquinon
Polimerization
Polymer
Melamin
(brown)
• Browning By enzymatic, its substrate role: p-difenol
(quinol), flavonoids, monofenol, katekol, tanin, kafeat
acid, klorogenat acid, protokatekoat acid
• In mechanics, browning because:
- Injured : (usually in the plant culture containing
the hidroksiphenol and tanin
-Heating: on the explant that contain a lot of sugar
• Chemical stimuli on browning  because on
explant environment available chemicals that
encourage formation of phenol
• for example: Auxin on young leaf of oil palm push
browning; the presence of oxidase enzymes phenols
•
• How to resolve of Browning
1. Removing phenol compounds  continuous
rinsing subculture, use of active charcoal
2. Modify the media redox potential
3. Reducing agents that cause the occurrence of
browning  reduces the number of carbohydrates
media in media, reducing contact with oxygen
4. Inhibits oxidase phenols enzyme  chelating
agents
5. Setting the low pH  optimum working at pH 6.5
polyphenols oxidase and decreases with
decreasing pH
6. Use of dark room  efectivity of polyphenols
oxidase work influenced light
Vitrification
• Vitrificati on  a term problem in cultures that
are marked:
- The emergence of growth and development
that is not normal
- The plant produced short or dwarf (often don't
have internodus)
- The growth of the stem tends towards the
addition of diameter
• Its leaves have a tendency on the part of the
base widens, eventually forming the leaf like
an arrow
• The leaves have chlorophyll which is less
than the normal
• Stalk and the leaves look translucent and
fragile
• Generally the leaves do not have palisade
- Vitrification  occure as effect from the failure or
absence of barriers in the process of the formation of
cell wall (parencym tissue ) & obstacles in the process
of formation of lignin
- How to Resolve:
• Raise the amount of agar and sucrose
• Add the pectin into media
• Move the culture at a temperature of 4 0C for 15
days
• Lowering the pH of the medium into 4
• The use of the compound CaCl2 on desicator
• Using semi solid media
Genetic Variability
• Become a constraint  if tissue culture for duplication
uniform plant effort uniform in large quantities and
not as an attempt of plant breeding
• Genetic variability may occur due to:
- The rate of multiplication is very high  mainly due
to recurring subculture uncontrolled
- The use of chemicals in certain levels  can be as
mutagenic
- The use of a technique  that perhaps occure the
variabillity
Growth & Development
 The main problem  If the explant be grown
stagnated, growing from the start up to a certain
period did not die but not growing
• Stagnation can be caused by:




The materials/explant are not meristematic
Sterilization is overload
Media does not match/suitable
The environment is not supported
Attempt:
• Avoid the use of explant which does not
meristematic
• Proper sterilization
• Suitable media  tend to precision both kinds
of materials and the size
• Modification of the media
• The combination of hormones
• Special treatments (cold stress, off light, light
periodization)
Pre- Treatment
• Pre treatment  is done for the purpose of eliminating
barriers, physical and biological khemikalis
 Chemicals barriers  the handling begin with the
introduction of active compounds, potential distractions,
trigger, the process of reaction and an alternative to
manage them
 Physical barriers  generally occurs on the explant
which having a physical strong patron (very hard skin)
 by eliminating the protective or parts that are not
needed in the culture
 Biological barriers  one relating to contamination,
juvenility of explant (with a trim plant stems for
pushing the emergence of buds)
Micro - Environment
Light
 The low intensity  can increasing of embryogenesis
and organogenesis.
• Ultra violet light can pus the growth and shoots
forming from tabacco callus on low intensity.
• The maximum callus formation more occure in dark
place
Temperature
• Optimum growth: 20 -30 0C.
• Endosperm callus growth: 25 0C.
Equipment, Electricity,
Water, Human
• Inadequate equipment, broken, can't wear, electric
water not drain dead, and carelessnes human 
cannot ignore because it will disturb with the
activity of tissue culture
• Necessary tools modifications, maintenance, tool
usage, courses, providing electric generators, a rule
or warning in the laboratory