Msc. Micro. M514

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Tissue Culture

• By
• Prof. Dr. Emad Eldin Abbas Ewais

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Introduction

• Tissue culture is the term used for the process
  of growing cells artificially in the laboratory
• Tissue Culture (also known as Micropropagation or
  In vitro culture) isThe growing of plant cells,
  tissues, organs, seeds or other plant parts in a
  sterile environment on a nutrient medium.
• Tissue culture involves both plant and animal
  cells
• Tissue culture produces clones, in which all
  product cells have the same genotype (unless
  affected by mutation during culture)

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Whats the Background?

• Tissue culture had its origins at the beginning
  of the 20th century with the work of Gottleib
  Haberlandt (plants) and Alexis Carrel (animals)
• The first commercial use of plant clonal
  propagation on artificial media was in the
  germination and growth of orchid plants, in the
  1920s

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• In the 1950s and 60s there was a great deal of
  research, but it was only after the development
  of a reliable artificial medium (Murashige
  Skoog, 1962) that plant tissue culture really
  took off commercially
• Plant tissue culture is a bit like the equivalent
  of Dolly the sheep but using plants.

Young cymbidium orchids
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• A more recent advance is the use of plant and
  animal tissue culture along with genetic
  modification using viral and bacterial vectors
  and gene guns to create genetically engineered
  organisms

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Both these processes use undifferentiated
cells
Cloning Plant Cells
Cloning Animal Cells
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What is needed?Tissue culture, both plant and
animal has several critical requirements

• Appropriate tissue (some tissues culture better
  than others)
• A suitable growth medium containing energy
  sources and inorganic salts to supply cell growth
  needs. This can be liquid or semisolid
• Aseptic (sterile) conditions, as microorganisms
  grow much more quickly than plant and animal
  tissue and can over run a culture

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What is Needed, II

• Growth regulators - in plants, both auxins
  cytokinins. In animals, this is not as well
  defined and the growth substances are provided in
  serum from the cell types of interest
• Frequent subculturing to ensure adequate
  nutrition and to avoid the build up of waste
  metabolites

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Culturing (micropropagating) Plant Tissue - the
steps

• Selection of the plant tissue (explant) from a
  healthy vigorous mother plant - this is often
  the apical bud, but can be other tissue
• This tissue must be sterilized to remove
  microbial contaminants

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The Steps, II

• Establishment of the explant in a culture medium.
  The medium sustains the plant cells and
  encourages cell division. It can be solid or
  liquid
• Each plant species (and sometimes the variety
  within a species) has particular medium
  requirements that must be established by trial
  and error

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The Steps, III

• Multiplication- The explant gives rise to a
  callus (a mass of loosely arranged cells) which
  is manipulated by varying sugar concentrations
  and the auxin (low) cytokinin (high) ratios to
  form multiple shoots
• The callus may be subdivided a number of times

Dividing shoots
Warmth and good light are essential
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The Steps, IV

• Root formation - The shoots are transferred to a
  growth medium with relatively higher auxin
  cytokinin ratios

The pottles on these racks are young banana
plants and are growing roots
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The Steps, V

• The rooted shoots are potted up (deflasked) and
  hardened off by gradually decreasing the
  humidity
• This is necessary as many young tissue culture
  plants have no waxy cuticle to prevent water loss

Tissue culture plants sold to a nursery then
potted up
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Culturing

• Take some meristematic cells from a plant.
• These cells are called an explant
• Place the explant on a sterile nutrient rich agar
• The explant grows into a ball of cells
• This ball of cells is called a callus
• The callus then develops roots, stem and leaves
• This is called a plantlet
• Transplant the plantlet into
• a traditional growing media

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What are undifferentiated cells

• What are undifferentiated cells
• In the human body we have lots of different types
  of cells
• Blood cells
• Muscle cells
• Bones cells
• Brain cells
• An undifferentiated cell is a cell that does not
  yet have a specialised job

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What are undifferentiated cells

• Undifferentiated cells in animals
• Stem cells
• Undifferentiated cells in plants
• ?

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Why do Plant Tissue Culture?

• A single explant can be multiplied into several
  thousand plants in less than a year - this allows
  fast commercial propagation of new cultivars
• Taking an explant does not usually destroy the
  mother plant, so rare and endangered plants can
  be cloned safely
• Once established, a plant tissue culture line can
  give a continuous supply of young plants
  throughout the year

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Why do Plant Tissue Culture, II

• In plants prone to virus diseases, virus free
  explants (new meristem tissue is usually virus
  free) can be cultivated to provide virus free
  plants
• Plant tissue banks can be frozen, then
  regenerated through tissue culture
• Plant cultures in approved media are easier to
  export than are soil-grown plants, as they are
  pathogen free and take up little space (most
  current plant export is now done in this manner)

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Why do Plant Tissue Culture, III

• Tissue culture allows fast selection for crop
  improvement - explants are chosen from superior
  plants, then cloned
• Tissue culture clones are true to type as
  compared with seedlings, which show greater
  variability

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Culturing Animal Tissue- the Steps

• Animal tissue is obtained either from a
  particular specimen, or from a tissue bank of
  cryo-preserved (cryo frozen at very low
  temperatures in a special medium)
• Establishment of the tissue is accomplished in
  the required medium under aseptic conditions

Culture vessels and medium for animal cell culture
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Culturing Animal Tissue, II

• Growing the cells / tissue requires an optimum
  temperature, and subculturing when required
• Human cells, for example are grown at 37degrees
  and 5 CO2

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Animal tissue/cell culture - differences from
plant tissue culture

• Animal cell lines have limited numbers of cell
  cycles before they begin to degrade
• Animal cells need frequent subculturing to remain
  viable
• Tissue culture media is not as fully defined as
  that of plants - in addition to inorganic salts,
  energy sources, amino acids, vitamins, etc., they
  require the addition of serum (bovine serum is
  very common, but others are used)

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Animal tissue/cell culture - differences from
plant tissue culture II

• Animal tissue cultures can pose biohazard
  concerns, and cultures require special
  inactivation with hypochlorite (e.g.
  Janola,Chlorox, etc.) and then incineration

Gloves and labcoat are always worn
The pipettes are disposable
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Uses of Animal Tissue Culture

• Growing viruses - these require living host cells
• Making monoclonal antibodies, used for diagnosis
  and research
• Studying basic cell processes
• Genetic modification analysis
• Knockout technology - inactivating certain
  genes and tracing their effects
• Providing DNA for the Human Genome Project (and
  other species genome projects)

Photo courtesy of Sigma Aldrich
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What Is plant tissue culture?

• Or in vitro culture?
• Or in vitro propagation?
• Or Micropropagation ?
• Definition
• the culture of plant seeds, organs, explants,
  tissues, cells, or protoplasts on nutrient media
  under sterile conditions.

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Basis for Plant Tissue Culture

• Two Hormones Affect Plant Differentiation
• Auxin Stimulates Root Development
• Cytokinin Stimulates Shoot Development
• Generally, the ratio of these two hormones can
  determine plant development
• ? Auxin ?Cytokinin Root Development
• ? Cytokinin ?Auxin Shoot Development
• Auxin Cytokinin Callus Development

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Factors Affecting Plant Tissue Culture

• Growth Media
• Minerals, Growth factors, Carbon source
• Environmental Factors
• Light, Temperature, Photoperiod
• Explant Source
• Types
• Usually, the younger, less differentiated the
  explant, the better for tissue culture
• Genetics
• 1. Different species show differences in
  amenability to tissue culture
• 2. In many cases, different genotypes within a
  species will have variable
• responses to tissue culture response to
  somatic embryogenesis has
• been transferred between melon cultivars
  through sexual
• hybridization

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Choice of explant

• Desirable properties of an explant
• Easily sterilisable
• Juvenile
• Responsive to culture

• Shoot tips
• Axillary buds
• Seeds
• Hypocotyl (from germinated seed)
• Leaves

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Media

• When you make an explant like an axillary bud,
  you remove it from the sources of many chemicals
  and have to re-supply these to the explants to
  allow them to grow.

• Shoot tip - Auxins
• and Gibberellin

Leaves - sugars, GAs
Roots - water, vitamins mineral salts and
cytokinins
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Medium constituents

• Inorganic salt formulations
• Source of carbohydrate
• Vitamins
• Water
• Plant hormones - auxins, cytokinins, GAs
• Solidifying agents
• Undefined supplements

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Carbohydrates

• Plants in culture usually cannot meet their needs
  for fixed carbon. Usually added as sucrose at
  2-3 w/v.
• Glucose or a mixture of glucose and fructose is
  occasionally used.
• For large scale cultures, cheaper sources of
  sugars (corn syrup) may be used.

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Photoautotrophic culture

• Growth without a carbon source. Therefore need to
  boost photosynthesis.
• High light intensities needed (90-150mMole/m2/s)
  compared to normal (30-50).
• Usually increase CO2 (1000ppm) compared to normal
  369.4ppm.
• Much reduced level of contamination and plants
  are easier to transfer to the greenhouse.

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Inorganic salt formulations

• Contain a wide range of Macro-elements (gtmg/l)
  and microelements (ltmg/l).
• A wide range of media are readily available as
  spray-dried powders.
• Murashige and Skoog Medium (1965) is the most
  popular for shoot cultures.
• Gamborgs B5 medium is widely used for cell
  suspension cultures (no ammonium).

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Vitamins

• A wide range of vitamins are available and may be
  used.
• Generally, the smaller the explant, the more
  exacting the vitamin requirement.
• A vitamin cocktail is often used (Nicotinic acid,
  glycine, Thiamine, pyridoxine).
• Inositol usually has to be supplied at much
  higher concentration (100mg/l)

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Plant hormones (Growth regulators)

• Auxins
• Cytokinins
• Gibberellic acids
• Ethylene
• Abscisic Acid
• Plant Growth Regulator-like compounds

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Auxins

• Absolutely essential (no mutants known)
• Only one compound, Indole-3-acetic acid. Many
  synthetic analogues (NAA, IBA, 2,4-D, 2,4,5-T,
  Pichloram) - cheaper more stable
• Generally growth stimulatory. Promote rooting.
• Produced in meristems, especially shoot meristem
  and transported through the plant in special
  cells in vascular bundles.

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Cytokinins

• Absolutely essential (no mutants known)
• Single natural compound, Zeatin. Synthetic
  analogues Benyzladenine (BA), Kinetin.
• Stimulate cell division (with auxins).
• Promotes formation of adventitious shoots.
• Produced in the root meristem and transported
  throughout the plant as the Zeatin-riboside in
  the phloem.

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Gibberellins (GAs)

• A family of over 70 related compounds, all forms
  of Gibberellic acid.
• Commercially, GA3 and GA49 available.
• Stimulate etiolation of stems.
• Help break bud and seed dormancy.
• Produced in young leaves.

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Abscisic Acid (ABA)

• Only one natural compound.
• Promotes leaf abscission and seed dormancy.
• Plays a dominant role in closing stomata in
  response to water stress.
• Has an important role in embryogenesis in
  preparing embryos for desiccation. Helps ensure
  normal embryos.

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Plant Growth Regulator-like substances

• Polyamines - have a vital role in embryo
  development.
• Jasmonic acid - involved in plant wound
  responses.
• Salicylic acid.
• Not universally acclaimed as plant hormones since
  they are usually needed at high concentrations.

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Undefined supplements

• Sources of hormones, vitamins and polyamines.
• e.g. Coconut water, sweetcorn extracts
• Not reproducible
• Do work.

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Fundamental abilities of plants

• Totipotency
• the potential or inherent capacity of a plant
  cell to develop into an entire plant if suitable
  stimulated.
• It implies that all the information necessary
  for growth and reproduction of the organism is
  contained in the cell
• Dedifferentiation
• The capacity of mature cells to return to
  meristematic condition and development of a new
  growing point, followed by redifferentiation
  which is the ability to reorganize into new
  organs
• Competency
• the endogenous potential of a given cell or
  tissue to develop in a particular way