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Plant Tissue Culture - PowerPoint PPT Presentation


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Plant Tissue Culture. 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). Why does it work?.

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what is it
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)
slide3

Why does it work?

  • Plant cells – Dedifferentiate
  • Plant cell division- Somatic cells are diploid
  • Mitosis – Chromosomes duplicate and form clones
  • Totipotency
slide4

What’s 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)
slide5

What’s 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
slide6

What’s 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
slide7

What’s 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
slide8

What’s 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
slide9

What’s 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
slide10

What’s 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.
slide11

What’s 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.
slide12

What’s the history?

  • 1962 - Development of MS medium. Murashige T. and Skoog F., Physiol. Plant., 15: 473-497
  • To be continued….
slide13

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
slide14

What is Needed

  • Growth regulators – discussed in depth later
  • Frequent subculturing to ensure adequate nutrition and to avoid the build up of waste metabolites
functions of medium
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
major components
Major Components
  • Salt Mixtures
  • Organic Substances
  • Natural Complexes
  • Inert Supportive Materials
  • Growth Regulators
slide19

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.
macroelements
Macroelements
  • Nitrogen (N)
    • nitrate ion (NO3- oxidized)
    • ammonium ion (NH4+ reduced)
    • 25-60 mM
    • organic
slide21
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.
macroelements1
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
slide23

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.
micronutrients
Micronutrients
  • Iron (Fe) 1 m M - Involved in respiration , chlorophyll synthesis and photosynthesis. FeNaEDTA = sodium salt ofEDTA 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.
micronutrients1
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)
organic compounds
Organic Compounds
  • Sugar – carbon source
    • sucrose
    • Others – fructose,glucose
    • 20 to 40 g/l, usually
organic compounds1
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
slide28

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
slide29

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
slide30

Natural Complexes - Undefined

  • -Coconut endosperm
  • -Fish emulsion
  • -Protein hydrolysates
  • -Tomato juice
  • -Yeast extracts
  • -Potato agar
slide31

Growth regulators - Hormones

  • -auxin - roots
  • -cytokinin - shoots
  • -gibberellin – cell enlargement
  • -abscisic acid – plant stress hormone
  • -ethylene – BAD!
support systems
Support Systems
  • Agar (from seaweed)
  • Agarose
  • Gelrite (Phytagel) (from bacteria)
  • Mixtures (Phytagar)
  • Mechanical (bridges, rafts)
  • Sand
media formulations
Media Formulations
  • Many available
  • Differ in salt concentrations
  • Differ in presence or absence of salts
  • M&S most widely used by far