PLANT TISSUE CULTURING Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient
known composition. A potion (explants) of desired plant is cultured in vitro (glass) on a defined medium, which promotes rapid multiplication of cells.
All plant tissue culture media are mostly synthetic or chemically defined. A few of them are organic complex nutrients e.g. Potato extract. Although, Tm Media has formulated a variety of media keeping our esteemed customer in mind. The composition of White, MS and B5 are the most commonly used media. The most common Plant Tissue Culture Media are: Murashige Skoog Medium (M.S. Medium) Banana Micropropagation Medium Orchid Maintenance Medium The basic nutrient requirements of cultured plant cells are very similar to those of whole plant. Culture media used for in vitro cultivation of plant cells are composed of following basic components: CONSTITUENTS
Complex mixture of salts
Vitamins, Amino Acids
Sucrose, Glucose, Fructose, Maltose
Plant Growth Regulators
Auxin, Cytokinins, Gibberellins, Abscisic Acid, Ethylene
Ampicillin, Tetracycline, Amphotericin B
NUTRIENTS: There are two types of nutrients, macronutrients which should be present in amount greater than 0.5 mM in a media and micronutrients which are required in less amount <0.5 mM. Macro element: Nitrogen, Phosphorous, Potassium, Magnesium, Calcium and Sulphur are These elements comprises at least 0.1% of dry weight of plants. The optimum concentration of each nutrient for achieving maximum growth rate varies considerably among species. B5 and MS Media are rich, especially for K and N. Microelement: These elements are required in trace amounts for plant growth and development. Manganese, Iodine, Copper, Cobalt, Boron, Molybdenum, Iron and Zinc are regarded as microelements, although other elements like Aluminium and Nickel are frequently found in some formulations. Manganese, Copper, Zinc are enzyme cofactors. Iron is a component of cytochrome and required for electron transfer. Generally, iron is added in the medium as Ferrous Sulphate, although Ferric citrate can also be used. Iron-EDTA complex is usually added to keep it available at higher pH. Uncomplexed Iron can precipitate out the medium as Ferric Oxide. Carbon sources: The plant tissue culture media, though sucrose is very frequently used (at the concentration 2-5%), there are many other carbohydrates that are also used, like lactose, galactose, maltose and starch but they are less effective than either sucrose and glucose. Glucose is more effective than fructose as it is used by the cells in the beginning. Organic Substances: Vitamins are needed for metabolic processes in tissue culture. Since tissue synthesize vitamins in low concentration therefore they have to be supplemented from outside. Thiamine, nicotinic acid, pyrodoxin and calcium pantothenate are the vitamins which are commonly supplemented.
Apart from this amino acid culture tissues are usually capable of synthesizing amino acid for various metabolic processes but we need to supplement them for stimulating cell growth in protoplast cultures and establishing cell lines. Glycine is most commonly used amino acid. However, Arginine, Asparagine, aspartic acid, Alanine, glutamine and proline are also used as source of amino acid. Gelling Agents: Growth of cultured tissues is greatly influenced by the hardness of culture medium. There are number of gelling agent used like agar, agarose and gellan gum. In plant tissue culture, agar has been used as a solidifying agent since long. There are several advantages of agar, over other gelling agent; like it get mixed in water, gets easily melt at the temperature range60 -100 C and forms a gel that is stable at all the feasible incubation temperature. It is inert and thus do not get reacted with media constituents and is not digested by plant enzymes.
Fig: TP 001A: MURASHIGE & SKOOG MEDIUM
Plant Growth Promotor: The growth regulators which are used in plant tissue culturing include: Auxins like Indole -3-acetic acid (IAA), Indole-3-butyric acid (IBA), Napthalene Acetic acid (NAA), (Naphthoxy Acetic Acid (NOA), 2,4-dicholorophenoxy acetic acid (2,4-D), to support cell division and callus growth (especially 2,4-D), somatic embryo induction and rooting.Cytokinins like kinetin, FAP(Furfurylamino purine),BAP (Benzylamino purine),TDZ (Thidiazuron) are employed to promote cell division , regeneration of shoot, somatic embryo induction and proliferation and growth of axillary buds. Abscisic Acid (ABA) promotes SE and shoot bud regeneration in many species and markedly improves SE maturation.Among 20 gibberellins known, GA3 is exclusively used to promote shoot elongation and SE germination.Ehylene is associated with controllong fruit ripening ams climatic fruits but is not widespread. The concentrations used of various growth regulators are as follows: auxins (0.1-3 mg/l);cytokinins (0.1-3mg/l);ABA (upto 0.2 mg/l); and GA3(0.1-1 mg/l). 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 tissue culture. B. Suppresses mold and yeast infections in cell cultures.
C. Eliminates Agrobacterium species after the transformation of plant tissue. These antibiotic can be divided into different classes on the basis of chemical structure and their mechanism of action: OSE
tors of bacterial cell wall cell permeability
am antibiotics, Penicillin and Cephalosporins
membrane acterial: Colistin Sulphate, Polymyxin B Sulphate, Gramicidin ungal: Amphotericin B, Nystatin, Pimaricin
iostatic Inhibitor of protein
Chloramphenicol, Chlortetracycline HCI, Lincomycin HCI, Oxytetracycline HCI, nomycin sulphate, Tetracycline HCI.
icide inhibitor of protein Aminoglycosides: Apramycin, Butriosine, Gentamicin, Kanamycin, Neomycin, Streptomycin, Tobramycin. synthesis Inhibitors of Nucleic Acid Metabolism: Rifampicin, Mitomycin C and Nalidixic acid Antimetabolites: Metronidazole, Miconazole, Nitrofurantoin, Trimethoprim and Sulpphomethoxazole. t enzymes essential for rouracil, Mercaptopurine DNA Synthesis Find