Plant Tissue Culture Media 1 Logical Basis For

Plant Tissue Culture Media 1

Logical Basis For healthy and vigorous growth, intact plants need to take up from soil of an essential elements. Essential elements (Epstein, 1971): 1. A plant grown in a medium adequately purged of that elements, failed to grow properly or to complete its life cycle 2. It is a constituent of a molecule that is known to be an essential metabolite 2

Essential element ü Macro element/major plant nutrition: Relatively large amount required a. Carbon (C) d. Nitrogen (N) b. Hydrogen (H) e. Calcium (Ca) c. Oxygen (O) f. Magnesium (Mg) g. Potassium (K) h. Phosphorus (P) i. Sulphur (S) ü Micro element/ minor plant nutrient/trace elements: Small quantities required a. Iron (Fe) b. Chlorine (Cl) c. Zinc (Zn) d. Copper (Cu) e. Nickel (Ni) f. Sodium (Na) g. Manganese (Mn) h. Boron (B) i. Molybdenum (Mo) 3

Why plant in vitro culture needs media? Functions of media: ü ü ü ü Provide water Provide mineral nutritional needs Provide vitamins Provide growth regulators Provide amino acids Provide sugars Access to atmosphere for gas exchange Removal of plant metabolite waste

Plant tissue culture media 1. Macronutrients (always employed) 2. Micronutrients (nearly always employed, although sometimes just one element, iron, has been used) 3. Vitamins (generally incorporated , although the actual number of compounds added, varies greatly) 4. Amino acids and other nitrogen supplements (usually omitted, but sometimes used with advantage) 5. Sugar (nearly always added, but omitted for some special purposes) 6. Undefined supplements (which, when used, contribute some above components, and also plant growth substances or regulants) 7. Buffers (have seldom been used in the past, but recently suggest that the additions of organic acids or buffers could be beneficial 5 in some circumstances)

Macronutrient 1. Macronutrients for plant tissue culture are provided from salt, however plant absorb entirely as ions 2. Nitrogen is mainly absorbed in the form of ammonium or nitrate 3. Phosphorus as the phosphate ions 4. Sulphur as sulphate ions 5. The most important step in deriving medium is the selection of macronutrient ions in the correct concentration and balanced 6. The salts normally used to provide macroelements also provide sodium and chlorine, however, plant cell tolerate high concentration of both ions without injury, these ions are frequently given little importance when contemplating 6 media changes

Quantity of the Macronutrient

Nitrogen 1. It is essential to plant life 2. Both growth and morphogenesis is markedly influenced by the availability of nitrogen and the form in which it is presented 3. Most media contain more nitrate than ammonium ions. Most intact plants, tissues and organ taken up nitrogen effectively, and grow more rapidly on nutrient solutions containing both nitrate and ammonium ions 4. Nitrate has to be reduced to ammonium before being utilized biosynthetically 5. Ammonium in high concentration is latent toxic 6. For most type of culture, nitrate needs to be presented together with the reduced form of nitrogen and tissue will usually fail to grow on a medium with nitrate as the only 8 nitrogen source

NH 4+ and NO 3 - Regulate Medium p. H and Root Morphogenesis of Rose Shoots

Amino acids 1. Amino acids can be added to satisfy the requirement for reduced nitrogen, but as they are expensive to purchase, they will only be used on media for mass propagation where this results in improved result 2. A casein hydrolysate, yeast extract which mainly consist of a mixture of amino acids substantially increased the yield of callus 3. Organic supplements have been especially beneficial for growth or morphogenesis when cells were cultured on media which do not contain ammonium ions 4. Glycine os an ingredient of many media. It is difficult to find hard evidence that glycine is really essential for so many tissue culture, but possible it helps to protect cell membranes from osmotic and temperature stress 10

Amino Acids The most common sources of organic nitrogen used in culture media are amino acid mixtures. n Its uptake more rapidly than in organic amino acids . (e. g. , casein hydrolysate), L-glutamine, L-asparagine, and adenine. When amino acids are added alone, they can be inhibitory to cell growth. . n

Beneficial effects of amino acids n n n n Rapid growth Protoplast cell division Conservation of ATP AS chelating agent Enhanced nitrogen assimilation Not toxic as ammonium As buffer

Phosphorous 1. It is a vital element in plant biochemistry 2. It occurs in numerous macromolecules such as nucleic acids, phospholipids and co-enzymes 3. It functions in energy transfer via pyrophosphate bond I ATP 4. Phosphate groups attached to different sugar provide energy in respiration and photosynthesis and phosphate bound to proteins regulate their activity 5. Phosphorous is absorbed into plants in the form of the primary or secondary orthophosphate anions by an active process which requires the expenditure of respiratory energy 6. Phosphate in not reduced in plants, but it remains in the oxydised form 7. It is used in plant as the fully oxydised orthophosphate form 13

Potassium 1. 2. 3. 4. It is not metabolized It is a major cation within the plants It contributes significantly to the osmotic potential of cells It is transported quickly across cell membrane and two of its major role is regulating the p. H and osmotic environment within the cells 5. Many protein show a high specificity for potassium which acting as a cofactor, alters their configuration so that it become active enzyme 6. It is also neutralize organic anions produce in the cytoplasm and so stabilize the p. H and osmotic potential of the cells 14

Sodium 1. It is taken up into plant but in most cases it is not required for growth and development 2. Many plants actively secret it from their roots to maintain a low internal concentration 3. It is only appeared to be essential to salt tolerance plant 15

Magnesium 1. It is an essential component of the chlorophyll molecules 2. It is also required non-specifically for the activity of many enzymes, especially in the transfer of phosphate 3. ATP synthesis has an absolute requirement for magnesium and it is a bridging element in the aggregation of ribosome sub-unit 4. It is the central atom in the phorphyrin structure of the chlorophyll molecules 16

Sulfur 1. It is mainly absorbed as sulfate 2. Its uptake is coupled to nitrogen assimilation 3. It is incorporated into chemical compounds mainly as reduced –SH, -S_ or –S-S groups 4. It is used in lipid synthesis and in regulating the structure of proline through the formation of S-S bridges 5. It acts as a ligand joining ion of iron, zinc, copper to metalloportein and enzymes 17

Calcium 1. It helps to balance anion within the plant 2. It is not readily mobile 3. It is involved in the structure and physiologically properties of cell membranes and the middle lamella of the cell walls 4. The enzyme -(1 -3)-glucan synthase depends on calcium ions 5. It is a cofactor in the enzymes responsible for the hydrolisis of ATP 18

Chlorine 1. It has been found to be essential for plant growth 2. It is sometimes considered as micro nutrient, because it is required in a small amount 3. It is required for water – splitting protein complex of photosystem II 4. It can function in osmoregulation in particular stomata guard cell 19

Micronutrients 1. Plant requirement for microelement have only been elucidated in the 19 th century 2. In the early of 20 th century, uncertainty still existed over the nature of the essential microelements 3. many tissue undoubtedly grown successfully because they were cultured on media prepared from impure chemicals or solidified with agar which acted as a micronutrient source 4. In the first instance, the advantage of adding micronutrients was mainly evaluated by their capability to improve the callus growth or root culture 5. Knudson (1922) incorporated Fe and Mn on very successful orchid seed media 6. Heller (1953) was first well demonstrated the advantages of microelement on tissue culture media 20

Why in the first development many tissue were undoubtedly grown successfully in tissue culture media without micronutrient? ü Media is solidified with agar which acted as a micronutrient source ü Plant cells are more demanding for micronutrients when undergoing morphogenesis 21

Quantity of the Micronutrient MS medium was formulated from the ash content of tobacco callus. The higher concentration of salts substantially enhanced cell division

Boron (B) 1. It is involved in plasma membrane integrity and function, probably by influencing membrane protein and cell wall intactness 2. It is required for the metabolism of phenolic acids, and for lignin biosynthesis 3. It is probably a component, or co-factor of the enzyme which converts p-coumaric acid to 5 -hydroxyferulate 4. It is necessary for the maintenance of meristematic activity, most likely because it is involved in the synthesis of Nbases 5. It is also thought to be involved in the maintenance of membrane structure and function, possibly by stabilizing natural metal chelates, which are important in wall and 23 membrane structure and function

Manganese (Mn) 1. It is the most important micro nutrients 2. It has similar properties to Magnesium, it is apparently able to replace magnesium is some enzyme systems 3. It is involved in respiration and photosynthesis as metalloprotein structure 4. It is known to be required for the activity of several enzymes 5. It is necessary for the maintenance of chloroplast ultra structure 6. It is involved in regulation of enzymes and growth hormones. 7. It assists in photosynthesis and respiration. 24

Zinc (Zn) 1. It is a component of stable metallo enzymes with many diverse function 2. It is required in more than 300 enzymes 3. Its deficient plants will suffer from reduced enzyme activities and as a consequent will diminute in protein, nucleic acid and chlorophyl synthesis 4. There is a close relationship between zinc concentration of plants and their auxin content 25

Copper (Cu) 1. Plant only contains a few part of million of Cu 2. It becomes attached to enzymes, many of which bind to and reach with oxygen 3. It occurs in plastocynain, a pigment participating in electron transport 4. Highly concentration of Cu can be toxic 26

Molybdenum (Mo) 1. It is utilized in the form of hexavalent Mo 2. It is absorbed as the molybdate ions 3. It is a component of several plant enzymes, two being nitrate reductase and nitrogenase, in which it is a cofactor together with iron 27

Cobalt (Co) 1. It is sometimes not regarded as an essential elements 2. It might have a role in regulating morhogenesis of higher plants 3. It is the metal component of vitamin B 12 which is concerned with nucleic acid synthesis, though evidence that it has any marked stimulatory effect on growth and morphogenesis is hard to find 4. It can have a protective action against metal chelate toxicity and it is able to inhibit oxidative reaction catalyzed by copper and iron 5. Cobalt can inhibit ethylene biosynthesis 28

Nickel (Ni) 1. It is a component of urease enzyme which convert urea to ammonia 2. It has been shown to be an essential micronutrient for some legumes 3. The presence of Ni strongly stimulate the cell growth in a medium containing urea as a nitrogen source 4. Agar contains relatively high levels of nickel and the possibility of urea toxicity may have been avoided because in tissue culture media, urea diffuses into the medium 29

Iodine (I) 1. It is not recognized as a essential element for plants, although it may be necessary for the growth of some algae and small amount was accumulated in higher plant 2. It has been added to many tissue culture media 3. In improve the in vitro root growth 4. It prevent the explant browning 5. It enhance the destruction and/or the lateral transport of auxin 30

Iron (Fe) 1. A key properties of iron is its capacity to be oxidized easily from the ferrous (Fe(II)) to the ferric (Fe(III)) state and for ferric compounds to be readily reduced back to the ferrous form 2. Iron is primarily used in the chloroplasts, mitochondria and peroxisomes for effecting oxidation/reduction reaction 3. It is a component of ferredoxin proteins which function as electron carriers in photosynthesis 4. Iron is an essential micronutrient for plant tissue culture and can be taken up as either ferrous or ferric ions 5. Iron may not be available to plant cells, unless the p. H falls sufficiently to bring free ions back to solutions 6. Iron can be chelated with EDTA 7. The addition of Fe-EDTA chelate greatly improved the availability of the element 31

Chelating agent 1. Some organic compounds are capable of forming complexes with metal cations, in which the metal is held with fairly tight chemical bonds 2. Metal can be bound (sequestered) by a chelating agent and held in solution under conditions where free ions would react with anions to form insoluble compounds, and some complexes can be more chemically reactive than the metals themselves 3. Chelating agents vary in their sequestering capacity according to chemical structure and their degree of ionisation, which changes with p. H of the solution 4. Naturally –occurring compounds can act as chelating agents such as proteins, peptides, carboxylic acids and amino acids 5. There also synthetic chelating agents with high avidity for divalent and trivalent ions 32

Chelating agents No. Chelating agents Chemical names 1. EDTA Ethylenediamine tetra acetic acid 2. EGTA Ethyleneglycol-bis(2 aminoethylether) tetra acetic acid 3. EDDHA Ethylenediamine-di(ohydroxyphenyl) acetic acid 4. DTPA Diethylenetriaminepentaacetic acid 5. DHPTA 1, 3 diamino-2 -hydroxypropanetetra acetic acid 33

Carbon Source n n n n Most plant tissue cultures are not highly autotrophic due to limitation of CO 2. Therefore, sugar is added to the medium as an energy source. Sucrose is the most common sugar added, although glucose, fructose, manitol and sorbitol are also used in certain instances. The concentration of sugars in nutrient media generally ranges from 20 to 40 g/l. Sugars also contribute to the osmotic potential in the culture The presence of sucrose specifically inhibits chlorophyll formation and photosynthesis, making autotrophic growth less feasible Sucrose in the culture media is usually hydrolyzed totally or partially into the component monosaccharides glucose and fructose The general superiority of sucrose over glucose may be on account of the more effective translocation of sucrose to apical meristems

Organic supplement a) Vitamins: Only thiamine (vitamin B 1) is essential for most plant cultures, it is required for carbohydrate metabolism and the biosynthesis of some amino acids u Thiamine (vitamin B 1) Essential as a coenzyme in the citric acid cycle u Nicotinic acid (niacin) and pyridoxine (B 6)

Organic supplement b) Myo-inositol Although it is not essential for growth of many plant species, its effect on growth is significant. Part of the B complex, in phosphate form is part of cell membranes, organelles and is not essential to growth but beneficial c) Complex organics Such as coconut milk, coconut water, yeast extract, fruit juices and fruit pulps.

Physical support agents A. Gelling agents When semi-solid or solid culture media are required, gelling agents are used. An example: Agar, agarose, gelrite, phytagel B. Structural supports Filter paper bridges, liquid permeable membrane support systems

Agar n n n Agar is the most commonly used gelling agent It is a natural product extracted from species of red algae, especially Gelidium amansii It is synthetic polysaccharide gelling agents Agar consists of 2 components 1. 2. n Agarose is an alternating D-galactose and 3, 6 -anhydro-L-galactose with side chains of 6 -methyl-D-galactose residues (50 -90%). Agaropectin is like agarose but additionally contains sulfate ester side chains and D-glucuronic acid. Agar tertiary structure is a double helix the central cavity of which can accommodate water molecules

ü Advantages: ü ü ü Agar is an inert component, form a gel in water that melt at 100 ° C and solidify at nearly 45 ° C Concentrations commonly used in plant culture media range between 0. 5% and 1% If necessary, agar can be washed to remove inhibitory organic and inorganic impurities. Gels are not digested by plant enzymes Agar does not strongly react with media constituent Disadvantages: ü ü Agar does not gel well under acidic conditions (p. H <4. 5). The inclusion of activated charcoal in media may also inhibit gelling of agar.

Agarose It is extracted from agar leaving behind agaropectin and its sulfate groups. n It is used when the impurities of agar are a major disadvantage. n

Gelrite™ n n Gelrite consists of a polysaccharide produced by the bacterium Pseudomonas elodea. It gives clear-solidified medium that leads to detection of contamination at an early stage. Gelrite requires more stirring than agar. Concentration of divalent cations such as calcium and magnesium must be within the range of 4 -8 m. M/L or the medium will not solidify

Phytagel™ It is an agar substitute produced from a bacterial substrate composed of glucuronic acid, rhamnose and glucose. n It produces a clear, colorless, high-strength gel, which aids in detection of microbial contamination. n It is used at a concentration of 1. 5 -2. 5 g/L. n It should be prepared with rapid stirring to prevent clumping. n

Commercial Media Formulations • Murashige and Skoog (MS) • Linsmaier and Skoog (LS) • White Medium • Gamborg medium • Schenk and Hildebrandt medium • Nitsch and Nitsch Medium • Lloyd and Mc. Cown Woody plant medium • Knudson’s medium

Hormone (the Greek word hormaein, meaning "to excite"). Small organic molecule that elicits a physiological response at very low concentrations Chemical signals that coordinate different parts of the organism Internal and external signals that regulate growth are mediated, at least in part, by growth-regulating substances, or hormones

Plant Hormone ü A natural substance which produced by plant and acts to control plant activities. ü Chemical messengers influencing many patterns of plant development ü Naturally occurring or synthetic compounds that affect plant growth and development Plant hormones differ from animal hormones in that: v No evidence that the fundamental actions of plant and animal hormones are the same. v Unlike animal hormones, plant hormones are not made in tissues specialized for hormone production. (e. g. , sex hormones made in the gonads, human growth hormone - pituitary gland) v Unlike animal hormones, plant hormones do not have definite target areas (e. g. , auxins can stimulate adventitious root development in a cut shoot, or shoot elongation or apical dominance, or differentiation of vascular tissue).

Characteristics 1. 2. 3. 4. 5. Synthesized by plants. Show specific activity at very low concentrations Display multiple functions in plants. Play a role in regulating physiological phenomena in vivo in a dose-dependent manner They may interact, either synergistically or antagonistically, to produce a particular effect.

Synthetic plant hormone Plant growth regulators üGrowth-inhibiting chemicals ü Growth-promoting chemicals ü Root-promoting chemicals

Auxin Cytokinin Gibberelin Classification of PGRs Abscisic acid Ethylene Other Jasmonates Salicylic acid Brassinosteroids

Plant hormones as “Chemical Messengers” üAuxins üCytokinins üGibberellins üEthylene

Auxins

Auxin ü Arpad Paál (1919) - Asymmetrical placement of cut tips on coleoptiles resulted in a bending of the coleoptile away from the side onto which the tips were placed (response mimicked the response seen in phototropism). ü Frits Went (1926) determined auxin enhanced cell elongation.

Auxins ü ü ü ü ü Absolutely essential (no mutants known) One compound: Indole-3 -acetic acid. Many synthetic analogues: NAA, IBA, 2, 4 -D, 2, 4, 5 -T, Picloram Cheaper & more stable Generally growth stimulatory. Promote rooting Stimulate cell elongation Increase the rate of transcription Mediate the response of bending in response to gravity or light Produced in meristems, especially shoot meristem and transported through the plant in special cells in vascular bundles.

Cytokinins

Cytokinin

Cytokinins

Discovery of cytokinins ü Gottlieb Haberlandt in 1913 reported an unknown compound that stimulated cellular division. ü In the 1940 s, Johannes van Overbeek, noted that plant embryos grew faster when they were supplied with coconut milk (liquid endosperm), which is rich in nucleic acids. ü In the 1950 s, Folke Skoog and Carlos Miller studying the influence of auxin on the growth of tobacco in tissue culture. When auxin was added to artificial medium, the cells enlarged but did not divide. Miller took herring-sperm DNA. Miller knew of Overbeek's work, and decided to add this to the culture medium, the tobacco cells started dividing. He repeated this experiment with fresh herring-sperm DNA, but the results were not repeated. Only old DNA seemed to work. Miller later discovered that adding the purine base of DNA (adenine) would cause the cells to divide.

Discovery of cytokinins ü Adenine or adenine-like compounds induce cell division in plant tissue culture. Miller, Skoog and their coworkers isolated the growth facto responsible for cellular division from a DNA preparation calling it kinetin which belongs to a class of compounds called cytokinins. ü In 1964, the first naturally occurring cytokinin was isolated from corn called zeatin. Zeatin and zeatin riboside are found in coconut milk. All cytokinins (artificial or natural) are chemically similar to adenine. ü Cytokinins move nonpolarly in xylem, phloem, and parenchyma cells. ü Cytokinins are found in angiosperms, gymnosperms, mosses, and ferns. In angiosperms, cytokinins are produced in the roots, seeds, fruits, and young leaves

Cytokinins ü ü ü ü ü Absolutely essential (no mutants known) Natural compound: Zeatin, 2 -isopentyl adenine (2 i. P) Synthetic analogues: Benyzladenine (BA), Kinetin. Stimulate cell division (with auxins). Promotes formation of adventitious shoots Stimulate cell division Stimulate dark germination Stimulate leaf expansion Produced in the root meristem and transported throughout the plant as the Zeatin-riboside in the phloem.

Auxin and Cytokinin Ratio

Interaction of cytokinin and auxin in tobacco callus (undifferentiated plant cells) tissue üOrganogenesis: Cytokinins and auxin affect organogenesis üHigh cytokinin/auxin ratios favor the formation of shoots üLow cytokinin/auxin ratios favor the formation of roots.

Gibberellin(GA’s)

Discovered of Gibbereline n n n In 1930's, Ewiti Kurosawa and colleagues were studying plants suffering from bakanae, or "foolish seedling" disease in rice. Disease caused by fungus called, Gibberella fujikuroi, which was stimulating cell elongation and division. Compound secreted by fungus could cause bakanae disease in uninfected plants. Kurosawa named this compound gibberellin. u Gibberella fujikuroi also causes stalk rot in corn, sorghum and other plants. u Secondary metabolites produced by the fungus include mycotoxins, like fumonisin, which when ingested by horses can cause equine leukoencephalomalacia - necrotic brain or crazy horse or hole in the head disease. u Fumonisin is considered to be a carcinogen.

Gibberellins n A family of over 70 related compounds, all forms of Gibberellic acid and named as GA 1, GA 2. . GA 110. Commercially, GA 3 and GA 4+9 available. Stimulate etiolation of stems. Help break bud and seed dormancy. Stimulate stem elongation by stimulation cell division and elongation Stimulate germination of pollen n Produced in young leaves n n n

Abscisic acid (ABA)

Discovery of abscisic acid ü In 1940 s, scientists started searching for hormones that would inhibit growth and development, what Hemberg called dormins. ü In the early 1960 s, Philip Wareing confirmed that application of a dormin to a bud would induce dormancy. ü F. T. Addicott discovered that this substance stimulated abscission of cotton fruit. he named this substance abscisin. (Subsequent research showed that ethylene and not abscisin controls abscission). ü Abscisin is made from carotenoids and moves nonpolarly through plant tissue.

Abscisic Acid n n n n Only one natural compound. Promotes leaf abscission and seed dormancy. Plays a dominant role in closing stomata in response to water stress Involved in the abscission of buds, flower and fruits Inhibit cell division and elongation Has an important role in embryogenesis in preparing embryos for desiccation. Helps ensure ‘normal’ embryos.

Ethylene H H / C = C / H H

Discovery of ethylene ü In the 1800 s, it was recognized that street lights that burned gas, could cause neighboring plants to develop short, thick stems and cause the leaves to fall off. In 1901, Dimitry Neljubow identified that a byproduct of gas combustion was ethylene gas and that this gas could affect plant growth. ü In R. Gane showed that this same gas was naturally produced by plants and that it caused faster ripening of many fruits. ü Synthesis of ethylene is inhibited by carbon dioxide and requires oxygen.

Ethylene n n n Gas - diffuses through tissues Stimulates abscission and fruit ripening Used in commercial ripening for bananas & green picked fruit Involved in leaf abscission & flower senescence Primarily synthesized in response to stress Regulate cell death programming

Brassinosteroids Promote shoot elongating ü Inhibit root growth ü Promote ethylene biosynthesis ü Enhance resistance to chilling, disease and herbicides ü

Salicylic acid Promote flowering üStimulate plant pathogenesis protein production ü Jasmonate Play an important role in plant defence mechanisms

Jasmonate Play an important role in plant defence mechanisms