Lecture 3

Dr Maged El-Sayed Mohamed
Summary of the last
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.
D. Hairy root cultures:
• 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.
Agrobacterium cell
Plant cell
Structure of Ri-plasmid
•Induction of hairy root cultures in vitro
•Advantages of hairy root cultures
•Application of hairy root cultures
Indirect somatic embryogenesis in carrot (dauctus carota)
2,4 D (1 mg/L)
Indirect Somatic
Abscisic acid
(0.025 mg/L)
Course outline
• Definitions
• Culture environment
A.Physical factors
B. Growth medium
C. Plant growth regulators
• Culture types
• Plant regeneration
• Micropropagation
• Definition: Rapid clonal in vitro propagation of plants from cells,
tissues or organs cultured aseptically on defined media under
controlled conditions of light and temperature.
• The asexual or vegetative propagation (multiplication) of plants in
• Applications:
1. Propagation of large numbers of uniform plants.
2. May allow faster production of plants that are slow to propagate
in vivo.
3. It may decrease the time needed for bulk-up of new cultivars
before they are introduced commercially.
• Stages of Micropropagation
Micropropagation is divided into 5 stages.
Stage 0: Preparative Stage:
• Donor plant selection and explant
• Explant quality is influenced by the
physiological condition of the donor plants.
• The explant should be Pathogen free and maintained in clean
Stage 1: Establishment of explant in culture:
• Surface-sterilization of explant tissues.
• Isolation of explant under sterile conditions.
• Medium must contain all components necessary to make the explant
perform as desired (medium composition and PGRs).
• Adjust the environmental conditions (Light, Temperature, Relative
humidity, etc)
Stage 2: Multiplication and proliferation of axillary shoots
• Repeated enhanced shoot production by high cytokinin in the
medium, alone or with a smaller amount of auxin.
• Shoots must be transferred to fresh medium at regular intervals.
• Number of subcultures possible from the original culture varies
with species/cultivar.
Stage 3: Pre-transplant (Rooting)
• It is rooting of shoots or shoot clusters in vitro.
• Auxins are important for root initiation in vitro
• Advantages of rooting after removal from culture
 Reduced costs
 Structurally and physiologically better
 Avoid damage to roots that occur during transplanting
Stage 4: transfer to natural environment:
Acclimatization: a process of physiologically and anatomically
adjustment from in vitro to ex vitro conditions.
Relatively slow process, may take weeks.
Must adjust from high to lower relative humidity (e.g. from 9899% to 20 - 60%): development of sufficient defenses to control
water loss.
Must adjust from low light to high light: from low
photosynthetic competence.
Advantages of Micropropagation:
• From one to many plants rapidly
• Multiplication in controlled lab conditions
• Continuous propagation year round
• Potential for disease-free plants
Limitation (Disadvantages)
• Equipment/facility intensive operation
• Technical expertise in management positions
• Protocols not optimized for all species
• It may be too expensive
General plant tissue culture laboratory design
1.Glassware washing and storage area
2.Media preparation and sterilization area
• Refrigerator/freezer
• Balances
• Hot plate/stirrer
• Ph meter
• Autoclave
3.Growth room
4.Aseptic transfer area
• Laminar flow hoods
Sterilization methods:
1. Micro-organism contamination can over grow the plant
culture resulting in culture death
2. Micro-organism contamination exhaust the nutrient media
3. Micro-organism can change in secondary metabolite
structure or produce other compounds .
The explant or culture
The vessels
The environment where handling is taking place
The media
The instruments
1. Heat sterilization:
a. Dry heat: 130-180 °C for 2-4 hours
Used for glassware, metal instruments
b. Autoclave: 121 °C, 1.06 kg/cm2 (15 Ib) for 15 to 20 minutes
Used for glassware, media and aqueous solutions and plastic caps
2. Sterilization by filtration: for heat labile aqueous solutions
3. Ethanol: used for handling surface sterilization and also for
4. Chemical sterilization: using sodium or calcium hypochlorite,
sliver nitrate, mercuric chloride or some other bactericidal
Secondary metabolites production
• Secondary metabolites could be produced from culture in
amounts more than or less than the original plant and this
depend on:
1.Origin of tissue
2.Physical factors (pH, temperature etc)
3.Media formulation (carbon source, PGRs and other elicitors)
an Elicitor may be defined as a substance
which, when introduced in small concentrations to a living
cell system, initiates or improves the biosynthesis of
specific compounds.
Classification of Elicitors
A) Biotic elicitors. From biological origin They include:
1. Enzymes and polysaccharides derived from microorganisms
2. Phytochemicals produced by plants in response to physical
damage, fungi or bacteria attack,
Classification of Elicitors
B) Abiotic elicitors are the substances of non-biological origin.
1. Chemicals such as inorganic salts, heavy metals, some chemicals
that disturb membrane integrity.
2. Physical factors like mechanical wounding, ultraviolet irradiation,
high salinity, high or low osmolarity, extreme temperature (freezing,
thawing), high pressure.
Dr Maged El-Sayed Mohamed