Tissue Culture Media

HOW MAKE CULTURE MEDIA 

One of the difficulties in plant tissue culture is the nutrient requirements for optimum growth is very different in each species, so there is no media that can be recommended for all crops. Research - intensive research on tissue culture during the last 50 years has been a lot of media developed, some of which have been used extensively in tissue culture at this time. This media is given in Table 12.1. Chemicals in the media are usually determined, meaning that only certain nutrients are incorporated into the media, or the media may also contain additional ingredients such as coconut water complex or orange juice containing plant growth regulators.
 12.1. Composition of Tissue Culture Media 
12.1.1. Inorganic nutrientThere are 12 mineral nutrients essential for plant growth and some nutrients that affect the reported growth in vitro. For normal growth in tissue culture, elements - essential elements should be included in the culture medium. 5 Comparison of media in Table 12.1 shows that the essential elements included on each - each is different because the media but his concentration is given in a different form. 
12.1.2. Organic nutrientPlants were grown under normal conditions are autotrophs and can synthesize all the organic material needs. Although in vitro plants can synthesize these compounds, suggesting that they do not produce adequate amounts of vitamins for healthy growth and one or more vitamins must be added to the media. Thiamine is an essential vitamin, besides nicotine acid, pyridoxine and inositol are usually added.In addition to the organic matter, complex materials are often added, including yeast extract, casein hydrolyzate, coconut water, orange juice, bananas networks, and others - others. The addition of this compound produces undefined media. With enough research, this complex material should be replaced with some substance, perhaps an additional vitamins or amino acids. 
12.1.3. Carbon sourcePlants grown in tissue culture and heterotrophic because they are not sufficiently synthesize carbon requirement, then the sucrose must be added to the media. The carbon source provides energy for plant growth as well as building blocks to produce larger molecules needed for growth.Usually sucrose at a concentration of 1-5% is used as a carbon source but other carbon sources such as glucose, maltose, galactose and lactose are also used. When autoclaved sucrose, hydrolysis to produce glucose and fructose that can be used more efficiently by plants in culture. 
12.1.4 In order toNetwork generally cultured on solid media made such as by using a gel or replacement in order to be just as Gelrite or Phytagel. Concentrations that were used ranged between 0.7 - 1.0%. At high concentrations to be very loud, very little water is available, so that the diffusion of nutrients to plants is very bad. Order with high quality such as Difco BiTek expensive but more pure, does not contain other ingredients that may interfere with growth. Such as gelatin substitute sometimes - sometimes used in a commercial lab.Synthetic gel is known to cause hyperhidration (vitrification) which is a physiological problem that occurs in culture. To overcome this problem, a new product has been manufactured ole bernaman Agargel Sigma. This product is a mixture of agar and synthetic gels and offers the advantages of both products at the same time reducing the problem of vitrification. This product can be created in the lab by mixing 1 g of Gelrite (Phytagel) with 4 g of agar as a thickening agent for 1 L medium. 
12.1.5 pHmedia pH is usually set in the range 5.6 - 5.8 but different plants may require different pH for optimum growth. If the pH is higher than 6.0, the media may be too harsh and if the pH is less than 5.2, so that can not be compacted. 
12.1.6. Substances Growth RegulatorIn the media generally added growth regulators. Growth regulators will be discussed separately at week 
13.12.1.7. WaterDistilata water typically used in tissue culture, and many labs use aquabides (double distilled water). Some lab, on economic grounds, using rain water, but the cause is difficult to control the content of organic matter and non-organic media. 
12.2. Selection of MediaIf there is no initial information, usually starting with MS medium (Murashige and Skoog 1962). This medium contains high concentrations of salts and nitrates are higher than other media, and has been successfully used in a variety of dicotyledonous plants. For callus initiation, 2,4-D was added to the medium with a concentration of 1-5 mgl-1. For shoot multiplication, cytokinins like BAP and also given the added auxin, such as NAA at low concentrations. For root initiation, IBA at a concentration of 1-2 mgl-1 was added. The most difficult factor determined in tissue culture is a growth regulator and usually need to do a little research to determine the best concentration that will be used. There are 2 approaches: pertaman approach is to use basic MS media and examines the range of two different growth regulators. See table 12.1.Table 
 12.1 The experimental approach to select the most appropriate concentration of BAP and NAA in addition to MS medium containing 2% sucrose and 0.8% agar, modified from Bhojwani and Razdan (1983).BAP (mg / L)NAA (mg / L) 0 0.5 2.5 5.00 1 2 3 40.5 5 6 7 82.5 9 10 11 125.0 13 14 15 16The second approach is to use a broader method according deFossard (1976) diaman 4 categories, minerals, auxin, cytokinins tested organic and respectively - each at 3 concentrations. This great experiment requires 81 different treatments and very time-consuming but may be required for some very difficult cultured plants.
 12.3. Preparation of MediaThe most widely used medium is Murashige and Skoog (1962). The easiest way to prepare the MS media is to buy prepacked media that are sold commercially.Here is the thing - the important thing is fundamental in creating the media:1. Before you begin, prepare a sheet of media and media determine what and how much you will make. Write this information on the worksheet and check each step while you work. Sign and write the date on a worksheet and place it on a notebook. You can write a comment about any unusual or important happens when you make the media.2. Wash glassware with distilled water before starting to prepare the media.3. Measure about - about 90% of the final volume of distilled water, for example, the final volume of 900 ml to 1 liter, and enter into the beaker.4. If you are going to heat the solution, make sure you use a heat resistant tool.5. Water while stirring, slowly insert MS powder and stir until completely - completely dissolved. Wash the inside of the MS package with distilled water to take a rest - the rest of the powder and put it into the media solution.6. Enter other heat resistant materials - GM stock, myo-inositol, sucrose, BA, stir well.7. Adjust the pH of the media using NaOH, HCl, or KOH.8. Make the final volume of the media by using pumpkin drinks9. If you use that, insert the media into the mix before autoclaved.10. Media must always be autoclaved in a container the size of 1 1/2 x or 2x greater than the volume of media that the media does not spill.11. Pour the appropriate media kebuthan before autoclaved or autoclaved after, depending on needs.12. Tutp container when autoclaved, but not too tightly, so that no air exchange.13. Sterilized media with mengautoklaf at 1 kg/cm2 (15 psi), 121 º C for about 30 minutes. Larger volume (200 ml or more) may require a longer time. Use a slow exhaust.14. Allow the medium to cool to 55 º C before adding materials - materials that are not heat resistant (acetosyringone, claforan, kanamycin).15. Media usually poured into a petri dish with a volume of 25 ml per petri. It will produce about 40 petri per liter of media.16. Chill in the laminar media. Do not move the filled petri petri the media until cold.17. Save the media that has been chilled in the refrigerator.