HRT4803

PLANT
MICROPROPAGATION

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 Last week lecture
Media
3 major components
• Inorganic - Major N P K Ca Mg & S
- Minor Fe Mn Zn B Cu Co Mo

• Organic - Carbohydrates, CHO (sucrose)

- Vitamins (Vit C, B complex)

• PGRs - Auxins – cell enlargement, root initiation,

adventitious bud formation - (2,4-D, IAA,
IBA, NAA, dicamba, picloram)
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 Last week lecture (cont.)
PGRs – cytokinins – cell division, shoot multiplication,
auxillary bud proliferation (BA, BAP, 2iP,
Kinetin, Zeatin)

• Undefined components
- Activated charcoal – absorb toxin
- Agar – solidifier
- Coconut milk, banana, potato – add on / supplements

• Medium nature – optimum pH – 5.5 – 5.8

before autoclave (↓ 0.3 – 0.5 after autoclave)
• Medium stock - why prepare stock? Infrequent
preparation, accurate, cheap, fast 3
Chapter 4

Explant

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 Learning Outcome
Scope:
1. Which plant part is suitable for TC?
2. Types of culture
3. Selection of explant for TC
Learning outcome:
• to learn how to select suitable explant for
TC based on the type of culture, vice
versa
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PLANT MICROPROPAGATION
2 major components in tissue culture :
> nutrient medium
> explant

Plant tissue culture is a marriage of

EXPLANT and NUTRIENT MEDIUM
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What is explant?
Explant
Part (cell, tissue or
organ) of a plant that
is used to start in vitro
culture

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 Types of Explant
Seed :
seed culture
Embryo :
immature embryo
Organ :
meristem, shoot tip, root tip, anther
Callus :
callus – unorganized mass of cells
Others :
cell, protoplast
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 Seed
culture

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Shoot tips from in
vitro culture

Embryos
developing from
callus

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Eurycoma longifolia –
Tongkat Ali

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 Types of culture
Embryo culture
Somatic embryogenesis
Cell culture
Callus Culture
Protoplast culture
Meristem culture
Shoot tip culture

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1. Embryo culture
Culture of immature embryo
Can mean : rescue of an embryo from
seed, sowing fine seed
For example : orchid
Seeds – fine like dust, no endosperm –
germination by symbiosis with mycorrhiza
Artificial germination – asymbiotic
germination on defined medium
Orchid seed germination  seed culture =
embryo culture 16
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2. Somatic embryogenesis
Cells from somatic tissues (= vegetative,
or asexual) induced to form embryos 
somatic embryos
Formation of embryos from somatic cells
 somatic embryogenesis

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Plant regeneration
through direct somatic
embryogenesis from
leaf explants of
Phalaenopsis amabilis

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3. Cell culture
Cultivation / multiplication of cells (in solid
or liquid medium)  cell culture
Cells cultivated / multiplied in liquid
medium  suspension culture or,
suspension cell culture
Source of the cell – callus culture

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4. Callus culture

Callus  mass of disorganized, mostly

undifferentiated or undeveloped cells
Cultivation / multiplication of callus (usually
on a solid medium)  callus culture

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5. Protoplast culture
Protoplast  cell without a cell wall, but has
a membrane
Protoplast culture  culture of protoplast,
usually in liquid medium
protoplast fusion  hybridizing two protoplast
to form a hybrid, cybrid (cytoplasmic hybrid)
Cybrid – cells or plants containing nucleus of
one species but cytoplasm from both the
parental species
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Protoplast fusion

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6. Meristem culture
Apical meristem  new group of
undifferentiated tissues at the microscopic
tip of a plant shoot

apical meristem  virus free

Culture of apical meristem  meristem
culture
Resulting plantlets  virus free 25
7. Shoot tip culture
Culture of shoot apices
Include : meristem tissue leaf primordia, etc

Shoot tip  not virus free

Easiest  most widely used method for
commercial micropropagation
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 Explant
Culture only pathogen-free explant
• Pathogen : fungus, bacteria, virus
• (introduction / identification of pathogen –
not covered in this course)
Preparation of explant before culture :
- isolation
- maintenance
- therapy
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 How to obtain explant
Stock / mother plant placed in isolation
(greenhouse / glasshouse)
• 2 weeks – 6 months before culture

Stock plant maintenance :

• Irrigate with clean water only
• Allow to dry
• Fertilise and irrigate at base of stock plant
only
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 How to obtain explant (cont.)
Choose juvenile part
• Younger  better response
Cut explant source bigger (size) than final
culture size
Maintain cleanliness from location of origin
to laboratory

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Problem associated with explant
Contamination by microorganism : fungus,
bacteria
Contamination : damage to culture

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 Explant treatment
Treatment of explant after selection ~
variables
Procedures depends on status of explant
General procedures :
• Keep under running water
• Alcohol : ethanol, iso-propanol
• Sodium hypochlorite, NaOCl
• Clorox® : 5.25% NaOCl
• + Teepol / Tween 20 ® - spreader to enhance
effect of sterilants 33
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African Violet

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THANK YOU

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