Dr Maged El-Sayed Mohamed
memohamed@zu.edu.eg
maged789@hotmail.com
Course outline
• Definitions
• Culture environment
 Physical factors
 Growth medium
 Plant growth regulators
• Culture types
• Plant regeneration
• Micropropagation
Summary of the last
lecture
Definitions
• Plant Tissue culture: It is the ability to in vitro generate a large
number of plant cells or even the whole plant from isolated plant
cells or tissue.
• Plasticity: A property that allows plants to alter their
metabolism, growth and development to best suit their
environment
• Totipotency: The ability to initiate cell division from any tissue
or cell of the plant in response to particular stimuli to regenerate
whole plant with maintenance of genetic potential.
The culture environment
A. The physical factors:
• Temperature
• pH
• The gaseous environment
• Light (quality and duration)
• Osmotic pressure
All these factors have to be maintained within acceptable
limits for the success of plant tissue culture
Course outline
• Definitions
• Culture environment
A.Physical factors
B.Growth medium
C.Plant growth regulators
• Culture types
• Plant regeneration
• Micropropagation
B: The growth
medium
Essential
elements
or mineral ions
Organic
supplement
Source of
carbon
Gelling
Agent
1) Essential elements
or mineral ions
Macroelements
(or Macronutrients)
•
•
•
•
•
•
Nitrogen (N)
Potassium (K)
Phosphorous (P)
Calcium (Ca)
Magnesium (Mg)
Sulfur (S)
Microelements
(or micronutrients)
•
•
•
•
•
•
•
•
Iron(Fe)
Manganese (Mn)
Zinc (Zn)
Boron (B)
Cupper(Cu)
Molybdenum(Mo)
Cobalt (Co)
Iodine (I)
B: The growth
medium
Essential
elements
or mineral ions
Organic
supplement
Source of
carbon
Gelling
Agent
2. Organic supplement
a) Vitamins:
• Only thiamine (vitamin B1) is essential for most plant
cultures, it is required for carbohydrate metabolism and the
biosynthesis of some amino acids,
b) Myo-inositol
• Although it is not essential for growth of many plant species,
its effect on growth is significant.
c) Complex organics
• Such as coconut milk, coconut water, yeast extract, fruit
juices and fruit pulps.
3. Source of carbon
 Sugars
•
Most plant tissue cultures are not highly autotrophic due to
limitation of CO2. Therefore, sugar is added to the medium as
an energy source.
•
Sucrose is the most common sugar added, although glucose,
fructose, and sorbitol are also used in certain instances.
•
The concentration of sugars in nutrient media generally
ranges from 20 to 40 g/l.
•
Sugars also contribute to the osmotic potential in the culture
B: The growth
medium
Essential
elements
or mineral ions
Organic
supplement
Source of
carbon
Gelling
Agent
4) Gelling agents
• When semi-solid or solid culture media are required, gelling
agents are used.
a. Agar
•Agar is the most commonly used gelling agent.
•Composition: Agar consists of 2 components
1.Agarose is an alternating D-galactose and 3,6-anhydro-L-
galactose with side chains of 6-methyl-D-galactose residues.
2.Agaropectin is like agarose but additionally contains sulfate
ester side chains and D-glucuronic acid.
•Agar tertiary structure is a double helix the central cavity of
which can accommodate water molecules
• Advantages:
Agar is an inert component, form a gel in water that melt at
100 ° C and solidify at nearly 45 ° C
Concentrations commonly used in plant culture media range
between 0.5% and 1%
If necessary, agar can be washed to remove inhibitory
organic and inorganic impurities.
• Disadvantages:
Agar does not gel well under acidic conditions (pH <4.5).
The inclusion of activated charcoal in media may also
inhibit gelling of agar.
b) Agarose
• It is extracted from agar leaving
behind agaropectin and its sulfate
groups.
• It is used when the impurities of
agar are a major disadvantage.
c) Gelrite™
• Gelrite consists of a polysaccharide
produced by the bacterium Pseudomonas elodea.
• It gives clear-solidified medium that
leads to detection of contamination at an
early stage.
• Gelrite requires more stirring than agar.
• Concentration of divalent cations such as calcium and
magnesium must be within the range of 4-8 mM/L or the
medium will not solidify
d) Phytagel™
• It is an agar substitute produced from a bacterial substrate
composed of glucuronic acid, rhamnose and glucose.
• It produces a clear, colorless, high-strength gel, which aids in
detection of microbial contamination.
• It is used at a concentration of 1.5-2.5 g/L.
• It should be prepared with rapid stirring to prevent clumping.
Commertial Media Formulations:
• Murashige and Skoog (MS)
• Linsmaier and Skoog (LS)
• White Medium
• Gamborg medium
• Schenk and Hildebrandt medium
• Nitsch and Nitsch Medium
• Lloyd and McCown Woody plant medium
• Knudson’s medium
Course outline
• Definitions
• Culture environment
A.Physical factors
B. Growth medium
C. Plant growth regulators
• Culture types
• Plant regeneration
• Micropropagation
C. Plant growth regulators:
Definition:
Plant hormone or
Phytohormone or
Plant growth regulator (PGR):
• They are small organic molecule that elicits a physiological
response at very low concentrations.
Characteristics:
1. Synthesized by plants.
2. Show specific activity at very low concentrations
3. Display multiple functions in plants.
4. Play a role in regulating physiological phenomena in vivo in a
dose-dependent manner
5. They may interact, either synergistically or antagonistically, to
produce a particular effect.
Break
Auxin
Cytokinin
Gibberelin
Classification of
PGRs
Abscisic acid
Ethylene
Other
Jasmonates
Salicylic acid
Brassinosteroids
PGR
Auxins
Examples
• Indole-3-acetic acid
(IAA)
• indole-acetyl-L- alanine
Effects
• Stimulate cell elongation
• Increase the rate of
transcription
• indole-acetyl-L-glycine
• Stimulate root initiation
• 2,4-
• Mediate the response of
Dichlorophenoxyacetic
bending in response to
acid (2,4-D)
gravity or light
PGR
Cytokinin
Examples
• Natural:
zeatin
2-isopentyl adenine
Effects
• Stimulate cell division
• Stimulate shoot initiation
and growth of lateral buds
(2iP)
• Stimulate dark
• Synthetic:
germination
kinetin
Benzylaminopurine
(BAP)
• Stimulate leaf expansion
Auxin and Cytokinin Ratio
PGR
Gibberellins
Examples
• Diterpenes
Effects
• Stimulate stem
synthesized via the
elongation by stimulation
mevalonic acid
cell division and
pathway
elongation
• They are more
• Break seeds dormancy
than 110 and
• Stimulate germination of
named as GA1,
GA2.... GA110
pollen
PGR
Examples
Abscisic Acid • It is a
(ABA)
sesquiterpene
Effects
• Involved in the abscission
of buds, flower and fruits
• Inhibit cell division and
elongation
Ethylene
•It is a gas produced • Regulate ripening of fruits
from L-methionine
• Inhibit flowering
• Regulate cell death
programming
Auxin
Cytokinin
Gibberelin
Classification of
PGRs
Abscisic acid
Ethylene
Other
Jasmonates
Salicylic acid
Brassinosteroids
PGR
Brassinosteroids
Effects
• Promote shoot elongating
• Inhibit root growth
• Promote ethylene biosynthesis
• Enhance resistance to chilling,
disease and herbicides
Salicylic acid
• Promote flowering
• Stimulate plant pathogenesis protein
production
Jasmonate
• Play an important role in plant defence
mechanisms
Course outline
• Definitions
• Culture environment
A.Physical factors
B. Growth medium
C. Plant growth regulators
• Culture types
• Plant regeneration
• Micropropagation
Culture Types
• Explants : Sterile pieces of a whole plant from which cultures
are generally initiated
• Types of explant:
Generally all plant cells can be used as an explant, however
young and rapidly growing tissue (or tissue at an early stage of
development) are preferred.
a) Root tip:
• Root cultures can be established from explants of the root tip of
either primary or lateral roots.
b) Shoot tip:
• The shoot apical meristem from either axillary or adventitious
buds can be cultured in vitro.
c) Embryo:
• Both immature and mature embryos can be used as explants to
generate callus cultures or somatic embryos.
• Immature, embryo-derived callus is the most popular method of
monocot plant regeneration.
d) Haploid tissue
• Male gametophyte (Pollen in anthers) or female gametophyte
(the ovule) can be used as an explant.
• Haploid tissue cultures can produce haploid or di-haploid
plants due to doubling of chromosomes during the culture
periods.
A. Callus:
• Definition: It is an unspecialized and
unorganized, growing and dividing
mass of cells, produced when explants
are cultured on the appropriate solid
medium, with both an auxin and a
cytokinin and correct conditions.
• During callus formation there is some
degree of dedifferentiation both in
morphology and metabolism, resulting
in the lose the ability to photosynthesis.
• This necessitates the addition of other components e.g.: vitamins
and, a carbon source to the culture medium, in addition to the usual
mineral nutrients.
• Habituation: it is the lose of the requirement for auxin and/or
cytokinin by the culture during long-term culture.
• Callus cultures may be compact or friable.
 Compact callus shows densely aggregated cells
 Friable callus shows loosely associated cells and the callus
becomes soft and breaks apart easily.
B. Cell-suspension cultures
•
When friable callus is placed into the appropriate liquid medium
and agitated, single cells and/or small clumps of cells are released
into the medium and continue to grow and divide, producing a
cell-suspension culture.
•
The inoculum used to initiate cell suspension culture should
neither be too small to affect cells numbers nor too large too
allow the build up of toxic products or stressed cells to lethal
levels.
•
Cell suspension culture techniques are very important for plant
biotransformation and plant genetic engineering.
Summary
• Gelling Agents and its types
Agar, Agarose, GilRite and Phytagel
• Plant growth regulators:
Auxin, Cytokinin, Gibberelin, Abscisic and ethylene
• Culture types: Callus and cell suspension cultures