MS mediumMurashige and Skoog medium or (MSO or MS0 (MS-zero)) MSO was invented by plant scientists Toshio Murashige and

Folke K. Skoog in 1962. MEDIA COMPONENTS 1.Inorganic Nutrients: Macronutrients Source Name Nitrogen

Form

Amount

Use

Potassium

Ammonium(NH4+)and nitrate Nitrates are usually (NO3-) ions(Ammonium supplied in the nitrate, Potassium nitrate). range of 25-20 mM; typical ammonium concentrations range between 2 and 20 mM. However, ammonium concentrations in excess of 8 mM may be deleterious to cell growth of certain species. Potassium nitrate, (KNO3) 1,900 mg/l Potassiumphosphate (KH2PO4) 170 mg/l

Important points responsible for Nitrate is superior healthy green leaf growth. to ammonium as the sole N source. Nitrogen deficiency will the ammonium show symptoms like ions will be stunted growth and pale utilized more green and yellow leaves rapidly and before the nitrate Necessary for formation of ions. amino acids, the building blocks of protein NH4+ checks the Essential for plant cell increase of pH division, vital for plant towards growth alkalinity responsible for chlorophyll formation which plays an important part in the strength of cells and encourages flower and fruit formation. Carbohydrate metabolism and the break down and translocation of starches. Potassium regulates the opening and closing of the stomata by a potassium ion pump. Since stomata are important in water regulation, potassium reduces water loss from the leaves and increasesdrought tolerance. responsible for cell development and the promotion of good root growth, As a component of ATP. playing a major role in biological excess of Potassium may tend to delay maturity

PHOSPHORUS Potassiumphosphate (KH2PO4)

170 mg/l

Involved in photosynthesis, respiration, energy storage and transfer, cell division, and

Calcium

Calcium chloride(CaCl2 2H2O)

440 mg/l

Magnesium

Magnesium sulfate (MgSO4 7H2O)

370 mg/l

Sulphur

Magnesium sulfate (MgSO4 7H2O)

370 mg/l

molecules such as DNA and RNA , Living cells also use phosphate to transport cellular energy in the form of adenosine triphosphate (ATP). Nearly every cellular process that uses energy obtains it in the form of ATP Calcium, an essential part of plant cell wall structure,Calcium regulates transport of other nutrients into the plant and is also involved in the activation of certain plant enzymes. Magnesium is an important part of chlorophyll, a critical plant pigment important in photosynthesis. It is important in the production of ATP through its role as an enzyme cofactor. Sulphur is a structural component of some amino acids and vitamins, and is essential in the manufacturing of chloroplasts. Sulphur is also found in the Iron Sulphur complexes of the electron transport chains in photosynthesis

enlargement

Calcium deficiency results in stunting.

It is immobile and deficiency therefore affects younger tissues first. Symptoms of deficiency include yellowing of leaves and stunted growth.

Micronutrients Source Name Iron Form Ferrous sulfate (FeSO4 7H2O) Manganese sulfate (MnSO4 4H2O) Amount 27.8 mg/l Use Iron is necessary for photosynthesis and is present as an enzyme cofactor in plants. Functions with enzyme systems involved in breakdown of carbohydrates, and nitrogen metabolism Important points Iron deficiency can result in interveinal chlorosis and necr osis. Manganese deficiency may result in coloration abnormalities, such as discolored spots on the foliage. A typical symptom of zinc deficiency- "little leaf"

Manganese

22.3 mg/l

Zinc

Zinc sulfate (ZnSO47H2 O) Boric acid (H3BO3)

8.6 mg/l

Boron

6.2 mg/l

Zinc is required in a large number of enzymes and plays an essential role in DNA transcription. Regulates consumption of sugars. Helps in the use of nutrients and regulates

other nutrients. Aids production of sugar and carbohydrates. Essential for seed and fruit development. Boron is important for binding of pectins in the RGII region of the primary cell wall, secondary roles may be in sugar transport, cell division, and synthesizing certain enzymes.

Copper

Cupric sulfate (CuSO4 5H2O)

0.025 mg /l

Copper is important for photosynthesis. Important for reproductive growth.Aids in root metabolism and helps in the utilization of proteins. Involved in the manufacture of lignin (cell walls)

Molybdenu m Sodium

Sodium molybdate (Na2Mo O4 2H2O) Sodium molybdate (Na2Mo O4 2H2O)

0.25 mg/l

Molybdenum is a cofactor to enzymes important in building amino acids. Sodium is involved in the regeneration of phosphoenolpyruvate in CAM and C4 plants

0.25 mg/l

EDTA(Ethylenediaminetetraacetic acid)-chelating a-gent- conjugate base mg or metal ions Sucrose-Carbon and Energy Source-20-60g/l.While autoclaving the medium, sucrose is hydrolysed to glucose and fructose which are then used up for growth. Fructose, if autoclaved is toxic. Other carbohydrates that have been tested include lactose, galactose, rafinose, maltose, and starch. Vitamins Normal plants synthesize the vitamins required for their growth and development. Vitamins are required by plants as catalysts in various metabolic processes. When plant cells and tissues are grown in vitro, some vitamins may become limiting factors for cell growth. Thiamine (B1), nicotinic acid (B3), pyridoxine(B6), pantothenic acid(B5) are commonly used vitamins of which thiamine (0.1 to 5mg/l) is essentially added to medium as it is involved in carbohydrate metabolism. Myo-inositol is a carbohydrate not a vitamin, it has been shown to stimulate growth in certain cell cultures. Its presence in the culture medium is not essential, but in small quantities myo-inositol stimulates cell growth in most species. Amino acids Amino acids provide plant cells with an immediately available source of nitrogen, which generally can be taken up by the cells more rapidly than inorganic nitrogen. The most common sources of organic nitrogen used in culture media are amino acid mixtures (e.g., casein hydrolysate- A preparation made from the milk protein casein, which is hydrolyzed to break it down into its constituent amino acids. Amino acids are the building blocks of proteins.), L-glutamine, L-asparagine, and adenine.

Undefined Organic Supplements Addition of a wide variety of organic extracts to culture media often results in favorable tissue responses. Supplements that have been tested include protein hydrolysates, coconut milk, yeast extracts, malt extracts, ground banana, orange juice, and tomato juice.

Activated charcoal The addition of activated charcoal (AC) to culture media may have a beneficial effect. The effect of AC is generally attributed to one of three factors: absorption of inhibitory compounds, absorption of growth regulators from the culture medium, or darkening of the medium. The inhibition of growth in the presence of AC is generally attributed to the absorption of phytohormones to AC. 1-Napthaleneacetic acid (NAA), kinetin, 6-benzylaminopurine (BA), indole-3-acetic acid (IAA), and 6---dimethylallylaminopurine (2iP) all bind to AC, with the latter two growth regulators binding quite rapidly. The stimulation of cell growth by AC is generally attributed to its ability to bind to toxic phenolic compounds produced during culture. Activated charcoal is generally acid-washed prior to addition to the culture medium at a concentration of 0.5-3.0 percent. It absorbs brown-black pigments and oxidized phenolics produced during culture and thus reduce toxicity. It also absorbs other organic compounds like PGRs, vitamins etc which may cause the inhibition of growth. Another feature of activated charcoal is that it causes darkening of medium and so helps root formation and growth. PGRs Auxins: Indole Acetic Acid (IAA)- 0.5 N NaOH Indole Butyric Acid (IBA)- 0.5 N NaOH Napthateneacetic Acid (NAA)- 0.5 N NaOH 2,4 Dichlorophenoxyacetic acid (2,4D)- 95% ethanol Cytokinins: kinetin (K)- 0.5 N HCl zeatin (Z)- hot, not boiling water (Do not autoclave, filter sterilize) benzylaminopurine (BA or BAP)- 0.5 N HCl 2-isopentyladenine (2iP)- 0.5 N HCl Others: Abscisic Acid (ABA)- 0.5 N NaOH Gibberelic Acid (GA) - hot, not boiling water (Do not autoclave, filter sterilize) Plant Growth Regulator Effects Auxins cell elongation cell wall relaxation increased RNA and protein synthesis direction of translocation prevents abscission enhanced ethylene production organ formation (with cytokinins) Cytokinins cell division cell enlargement organ formation (with auxins) overcomes dormancy lateral bud development

retards senescence mobilization of nutrients 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. Gibberellins cell elongation at apex Cell division enzyme induction flowering (long day) overcomes dormancy (anatagonist to ABA) inhibits organ formation Abscisic Acid dormancy gibberellin anatagonist flowering (short day) stomate closure during water stress Ethylene enhanced production with high auxin concentrations epinasty geotrophism fruit ripening flowering of mangos and bromeliads senescence abscission CytokininAdenine derivatives (amino purines) Synthesis in root,Transport via xylem Auxin The four naturally occurring (endogenous) auxins are IAA, 4-chloroindole-3-acetic acid, phenylacetic acid and indole-3butyric acid; Synthetic auxin analogs include 1-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), Hormone-natural or chemical messenger transport to target cells

Certain items, e.g., IAA, must be prepared and stored in amber bottles to prevent photodecomposition.
Any photochemical reaction that results in the decomposition of a substance

Phenolic acids are plant metabolites widely spread throughout the plant kingdom. Recent interest in phenolic acids stems from their potential protective role, through ingestion of fruits and vegetables, against oxidative damage diseases (coronary heart disease, stroke, and cancers). Phenolic compounds are essential for the growth and reproduction of plants, and are produced as a response for defending injured plants against pathogens. The importance of antioxidant activities of phenolic compounds and their possible usage in processed foods as a natural antioxidant have reached a new high in recent years. Phenolics in Plants Phenolic acid compounds seem to be universally distributed in plants. They have been the subject of a great number of chemical, biological, agricultural, and medical studies. They form a diverse group that includes the widely distributed hydroxybenzoic and hydroxycinnamic acids. Hydroxycinnamic acid compounds occur most frequently as simple esters with hydroxy carboxylic acids or glucose Hydroxybenzoic acid compounds are present mainly in the form of glucosides. Secondary metabolites are chemicals produced by plants for which no role has yet been found in growth, photosynthesis, reproduction, or other "primary" functions. These chemicals are extremely diverse; many thousands have been identified in several major classes. Each plant family, genus, and species produces a characteristic mix of these chemicals, and they can sometimes be used as taxonomic characters in classifying plants. Humans use some of these compounds as medicines, flavorings, or recreational drugs. Secondary metabolites are organic compounds that are not directly involved in the normal growth, development, or reproduction of an organism.[1] Unlike primary metabolites, absence of secondary metabolities does not result in immediate death, but rather in long-term impairment of the organism's survivability, fecundity, or aesthetics, or perhaps in no

significant change at all. Secondary metabolites are often restricted to a narrow set of species within a phylogenetic group.[2] Secondary metabolites often play an important role in plant defense against herbivory[3] and other interspecies defenses. Humans use secondary metabolites as medicines, flavorings, and recreational drugs. An antioxidant is a molecule capable of inhibiting the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons or hydrogen from a substance to an oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions. When the chain reaction occurs in a cell, it can cause damage or death to the cell. The phenolic compounds in pulses are generally polyphenols and include tannins, phenolic acids, and flavonoids (46). Antioxidant activity is related to total phenolic content (48). Their potential role in weight management is unclear, although studies indicate that certain phenolics interfere with enterocyte glucose absorption through interference with the glucose transporters (49,50). Class Example Compounds NITROGEN-CONTAINING Alkaloids nicotine cocaine theobromine NITROGEN-AND SULFUR-CONTAINING Glucosinolates TERPENOIDS Monoterpenes menthol linalool mint and relatives, many plants Sesquiterpenes parthenolid Parthenium and relatives ( Asteraceae ) Diterpenes Triterpenes, cardiac glycosides Tetraterpenoids carotene many plants antioxidant; orange coloring gossypol digitogenin cotton Digitalis (foxglove) block phosphorylation; toxic stimulate heart muscle, alter ion transport interfere with neurotransmission, block ion transport, anesthetic contact dermatitis sinigrin cabbage, relatives tobacco coca plant chocolate (cocao) interfere with neurotransmission, block enzyme action Example Sources Some Effects and Uses

Class

Example Compounds

Example Sources

Some Effects and Uses

Terpene polymers

rubber

Hevea (rubber) trees, dandelion

gum up insects; airplane tires

Sterols PHENOLICS Phenolic acids

spinasterol

spinach

interfere with animal hormone action

caffeic, chlorogenic all plants

cause oxidative damage, browning in fruits and wine

Coumarins Lignans Flavonoids

umbelliferone

carrots, parsnip

cross-link DNA, block cell division cathartic, vomiting, allergic dermatitis flower, leaf color; inhibit enzymes, antiand pro-oxidants, estrogenic

podophyllin urushiol mayapple poison ivy anthocyanin, catechin almost all plants

Tannins

gallotannin, condensed tannin

oak, hemlock trees, birdsfoot trefoil, legumes all land plants

bind to proteins, enzymes, block digestion, antioxidants structure, toughness, fiber

Lignin

lignin