Plant Tissue Culture Medium Components PDF

Nutritional requirements of in vitro cultures Culture medium Basal media In vitro response of an explant largely depends on the composition of the culture medium. The Knop's (1865) mineral solution was the widely used medium by early investigators (Hydroponics). Currently there are several univ...

Nutritional requirements of in vitro cultures Culture medium Basal media In vitro response of an explant largely depends on the composition of the culture medium. The Knop's (1865) mineral solution was the widely used medium by early investigators (Hydroponics). Currently there are several universal basal media available. In general, culture medium consists of salts of major and minor nutrient elements, vitamins, amino acids, plant growth substances and a source of carbon. The following are the most commonly used basal media: Murashige and Skoog medium This is one of the most extensively used basal media in plant tissue culture. It was evolved by Murashige and Skoog (1962) primarily for the culture of tobacco callus. Murashige and Skoog medium is characterised by high concentration of mineral salts. It contains iron in the chelated form of ferrous EDTA (ethylene diamine tetra acetic acid), ensuring the availability of iron at pH values upto 9.0 and throughout the growth of the cultures. B5 medium This was originally developed by Gamborg et al. (1968) for the culture of soybean cell suspension. Schenk and Hildebrandt medium This medium developed by Schenk and Hildebrandt (1972) is being used for the tissue culture of a number of dicotyledonous plants. It is also regarded as a high salt medium. It contains iron in the chelated form of ferrous EDTA. N6 medium N6 medium, formulated by Zhu et al. (1975) is extensively used in anther culture of cereal crops. White’s medium This medium developed by White (1943) is low in inorganic salts. It has been used for root culture studies and for the in vitro rooting of microshoots. Woody plant medium  The Woody plant medium developed by Lloyd and McCown (1980) is commonly used for the in vitro propagation of woody perennials. It contains low ionic strength, ideal for woody plants. Knudson’s and Vacin and Went media The media developed by Knudson (1946) and Vacin and Went (1949) are extensively used for orchid tissue culture. Selection of basal medium The requirement of a basal medium may be influenced by the composition of the plant tissue. One milli litre of the medium has to provide approximately the same quantity of mineral nutrients present in 15 mg of dry tissue. Nutrient elements Major and minor nutrient elements are provided in the culture medium. They are essential for stress free response of explants. Macronutrients Macronutrients (nitrogen, phosphorus, potassium, calcium, sulphur and magnesium) are required in larger quantities as against micronutrients. Nitrogen is an integral part of nucleic acids, amino acids and plant growth substances. It is required by the cultures in the range of 25 to 60 mM. It is provided in the nitrate, ammoniacal and organic forms. The form as well as absolute quantity of nitrogen influence the in vitro morphogenesis of cultures. The effects of different forms vary. There is difference in the absolute requirements of the various forms. Nitrate is used in the range of 25 to 40 mM. ammonium nitrate and ammonium ions depends on the pH of the culture medium. An acid pH favours the uptake of nitrate ions. Removal of nitrate ions finally leads to an increase in the pH. Uptake of ammonium ions, on the other hand, is provided by a higher pH and will lead to a drop in the pH. Asssimilation of nitrate and ammonium ions is an energy requiring process. Nitrate and nitrite reductase enzymes are required for the assimilation of nitrate ions. Ammonium ion is generally toxic to plant cells. It cannot be accumulated in the cytoplasm. Ammonium ions can be assimilated directly into amino acids, depending on the availability of photosynthates and keto acids. Organic nitrogen is readily assimilated. It is not an energy requiring process. Organic nitrogen supports the cultures when there is delay in the assimilation of ammonium and nitrate ions. Amino acids and casein hydrolysate are the common sources of organic nitrogen in culture media. Potassium is required for the formation of high energy phosphate molecules. It is concerned with osmoregulation and enzyme activation. It is needed in the concentration of about 20 mM in the culture medium. The requirement of phophorous, calcium, sulphur and magnesium ranges between 1.0 and 3.0 mM. Phosphorus is essential for the storage and transfer of energy. High concentration of dissolved phosphate may reduce the uptake of micronutrients like zinc, iron and copper. Calcium is responsible for membrane permeability and transport of ions. It helps in maintaining cell integrity as well. It influences cell elongation and division. Magnesium is the primary constituent of chlorophyll. It is the structural component of ribosomes as well. Sulphur is essential for the synthesis of sulphur containing amino acids, coenzyme A, biotin and thiamin. Micronutrients Micronutrients (zinc, boron, copper, molybdenum and cobalt) are required in micro molar concentration. Aluminum, nickel and iodine are used in rare cases. Micronutrients are components of plant cell proteins. They are required for chlorophyll synthesis and chloroplast function. They are associated with plant growth substances also. Iron is involved in a number of redox processes. It is present in nitrate reductase, photosystem I, ferritin and cytochromes. It is usually supplied in the chelated form in order to prevent precipitation and reduced availability. This will ensure the availability of iron upto pH 9.0. Like iron, copper is also involved in many redox processes. Cupro-proteins like cytochrome oxidases are present in cells. Requirement for copper is more for juvenile explants. Zinc is an integral part of enzymes like RNA polymerase, aspartate transcarbamylase and dehydrogenases. It is essential for the synthesis of auxin. Manganese is important for photosynthetic reactions. It can substitute for magnesium in many enzyme reactions.  Cobalt is included in micromolar concentrations (0.1 M) in various culture media. It is a component of vitamin B12 analogues, which are concerned with nucleic acid synthesis. Molybdynum is required in concentrations of about 1.0 M. It is present in enzymes like nitrate reductase. Sodium and chlorine are indirectly being supplied to the medium along with the salts of the macronutrients. The cells can tolerate comparatively high concentrations of these elements. Chlorine in small quantities is essential for cell growth. It is involved in photosynthesis and stomatal closure mechanism. Sodium is beneficial in rare instances. It can sometimes substitute for potassium. Boron is involved in the control of auxin metabolism and functioning of cell membrane. Although iodine is not considered essential, it is used in many tissue culture media. Carbon source Cultures have an absolute requirement for carbohydrates. They function as sources of inorganic carbon and energy and as an osmotic regulant. The commonly used carbohydrates are sucrose, glucose, fructose, maltose, galactose, mannose and lactose. Sucrose is the most widely used one, followed by glucose. They are provided in the range of 20.0 to 60.0 g/l. The other types of carbohydrates are generally used as supplements to sucrose or glucose. Sometimes sugar alcohol like sorbitol and mannitol are used. Hexitols like inositol and sorbitol help in preventing precocious germination of somatic embryoids. Inositol is used at the concentration of 100 mg/l. Sucrose undergoes partial hydrolysis during the autoclaving of the culture medium. This results in the formation of glucose and fructose. Carbohydrates help provide and maintain the required osmolarity of the culture medium. Sometimes a combination of metabolically active (sucrose; glucose) and metabolically inactive (mannose; sorbitol; mannitol) carbohydrates is used for maintaining optimum osmotic potential throughout the culture period. Myo-inositol is also used as a carbon source and as a vitamin in culture media. Vitamins Isolated plant tissues in culture do not synthesise vitamins in required quantities. They are essential in performing certain catalytic functions. Hence vitamins are supplemented in the media. They are generally used in the range of 0.1 to 10.0 mg/l. Among them thiamine (B1) is essential. Nicotinic acid (niacin), pyridoxine (B6), biotin, pantothenic acid, riboflavin, folic acid, choline chloride, and para amino benzoic acid are found beneficial for the growth and development of the cultures. Amino acids Glycine, glutamine, asparagine, arginine and cysteine are the amino acids commonly supplemented in the culture medium. These form a specific source of organic nitrogen as well. They are used in the concentration range of 2 to 600 mg/l. Protein digests like casein hydrolysat are also used sometimes, as a non-specific source of amino acids. In certain instances, amino acids are found to be inhibitory for in vitro response of explant. Hence, care should be taken in the selection of amino acids suitable for specific plant species. Plant growth substances There are five major groups of plant growth substances, namely, auxins, cytokinins, gibberellins, ethylene and abscisins. Synergistic or antagonistic interactions are observed among the plant growth substance. Auxins and cytokinins are the most frequently used groups. Skoog and Miller (1957) showed that the ratio of auxins to cytokinins was critical in the regulation of in vitro morphogenesis of callus. Additional substances gaining recognition as hormones in plant tissue culture are: polyamines, jasmonates, salicylic acid and brassinosteroids. Auxins Auxins control cell elongation, apical dominance and adventitious root formation. They are also capable of inducing somatic embryogenesis. At higher concentrations they can inhibit embryo formation in cell suspension cultures and bring about mitotic irregularities. At optimum concentrations auxins are used to improve the growth of shoot cultures. The induction and initiation of adventitious roots are favoured by auxins. However, the continued presence of auxins in the root induction medium is often inhibitory for the elongation of the roots initiated. The important auxins used in tissue culture media are indole acetic acid (IAA), naphthalene acetic acid (NAA), indole butyric acid (IBA), 2,4 dichlorophenoxyacetic acid (2,4-D) and 2,4,5 trichlorophenoxyacetic acid (2,4,5-T). Indole acetic acid is the naturally occurring auxin. The others are synthetic. Indole acetic acid is rapidly metabolised in plants. It is inactivated by light and IAA oxidase activity. It is heat labile and hence should be filter sterilized while preparing culture media. Autoclaving is assumed to destroy approximately nine tenths of its activity. Synthetic auxins are stable. Auxins are being used in the concentration range of 0.05 to 100 mg/l. Synergistic effects of auxins are observed in the induction of callus and roots. 2,4-D is very often used for the induction of somatic embryoids. Cytokinins Cytokinins support cell division. They can retard the cell senescence process. They are used for counteracting the apical dominance caused by auxins. They are most often used in the concentration range of 0.05 to 10.0 mg/l. Along with auxins cytokinins are used for the initiation and maintenance of callus. Cytokinins favour the enhanced release of axillary buds. They are vital for the induction of multiple shoot formation in the clonal propagation of plant species. Adventitious bud can also be induced from various explants using appropriate cytokinins.Toxic hypersensitivity symptoms due to specific cytokinins are exhibited by some species and explant. Cytokinins can be inhibitory to the formation of adventitious roots. The residual effect of the cytokinins used for the enhanced release of axillary buds can be inhibitory for the rooting of the microshoots later. Physiological disorders like nitrification and fasciation of somatic embryoids and adventitious shoots due to cytokinins are sometimes observed. Synergistic effects of cytokinins are observed in the enhanced release of axillary buds. A lower concentration of cytokinin is required for supporting the further growth of the multiple shoots induced. The commonly used cytokinins in tissue culture media are 6 benzyl amino purine (BAP) or 6 benzyl adenine (BA), 6 furfuryl amino purine (kinetin) and 2 isopentenyl adenine (2iP). Zeatin, the naturally occurring cytokinin has been shown to be more effective, though expensive. Thidiazuron (TDZ) is a non-purine cytokinin. Chemicals like adenine and adenine sulphate, having cytokinin like activities are sometimes used as complementary to the cytokinins in culture medium. Gibberellins Gibberellins support cell division and elongation. They are capable of stimulating the synthesis of hydrolysing enzymes like  amylase, involved in the hydrolysis of reserved carbohydrates. They can counteract the effects of abscisic acid. Gibberellins are used for meristem culture, culture establishment of explants and germination of somatic embryos. GA3 is the most commonly used gibberellin. GA4 and GA7 are also being used. Gibberellic acid is generally used in the concentration range of 0.1 to 10.0 mg/l in culture media. It is heat labile and has to be filter sterilized while preparing media, for precise effects. Retention of approximately one tenth activity is expected after autoclaving. Abscisic acid Abscisic acid (ABA) is a naturally occurring growth inhibitor. It is useful for preventing the formation of aberrant and secondary somatic embryoids. Precocious germination of embryoids is also prevented. ABA increases the accumulation of storage lipids and proteins in embryoids. Generally, 0.05 to 10.0 mg/l ABA is used for somatic embryogenesis. It is heat labile and hence should be filter sterilized. Ethylene Ethylene is a gaseous hormone. It promotes cell senescence. It is produced in appreciable quantity in cell and tissue cultures, especially those from mature phase woody perennials. Depending on the type of cell, tissue or organ and the physiological state of the explants there can be marked difference in the rate of ethylene production in vitro. Cultures subjected to stress conditions will have increased production of ethylene. It may enter the culture vessels while flaming their rims. Concentration of ethylene gas that develop in culture flasks, especially when tightly closed, can be sufficient to modify plantlet development. Ethylene is most often inhibitory to the in vitro response of explants. Ethylene is known to interfere with in vitro shoot proliferation and rooting. Modulation of ethylene biosynthesis and action may be used to minimize the inhibitory action of ethylene. Silver nitrate and norbornadiene are inhibitors of ethylene action. Aminoethoxyvinyl glycine (AVG) and cobalt chloride inhibit ethylene action. These agents can be incorporated in the culture medium. Brassinosteroids It promotes shoot elongation at low concentrations and strongly inhibits root growth and development. It also promotes ethylene biosynthesis and epinasty. Jasmonates Jasmonates are represented by jasmonic acid and it is a methyl ester. Jasmonic acid is considered to be a new class of plant growth substance. It inhibits many processes such as embryogenesis, seed germination, pollen germination, flower bud formation, chlorophyll formation. It is involved in differentiation, adventitious root formation, breaking of seed dormancy and pollen germination. Polyamines There is some controversy as to whether these compounds should be classified with hormones. They appear to be essential in growth and cell division. Salicylic acid It is thought to be a new class of plant growth substances. It promotes flowering, inhibits ethylene biosynthesis and reverses the effects of ABA. Natural complexes Natural complexes like coconut water, tomato juice, orange juice, yeast extract and malt extract are sometimes used in culture media. Coconut water (also denoted by some authors as coconut milk) is the most commonly used natural complex. It contains sugars, amino acids, vitamins, plant growth substances and other beneficial growth factors. It is used in the concentration range of 100 to 200 ml in the medium. It can be autoclaved without losing much of its active principles. Potato extract, maize kernel extract, banana extract, tomato juice, barley endosperm and watermelon juice are also used in culture media. The use of natural complexes are generally discouraged. The composition of natural complexes is highly variable and not always well defined. Their use in culture medium can interfere with the reproducibility of results. Substitution of natural complexes, in beneficial instances, with plant growth factors is recommended. pH of the culture medium pH of the culture medium is important in ensuring the availability of the components to the cultures. The pH is corrected before autoclaving using dilute alkali (KOH or NaOH) or acid (HCl). Autoclaving is found to lower the pH value. The optimum pH values have been specified for different basal media. Low pH values can cause difficulty in the gelling of agar, due to acid hydrolysis. Solidifying agents Agar is the most commonly used solidifying agent in culture media and is obtained form red-purple sea weeds of Rhodophyceae. It is a mixture of polysaccharides. Agar is insoluble in cold water; but soluble in hot water. Due to its hydrophylic nature, as the concentration of agar increases, more and more water molecules become in bound form. This results in reduced water availability to the explants. Agar is used in the concentration range of 0.45 to 1.50 per cent. Poor aeration of the culture medium is a disadvantage while using agar. Slow rate of diffusion of toxic metabolites from the explants may also result. Prolonged heating or autoclaving can break down the agar and affect the gelling process. At low pH values also the gelling is affected. Gelrite is sometimes used as a substitute for agar. It is a self gelling hydrocolloid. It forms rigid and transparent gels in the presence of soluble salts. Bacterial contamination can easily be observed. Polyacrylamides, silica gel, glass wool and glass beads are also used as substitutes for agar. Activated charcoal Activated charcoal can adsorb inhibitors that affect the growth of cultures. However, it adsorbs growth promoters like plant growth substances as well. It is a common antioxidant used for overcoming the problem of polyphenol interference in cultures. It is found to promote somatic embryogenesis in several instances. It is usually used in the concentration range of 0.05 to 2.00 per cent. Sterilization techniques Sterilization refers to the complete destruction of all microorganisms including spores. Different methods of sterilization are followed in a tissue culture laboratory.  Dry heat sterilization Red heat flaming destroys all vegetative microorganism. This is suitable for glassware, forceps, scalpel which is not affected by very high temperature. A hot air oven is also used for dry heat sterilisation. Commonly used time-temperature relations for sterilization hold time are 20 min at 180◦ C, 40 min at 170◦ C and 60 min at 160◦ C. Moist heat sterilization The plant tissue culture media and other liquids can be sterilized by steam under pressure (autoclaving). Dry saturated steam penetrates deep and kills microorganisms be denaturing their protein. Autoclave is operated at a pressure of 1.05 kg/cm2 and temperature of 121◦C. Filtration This is used to sterilize thermo labile constituents of culture medium. Filters function by entrapping microorganisms within the porous structure of the filter matrix. Membrane filters are made from polymeric materials such as cellulose diacetate, polycarbonate and polyesters. These filters are available in 0.2 µm pore size to 10 µm sizes. Air filters are commonly used in inoculation rooms for sterilizing large volumes of air to create a sterile work area in Laminar Air flow chamber, The air is passed through HEPA filters (High Efficiency Particle Air Filters) and air free of pathogens are blowed in the work area for inoculation of explants. Radiation Ultraviolet radiation in the wavelength of 250-260nm are bactericidal and to a lesser extent sporicidal. Irradiation of laminar air flow chambers for 15-20 min with UV light is sufficient enough to get a sterile work area. The skin and eyes should be protected from UV radiation.