Plant Tissue Culture

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Plant Tissue Culture
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What is it?

• Tissue culture is the term used for the process
  of growing cells artificially in the laboratory
• Tissue culture produces clones, in which all
  product cells have the same genotype (unless
  affected by mutation during culture)

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Why does it work?

• Plant cells Dedifferentiate
• Plant cell division- Somatic cells are diploid
• Mitosis Chromosomes duplicate and form clones
• Totipotency

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Whats the history?

• Cell theory, suggesting totipotentiality of
  cells. Schleiden M. J., Arch. Anat., Physiol. U.
  wiss. Med. (J. Muller), 1838 137-176 Schwann
  T., W. Engelman, No. 176 (1910)

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Whats the history?

• 1902 - First but unsuccessful attempt of tissue
  culture using monocots. Haberlandt G.,
  Sitzungsber Akad. Wiss. Wien, Math.-Naturwiss.
  Kl., 111 69-92.
• 1926 - FW Went demonstrated that there were
  growth substances in coleoptiles from Avena

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Whats the history?

• 1934 - White generated continuously growing
  culture of meristematic cells of tomato on medium
  containing salts,yeast extract and sucrose and 3
  vitamins (pyridoxine, thiamine, nicotinic acid)
  established the importance of additives

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Whats the history?

• 1939 - Successful continuously growing cambial
  cultures of carrot and tobacco. Gautheret R. J.,
  C. R. Acad. Sci. (Paris), 208 118-120 Nobecourt
  P., C. R. Soc. Biol. (Paris), 130 1270-1271
  White P. R., Am. J. Bot., 26 59-64

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Whats the history?

• 1943-1950 - Tumor-inducing principle of crown
  gall tumors identified. Braun A. C. Phytopathol.
  33 85-100 P. N. A. S. USA 45 932-938
• 1948 - Formation of adventitious shoots and roots
  in tobacco. Skoog F. and Tsui C., Am. J. Bot.,
  355 782-787

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Whats the history?

• 1952 - Virus-free Dahlia through meristem
  culture. Morel G. and Martin C., C. R. Hebd.
  Seances Acad. Sci. (Paris), 235 1324-1325.
• 1952 - First successful micro-grafts. Morel G.
  and Martin C., C. R. Acad. Sci. (Paris), 235
  1324-1325

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Whats the history?

• 1957 - Discovery that root or shoot formation in
  culture depends on auxin cytokinin ratio. Skoog
  F. and Miller C. O., In vitro Symp. Soc. Exp.
  Biol., No. 11 118-131
• 1958 - Pro-embryo formation in callus clumps and
  cell suspension of carrot. Reinert J. and Steward
  F. C., Naturwiss., 45 344-345.

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Whats the history?

• 1960 - Enzymatic degradation of cell wall for
  protoplast formation. Cocking E. C., Nature, 187
  927-929.
• 1960 - Vegetative propagation of orchids by
  meristem culture. Morel G., Am. Orchid Soc.
  Bull., 29 495-497.

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Whats the history?

• 1962 - Development of MS medium. Murashige T. and
  Skoog F., Physiol. Plant., 15 473-497
• To be continued.

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What is needed?

• 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

• Growth regulators discussed in depth later
• Frequent subculturing to ensure adequate
  nutrition and to avoid the build up of waste
  metabolites

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Nutrient Media for Plant Tissue Cultures
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Functions of medium

• Provide water
• Provide mineral nutritional needs
• Provide vitamins
• Provide growth regulators
• Access to atmosphere for gas exchange
• Removal of plant metabolite waste

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Major Components

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

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Mineral Elements
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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 Abundant 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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Macroelements

• Nitrogen (N)
• nitrate ion (NO3- oxidized)
• ammonium ion (NH4 reduced)
• 25-60 mM
• organic

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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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Macroelements

• Potassium (K) 20 -30 mM
• Phosphorous (P) 1-3 mM
• Calcium (Ca) 1-3 mM
• Magnesium (Mg) 1-3 mM
• Sulfur (S) 1-3 mM

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• 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)
• 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.

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Micronutrients

• Iron (Fe) 1 m M - Involved in respiration ,
  chlorophyll synthesis and photosynthesis.
  FeNaEDTA sodium salt of EDTA sequesters iron,
  making it available to plants.
• Manganese (Mn) 5-30 m M - Involved in regulation
  of enzymes and growth hormones. Assists in
  photosynthesis and respiration.

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Micronutrients

• Zinc (Zn)
• Boron (B)
• Copper (Cu) 0.1 m M
• Molybdenum (Mo) 1 m M
• Cobalt (Co) 0.1 m M
• Iodine (I) Nickel (Ni), aluminum (Al), and
  silicon (Si)

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Organic Compounds

• Sugar carbon source
• sucrose
• Others fructose,glucose
• 20 to 40 g/l, usually

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Organic Compounds

• Vitamins
• thiamine (vitamin B1) - essential as a coenzyme
  in the citric acid cycle
• nicotinic acid (niacin) and pyridoxine (B6)
• myo-inositol - part of the B complex, in
  phosphate form is part of cell membranes,
  organelles and is not essential to growth but
  beneficial

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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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Charcoal

• 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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Natural Complexes - Undefined

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

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Growth regulators - Hormones

• -auxin - roots
• -cytokinin - shoots
• -gibberellin cell enlargement
• -abscisic acid plant stress hormone
• -ethylene BAD!

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Support Systems

• Agar (from seaweed)
• Agarose
• Gelrite (Phytagel) (from bacteria)
• Mixtures (Phytagar)
• Mechanical (bridges, rafts)
• Sand

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Media Formulations

• Many available
• Differ in salt concentrations
• Differ in presence or absence of salts
• MS most widely used by far

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