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 1
• References
2
Plant tissue culture Plant tissue culture broadly refers to the in vitro cultivation of plants, seeds and various plant parts of the plants plants viz. viz. organs, organs, embryos, embryos, tissues, tissues, single single cells, cells, protoplasts protoplasts.. Plant cell cell have Ability 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 pollen sacs or pollen grains grains or embryo or or embryoids”. embryoids”.
Historical Resume Year
Authors
Results
Species
1892
Klercker
First attempts to isolate protoplasts
-
mechanically. 1902
Haberlandt
First cultivation experiments with
Tradescantia
isolated plant cells; cell growth, but no cell division obtained. 1904
Hanning
Establishment of embryo culture for the
Cochleria raphanus
first time 1909
Kuster
First observation of fusing cells
-
1922
Kotte, Ro Robins
In vitro vitro cultivation of root tips, no
Zea, Pisum
permanent permanent cultures cultures obtained obtained 1924
Dieterich
Embryo rescue- "artificial premature
Linum
3
1925
Laibach
birth"
1934 Gautheret Nobecourt First permanent callus culture using Bvitamins and auxins 1942
Gautheret
Daucus, Nicotiana glauca x N. longsdorffii
Observation of secondary metabolites in
-
plant callus culture 1946
Ball
1952
Morel et al.
Micropropagation: first development of stem tips and sub adjacent regions:
Lupinus
1934 Gautheret Nobecourt First permanent callus culture using Bvitamins and auxins 1942
Gautheret
Tropaeolum
Daucus, Nicotiana glauca x N. longsdorffii
Observation of secondary metabolites in
-
plant callus culture 1946
Ball
1952
Morel and Martin
Micropropagation: first development of Tropaeolum , Lupinus Stem tips and sub adjacent regions:
Dahlia
plants free of viruses. 1954
Morel et al.
First suspension cultures of single cells
Tagetes, Nicotiana,
or cell aggregates. Nurse culture
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
Phaseolus
production of substances from plant tissue culture. 1958
Wickson and
Establishment of axillary’s branching
Thimann Reinert,
Somatic embryogenesis in tissue
Daucus
4
Steward et al.
cultures
1959 Tukecke and Nickell First report of large-scale (1341) culture
1960
Bergmann
Ginkgo,Lolium,
of plant cells: carboy system
Rosa,llex
Cell clones obtained from single
Nicotiana, Phaseolus
cultured cells plated in an agar medium 1960
Jones et al.
Hanging drop culture in conditioned
Nicotiana
medium 1960
Cocking
Method for obtaining large number of
Lycopersicon
protoplasts from plant tissue 1965
Morel
Clonal multiplication of horticultural
Cymbidium
plant (orchid) through tissue droplet 1965
1966
Vasil and
Regeneration of a plant from one single
Hildebrandt
cell cultivated in a hanging droplet
Kohlenbach
First cell division and culture of
Nicotiana
Macleaya
differentiated mesophyll cells. 1967
Kaul and stabe
Reports of the yields of certain of
Ammi
differentiated mesophyll cells. 1967 Bourgin and Nisoh
1970
In vitro production of haploid plant
Guha and
from immature pollen with cultured
Maheshwari
anthers.
Carlson
Isolation of auxotrophic mutants from
Nicotiana
Datura
Nicotiana
cultured cells
1977 Noguchi
et al.
Cultivation of tobacco cells in
Nicotiana
200001reactors
5
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
Lithospermum
plant products by suspension cultures
1983
Barton Brill
Insertion of foreign genes attached to a
-
plasmid 1983
Chilton
Production of transformed tobacco
Tobacco
plants following single cell transformation
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.
6
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
7
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 Gautheret’s 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
8
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.
•
1µM - 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 can’t be done when the concentration are in mass values.
9
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
NO3-
from NH4 NO3 and KNO3
SO4-
from MNSO4.4H2O andFESO4.7H2O
K +
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 culturesin vitro.
Element
Function
Nitrogen
Component of proteins, nucleic acids, and some coenzymes; Element equired in the greatest amounts Regulates osmotic potential; principal
Potassium
inorganic cation Manganese
Enzyme cofactor
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Magnesium
Enzyme cofactor, component of chlorophyll
Phosphorus
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
Cell-wall synthesis, membrane function, cell signalling Component of some vitamins
Copper
Enzyme cofactor; electron-transfer reactions
Zinc
Enzyme cofactor; chlorophyll biosynthesis
Molybdenum
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 KNO 3) and ammonium ions (from NH4 NO3). 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.
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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
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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 can’t 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
-
Sucrose
Monocotyledons Roots
-
Dextrose (glucose)
Malus Pumila
-
Few plants
-
Maltose, Galactose, Mannose, Lactose
Sequioa and
-
Starch
Sorbitol/Sucrose/Glucose
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.
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 µ 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;
major
(2) cytokinins; (3) gibberellins; (4) abscisic acid;
minor
(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
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2,4-D 2,4,5-T Dicamba IAA IBA MCPA NAA NOA Picloram
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
Chemical structure of auxins
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)
NOA (2- napthoxy acetic acid)
p-CPA(parachlorophenoxyacetic 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 N 6-(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
Chemical name
BAP 2iP (or IPA) Kinetin Thidiazuron
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-2 butenylaminopurine (natural)
Zeatin
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
23
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 algae-
Gelidium 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.
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(ii)
Agarose-
Agarose
consist
of β-D(1,3)galactopyranose
and
3,6anhydro-α-
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 agent Alginate 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.
26
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. β-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
27
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,
Tobramycin.
(b) Inhibitors of Nucleic Acid Metabolism: e.g. Rifampicin, Mitomycin C and Nalidixic acid. (c) Antimetabolites: Antibiotics, which block specific metabolic steps that are essential to
microorganisms e.g. Metronidazole, Miconazole, Nitrofurantoin,
Trimethoprim and
Sulphomethoxazole.
(d) Nucleic Acid Analogs, which inhibit enzymes essential for DNA synthesis. e.g. 5-Fluorouracil,
Mercaptopurine
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 µ 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,
(3).Cytokinin and (4). Organic nutrients
(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.
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.
29
Nutritional component of some plant tissue culture media Table 6 - Media for Plant Tissue and Cell Cultures (mg/L) Components
(NH4)2SO4
Murashige- White Skoog (1963 (1962)
Gamborg Nitsch Heller (1968) (1951) (1953)
Schenk Hildebrandt (1972)
Nitsch Nitsch (1967)
Kohlenbach Knop (1865) Schmidt (1975)
-
-
134
-
-
-
-
-
-
370
720
500
250
250
400
125
185
250
Na2SO4
-
200
-
-
-
-
-
-
-
KC1
-
65
-
1,500
750
-
-
-
-
440
-
150
25
75
200
-
166
-
-
-
-
-
600
-
-
-
-
1,900
80
3,000
2,000
-
2,500
125
950
250
-
300
-
-
-
-
500
-
1,000
1,650
-
-
-
-
-
-
720
-
NaH2PO4× H2O
-
16.5
150
250
125
-
-
-
-
NH4H2PO4
-
-
-
-
-
300
-
-
-
170
-
-
-
_
-
125
68
250
FeSO4× 7H2)
27.8
-
27.8
-
-
15
27.85
27.85
-
Na2EDTA
37.3
-
37.3
-
-
20
37.25
37.25
-
MnSO4× 4H2O
22.3
7
10 (1 H2O)
3
0.1
10
25
25
-
ZnSO4× 7H2O
8.6
3
2
0.5
1
0.1
10
10
-
CuSO4× 5H2O
0.025
-
0.025
0.025
0.03
0.2
0.025
0.025
-
H2SO4
-
-
-
0.5
-
-
-
-
-
Fe2(SO4)3
-
2.5
-
-
-
-
-
-
-
NiC12× 6H2O
-
-
-
-
0.03
-
-
-
-
CoC12× 6H2O
0.025
-
0.025
-
-
0.1
0.025
-
-
A1C13
-
-
-
-
0.03
-
-
-
-
FeC13× 6H2O
-
-
-
-
1
-
-
-
-
FeC6O5H7× 5H2O
-
-
-
10
-
-
-
-
-
0.83
0.75
0.75
0.5
0.01
1.0
-
-
-
6.2
1.5
3
0.5
1
5
10
10
-
0.25
-
0.25
0.25
-
0.1
0.25
0.25
-
30,000
20,000
20,000
30,000
20,000~30,000
10,000
-
MgSO4× 7H2O
CaC12× 2H2O NaNO3 KNO3 Ca(NO3)2× 4H2O NH NO 4 3
KH2PO4
K1 H3BO3 Na M 2 0O4× 2H2O Sucrose
50,000 20,000
30
Glucose
-
-
-
or 36,000
-
-
-
-
-
Myo-Inositol
100
-
100
-
-
1,000
100
100
-
Nicotinic Acid
0.5
0.5
1.0
-
-
0.5
5
5
-
Pyridoxine HC1
0.5
0.1
1.0
-
-
0.5
0.5
0.5
-
Thiamine HC1
0.1-1
0.1
10
1
1
5
0.5
0.5
-
CaPantothenate
-
1
-
-
-
-
-
-
-
Biotin
-
-
-
-
-
-
0.05
0.05
-
Glycine
2
3
-
-
-
-
2
2
-
Cysteine HC1
-
1
-
10
-
-
-
-
-
Folic Acid
-
-
-
-
-
-
0.5
0.5
-
Glutamine
-
-
-
-
-
-
-
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 plant’s 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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