Plant Tissue Culture owes its origin to the ideas of the German Scientist, Haberlandt, in the beginning of the 20th century. This was just the beginning of the tissue culture; thereafter in 70’s began the commercialization of the technology. Currently the world is revolving around it due to predicated future grain shortage; green house effect and total environmental disbalance.
Keeping all these factors in mind and to support mankind Titan Media had taken steps and introduced an extensive range of Ready to use PTC Medium; PTC Medium Ingredients like Plant Growth Regulators or Phytoharmones (Auxins; Cytokinins; Gibberellins; Abscisic and & Others), Macro & Micro Nutrients (Nitrogen; Potasssium; Phosphorus; Sulphur; Magnesium; Calcium; Boron; Zinc; Iron; Iodine; Vitamins; Amino acids; Carbohydrates & Others), Gelling Agents (Gelrite; Agar Agar & Others); Adsorbing Agents; Buffering Agents; Pure Antibiotics and range of products used in the process.
Plant Tissue Culture Media Preparation is based on the unique property of the cell-totipotency. The cell-totipotency is the ability of the plant cell to regenerate into whole plant. In this process the excised bud is transferred into a tube containing a sterile nutrient medium. The success of tissue culture depends very much on the stage of explant selected, the sterilization period and the type of culture media used; different types of plants require different sets of culture media. Plant tissues are grown in vitro on artificial media, which supply the nutrients necessary for growth. The success of plant culture as a means of plant propagation is greatly influenced by the nature of the culture medium used. The rich tissue culture media provides a good nutrient source for bacteria and fungi, therefore precautions against microbial contamination must be taken in all in vitro procedures.Tissue culture media used for in vitro cultivation of plant cells are composed of following basic components:
Complex mixture of salts is Inorganic nutrients (both micro- and macro-elements : C, H, O, N, P, S, Ca, K, Mg, Fe, Mn, Cu, Zn, B, Mb). For healthy and vigorous growth, intact plants need to take up from soil: relatively large amounts of some inorganic elements (the major plant nutrients): ions of Nitrogen (N), Potassium (K), Calcium (Ca), Phosphorous (P), Magnesium (Mg) and Sulphur (S). And some other elements in small quantities (minor plant nutrients or trace elements): Iron (Fe), Nickel (Ni), Chlorine (Cl), Manganese (Mn), Zinc (Zn), Boron (B), Copper (Cu), and Molybdenum (Mo). Among the micronutrients, iron requirement is very critical. Chelated form of Iron and Copper are commonly used in culture media.
Organic supplement: Vitamins and/or Amino acids (e.g. Nicotinic acid, Thiamine, Pyridoxine and myo-Inositol (Viamin B), Amino acids (e.g. Arginine) are essential for the culture of plant cells in vitro. The plant cells in culture are able to synthesize vitamins just like natural plants, but in suboptimal quantities which does not support proper growth of cells in culture. Therefore the medium is supplemented with vitamins to achieve good growth of cells. Similarly amino acids are added to the cell cultures to stimulate the cell growth and estabilish the cell lines. The most commonly used amino acid is glycine. However, Asparagine, Aspartic acid, Alanine, Glutamic acid, Glutamine and Proline are also used. Amino acids provide a source of reduced nitrogen and, like ammonium ions, uptake causes acidification of the medium. Organic acids especially the intermediates of krebs cycle e.g. Citrate, Malate, Succinate, Pyruvate also enhances the growth of plant cells. Sometimes antibiotics (e.g. Streptomycin, Kanamycin) are also added to the medium to prevent the growth of the microorganisms.
Carbon sources: usually sugar Sucrose (usually sucrose) - However, plant cells and tissues in the culture medium are heterotrophic and therefore depend on the external carbon for energy. During the sterilization of the medium, Sucrose gets hydrolyzed to glucose and fructose and the plant cells utilize first the glucose and than the fructose. The other carbohydrates such as Lactose, Maltose, Galactose etc., have been used in culture media but with limited success.
Gelling or Solidifying agents like Agar. Plant tissue culture media can be used in either liquid or ‘solid’ forms, depending on the type of culture being grown. Generally, a gelling agent Agar (a polysaccharide obtained from red algae, Gelidium amansil) is added to the liquid medium for its solidification. The agar obtained from seaweeds provides solid surface for the growth of cells because in the liquid medium, the tissue will be submerged and die due to lack of oxygen. Cells are grown in suspension medium without agar but such cultures are aerated regularly either by bubbling sterile air or by gentle agitation. Some other less frequently used solidifying agents are Gelrite, Biogel (polyacrlyamide pellets), Phytagel, and purified Agarose, as can a variety of Gellan gums.
Growth regulators (e.g. Auxins, Cytokinins and Gibberellins) - Plant hormones play an important role in growth and differentiation of cultured cells and tissues. There are many classes of plant growth regulators used in culture media involves namely: Auxins, Cytokinins, Gibberellins, Abscisic acid, Ethylene, 6 BAP (6 Benzyladenine), I.A.A (Indole Acetic Acid), I.B.A (Indole - -3- Butyric Acid), Zeatin, Zeatin Riboside.
- The Auxins facilitate the cell division and root differentiation. Auxins induce cell division, cell elongation, and formation of callus in cultures. For eg., 2,4-dichlorophenoxy acetic acid is one of the most commonly added auxins in plant cell cultures.
- The Cytokinins induce cell division and differentiation. Cytokinins, promotes RNA synthesis and stimulate protein and enzyme activities in tissues. Kinetin and benzyl-aminopurine are the most frequently used cytokinins in plant cell cultures.
- The Gibberellins is mainly used to induce plantlet formation from adventive embryos formed in culture.
- Abscisic acid is used in plant tissue culture to promote distinct developmental pathways such as somatic embryogenesis. Abscisic acid (ABA) inhibits cell division.
- Ethylene is associated with controlling fruit ripening in climacteric fruits, and its use in plant tissue culture is not widespread. Some plant cell cultures produce ethylene, which, if it builds up sufficiently, can inhibit the growth and development of the culture.
Plant growth promoters are α - Naphthalene acetic acid, Humic acid (granular/ powder) etc. Plant growth promoters (PGP) are those substances used for better management of nutrients and plant growth. Many times some crops fail to produce optimum yields in spite of proper nutrient supply. Physiological inefficiency in plants is responsible for such effects. Therefore, these PGA play a major role in seed germination, fruit ripening, enhances uptake of nutrients, boost protein synthesis, augment immunity and helps to withstand stress conditions, reduce flowering and fruiting drop and help in better plant growth.
Complex organics like Casein hydrolysate, Coconut milk, Malt extract, Yeast extract, Tomato juice, etc. may be added for specific purposes. Casein hydrolysate has given significant success in tissue culture and potato extract also has been found useful for anther culture. However, these natural extracts are avoided as their composition is unknown and vary from lot to lot and also vary with age affecting reproducibility of results.
Activated charcoal acts both in promotion and inhibition of culture growth depending upon plant species being cultured. It is reported to stimulate growth and differentiation in orchids, carrot, ivy and tomato whereas inhibits tobacco, soybean etc. It absorbs brown-black pigments and oxidized phenolics produced during culture and thus reduce toxicity. It also absorbs other organic compounds like PGRs, vitamins etc which may cause the inhibition of growth. Another feature of activated charcoal is that it causes darkening of medium and so helps root formation and growth.
pH - affects absorption of ions and also solidification of gelling agent. An optimum pH for culture media is 5.8 before sterilization. Values of pH lower than 4.5 or higher that 7.0 greatly inhibit growth and development in vitro. The pH of culture media generally drops by 0.3 to 0.5 units after autoclaving and keeps changing through the period of culture due to oxidation and also differential uptake and secretion of substances by growing tissue. At pH higher than 7.0 and lower than 4.5, the plant cells stop growing in the medium.
Antibiotics used as Antmicrobial agents in Plant tissue culture media. The combination of antibiotics and chemical biocides has been tested for their ability to inhibit microbial contamination in plant cultures. For example, Phillips, R., et al., Plant Sci. Lett., 21: 235-240 (1981), describe tests of six antibiotics, i.e., Benzyl penicillin, Phosphomycin, Chloramphenicol, Streptomycin, Rifampicin and Nalidixic acid, on preventing bacterial infection in cultures of Jerusalem artichoke (Helianthus tuberosus). Only Rifampicin was able to control bacterial contamination without affecting rates of plant cell division, cell differentiation or DNA synthesis in cultured explants. However, Rifampicin triggered an increase in protein synthesis.
Antibiotics help in suppressing the bacterial infections in plant cell and tissue culture. Also helps in suppressing mould and yeast infections in cell cultures. Whereas, helps in elimination of Agrobacterium species after the transformation of plant tissue. The ability of Agrobacterium to transfer genes to plants and fungi is used in biotechnology, in particular, genetic engineering for plant improvement. The genes to be introduced into the plant are cloned into a plant transformation vector that contains the T-DNA region of the disarmed plasmid, together with a selectable marker (such as antibiotic resistance) to enable selection for plants that have been successfully transformed. Plants are grown on media containing antibiotic following transformation, and those that do not have the T-DNA integrated into their genome will die. An alternative method is agroinfiltration.
The elements listed above are - together with Carbon (C), Oxygen (O) and Hydrogen (H) - the 17 essential elements. Certain others, such as Cobalt (Co), Aluminium (Al), Sodium (Na) and Iodine (I), are essential or beneficial for some species but their widespread essentiality has still to be established.According to Epstein (1971), elements can be considered to be essential for plant growth if :
- A plant fails to complete its life cycle without it
- Its action is specific and cannot be replaced completely by any other element
- Its effect on the organism is direct, not indirect on the environment
- It is a constituent of a molecule that is known to be essential.
For 1liter of growth medium, this is enough to prepare about 100 growing tubes.