More Related Content
Similar to Function of medium
Function of medium
- 1. RITASREE SARMA PLANT TISSUECULTURE MEDIA
- 2. MEDIA Media Environment providingcorrect ‘necessities’ for plant growth Artificial media must provide Minerals Vitamins Growth regulators Water Removal of plant metabolic waste
- 3. Complex mixtureof inorganic salts Organic supplement: vitamins and/or amino acids Carbon source Plant Growth Regulators Gelling agents Water
- 4. Inorganic salts Macronutrients Nitrogen Essential inplant nucleic acid, protein, chlorophyll, hormone In inorganic form, used as Nitrate or/and ammonia Phosphorus • Essential component of nucleic acid, phospholipids, enzyme cofactor • Used as mono or dihydrogen phosphate Potassium • Essential in cell division, stomatal regulation,pathway for chlorophyll, carbohydrate, protein synthesis
- 5. Calcium • Formation ofcell walls and root and shoot development. Translocation of sugars as part of glycan synthase, amino acids • Supplied as calcium chloride and calcium nitrate Magnesium • Involve in photosynthetic and respiration system (ATP synthesis) • Supplied as magnesium sulphate Sulphur • Structural component of amino acid and enzyme, detoxification of oxygen radical as a part of glutathion • Supplied as magnesium Sulphate and Potassium Sulphate.
- 6. Micronutrients Iron • Involve inphotosynthesis, redox reaction • Iron is usually added in along with a chelator • EDTA is usually used in conjunction with the iron sulphate. Copper • Important role in photosynthesis, respiration,enzyme cofactor for SOD, cytochrome oxidase • Supplied as Cupric Sulphate
- 7. Zinc • Zn playsan active role in protein synthesis, enzyme cofactor, tryphophan synthesis • Used as Zinc Sulphate Manganese • Plays an important role in the Hill reaction of photosynthesis. • Required in many enzymatic activities • Supplied as Manganese Sulphate Molybdenum • Essential for conversation of Nitrate to Ammonium • Supplied as Sodium molybdate
- 8. Vitamins • Important forplant growth and metabolism • Required in very small amounts, thiamine (B1 ), is more commonly used • Involve in (CH2O)n metabolism, biosynthesis of amino acid • Other vitamins used are nicotinic acid, pyridoxine (B6), Inositol Nicotinic acid act as respiratory coenzyme Pyridoxine in metabolic reaction Inositol involve in phospholipid,pectin synthesis Amino Acids • Glycine is the most commonly used • Involve in purine synthesis, porphyrin structure of chrolophyll Contd…
- 9. Growth regulators Auxins • Auxinspromote both cell division and cell growth. • IAA ,naturally occurring auxin is limited use in media as unstable to both heat and light. Cytokinins • Cytokinins promote cell division, stimulate tissue growth • Embryogenesis and inhibit root formation. • Naturally occurring cytokinins, only zeatin and 2iP (2- isopentyl adenine and synthetic analogues kinetin and BAP (benzylaminopurine), are used more frequently.
- 10.
- 11. Gibberellins • Regulating cellelongation, determining plant height and fruit-set • GA3 are used in plant tissue culture media • Used in meristem culture for plant regeration and elogtation Abscisic acid • Promote distinct developmental pathways such as somatic embryogenesis
- 12. Carbon sources • Plantcells lack autotrophic ability in culture media • The most commonly used carbon source is sucrose. • It is readily assimilated and relatively stable. Activated Charcoal • Very fine network of pores with large inner surface area • Irreversible adsorption of inhibitory compounds, decreasing the toxic metabolites, phenolic exudation and brown exudate accumulation
- 13. Undefined organic supplements •Protein hydrolysates Source for calcium, phosphorus, several microelement, mixture 18 amino acid • Coconut milk Rich in diphenylurea,kinetin like compound • Yeast extract Source for amino acid,inositol,thiamine • Ground banana Stabilize pH of medium • Potato extract Source for(CH2O)n, amino acid,vitamin B1,B6
- 14. Agar Gelatinous substance derivedfrom the seaweeds It does not react with any components of the medium and is not digested by enzymes from the plant tissue. Agarose Purified extract of agar without the agaropectin fraction and sulphate groups. Mostly use for culturing protoplasts. Gellan gum Composed of glucouronic acid,rhamnose and glucose Facilitating light microscopical analyses of the cells and tissue
- 15. Gelrite Produced byPseudomonas elodea Medium produced is very clear so easily detected contamination No gelling of media if concentration of Ca,Mg is beyond 4-8mM/L Water • Distilled water is used • Distilled water of first 10-15 min should not be used as early vaporization have volatile organic compound
- 16. Types of media Murashige & Skoog Medium (MS) White Medium Nitsch Medium CHU (N6 ) Medium B5 medium Schenk & Hidebrandt Medium (SH) Woody plant media
- 17. Murashige & SkoogMedium • Murashige & Skoog Medium (MS) is established by Murashige & Skoog (1962) for in vitro callus culture of Nicotiana tabacum • Used as basal media • A high concentration of nitrate,potassium and ammonium compared to other • Total ion concentration of N is 60.13mM which is five fold higher than earlier media • K concentration is 20mM • 07 Artigo MS 1962.pdf
- 18. Composition of MSmedia • Macroelements (mg/l) • Potassiumnitrate 1900.00 Ammoniumsulphate 1650.00 • Calciumchlorideanhydrous 332.16 • Magnesiumsulphate 180.69 • Potassiumphosphatemonobasic 170.00 • Microelements • Manganese sulphate.H2O 16.90 • Boric Acid 6.20
- 19. Composition of MSmedia • Copper sulphate.5H O 0.03 • Cobalt chloride.6H O 0.03 • Ferrous sulphate.7H O 27.80 • Na .EDTA 37.30 • Vitamins • Myo-Inositol 0.83 • Potassium iodide 0.25 • Molybdic acid (sodium salt).2H O 8.60 • Zinc sulphate.7H O 100 • Thiamine HCL 0.10 • Pyridoxine HCL 0 .50
- 20. Contd.. Amino Acid • Glycine(Free base) 2.00 Carbohydrate • Sucrose 30000 Plant Growth Regulators • IAA 3.00 • 6-Benzyl amino purine(BAP) 5.00 Gelling Agent • Agar 8000
- 21. Kinds of media •White Medium • White Medium was established by White (1963) for root culture of tomato • This is one of the earliest media with low salt formulation. • Nitrate concentration is 19% less than MS media • Prominence was given to MgSO4 • Nitsch Medium • Nitsch Medium is established by Nitsch J.P. (1969) for in vitro anther culture of Nicotiana (Family-Solanaceae). • It contains low concentration of microelement to MS media but higher concentration of salt compared to white media
- 22. • Gamborg B5Medium • B5 Medium was established by by Gamborg O.L. (1968) for callus and cell suspension culture of Glycine max • This medium generally contains greater proportion of nitrate and potassium but low concentration of ammonia • Schenk & Hidebrandt Medium (SH) • Establised by Schenk R.U. & Hilderbrandt a.C.(1972)for in vitro callus culture of monocotyledonous and dicotyledonous plants • Copper content and Myo inositol concentration was increased in the medium as it shows stimulatory effect in monocotyledonous and dicotyledonous callus
- 23. CHU(N6) Medium • CHU(N6) Medium was developed by Chu C.C. et al. (1975) • 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. • 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
- 24. Constituents of differenttissue culture media(mg/l) CONSTITUENTS WHITE NITSCH& NITSCH GAMBOR G’s B5 Schenk & Hidebrandt Medium (SH) N6 KCl 65 1500 - - - NaH2PO4.H2O 16.5 250 150 300 - CaCl2.2H2O - - 150 151 166 Na2SO4 200 - - - - MgSO4.7H2O 720 250 - 400 - KH2PO4 - - - - - Ca(NO3)2.4H2O 300 - - - - (NH4)2SO4 - - 134 - 463 NiSO4 - - - - - FeSO4.7H2O - - - 15 27.8 MnSO4.4H2O 7 3 - 10 -
- 25. Constituents of differenttissue culture media(mg/l) CONSTITUENTS WHITE NITSCH& NITSCH GAMBORG’s B5 Schenk & Hidebrandt Medium (SH) N6 KI 0.75 - 0.75 - 0.8 CoCl2.6H2O - - 0.025 0.1 - Ti(SO4)3 - - - - - ZnSO4.7H2O 3 0.5 2 1 1.5 CuSO4.5H2O - 0.025 0.025 0.2 - H3BO3 1.5 0.5 3 5 1.6 H2SO4 - - - - - Na2.MoO4.2H2O - 0.025 .25 0.1 - Fe2(SO4)3 - - - - 37.3
- 26. Constituents of differenttissue culture media(mg/l) CONSTITUENTS WHITE NITSCH& NITSCH GAMBORG’s B5 Schenk & Hidebrandt Medium (SH) N6 EDTA-Disodium salt - - - 20.1 37.3 EDTA-Na Ferric salt - - 43 - - M-INOSITOL - - 100 1000 - THIAMINE 0.1 - 1.0 0.5 1.0 NICOTINIC ACID 0.5 1.0 5 0.5 GLYCINE 3 - - - - CYSTIENE 1.0 - 10 - - SUCROSE 20,000 34,000 20,000 10,000 30,000
- 27. Effect of media Varietyused: Thai Aromatic Rice KDM105 Culture media : MS, Gamborg-B5 (B5), Linsmair and Skoog (LS) and Chu medium (N6) and concentration of 2,4-D on growth of rice callus Source: Summart et al. world applied sciences J 5 (2): 246-251, 2008
- 28. Source: Summart etal. world applied sciences J 5 (2): 246-251, 2008
- 29. Woody plant material •Woody Plant medium was establised by Lloyd and Mc Crown (1981) for Kalmia latifolia • It has been specially formulated for plant cell, tissue and organ cultures • Here ionic strength of salts are less than MS media but sulphate is higher than average
- 30. • Ammonium nitrate400.00 • Calcium chloride.2H2O 96.00 • Calcium nitrate 386.34 • Magnesium sulphate 180.69 • Potassium phosphate monobasic 170.00 • Potassium sulphate 990.00 • Manganese sulphate.H2O 22.30 • Boric acid 6.20 • Molybdic acid (sodium salt).2H2O 0.25 • Zinc sulphate.7H2O 8.60 • Copper sulphate.5H2O 0.25 • Ferrous sulphate.7H2O 27.80 • DTA disodium salt.2H2O 37.30 • myo - Inositol 100.00 • Thiamine hydrochloride 1.00 • Pyridoxine hydrochloride 0.50 • Nicotinic acid (Free acid) 0.50 • Glycine (Free base) 2
- 31. Effect of media •Plant used: Sweet and sour cherry • Medium used: WPM,MS,QL,DKW medium • Concentration of NAA and BAP on plant regeneration from leaf Source:
- 33. Banana medium • Bananamedium is used for in vitro micropropagation of Musa (Family – Musaceae • Banana medium is the modification of Murashige and Skoog medium(1962) • 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. • The medium contains increased concentration of glycine • Indole-3-acetic acid (IAA) is used in the medium for improved growth and quanlity of banana plantlets. 6-Benzyl amino purine (BAP) induces shoot proliferation
- 34. Composition • Macroelements • Potassiumnitrate 1900.00 • Ammonium nitrate 1650.00 • Calcium chloride anhydrous 332.16 • Magnesium phosphate anhydrous 180.69 • Potassium phosphate monobasic 170.00 • Sodium phosphate monobasic 221.00 • Microelements • Manganese sulphate.H2 O 16.90 2 • Boric Acid 6.20 • Potassium iodide 0.83 • Molybdic acid (sodium salt).2H2 O 0.25 2 • Zinc sulphate.7H2 O 8.60 2 • Copper sulphate.5H2 O 0.03 2
- 35. • Cobalt chloride.6H2O 0.03 • Ferrous sulphate.7H2 O 27.80 • Na .EDTA 37.26 • Myo-Inositol 100.00 • Thiamine HCL 0.10 • Carbohydrate • Sucrose 30000.00 • Organic supplement • Casein enzymic hydrolysate 10.00 • Plant Growth Regulators • Indole-3-acetic acid 0.18 • Gelling agent • Agar 8000.00
- 36. Orchid medium • OrchidMedium for in vitro cultivation of Orchids. • The macroelements are half the concentration of the Murashige and Skoog medium. • Potassium dihydrogen phosphate which increases the growth rate of anther callus • Thiamine content has been increased in the medium which is • beneficial for the growth of orchid seedlings. • Banana powder serve as a complex source of carbohydrate in the medium. • Meat peptone and tryptone are the forms of nitrogen, supplied to the medium
- 37. Composition(mg/ml) • Macroelements • Potassiumnitrate 950.00 • Ammonium sulphate 825.00 • Calcium chloride anhydrous 166.08 • Magnesium sulphate 90.34 • Potassium phosphate monobasic 85.00 • Microelements • Manganese sulphate.H2O 8.45 • Boric Acid 3.10 • Potassium iodide 0.42 • Molybdic acid (sodium salt).2H2O 0.12 • Zinc sulphate.7H2O 5.30 • Copper sulphate.5H2O 0.013 • Cobalt chloride.6H2O 0.013
- 38. • Na .EDTA37.30 • Vitamins • Myo-Inositol 100.00 • Thiamine HCL 10.00 • Pyridoxine HCL 1.00 • Nicotinic acid (Free acid) 1.00 • Carbohydrate • Sucrose 20000.00 • Banana Powder 30000.00 • Organic Supplement • Peptone from meat 2000.00 • Tryptone 2000.00 • Buffering Agent: • MES (Free acid) 1000.00 • Adsorbing Agent: • Activated Charcoal 2000.00 • Gelling Agent: • Agar 8000.00
- 39. References Plant propagationby plant tissue culture. Edwin F Goerge, Michae A. Hall, Gretan Da Klerk. vol I.2nd ed.2008. Introduction to plant biotechnology.H.S Chawala.3rd ed.2009. Oxford&IBH Plant tissue culture thoery and practise. .B.N.Sathyanarayana.Dalia B.Varghese.2007.I.K.International Summart J, Sanha Panichajakul, Prathepha.P and Thanonkeo.P. Callus Induction and Influence of Culture Condition and Culture Medium on Growth of Thai Aromatic Rice, Khao Dawk Mali 105. Cell Culture. World Applied Sciences Journal 5 (2): 246-251. 2008 Tang.H.Rang.Z.Reustle.G.Krczal.G.Plant regeration fron sweet and sour cherry cultivar.Scientia hort.2002.93:235-244 Toshio Murasnige and Folke skoog. A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cultures. physiologia plantarum, Vol. 15. 1962 Thorpe T. History of plant tissue culture. J. Mol. Microbial Biotechnol. 2007; 37: 169-180. www.tulipgroup.com/MicroExpress/Accumix/comp_profile.htm
- 40.