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: 118-131 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 Provide water Provide mineral nutritional needs Provide vitamins Provide growth regulators Access to atmosphere for gas exchange Removal of plant metabolite waste

Major Components 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 Phenolic compounds 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 -Coconut endosperm -Fish emulsion -Protein hydrolysates -Tomato juice -Yeast extracts -Potato agar

Growth regulators - Hormones -auxin - roots -cytokinin - shoots -gibberellin – cell enlargement -abscisic acid – plant stress hormone -ethylene – BAD!

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

Media Formulations Many available Differ in salt concentrations Differ in presence or absence of salts M&S most widely used by far