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
B: The growth
medium
Essential
elements
or mineral ions
Organic
supplement
Source of
carbon
Gelling
Agent
a. Agar
• Composition:
1. Agarose
2. Agaropectin
• Agar tertiary structure
• Advantages
• Disadvantages
b) Agarose
c) Gelrite™
d) Phytagel™
C. Plant growth regulators:
Definition
Characteristics
Auxin
Cytokinin
Gibberelin
Classification of
PGRs
Abscisic
acid
Ethylene
Other
Jasmonates
Salicylic acid
Brassinosteroids
Course outline
• Definitions
• Culture environment
A.Physical factors
B. Growth medium
C. Plant growth regulators
• Culture types
• Plant regeneration
• Micropropagation
Culture Types
• Explants
• Types of explant:
a) Root tip
b) Shoot tip
c) Embryo
d) Haploid tissue
A. Callus:
•
Definition: It is an unspecialized and
unorganized, growing and dividing
mass of cells,.
•
During callus formation there is some
degree of dedifferentiation both in
morphology and metabolism, resulting
in the lose the ability to photosynthesis.
• Habituation:
• Compact callus
• Friable callus
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.
C. Protoplasts
Definition: They are plant cells with the cell wall removed.
Source: from either leaf mesophyll cells or callus or cell suspensions.
Production : Removing the cell wall is achieved:
1. Mechanically: by Scissors and forceps, but low yield due to
damaged cells
2. Enzymatically: Cell wall is removed using degrading enzymes
(cellulase and pectinase) in a simple salt solution with a high
osmotic potential to maintain the cells.
•
Liquid medium is not agitated so it must be shallow enough to
allow aeration in the absence of agitation.
Applications:
1. Ideal target for transformation and genetic engineering.
2. Can be developed to callus on solid medium
3. Used for ultra-structure studies of plant cells
4. Isolation of subcellular components such as nuclei and
chromosomes
D. Hairy root cultures:
Definition:
• It is the culture produced after the infection of explants or cultures
by the gram negative soil bacterium Agrobacterium rhizogenes.
• This processes take advantage of the naturally occurring hairy root
disease in Dicotyledons.
Break
Production of hairy roots in vivo:
a) Agrobacterium recognizes some signal molecules exuded by
wounded plant cells and becomes attached to it.
b) The bacteria contain the Root inducing plasmid (Ri-plasmid)
c) The bacteria genetically transfer part of the Ri-plasmid called the
transfer DNA (T-DNA) to the plant genome, where it is get
expressed and make the plant cell to:
1. Proliferate by increasing the rate of cell division (cytokinie
expession) and cell elongation (auxin expression) to produce the
hairy roots. why?
2. Produce the opines which is a type of unusual amino acids
(octopine, agropine,nopaline, mannopine, and cucumopine) which
is used by the bacterium as a carbon, nitrogen and energy source
Agrobacterium cell
Ri-plasmid
Plant cell
Structure of Ri-plasmid
Ri-Plasmid
Induction of hairy root cultures in vitro:
1. Explants are wounded and then inoculated with Agrobacterium
rhizogenes.
2. Usually two or three days later, the explant can be transferred into
solid media with antibiotics, such as cefotaxime, vancomycin or
ampicillin to kill or eliminate redundant bacteria.
3. The hairy roots will be induced within a short period of time,
which varies from one week to over a month depending on
different plant species.
4. The decontaminated hairy roots can be subcultured on
phytohormone-free medium.
Advantages of hairy root cultures:
1. The hairy root system is genetically and biosynthetically stable
2. High production of secondary metabolites
3. The culture can grow under phyto-hormone-free conditions.
4. The culture shows fast growth which reduce the culture time and
easy the handling
Application of hairy root cultures:
1. Functional analysis of genes
2. Expressing foreign proteins
3. Production of secondary metabolites
4. The culture may Produce compounds which is not found in
untransformed roots
5. The culture may change the composition of metabolites
6. The culture can be used to regenerate a whole plants
Course outline
• Definitions
• Culture environment
A.Physical factors
B. Growth medium
C. Plant growth regulators
• Culture types
• Plant regeneration
• Micropropagation
Plant
Regeneration
Somatic
embryogenesis
Organogenesis
A. Somatic embryogenesis
Definition: A process in which embryo-like structures are formed
from somatic tissues and developed into a whole plant.
Types:
1.Direct somatic embryogenesis:
• The embryo is formed directly from a cell or small group of
cells such as the nucellus, styles or pollen without the
production of an intervening callus.
• Direct somatic embryogenesis is generally rare
2.Indirect somatic embryogenesis
• Callus is first produced from the explant and then embryos are
produced from the callus tissue or from a cell suspension
cultures.
Indirect somatic embryogenesis in carrot (dauctus carota)
2,4 D (1 mg/L)
Indirect Somatic
embryogenesis
Abscisic acid
(0.025 mg/L)
B. Organogenesis
Definition: The production of organs, either directly from an
explant or from a callus culture.
• Organogenesis depend on adventitious organs arising either
from a callus culture or directly from an explant or on the
formation of axillary bud to regenerate whole plants from
some types of tissue culture.
Summary
• Protoplast cultures
• Hairy root cultures
• Plant regeneration:
1. Somatic embryogenesis
2. Organogenesis