Anshika Pandey (M.Sc.

) Pandit Ravishankar Shukla University

Plant Tissue Culture Media Constituents And Their Role Synopsis Introduction to plant tissue culture Historical Background of plant tissue culture Media Units used in tissue culture literature Media constituents 1. Inorganic Nutrients 2. Organic Nutrients 3. Growth regulator 4. Gelling Agent 5. Antibiotics Media selection Media Preparation Composition of different plant tissue culture media Companies manufacturing plant tissue culture media Conclusion
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 References

Plant tissue culture

Plant tissue culture broadly refers to the in vitro cultivation of plants, seeds and various plant parts of the plants viz. organs, embryos, tissues, single cells, protoplasts. Plant cell have Ability to regenerate complete plant known as totiopotency. Plant cell and tissue culture is the cultivation of plant cells, tissue, and organs under aseptic condition in controlled environment. Tissue culture is an experimental process through which a mass of cells (callus) is produced from an explant tissue and the callus produced in this way can be used directly either to regenerate plantlets or to extract to some primary and secondary metabolites. In other words, tissue culture is a collection of that experimental process through which plants are developed in controlled and aseptic condition from isolated cells or tissues or organs (like pollen sacs or pollen grains or embryo or embryoids.

Historical Resume
Year 1892 Authors Klercker Results First attempts to isolate protoplasts mechanically. 1902 Haberlandt First cultivation experiments with isolated plant cells; cell growth, but no cell division obtained. 1904 Hanning Establishment of embryo culture for the first time 1909 1922 Kuster Kotte, Robins First observation of fusing cells In vitro cultivation of root tips, no permanent cultures obtained 1924 Dieterich Embryo rescue- "artificial premature Linum Zea, Pisum Cochleria raphanus Tradescantia Species -

1925

Laibach

birth" Daucus, Nicotiana glauca x N. longsdorffii -

1934 Gautheret Nobecourt First permanent callus culture using Bvitamins and auxins 1942 Gautheret Observation of secondary metabolites in plant callus culture 1946 1952 Ball Morel et al. Micropropagation: first development of stem tips and sub adjacent regions:

Tropaeolum Lupinus

1934 Gautheret Nobecourt First permanent callus culture using Bvitamins and auxins 1942 Gautheret Observation of secondary metabolites in plant callus culture 1946 Ball

Daucus, Nicotiana glauca x N. longsdorffii -

Micropropagation: first development of Tropaeolum , Lupinus Stem tips and sub adjacent regions: plants free of viruses. Dahlia

1952 Morel and Martin

1954

Morel et al.

First suspension cultures of single cells or cell aggregates. Nurse culture

Tagetes, Nicotiana, Daucus, Picea, Phaseolus

1955

Mothes and Kala

First reports of secondary metabolite production in liquid media

1956 Routien and Nickell

US patent No.2747334 for the production of substances from plant tissue culture.

Phaseolus

1958

Wickson and Thimann Reinert,

Establishment of axillarys branching Somatic embryogenesis in tissue

Daucus

Steward et al.

cultures Ginkgo,Lolium, Rosa,llex Nicotiana, Phaseolus

1959 Tukecke and Nickell First report of large-scale (1341) culture of plant cells: carboy system 1960 Bergmann Cell clones obtained from single cultured cells plated in an agar medium 1960 Jones et al. Hanging drop culture in conditioned medium 1960 Cocking Method for obtaining large number of protoplasts from plant tissue 1965 Morel Clonal multiplication of horticultural plant (orchid) through tissue droplet 1965 Vasil and Hildebrandt 1966 Kohlenbach Regeneration of a plant from one single cell cultivated in a hanging droplet First cell division and culture of differentiated mesophyll cells. 1967 Kaul and stabe Reports of the yields of certain of differentiated mesophyll cells. 1967 Bourgin and Nisoh Guha and Maheshwari 1970 Carlson In vitro production of haploid plant from immature pollen with cultured anthers. Isolation of auxotrophic mutants from cultured cells 1977 Noguchi et al. Cultivation of tobacco cells in 200001reactors

Nicotiana

Lycopersicon

Cymbidium

Nicotiana

Macleaya

Ammi

Nicotiana

Datura

Nicotiana

Nicotiana

1978

Zenk

Manifold increase in product yields by selection over parent plant documented for a variety of plant metabolites

1979

Brodelius et al. Alginate beads used to immobilize plant cells for biotransformation and secondary metabolite production

1981

Shuler

Use of hollow reactor for secondary metabolite production

1983 Mitsui petrochemi. First industrial production of secondary plant products by suspension cultures

Lithospermum

1983

Barton Brill

Insertion of foreign genes attached to a plasmid

1983

Chilton

Production of transformed tobacco plants following single cell transformation

Tobacco

Contribution of Indian scientist in plant tissue cultureP. Maheshwari was the first to work on plant tissue culture in India. He started his work in Delhi University. In 1966, Guha and Maheshwari demonstrated the possibility of raising large numbers of androgenic haploids plantlets from the pollen grains of Datura innoxia by culturing immature anthers Indra K Vasil in 1965 collabration with Hildebrandt raised whole plant starting from single cells of tobacco. In 1972 , Saunders and Bingham reported that different cultivars of alfalfa varied considerably in their regenaeration potential under a culture regime. Through more detailed study they conclude that regeneration in tissue culture is a genetically controlled phenomenon.

Regeneration of plants from carrot cells frozen at the temperature of liquid nitrogen was reported by Nag and Street in 1973. Bhozwani and Razdan discovered the property of isolated protoplast to take up the organelles and macromolecules

Media
The nutrient preparation on or in which a culture (i.e. population of cells) is grown in the laboratory is called a culture medium. Chemicals composition that provide nutrition for culturing plant cell in vitro. It consist all the nutrient requirement viz.inorganic nutrients ,organic nutrienrs , gelling agent , PGRetc. Tissue from different parts of a plant may have different requirements for satisfactory growth - (Muashige and Skoog 1962.) Needs to culture diverse tissues and organs has led to the development of several resipices

Roots culturemedium of White (1943) , Callus culture medium Gavtheret(1939) were developed from nutrient solution previously used for culturing whole plant. White media from Uspenski and uspenskiash medium (1925) for algae and Gautherets medium is based on knops (1965) salt solution

All subsequent formation are based on White and Gautheret media.

Some calli (carrot tissue , blackberry tissue , most tumour tissue ) - requiqe only inorganic salts and utilize sugar while others require vitamins, amino acid, growth substance in different qualitative and quantitative combinations.

Types of media

Synthetic media :- Medium containing only chemically defined compounds is known as synthetic media. Natural media:-medium which consist natural products (coconut milk etc) for culturing whose constituents are not chemically defined are known as synthetic defined Media

When cultured in vitro, all the needs of the plant cells, both chemical and physical, have to met by the culture vessel, the growth medium, and the external environment (light, temperature, etc.). The growth medium has to supply all the essential mineral ions required for growth and development. In many cases (as the biosynthetic capability of cells cultured in vitro may not replicate that of the parent plant), it must also supply additional organic supplements such as amino acids and vitamins. Many plant cell cultures, as they are not photosynthetic, also require the addition of a fixed carbon source in the form of a sugar (most often sucrose). One other vital component that must also be supplied is water, the principal biological solvent. Physical factors, such as temperature, pH, the gaseous environment, light (quality and

duration), and osmotic pressure, also have to be maintained within acceptable limits.

UNITS USED IN TISSUE CULTURE LITERATURE

Inorganic and organic constituents of the medium are generally expressed in mass

values mg / liter or ppm (parts per million)

Nowadays mg/liter is acceptable.

mg = 10-6 = 1mg/liter or 1ppm 10-7 = 0.1 mg/liter 10-9= 0.001 mg/liter Internationally association for plant physiology has recommended the use of moles.

Mole: - Mole is an abbreviation for gram molecular weight which is the formula weight of a substance in grams. Formula weight: - Sum of the weights of the atoms in the formula of a substance.

1M - 1 mole of substance in 1 liter of solution. 1mM molecular weight in mg/liter or 10-3 M. 1M - molecular weight in mg/liter or 10-6 M.

According to international association of plant physiology

mMol/liter is used for expressing the concentration of macronutrient and organic nutrient. Mol/liter is used for expressing the concentration of micronutrient, hormones, vitamins and other organic constituents in the plant tissue culture.

Reason for using mole values is that the number of molecules per mole is constant for

all compounds. Mole values can be used irrespective of number of water molecules in the sample of salts. This cant be done when the concentration are in mass values.

Media Constituents MEDIA CONSTITUENTS

INORGANIC NUTRIENTS Macro nutrient Micro nutrient ORGANIC NUTRIENTS Vitamins Amino Acids Carbon source Other nutrient complex GROWTH HARMONES Auxin Cytokynin Gebberellis Ethylene Other ABA GELLING AGENT Agar Aagaioze crelerite

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1. Inorganic Nutrients
Mineral elements are a very important in life of a plant For example, magnesium is a part of chlorophyll molecule, and Calcium is a constituent of the cell wall, nitrogen is an important source of amino acid, proteins, and nucleic acids. Similarly, Iron, zinc, molybdenum are parts of certain enzymes. Essential elements besides C,H,O there are other twelve elements required for plant growth they are nitrogen (N), Phosphorus(P), potassium(K), sulfur(S), Calcium(Ca), Magnesium(Mg), iron(Fe), molybdenum(mo), copper(Cu), Zinc(Zn), manganese(Mn), Boron(B). The 15 element found important for whole plant growth have also proved necessary for tissue cultures. Qualitatively the inorganic nutrients required for various plant tissue appears to be fairly constant. When mineral salts are dissolved in water they undergo dissociation and ionization .The active factor in the medium is the ion of different types rather than the compounds. One type of ion may be contributed by more than one salt. For e.g NO3SO4K+ from NH4NO3 and KNO3 from MNSO4.4H2O andFESO4.7H2O from KNO3 and KH2PO4.

Therefore a useful comparison between the two media can be made by looking into total concentration of different types of ions in them. Table2- Some elements important plant nutrition and their physiological function, supplied by the culture medium to support the growth of healthy cultures in vitro.

Element
Nitrogen Potassium

Function
Component of proteins, nucleic acids, and some coenzymes; Element equired in the greatest amounts Regulates osmotic potential; principal inorganic cation

Manganese

Enzyme cofactor

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Magnesium Phosphorus

Enzyme cofactor, component of chlorophyll Component of nucleic acids; energy transfer; component

Sulphur

Component of some amino acids (methionine, cysteine) and some cofactor

Chlorine Iron

Required for photosynthesis Electron transfer as a component of cytochromes

Calcium Cobalt Copper Zinc Molybdenum

Cell-wall synthesis, membrane function, cell signalling Component of some vitamins Enzyme cofactor; electron-transfer reactions Enzyme cofactor; chlorophyll biosynthesis Enzyme cofactor; component of nitrate reductase

The essential elements are further divided into the following categories: A. macroelements (or macronutrients); B. microelements (or micronutrients);

A. Macronutrients
According to the recommendation of then international association for plant physiology the elements required by plants in concentration greater than 0.5 mMol/liter are referred to as 12

macronutrients. Nitrogen, phosphorus, potassium, magnesium, calcium, and sulphur (and carbon, which is added separately) are usually regarded as macroelements. These elements usually comprise at least 0.1% of the dry weight of plants. (i) Nitrogen - main element contributing to the growth in plant in vitro and in vivo. Nitrogen is the main constituents of amino acid, proteins certain hormones and chlorophyll. The source of nitrogen in vitro could be organic or inorganic. Nitrogen is most commonly supplied as a mixture of nitrate ions (from KNO3) and ammonium ions (from NH4NO3). Theoretically, there is an advantage in supplying nitrogen in the form of ammonium ions, as nitrogen must be in the reduced form to be incorporated into macromolecules. Nitrate ions therefore need to be reduced before incorporation. However, at high concentrations, ammonium ions can be toxic to plant cell cultures and uptake of ammonium ions from the medium causes acidification of the medium. For ammonium ions to be used as the sole nitrogen source, the medium needs to be buffered. High concentrations of ammonium ions can also cause culture problems by increasing the frequency of vitrification (the culture appears pale and glassy and is usually unsuitable for further culture). Using a mixture of nitrate and ammonium ions has the advantage of weakly buffering the medium as the uptake of nitrate ions causes OH ions to be excreted. If there is deficiency of nitrate in media it leads to development of anthrocyanin pigment. Role Constituents of various biological macromolecules Essential for protein synthesis Essential for DNA, RNA synthesis Porphyrins are the important constituents of chlorophyll and cytochrome enzyme contain nitrogen. (ii) Phosphorus - phosphorus is vital for cell division as well as in storage and transfer of energy in plants. Usually supplied in form of phosphate ion from salts such as ammonium, sodium, potassium dihydrogen orthophosphate.

13

The concentration of phosphates in plant tissue culture media ranges from 1 to 3 mM. Its higher concentration may inhibit the growth, presumably by causing the precipitation of other elements, or by the formation of insoluble calcium phosphates. Role Important constituents of phospholipids, sugar phosphates, nucleic acid or nucleotide Co enzymes such as NAD, NADP. Regulates the activity of various enzymes Play a key role in the transfer of energy

(iii)Potassium- Potassium is supplied as the chloride, nitrate or orthophosphate salt in the plant tissue culture media. Their concentration ranges from 2 to 25 mM.high level may cause harmful effect on some plants viz. Rhododendron species. Role It is the most important cellular cation and has major roles in cellular homeostatsis, such as pH regulation and osmotic regulation. Also regulate the activity of various enzymes. Pholem transport and changes in stomatal guard cell turgor also rely K and its ability to be transported across plant cell membrane. Essential for respiration.

(iv)Magnesium Magnesium is usually supplied as magnesium sulfate. Its concentration level ranges from 1 to 3 mM. Role Component of chlorophyll It is important biochemically as an enzyme cofactor Structural component of ribosome. Activate the enzyme of carbohydrate metabolism and nucleic acid. It can also fulfill some of the pH and osmotic regulatory function potassium.

(v) Calcium-

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Calcium is usually provided as calcium chloride although calcium nitrate is also used in some media formulations. It is generally used in the range of 1 to 3 mM. The concentration of calcium in fresh medium is often several times greater than that found with in cell protoplast due to active removal of calcium from calls against a concentration gradient. Removal of calcium from the protoplast is important if precipitation of other elements and the formation of calcium phosphate are to be avoided. In high concentration of calcium in culture medium, promote callose deposition as a result inhibits cell extension. The plants grown in high calcium presence have open stomata. Role As calcium pectate is an integral part of the walls of plant cells and helps in maintaining the integrity of membranes. Calcium could be having a pre-emptive role in morphogenesis. Also function as second messenger. Important enzyme co-factor and enzyme regulator.

(vi) Sulphur- Sulphur is usually supplied as sulphate. It is used in range from 1 to 3 mM. Role Component of vitamin and various coenzymes Important part of amino acid and also involved in determining protein structure by the formation of disulfide bridge.

B. Micronutrients
Elements which are required in concentration less than 0.5 mMol/liter are referred as micronutrients or microelements. Micronutrients are essential as catalyst for many biochemical reactions. Manganese, iodine, copper, cobalt, boron, molybdenum, iron, and zinc usually comprise the microelements, although other elements such as nickel and aluminum are found frequently in some formulations. Role Iron is the micronutrient present in the largest quantities and is considered the most important micronutrient. It is required for the formation of several chlorophyll precursors

15

and is a component of ferrdoxins which are important oxidation: reduction reagents. Iron is also important components of proteins which carry out oxidation and reduction reaction. Manganese is required to maintain chloroplast ultra structure and for photosynthesis. It is required in less amount then iron as most of the culture in vitro are not autotrophic. Copper and Zinc are the important components of some type of enzyme, such as oxidases and superoxide dismutase which help to prevent damage to tissue due to superoxide radicals. Molybdenum and iron important in nitrogen metabolism as they are part of the nitrate reductase and nitrogenase enzymes. Cobalt is a component of vitamin B12. Boron is required for continued cell division and is used in the formation of DNA and RNA bases. Iodine is not thought to be essential for the growth of plant cell cultures, but is usually included in micronutrients formulations.

2. Organic Nutrients
Normal plant synthesizes the vitamin required for the growth and development but plant cell in culture for their better growth require a high amount of organic nutrients which they cant synthesizes through themselves hence requires extra supplements in media. Organic nutrients are supplied in various forma some of them are: (i) Vitamins- Plant cell culture needs to be supplemented with certain vitamins. Most widely used vitamins are Thiamine (vitamin B1), Niacin (vitamin B3), pyridoxine (vitaminB6), myo-inositol(a member of vitamin B complex). Other vitamins having specific uses are pantothenic acid, vitamin C, vitamin D and vitamin E. In MS media four vitamin myo-inositol, thiamine, pyridoxine, nicotinic acid are used. There are no firm rules as to what vitamins are essential for plant tissue and cell cultures. Many vitamins are added to plant cell culture media formulations as they were used previously. The only two vitamins that are considered to be essential are myo-inositol and thiamine. Myo-inositol has many and diverse role in cellular metabolism and physiology. Inositol forms a part of the phosphoinositoids and phosphotidylinositol which are important factors

16

in processes such as cell division and act as intra cellular messengers and enzyme activators. Inositol is also involved in the biosynthesis of vitamin C. Myo-inisitol probably has a role as a carrier and in storage of IAA as IAAmyoinositolester. Myoinositol in culture media could be the precursor in the biosynthetic pathways leading to the formation of pectin and hemicellulose needed in the cell wall synthesis and may have role in the uptake and utilization of ions. (Wood and Braun (1962) and Verma and Dougall (1978)) Thiamine it is involved in the direct biosynthesis of certain amino acid and is an essential cofactor in carbohydrate metabolism. Thiamine could be having a synergistic interaction with cytokinins (reported by Digby and Skoog 1966). Vitamin E is used as an antioxidant. Vitamin C (ascorbic acid) also used as an antioxidant and prevent blackening during explant isolation. Vitamin D (calciferol) has growth regulatory effects. Riboflavin inhibits the callus formation and improves growth and quality of shoots (reported by Drew and Smith in 1986). (ii) Amino acids- There are less substantive evidence for the role and necessity of amino acid in plant tissue culture media. Skoog and Linsmaier in 1965 demonstrated the glycine use for the sustained growth of tobacco callus but it may have inhibitory effect at high concentration. Amino acid can be used directly by plant cell or as a source of nitrogen. Commonly used amino acids are arginine, glycine, and cysteine although asparagines, aspartic acid, alanine, praline and glutamic acid are also used. Cysteine used in media as an antioxidant to control the oxidation of phenolics and prevent blackening or browning of tissues. Arginine used in the media for apple. In vitro produced shoots of dwarf apple rootstocks formed more roots in presence of arginine.

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(iii) Carbon source- In tissue culture the tissues which are initially green gradually lose their green pigment in culture and depend on external source of carbon. Even those tissues which acquire pigments through sudden changes or under special conditions during culture e period are not autotrophs for carbon. Fully organized green shoots in cultures also show better growth and proliferation with the addition of a suitable carbon source in the medium. Thus it is essential to add a utilizable source of carbon to the culture medium. Concentration of carbon source is 2 to 5 % of media. Glucose and fructose are suitable for supporting growth but sucrose used mostly. Ball (1953, 1955) observed that autoclaved sugar was better than filter sterilized sucrose fir the growth of Sequiopa callus. Autoclaving seems to bring about hydrolysis of sucrose into more efficiently utilizable sugar such as glucose and fructose. Sucrose is necessary for various metabolic activities. It is required for differentiation of xylem and phloem in cultured cells (Aloni 1980). Sugar also represent major component of medium. Requirement of carbon source differ according the plant needDicotyledons roots Monocotyledons Roots Malus Pumila Few plants Sequioa and maize endosperm (v) Unidentified supplements- Casein hydrolysates(CH), Coconut milk (CM), Corn milk, Malt extract(ME), Tomato juice(TJ), and Yeast extract (YE) are complex nutritive mixture of undefined composition which is used to promote the growth of certain calli and organs. Before using these substance a preliminary test in range of 0.1 to 1.0 gm/liter to asses its effect on growth. These undefined supplements were avoided because The quality and quantity of growth promoting constituents in these extracts often vary with the age of the tissue and the variety of donor organisms. If used at higher concentration, the complex substance may adversely affect the cell growth. Sucrose Dextrose (glucose) Sorbitol/Sucrose/Glucose Maltose, Galactose, Mannose, Lactose Starch

3. Growth regulators -

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Hormones are organic compounds naturally synthesized in higher plants, which influences the growth and development. They are active at a site different from they are produced and are only present and active in very small quantities. Apart from natural compounds, synthetic compounds have been developed which correspond to the natural ones, these are called growth regulator. They are usually used in mol/liter concentration. Classes of plant growth regulator There are five main classes of plant growth regulator used in plant cell culture, namely: (1) auxins; (2) cytokinins; (3) gibberellins; (4) abscisic acid; (5) ethylene. (i) Auxin Weakly saturated growth regulator having an unsaturated ring structure and capable of promoting of cell elongation of stem and internodes, tropism, apical dominance, abscission, root differentiation in nature. In plant tissue culture auxin induces cell division, formation of callus and inhibits adventitious or axillary shoot formation. In media low concentration of auxin leads to the formation adventitious roots while in high concentration, callus formation result and root formation fails to occur Auxins promote both cell division and cell growth. The most important naturally occurring auxin is indole-3-acetic acid (IAA), but its use in plant cell culture media is limited because it is unstable to both heat and light. Occasionally, amino acid conjugates of IAA (such as indole-acetyl-l-alanine and indole-acetyl-l-glycine), which are more stable, are used to partially alleviate the problems associated with the use of IAA. It is more common, though, to use stable chemical analogues of IAA as a source of auxin in plant cell culture media. 2,4-Dichlorophenoxyacetic acid (2,4-D) is the most commonly used auxin and is extremely effective in most circumstances. Other auxins are available, and some may be more effective or potent than 2,4-D in some instances. Auxins commonly used in plant tissue culture3- indolebutyreic acid and IAA used for rooting and in interaction with a cytokinin for shoot proliferation. 2,4-D and 2,4-T used for induction and growth of callus. Table3 Commonly used auxins, their abbreviations and chemical names Abbreviation/name Chemical name 19 minor major

2,4-D 2,4,5-T Dicamba IAA IBA MCPA NAA NOA Picloram Chemical structure of auxins

2,4-Dichlorophenoxyacetic acid 2,4,5-Trichlorophenoxyacetic acid 2-Methoxy-3,6-dichlorobenzoic acid Indole-3-acetic acid Indole-3-butyric acid 2-Methyl-4-chlorophenoxyacetic acid 1-Naphthylacetic acid 2-Naphthyloxyacetic acid 4-Amino-2,5,6-trichloropicolinic acid

2,4-D(2,4-dichlorophenoxy acetic acid)

IBA(3-indolebutyric acid)

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2,4,5-T (2,4,5-Trichlorophenoxy acetic acid)

p-CPA(parachlorophenoxyacetic acid)

NOA (2- napthoxy acetic acid)

PICLORAM (4-amino-2, 5,6trichloropicolinic acid)

(ii) Cytokinins- Cytokinins are also considered to be promotes of growth. It is a growth regulating substances responsible for cell division alone or in conjunction with auxins. Basic in nature either in amino purine or phenyl urea derivative. At least 25 structurally related compounds are important naturally. Of the naturally occurring cytokinins, two have some use in plant tissue culture media zeatin and N6-(2-isopentyl)adenine (2iP). Their use is not widespread as they are expensive(particularly zeatin) and relatively unstable. The synthetic analogues kinetin and 6-benzylaminopurine (BAP) are therefore used more frequently. Non-purine-basedchemicals, such as substituted phenylureas, are also used as cytokinins in plant cell culture media. These substituted phenylureas can also substitute for auxin in some culture systems.

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Table4 Commonly used cytokinins, their abbreviations and chemical names Abbreviation/name BAP 2iP (or IPA) Kinetin Thidiazuron Zeatin Chemical name 6-Benzylaminopurine (synthetic) N6-(2-Isopentyl)adenine (natural) 6-Furfurylaminopurine(synthetic) 1-Phenyl-3-(1,2,3-thiadiazol-5-yl)urea (phenylurea type cytokinin) 4-Hydroxy-3-methyl-trans-2butenylaminopurine (natural)

Chemical structure of cytokinin-

(BAP) 6-Benzylaminopurine

Thidiazuron

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2iP (or IPA) N6-(2-Isopentyl)adenine (iii) Gibberellins- Gibberellins are weakly acidic growth hormones having gibbane ring structure which cause cell elongation of intact plants. There are about of 100s of gibberellins are known among them 20 are considered important. Gibberellin A3 GA3 being the most widely used. Role Responsible for normal development of plantlets from in vitro formed adventive embryos. Gibberellins induce elongation of internodes and the growth of meristem or buds in vitro. Inhibits adventitious root and shoot formation. Exception in Arabidopsis and Chrysanthemum GA3 induces shoot regeneration. Chemical structure of gibberellins

(iv) Ethylene- Ethylene is a gaseous, naturally occurring, plant growth regulator most commonly associated with controlling fruit ripening in climacteric fruits, and its use in plant tissue culture is not widespread. It does, though, present a particular problem for plant

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tissue culture. Some plant cell cultures produce ethylene, which, if it builds up sufficiently, can inhibit the growth and development of the culture. The type of culture vessel used and its means of closure affect the gaseous exchange between the culture vessel and the outside atmosphere and thus the levels of ethylene present in the culture.

Ethylene

Ethephone

(v) Abscisic acid - Abscisic acid (ABA) also called stress hormone because naturally produce in drought, water lodging and other adverse environmental condition. Mildly acidic growth hormone which function as general growth inhibitor by counteraction other hormones (auxin, cytokinin, gibberellins) or reaction mediated by them. ABA moat often required for normal growth and development of somatic embryos. In presence of ABA somatic embryos resembles to zygotic embryos. It also induces morphogenesis in Begonia cultures. Chemical structure of abscisic acid

Abscisic acid (ABA) Growth retardant (i) Paclobutarol- During acclimatization stage of micropropagation to reduce hyperhydracity and regulate leaf growth and function in relation to control water stress (reported by Smith and Krikorin 1990) (ii) Ancymidol- It is used to inhibit leaf formation and promote shoot formation in Gladiolus (by Ziv 1989).

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Paclobutrol Fig : chemical structure of growth retardant

4.Gelling agentMedia for plant cell culture in vitro can be used in either liquid or solid forms, depending on the type of culture being grown. For any culture types that require the plant cells or tissues to be grown on the surface of the medium, it must be solidified (more correctly termed gelled). Properties of gelling agent1. It should withstand sterilization by autoclaving. 2. Medium should be liquid when hot and form a semisolid gel when cool. 3. It must adequate amount of water to the cells. Most of the gelling agents are natural product such as agarose, agar, gelerite etc. (i)Agar- Agar is the most widely used substance for gelling. Agar obtained from red algaeGelidium amansii. Agar is a complex of related polysaccharides built up from the sugar galactose. It includes the neutral polymer fraction agarose, which give strength to the gel and the highly charged anionic polysaccharides agropectins which give agar its viscosity. Its quality and purity vary from batch to batch as it depends a lot on the culture condition of the algae and the varying degrees of processing and purification. The hardness of the medium produced depends on the concentration of agar used and the pH of the medium. Low pH values inhibit the gelling of the agar. Concentrations of 0.8% to 1% are usually used. High concentration of agar has adverse effect on in vitro growth as the medium become hard and does not allow diffusion of nutrients into the tissues.

25

(ii)

Agarose-

Agarose

consist

of

-D(1,3)galactopyranose

and

3,6anhydro--

L(1,4)galactopyranose linked into a polymer chain of 20 60 monosaccharide units. It is obtained by purifying agar to remove agropectins with its sulphide side groups. This purification process is tedious; this is why agarose is much costlier than agar. Agarose is used where high gels strength is required viz. single cell culture and protoplast culture. Concentration of 0.4% is usually used. (iii) Gelerite- Gelerite or phytagel, a gellan gum is a linear polysaccharide produced by the bacterium Pseudomonas elodea. It comprises of linked K-glucuronate, rhamnose and cellobiose molecules. Commercial product consist significant quantities of K, Ca, Na, Mg but free from organic impurities found in agar. Gelerite require less number or minimum level of cations in the solution for gelling. It can be prepared in cold solution and very less concentration is required 0.1% to 0.2% is sufficient.on solidification it sets as a clear gel and assists easy observation of cultures and their possible contamination. It also remains unaffected over a wide range of pH. Because of above positive points it is considered as a most appropriate gelling agent. Adverse effect of geleriteIt causes hyperhydracity in some plants. This problem can be rectified by mixing small quantities of agar in gelerite. According to Kyte when gelerite and agar is used in 3:1 ratio leads better result. (iv) Other gelling agentAlginate gels- alginate solution have the property of setting quickly when mixed with a solution containing many divalent cations (such as calcium) and of returning to a fluid form when these cations are removed. These cations can be removed by the use of chelating agents such as EDTA. The most common use of alginate has thus been in the encapsulation of cells to produce secondary metabolites, or somatic embryos to produce synthetic (somatic) seeds. Polyacrylamide gels (produced at room temperature by a catalyst) have also been used for such purposes, but suffer from the disadvantage that acrylamide monomer from which gel is formed is highly toxic to plant cell cultures.

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5. pHThe pH of the medium is usually adjusted between 5.0 and 6.0 before sterilization. In general, a pH higher than 6.0 gives fairly hard medium and a pH below 5.0 does not allow satisfactory gelling of the agar. The pH of the medium changes at various stages of preparation and culture. pH of the medium set after the addition of the gelling agent shows a remarkable drop on autoclaving. The pH of the medium further changes once plant tissue placed on it. The plant tissue and medium interact to adjust the pH to an equilibrium irrespective of the initial pH adjusted. The ratio of NH4+ and NO3- ions in the medium also influences the pH. When NH4+ is predominantly taken up the medium gets acidified due to liberation of H+ ions, while uptake of NO3-ions increases pH due to liberation of OH- ions (reported by- Dougall 1980, Congrad et al 1986). Such pH changes then influence the availability of various mineral ions in the medium and their uptake by the plant tissue. Other additional substances in media Antibiotics Antibiotics are substances produced by certain microorganisms that suppress the growth of other microorganisms and eventually destroy them. Their applications include: a. Suppresses bacterial infections in plant cell and tissue culture. b. Suppresses mould and yeast infections in cell cultures. c. Eliminates Agrobacterium species after the transformation of plant tissue. These antibiotics can be divided into different classes on the basis of chemical structure and their mechanism of action: 1. Inhibitors of Bacterial Cell Wall Synthesis. e.g. -lactam antibiotics, Penicillins and Cephalosporins. 2. Antibiotics that affect Cell Membrane permeability. (a) Antibacterial e.g. Colistin Sulphate, Polymixin B Sulphate, Gramicidin (b) Antifungal e.g. Amphotericin B, Nystatin, Pimaricin 3. Bacteriostatic Inhibitors of Protein Antibiotics that affect the function of 30 S or 50 S ribosomal subunits to cause a

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reversible inhibition of protein synthesis. e.g. Chloramphenicol, Chlortetracycline HCl, Clindamycin HCl, Doxycycline HCl, Erythromycin, Lincomycin HCl, Oxytetracycline HCl, Spectinomycin sulphate, Tetracycline HCl, Tylosin tartrate, Lincomycin HCl 4. Bactericide Inhibitors of Protein Synthesis Antibiotics that bind to the 30 S ribosomal subunit and alter protein synthesis which eventually leads to cell death. This group includes: (a) Aminoglycosides: Apramycin, Butirosine, Gentamicin, Kanamycin, Neomycin, Streptomycin, Nalidixic acid. (c) Antimetabolites: Antibiotics, which block specific metabolic steps that are essential to microorganisms e.g. Metronidazole, Miconazole, Nitrofurantoin, Sulphomethoxazole. Mercaptopurine Trimethoprim and 5-Fluorouracil, Tobramycin. (b) Inhibitors of Nucleic Acid Metabolism: e.g. Rifampicin, Mitomycin C and

(d) Nucleic Acid Analogs, which inhibit enzymes essential for DNA synthesis. e.g.

Media selection
There is no ideal approach to formulate a suitable medium for a new system. A convenient approach could be to select three media from the available recepies that represent high, medium and low salt media and combine them factorially with different levels of plant growth regulators suitable for the desired response. Example- (1). For shoot proliferation or adventitious shoot bud differentiation a commonly used auxin (NAA) and cytokinin(BAP) may be used each at five concentration (0, 0.5, 2.5, 5.0,10 mol/lit). (2). All possible combination of the five concentration of the two substances would lead to an experiment with 25 treatments with each basal medium (3). Select the best of the 75 treatment and test some of the available auxins and cytokinin at that concentration. While varying the cytokinins , keep the auxin concentration constant and vice versa.

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(4). Test a range of sucrose concentration 2 to 6% to decide its optimal level. However there are limitless opportunities to further improve the selected medium by manipulating its nutrient salts and plant growth regulators. Broad spectrum experiment Proposed by De Fossard et al in 1974 for selecting a suitable medium for an untested system. (1). In this approach all the components of the medium are divided into four broad categories- (1.). Minerals, (2).Auxins, (sucrose, amino acid etc) (2). For each group substances three concentration are choosen: Low (L), Medium (M) and High (H). (3). Trying various combination of the four substances at three different concentration leads to an experiment with 81 treatments. (4). The best of 81 treatments is denoted by a four letter code. For example, treatment with medium salts, low auxin, medium cytokinin and high organic nutrients would be represented as MLMH. (5).On reaching this stage it would be desirable to test different auxins and cytokinin to find best types. (3).Cytokinin and (4). Organic nutrients

Media preparation
The main components can be prepared as stock solution, by dissolving the required amounts of each salt or other compound in water. These stock solution can be stored at 4o Celsius with the exception of vitamin stock solution which is stored at -20o Celsius. To produce the medium at the final desired concentration for plant cell or tissue growth, these stocks solution must be mixed in the correct proportion with water. Sucrose is then added and dissolved, and water added to 900 ml. The medium adjusted to correct pH 5.7 and volume were making up to 1000ml. Then autoclaved the media at 15 psi pressure for 15 minutes.

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Nutritional component of some plant tissue culture media

Table 6 - Media for Plant Tissue and Cell Cultures (mg/L)
Components MurashigeSkoog (1962) 370 440 1,900 1,650 170 27.8 37.3 22.3 8.6 0.025 0.025 0.83 6.2 0.25 30,000 White (1963 Gamborg Nitsch Heller Schenk (1968) (1951) (1953) Hildebrandt (1972) 134 500 150 3,000 150 27.8 37.3 10 (1 H2O) 2 0.025 0.025 0.75 3 0.25 20,000 250 1,500 25 2,000 250 3 0.5 0.025 0.5 10 0.5 0.5 0.25 250 750 75 600 125 _ 0.1 1 0.03 0.03 0.03 1 0.01 1 400 200 2,500 300 15 20 10 0.1 0.2 0.1 1.0 5 0.1 30,000 Nitsch Nitsch (1967) 125 125 500 125 27.85 37.25 25 10 0.025 0.025 10 0.25 20,000~30,000 Kohlenbach Knop (1865) Schmidt (1975) 185 166 950 720 68 27.85 37.25 25 10 0.025 10 0.25 10,000 250 250 1,000 250 -

(NH4)2SO4 MgSO4 7H2O Na2SO4 KC1 CaC12 2H2O NaNO3 KNO3 Ca(NO3)2 4H2O NH4NO3 NaH2PO4 H2O NH4H2PO4 KH2PO4 FeSO4 7H2) Na2EDTA MnSO4 4H2O ZnSO4 7H2O CuSO4 5H2O H2SO4 Fe2(SO4)3 NiC12 6H2O CoC12 6H2O A1C13 FeC13 6H2O FeC6O5H7 5H2O K1 H3BO3 Na2M0O4 2H2O Sucrose

720 200 65 80 300 16.5 7 3 2.5 0.75 1.5 20,000

50,000 20,000

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Glucose Myo-Inositol Nicotinic Acid Pyridoxine HC1 Thiamine HC1 CaPantothenate Biotin Glycine Cysteine HC1 Folic Acid Glutamine

100 0.5 0.5 0.1-1 2 -

0.5 0.1 0.1 1 3 1 -

100 1.0 1.0 10 -

or 36,000 1 10 -

1 -

1,000 0.5 0.5 5 -

100 5 0.5 0.5 0.05 2 0.5 -

100 5 0.5 0.5 0.05 2 0.5 14.7

Conclusion
Plant cell culture media are complex mixture of inorganic and organic compounds designed to provide all the essential elements required for the growth and development of cultures in vitro. There is no universal media for all plants cell and tissue cultures, different plants have different requirement. By varying the concentration of salts, plant growth regulators and carbon source a new formulation can be designed. So it provide a lot of opportunities to designed new formulation by varying there concentration.

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References
Plant tissue culture theory and practice- Bhojwani,S.S. and RazdaN,M.K. Introduction to plant biotechnology- Chawla,H.S. In vitro cultivation of plant cells- Butterworth and Heinmann Plant cell culture- Evans, Coleman and Kearns Dodds, J. H., and L. W. Roberts. 1985. Experiments in plant tissue culture. Second edition.Cambridge University Press, New York, 232 pages. Street, H. E. 1973. Plant tissue and cell culture. Blackwell Scientific Publications, Oxford, 320pages. Plant Tissue Culture Techniques- Lorraine Mineo, Department of Biology,Lafayette College- Easton, Pennsylvania 18042 www.springerlink.com www.phytotechlab.com www.fao.org www.sigmaaldrich.com www.molecular-plant-biotechnology-info/planttissueculture/ www.plant-genetics.com www.biotechnology4u.com www.scribd.com www.planttissueculturemedia.com www.oxfordtextbooks.co.uk/orc/slaterplants2e/

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