Setting Up a Tissue Culture Lab
  Any laboratory, in which tissue culture techniques are performed, regardless of 
  the specific purpose, must contain a number of basic facilities. These usually 
  include the following:
  • A general washing area
  • A media preparation, sterilization, and storage area
  • An aseptic transfer area 
  • Environmentally controlled incubators or culture rooms 
  • An observation/data collection area. 
    Nutrient media: 
    The type and composition of culture media very strongly govern 
    the growth and morphogenesis of plant tissues. The choice of 
    tissue culture medium largely depends upon the species to be 
    cultured. For e.g. some species are sensitive to high salts or have 
    different requirements for PGRs. Some tissues show better 
    response on solid medium while others prefer a liquid medium. 
    Therefore, development of culture medium formulations is result of 
    systematic trial and experimentation considering specific 
    requirements of a particular culture system. White’s medium is 
    one of the earliest plant tissue culture media originally formulated 
    for root culture. Murashige and Skoog (MS) medium is the most 
    suitable and commonly used medium for plant regeneration from 
    tissues and callus. This is a high salt medium due to its content of 
    potassium and nitrogen salts. B5 medium works well 7 for 
    protoplast culture. It has lesser amounts of nitrate and particularly 
    ammonium salts than MS medium. Nitsch’s medium developed for 
    anther culture contains salt concentration intermediate to MS and 
    White. 
  Components of Tissue Culture Medium: 
  1.In organic Nutrients: 
  In vitro growth of plants also requires combination of macro and micronutrients like in 
  vivo growth.
   Macronutrients are classified as those elements which are required in concentration 
  greater than 0.5 mM/l. They include nitrogen, potassium, phosphorus, calcium, 
  magnesium and sulphur in form of salts in media. Macro nutrients have structural and 
  functional role in protein synthesis, cell wall synthesis enzyme Co-factors and membrane 
  integrity. 
  Nitrogen: Nitrogen is major component of all plant tissue culture media. Nitrogen helps 
  to synthesis complex organic molecule. Nitrogen is usually supplied in form of 
  ammonium (NH4 + ) and nitrate (NO3 - ) ions. Nitrate is superior to ammonium as the 
  sole N source but use of NH4 + checks the increase of pH towards alkalinity. Potassium K 
  ion is present in high concentration in the cytoplasm (100-200 mM) and in 
  chloroplast(20-200 mM). K+ is essential for maintaining the ion balancing, activation of 
  many enzymes. Maintaining osmotic pressure and osmotic regulation of cells.
  Culture media should contain atleast 25mM/l nitrogen and potassium. Other major 
  elements are adequate in concentration range of 1-3mM/l. 
  Calcium  Calcium functions with different enzymes as Cofactor and bound to the cell wall 
  and cell membrane.  It gives strength to cell wall.  It helps in the regulation of the the cell 
  membrane structure.  Deficiency causes disintegration of the membrane and shoot tip 
  necrosis.  Important in cell and root multiplication.  Supplied as calcium chloride and 
  calcium nitrate.
  Phosphorus  Phosphorus  Very important for energy metabolism.  Essential element for 
  DNA & RNA.  Deficiency may cause delayed growth and dark green coloure of leaves.  
  Supplied as sodium hydrogen phosphate or potassium hydrogen phosphate.
  Magnesium  Essential for enzymatic reactions, energy metabolism(ATP synthesis).  
  Supplied as magnesium sulphate.
  Sulphur  Important substance.  Deficiency of Sulphur inhibits protein synthesis and 
  decreases Chlorophyll in leaves.  Supplied as magnesium Sulphate and Potassium 
  Sulphate.
  Micronutrients  are  those  elements  which  are  required  at  a  concentration  less  than 
  0.05mM/l or 30ppm(mg/l). These include iron, manganese, zinc, boron, copper and 
  molybdenum. Concentration is always in uM.These inorganic elements although required 
  in small quantity are essential for plant growth, most critical of them being iron which is 
  not  available  at  low  pH.  Therefore,  it  is  provided  as  iron  EDTA  complex  to  make  it 
  available at wide range of pH.
  Zinc:  Zn plays an active role in protein synthesis and in the synthesis of tryptophan.  
  Supplied as Zinc Sulphate.
  Manganese:  Plays an important role in the Hill reaction of photasynthesis.  Required in 
  many enzymatic activities.  Supplied as Manganese Sulphate.
  Copper:  Copper plays important role in photosynthesis.  Intermediate of the electron 
  transport  chain  between  photo  system  1  &  2    Deficiency  leads  to  decrease  in 
  photosynthesis.  Supplied as Copper Sulphate.
  Molybdenum:    Essential  for  conversation  of  Nitrate  to  Ammonium.    Supplied  as 
  Sodium molybdate.
  Boron: Involves in different enzymatic activities.  Supplied as Boric acid.