Plant Tissue Culture Media

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Plant Tissue Culture Media

• Whats really important?

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Plant Tissue CultureWhy does it work?

• Plant cells Dedifferentiate
• Plant cell division- Somatic cells are diploid
• Mitosis Chromosomes duplicate and form clones
• Meiosis The process of forming sex cells, 2n
  splits and become 1n gametes

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Major ConstituentsWhy are these things in media?

• Salt Mixtures
• Organic Substances
• Natural Complexes
• Inert Supportive Materials
• Growth Regulators

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Macro-nutrient saltsWhat the ?

• NH4NO3 Ammonium nitrate
• KNO3 Potassium nitrate
• CaCl2 -2 H2O Calcium chloride (Anhydrous)
• MgSO4 -7 H2O Magnesium sulfide (Epsom Salts)
• KH2PO4 Potassium hypophosphate
• FeNaEDTA Fe/Na ethylene-diamine-tetra acetate
• H3BO3 Boric Acid
• MnSO4 - 4 H2O Manganese sulfate
• ZnSO4 - 7 H2O Zinc sulfate
• KI Potassium iodide
• Na2MoO4 - 2 H2O Sodium molybdate
• CuSO4 - 5 H2O Cupric sulfate
• CoCl2 - H2O Cobaltous sulfide

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Macronutrient saltsFunction of nutrients in
plant growth

• Nitrogen Influences plant growth rate,
  essential in plant nucleic acids (DNA), proteins,
  chlorophyll, amino acids, and hormones.
• Phosphorus Abundent in meristimatic and fast
  growing tissue, essential in photosynthesis,
  respiration,
• Potassium Necessary for cell division,
  meristematic tissue, helps in the pathways for
  carbohydrate, protein and chlorophyll synthesis.

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Macronutrient saltsFunction of nutrients in
plant growth

• Calcium - Involved in formation of cell walls and
  root and leaf development. Participates in
  translocation of sugars, amino acids, and ties up
  oxalic acid (toxin)
• Iron - Involved in respiration , chlorophyll
  synthesis and photosynthesis. FeNaEDTA sodium
  salt of EDTA sequesters iron, making it available
  to plants.
• Magnesium - Involved in photosynthetic and
  respiration system. Active in uptake of phosphate
  and translocation of phosphate and starches.
• Sulfur - Involved in formation of nodules and
  chlorophyll synthesis, structural component of
  amino acids and enzymes.
• Manganese - Involved in regulation of enzymes and
  growth hormones. Assists in photosynthesis and
  respiration.

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Macronutrient saltsFunction of nutrients in
plant growth

• Molybdenum - Involved in enzymatic reduction of
  nitrates to ammonia. Assists in conversion of
  inorganic phosphate to organic form.
• Zinc - Involved in production of growth hormones
  and chlorophyll. Active in respiration and
  carbohydrate synthesis.
• Boron - Involved in production of growth hormones
  and chlorophyll. Active in respiration and
  carbohydrate synthesis.
• Copper -Involved in photosynthetic and
  respiration systems. Assists chlorophyll
  synthesis and used as reaction catalyst.

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Organic Compounds

• Carbon Sources Sucrose, Glucose (Sometimes used
  with monocots), Fructose (Plants Need Carbon)
• Vitamins
• Adenine part of RNA and DNA
• Inositol part of the B complex, in phosphate
  form is part of cell membranes, organelles and is
  not essential to growth but benificial
• Thiamine essential as a coenzyme in the citric
  acid cycle.

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• Amino Acids -The most common sources of organic
  nitrogen used in culture media are amino acid
  mixtures, (e.g., casein hydrolysate),
  L-glutamine, L-asparagine, and adenine. When
  amino acids are added alone, they can be
  inhibitory to cell growth. Tyrosine has been used
  to stimulate morphogenesis in cell cultures but
  should only be used in an agar medium.
  Supplementation of the culture medium with
  adenine sulfate can stimulate cell growth and
  greatly enhance shoot formation. L-tyrosine -
  stimulates shoot formation.

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Still other organics

• Organic Acids
• Citric acid (150 mg/l) typically used with
  ascorbic acid (100 mg/l) as an antioxidant.
• Can also use some of Kreb Cycle acids
• Phenolic compounds
• Phloroglucinol - Stimulates rooting of shoot
  sections

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Natural ComplexesHome Made Bombs

• -Coconut endosperm
• -Fish emulsion
• -Protein hydrolysates
• -Tomato juice
• -Yeast extracts
• -Potato agar

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CharcoalWheres the toilet?

• Activated charcoal is used as a detoxifying
  agent. Detoxifies wastes from plant tissues,
  impurities
• Impurities and absorption quality vary
• Concentration normally used is 0.3 or lower
• Charcoal for tissue culture
• acid washed and neutralized
• never reuse

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Growth regulatorsBody building Plants

• -auxin - Roots
• -cytokinin - Shoots
• -gibberellin Cell Enlargement
• -abscisic acid Plant stress hormone
• -ethylene BAD!

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Auxins

• Order of effectiveness in callus formation,
  rooting of cuttings, and the induction of
  adventive embryogenesis
• IAA
• IBA
• NAA
• 2,4-D
• 2,4,5-T
• Pictoram

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Auxins

• The various auxins differ in their physiological
  activity and in the extent to which they move
  through tissue, are bound to the cells, or
  metabolized.

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Auxins

• Auxins are generally included in a culture medium
  to stimulate callus production and cell growth,
  to initiate shoots, particularly roots, and to
  induce somatic embryogenesis and stimulate growth
  from shoot apices and shoot tip cultures.

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Auxins

• Naturally occurring IAA has been shown to have
  less physiological activity than synthetic
  auxins. 2,4- D has eight to twelve times the
  activity,
• 2,4,5-T has four times the activity,
• PCPA and Picloram have two to four times the
  activity, and NAA has two times the activity of
  IAA. Although 2,4-D, 2,4,5-T, PCPA, and Picloram
  are often used to induce rapid cell
  proliferation, exposure to high levels or
  prolonged exposure to these auxins, particularly
  2,4-D, results in suppressed morphogeneic
  activity

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Cytokinins

• -Enhances adventitious shoot formation
• BA
• 2iP
• Kinetin
• Zeatin
• PBA

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Cytokinis

• Zeatin and 2iPare considered to be naturally
  occurring cytokinins, while BA and kinetin are
  synthetically derived cytokinins. Adenine,
  another naturally occurring compound, has a base
  structure similar to that of the cytokinins and
  has shown cytokinin-like activity in some cases.

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Cytokins

• Many plant tissues have an absolute requirement
  for a specific cytokinin for morphogenesis to
  occur, whereas some tissue are considered to be
  cytokinin independent, i.e., no cytokinin or a
  specific cytokinin may be required for
  organogenesis. The cytokinins are generally added
  to a culture medium to stimulate cell division,
  to induce shoot formation and axillary shoot
  proliferation, and to inhibit root formation.

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Auxins and Cytokinis

• The type of morphogenesis that occurs in a plant
  tissue culture largely depends upon the ratio and
  concentrations of auxins and cytokinins present
  in the medium. Root initiation of plantlets,
  embryogenesis, and callus initiation all
  generally occur when the ration of auxin to
  cytokinin is high, whereas adventitious and
  axillary shoot proliferation occur when the
  ration is low. The concentrations of auxins and
  cytokinins are equally as important as their
  ratio.

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Gibberellin

• Not generally used in tissue culture
• Tends to suppress root formation and adventitious
  embryo formation

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Abscisic Acid

• Primarily a growth inhibitor but enables more
  normal development of embryos, both zygotic and
  adventitious

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Ethylene

• Question is not how much to add but how to get
  rid of it in-vitro
• Natural substance produced by tissue cultures at
  fairly high levels especially when cells are
  under stress
• Enhances senescense
• Supresses embryogenesis and development in general

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Hormone Combinations

• Callus development
• Adventitious embryogenesis
• Rooting of Shoot cuttings
• Adventitious shoot and root formation

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Callus development

• Auxin alone
• Pictoram 0.3 to 1.9 mg/l
• 2,4-D 1.0 to 3.0 mg/l
• Auxin and Cytokinin
• IAA 2.0 to 3.0 mg/ l
• 2iP 0.1 mg/l
• NAA 0.1 mg/l
• 2iP 0.1 mg/l

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Adventitious Embryogenesis

• Induction is the first step (biochemical
  differentiation
• High auxin in media
• Development is the second step which includes
  cell and tissue organization, growth and
  emergence of organ or embryo
• no or very low auxin. Can also add ABA 10 mg/l,
  NH4 and K (dont know which step)

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Rooting of Shoot Cutttings

• Induction need high auxin, up to 100 mg/l for
  3-14 days
• Development no auxin, in fact auxin may inhibit
  growth
• Can also add phloroglucinol and other phenolics
  but we dont know for sure how they fit in

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Adventitious shoot and root development

• Skoog and Millers conclusions
• -formation of shoots and roots controlled by a
  balance between auxin and cytokinin
• -high auxin/low cytokinin root development
• -low auxin/high cytokinen shoot development
• -concept applies mainly to herbaceous genera and
  easy to propagate plants
• -we lump together induction and development
  requirements

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Summary

• Auxin Cytokinin
• callus high low
• axillary shoots low to none very high
• adventi. shoots equal equal
• rooting high low
• embryogenesis high low