Plant Tissue Culture

Basic Principles
of Plant Tissue Culture

Plant Tissue Culture

ure of plant protoplast, cells, tissues, organs, seeds or other plant parts in a sterile environment on a nutrient

Plant Tissue Culture (PTC) is a form of asexual propagation of plants under laboratory conditions
artificial vegetative propagation

PTC is widely used to produce clones of a plant in a method known as micropropagation

1
 Plant Tissue Culture

The explant grows into a callus

The callus develops into plantlet

Plant Tissue Culture

Take some meristematic cells from a plant (explant)
Place the explant on a sterile nutrient rich agar
The explant grows into a callus
The callus then develops roots, stem and leaves (plantlet)
Transplant the plantlet into a traditional growing media
 Callus
An amorphous mass of loosely
arranged thin-walled parenchyma cells arising from the proliferating cells of the parent tissue cultu

Dedifferentiation: the phenomenon of mature cells reverting to a meristematic

state and forming undifferentiated callus tissue.

Redifferentiation: The ability of the component cells of the callus to differentiate into a whole plant or organ

damental principles
t Tissue Culture depends upon
potency: It is the ability of plant cells to regenerate into a whole plant (when given the correct condition)

ticity: It is the ability of plants to alter their metabolism, growth and development to best suit their environm
 Totipotency
the ability of plant cells to regenerate into a whole plant

Phenotypic plasticity of Arabidopsis thaliana rosettes in

different growth regimes.

Plants were grown in climate chambers under

Low, Normal or High Light (LL, NL and HL; 30,
300 and 600 μmol quanta m-2 s-1, respectively) and under field conditions.
 History of Plant Tissue Culture

1838-39
Cellular theory (cell is autonomy and totipotent) Schleiden-Schwann)
 Haberlandt 1902 (the German
The father of plant tissue culture

The ability of a single starting cell or nucleus to support al

Explant: pallisade cells,

pith cells, stamen hairs, stomatal guard cells Medium: in
Unsucessful:?
did not divide
but were maintained in a living state
for several weeks.

# to recognise that the meristematic cells of the plant body are basically heterotrophic # know that the dedifferentiati

HISTORY OF PLANT TISSUE CULTURE

1902 Haberlandt The father of Plant Tissue Culture

1922 Kotte & growth of root tips for a periods of up to

Robbins 2 weeks
1934 White obtain indefinite cultures with roots from
plant tomato

1954 Street development of various procedures

cell suspension culture
1956 Nickel A pioneer in the development of tissue
production of
culture, especially the
secondary metabolites in culture
1957 Skoog & the concept of hormonal control
Miller of organ formation. Root or shoot formation
in culture depends on auxin : cytokinin
ratio
 1960 Cocking developed the enzymatic
isolation
& culture of protoplasts
1960s Morel applying the technique of shoot-tip culture for
raising virus-free individuals of an orchid

1962 Murashige & Murashige and Skoog Medium (MS

Skoog medium)
1964 Guha Culture of pollen …have the potential
&Maheswari to produce haploid embryo
1970’s Tissue culture is promoted for commercial
plant production
1978 Melchers produced a hybrid plant from the
fusion
of potato and tomato protoplasts
1981 Larkin Somaclonal variation
&Scowcroft
1983 PTC technique to produce
transgenic plant

Lay-out of a lab
Corridor/lorong luas u/ transport Bersih
Hygiene: jas lab, masker, tutup kepala, shoe covers

Clean area: medium storage, transfer

Gray
room,
area:
growth roomsDirty
preparation room,area: offices, reception and shipping
sterilization

Transfer room
Growth room
Medium storage
Equipment and material room
Preparation and sterilization room
Schematic overview of the different areas in a lab
 What conditions do plant cells need to multiply in vitro?
Tissue culture has several critical requirements:

Aseptic (sterile) conditions, as microorganism grow much more quickly than plan
a culture

A suitable growth medium containing energy sources and in organic salts to supp

Appropriate tissue (some tissue culture better than others)

Growth regulators- in plant, both auxins and cytokinins

Aseptic (sterile) conditionsWhy?

PTC media are rich in nutrients, that support the growth of many micro-organisms (bacteria, fungi)

They grow much faster than the cultured tissue & finally kill it

The contaminants may also give out metabolic wastes which are toxic to the plant tissue
Aseptic (sterile) conditions
Tissue culture is an aseptic technique
Sterilization condition essential for success in tissue culture All materials must be fre
Sterilization of
Tissue culture containers, working tools and environment (culture and transfer room
Culture media
Plant material (explants)

Sterilization Techniques

Steam sterilization (Autoclave)

Use of steam at 121 oC with pressure 15 psi for 20 min
Dry sterilization
Heat at 160-180 oC for 3 hours
Filter sterilization
Membrane filter of size 0.45-0.22 um
Flame sterilization
Chemical sterilization
 Sterilization of tissue culture containers,
working tools and environment
Glassware, scissors, tweezers, scalpel, handles, filter paper, needles
Glassware: autoclaving (121 oC at 15 psi/1 atm for 30-60 minutes), dry heat (160-180
Disposable containers: radiation Paper: dry heat
Working tools: flaming or heat (flame, glass beads)
Bench surface: swapping with alcohol
environment: laminar flow cabinet (HEPA-filtered)

sterilizing paper:
dry heatsterilizing toolslaminar Air Flow Autoclave

tic manipulations related to plant tissue culture. This is equipment fitted with High Efficiency Particulate Air (HEPA) Filters, which

Laminar flow hoods are available in horizontal or vertical airflow configurations.

A horizontal flow hood will move air from the back of the unit through HEPA or ULPA filters and to the front of the work surface.
A vertical flow hood will move air from the top of the unit through filters and down to the work surface.
Sterilization of culture media
Thermostabile ingredients are autoclaved; thermolabile ones are filter sterilized

Autoclaving: 121 oC at 15 psi/1 atm for 30-60 minutes

Filter sterilization: the filter pore size is

not greater than 0.22µm

Method of sterilizing small

volume of liquid

Apparatus for filter-

sterilizing large volumes of
media components
 Sterilization of plant material/explants
Explants sterilization is the process of making explants contamin

Surface sterilization of explant is a process which involves the

immersion of explants into appropriate concentration of chemical sterilant(s) or disinfectant(s) for

How do you sterilize explants?

Surface sterilization of plant material

Cut the plant material to an appropriate size to fit the container

which will be used during the sterilization procedure Rinse plant material under running tap water
Shake for a few seconds in alcohol

HgCl2 (0.1 - 1%) + Teepol 2 drops/100ml: 3-5 min Rinse in autoclaved distilled water
Commercial bleach 7-15% + Teepol 2 drops/100ml: 10-30 min

Rinse several times in autoclaved distilled water

 Agents used for surface sterilization

One method of sterilising explants

Steps 4 through 7 should be carried out in a sterile environment, such as the bench of a laminar flow cabinet
A suitable growth medium Culture Medium Constituents
Inorganic salt formulations

Source of carbohydrate

Vitamins

Water

Plant hormones-auxins, cytokinins, GA’s

Solidifyng agents
 Functions of medium
Provide water
Provide mineral nutritional needs
Provide vitamins
Provide growth regulators
Access to atmosphere for gas exchange
Removal of plant metabolite waste

Natural Complexes - Undefined

-Coconut endosperm
(Rich in diphenylurea, kinetin like compoud)

-Protein hydrolysates
(Source for calcium, phosphorous, several microelement, mixture 18 amino acid)

-Yeast extracts
(Source for amino acid, inositol, thiamin)

-Potato agar
(Source for (CH2O)n, amino acid, vitamin B1, B6)
 pH
pH is normally adjusted 5.5 - 6.0 before autoclaving
it governs the solubility of the salts Influences the uptake of medium ingredients
>6.0 gives a hard medium

Affects the gelling efficiency of agar.

<5.0 no gelling of the agar

pH is altered by autoclaving and during the culture period

Media Formulations

Many available
Differ in salt concentrations
Differ in presence or absence of salts
MS most widely used by far
Plant Tissue Culture Medium:

Murashige and Skoog (1962): MS : most widely used for many

types of culture system
- total N >>>

White’s medium (1963): - low salt

- root culture.

Gamborg et al.(1968): B5 - greater Ammonium and Nitrate ion

- cell suspention or callus culture.

Nitsch and Nitsch (1969): N6: - low salt concentration

- anther culture.

Lloyd & McCown (1981): WPM - very low salt

- tree sp.

Composition of Five Commonly Used Tissue Culture Media in Milligrams per Liter

Compounds Murashige Gamborg WPMNitsch and Schenk andWhite

and SkoogB-5NitschHildebrandt

NH4NO3 1650 _ 400 _ _ _

NH4H2PO4 _ _ _ _ 300 _
NH4SO4 _ 134 _
Macronutrients in mg/L _ _ _
CaCl2.2H2O 332.2 150 96 166 151 _

Ca(NO3)2.4H2O _ _ 556 _ _ 288

MgSO4.7H2O 370 250 370 185 400 737
KCl _ _ 990 _ _ _
KNO3 1900 2500 _ 950 2500 80
K2SO4 _ _ 990 _ _ _
KH2PO4 170 _ 170 68 _ _
NaH2PO4 _ 130.5 _ _ _ 16.5
Na2SO4 _ _ _ _ _ 200

Micronutrients in mg/L
H3BO3 6.2 3 6.2 10 5 1.5
CoCl2.6H2O 0.025 0.025 _ _ 0.1 _
CuSO4.5H2O 0.025 0.025 0.25 0.025 0.2 0.01
Na2EDTA 37.3 37.3 37.8 37.3 20.1 _
Fe(SO4)3 _ _ _ _ _ 2.5
FeSO4.7H2O 27.8 27.8 27.8 27.8 15 _
MnSO4.H2O 16.9 10 22.3 18.9 10 5.04
KI 0.83 0.75 _ _ 0.1 0.75
NaMoO3 _ _ _ _ _ 0.001
Na2MoO4 0.25 0.25 0.25 0.25 0.1 _
ZnSO4.7H2O 8.6 2 8.6 8.6 1 2.67

Organics in mg/L
Myo-inositol 100 100 100 100 1000 _
Glycine 2 _ 2 2 _ 3
Nicotinic acid 0.5 1 0.5 5 5 0.5
Pyridoxine HCl 0.5 0.1 0.5 0.5 0.5 0.1
Thiamin HCL 0.1 10.1 1 0.5 5 0.1
Biotin _ _ _ 0.2 _ _
 Compounds Murashige
and Skoog

NH4NO3 1650
CaCl2.2H2O 332.2
MgSO4.7H2O 370
KNO3 1900
KH2PO4 170

H3BO3 6.2
CoCl2.6H2O 0.025
CuSO4.5H2O 0.025
Na2EDTA 37.3
FeSO4.7H2O 27.8
MnSO4.H2O 16.9
KI 0.83
Na2MoO4 0.25
ZnSO4.7H2O 8.6

Myo-inositol 100
Glycine 2
Nicotinic acid 0.5
Pyridoxine HCl 0.5
Thiamin HCL 0.1

Alternative methods of preparing MS medium (Helgeson 1979)

 PEMBUATAN LARUTAN STOK MEDIUM MS
Larutan STOK Bahan Berat (mg/L) Berat (g) untuk Pengambilan untuk
volume 100 ml 1 liter media
A (5x) NH4NO3 1650 8.25 20 ml
B (5x) KNO3 1900 9.50 20 ml
C (10x) CaCl2.H2O 332.2 4.40 10 ml
D (10x) MgSO4.7H2O 370 3.70 10 ml
KH2PO4 170 1.70
E (20x) FeSO4.7H2O 27.8 0.56 5 ml
Na2EDTA.2H2O 37.3 0.75
F (20x) H3BO3 6.2 0.12 5 ml
MnSO4.4H2O 16.9 0.34
ZnSO4.7H2O 8.6 0.17
G (200x) KI 0.83 0.20 0.5 ml
Na2MoO4.2H2O 0.25 0.05
CuSO4.5H2O 0.025 0.005
CoCl2.6H2O 0.025 0.005
Vitamin Nicotinic acid 0.5 0.05 1 ml
(100x) Pyridoxine-HCl 0.5 0.05
Thiamine-HCl 0.1 0.01
Glycine 2 0.20
Myo (10x) Myo-Inositol 100 1.00 10 ml
Zpt Auksin/sitokinin 0.10 1ml untuk 1 ppm

* Label: Stok A (NH4NO3) (5x) 20 ml/l

Medium preparation

Suggested steps in preparing a medium from a ready-packaged mixture

Media sterilization should not be too long because it can cause:
The decomposition of sugar
The degradation of vitamins and amino acids
Inactivation of cytokinin (zeatin riboside)
Change in pH  cause depolymerization of compactor

The suggestion of a minimum time for media sterilization

Media Volume (ml) Time

20 - 50 15 minutes
75 20 minutes
250 - 500 25 minutes
1000 30 minutes
1500 35 minutes
2000 40 minutes

3.Appropriate tissue (explant)

Explant
Cell, tissue or organ of a plant that is used to start in
vitro cultures
Many different explants can be used for
micropropagation, but axillary buds and meristems are most commonly used
 Mother or donor plant
The mother plant is the plant used
as source of explant/which is to be propogate

It should have a desirable characteristic, such as good quality fruits, high growth rate

This plant should be healthy and disease-free

Factors affecting explant’s tissue culture response are:

Genotype
Physiological stage of donor plant
Explant source
Explant age
Explant size
Explant position in donor plant
Explant density
 Explant
Aerial plant parts are “cleaner” than underground parts Inner tissues are less contaminated than
The smaller the explant the better the chances to overcome specific phytopathological problems (v
The explant size has an effect on the response of the tissue.
Generally, the smaller the explant, the harder it is to culture.
The culture medium usually has to have additional components.
The larger explants probably contain more nutrient reserves and plant growth regulators to sustain

and fl oral parts of saff ron have been used for micropropagation, but corm segments, which have eye buds, usually respo
 4. Growth regulators

Two hormones affect plant differentiation:

-Auxin: stimulates root development
-Cytokinin: stimulates shoot development

Generally, the ratio of these two

hormones can determine plant development.
AuxinCytokinin = root development
CytokininAuxin = shoot development
Auxin = Cytokinin = callus development
TYPES OF PLANT TISSUE CULTURE
Seed Culture
Embryo culture
Pollen/anther culture
Ovary/ovule culture
Meristem culture
Organ culture
Callus culture
Cell suspension culture
Protoplast isolation, culture and fusion
Somaclonal variation
In vitro mutagenesis
Culture of hairy roots
Cryopreservation
 22-28 °C

2000-2500 lux16 hours light cycle

60% RH

high humidity may cause fungal growth in the environment and on a

various articles.
 Advantages of plant tissue culture:

Genetically identical
Virus - indexed
Germplasm maintenance
Hybrid production for incompatible species
Haploid plants
Year round production
Difficult to propagate species
New variants
Research tool

Disadvantages:

Requires expensive facilities

Particular skills are required
Appearance of an unobserved mutant
Protocols not optimized for all species
Propagule may be too expensive