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Premium member Presentation Transcript NUTRIENT MEDIA FOR PLANT TISSUE CULTURES : NUTRIENT MEDIA FOR PLANT TISSUE CULTURES BY- JWALANT LOHIYA NATIONAL INSTITUTE OF TECHNOLOGY,WARANGALPLANT CELL CULTURE MEDIA: PLANT CELL CULTURE MEDIA Culture media used for the in vitro cultivation of plant cells are composed of three basic components: (1).essential elements , or mineral ions, supplied as a complex mixture of salts; (2).an organic supplement supplying vitamins and/or amino acids;& (3).a source of fixed carbon; usually supplied as the sugar sucrose.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 wasteMajor Components: Major Components Salt Mixtures Organic Substances Natural Complexes Inert Supportive Materials Growth RegulatorsMINERAL ELEMENTS: MINERAL ELEMENTSMacroelements: Macroelements Nitrogen (N) nitrate ion (NO 3- oxidized) ammonium ion (NH 4+ reduced) 25-60 mM organicMacroelements: Macroelements Potassium (K) 20 -30 mM Phosphorous (P) 1-3 mM Calcium (Ca) 1 and 3 mM Magnesium (Mg) 1-3 mM Sulfur (S) 1-3 mMSlide 8: Macronutrient salts Function … 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.Slide 9: 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. Molybdenum - Involved in enzymatic reduction of nitrates to ammonia. Assists in conversion of inorganic phosphate to organic form.Slide 10: Zinc - Involved in production of growth hormones and chlorophyll. Active in respiration and carbohydrate synthesis. Copper - Involved in photosynthetic and respiration systems. Assists chlorophyll synthesis and used as reaction catalyst.Micronutrients: Micronutrients Iron (Fe) 1 m M Manganese (Mn) 5-30 m M 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)Micronutrients Function: Micronutrients Function 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.ORGANIC COMPOUNDS: ORGANIC COMPOUNDS Sugar sucrose others 20 to 40 g/l Vitamins thiamine (vitamin B1) Nicotinic acid (niacin) and pyridoxine (B6) OthersORGANIC COMPOUNDS …: ORGANIC COMPOUNDS … Myo-inositol Complex organics Activated charcoalOrganic Compounds Function: Organic Compounds Function 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 beneficialAmino Acids: 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.Slide 17: 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 sectionsSlide 18: 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 reuseSlide 19: Natural Complexes Undefined -Coconut endosperm -Fish emulsion -Protein hydrolysates -Tomato juice -Yeast extracts -Potato agarSlide 20: Growth regulators Hormones -Auxin - roots -Cytokinin - shoots -Gibberellin – cell enlargement -Abscisic acid – plant stress hormone -Ethylene – BAD!Auxins: Auxins Order of effectiveness in callus formation, rooting of cuttings, and the induction of adventive embryogenesis IAA IBA NAA 2,4-D 2,4,5-T PicloramSlide 22: The various auxins differ in their physiological activity and in the extent to which they move through tissue, are bound to the cells, or metabolized. Auxins are generally included in a culture medium to stimulate callus production and cell growth, to initiate shoots, particularly roots, and to induce somatic embryogenesis and stimulate growth from shoot apices and shoot tip cultures.Slide 23: Naturally occurring IAA has been shown to have less physiological activity than synthetic auxins. 2,4- D has eight to twelve times the activity, 2,4,5-T has four times the activity, PCPA and Picloram have two to four times the activity, and NAA has two times the activity of IAA. High levels or prolonged exposure to 2,4-D, 2,4,5-T, PCPA, and Picloram, particularly 2,4-D, results in suppressed morphogeneic activityCytokinins: Cytokinins -Enhances adventitious shoot formation BA 2iP Kinetin Zeatin PBASlide 25: Zeatin and 2iPare considered to be naturally occurring cytokinins BA and kinetin are synthetically derived cytokinins. Adenine, another naturally occurring compound, has a base structure similar to that of the cytokinins and has shown cytokinin-like activity in some cases.Slide 26: Many plant tissues have an absolute requirement for a specific cytokinin for morphogenesis to occur, Some tissue are considered to be cytokinin independent, i.e., no cytokinin or a specific cytokinin may be required for organogenesis. The cytokinins are generally added to a culture medium to stimulate cell division, to induce shoot formation and axillary shoot proliferation, and to inhibit root formation .Auxins and Cytokinins: Auxins and Cytokinins The type of morphogenesis that occurs in a plant tissue culture largely depends upon the ratio and concentrations of auxins and cytokinins present in the medium. Root initiation of plantlets, embryogenesis, and callus initiation all generally occur when the ration of auxin to cytokinin is high Adventitious and axillary shoot proliferation occur when the ration is low. The concentrations of auxins and cytokinins are equally as important as their ratio.Gibberellin: Gibberellin Not generally used in tissue culture Tends to suppress root formation and adventitious embryo formationAbscisic Acid: Abscisic Acid Primarily a growth inhibitor Enables more normal development of embryos, both zygotic and adventitiousEthylene: Ethylene Question is not how much to add but how to get rid of it in-vitro Natural substance produced by tissue cultures at fairly high levels especially when cells are under stress Enhances senescense Supresses embryogenesis and development in generalHormone Combinations: Hormone Combinations Callus development Adventitious embryogenesis Rooting of Shoot cuttings Adventitious shoot and root formationCallus development: Callus development Auxin alone: Picloram 0.3 to 1.9 mg/l 2,4-D 1.0 to 3.0 mg/l Auxin and Cytokinin: IAA 2.0 to 3.0 mg/ l 2iP 0.1 mg/l NAA 0.1 mg/lAdventitious Embryogenesis: Adventitious Embryogenesis Induction is the first step (biochemical differentiation High auxin in media Development is the second step which includes cell and tissue organization, growth and emergence of organ or embryo no or very low auxin. Can also add ABA 10 mg/l, NH4 and K (don’t know which step)Rooting of Shoot Cutttings: Rooting of Shoot Cutttings Induction: need high auxin, up to 100 mg/l for 3-14 days Development: no auxin, in fact auxin may inhibit growth Can also add phloroglucinol and other phenolics but we don’t know for sure how they fit inAdventitious shoot and root development: Adventitious shoot and root development Skoog and Miller’s conclusions - formation of shoots and roots controlled by a balance between auxin and cytokinin -high auxin/low cytokinin = root development -low auxin/high cytokinin = shoot development -concept applies mainly to herbaceous genera and easy to propagate plants -we lump together induction and development requirementsSlide 36: A uxin Cytokinin callus high low axillary shoots low to none very high adventi . shoots equal equal rooting high low embryogenesis high low Hormone Combinations …Support Systems: Support Systems Agar (from seaweed) Agarose Gelrite (Phytagel) (from bacteria) Mixtures (Phytagar) Mechanical (bridges, rafts) SandMedia Formulations: Media Formulations Many available Differ in salt concentrations Differ in presence or absence of salts M&S most widely used by farSlide 39: Thank You You do not have the permission to view this presentation. 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