2 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)
3 Why does it work? Plant cells – Dedifferentiate Plant cell division- Somatic cells are diploidMitosis – Chromosomes duplicate and form clonesTotipotency
4 What’s the history?Cell theory, suggesting totipotentiality of cells. Schleiden M. J., Arch. Anat., Physiol. U. wiss. Med. (J. Muller), 1838: ; Schwann T., W. Engelman, No. 176 (1910)
5 What’s the history?First but unsuccessful attempt of tissue culture using monocots. Haberlandt G., Sitzungsber Akad. Wiss. Wien, Math.-Naturwiss. Kl., 111:FW Went demonstrated that there were growth substances in coleoptiles from Avena
6 What’s the history?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
7 What’s the history?Successful continuously growing cambial cultures of carrot and tobacco. Gautheret R. J., C. R. Acad. Sci. (Paris), 208: ; Nobecourt P., C. R. Soc. Biol. (Paris), 130: ; White P. R., Am. J. Bot., 26: 59-64
8 What’s the history?Tumor-inducing principle of crown gall tumors identified. Braun A. C. Phytopathol. 33: & P. N. A. S. USA 45:Formation of adventitious shoots and roots in tobacco. Skoog F. and Tsui C., Am. J. Bot., 355:
9 What’s the history?Virus-free Dahlia through meristem culture. Morel G. and Martin C., C. R. Hebd. Seances Acad. Sci. (Paris), 235:First successful micro-grafts. Morel G. and Martin C., C. R. Acad. Sci. (Paris), 235:
10 What’s the history?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:Pro-embryo formation in callus clumps and cell suspension of carrot. Reinert J. and Steward F. C., Naturwiss., 45:
11 What’s the history?Enzymatic degradation of cell wall for protoplast formation. Cocking E. C., Nature, 187:Vegetative propagation of orchids by meristem culture. Morel G., Am. Orchid Soc. Bull., 29:
12 What’s the history?Development of MS medium. Murashige T. and Skoog F., Physiol. Plant., 15:To be continued….
13 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 semisolidAseptic (sterile) conditions, as microorganisms grow much more quickly than plant and animal tissue and can over run a culture
14 What is Needed Growth regulators – discussed in depth later Frequent subculturing to ensure adequate nutrition and to avoid the build up of waste metabolites
19 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.
20 Macroelements Nitrogen (N) nitrate ion (NO3- oxidized) ammonium ion (NH4+ reduced)25-60 mMorganic
21 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.
22 Macroelements Potassium (K) 20 -30 mM Phosphorous (P) 1-3 mM Calcium (Ca) 1-3 mMMagnesium (Mg) 1-3 mMSulfur (S) 1-3 mM
23 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.
24 MicronutrientsIron (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.
25 Micronutrients Zinc (Zn) Boron (B) Copper (Cu) 0.1 m M Molybdenum (Mo) 1 m MCobalt (Co) 0.1 m MIodine (I) Nickel (Ni), aluminum (Al), and silicon (Si)
26 Organic Compounds Sugar – carbon source sucrose Others – fructose,glucose20 to 40 g/l, usually
27 Organic Compounds Vitamins thiamine (vitamin B1) - essential as a coenzyme in the citric acid cyclenicotinic 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
28 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 acidsPhenolic compoundsPhloroglucinol - Stimulates rooting of shoot sections
29 CharcoalActivated charcoal is used as a detoxifying agent. Detoxifies wastes from plant tissues, impuritiesImpurities and absorption quality varyConcentration normally used is 0.3 % or lowerCharcoal for tissue cultureacid washed and neutralizednever reuse