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Plant Tissue Culture

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Agrobacterium cell Plant cell Ri-plasmid ... Course outline Definitions Culture environment Physical factors Growth medium Plant growth regulators Culture types ... – PowerPoint PPT presentation

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Title: Plant Tissue Culture


1
Plant Tissue Culture
Dr Maged El-Sayed Mohamed memohamed_at_zu.edu.eg mage
d789_at_hotmail.com
2
Summary of the last lecture
3
  • 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
  • Mechanically by Scissors and forceps, but low
    yield due to damaged cells
  • 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.

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  • 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.

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7
Structure of Ri-plasmid
8
  • Induction of hairy root cultures in vitro
  • Advantages of hairy root cultures
  • Application of hairy root cultures

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10
Indirect somatic embryogenesis in carrot (dauctus
carota)
2,4 D (1 mg/L)
Abscisic acid (0.025 mg/L)
11
Course outline
  • Definitions
  • Culture environment
  • Physical factors
  • Growth medium
  • Plant growth regulators
  • Culture types
  • Plant regeneration
  • Micropropagation

12
  • 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 vitro
  • Applications
  • Propagation of large numbers of uniform plants.
  • May allow faster production of plants that are
    slow to propagate in vivo.
  • It may decrease the time needed for bulk-up of
    new cultivars before they are introduced
    commercially.

13
  • Stages of Micropropagation
  • Micropropagation is divided into 5 stages.
  • Stage 0 Preparative Stage
  • Donor plant selection and explant
  • preparation
  • Explant quality is influenced by the
  • physiological condition of the donor plants.
  • The explant should be Pathogen free and
    maintained in clean conditions.

14
  • 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)

15
  • 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.

16
  • 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

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18
  • 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 98-99 to 20 - 60) development of
    sufficient defenses to control water loss.
  • Must adjust from low light to high light from
    low photosynthetic competence (heterotrophic
    nutrition) to photosynthetic competence.

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  • Advantages of Micropropagation
  • From one to many plants rapidly
  • Multiplication in controlled lab conditions
  • Continuous propagation year round
  • Potential for disease-free plants

21
  • Limitation (Disadvantages)
  • Equipment/facility intensive operation
  • Technical expertise in management positions
  • Protocols not optimized for all species
  • It may be too expensive

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Break
24
  • General plant tissue culture laboratory design
  • Glassware washing and storage area
  • Media preparation and sterilization area
  • Refrigerator/freezer
  • Balances
  • Hot plate/stirrer
  • Ph meter
  • Autoclave
  • Growth room
  • Aseptic transfer area
  • Laminar flow hoods

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27
  • Sterilization methods
  • Why?
  • Micro-organism contamination can over grow the
    plant culture resulting in culture death
  • Micro-organism contamination exhaust the nutrient
    media
  • Micro-organism can change in secondary metabolite
    structure or produce other compounds .
  • What?
  • The explant or culture
  • The vessels The media The instruments
  • The environment where handling is taking place

28
  • How?
  • Heat sterilization
  • a. Dry heat 130-180 C for 2-4 hours
  • Used for glassware, metal instruments
  • Autoclave 121 C, 1.06 kg/cm2 (15 Ib) for 15 to
    20 minutes
  • Used for glassware, media and aqueous solutions
    and plastic caps
  • Sterilization by filtration for heat labile
    aqueous solutions
  • Ethanol used for handling surface sterilization
    and also for explant.
  • Chemical sterilization using sodium or calcium
    hypochlorite, sliver nitrate, mercuric chloride
    or some other bactericidal chemicals

29
Secondary metabolites production
  • Secondary metabolites could be produced from
    culture in amounts more than or less than the
    original plant and this depend on
  • Origin of tissue
  • Physical factors (pH, temperature etc)
  • Media formulation (carbon source, PGRs and other
    elicitors)

30
Elicitation Difinition 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.
31
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,
32
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.
33
Thank you
Dr Maged El-Sayed Mohamed memohamed_at_zu.edu.eg mage
d789_at_hotmail.com
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