Every accomplishment starts with the decision to succeed

Transcription

1 Every accomplishment starts with the decision to succeed

2 Did you ever have a plant that was so unique or so beautiful that you wished you had hundreds or thousands of them to enjoy or to sell? Plant tissue culture (micropropagation) is a technique which will do just that for us. It is a fascinating and useful tool which allows the rapid production of many genetically identical plants using relatively small amounts of space, supplies and time. Basically the technique consists of taking a piece of a plant (such as a stem tip, node, meristem, embryo, or even a seed) and placing it in a sterile, (usually gel-based) nutrient medium where it multiplies. The formulation of the growth medium is changed depending upon whether you are trying to get the plant to produce undifferentiated callus tissue, multiply the number of plantlets, grow roots, or multiply embryos for "artificial seed". Plant tissue culture research has become a thrust area during the last decade due to the renewed emphasis it has received in all areas of crop improvement programmes. In several publications, including books, and in syllabi prescribed for biotechnology courses of our universities, plant tissue culture along with plant genetic engineering have become synonymous with plant biotechnology. Plant Tissue Culture refers to the technique of growing plant cells, tissues, organs, seeds or other plant parts in a sterile environment on a nutrient medium. Culture media used for in vitro cultivation of plant cells are composed of following basic components: 1. Complex mixture of salts: Essential elements, or mineral ions.. Organic supplement: vitamins and/or amino acids. 3. Carbon source: usually sugar sucrose. 4. Gelling agents 5. Plant Growth Regulators 6. Antibiotics Complex mixture of salts: These include essential elements or mineral ions important for plant nutrition and their physiological function. The essential elements can further be divided into the following categories: a. (or macronutrients) b. (or micronutrients) c. Iron Source : These elements are required in large amounts for plant growth and development. Nitrogen, phosphorus, potassium, magnesium, calcium and sulphur (and carbon, which is added separately) are regarded as macroelements. These elements comprise at least 0.1% of the dry weight of plan : 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. Iron Source: Iron is usually added in the medium as iron sulphate, although iron citrate can also be used. Ethylenediaminetetraacetic acid (EDTA) is usually used in conjunction with the iron sulphate. The EDTA complexes with the iron so as to allow the slow and continuous release of iron into the medium. Uncomplexed iron can precipitate out of the medium as ferric oxide. Organic supplements: BASIC REQUIREMENT OF A PLANT TISSUE CULTURE MEDIA These include vitamins and amino acids. Two vitamins, i.e., thiamine (vitamin B1) and myoinositol (a vitamin B) are essential for the culture of plant cells in vitro. However, other vitamins are often added to for historical reasons. The most commonly used amino acid is glycine. However, arginine, 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. Casein hydrolysate can be used as a source of a mixture of amino acids. 464

3 Carbon source: The most commonly used carbon source is sucrose. It is readily assimilated and relatively stable. Other carbohydrates like glucose, maltose, galactose and sorbitol can also be used and may prove better than sucrose in specialized circumstances. Gelling agents: Plant tissue culture media can be used in either liquid or 'solid' forms, depending on the type of culture being grown. Agar, produced from seaweed, is the most common type of gelling agent, and is ideal for routine applications. For more demanding applications, a range of purer gelling agents are available. Purified agar or agarose can be used, as can a variety of gellan gums. Agar is a gelatinous substance derived from the seaweeds. It is an unbranched high molecular weight polysacchride obtained from the cell walls of some species of the red algae, primarily from the genra Gelidium and Gracilaria. Agar is insoluble in cold water but forms relatively inert gel that melts at about 100 C and solidifies at around 45 C. Using Agar as a gelling agent main advantage is that agar does not react with any components of the medium and is not digested by enzymes from the plant tissue. If necessary, agar can be washed to remove inhibitory impurities. Agarose is a purified extract of agar without the agaropectin fraction and sulphate groups. It is required in lesser quantities since it has a higher gelling strength. Agarose is used for more demanding procedures like culturing protoplasts. It consists of â-d(1-3) galactopyranose and 3,6-anhydro-á-L(1-4) galactopyranose linked into polymer chains of monosaccharides units. Gellan gum, also known commercially as Phytagel or Gelrite or Gelwell, is used primarily as a gelling agent, alternative to agar, in microbiological culture. It is able to withstand 10 C heat, making it especially useful in culturing thermophilic organisms. One needs only approximately half the amount of gellan gum as agar to reach an equivalent gel strength, though the exact texture and quality depends on the concentration of divalent cations present. Gellan gum is used as gelling agent in plant cell culture on petri-dishes, as it provides a very clear gel, facilitating light microscopical analyses of the cells and tissue. Plant growth regulators: Specific media manipulations can be used to direct the development of plant cells in culture due to plasticity and totipotency. Plant growth regulators are the critical media components in determining the developmental pathway of the plant cells. There are five main classes of plant growth regulator used in plant cell culture, namely: a. Auxins b. Cytokinins c. Gibberellins d. Abscisic acid e. Ethylene Auxins: Auxins promote both cell division and cell growth. IAA (indole-3-acetic acid) is the most important naturally occurring auxin but its use in plant tissue culture media is limited because it is unstable to both heat and light.,4-dichlorophenoxyacetic acid (,4-D) is the most commonly used auxin and is extremely effective in most circumstances. Cytokinins: Cytokinins promote cell division. Of the naturally occurring cytokinins, only zeatin and ip (-isopentyl adenine have some use in plant tissue culture media. The synthetic analogues, kinetin and BAP (benzylaminopurine), are used more frequently. Non-purine-based chemicals, such as substituted phenylureas, are also used as cytokinins in plant tissue culture media. Gibberellins: Gibberellins are involved in regulating cell elongation, in determining plant height and fruit-set. Only a few of the gibberellins like GA3 are used in plant tissue culture media. Abscisic acid: It is used in plant tissue culture to promote distinct developmental pathways such as somatic embryogenesis. Abscisic acid (ABA) inhibits cell division. Ethylene: 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. 465

4 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. d. As a selective agent in plant transformation experiments. Antimicrobial Compound Mechanism of Action Nature Amoxycillin Inhibits synthesis of the bacterial peptidoglycan cell wall Bactericidal Amphotericin B solubilized powder Act by increasing membrane permeability by binding to sterol moiety, Antifungal primarily ergosterol present in the cell membrane of sensitive fungi Amphicillin sodium salt Inhibit synthesis of the bacterial peptidoglycan cell wall Bactericidal Bacitracin Inhibit synthesis of the bacterial peptidoglycan cell wall Bactericidal Carbenicillin,disodium salt Inhibit synthesis of the bacterial peptidoglycan cell wall Bactericidal Cephotaxime, sodium salt Inhibit bacterial cell wall synthesis Bactericidal Chloramphenicol Inhibits protein synthesis by acting on 50S ribosomes Bactericidal Ciprofloxacin hydrochloride Interfere with DNA replication by inhibition of DNA gyrase Bactericidal Erythromycin Inhibit protein synthesis by acting on 50S ribosomes Bactericidal Gentamycin sulphate Inhibit protein synthesis by binding to 30S ribosomes and Bactericidal decreasing the fidelity of translation of m RNA Geneticin disulphate Inhibit prokaryotic and eukaryotic protein synthesis Bactericidal Hygromycin B Inhibit bacterial cell wall synthesis Bactericidal Kanamycin acid sulphate Inhibit protein synthesis by interaction with 30s, 50S ribosomes Bactericidal Mycostain Bind to sterols in fungal cell membrane, changing the cell Antifungal wall permeability allowing for leakage of cellular contents Penicillin, benzyl sodium salt Inhibit bacterial cell wall synthesis Bactericidal Vancomycin hydrochloride Interferes bacterial cell wall synthesis for Gram+ only Bactericidal Rifampicin Rifampicin inhibits DNA-dependent RNA polymearase in Bactericidal bacterial cells by binding its beta subunit, thus preventing transcription of messenger RNA (mrna) and subsequent translation to Proteins. Neomycin sulphate Inhibits protein synthesis Bactericidal 466

5 Banana Medium BM Medium CHU (N ) Medium 6 Gamborg B5 Medium Murashige & Skoog Medium (MS) Plant Tissue Culture Media Musrashige & SkoogModified Medium (MS) Nitsch Medium Orchid Medium Schenk & Hidebrandt Medium (SH) Use Banana Medium Banana medium is used for in vitro micropropagation of Musa (Family Musaceae). Banana medium is the modification of Murashige and Skoog medium(196), which provides all essential elements to grow Banana in vitro. Banana medium provides all essential,,, Amino acid & Plant growth regulators for the growth of Banana in vitro. : Potassium nitrate, ammonium nitrate provide nitrogen source to this media. This mixture of cation and anion balances the ph of the medium. Also this ratio play a very important role in plant growth. : Manganese, molybdenum, zinc, boron, copper and iron play an important role in plant metabolism while manganese plays a vital catalytic role in nitrogen metabolism. Iron used as a FeEDTA to overcome the problem of precipitation in the medium. : Myo inositol concentartion was increased as it shows stimulatory effect in tobacco callus. Increased concentration of thiamine, pyridoxine and nicotinic acid have a stimulatory effect. Thiamine in the medium is a key elements of carbohydrate metabolism and biosynthesis of some amino acids. Ascorbic acid is provided to control phenol production in the medium. Amino acid: The medium contains increased concentration of glycine (mg/l). Plant growth regulators: Indole-3-acetic acid (IAA) is used in the medium forimproved growth and quanlity of banana plantlets. 6-Benzyl amino purine(bap) induces shoot proliferation. APM1001/APM5001 Ingredients in Grams/Litre Potassium nitrate Ammonium nitrate Calcium chloride anhydrous Magnesium phosphate anhydrous Potassium phosphate monobasic Sodium phosphate monobasic 1.00 Manganese sulphate.ho Boric Acid 6.0 Potassium iodide 0.83 Molybdic acid (sodium salt).h O 0.5 Zinc sulphate.7h O 8.60 Copper sulphate.5h O 0.03 Cobalt chloride.6h O 0.03 Ferrous sulphate.7h O 7.80 Na.EDTA 37.6 Myo-Inositol Thiamine HCL 0.10 Carbohydrate Sucrose Organic supplement Casein enzymic hydrolysate Plant Growth Regulators Indole-3-acetic acid 0.18 Gelling agent Agar Store at -8 C and prepared medium at -8 C. Use before expiry date as mentioned on the label 467

6 USE BM Medium BM Media is used for seed culture and micropropagation of orchids. Van waes, (1986) has developed BM medium for in vitro cultivation of Protocorms from orchid seeds. BM medium provides all essential,,, Amino acid & Plant growth regulators for the growth of Orchid in vitro. This medium is especially suitable for terrestrial orchids. : Potassium dihydrogen phosphate serves as a source of phosphate. This medium lacks in inorganic nitrogen. : Zinc and boron content in the medium is increased to provide proper nourishment to developing protocomes. : Thiamine content had been increased (0.5mg/l) in the medium. It is a most important element in carbohydrte metabolism and some amino acids biosynthesis. Biotin and folic acid along with other vitamins facilitates in vitro development of Protocorm. Amino acid: Glycine and glutamine serve as reduced nitrogen source. Carbohydrte: Sucrose serves as a carbon source. Organic supplements: Casein hydrolysate used as a supplement, which is a sources of free amino acid. Plant growth regulators: APM100/APM500 6-Benzyl amino purine(bap) induces shoot proliferation. Ingredients in Grams/Litre Potassium nitrate Ammonium sulphate Calcium chloride anhydrous Magnesium sulphate Potassium phosphate monobasic Manganese sulphate.ho 3.33 Boric Acid 1.60 Potassium iodide 0.80 Zinc sulphate.7h O 1.50 Ferrous sulphate.7h O 7.80 Na.EDTA 37.6 Thiamine HCL 1.00 Pyridoxine HCL 0.50 Nicotinic acid (Free acid) 0.50 Amino acid Glycine (free base).00 Store at -8 C and prepared medium at -8 C. Use before expiry date as mentioned on the label. Chu C.C., et. al., Scientia Sinic., 18, USE CHU (N ) Medium 6 APM1003/APM5003 CHU (N ) Medium is used to promote for organ culture and cell suspension culture. 6 Chu C.C. et al. (1975) has developed CHU (N 6) Medium for in vitro anther culture of Oryza sativa (Family-Graminae). It is useful to generate new useful genetic varieties in gramineous plants by the initiation, growth, and differentiation of callus from rice pollen culture.. CHU (N ) medium provides all essential,, 6 & Amino acid for the growth of Orchid in vitro. This medium is not only to culture of rice anther but also for in vitro studies of other graminous plants. : Potassium nitrate serves as a source of nitrate. High concentration of ammonium ions has an inhibitory effect on the growth and quality of rice callus. The increased concentration of phosphate has a beneficial effect on the growth of rice callus. : Boron, Managanese and zinc improves the quality of anther callus. : Thiamine has been found to be beneficial for the growth and quality of rice callus. It is a most important elements of carbohydrate metabolism and biosynthesis of some amino acids. 468

7 Ingredients in Grams/Litre Potassium nitrate Ammonium sulphate Calcium chloride anhydrous Magnesium sulphate Potassium phosphate monobasic Manganese sulphate.ho 3.33 Boric Acid 1.60 Potassium iodide 0.80 Zinc sulphate.7h O 1.50 Ferrous sulphate.7ho 7.80 Na..EDTA 37.6 Thiamine HCl 1.00 Pyridoxine HCl 0.50 Nicotinic acid (Free acid) 0.50 Amino acid Glycine (free base).00 Store at -8 C and prepared medium at -8 C. Use before expiry date as mentioned on the label. Chu C.C., et. al., Scientia Sinic., 18, Use Gamborg B5 Medium Gamborg B5 Medium is used for callus culture and cell suspension culture. Gamborg B5 Medium is established by Gamborg O.L. (1968) for callus and cell suspension culture of Glycine max (Family- Fabaceae). This medium is widely used for in vitro plant cell, tissue and organ culture. Gamborg B5 Medium provides all essential,, & for the growth of plant cell, tissue and organ culture in vitro. : Increased nitrate content was found to beneficial for soyabean root callus. Potassium nitrate served as the sole source of nitrate in the medium. Ammonium sulphate fulfilled the ammonium requirement in the medium without altering cell growth. Sodium dihydrogen phosphate served as the source of phosphate in the medium. : Boron, Managanese and zinc provided for proper nourishment. Molybdenum, copper and cobalt were not included in the medium. : Thiamine content had been increased in the medium which supported the growth of soyabean cell suspension culture. The medium lacks glycine. Ingredients in mg per liter Potassium nitrate APM1004/APM5004 Ammonium sulphate Calcium chloride anhydrous Magnesium sulphate Sodium phosphate monobasic Manganese sulphate.ho Boric Acid 3.00 Potassium iodide 0.75 Molybdic acid (sodium salt).h O 0.1 Zinc sulphate.7h O.00 Copper sulphate.5h O 0.05 Cobalt chloride.6h O 0.05 Ferrous sulphate.7h O 7.80 Na.EDTA 37.6 Myo-Inositol Thiamine HCL Pyridoxine HCL 1.00 Nicotinic acid (Free acid) 1.00 Gelling Agent Agar Store at -8 C and prepared medium at -8 C. Use before expiry date as mentioned on the label. Gamborg O.L., Miler R.A. And Ojima K., Exp. Cell Res., 50,

8 Use Use Murashige & Skoog Medium (MS) Murashige & Skoog Medium (MS) is used for micropropagation, organ culture, callus culture and cell suspension culture.. Murashige & Skoog Medium (MS) is established by Murashige & Skoog (196) for in vitro callus culture of Nicotiana tabacum (family- Solanaceae). Murashige & Skoog Medium (MS) provides all essential,, & for the growth of plant cell, tissue and organ culture in vitro. Medium with high concentration of salts is used for cultivating plant cell, tissue and organ culture. : In this medium nitrogen is supplied as ammonium and nitrate ions. This mixture of cation and anion balances the ph of the medium. Also plays a important role in plant growth. Potassium dihydrogen phosphate serves as a source of phosphate in medium. : Boron, Managanese, molybdenum, copper, iron and zinc plays a vital catalytic role in plant metabolism. Boron plays a key role in carbohydrate metabolism in plant cells. : Thiamine, pyridoxin and nicotinic acid content had been increased in the medium which have a stimulatory effect. Amino acid: The medium contains increased concentration of glycine. Ingredients in mg per liter Potassium nitrate Ammonium sulphate Murashige & Skoog Modified Medium Murashige & Skoog Medium (MS) is used for micropropagation, organ culture, callus culture and cell suspension culture.. Murashige & Skoog Medium (MS) is established by Murashige & Skoog (196) for in vitro callus culture of Nicotiana tabacum (family- Solanaceae). This medium was modified by incorporating plant growth regulators for the development of micropropagated explants in vitro. APM1006/APM5006 Murashige & Skoog Medium (MS) provides all essential,, & for the growth of plant cell, tissue and organ culture in vitro. : APM1005/APM5005 Calcium chloride anhydrous Magnesium sulphate Potassium phosphate monobasic Manganese sulphate.ho Boric Acid 6.0 Potassium iodide 0.83 Molybdic acid (sodium salt).h O 0.5 Zinc sulphate.7h O 8.60 Copper sulphate.5h O 0.03 Cobalt chloride.6h O 0.03 Ferrous sulphate.7h O 7.80 Na.EDTA Myo-Inositol Thiamine HCL 0.10 Pyridoxine HCL 0.50 Nicotinic acid (Free acid) 0.50 Amino Acid Glycine (Free base).00 Carbohydrate Sucrose Buffering Agent MES (Free acid) Plant Growth Regulators 6-Benzyl amino purine(bap) 5.00 Gelling Agent Agar Store at -8 C and prepared medium at -8 C. Use before expiry date as mentioned on the label. Murashige T. and Skoog F., 196. Physio. Plant., 15, In this medium nitrogen is supplied as ammonium and nitrate ions. This mixture of cation and anion balances the ph of the medium. Also plays a important role in plant growth. Potassium dihydrogen phosphate serves as a source of phosphate in medium. 470

9 : Boron, Managanese, molybdenum, copper, iron and zinc plays a vital catalytic role in plant metabolism. Boron plays a key role in carbohydrate metabolism in plant cells. Manganese to iron ratio is very important for plant metabolism. Zinc is essential for synthesis of tryptophan which is a precursor of indole-3-acetic acid. Iron is supplemented as FeEDTA to overcome the problem of precipitation in the medium. : Thiamine, pyridoxin and nicotinic acid content had been increased in the medium which have a stimulatory effect. Amino acid: The medium contains increased concentration of glycine. Plant Growth Regulators: Indole 3- butyric acid and a- naphthalene acetic acid are promote the elongation of cell or tissue and mainly roots. Adenine sulphate stimulates axillary bud growth and help for shoot growth. Indole -3- acetic acid is a naturally occuring plant growth regulators. IIngredients in mg per liter Potassium nitrate Ammonium sulphate Calcium chloride anhydrous Magnesium sulphate Potassium phosphate monobasic Sodium phosphate monobasic Manganese sulphate.ho Use Nitsch Medium Nitsch Medium is used for Anther callus culture.. Nitsch Medium is established by Nitsch J. P. (1969) for in vitro anther culture of Nicotiana (Family-Solanaceae). Nitsch Medium provides all essential,, & for anther callus culture in vitro. : In this medium nitrogen is supplied as ammonium and nitrate ions. This mixture of cation and anion balances the ph of the medium. Also plays a important role in plant growth. Potassium dihydrogen phosphate which increases the growth rate of anther callus. Boric Acid 6.0 Potassium iodide 0.83 Molybdic acid (sodium salt).h O 0.5 Zinc sulphate.7h O 8.60 Copper sulphate.5h O 0.05 Cobalt chloride.6h O 0.05 Ferrous sulphate.7h O 7.80 Na.EDTA Myo-Inositol Thiamine HCL 0.40 Pyridoxine HCL 0.50 Nicotinic acid (Free acid) 0.50 Amino Acid Glycine (Free base).00 Carbohydrate Sucrose Plant Growth Regulators Indole-3-acetic acid 0.30 Indole-3-butyric acid 1.75 a- naphthalene acetic acid 1.75 Adenine hemisulphate (g,g-Dimethylallylamino) purine Store at -8 C. and prepared medium at -8 C. Use before expiry date as mentioned on the label Murashige T. and Skoog F., 196. Physio. Plant., 15, : APM1007/APM5007 Boron, Managanese, molybdenum, copper, iron and zinc plays a vital catalytic role in plant metabolism. Boron plays a key role in carbohydrate metabolism in plant cells. Manganese to iron ratio is very important for plant metabolism. Zinc is essential for synthesis of tryptophan which is a precursor of indole-3-acetic acid. Iron is supplemented as FeEDTA to overcome the problem of precipitation in the medium. : The medium contains essential vitamins for proper growth and development of anther callus. Thiamine content has been increased in the medium. Folic acid serves as a coenzyme. Biotin in the medium is an essential cofactor in fat, protein and carbohydrate metabolism. 471

10 Amino acid: The medium contains increased concentration of glycine. Carbohydrate: Sucrose serve as the source of carbohydrate. Ingredients in mg per liter Potassium nitrate Ammonium sulphate Calcium chloride anhydrous Magnesium sulphate Potassium phosphate monobasic Manganese sulphate.ho Boric Acid Molybdic acid (sodium salt).h O 0.5 Zinc sulphate.7h O Copper sulphate.5h O 0.03 Ferrous sulphate.7ho 7.85 Na.EDTA 37.5 Use Orchid Medium Orchid Medium is used for Orchid culture.. Orchid Medium for in vitro cultivation of Orchids. Orchid Medium provides all essential,, & for Orchid culture in vitro. : The macroelements are half the concentration of the Murashige and Skoog medium. In this medium nitrogen is supplied as ammonium and nitrate ions. This mixture of cation and anion balances the ph of the medium. Also plays a important role in plant growth. Potassium dihydrogen phosphate which increases the growth rate of anther callus. : Boron, Managanese, molybdenum, copper, iron and zinc plays a vital catalytic role in plant metabolism. Boron plays a key role in carbohydrate metabolism in plant cells. Manganese to iron ratio is very important for plant metabolism. Zinc is essential for synthesis of tryptophan which is a precursor of indole-3-acetic acid. Iron is supplemented as FeEDTA to overcome the problem of precipitation in the medium. Myo-Inositol Thiamine HCL 0.50 Pyridoxine HCL 0.50 Nicotinic acid (Free acid) 5.00 Folic acid 0.50 D-Biotin 0.05 Amino Acid Glycine (Free base).00 Carbohydrate Sucrose Store at -8 C and prepared medium at -8 C. Use before expiry date as mentioned on the labe Nitsch J.P. And Nitsch C., Science, 163, Indole 3- butyric acid and a- naphthalene acetic acid are promote the elongation of cell or tissue and mainly roots. Adenine sulphate stimulates axillary bud growth and help for shoot growth. Indole -3- acetic acid is a naturally occuring plant growth regulators. : APM1008/APM5008 The medium contains essential vitamins for proper growth and development of Orchid culture. Thiamine content has been increased in the medium which is beneficial for the growth of orchid seedlings. Carbohydrate: Sucrose serve as the source of carbohydrate. Banana powder serve as a complex source of carbohydrate in the medium. Organic supplements: Meat peptone and tryptone are the forms of nitrogen, supplied to the medium. Buffering Agent: -(N-morpholino) ethanesulfonic acid (MES) is commonly used as a buffering agent. This prevents acidification by buffering the medium. Adsorbent Agent: Activated charcoal in the medium adsorbs toxic phenolics. Also used in rooting medium to adsorb root inhibiting agents. IIngredients in mg per liter Potassium nitrate Ammonium sulphate Calcium chloride anhydrous

11 Magnesium sulphate Potassium phosphate monobasic Manganese sulphate.h O 8.45 Boric Acid 3.10 Potassium iodide 0.4 Molybdic acid (sodium salt).h O 0.1 Zinc sulphate.7h O 5.30 Copper sulphate.5h O Cobalt chloride.6h O Ferrous sulphate.7h O 7.80 Na.EDTA Myo-Inositol Thiamine HCL Pyridoxine HCL 1.00 Nicotinic acid (Free acid) 1.00 Carbohydrate Sucrose Banana Powder Organic Supplement Peptone from meat Tryptone Buffering Agent: MES (Free acid) Adsorbing Agent: Activated Charcoal Gelling Agent: Agar Store at -8 C and prepared medium at -8 C. Use before expiry date as mentioned on the label. Nitsch J.P. And Nitsch C., Science, 163, Indole 3- butyric acid and á- naphthalene acetic acid are promote the elongation of cell or tissue and mainly roots. Adenine sulphate stimulates axillary bud growth and help for shoot growth. Indole -3- acetic acid is a naturally occuring plant growth regulators. Use Schenk & Hidebrandt Medium (SH) Schenk & Hidebrandt Medium (SH) used for callus and cell suspension culture. Schenk & Hidebrandt Medium (SH) was established by Schenk R.U & Hilderbrandt A.C. (197) for in vitro callus culture of monocotyledonous and dicotyledonous plants. Medium supports the growth of both monocotyledonous and dicotyledonous plant tissues by providing all essential nutrients. : Nitrogen is supplied as ammonium and nitrate ions. This mixture of cation and anion balances the ph of the medium. Also plays a important role in plant growth. Potassium dihydrogen phosphate which increases the growth rate of anther callus. : Boron, Managanese, molybdenum, copper, iron and zinc plays a vital catalytic role in plant metabolism. Boron plays a key role in carbohydrate metabolism in APM1009/APM5009 plant cells. Manganese to iron ratio is very important for plant metabolism. Zinc is essential for synthesis of tryptophan which is a precursor of indole-3-acetic acid. Iron is supplemented as FeEDTA to overcome the problem of precipitation in the medium. Copper content was increased as it shows stimulatory effect in monocotylenous callus. Iron is supplemented as ferric citrate to overcome the problem of precipitation in the medium. Ferric is converted to the active ferrous forms. : Myo inositol concentration was increased in the medium as it shows stimulatory effect in monocotyledonous and dicotyledonous callus. Buffering Agent: -(N-morpholino) ethanesulfonic acid (MES) is commonly used as a buffering agent. This prevents acidification by buffering the medium. Adsorbent Agent: Activated charcoal in the medium adsorbs toxic phenolics. Also used in rooting medium to adsorb root inhibiting agents. Indole 3- butyric acid and a- naphthalene acetic acid are promote the 473

12 elongation of cell or tissue and mainly roots. Adenine sulphate stimulates axillary bud growth and help for shoot growth. Indole -3- acetic acid is a naturally occuring plant growth regulators. Ingredients in mg per liter Potassium nitrate Ammonium phosphate monobasic Calcium chloride anhydrous Magnesium sulphate Manganese sulphate.h O Boric Acid 5.00 Potassium iodide 1.00 Molybdic acid (sodium salt).h O 0.10 Zinc sulphate.7h O 1.00 Copper sulphate.5h O 0.0 Cobalt chloride.6h O 0.10 Ferrous sulphate.7h O Na.EDTA 0.00 Myo-Inositol Thiamine HCL 5.00 Pyridoxine HCL 0.50 Nicotinic acid (Free acid) 5.00 Store at -8 C and prepared medium at -8 C. Use before expiry date as mentioned on the label. Schenk R.U. & Hilderbrandt a.c., 197. Can. J. Bot., 50,

13 Ammonium nitrate Plant Tissue Culture Chemicals Macro Elements APCM10011 Potassium nitrate APCM Kg APCM10015 APCM Kg Calcium chloride APCM1001 APCM1001-5Kg Potassium phosphate monobasic APCM10016 APCM Kg Magnesium sulphate heptahydrate APCM10013 APCM Kg Sodium phosphate monobasic APCM10017 APCM Kg Potassium chloride APCM10014 Sodium Nitrate Micro Elements APCM10018 APCM Kg Boric acid APCM50101 Ferrous sulphate heptahydrate APCM50106 APCM Kg Copper sulphate pentahydrate (Cupric sulphate pentahydrate) Cobalt chloride hexahydrate EDTA Ferric Mono Sodium Salt EDTA Disodium salt dihydrate D-Biotin APCM5010 Manganese sulphate monohydrate Potassium iodide APCM10103 Sodium Molybdate dihydrate APCM50104 Zinc sulphate heptahydrate APCM50105 APV0001 L-Ascorbic acid (Vitamin C) APCM50107 APCM10108 APCM10109 APCM50111 APCM K APV0003 Folic acid APV000 APV000-10gm Myo-Inosotol or (Meso-Inositol) APV

14 Nicotinic acid or Niacin(Free acid) APV0005 Thiamine Hydrochloride APV0007 APV1007 Pyridoxine Hydrochloride APV0006 APV0006-5gm Amino Acid L-Glycine (Free base) APA0001 L-Tyrosine APA0003 APA1003 L-Glutamine APA000 APA100 Carbohydrate D-Glucose APC5001 Sucrose APC500 Plant Growth Regulators Adenine Sulphate APR0001 Indole-3-Acetic acid APR Benzy laminopurine (BAP) APR000 Kinetin (6-Furfurylaminopurine) APR0005 APR0005-5gm Indole- 3-Butyric acid (IBA) APR0003 a- Naphthalene acetic acid APR0006 Adsorbent Activated Charcoal APG10001 APG50001 Sucrose APC