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Plant Tissue Culture Media


Plant Tissue Culture Media * Abscisic Acid Only one natural compound. Promotes leaf abscission and seed dormancy. Plays a dominant role in closing stomata in response ... – PowerPoint PPT presentation

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Title: Plant Tissue Culture Media

Plant Tissue Culture Media
Logical Basis
For healthy and vigorous growth, intact plants
need to take up from soil of an essential
  • Essential elements (Epstein, 1971)
  • A plant grown in a medium adequately purged of
    that elements, failed to grow properly or to
    complete its life cycle
  • It is a constituent of a molecule that is known
    to be an essential metabolite

Essential element
  • Macro element/major plant nutrition
  • Relatively large amount required
  • a. Carbon (C) d. Nitrogen (N) g. Potassium (K)
  • b. Hydrogen (H) e. Calcium (Ca) h. Phosphorus
  • c. Oxygen (O) f. Magnesium (Mg) i. Sulphur (S)
  • Micro element/ minor plant nutrient/trace
  • Small quantities required
  • a. Iron (Fe) f. Sodium (Na)
  • b. Chlorine (Cl) g. Manganese (Mn)
  • c. Zinc (Zn) h. Boron (B)
  • d. Copper (Cu) i. Molybdenum (Mo)
  • e. Nickel (Ni)

Functions of media
Why plant in vitro culture needs media?
  • Provide water
  • Provide mineral nutritional needs
  • Provide vitamins
  • Provide growth regulators
  • Provide amino acids
  • Provide sugars
  • Access to atmosphere for gas exchange
  • Removal of plant metabolite waste

Plant tissue culture media
  • Macronutrients (always employed)
  • Micronutrients (nearly always employed, although
    sometimes just one element, iron, has been used)
  • Vitamins (generally incorporated , although the
    actual number of compounds added, varies greatly)
  • Amino acids and other nitrogen supplements
    (usually omitted, but sometimes used with
  • Sugar (nearly always added, but omitted for some
    special purposes)
  • Undefined supplements (which, when used,
    contribute some above components, and also plant
    growth substances or regulants)
  • Buffers (have seldom been used in the past, but
    recently suggest that the additions of organic
    acids or buffers could be beneficial in some
  • A solidifying agent (used when a semi solid
    medium is required)

  • Macronutrients for plant tissue culture are
    provided from salt, however plant absorb entirely
    as ions
  • Nitrogen is mainly absorbed in the form of
    ammonium or nitrate
  • Phosphorus as the phosphate ions
  • Sulphur as sulphate ions
  • The most important step in deriving medium is the
    selection of macronutrient ions in the correct
    concentration and balanced
  • The salts normally used to provide macroelements
    also provide sodium and chlorine, however, plant
    cell tolerate high concentration of both ions
    without injury, these ions are frequently given
    little importance when contemplating media

Quantity of the Macronutrient
  • It is essential to plant life
  • Both growth and morphogenesis is markedly
    influenced by the availability of nitrogen and
    the form in which it is presented
  • Most media contain more nitrate than ammonium
    ions. Most intact plants, tissues and organ taken
    up nitrogen effectively, and grow more rapidly on
    nutrient solutions containing both nitrate and
    ammonium ions
  • Nitrate has to be reduced to ammonium before
    being utilized biosynthetically
  • Ammonium in high concentration is latent toxic
  • For most type of culture, nitrate needs to be
    presented together with the reduced form of
    nitrogen and tissue will usually fail to grow on
    a medium with nitrate as the only nitrogen source

NH4 and NO3- Regulate Medium pH and Root
Morphogenesis of Rose Shoots
Amino acids
  • Amino acids can be added to satisfy the
    requirement for reduced nitrogen, but as they are
    expensive to purchase, they will only be used on
    media for mass propagation where this results in
    improved result
  • A casein hydrolysate, yeast extract which mainly
    consist of a mixture of amino acids substantially
    increased the yield of callus
  • Organic supplements have been especially
    beneficial for growth or morphogenesis when cells
    were cultured on media which do not contain
    ammonium ions
  • Glycine os an ingredient of many media. It is
    difficult to find hard evidence that glycine is
    really essential for so many tissue culture, but
    possible it helps to protect cell membranes from
    osmotic and temperature stress

Amino Acids
  • The most common sources of organic nitrogen used
    in culture media are amino acid mixtures.
  • Its uptake more rapidly than in organic amino
  • . (e.g., casein hydrolysate), L-glutamine,
    L-asparagine, and adenine.
  • When amino acids are added alone, they can be
    inhibitory to cell growth.
  • .

Beneficial effects of amino acids
  • Rapid growth
  • Protoplast cell division
  • Conservation of ATP
  • AS chelating agent
  • Enhanced nitrogen assimilation
  • Not toxic as ammonium
  • As buffer

  • It is a vital element in plant biochemistry
  • It occurs in numerous macromolecules such as
    nucleic acids, phospholipids and co-enzymes
  • It functions in energy transfer via pyrophosphate
    bond I ATP
  • Phosphate groups attached to different sugar
    provide energy in respiration and photosynthesis
    and phosphate bound to proteins regulate their
  • Phosphorous is absorbed into plants in the form
    of the primary or secondary orthophosphate anions
    by an active process which requires the
    expenditure of respiratory energy
  • Phosphate in not reduced in plants, but it
    remains in the oxydised form
  • It is used in plant as the fully oxydised
    orthophosphate form

  • It is not metabolized
  • It is a major cation within the plants
  • It contributes significantly to the osmotic
    potential of cells
  • It is transported quickly across cell membrane
    and two of its major role is regulating the pH
    and osmotic environment within the cells
  • Many protein show a high specificity for
    potassium which acting as a cofactor, alters
    their configuration so that it become active
  • It is also neutralize organic anions produce in
    the cytoplasm and so stabilize the pH and osmotic
    potential of the cells

  • It is taken up into plant but in most cases it is
    not required for growth and development
  • Many plants actively secret it from their roots
    to maintain a low internal concentration
  • It is only appeared to be essential to salt
    tolerance plant

  • It is an essential component of the chlorophyll
  • It is also required non-specifically for the
    activity of many enzymes, especially in the
    transfer of phosphate
  • ATP synthesis has an absolute requirement for
    magnesium and it is a bridging element in the
    aggregation of ribosome sub-unit
  • It is the central atom in the phorphyrin
    structure of the chlorophyll molecules

  • It is mainly absorbed as sulfate
  • Its uptake is coupled to nitrogen assimilation
  • It is incorporated into chemical compounds mainly
    as reduced SH, -S_ or S-S groups
  • It is used in lipid synthesis and in regulating
    the structure of proline through the formation of
    S-S bridges
  • It acts as a ligand joining ion of iron, zinc,
    copper to metalloportein and enzymes

  • It helps to balance anion within the plant
  • It is not readily mobile
  • It is involved in the structure and
    physiologically properties of cell membranes and
    the middle lamella of the cell walls
  • The enzyme ?-(1-3)-glucan synthase depends on
    calcium ions
  • It is a cofactor in the enzymes responsible for
    the hydrolisis of ATP

  • It has been found to be essential for plant
  • It is sometimes considered as micro nutrient,
    because it is required in a small amount
  • It is required for water splitting protein
    complex of photosystem II
  • It can function in osmoregulation in particular
    stomata guard cell

  • Plant requirement for microelement have only been
    elucidated in the 19th century
  • In the early of 20th century, uncertainty still
    existed over the nature of the essential
  • many tissue undoubtedly grown successfully
    because they were cultured on media prepared
    from impure chemicals or solidified with agar
    which acted as a micronutrient source
  • In the first instance, the advantage of adding
    micronutrients was mainly evaluated by their
    capability to improve the callus growth or root
  • Knudson (1922) incorporated Fe and Mn on very
    successful orchid seed media
  • Heller (1953) was first well demonstrated the
    advantages of microelement on tissue culture

Why in the first development many tissue were
undoubtedly grown successfully in tissue culture
media without micronutrient?
  • Media is solidified with agar which acted as a
    micronutrient source
  • Plant cells are more demanding for micronutrients
    when undergoing morphogenesis

Quantity of the Micronutrient
Boron (B)
  • It is involved in plasma membrane integrity and
    function, probably by influencing membrane
    protein and cell wall intactness
  • It is required for the metabolism of phenolic
    acids, and for lignin biosynthesis
  • It is probably a component, or co-factor of the
    enzyme which converts p-coumaric acid to
  • It is necessary for the maintenance of
    meristematic activity, most likely because it is
    involved in the synthesis of N-bases
  • It is also thought to be involved in the
    maintenance of membrane structure and function,
    possibly by stabilizing natural metal chelates,
    which are important in wall and membrane
    structure and function

Manganese (Mn)
  • It is the most important micro nutrients
  • It has similar properties to Magnesium, it is
    apparently able to replace magnesium is some
    enzyme systems
  • It is involved in respiration and photosynthesis
    as metalloprotein structure
  • It is known to be required for the activity of
    several enzymes
  • It is necessary for the maintenance of
    chloroplast ultra structure
  • It is involved in regulation of enzymes and
    growth hormones.
  • It assists in photosynthesis and respiration.

Zinc (Zn)
  • It is a component of stable metallo enzymes with
    many diverse function
  • It is required in more than 300 enzymes
  • Its deficient plants will suffer from reduced
    enzyme activities and as a consequent will
    diminute in protein, nucleic acid and chlorophyl
  • There is a close relationship between zinc
    concentration of plants and their auxin content

Copper (Cu)
  • Plant only contains a few part of million of Cu
  • It becomes attached to enzymes, many of which
    bind to and reach with oxygen
  • It occurs in plastocynain, a pigment
    participating in electron transport
  • Highly concentration of Cu can be toxic

Molybdenum (Mo)
  • It is utilized in the form of hexavalent Mo
  • It is absorbed as the molybdate ions
  • It is a component of several plant enzymes, two
    being nitrate reductase and nitrogenase, in which
    it is a cofactor together with iron

Cobalt (Co)
  • It is sometimes not regarded as an essential
  • It might have a role in regulating morhogenesis
    of higher plants
  • It is the metal component of vitamin B12 which is
    concerned with nucleic acid synthesis, though
    evidence that it has any marked stimulatory
    effect on growth and morphogenesis is hard to
  • It can have a protective action against metal
    chelate toxicity and it is able to inhibit
    oxidative reaction catalyzed by copper and iron
  • Cobalt can inhibit ethylene biosynthesis

Nickel (Ni)
  • It is a component of urease enzyme which convert
    urea to ammonia
  • It has been shown to be an essential
    micronutrient for some legumes
  • The presence of Ni strongly stimulate the cell
    growth in a medium containing urea as a nitrogen
  • Agar contains relatively high levels of nickel
    and the possibility of urea toxicity may have
    been avoided because in tissue culture media,
    urea diffuses into the medium

Iodine (I)
  • It is not recognized as a essential element for
    plants, although it may be necessary for the
    growth of some algae and small amount was
    accumulated in higher plant
  • It has been added to many tissue culture media
  • In improve the in vitro root growth
  • It prevent the explant browning
  • It enhance the destruction and/or the lateral
    transport of auxin

Iron (Fe)
  • A key properties of iron is its capacity to be
    oxidized easily from the ferrous (Fe(II)) to the
    ferric (Fe(III)) state and for ferric compounds
    to be readily reduced back to the ferrous form
  • Iron is primarily used in the chloroplasts,
    mitochondria and peroxisomes for effecting
    oxidation/reduction reaction
  • It is a component of ferredoxin proteins which
    function as electron carriers in photosynthesis
  • Iron is an essential micronutrient for plant
    tissue culture and can be taken up as either
    ferrous or ferric ions
  • Iron may not be available to plant cells, unless
    the pH falls sufficiently to bring free ions
    back to solutions
  • Iron can be chelated with EDTA
  • The addition of Fe-EDTA chelate greatly improved
    the availability of the element

Chelating agent
  • Some organic compounds are capable of forming
    complexes with metal cations, in which the metal
    is held with fairly tight chemical bonds
  • Metal can be bound (sequestered) by a chelating
    agent and held in solution under conditions where
    free ions would react with anions to form
    insoluble compounds, and some complexes can be
    more chemically reactive than the metals
  • Chelating agents vary in their sequestering
    capacity according to chemical structure and
    their degree of ionisation, which changes with pH
    of the solution
  • Naturally occurring compounds can act as
    chelating agents such as proteins, peptides,
    carboxylic acids and amino acids
  • There are also synthetic chelating agents with
    high avidity for divalent and trivalent ions

Chelating agents
Carbon Source
  • 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, manitol 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
  • The presence of sucrose specifically inhibits
    chlorophyll formation and photosynthesis, making
    autotrophic growth less feasible
  • Sucrose in the culture media is usually
    hydrolyzed totally or partially into the
    component monosaccharides glucose and fructose
  • The general superiority of sucrose over glucose
    may be on account of the more effective
    translocation of sucrose to apical meristems

Organic supplement
  • Vitamins
  • Only thiamine (vitamin B1) is essential for most
    plant cultures, it is required for carbohydrate
    metabolism and the biosynthesis of some amino
  • Thiamine (vitamin B1)
  • Essential as a coenzyme in the citric acid
  • Nicotinic acid (niacin) and pyridoxine (B6)

Organic supplement
  • Myo-inositol
  • Although it is not essential for growth of many
    plant species, its effect on growth is
  • Part of the B complex, in phosphate form is part
    of cell membranes, organelles and is not
    essential to growth but beneficial
  • Complex organics
  • Such as coconut milk, coconut water, yeast
    extract, fruit juices and fruit pulps.

Physical support agents
  • A. Gelling agents
  • When semi-solid or solid culture media are
    required, gelling agents are used.
  • An example
  • Agar, agarose, gelrite, phytagel
  • Structural supports
  • Filter paper bridges, liquid permeable
    membrane support systems

  • Agar is the most commonly used gelling agent
  • It is a natural product extracted from species of
    red algae, especially Gelidium amansii
  • It is synthetic polysaccharide gelling agents
  • Agar consists of 2 components
  • Agarose is an alternating D-galactose and
    3,6-anhydro-L-galactose with side chains of
    6-methyl-D-galactose residues (50 -90).
  • 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

  • Advantages
  • Agar is an inert component, form a gel in water
    that melt at 100 C and solidify at nearly 45
  • 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.
  • Gels are not digested by plant enzymes
  • Agar does not strongly react with media
  • Disadvantages
  • Agar does not gel well under acidic conditions
    (pH lt4.5).
  • The inclusion of activated charcoal in media may
    also inhibit gelling of agar.

  • It is extracted from agar leaving behind
    agaropectin and its sulfate groups.
  • It is used when the impurities of agar are a
    major disadvantage.

  • 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

  • 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
  • It is used at a concentration of 1.5-2.5 g/L.
  • It should be prepared with rapid stirring to
    prevent clumping.

Commercial 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
  • Knudsons medium

  • (the Greek word hormaein, meaning "to excite").
  • Small organic molecule that elicits a
    physiological response at very low concentrations
  • Chemical signals that coordinate different parts
    of the organism
  • Internal and external signals that regulate
    growth are mediated, at least in part, by
    growth-regulating substances, or hormones

Plant Hormone
  • A natural substance which produced by plant and
    acts to control plant activities.
  • Chemical messengers influencing many patterns of
    plant development
  • Naturally occurring or synthetic compounds that
    affect plant growth and development
  • Plant hormones differ from animal hormones in
  • No evidence that the fundamental actions of plant
    and animal hormones are the same.
  • Unlike animal hormones, plant hormones are not
    made in tissues specialized for hormone
    production. (e.g., sex hormones made in the
    gonads, human growth hormone - pituitary gland) 
  • Unlike animal hormones, plant hormones do not
    have definite target areas (e.g., auxins can
    stimulate adventitious root development in a cut
    shoot, or shoot elongation or apical dominance,
    or differentiation of vascular tissue). 

  • Synthesized by plants.
  • Show specific activity at very low concentrations
  • Display multiple functions in plants.
  • Play a role in regulating physiological phenomena
    in vivo in a dose-dependent manner
  • They may interact, either synergistically or
    antagonistically, to produce a particular effect.

Synthetic plant hormone
Plant growth regulators
  • Growth-inhibiting chemicals
  • Growth-promoting chemicals
  • Root-promoting chemicals

(No Transcript)
Plant hormones as Chemical Messengers
  • Auxins
  • Cytokinins
  • Gibberellins
  • Ethylene

  • Arpad Paál (1919) - Asymmetrical placement of cut
    tips on coleoptiles resulted in a bending of the
    coleoptile away from the side onto which the tips
    were placed (response mimicked the response seen
    in phototropism). 
  • Frits Went (1926) determined auxin enhanced cell

  • Absolutely essential (no mutants known)
  • One compound Indole-3-acetic acid.
  • Many synthetic analogues
  • NAA, IBA, 2,4-D, 2,4,5-T, Picloram
  • Cheaper more stable
  • Generally growth stimulatory.
  • Promote rooting
  • Stimulate cell elongation
  • Increase the rate of transcription
  • Mediate the response of bending in response to
    gravity or light
  • Produced in meristems, especially shoot meristem
    and transported through the plant in special
    cells in vascular bundles.

Discovery of cytokinins
  • Gottlieb Haberlandt in 1913 reported an unknown
    compound that stimulated cellular division. 
  • In the 1940s, Johannes van Overbeek, noted that
    plant embryos grew faster when they were supplied
    with coconut milk (liquid endosperm), which is
    rich in nucleic acids.
  • In the 1950s, Folke Skoog and Carlos Miller
    studying the influence of auxin on the growth of
    tobacco in tissue culture. When auxin was added
    to artificial medium, the cells enlarged but did
    not divide. Miller took herring-sperm DNA.
    Miller knew of Overbeek's work, and decided to
    add this to the culture medium, the tobacco cells
    started dividing. He repeated this experiment
    with fresh herring-sperm DNA, but the results
    were not repeated. Only old DNA seemed to work.
    Miller later discovered that adding the purine
    base of DNA (adenine) would cause the cells to

Discovery of cytokinins
  • Adenine or adenine-like compounds induce cell
    division in plant tissue culture. Miller, Skoog
    and their coworkers isolated the growth facto
    responsible for cellular division from a DNA
    preparation calling it kinetin which belongs to a
    class of compounds called cytokinins. 
  • In 1964, the first naturally occurring cytokinin
    was isolated from corn called zeatin. Zeatin and
    zeatin riboside are found in coconut milk. All
    cytokinins (artificial or natural) are chemically
    similar to adenine. 
  • Cytokinins move nonpolarly in xylem, phloem, and
    parenchyma cells.
  • Cytokinins are found in angiosperms, gymnosperms,
    mosses, and ferns. In angiosperms, cytokinins are
    produced in the roots, seeds, fruits, and young

  • Absolutely essential (no mutants known)
  • Natural compound Zeatin, 2-isopentyl adenine
  • Synthetic analogues Benyzladenine (BA), Kinetin.
  • Stimulate cell division (with auxins).
  • Promotes formation of adventitious shoots
  • Stimulate cell division
  • Stimulate dark germination
  • Stimulate leaf expansion
  • Produced in the root meristem and transported
    throughout the plant as the Zeatin-riboside in
    the phloem.

Auxin and Cytokinin Ratio
Interaction of cytokinin and auxin in tobacco
callus (undifferentiated plant cells) tissue
  • Organogenesis Cytokinins and auxin affect
  • High cytokinin/auxin ratios favor the formation
    of shoots
  • Low cytokinin/auxin ratios favor the formation of

Discovered of Gibbereline
  • In 1930's, Ewiti Kurosawa and colleagues were
    studying plants suffering from bakanae, or
    "foolish seedling" disease in rice.
  • Disease caused by fungus called, Gibberella
    fujikuroi, which was stimulating cell elongation
    and division.
  • Compound secreted by fungus could cause bakanae
    disease in uninfected plants. Kurosawa named this
    compound gibberellin. 
  • Gibberella fujikuroi also causes stalk rot in
    corn, sorghum and other plants.
  • Secondary metabolites produced by the fungus
    include mycotoxins, like fumonisin, which when
    ingested by horses can cause equine
    leukoencephalomalacia - necrotic brain or crazy
    horse or hole in the head disease.
  • Fumonisin is considered to be a carcinogen.

  • A family of over 70 related compounds, all forms
    of Gibberellic acid and named as GA1, GA2....
  • Commercially, GA3 and GA49 available.
  • Stimulate etiolation of stems.
  • Help break bud and seed dormancy.
  • Stimulate stem elongation by stimulation cell
    division and elongation
  • Stimulate germination of pollen
  • Produced in young leaves

Abscisic acid (ABA)
Discovery of abscisic acid
  • In 1940s, scientists started searching for
    hormones that would inhibit growth and
    development, what Hemberg called dormins.
  • In the early 1960s, Philip Wareing confirmed that
    application of a dormin to a bud would induce
  • F.T. Addicott discovered that this substance
    stimulated abscission of cotton fruit. he named
    this substance abscisin. (Subsequent research
    showed that ethylene and not abscisin controls
  • Abscisin is made from carotenoids and moves
    nonpolarly through plant tissue. 

Abscisic Acid
  • Only one natural compound.
  • Promotes leaf abscission and seed dormancy.
  • Plays a dominant role in closing stomata in
    response to water stress
  • Involved in the abscission of buds, flower and
  • Inhibit cell division and elongation
  • Has an important role in embryogenesis in
    preparing embryos for desiccation.
  • Helps ensure normal embryos.

H H \ / C C / \ H H
Discovery of ethylene
  • In the 1800s, it was recognized that street
    lights that burned gas, could cause neighboring
    plants to develop short, thick stems and cause
    the leaves to fall off. In 1901, Dimitry Neljubow
    identified that a byproduct of gas combustion was
    ethylene gas and that this gas could affect plant
  • In R. Gane showed that this same gas was
    naturally produced by plants and that it caused
    faster ripening of many fruits. 
  • Synthesis of ethylene is inhibited by carbon
    dioxide and requires oxygen. 

  • Gas - diffuses through tissues
  • Stimulates abscission and fruit ripening
  • Used in commercial ripening for bananas green
    picked fruit
  • Involved in leaf abscission flower senescence
  • Primarily synthesized in response to stress
  • Regulate cell death programming

  • Promote shoot elongating
  • Inhibit root growth
  • Promote ethylene biosynthesis
  • Enhance resistance to chilling, disease and

Salicylic acid
  • Promote flowering
  • Stimulate plant pathogenesis protein production

Play an important role in plant defence mechanisms
  • Play an important role in plant defence mechanisms

Thank you