Download Aseptic Technique

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?
• Plant cells – Dedifferentiate
• Plant cell division- Somatic cells are
diploid
• Mitosis – Chromosomes duplicate and
form clones
• Totipotency
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)
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
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
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
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
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
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: 118131
• 1958 - Pro-embryo formation in callus
clumps and cell suspension of carrot.
Reinert J. and Steward F. C., Naturwiss.,
45: 344-345.
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.
What’s the history?
• 1962 - Development of MS medium.
Murashige T. and Skoog F., Physiol.
Plant., 15: 473-497
• To be continued….
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
What is Needed
• Growth regulators – discussed in
depth later
• Frequent subculturing to ensure
adequate nutrition and to avoid the build
up of waste metabolites
Nutrient Media for Plant
Tissue Cultures
Functions of medium
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Provide water
Provide mineral nutritional needs
Provide vitamins
Provide growth regulators
Access to atmosphere for gas
exchange
• Removal of plant metabolite waste
Major Components
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Salt Mixtures
Organic Substances
Natural Complexes
Inert Supportive Materials
Growth Regulators
Mineral Elements
Macronutrient salts
Function 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
• Nitrogen (N)
– nitrate ion (NO3- oxidized)
– ammonium ion (NH4+ reduced)
– 25-60 mM
– organic
• 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.
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
• 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
– 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.
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
• Sugar – carbon source
– sucrose
– Others – fructose,glucose
– 20 to 40 g/l, usually
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
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
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
Natural Complexes - Undefined
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-Coconut endosperm
-Fish emulsion
-Protein hydrolysates
-Tomato juice
-Yeast extracts
-Potato agar
Growth regulators - Hormones
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-auxin - roots
-cytokinin - shoots
-gibberellin – cell enlargement
-abscisic acid – plant stress hormone
-ethylene – BAD!
Support Systems
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Agar (from seaweed)
Agarose
Gelrite (Phytagel) (from bacteria)
Mixtures (Phytagar)
Mechanical (bridges, rafts)
Sand
Media Formulations
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Many available
Differ in salt concentrations
Differ in presence or absence of salts
M&S most widely used by far