permasalahan kultur jaringan egl by Q4r3Y1

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									       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          12
     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            Material     Concentration      Long Soaking
 1    Kalsium hipoklorid   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 klorid-
   aquadest 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

								
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