HRT 4701 : PLANT MICROPROPAGATION

HRT4701
PLANT
MICROPROPAGATION

Semester 1, 2016-2017

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HRT 4701 : PLANT MICROPROPAGATION Chapter 4 Explant

Previous lecture
Explant – What is explant?
Part (cell, tissue or organ) of a plant that is
used to start in vitro culture
Type of explant?
Seed, embryo, organ, callus, etc
Types of culture
Embryo culture, somatic embryogenesis,
meristem culture, shoot tip culture, callus and
cell suspension culture, protoplast culture
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HRT 4701 : PLANT MICROPROPAGATION Chapter 5 General in vitro technique

Chapter 5

General in vitro technique

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HRT 4701 : PLANT MICROPROPAGATION Chapter 5 General in vitro technique

Learning Outcome
Scope:
1. What is the steps involved in TC?
2. How to sterilize?
Learning outcome:
• To learn basic technique in TC and
sterilization method

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HRT 4701 : PLANT MICROPROPAGATION Chapter 5 General in vitro technique

General in vitro technique

Basic skill and technique required in
tissue culture :
Explant isolation / excision
Sterilisation technique
Inoculation / culture
Subculture
Decontamination
Laboratory safety
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HRT 4701 : PLANT MICROPROPAGATION Chapter 5 General in vitro technique

1. Explant Isolation / Excision

Stock plant isolation in controlled
environment – glasshouse pre-treatment :
Avoid too high humidity
Control irrigation
Pest / disease control
Explant isolation – difficult. Why?
Explant exposed to myriads of micro-
organisms
Micro-organism – contamination
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HRT 4701 : PLANT MICROPROPAGATION Chapter 5 General in vitro technique

1. Explant Isolation / Excision (cont.)

Explant – shoot tip, cutting
Clean of all debris
Wash with kitchen detergent – rinsed under
running tap water
Cut as recommended for species
Transfer explant to medium after sterilisation

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HRT 4701 : PLANT MICROPROPAGATION Chapter 5 General in vitro technique

2. Sterilization technique
To prevent bacterial, fungal and viral
contamination
Technique for sterilisation of glasswares,
equipment, liquid, explant :
Dry heat sterilisation
Steam sterilisation
Filter sterilisation
Chemical sterilisation
Other method / technique 8
HRT 4701 : PLANT MICROPROPAGATION Chapter 5 General in vitro technique

a) Dry heat sterilisation

For glassware, equipment, others which
resist high temperature
Cotton, paper, plastic – cannot be
sterilised in dry heat
Using hot air with no or minimal moisture
Wrap in aluminium foil, oven 4 hr (160°C
(320°F))

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HRT 4701 : PLANT MICROPROPAGATION Chapter 5 General in vitro technique

b) Steam sterilisation
Use of autoclave / pressure cooker
Steam under high pressure
Temperature : 121°C
Pressure : 15 p.s.i
Time : 15 – 30 minutes (depend on what
item)
Autoclave also used to sterilise
contaminated cultures and old cultures

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HRT 4701 : PLANT MICROPROPAGATION Chapter 5 General in vitro technique

Pressure cooker Common autoclave in lab Autoclave tape

Dental autoclave Autoclave for industry

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HRT 4701 : PLANT MICROPROPAGATION Chapter 5 General in vitro technique

c) Filter sterilisation
Ultra-filter/ micro-filter sterilisation
Specific for hormones, enzymes, vitamins
Heat labile
Eg: GA3, IAA
Sterilisation at room temperature
Use of filter : Millipore®
Membrane pore size : 0.22 µm
Prevent passage of micro-organisms
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HRT 4701 : PLANT MICROPROPAGATION Chapter 5 General in vitro technique

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HRT 4701 : PLANT MICROPROPAGATION Chapter 5 General in vitro technique

d) Chemical sterilisation
Surface sterilisation of explant
Using disinfectant :
Alcohol (methanol, ethanol, iso-propanol)
Sodium hypochlorite, NaOCl
Calcium hypochlorite, CaOCl2
Mercury chloride, HgCl2
Clorox® : 5.25% NaOCl
+ Teepol / Tween 20®
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HRT 4701 : PLANT MICROPROPAGATION Chapter 5 General in vitro technique

Other - Antibiotic (antibacterial)

Internal infection (bacteria)
Antibiotic added to medium
Initial stages of culture
Not highly recommended because it can
modify growth of explant
Examples - Penicillin G (100 mg/L)
- Aureomycin (50 mg/L)
- Gentamycin (50 mg/L)
- Streptomycin (100 mg/L)
- Nystatin (25 mg/L) 15
HRT 4701 : PLANT MICROPROPAGATION Chapter 5 General in vitro technique

Other – Radiation
Seldom used – high cost, trained personel
Use for heat labile equipments
Disposable wares (syringe, needle, swabs)
Eg: UV : in laminar air flow
X-ray
gamma ray (60Cobalt)

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HRT 4701 : PLANT MICROPROPAGATION Chapter 5 General in vitro technique

3. Inoculation
Transfer of explant on to medium in
laminar air flow
Sterilise equipment / work area
Use UV light before start
Sterilise bench surface
Sterilise using 70% ethanol
equipment using 70% ethanol

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HRT 4701 : PLANT MICROPROPAGATION Chapter 5 General in vitro technique

4. Subculture
Transfer of explant to fresh medium when:
Medium depleted / contaminated /
overcrowded
Medium changes in form (colour, dry)
Every 2-6 weeks

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HRT 4701 : PLANT MICROPROPAGATION Chapter 5 General in vitro technique

5. Decontamination
Sterilising old cultures / contaminated
cultures
Autoclave prior to cleaning
Culture vessels may be re-used

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HRT 4701 : PLANT MICROPROPAGATION Chapter 5 General in vitro technique

6. Laboratory safety
Use with care flammable products
Gas source closed after use
Avoid exposure to UV source
Care in using sterilants (disinfectant)
Use gloves/mask when necessary
Care when using autoclave

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THANK YOU

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